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## **Meet the editor**

José Antonio Morales-González carried out his undergraduate rotating internship year at the Hospital de Jesús Public Welfare Institution (IAP), graduating as Surgeon Physician in 1997 from the Faculty of Higher Education (FES)-Iztacala National Autonomous University of Mexico (UNAM). He engaged in Doctoral studies in Biological Sciences (with a specialty in Cellular Bi-

ology) at the UNAM (1997-2001). Dr. Morales-Gonzalez has been awarded diverse recognitions: Hospital de Jesus IAP awards (1995 and 1996); Gustavo Baz Prada medal by the UNAM (1997); the Alfonso Caso medal for academic merit by the UNAM (2004); distinguished by the National System of Researchers (SNI) as National Researcher level 1 (2010-2016). He is the author of 42 internationally and nationally published articles. He is also editor and coordinator of 20 specialized books. Dr. Morales-González is a Full-time Titular Professor-Researcher C at the Area Académica de Farmacia, ICSa-UAEH, Mexico.

### Contents

**Preface XIII**

### **Section 1 Cell Biology, Chemical Free Radicals and Antioxidant Defenses 1**

### Chapter 1 **Cell Nanobiology 3**

María de Lourdes Segura-Valdez, Lourdes T. Agredano-Moreno, Tomás Nepomuceno-Mejía, Rogelio Fragoso-Soriano, Georgina Álvarez-Fernández, Alma Zamora-Cura, Reyna Lara-Martínez and Luis F. Jiménez-García

### Chapter 2 **The Exogenous Antioxidants 33** Alejandro Chehue Romero, Elena G. Olvera Hernández, Telma Flores Cerón and Angelina Álvarez Chávez


Chapter 6 **Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion: A Review 131**

Antonio Cilla, Amparo Alegría, Reyes Barberá and María Jesús Lagarda


Eduardo Madrigal-Santillán, Eduardo Madrigal-Bujaidar, Sandra Cruz-Jaime, María del Carmen Valadez-Vega, María Teresa Sumaya-Martínez, Karla Guadalupe Pérez-Ávila and José Antonio Morales-González

Chapter 8 **Inflammatory Environmental, Oxidative Stress in Tumoral Progression 187**

César Esquivel-Chirino, Jaime Esquivel-Soto, José Antonio Morales-González, Delina Montes Sánchez, Jose Luis Ventura-Gallegos, Luis Enrique Hernández-Mora and Alejandro Zentella-Dehesa


Contents **VII**

Chapter 6 **Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion:**

**Section 2 Cell Damage by Free Radicals - Oxidative Stress**

Chapter 7 **The Chemoprevention of Chronic Degenerative Disease**

Chapter 8 **Inflammatory Environmental, Oxidative Stress in Tumoral**

**Through Dietary Antioxidants: Progress, Promise and**

Eduardo Madrigal-Santillán, Eduardo Madrigal-Bujaidar, Sandra Cruz-Jaime, María del Carmen Valadez-Vega, María Teresa Sumaya-Martínez, Karla Guadalupe Pérez-Ávila and José Antonio Morales-

César Esquivel-Chirino, Jaime Esquivel-Soto, José Antonio Morales-González, Delina Montes Sánchez, Jose Luis Ventura-Gallegos, Luis

Maria-Luisa Lazo-de-la-Vega-Monroy and Cristina Fernández-Mejía

Enrique Hernández-Mora and Alejandro Zentella-Dehesa

Chapter 9 **Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins**

Chapter 10 **Oxidative Stress and Antioxidant Therapy in Chronic Kidney**

Chapter 11 **Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche 265**

Chapter 13 **Oxidative Stress in Periodontal Disease and Oral Cancer 317**

Fernández and Torres-Carmona Marco

Claudia Camelia Calzada Mendoza and Carlos Alberto Jiménez

Mario Nava-Villalba, German González-Pérez, Maribel Liñan-

Antonio Cilla, Amparo Alegría, Reyes Barberá and María Jesús

**A Review 131**

**in Disease 153**

**Evidences 155**

**Progression 187**

Nalini Rajamannan

Zamarripa

**with Antioxidant Actions 209**

**and Cardiovascular Disease 233** David M. Small and Glenda C. Gobe

Chapter 12 **Menopause Induces Oxidative Stress 289**

González

Lagarda

### **Section 3 Aging 329**

Chapter 14 **Aging, Oxidative Stress and Antioxidants 331** B. Poljsak and I. Milisav

#### **Section 4 Disease and Therapy - A Role for Antioxidants 355**


José Luis Silencio Barrita and María del Socorro Santiago Sánchez

Chapter 19 **Protective Effect of Silymarin on Liver Damage by Xenobiotics 485** José A. Morales-González, Evila Gayosso-Islas, Cecilia Sánchez-

Moreno, Carmen Valadez-Vega, Ángel Morales-González, Jaime Esquivel-Soto, Cesar Esquivel-Chirino, Manuel García-Luna y González-Rubio and Eduardo Madrigal-Santillán

Preface

pathologies.

rate, worldwide.

Aerobic organisms, such as humans, possess several metabolic pathways in the process of obtaining energy, utilizing oxygen as final electron acceptor. Thus, this is indispensable for the condition of life to exist. Paradoxically, under certain conditions in which reduction in‐ termediaries, mainly of oxygen, accumulate, Free radicals (FR) are produced, which are ca‐ pable of producing damage at a molecular level, which manifests in diverse chronic

Oxidative stress constitutes an alteration produced by disequilibrium between generation of FR and the antioxidant system, which can lead to a damage state, in particular of the biomo‐ lecules. Due to the wide variety of reactions produced by FR, these are related with the de‐ velopment and evolution of diverse illnesses such as the chronic degenerative diseases, for example, atherosclerotic disease, high blood pressure, renal disorders, and obesity, in which FR plays a primordial role in the development of long-term complications of these illnesses. Diet and nutrition are very important in the promotion and maintenance of health through‐ out life. For some time, the function has been recognized of diet and nutrition as determin‐ ing factors of non-transmittable diseases; thus, a healthy diet is one of the pillars of health that has become consolidated in our lifestyle over the past several years. A search has been conducted for all of the properties of foods that are beneficial in increasing or maintaining our state of health. Likewise, there are natural and synthetic molecules that are capable of inactivating FR; these FR scavengers are found classified in groups or families of com‐ pounds that are in general denominated "antioxidants". The main objective of mechanisms of defense is that of transforming FR into less-damaging products or to neutralize their ma‐ lignancy completely, performing this by means of a mechanism called redox potential, through oxido-reduction reactions in which a reducer agent participates that donates elec‐

There are elements in the diet, in addition to their nutritional characteristics, in which the property is recognized for being antioxidant agents. Among the many studied, we find vita‐ min C, vitamin E, vitamin D, vitamin A, some amino acids, the flavonoids, and some oli‐ goelements. All of these antioxidant elements represent an alternative for the treatment and prevention of chronic degenerative diseases, which represent a very high morbid-mortality

The work presented here responds to the need of finding, in a sole document, the effect of oxidative stress at different levels, as well as treatment with antioxidants to revert and di‐ minish damage. On the other hand, it is noteworthy that the work will be published for health professionals and researchers who are expert in the theme, and that it contains cur‐ rent, scientifically based information. Thus, I am convinced that the work carried out will be

trons or through an oxidating agent that removes electrons from FR.

### Preface

Aerobic organisms, such as humans, possess several metabolic pathways in the process of obtaining energy, utilizing oxygen as final electron acceptor. Thus, this is indispensable for the condition of life to exist. Paradoxically, under certain conditions in which reduction in‐ termediaries, mainly of oxygen, accumulate, Free radicals (FR) are produced, which are ca‐ pable of producing damage at a molecular level, which manifests in diverse chronic pathologies.

Oxidative stress constitutes an alteration produced by disequilibrium between generation of FR and the antioxidant system, which can lead to a damage state, in particular of the biomo‐ lecules. Due to the wide variety of reactions produced by FR, these are related with the de‐ velopment and evolution of diverse illnesses such as the chronic degenerative diseases, for example, atherosclerotic disease, high blood pressure, renal disorders, and obesity, in which FR plays a primordial role in the development of long-term complications of these illnesses.

Diet and nutrition are very important in the promotion and maintenance of health through‐ out life. For some time, the function has been recognized of diet and nutrition as determin‐ ing factors of non-transmittable diseases; thus, a healthy diet is one of the pillars of health that has become consolidated in our lifestyle over the past several years. A search has been conducted for all of the properties of foods that are beneficial in increasing or maintaining our state of health. Likewise, there are natural and synthetic molecules that are capable of inactivating FR; these FR scavengers are found classified in groups or families of com‐ pounds that are in general denominated "antioxidants". The main objective of mechanisms of defense is that of transforming FR into less-damaging products or to neutralize their ma‐ lignancy completely, performing this by means of a mechanism called redox potential, through oxido-reduction reactions in which a reducer agent participates that donates elec‐ trons or through an oxidating agent that removes electrons from FR.

There are elements in the diet, in addition to their nutritional characteristics, in which the property is recognized for being antioxidant agents. Among the many studied, we find vita‐ min C, vitamin E, vitamin D, vitamin A, some amino acids, the flavonoids, and some oli‐ goelements. All of these antioxidant elements represent an alternative for the treatment and prevention of chronic degenerative diseases, which represent a very high morbid-mortality rate, worldwide.

The work presented here responds to the need of finding, in a sole document, the effect of oxidative stress at different levels, as well as treatment with antioxidants to revert and di‐ minish damage. On the other hand, it is noteworthy that the work will be published for health professionals and researchers who are expert in the theme, and that it contains cur‐ rent, scientifically based information. Thus, I am convinced that the work carried out will be

of utmost usefulness for the active health professional as well as for the health professional in training, with the purpose of creating a novel panorama on study of the theme as well as showing an alternative in the treatment of the chronic diseases that are affecting our popula‐ tion to such a great extent.

Therefore, the objective of this book is to understand the mechanism by which free radicals contribute to the development of complications in chronic degenerative diseases. On knowing how FR are generated, the interaction of FR with cells, and the reactions in which they are involved, it will be possible to establish novel treatment alternatives, which could comprise antioxidants, providing the scientific community with the opportunity to make their research‐ es known and enabling the readers to deepen or broaden their knowledge in this field.

Finally, I dedicate this book to Julia González-González, with all my love for your teachings and example.

> **Prof. Dr. José Antonio Morales-González** Área Académica de Farmacia, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, México

**Section 1**

**Cell Biology, Chemical Free Radicals and**

**Antioxidant Defenses**

**Cell Biology, Chemical Free Radicals and Antioxidant Defenses**

of utmost usefulness for the active health professional as well as for the health professional in training, with the purpose of creating a novel panorama on study of the theme as well as showing an alternative in the treatment of the chronic diseases that are affecting our popula‐

Therefore, the objective of this book is to understand the mechanism by which free radicals contribute to the development of complications in chronic degenerative diseases. On knowing how FR are generated, the interaction of FR with cells, and the reactions in which they are involved, it will be possible to establish novel treatment alternatives, which could comprise antioxidants, providing the scientific community with the opportunity to make their research‐

Finally, I dedicate this book to Julia González-González, with all my love for your teachings

**Prof. Dr. José Antonio Morales-González**

Universidad Autónoma del Estado de Hidalgo, México

Área Académica de Farmacia, Instituto de Ciencias de la Salud,

es known and enabling the readers to deepen or broaden their knowledge in this field.

tion to such a great extent.

and example.

X Preface

**Chapter 1**

**Cell Nanobiology**

http://dx.doi.org/10.5772/52003

**1. Introduction**

**1.1. Cell nanobiology**

María de Lourdes Segura-Valdez, Lourdes T. Agredano-Moreno, Tomás Nepomuceno-Mejía, Rogelio Fragoso-Soriano,

Georgina Álvarez-Fernández, Alma Zamora-Cura, Reyna Lara-Martínez and Luis F. Jiménez-García

We define cell nanobiology as an emergent scientific area trying to approach the study of the *in situ* cell processes ocurring at the nanoscale. Therefore, it is part of cell biology but mainly deals with an interphase between analytical methods such as X-ray crystallography producing models at atomic or molecular resolution, and direct nanoscale imaging with high resolution microscopes such as scanning probe microscopes, electron microscopes and super-resolution

The main flow of genetic information represented as the so-called central dogma of molecular

During this pathway, nanoscale particles represent substrates of different moments. During transcription, nuclear particles are involved in transcription and processing of RNA, both, premRNA and pre-rRNA. pre-mRNA is transcribed and processed in the nucleoplasm while prerRNA is transcribed and processed within the nucleolus, the major known ribonucleoproteins structure where ribosome biogenesis and other functions of eukaryotic cell take place. Once in the cytoplasm, translation takes place in the ribosome, also a major ribonucleoprotein

> © 2013 Segura-Valdez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

microscopes. Several cell structures are involved in nanoscale processes (Figure 1).

*1.1.1. An overview of cell structure under a genomic approach of gene expression*

biology *in situ*, illustrates the major secretory pathway in the cell (Figure 2).

Additional information is available at the end of the chapter

**Chapter 1**

### **Cell Nanobiology**

María de Lourdes Segura-Valdez, Lourdes T. Agredano-Moreno, Tomás Nepomuceno-Mejía, Rogelio Fragoso-Soriano, Georgina Álvarez-Fernández, Alma Zamora-Cura, Reyna Lara-Martínez and Luis F. Jiménez-García

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52003

**1. Introduction**

### **1.1. Cell nanobiology**

We define cell nanobiology as an emergent scientific area trying to approach the study of the *in situ* cell processes ocurring at the nanoscale. Therefore, it is part of cell biology but mainly deals with an interphase between analytical methods such as X-ray crystallography producing models at atomic or molecular resolution, and direct nanoscale imaging with high resolution microscopes such as scanning probe microscopes, electron microscopes and super-resolution microscopes. Several cell structures are involved in nanoscale processes (Figure 1).

### *1.1.1. An overview of cell structure under a genomic approach of gene expression*

The main flow of genetic information represented as the so-called central dogma of molecular biology *in situ*, illustrates the major secretory pathway in the cell (Figure 2).

During this pathway, nanoscale particles represent substrates of different moments. During transcription, nuclear particles are involved in transcription and processing of RNA, both, premRNA and pre-rRNA. pre-mRNA is transcribed and processed in the nucleoplasm while prerRNA is transcribed and processed within the nucleolus, the major known ribonucleoproteins structure where ribosome biogenesis and other functions of eukaryotic cell take place. Once in the cytoplasm, translation takes place in the ribosome, also a major ribonucleoprotein

particle of 10-15 nm in diameter. When the synthesized protein contains a signal peptide, it is translocated into the rough endoplasmic reticulum, helped by the signal recognition particle or SRP, another major and conserved ribonucleoprotein. The transport to Golgi apparatus by the intermediated zone and the TGN producing the three derivatives from the Golgi apparatus are mediated by vesicles [see 1].

**a.** Semenogelin

Semenogelin is the most abundant protein in the semen of mammals. It is a glycosylated protein that is responsible for properties such as density. As an example, the semenogelin of the tamarin *Saguinus oedipus* is used to show how the signals in the nucleic acids and proteins determine the intracellular pathways associated to that expression. Its expression includes intranuclear events as transcription by RNA polymerase II from a split gene consisting of 3 exones and 2 intrones, processing of the transcript as 5' end methylation, 3' polyadenylation and splicing. All of them are associated to nuclear particles. Once in the cytoplasm, the mature transcript or mRNA associates to a ribosome that in turns translates the transcript. If the resulting protein contains a signal peptide, the signal recognition particle or SRP -a very well conserved RNA+protein complex- binds to it and associates to the rough endoplasmic reticulum, giving rise to the translocation process that introduces that protein to the lumen. Once there, N-glycosylation takes place at several asparagine residues following the basic rule of adjacent aminoacids showing a basic rule as Asn-X(except proline)-Ser or Asn. In *S. oedipus* semenogelin, there are 14 N-glycosylation sites. The protein then continues flowing through the Golgi apparatus or complex and at the TGN a secretory vesicle forms containing the protein that finally is secreted by the epithelial cell of seminal vesicle. The analysis of the gene sequences, as well as the transcription, processing, translation and post-translational

Cell Nanobiology

5

http://dx.doi.org/10.5772/52003

**Figure 2.** A general overview of the cell structure and function. The diagram illustrates the *in situ* flow of genetic infor‐ mation of a secretory protein encoded in the genome within the cell nucleus. A gene is copied into a pre-mRNA that is processed to mRNA within the nucleus. mRNA in the cytoplasm may contain a signal sequence that allows entrance to rough endoplasmic reticulum and further to Golgi complex. The protein inside a vesicle is secreted out of the cell.

products can predict the cell structures involved in the process [see 1].

**Figure 1.** Cell nanobiology proposes to study cell structures using *in situ* high resolution microscopical approaches as electron and atomic force microscopy that could complement molecular and biochemical data to better understand a physiological role at the nanoscale.

#### **a.** Semenogelin

particle of 10-15 nm in diameter. When the synthesized protein contains a signal peptide, it is translocated into the rough endoplasmic reticulum, helped by the signal recognition particle or SRP, another major and conserved ribonucleoprotein. The transport to Golgi apparatus by the intermediated zone and the TGN producing the three derivatives from the Golgi apparatus

**Figure 1.** Cell nanobiology proposes to study cell structures using *in situ* high resolution microscopical approaches as electron and atomic force microscopy that could complement molecular and biochemical data to better understand a

are mediated by vesicles [see 1].

4 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

physiological role at the nanoscale.

Semenogelin is the most abundant protein in the semen of mammals. It is a glycosylated protein that is responsible for properties such as density. As an example, the semenogelin of the tamarin *Saguinus oedipus* is used to show how the signals in the nucleic acids and proteins determine the intracellular pathways associated to that expression. Its expression includes intranuclear events as transcription by RNA polymerase II from a split gene consisting of 3 exones and 2 intrones, processing of the transcript as 5' end methylation, 3' polyadenylation and splicing. All of them are associated to nuclear particles. Once in the cytoplasm, the mature transcript or mRNA associates to a ribosome that in turns translates the transcript. If the resulting protein contains a signal peptide, the signal recognition particle or SRP -a very well conserved RNA+protein complex- binds to it and associates to the rough endoplasmic reticulum, giving rise to the translocation process that introduces that protein to the lumen. Once there, N-glycosylation takes place at several asparagine residues following the basic rule of adjacent aminoacids showing a basic rule as Asn-X(except proline)-Ser or Asn. In *S. oedipus* semenogelin, there are 14 N-glycosylation sites. The protein then continues flowing through the Golgi apparatus or complex and at the TGN a secretory vesicle forms containing the protein that finally is secreted by the epithelial cell of seminal vesicle. The analysis of the gene sequences, as well as the transcription, processing, translation and post-translational products can predict the cell structures involved in the process [see 1].

**Figure 2.** A general overview of the cell structure and function. The diagram illustrates the *in situ* flow of genetic infor‐ mation of a secretory protein encoded in the genome within the cell nucleus. A gene is copied into a pre-mRNA that is processed to mRNA within the nucleus. mRNA in the cytoplasm may contain a signal sequence that allows entrance to rough endoplasmic reticulum and further to Golgi complex. The protein inside a vesicle is secreted out of the cell.

### *1.1.2. Some nanoscale cell structures*

There are many cell structures or products made by cells that could be analyzed under the present approach. Some of them are indicted in Figure 1, but there are others as extracellular matriz components, cytoskeleton elements, etc.; virus are also nanometric structures associ‐ ated always to cell organelles. Here we will give an overview of some of the cell components, as examples.

#### **a.** Nuclear particles

In eukaryote cells, transcription and processing mainly takes place within the cell nucleus, associated to nuclear particles that are well known since a method for ribonucleoprotein (RNP) structures was described in 1969 [2]. These particles are few nanometers in diameter or lengh. To date, several nuclear RNPs have been described including involved in mRNA metabolism: perichromatin fibers, perichromatin granules, interchromatin granules in mammals. In insects, Balbiani ring granules are well known structures [3]. In 1992 Lacandonia granules were described for some plants [4]. In addition, other nuclear bodies around 300-400 nm in diameter have been described involved in gene expression. As for rRNA transcription and processing, the nucleolus is a nuclear organelle containing pre-ribosomes in the granular component that are about 10-20 nm in diameter.

> **Figure 3.** Lacandonia granules are nanoscale (32 nm in diameter) and abundant particles (arrows) in the nucleoplasm of the plant *Lacandonia schismatica*. Transmission electron microscopy image of a cell in the teguments of the flower.

Cell Nanobiology

7

http://dx.doi.org/10.5772/52003

Snapshots of ribosome intermediates provided by cryo-EM and x-ray crystallography, associated translation factors, and transfer RNA (tRNA) have allowed dynamic aspects of protein translation to be reconstructed. For example, recent cryo-EM reconstructions of translating ribosomes allowed direct visualization of the nascent polypeptide chain inside the ribosomal tunnel at subnanometer resolution [13-15]. The dimension of the ribosomal tunnel in bacterial, archaeal, and eukaryotic cytoplasmic ribosomes is conserved in evolution [16 -18]. The ribosomal tunnel in the large ribosomal subunit is ~80 Å long, 10–20 Å wide, and pre‐ dominantly composed of core rRNA [19]. The tunnel is clearly not just a passive conduit for the nascent chain, but rather a compartment in a dynamic molecular dialogue with the nascent chain. This interplay might not only affect the structure and function of the ribosome and associated factors, but also the conformation and folding of the nascent chain [20]. As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large

Peptide bond formation on the bacterial ribosome and perhaps on the ribosomes from all organisms is catalyzed by ribosomal RNA as well as ribosomal protein and also by the 2'-OH group of the peptidyl-tRNA substrate in the P site. The high resolution crystal structures of two ribosomal complexes from T. thermophilus [21] revealed that ribosomal proteins L27 and L16 of the 50S subunit stabilize the CCA-ends of both tRNAs in the peptidyl-transfer reaction,

subunit and emerges at the solvent side, where protein folding occurs.

Cc, compact chromatin.


Ribosomes are the universal ribonucleoprotein particles that translate the genetic code into proteins. The shape and dimensions of the ribosome were first visualized by electron micro‐ scopy [6-8]. Ribosomes have diameters of about 25 nanometers in size and are roughly twothirds RNA and one-third protein. All ribosomes have two subunits, one about twice the mass of the other. The ribosome basic structure and functions are well-known. There are 70S ribosomes common to prokaryotes and 80S ribosomes common to eukaryotes. The bacterial ribosome is composed of 3 RNA molecules and more than 50 proteins. In humans, the small ribosome unit has 1 large RNA molecule and about 32 proteins; the large subunit has 3 RNA molecules, and about 46 proteins. Each subunit has thousands of nucleotides and amino acids, with hundreds of thousands of atoms. The small subunit (0.85 MDa) initiates mRNA engage‐ ment, decodes the message, governs mRNA and tRNA translocation, and controls fidelity of codon–anticodon interactions and the large subunit catalyzes peptide bond formation.

In 1980, the first three-dimensional crystals of the ribosomal 50S subunit from the thermophile bacterium *Geobacillus stearothermophilus* were reported [9]. Since then, ribosome crystallogra‐ phy advanced rapidly. To date crystal structures have been determined for the large ribosomal subunit from the archaeon *Haloarcula marismortui* at 2.4 Å [10] and the 30S ribosomal subunit from *Thermus thermophilus*[11]. The structure of the entire 70S ribosome in complex with tRNA ligands (at 5.5Å resolution) emerged shortly after the structures of the initial subunits [12].

*1.1.2. Some nanoscale cell structures*

6 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

as examples.

**a.** Nuclear particles

are about 10-20 nm in diameter.

**i.** The ribosome

**b.** Rough endoplasmic reticulum particles

There are many cell structures or products made by cells that could be analyzed under the present approach. Some of them are indicted in Figure 1, but there are others as extracellular matriz components, cytoskeleton elements, etc.; virus are also nanometric structures associ‐ ated always to cell organelles. Here we will give an overview of some of the cell components,

In eukaryote cells, transcription and processing mainly takes place within the cell nucleus, associated to nuclear particles that are well known since a method for ribonucleoprotein (RNP) structures was described in 1969 [2]. These particles are few nanometers in diameter or lengh. To date, several nuclear RNPs have been described including involved in mRNA metabolism: perichromatin fibers, perichromatin granules, interchromatin granules in mammals. In insects, Balbiani ring granules are well known structures [3]. In 1992 Lacandonia granules were described for some plants [4]. In addition, other nuclear bodies around 300-400 nm in diameter have been described involved in gene expression. As for rRNA transcription and processing, the nucleolus is a nuclear organelle containing pre-ribosomes in the granular component that

Ribosomes are the universal ribonucleoprotein particles that translate the genetic code into proteins. The shape and dimensions of the ribosome were first visualized by electron micro‐ scopy [6-8]. Ribosomes have diameters of about 25 nanometers in size and are roughly twothirds RNA and one-third protein. All ribosomes have two subunits, one about twice the mass of the other. The ribosome basic structure and functions are well-known. There are 70S ribosomes common to prokaryotes and 80S ribosomes common to eukaryotes. The bacterial ribosome is composed of 3 RNA molecules and more than 50 proteins. In humans, the small ribosome unit has 1 large RNA molecule and about 32 proteins; the large subunit has 3 RNA molecules, and about 46 proteins. Each subunit has thousands of nucleotides and amino acids, with hundreds of thousands of atoms. The small subunit (0.85 MDa) initiates mRNA engage‐ ment, decodes the message, governs mRNA and tRNA translocation, and controls fidelity of codon–anticodon interactions and the large subunit catalyzes peptide bond formation.

In 1980, the first three-dimensional crystals of the ribosomal 50S subunit from the thermophile bacterium *Geobacillus stearothermophilus* were reported [9]. Since then, ribosome crystallogra‐ phy advanced rapidly. To date crystal structures have been determined for the large ribosomal subunit from the archaeon *Haloarcula marismortui* at 2.4 Å [10] and the 30S ribosomal subunit from *Thermus thermophilus*[11]. The structure of the entire 70S ribosome in complex with tRNA ligands (at 5.5Å resolution) emerged shortly after the structures of the initial subunits [12].

**Figure 3.** Lacandonia granules are nanoscale (32 nm in diameter) and abundant particles (arrows) in the nucleoplasm of the plant *Lacandonia schismatica*. Transmission electron microscopy image of a cell in the teguments of the flower. Cc, compact chromatin.

Snapshots of ribosome intermediates provided by cryo-EM and x-ray crystallography, associated translation factors, and transfer RNA (tRNA) have allowed dynamic aspects of protein translation to be reconstructed. For example, recent cryo-EM reconstructions of translating ribosomes allowed direct visualization of the nascent polypeptide chain inside the ribosomal tunnel at subnanometer resolution [13-15]. The dimension of the ribosomal tunnel in bacterial, archaeal, and eukaryotic cytoplasmic ribosomes is conserved in evolution [16 -18]. The ribosomal tunnel in the large ribosomal subunit is ~80 Å long, 10–20 Å wide, and pre‐ dominantly composed of core rRNA [19]. The tunnel is clearly not just a passive conduit for the nascent chain, but rather a compartment in a dynamic molecular dialogue with the nascent chain. This interplay might not only affect the structure and function of the ribosome and associated factors, but also the conformation and folding of the nascent chain [20]. As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large subunit and emerges at the solvent side, where protein folding occurs.

Peptide bond formation on the bacterial ribosome and perhaps on the ribosomes from all organisms is catalyzed by ribosomal RNA as well as ribosomal protein and also by the 2'-OH group of the peptidyl-tRNA substrate in the P site. The high resolution crystal structures of two ribosomal complexes from T. thermophilus [21] revealed that ribosomal proteins L27 and L16 of the 50S subunit stabilize the CCA-ends of both tRNAs in the peptidyl-transfer reaction, suggesting that peptide chains from both these proteins take part in the catalytic mechanism of peptide bond formation.

nucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degra‐ dation of the incomplete proteins. The cryo-electron microscopy structures of tmRNA– SmpB accommodated or translocated into stalled ribosomes demonstrate how tmRNA– SmpB crosses the ribosome and how as the problematic mRNA is ejected, the tmRNA re‐ sume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence [22]. Recently, the crystal structure of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate

Cell Nanobiology

9

http://dx.doi.org/10.5772/52003

**Figure 5.** Representation of a prokaryotic (a) and a eukaryotic ribosome (a). Each one is an RNP is constituted by two subunits, each containing rRNA and proteins. b) a model to show the nanoscale morphology of a mammalian cyto‐

The structure of the ribosome at high resolution reveals the molecular details of the antibioticbinding sites, explain how drugs exercise their inhibitory effects. Also, the crystal structures help us to speculate about how existing drugs might be improved, or novel drugs created, to circumvent resistance [24]. Recently, ribosome engineering has emerged as a new tool to promote new crystal forms and improve our knowledge of protein synthesis. To explore the crystallization of functional complexes of ribosomes with GTPase, a mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic

In contrast to their bacterial counterparts, eukaryotic ribosomes are much larger and more complex, containing additional rRNA in the form of so-called expansion segments (ES) as well as many additional r-proteins and r-protein extensions [26]. The first structural models for the eukaryotic (yeast) ribosome were built using 15-A° cryo–electron microscopy (cryo-EM) maps fitted with structures of the bacterial SSU [11] and archaeal LSU [10], thus identifying the location of a total of 46 eukaryotic r-proteins with bacterial and/or archaeal homologs as well

plasmic ribosome (small [S] and large [L] subunits).

acid in a previously unobserved crystal form [25].

as many ES [27].

decoding in the absence of an mRNA codon in the A site of the ribosome [23].

**Figure 4.** Nucleolus of a PtK2 cell. Within the cell nucleus (N), the nucleolus displays three different components named fibrillar center (fc), dense fibrillar component (dfc) and granular components (g). In the inset, a high magnifica‐ tion of the nucleolus shows granular particles or pre-ribosomes in the granular component (g). In the cytoplasm (c), ribosomes are also visible (arrow).

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalyzing peptide bond formation between amino acids. When bacte‐ rial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA–SmpB ribo‐ nucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degra‐ dation of the incomplete proteins. The cryo-electron microscopy structures of tmRNA– SmpB accommodated or translocated into stalled ribosomes demonstrate how tmRNA– SmpB crosses the ribosome and how as the problematic mRNA is ejected, the tmRNA re‐ sume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence [22]. Recently, the crystal structure of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome [23].

suggesting that peptide chains from both these proteins take part in the catalytic mechanism

**Figure 4.** Nucleolus of a PtK2 cell. Within the cell nucleus (N), the nucleolus displays three different components named fibrillar center (fc), dense fibrillar component (dfc) and granular components (g). In the inset, a high magnifica‐ tion of the nucleolus shows granular particles or pre-ribosomes in the granular component (g). In the cytoplasm (c),

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalyzing peptide bond formation between amino acids. When bacte‐ rial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA–SmpB ribo‐

of peptide bond formation.

8 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ribosomes are also visible (arrow).

**Figure 5.** Representation of a prokaryotic (a) and a eukaryotic ribosome (a). Each one is an RNP is constituted by two subunits, each containing rRNA and proteins. b) a model to show the nanoscale morphology of a mammalian cyto‐ plasmic ribosome (small [S] and large [L] subunits).

The structure of the ribosome at high resolution reveals the molecular details of the antibioticbinding sites, explain how drugs exercise their inhibitory effects. Also, the crystal structures help us to speculate about how existing drugs might be improved, or novel drugs created, to circumvent resistance [24]. Recently, ribosome engineering has emerged as a new tool to promote new crystal forms and improve our knowledge of protein synthesis. To explore the crystallization of functional complexes of ribosomes with GTPase, a mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form [25].

In contrast to their bacterial counterparts, eukaryotic ribosomes are much larger and more complex, containing additional rRNA in the form of so-called expansion segments (ES) as well as many additional r-proteins and r-protein extensions [26]. The first structural models for the eukaryotic (yeast) ribosome were built using 15-A° cryo–electron microscopy (cryo-EM) maps fitted with structures of the bacterial SSU [11] and archaeal LSU [10], thus identifying the location of a total of 46 eukaryotic r-proteins with bacterial and/or archaeal homologs as well as many ES [27].

Ribosome biogenesis is regulated by the conserved protein kinase TOR (target of rapamycin), a member of the ATM-family protein. TOR up-regulates transcription of rRNA and mRNA for ribosomal proteins in both yeast and mammals [28-30]. Recent results indicate that in yeast, conserved kinases of the LAMMER/Cdc-like and GSK-3 families function downstream of TOR complex 1 to repress ribosome and tRNA synthesis in response to nutrient limitation and other types of cellular stress [31].

chain complex (RNC) to the protein-conducting channel [50]. Despite the diversity of signal sequences, SRP productively recognizes and selectively binds them, and this binding event serves as the critical sorting step in protein localization within the cell. The structural details that confer on SRP this distinctive ability are poorly understood. SRP signal sequences are characterized by a core of 8–12 hydrophobic amino acids that preferentially form an α-helix, but are otherwise highly divergent in length, shape, and amino acid composition [51-52]. This and the unusual abundance of methionine in the SRP54 M-domain led to the 'methionine bristle' hypothesis, in which the flexible side chains of methionine provide a hydrophobic

Cell Nanobiology

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http://dx.doi.org/10.5772/52003

environment with sufficient plasticity to accommodate diverse signal sequences [53].

**Figure 6.** The signal recognition particle. During protein synthesis of the secretory pathway, the signal peptide binds to SRP, an RNP particle containing a small RNA and 6 different proteins (b, [modified from 60]). A model for SRP at

nanoscale is shown in (c).

### **ii.** The signal recognition particle (SRP)

The Signal Recognition Particle (SRP) is an evolutionarily conserved rod-shaped 11S ribonu‐ cleoprotein particle, 5–6 nm wide and 23–24 nm long [32]. It comprises an essential component of the cellular machinery responsible for the co-translational targeting of proteins to their proper membrane destinations [33].

Although SRP is essential and present in all kingdoms of life maintaining its general function, structurally it shows high diversity. Vertebrates SRP consists of a single ~ 300-bp RNA (SRP RNA or 7S RNA) and six polypeptides designated SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72. It can be divided into two major functional domains: the Alu domain (comprising the proteins SRP9 and -14) and the S domain (SRP19, -54, -68, and -72). The S domain functions in signal sequence recognition and SR interaction, whereas the Alu domain is required for translational arrest on signal sequence recognition [34]. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA [35]. SRP54, comprises an N-terminal domain (N, a four-helix bundle), a central GTPase domain [G, a ras-like GTPase fold, with an additional unique α-β- α insertion box domain (IBD)], and a methionine-rich Cterminal domain [36-37]. The N and G domains are structurally and functionally coupled; together, they build the NG domain that is connected to the M domain through a flexible linker [38]. The M domain anchors SRP54 to SRP RNA and carries out the principal function of signal sequence recognition [39-41]. The NG domain interacts with the SR in a GTP-dependent manner [43].

SRP is partially assembled in the nucleus and partially in the nucleolus. In agreement with that, nuclear localization for SRP proteins SRP9/14, SRP68, SRP72 and SRP19 has been determined [44]. After the transport into the nucleus the subunits bind SRP RNA and form a pre-SRP which is exported to the cytoplasm where the final protein, Srp54p, is incorporated [45-47]. Although this outline of the SRP assembly pathway has been determined, factors that facilitate this and/or function in quality control of the RNA are poorly understood [48]. SRP assembly starts during 7S RNA transcription by RNA polymerase III in the nucleolus, by binding of the SRP 9/14 heterodimer and formation of Alu-domain. Prior to transportation to the nucleus SRP9 and SRP14 form the heterodimer in the cytoplasm, a prerequisite for the binding to 7S RNA [49].

The signal recognition particle displays three main activities in the process of cotranslational targeting: (I) binding to signal sequences emerging from the translating ribosome, (II) pausing of peptide elongation, and (III) promotion of protein translocation through docking to the membrane-bound SRP receptor (FtsY in prokaryotes) and transfer of the ribosome nascent chain complex (RNC) to the protein-conducting channel [50]. Despite the diversity of signal sequences, SRP productively recognizes and selectively binds them, and this binding event serves as the critical sorting step in protein localization within the cell. The structural details that confer on SRP this distinctive ability are poorly understood. SRP signal sequences are characterized by a core of 8–12 hydrophobic amino acids that preferentially form an α-helix, but are otherwise highly divergent in length, shape, and amino acid composition [51-52]. This and the unusual abundance of methionine in the SRP54 M-domain led to the 'methionine bristle' hypothesis, in which the flexible side chains of methionine provide a hydrophobic environment with sufficient plasticity to accommodate diverse signal sequences [53].

Ribosome biogenesis is regulated by the conserved protein kinase TOR (target of rapamycin), a member of the ATM-family protein. TOR up-regulates transcription of rRNA and mRNA for ribosomal proteins in both yeast and mammals [28-30]. Recent results indicate that in yeast, conserved kinases of the LAMMER/Cdc-like and GSK-3 families function downstream of TOR complex 1 to repress ribosome and tRNA synthesis in response to nutrient limitation and other

The Signal Recognition Particle (SRP) is an evolutionarily conserved rod-shaped 11S ribonu‐ cleoprotein particle, 5–6 nm wide and 23–24 nm long [32]. It comprises an essential component of the cellular machinery responsible for the co-translational targeting of proteins to their

Although SRP is essential and present in all kingdoms of life maintaining its general function, structurally it shows high diversity. Vertebrates SRP consists of a single ~ 300-bp RNA (SRP RNA or 7S RNA) and six polypeptides designated SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72. It can be divided into two major functional domains: the Alu domain (comprising the proteins SRP9 and -14) and the S domain (SRP19, -54, -68, and -72). The S domain functions in signal sequence recognition and SR interaction, whereas the Alu domain is required for translational arrest on signal sequence recognition [34]. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA [35]. SRP54, comprises an N-terminal domain (N, a four-helix bundle), a central GTPase domain [G, a ras-like GTPase fold, with an additional unique α-β- α insertion box domain (IBD)], and a methionine-rich Cterminal domain [36-37]. The N and G domains are structurally and functionally coupled; together, they build the NG domain that is connected to the M domain through a flexible linker [38]. The M domain anchors SRP54 to SRP RNA and carries out the principal function of signal sequence recognition [39-41]. The NG domain interacts with the SR in a GTP-dependent

SRP is partially assembled in the nucleus and partially in the nucleolus. In agreement with that, nuclear localization for SRP proteins SRP9/14, SRP68, SRP72 and SRP19 has been determined [44]. After the transport into the nucleus the subunits bind SRP RNA and form a pre-SRP which is exported to the cytoplasm where the final protein, Srp54p, is incorporated [45-47]. Although this outline of the SRP assembly pathway has been determined, factors that facilitate this and/or function in quality control of the RNA are poorly understood [48]. SRP assembly starts during 7S RNA transcription by RNA polymerase III in the nucleolus, by binding of the SRP 9/14 heterodimer and formation of Alu-domain. Prior to transportation to the nucleus SRP9 and SRP14 form the heterodimer in the cytoplasm, a prerequisite for the

The signal recognition particle displays three main activities in the process of cotranslational targeting: (I) binding to signal sequences emerging from the translating ribosome, (II) pausing of peptide elongation, and (III) promotion of protein translocation through docking to the membrane-bound SRP receptor (FtsY in prokaryotes) and transfer of the ribosome nascent

types of cellular stress [31].

manner [43].

binding to 7S RNA [49].

**ii.** The signal recognition particle (SRP)

10 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

proper membrane destinations [33].

**Figure 6.** The signal recognition particle. During protein synthesis of the secretory pathway, the signal peptide binds to SRP, an RNP particle containing a small RNA and 6 different proteins (b, [modified from 60]). A model for SRP at nanoscale is shown in (c).

In the SRP pathway, SRP binds to the ribosome synthesizing the polypeptide, and subse‐ quently also binds an SRP receptor, located next to the machinery that transfers proteins across the membrane and out of the cell. This process begins when a nascent polypeptide carrying a signal sequence emerges from the translating ribosome and is recognized by the SRP. The ribosome-nascent chain complex is delivered to the target membrane via the interaction of SRP with the SRP receptor. There, the cargo is transferred to the Sec61p (or secYEG in archaea and bacteria) translocon, which translocates the growing polypeptide across the membrane or integrates it into the membrane bilayer. SRP and SR then dissociate from one another to enter subsequent rounds of targeting.

Peroxisomal membrane-bound PEX proteins, also encoded in the nuclear genome, are synthetized by ribosomes associated to rough endoplasmic reticulum since they display signal peptide. Therefore the peroxisome as an organelle derives from the rough endoplasmic reticulum. These organelles participate in ROS generation, as H2O2, but also in cell rescue from oxidative stress by catalase activity. In several biological models for pathological processes involving oxygen metabolites, the role of peroxisomes in prevention of oxidative stress is strongly suggested by de co-localization of catalase and H2O2, and the induction of peroxi‐

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To support life, cells must be continuously supplied with external energy in form of light or nutrients and must be equipped with chemical devices to convert these external energy sources into adenosine triphosphate (ATP). ATP is the universal energy currency of living cells and as such is used to drive numerous energy-consuming reactions, e.g., syntheses of biomolecules, muscle contraction, mechanical motility and transport through membranes, regulatory networks, and nerve conduction. When performing work, ATP is usually converted to ADP and phosphate. It must therefore continuously be regenerated from these compounds to continue the cell energy cycle. The importance of this cycle can be best illustrated by the

Prokaryotes use their plasma membrane to produce ATP. Eukaryotes use instead the special‐ ized membrane inside energy-converting organelles, mitochondria and chloroplasts, to produce most of their ATP. The mitochondria are present in the cells of practically all eukary‐ otic organisms (including fungi, animals, plants, algae and protozoa), and chloroplasts occur only in plants and algae. The most striking morphological feature of both organelles, revealed by electron microscopy, is the large amount of internal membrane they contain. This internal membrane provides the framework for an elaborate set of electron-transport processes, mediated by the enzymes of the Respiratory Chain that are essential to the process of Oxidative

In eukaryotes, oxidative phosphorylation occurs in mitochondria and photophosphorylation in chloroplasts. In the mitochondria, the energy to drive the synthesis of ATP derive from the oxidative steps in the degradation of carbohydrates, fats and amino acids; whereas the

Our current understanding of ATP synthesis in mitochondria and chloroplasts is based on the chemiosmotic model proposed by Peter Mitchell in 1961 [60], which has been accepted as one of the great unifying principles of twentieth century. According with this model, the electro‐ chemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane (the proton motive force) drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with the

chloroplasts capture the energy of sunlight and harness it to make ATP [60].

somes proliferation [63].

**d.** Mitochondrion and chloroplast particles

**i.** ATP synthase: A rotary molecular motor

demand of 50 Kg of ATP in a human body on average [64].

Phosphorylation which generate most of the cell's ATP.

**ii.** The Chemiosmotic Model of Peter Mitchell

ATP synthase (Fig. 7).

During the last years, several structures have been solved by crystallography and cryo-electron microscopy that represent distinct functional states of the SRP cycle. On this basis, the first structure-based models can be suggested that explain important aspects of protein targeting, such as the SRP–ribosome [54], SRP-SRP receptor [55] and SRP–SR interactions. The snapshots obtained by single-particle EM reconstructions enable us to follow the path of a nascent protein from the peptidyl-transferase center, through the ribosomal tunnel, to and across the translo‐ con in the membrane. With new developments in image processing techniques it is possible to sort a biological homogenous sample into different conformational states and to reach subnanometer resolution such that folding of the nascent chain into secondary structure elements can be directly visualized [56].

Molecular biology, biochemistry, and cryo-electron microscopy, have been combined to study the ribosome-protein complexes involved in protein assembly, folding and targeting. These approaches led to obtain structural snapshots of entire pathways by which proteins are synthesized and targeted to their final positions. The link between SRP and its receptor is usually transient and chemically unstable, for this reason, engineered SRP receptor bind more stably to SRP, then introduced to ribosomes and observed the resulting complexes using cryoelectron microscopy (cryo-EM). Cryo-EM can be performed in roughly physiological condi‐ tions, providing a picture that closely resembles what happens in living cells. This picture can then be combined with higher-resolution crystallography data and biochemical studies [57-58].

#### **c.** Peroxisomes

The oxidative stress (EO) is a disorder where reactive oxygen species (ROS) are produced. These compounds, that include free radicals and peroxides, play important roles in cell redox signaling. However, disturbances in the balance between the ROS production and the biological system can be particularly destructive. For example, the P450 oxide reductase activity produces H2O2 as a metabolite. This enormous family of enzymes is present in the mitochondrial and smooth endoplasmic reticulum (SER) membranes and catalyzes several reactions in the pathway of the biogenesis of steroid hormones [59] and in the detoxification process or in the first stage of drugs or xenobiotics hydrolysis, converting them in the SER, in water-soluble compounds for its excretion in the urine [60-62].

Peroxisomes are single membrane organelles present in practically every eukaryotic cell. Matrix proteins of peroxisomes synthesized in free polyribosomes in the cytoplasm and imported by a specific signal, are encoded in genes present in the cell nucleus genome. Peroxisomal membrane-bound PEX proteins, also encoded in the nuclear genome, are synthetized by ribosomes associated to rough endoplasmic reticulum since they display signal peptide. Therefore the peroxisome as an organelle derives from the rough endoplasmic reticulum. These organelles participate in ROS generation, as H2O2, but also in cell rescue from oxidative stress by catalase activity. In several biological models for pathological processes involving oxygen metabolites, the role of peroxisomes in prevention of oxidative stress is strongly suggested by de co-localization of catalase and H2O2, and the induction of peroxi‐ somes proliferation [63].

**d.** Mitochondrion and chloroplast particles

In the SRP pathway, SRP binds to the ribosome synthesizing the polypeptide, and subse‐ quently also binds an SRP receptor, located next to the machinery that transfers proteins across the membrane and out of the cell. This process begins when a nascent polypeptide carrying a signal sequence emerges from the translating ribosome and is recognized by the SRP. The ribosome-nascent chain complex is delivered to the target membrane via the interaction of SRP with the SRP receptor. There, the cargo is transferred to the Sec61p (or secYEG in archaea and bacteria) translocon, which translocates the growing polypeptide across the membrane or integrates it into the membrane bilayer. SRP and SR then dissociate from one another to enter

During the last years, several structures have been solved by crystallography and cryo-electron microscopy that represent distinct functional states of the SRP cycle. On this basis, the first structure-based models can be suggested that explain important aspects of protein targeting, such as the SRP–ribosome [54], SRP-SRP receptor [55] and SRP–SR interactions. The snapshots obtained by single-particle EM reconstructions enable us to follow the path of a nascent protein from the peptidyl-transferase center, through the ribosomal tunnel, to and across the translo‐ con in the membrane. With new developments in image processing techniques it is possible to sort a biological homogenous sample into different conformational states and to reach subnanometer resolution such that folding of the nascent chain into secondary structure

Molecular biology, biochemistry, and cryo-electron microscopy, have been combined to study the ribosome-protein complexes involved in protein assembly, folding and targeting. These approaches led to obtain structural snapshots of entire pathways by which proteins are synthesized and targeted to their final positions. The link between SRP and its receptor is usually transient and chemically unstable, for this reason, engineered SRP receptor bind more stably to SRP, then introduced to ribosomes and observed the resulting complexes using cryoelectron microscopy (cryo-EM). Cryo-EM can be performed in roughly physiological condi‐ tions, providing a picture that closely resembles what happens in living cells. This picture can then be combined with higher-resolution crystallography data and biochemical studies [57-58].

The oxidative stress (EO) is a disorder where reactive oxygen species (ROS) are produced. These compounds, that include free radicals and peroxides, play important roles in cell redox signaling. However, disturbances in the balance between the ROS production and the biological system can be particularly destructive. For example, the P450 oxide reductase activity produces H2O2 as a metabolite. This enormous family of enzymes is present in the mitochondrial and smooth endoplasmic reticulum (SER) membranes and catalyzes several reactions in the pathway of the biogenesis of steroid hormones [59] and in the detoxification process or in the first stage of drugs or xenobiotics hydrolysis, converting them in the SER, in

Peroxisomes are single membrane organelles present in practically every eukaryotic cell. Matrix proteins of peroxisomes synthesized in free polyribosomes in the cytoplasm and imported by a specific signal, are encoded in genes present in the cell nucleus genome.

water-soluble compounds for its excretion in the urine [60-62].

subsequent rounds of targeting.

12 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

elements can be directly visualized [56].

**c.** Peroxisomes

**i.** ATP synthase: A rotary molecular motor

To support life, cells must be continuously supplied with external energy in form of light or nutrients and must be equipped with chemical devices to convert these external energy sources into adenosine triphosphate (ATP). ATP is the universal energy currency of living cells and as such is used to drive numerous energy-consuming reactions, e.g., syntheses of biomolecules, muscle contraction, mechanical motility and transport through membranes, regulatory networks, and nerve conduction. When performing work, ATP is usually converted to ADP and phosphate. It must therefore continuously be regenerated from these compounds to continue the cell energy cycle. The importance of this cycle can be best illustrated by the demand of 50 Kg of ATP in a human body on average [64].

Prokaryotes use their plasma membrane to produce ATP. Eukaryotes use instead the special‐ ized membrane inside energy-converting organelles, mitochondria and chloroplasts, to produce most of their ATP. The mitochondria are present in the cells of practically all eukary‐ otic organisms (including fungi, animals, plants, algae and protozoa), and chloroplasts occur only in plants and algae. The most striking morphological feature of both organelles, revealed by electron microscopy, is the large amount of internal membrane they contain. This internal membrane provides the framework for an elaborate set of electron-transport processes, mediated by the enzymes of the Respiratory Chain that are essential to the process of Oxidative Phosphorylation which generate most of the cell's ATP.

In eukaryotes, oxidative phosphorylation occurs in mitochondria and photophosphorylation in chloroplasts. In the mitochondria, the energy to drive the synthesis of ATP derive from the oxidative steps in the degradation of carbohydrates, fats and amino acids; whereas the chloroplasts capture the energy of sunlight and harness it to make ATP [60].

**ii.** The Chemiosmotic Model of Peter Mitchell

Our current understanding of ATP synthesis in mitochondria and chloroplasts is based on the chemiosmotic model proposed by Peter Mitchell in 1961 [60], which has been accepted as one of the great unifying principles of twentieth century. According with this model, the electro‐ chemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane (the proton motive force) drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with the ATP synthase (Fig. 7).

Under aerobic conditions, the major ATP synthesis pathway is oxidative phosphorylation of which the terminal reaction is catalyzed by *F <sup>O</sup> F <sup>1</sup>*-ATP synthase. This enzyme is found widely in the biological world, including in thylakoid membranes, the mitochondrial inner membrane and the plasma membrane of bacteria, and is the central enzyme of energy metabolism in most organisms [65].

Mitochondrial ATP synthase is an F-type ATPase similar in structure and mechanism to the ATP synthases of chloroplasts and bacteria. This large complex of the inner mitochondrial membrane, also called Complex V, catalyzes the formation of ATP from ADP and Pi, accom‐ panied by the flow of protons from P (positive) side to N (negative) side of the membrane [66].

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ATP synthase is a supercomplex enzyme with a molecular weight of 500 kDal and consists of two rotary motors. One is *F <sup>1</sup>* subcomplex (~380 kDal), which is the water-soluble part of ATP synthase. *F <sup>1</sup>* was identified and purified by Efraim Racker and his colleagues in the early 1960s. When isolated from the membrane portion, it acts as an ATP-driven motor: it rotates its inner subunit to hydrolyze ATP and is therefore term *F <sup>1</sup>* ATPase. The other rotary motor of ATP synthase is the membrane-embedded Fo subcomplex (~120 kDal) through which the protons

In the simplest form of the enzyme, in bacteria like *Escherichia coli*, *F <sup>1</sup>* is composed of five different subunits, in a stoichiometry of α3β3γδε, and *F <sup>O</sup>* consists of three distinct subunits in a stoichiometry of *ab <sup>2</sup> c 10-15*. A newer more mechanically-based division differentiates between the "rotor" (in *E. coli*,γεcn) and the "stator" *(α <sup>3</sup> β <sup>3</sup> δab <sup>2</sup>*). The α3α<sup>3</sup> ring of the stator contains the three catalytic nucleotide sites, on the β subunits at the interphase to the adjacent α subunit. The *a* subunit contains the static portion of the proton traslocator machinery. α3β3 and *a* are

The crystallographic determination of the *F <sup>1</sup>* structure by John Walker and colleagues [67] revealed structural details very helpful in explaining the catalytic mechanism of the enzyme. The three α- and β- subunits that constitute the hexameric stator ring are alternately arranged like the sections of an orange. The rotor shaft is the γ-subunit, which is accommodated in the central cavity of the α3β3-ring. The ε-subunit binds onto the protruding part of the γ-subunit and provides a connection between the rotor parts of *F <sup>1</sup>* and *F <sup>O</sup>*. The δ-subunit acts as a

Catalytic reaction centers for ATP hydrolysis/synthesis reside at the three of the α-β interfaces, whereas the non-catalytic ATP-binding sites reside on the other α/β interfaces. While the catalytic site is formed mainly with amino acid residues from γ-subunit, the non- catalytic sites are primarily within the α-subunit. Upon ATP hydrolysis on the catalytic sites, *F <sup>1</sup>* rotates the

As mentioned before, *F <sup>O</sup>* subcomplex (*o* denoting oligomycin sensitive) consists of *ab <sup>2</sup> c 10-15* subunits. The number of *c* subunits varies among the species and form a ring complex by aligning in a circle. It is widely thought that the *c*-ring and the *a* subunit form a proton pathway. With the downhill proton flow through the proton channel, the *c*-ring rotates against the *ab <sup>2</sup>* subunits in the opposite direction of the γ-subunit of the *F <sup>1</sup>* motor [69]. Thus, in the *F <sup>O</sup> F <sup>1</sup>* complex, *F <sup>O</sup>* and *F <sup>1</sup>* push each other in the opposite direction. Under physiological condition where the electrochemical potential of the protons is large enough to surpass the free energy of ATP hydrolysis, *F <sup>O</sup>* forcibly rotates the γ-subunit in the clockwise direction and then *F <sup>1</sup>* catalyzes the reverse reaction*, i.e.* ATP synthesis which is the principle function of ATP

held together by the "stator stalk" (or "peripheral stalk"), consisting of *b <sup>2</sup>*γ [65].

connector between *F <sup>1</sup>* and *F <sup>O</sup>* that connects the stator parts.

γ-subunit in the anticlockwise direction viewed from the *F <sup>O</sup>* side [68].

**iii.** *F*O*F1*-ATP Synthase Structure and Function

flow.

**Figure 7.** In the mitochondrion (a), ATP synthase (arrow in a; b) is part of the respiratory chain.

Like many transporters the *F <sup>O</sup> F <sup>1</sup>*-ATP synthase (or F-type ATPase) has been fascinating subject for study of a complex membrane-associated process. This enzyme catalyzes ATP synthesis from adenosine diphosphate (ADP) and inorganic phosphate (Pi), by using the electrochemical potential of protons (or sodium ions in some bacteria) across the membrane, i.e. it converts the electrochemical potential into its chemical form. ATP synthase also functions in the reverse direction (ATPase) when the electrochemical potential becomes insufficient: it catalyzes proton pumping to form an electrochemical potential to hydrolyze ATP into ADP and Pi. Proton translocation and ATP synthesis (or hydrolysis) are coupled by a unique mechanism, subunit rotation. Electrochemically energy contained in the proton gradient is converted into mechan‐ ical energy in form of subunit rotation, and back into chemical energy as ATP (Nakamoto RK, et al, 2008).

Mitochondrial ATP synthase is an F-type ATPase similar in structure and mechanism to the ATP synthases of chloroplasts and bacteria. This large complex of the inner mitochondrial membrane, also called Complex V, catalyzes the formation of ATP from ADP and Pi, accom‐ panied by the flow of protons from P (positive) side to N (negative) side of the membrane [66].

### **iii.** *F*O*F1*-ATP Synthase Structure and Function

Under aerobic conditions, the major ATP synthesis pathway is oxidative phosphorylation of which the terminal reaction is catalyzed by *F <sup>O</sup> F <sup>1</sup>*-ATP synthase. This enzyme is found widely in the biological world, including in thylakoid membranes, the mitochondrial inner membrane and the plasma membrane of bacteria, and is the central enzyme of energy metabolism in most

14 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 7.** In the mitochondrion (a), ATP synthase (arrow in a; b) is part of the respiratory chain.

Like many transporters the *F <sup>O</sup> F <sup>1</sup>*-ATP synthase (or F-type ATPase) has been fascinating subject for study of a complex membrane-associated process. This enzyme catalyzes ATP synthesis from adenosine diphosphate (ADP) and inorganic phosphate (Pi), by using the electrochemical potential of protons (or sodium ions in some bacteria) across the membrane, i.e. it converts the electrochemical potential into its chemical form. ATP synthase also functions in the reverse direction (ATPase) when the electrochemical potential becomes insufficient: it catalyzes proton pumping to form an electrochemical potential to hydrolyze ATP into ADP and Pi. Proton translocation and ATP synthesis (or hydrolysis) are coupled by a unique mechanism, subunit rotation. Electrochemically energy contained in the proton gradient is converted into mechan‐ ical energy in form of subunit rotation, and back into chemical energy as ATP (Nakamoto RK,

organisms [65].

et al, 2008).

ATP synthase is a supercomplex enzyme with a molecular weight of 500 kDal and consists of two rotary motors. One is *F <sup>1</sup>* subcomplex (~380 kDal), which is the water-soluble part of ATP synthase. *F <sup>1</sup>* was identified and purified by Efraim Racker and his colleagues in the early 1960s. When isolated from the membrane portion, it acts as an ATP-driven motor: it rotates its inner subunit to hydrolyze ATP and is therefore term *F <sup>1</sup>* ATPase. The other rotary motor of ATP synthase is the membrane-embedded Fo subcomplex (~120 kDal) through which the protons flow.

In the simplest form of the enzyme, in bacteria like *Escherichia coli*, *F <sup>1</sup>* is composed of five different subunits, in a stoichiometry of α3β3γδε, and *F <sup>O</sup>* consists of three distinct subunits in a stoichiometry of *ab <sup>2</sup> c 10-15*. A newer more mechanically-based division differentiates between the "rotor" (in *E. coli*,γεcn) and the "stator" *(α <sup>3</sup> β <sup>3</sup> δab <sup>2</sup>*). The α3α<sup>3</sup> ring of the stator contains the three catalytic nucleotide sites, on the β subunits at the interphase to the adjacent α subunit. The *a* subunit contains the static portion of the proton traslocator machinery. α3β3 and *a* are held together by the "stator stalk" (or "peripheral stalk"), consisting of *b <sup>2</sup>*γ [65].

The crystallographic determination of the *F <sup>1</sup>* structure by John Walker and colleagues [67] revealed structural details very helpful in explaining the catalytic mechanism of the enzyme. The three α- and β- subunits that constitute the hexameric stator ring are alternately arranged like the sections of an orange. The rotor shaft is the γ-subunit, which is accommodated in the central cavity of the α3β3-ring. The ε-subunit binds onto the protruding part of the γ-subunit and provides a connection between the rotor parts of *F <sup>1</sup>* and *F <sup>O</sup>*. The δ-subunit acts as a connector between *F <sup>1</sup>* and *F <sup>O</sup>* that connects the stator parts.

Catalytic reaction centers for ATP hydrolysis/synthesis reside at the three of the α-β interfaces, whereas the non-catalytic ATP-binding sites reside on the other α/β interfaces. While the catalytic site is formed mainly with amino acid residues from γ-subunit, the non- catalytic sites are primarily within the α-subunit. Upon ATP hydrolysis on the catalytic sites, *F <sup>1</sup>* rotates the γ-subunit in the anticlockwise direction viewed from the *F <sup>O</sup>* side [68].

As mentioned before, *F <sup>O</sup>* subcomplex (*o* denoting oligomycin sensitive) consists of *ab <sup>2</sup> c 10-15* subunits. The number of *c* subunits varies among the species and form a ring complex by aligning in a circle. It is widely thought that the *c*-ring and the *a* subunit form a proton pathway. With the downhill proton flow through the proton channel, the *c*-ring rotates against the *ab <sup>2</sup>* subunits in the opposite direction of the γ-subunit of the *F <sup>1</sup>* motor [69]. Thus, in the *F <sup>O</sup> F <sup>1</sup>* complex, *F <sup>O</sup>* and *F <sup>1</sup>* push each other in the opposite direction. Under physiological condition where the electrochemical potential of the protons is large enough to surpass the free energy of ATP hydrolysis, *F <sup>O</sup>* forcibly rotates the γ-subunit in the clockwise direction and then *F <sup>1</sup>* catalyzes the reverse reaction*, i.e.* ATP synthesis which is the principle function of ATP synthase. In contrast, when the electrochemical potential is small or decreases, *F <sup>1</sup>* forces *F <sup>O</sup>* to rotate the *c-*ring in the reverse direction to pump protons against the electrochemical potential.

One strong prediction of the binding-change model of Boyer is that the γ subunit should rotate in one direction when *F <sup>O</sup> F <sup>1</sup>* is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita Jr. [74]. The rotation of γ in a single *F <sup>1</sup>* molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to γ and watching it move relative to *α <sup>3</sup> β <sup>3</sup>* immobilized on a microscope slide, as ATP was hydrolyzed [see 75]. Lately the unidirectional γ rotation was visualized in simultaneous imaging of the

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The Atomic Force Microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and place in aqueous solution. This technology allows visualization of biomolecules under physiological conditions. However, it is limited by the speed at which it can successively record highly resolved images. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, allowing single biomolecules to be watch in action in real time. Toshio Ando and his coworkers at Kanazawa University have been leading innovators in this so-called High-Speed Atomic Force Microscope (HS-AFM) technology [76]. This technology allows direct visualization of dynamic structural changes and dynamic processes of functioning biological molecules in physiological solutions, at high spatial-temporal resolution. Dynamic molecular events appear in detail in an AFM movie, facilitating our understanding of how biological molecules operate to function.

In this regard, the Ando group showed a striking example of molecular motor action in their AFM movies of the isolated subcomplex of the rotary motor protein *F <sup>1</sup>*-ATPase. Previous sin‐ gle-molecule experiments on parts of this enzyme had measured rotation, but they could only be done if at least one subunit of the rotor was attached. The AFM, however, could visualize the conformational change that the β subunits of the stator undergo when they bind ATP. By imag‐ ing at 12.5 frames/s, the authors followed the time dependence of these conformational changes, leading to the surprising conclusion that, contrary to what was widely assumed be‐ fore, the catalysis on the enzyme maintains its sequential rotary order even in absence of the ro‐

"To directly observe biological molecules at work was a holy grail in biology. Efforts over the last two decades at last materialized this long-quested dream. In high-resolution AFM movies, we can see how molecules are dynamically behaving, changing their structure and interacting with other molecules, and hence we can quickly understand in stunning detail how molecules operate to function. This new approach will spread over the world and widely applied to a vast array of biological issues, leading to a number of new discoveries. The extension of highspeed AFM to a tool for imaging live cells, which allows direct in situ observation of dynamic processes of molecules and organelles, remains an exciting challenge but will be made in the

Cell membranes are dynamic assemblies of a variety of lipids and proteins. They form a protective layer around the cell and mediate the communication with the outside world. The

conformational change of the β-subunit and the γ rotation.

near future because it is a right and fruitful goal" [77].

tor subunits.

**e.** Lipid rafts

**v.** New approaches for studying biological macromolecules.

The *c* subunit of the *F <sup>O</sup>* complex is a small (Mr 8,000), very hydrophobic polypeptide, consisting almost entirely of two membrane-spanning α-helices, that are connected by a small loop extending from the matrix side of the membrane. The crystal structure of the yeast *F <sup>O</sup> F <sup>1</sup>*, solved in 1999, shows the arrangement of the subunits. The yeast complex has 10 *c* subunits, each with two transmembrane helices roughly perpendicular to the plane of the membrane and arranged in two concentric circles. The inner circle is made up of the amino-terminal helices of each *c* subunit; the outer circle, about 55 Å in diameter, is made up of the carboxylterminal helices. The ε andγ subunits of *F <sup>1</sup>* form a leg-and-foot that projects from the bottom (membrane) side of *F <sup>1</sup>* and stands firmly on the ring of *c* subunits. The *a* subunit is a very hydrophobic protein that in most models is composed of five transmembrane helices. Ion translocation takes place through subunit *a* and its interface with subunit *c*. The *b* subunits are anchored within the membrane by an N-terminal α-helix and extend as a peripheral stalk all the way to the head of the *F <sup>1</sup>* domain. According to cross-linking studies, the *b* subunits contact de C-terminal part of the c subunit and the loop between helices 4 and 5 of the *a* subunit at the periplasmic surface. The δ-subunit forms a strong complex with the α-subunit. In mitochon‐ dria, the peripheral stalk consists of more subunits named OSCP (Oligomycin Sensitive Conferring Protein), *b*, δ and F6 [64].

**iv.** Structure of *F <sup>1</sup>* and binding-change mechanism for ATP Synthesis.

The classic working model for F1 is the "binding-change mechanism" proposed by Paul Boyer [70]. The early stage of this model postulated an alternating transition between two chemical states, assuming two catalytic sites residing on *F <sup>1</sup>*. It was later revised to propose the cyclic transition of the catalytic sites based on the biochemical and electron microscopic experiments that revealed that *F <sup>1</sup>* has the three catalytic sites [71-73]. One important feature of this model is that the affinity for nucleotide in each catalytic site is different from each other at any given time, and the status of the three β-subunits cooperatively change in one direction accompa‐ nying γ rotation. This hypothesis is strongly supported by X-ray crystallographic studies performed by Walker's group [67] that first resolved crystal structure of *F <sup>1</sup>*, which revealed many essential structural features of *F <sup>1</sup>* at atomic resolution. Importantly, the catalytic βsubunits differ from each other in conformation and catalytic state: one binds to an ATP analogue, adenosine 5'-(β,γ-imino)-triphosphate (AMP-PNP), the second binds to ADP and the third site is empty. Therefore, these sites are termed βTP, βDP and βEmpty, respectively. While βTP and βDP have a close conformation wrapping bound nucleotides on the catalytic sites, βEmpty has an open conformation swinging the C-terminal domain away from the binding site to open the cleft of the catalytic site. These features are consistent with the bindingchange mechanism. Another important feature found in the crystal is that while the N-terminal domains of the α- and β-subunits form a symmetrical smooth cavity as the bearing for γ rotation at the bottom of the α3β3-ring, the C-terminal domains of the β-subunit show distinct asymmetric interactions with the γ-subunit. Therefore, the most feasible inference is that the open-to-closed transition of the β-subunits upon ATP binding pushes γ, and the sequential conformational change among β- subunits leads the unidirectional γ rotation.

One strong prediction of the binding-change model of Boyer is that the γ subunit should rotate in one direction when *F <sup>O</sup> F <sup>1</sup>* is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita Jr. [74]. The rotation of γ in a single *F <sup>1</sup>* molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to γ and watching it move relative to *α <sup>3</sup> β <sup>3</sup>* immobilized on a microscope slide, as ATP was hydrolyzed [see 75]. Lately the unidirectional γ rotation was visualized in simultaneous imaging of the conformational change of the β-subunit and the γ rotation.

**v.** New approaches for studying biological macromolecules.

The Atomic Force Microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and place in aqueous solution. This technology allows visualization of biomolecules under physiological conditions. However, it is limited by the speed at which it can successively record highly resolved images. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, allowing single biomolecules to be watch in action in real time. Toshio Ando and his coworkers at Kanazawa University have been leading innovators in this so-called High-Speed Atomic Force Microscope (HS-AFM) technology [76]. This technology allows direct visualization of dynamic structural changes and dynamic processes of functioning biological molecules in physiological solutions, at high spatial-temporal resolution. Dynamic molecular events appear in detail in an AFM movie, facilitating our understanding of how biological molecules operate to function.

In this regard, the Ando group showed a striking example of molecular motor action in their AFM movies of the isolated subcomplex of the rotary motor protein *F <sup>1</sup>*-ATPase. Previous sin‐ gle-molecule experiments on parts of this enzyme had measured rotation, but they could only be done if at least one subunit of the rotor was attached. The AFM, however, could visualize the conformational change that the β subunits of the stator undergo when they bind ATP. By imag‐ ing at 12.5 frames/s, the authors followed the time dependence of these conformational changes, leading to the surprising conclusion that, contrary to what was widely assumed be‐ fore, the catalysis on the enzyme maintains its sequential rotary order even in absence of the ro‐ tor subunits.

"To directly observe biological molecules at work was a holy grail in biology. Efforts over the last two decades at last materialized this long-quested dream. In high-resolution AFM movies, we can see how molecules are dynamically behaving, changing their structure and interacting with other molecules, and hence we can quickly understand in stunning detail how molecules operate to function. This new approach will spread over the world and widely applied to a vast array of biological issues, leading to a number of new discoveries. The extension of highspeed AFM to a tool for imaging live cells, which allows direct in situ observation of dynamic processes of molecules and organelles, remains an exciting challenge but will be made in the near future because it is a right and fruitful goal" [77].

### **e.** Lipid rafts

synthase. In contrast, when the electrochemical potential is small or decreases, *F <sup>1</sup>* forces *F <sup>O</sup>* to rotate the *c-*ring in the reverse direction to pump protons against the electrochemical potential.

16 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

The *c* subunit of the *F <sup>O</sup>* complex is a small (Mr 8,000), very hydrophobic polypeptide, consisting almost entirely of two membrane-spanning α-helices, that are connected by a small loop extending from the matrix side of the membrane. The crystal structure of the yeast *F <sup>O</sup> F <sup>1</sup>*, solved in 1999, shows the arrangement of the subunits. The yeast complex has 10 *c* subunits, each with two transmembrane helices roughly perpendicular to the plane of the membrane and arranged in two concentric circles. The inner circle is made up of the amino-terminal helices of each *c* subunit; the outer circle, about 55 Å in diameter, is made up of the carboxylterminal helices. The ε andγ subunits of *F <sup>1</sup>* form a leg-and-foot that projects from the bottom (membrane) side of *F <sup>1</sup>* and stands firmly on the ring of *c* subunits. The *a* subunit is a very hydrophobic protein that in most models is composed of five transmembrane helices. Ion translocation takes place through subunit *a* and its interface with subunit *c*. The *b* subunits are anchored within the membrane by an N-terminal α-helix and extend as a peripheral stalk all the way to the head of the *F <sup>1</sup>* domain. According to cross-linking studies, the *b* subunits contact de C-terminal part of the c subunit and the loop between helices 4 and 5 of the *a* subunit at the periplasmic surface. The δ-subunit forms a strong complex with the α-subunit. In mitochon‐ dria, the peripheral stalk consists of more subunits named OSCP (Oligomycin Sensitive

**iv.** Structure of *F <sup>1</sup>* and binding-change mechanism for ATP Synthesis.

conformational change among β- subunits leads the unidirectional γ rotation.

The classic working model for F1 is the "binding-change mechanism" proposed by Paul Boyer [70]. The early stage of this model postulated an alternating transition between two chemical states, assuming two catalytic sites residing on *F <sup>1</sup>*. It was later revised to propose the cyclic transition of the catalytic sites based on the biochemical and electron microscopic experiments that revealed that *F <sup>1</sup>* has the three catalytic sites [71-73]. One important feature of this model is that the affinity for nucleotide in each catalytic site is different from each other at any given time, and the status of the three β-subunits cooperatively change in one direction accompa‐ nying γ rotation. This hypothesis is strongly supported by X-ray crystallographic studies performed by Walker's group [67] that first resolved crystal structure of *F <sup>1</sup>*, which revealed many essential structural features of *F <sup>1</sup>* at atomic resolution. Importantly, the catalytic βsubunits differ from each other in conformation and catalytic state: one binds to an ATP analogue, adenosine 5'-(β,γ-imino)-triphosphate (AMP-PNP), the second binds to ADP and the third site is empty. Therefore, these sites are termed βTP, βDP and βEmpty, respectively. While βTP and βDP have a close conformation wrapping bound nucleotides on the catalytic sites, βEmpty has an open conformation swinging the C-terminal domain away from the binding site to open the cleft of the catalytic site. These features are consistent with the bindingchange mechanism. Another important feature found in the crystal is that while the N-terminal domains of the α- and β-subunits form a symmetrical smooth cavity as the bearing for γ rotation at the bottom of the α3β3-ring, the C-terminal domains of the β-subunit show distinct asymmetric interactions with the γ-subunit. Therefore, the most feasible inference is that the open-to-closed transition of the β-subunits upon ATP binding pushes γ, and the sequential

Conferring Protein), *b*, δ and F6 [64].

Cell membranes are dynamic assemblies of a variety of lipids and proteins. They form a protective layer around the cell and mediate the communication with the outside world. The original fluid mosaic model [78] of membranes suggested a homogenous distribution of proteins and lipids across the two-dimensional surface, but more recent evidence suggests that membranes themselves are not uniform and that microdomains of lipids in a more ordered state exist within the generally disorder lipid milieu of the membrane. These clusters of ordered lipids are now referred to as lipid rafts [79] (Pike LJ 2009).

Different subtypes of lipid rafts can be distinguished according to their protein and lipid composition. Caveolae are types of rafts that are rich in proteins of the caveolin family (caveolin-1, -2 and -3) which present a distinct signaling platform [96]. The caveolae are enriched in cholesterol, glycosphingolipids, and sphingomyelin. They are the site of several important protein–protein interactions, for example, the neurotrophin receptors, TrkA and p75(NTR), whose respective interactions with caveolin regulates neurotrophin signaling in the

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The most important role of rafts at the cell surface may be their function in signal transduction. Lipid rafts have been implicated as the sites for a great number of signaling pathways.They form concentrating platforms for individual receptors, activated by ligand binding [86]. If receptor activation takes place in a lipid raft, the signaling complex is protected from non-raft enzymes such as membrane phosphatases that otherwise could affect the signaling process. In general, raft binding recruits proteins to a new micro-environment, where the phosphory‐ lation state can be modified by local kinases and phosphatases, resulting in downstream signalling. Individual signaling molecules within the raft are activated only for a short period

Immobilization of signaling molecules by cytoskeletal actin filaments and scaffold proteins may facilitate more efficient signal transmission from rafts [97]. Current evidence supports a role for lipid rafts in the initiation and regulation of The B-cell receptor signaling and antigen trafficking [98-100]. The importance of lipid raft signalling in the pathogenesis of a variety of conditions, such as Alzheimer's, Parkinson's, cardiovascular and prion diseases, systemic lupus erythematosus and HIV, has been elucidated over recent years[ 101] and makes these specific membrane domains an interesting target for pharmacological approaches in the cure and prevention of these diseases [102]. Rafts serve as a portal of entry for various pathogens and toxins, such as human immunodeficiency virus 1 (HIV-1). In the case of HIV-1, raft microdomains mediate the lateral assemblies and the conformational changes required for fusion of HIV-1 with the host cell [103]. Lipid rafts are also preferential sites of formation for pathological forms of the prion protein (PrPSc) and of the β-amyloid peptide associated with

Plasma membranes typically contain higher concentrations of cholesterol and sphingomyelin than do internal membranous organelles [105-106]. Thus, along the secretion pathway, there are very low concentrations of cholesterol and sphingolipids in the endoplasmic reticulum, but the concentrations of these lipids increase from the cis-Golgi to the trans-Golgi and then to the plasma membrane [107-108]. On the contrary, recent evidence suggests that mitochon‐ dria do not contain lipid rafts, and lipid rafts do not contain mitochondrial proteins [109]. Lipid raft domains play a key role in the regulation of exocytosis [110]. The association of SNAREs protein complexes with lipid rafts acts to concentrate these proteins at defined sites

of the plasma membrane that are of functional importance for exocytosis [111-114].

The cell nucleus contains different compartments that are characterized by the absence of delineating membranes that isolate it from the rest of the nucleoplasm [5]. Due to the high

brain. Caveolins also regulate G-proteins, MAPK, PI3K, and Src tyrosine kinases.

of time.

Alzheimer's disease {104].

**f.** The nucleolus

Lipid rafts (LRs), consist of cell membrane domains rich in cholesterol, sphingolipids and lipidanchored proteins in the exoplasmic leaflet of the lipid bilayer. Because of their ability to sequester specific lipids and proteins and exclude others, rafts have been postulated to perform critical roles in a number of normal cellular processes, such as signal transduction [80], membrane fusion, organization of the cytoskeleton [81-83], lipid sorting, and protein traffick‐ ing/recycling, as well as pathological events [84].

LRs are too small to be resolved by standard light microscopy - they range from 10 to about 200 nm - with a variable life span in the order of milliseconds (msec). Detergent resistant membranes, containing clusters of many rafts, can be isolated by extraction with Triton X-100 or other detergents on ice. However, this method involves breaking up the membrane and has limitations in terms of defining the size, properties, and dynamics of intact microdomains [85-88].Thus, a variety of sophisticated techniques have recently been used to analyze in detail open questions concerning rafts in cell and model membranes including biochemical, bio‐ physical, quantitative fluorescence microscopy, atomic force microscopy and computational methodologies [89-90].

**Figure 8.** Components of a lipid raft. (1) non raft membrane, (2) lipid raft, (3) lipid raft associated transmembrane pro‐ tein, (4) GPI-anchored protein, (5) glycosylation modifications (glycoproteins and glycolipids).

The raft affinity of a given protein can be modulated by intra- or extracellular stimuli. Saturated fatty acids are preferentially enriched in the side chains of the membrane phospholipids, which allows closer packing and thus increased rigidity, more order and less fluidity of the LRs compared to the surrounding membrane [91-92]. Proteins with raft affinity include glycosyl‐ phosphatidylinositol (GPI)-anchored proteins [93-94], doubly acylated proteins, such as Srcfamily kinases or the α-subunits of heterotrimeric G proteins8, cholesterol-linked and palmitoylated proteins such as Hedgehog9, and transmembrane proteins, particularly palmitoylated ones [92-95].

Different subtypes of lipid rafts can be distinguished according to their protein and lipid composition. Caveolae are types of rafts that are rich in proteins of the caveolin family (caveolin-1, -2 and -3) which present a distinct signaling platform [96]. The caveolae are enriched in cholesterol, glycosphingolipids, and sphingomyelin. They are the site of several important protein–protein interactions, for example, the neurotrophin receptors, TrkA and p75(NTR), whose respective interactions with caveolin regulates neurotrophin signaling in the brain. Caveolins also regulate G-proteins, MAPK, PI3K, and Src tyrosine kinases.

The most important role of rafts at the cell surface may be their function in signal transduction. Lipid rafts have been implicated as the sites for a great number of signaling pathways.They form concentrating platforms for individual receptors, activated by ligand binding [86]. If receptor activation takes place in a lipid raft, the signaling complex is protected from non-raft enzymes such as membrane phosphatases that otherwise could affect the signaling process. In general, raft binding recruits proteins to a new micro-environment, where the phosphory‐ lation state can be modified by local kinases and phosphatases, resulting in downstream signalling. Individual signaling molecules within the raft are activated only for a short period of time.

Immobilization of signaling molecules by cytoskeletal actin filaments and scaffold proteins may facilitate more efficient signal transmission from rafts [97]. Current evidence supports a role for lipid rafts in the initiation and regulation of The B-cell receptor signaling and antigen trafficking [98-100]. The importance of lipid raft signalling in the pathogenesis of a variety of conditions, such as Alzheimer's, Parkinson's, cardiovascular and prion diseases, systemic lupus erythematosus and HIV, has been elucidated over recent years[ 101] and makes these specific membrane domains an interesting target for pharmacological approaches in the cure and prevention of these diseases [102]. Rafts serve as a portal of entry for various pathogens and toxins, such as human immunodeficiency virus 1 (HIV-1). In the case of HIV-1, raft microdomains mediate the lateral assemblies and the conformational changes required for fusion of HIV-1 with the host cell [103]. Lipid rafts are also preferential sites of formation for pathological forms of the prion protein (PrPSc) and of the β-amyloid peptide associated with Alzheimer's disease {104].

Plasma membranes typically contain higher concentrations of cholesterol and sphingomyelin than do internal membranous organelles [105-106]. Thus, along the secretion pathway, there are very low concentrations of cholesterol and sphingolipids in the endoplasmic reticulum, but the concentrations of these lipids increase from the cis-Golgi to the trans-Golgi and then to the plasma membrane [107-108]. On the contrary, recent evidence suggests that mitochon‐ dria do not contain lipid rafts, and lipid rafts do not contain mitochondrial proteins [109].

Lipid raft domains play a key role in the regulation of exocytosis [110]. The association of SNAREs protein complexes with lipid rafts acts to concentrate these proteins at defined sites of the plasma membrane that are of functional importance for exocytosis [111-114].

**f.** The nucleolus

original fluid mosaic model [78] of membranes suggested a homogenous distribution of proteins and lipids across the two-dimensional surface, but more recent evidence suggests that membranes themselves are not uniform and that microdomains of lipids in a more ordered state exist within the generally disorder lipid milieu of the membrane. These clusters of ordered

Lipid rafts (LRs), consist of cell membrane domains rich in cholesterol, sphingolipids and lipidanchored proteins in the exoplasmic leaflet of the lipid bilayer. Because of their ability to sequester specific lipids and proteins and exclude others, rafts have been postulated to perform critical roles in a number of normal cellular processes, such as signal transduction [80], membrane fusion, organization of the cytoskeleton [81-83], lipid sorting, and protein traffick‐

LRs are too small to be resolved by standard light microscopy - they range from 10 to about 200 nm - with a variable life span in the order of milliseconds (msec). Detergent resistant membranes, containing clusters of many rafts, can be isolated by extraction with Triton X-100 or other detergents on ice. However, this method involves breaking up the membrane and has limitations in terms of defining the size, properties, and dynamics of intact microdomains [85-88].Thus, a variety of sophisticated techniques have recently been used to analyze in detail open questions concerning rafts in cell and model membranes including biochemical, bio‐ physical, quantitative fluorescence microscopy, atomic force microscopy and computational

**Figure 8.** Components of a lipid raft. (1) non raft membrane, (2) lipid raft, (3) lipid raft associated transmembrane pro‐

The raft affinity of a given protein can be modulated by intra- or extracellular stimuli. Saturated fatty acids are preferentially enriched in the side chains of the membrane phospholipids, which allows closer packing and thus increased rigidity, more order and less fluidity of the LRs compared to the surrounding membrane [91-92]. Proteins with raft affinity include glycosyl‐ phosphatidylinositol (GPI)-anchored proteins [93-94], doubly acylated proteins, such as Srcfamily kinases or the α-subunits of heterotrimeric G proteins8, cholesterol-linked and palmitoylated proteins such as Hedgehog9, and transmembrane proteins, particularly

tein, (4) GPI-anchored protein, (5) glycosylation modifications (glycoproteins and glycolipids).

lipids are now referred to as lipid rafts [79] (Pike LJ 2009).

18 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ing/recycling, as well as pathological events [84].

methodologies [89-90].

palmitoylated ones [92-95].

The cell nucleus contains different compartments that are characterized by the absence of delineating membranes that isolate it from the rest of the nucleoplasm [5]. Due to the high concentration of RNA and proteins that form it, the nucleolus is the most conspicuous nuclear body in cycling cells observed by light and electron microscopy. Nucleoli are formed around nucleolar organizer regions (NORs), which are composed of cluster of ribosomal genes (rDNA) repeat units [115-121]. The number of NOR-bearing chromosomes varies depending on the species, can be found 1 in haploid yeast cells to 10 in human somatic cells (short arms of chromosomes 13, 14, 15, 21 and 22). Nucleolus is the organelle of rDNA transcription by RNA polymerase I, whose activity generates a long ribosomal precursor (pre-rRNA), this molecule is the target of an extensive process that includes removing or cutting the spacers and 2'-Omethylation of riboses and coversions of uridine residues into pseudouridines. The net result of these reactions is the release of mature species of ribosomal RNA (rRNA) 18S, 5.8S and 28S. These particles are assembled with approximately 82 ribosomal proteins and rRNA 5S (synthesized by RNA polymerase III) to form the 40S and 60S subunits; both of these subunits are then exported separately to the cytoplasm and are further modified to form mature ribosomal subunits. Currently, it is widely accepted that nucleolar transcription and early prerRNA processing take place in the fibrillar portion of nucleolus while the later steps of processing and ribosome subnits assembly occurs mainly in the granular zone. The architec‐ ture of the nucleolus reflects the vectorial maturation of the pre-ribosomes. The nucleolar structure is organized by three canonical subdomains that are morphologically and biochem‐ ically different. The fibrillar centers (FC), dense fibrillar component (DFC) and granular component (GC). The FCs are structures with a low electron density, often circular shape of ~0.1 to 1µm in diameter. The FCs are enriched with rDNA, RNA polymerase I, topoisomerase I and upstream binding factor (UBF). DFCs are a compact fibrillar region containing a high concentration of ribonucleoprotein molecules that confer a high electrodensity. This compo‐ nent entirely or partially surrounds the FCs. DFCs contains important proteins such as fibrillarin and nucleolin as well as small nucleolar RNAs, pre-rRNA and some transcription factors. FCs and DFCs are embedded in the GC, composed mainly of granules of 15 to 20nm in diameter with a loosely organized distribution. In the GC are located B23/nucleophosmin, Nop 52, r-proteins, auxiliary assembly factors, and the 40S and 60S subunits that the GC is itself composed of at least two distintc molecular domains. Considering the species, cell type and physiological state of the cell, there is considerable diversity in the prevalence and arrangement of the three nucleolar components.

*1.1.3. Microscopy*

associated particles (arrow).

Fundamental to approach the cell at the nanoscale in cell nanobiology are the classical and also remarkably new types of microscopy. Three different epochs characterize microscopy: 1) Light microscopy, developed since ca. 1500, where glass lenses and light as source of illumination are used to get resolution of up to 0.2 µm. Different types such as bright field, phase contrast, differential interference contrast (Nomarsky), dark field, polarization, fluorescence, confocal, and super-resolution, are variants of this type of microscopy. 2) Electron microscopy, devel‐ oped since early 1930s, where electromagnetic lenses and electrons as source of illumination are used to get resolution of up to nm or A°. Transmission and scanning electron microscopy –including the environmental and high resolution modes- are the two forms of this microsco‐ py. 3) Scanning probe microscopy, developed in the early 1980s, where no lenses or illumina‐ tions are used, but instead the microscope consists of a fine tip interacting with the samples to potentially obtaining atomic resolution. Scanning tunneling microscopy and atomic force microscopy are the major variants of this type of modern microscopy. Because atomic force microscopy may produce images at high resolution even under liquid, we have been using such microscopy for imaging the cell components. To test this approach, we used several cell types and generated images at low magnification (Figure 9a). Nuclear particles *i.e*. Lacandonia

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b a

**Figure 9.** a) Atomic force microscopy image of a cell from the tegument of the plant *Lacandonia schismatica*. Cell wall (CW), vacuole (V), cytoplasm (Cy), Within the nucleus (N), compact chromatin (cc), nucleolus (nu), nucleolar organizer (small arrow) and nuclear pore (large arrow). b) Atomic force microscopy of Lacandonia granules within the nucleus of a tegument cell of the plant *Lacandonia schismatica*. Three dimensional displaying shows compact chromatin (cc) and

granules were already visualized using this approach (Figure 9b).

On the other hand, the current eukaryotic nucleolus is involved in the ribosomal biogenesis but has been described as a multifunctional entity. Extra ribosomal functions include biogen‐ esis and/or maturation of other ribonucleoprotein machines, including the signal recognition particle, the spliceosomal small nuclear RNPs and telomerase, processing or export of some mRNAs and tRNA, cell cycle and cell proliferation control, stress response and apoptosis [116]. The plurifunctional nucleolus hypothesis is reinforced by the description of nucleolar pro‐ teome of several eukaryotes. A proteomic analysis has identified more than 200 nucleolar proteins in Arabidopsis and almost 700 proteins in the nucleolus of HeLa cells. A comparison of nucleolar proteome from humans and budding yeast showed that ~90% of human nucleolar proteins have yeast homologues. Interestingly, only 30% of the human nucleolar proteome is intended for ribosomal biogenesis [120, 122].

### *1.1.3. Microscopy*

concentration of RNA and proteins that form it, the nucleolus is the most conspicuous nuclear body in cycling cells observed by light and electron microscopy. Nucleoli are formed around nucleolar organizer regions (NORs), which are composed of cluster of ribosomal genes (rDNA) repeat units [115-121]. The number of NOR-bearing chromosomes varies depending on the species, can be found 1 in haploid yeast cells to 10 in human somatic cells (short arms of chromosomes 13, 14, 15, 21 and 22). Nucleolus is the organelle of rDNA transcription by RNA polymerase I, whose activity generates a long ribosomal precursor (pre-rRNA), this molecule is the target of an extensive process that includes removing or cutting the spacers and 2'-Omethylation of riboses and coversions of uridine residues into pseudouridines. The net result of these reactions is the release of mature species of ribosomal RNA (rRNA) 18S, 5.8S and 28S. These particles are assembled with approximately 82 ribosomal proteins and rRNA 5S (synthesized by RNA polymerase III) to form the 40S and 60S subunits; both of these subunits are then exported separately to the cytoplasm and are further modified to form mature ribosomal subunits. Currently, it is widely accepted that nucleolar transcription and early prerRNA processing take place in the fibrillar portion of nucleolus while the later steps of processing and ribosome subnits assembly occurs mainly in the granular zone. The architec‐ ture of the nucleolus reflects the vectorial maturation of the pre-ribosomes. The nucleolar structure is organized by three canonical subdomains that are morphologically and biochem‐ ically different. The fibrillar centers (FC), dense fibrillar component (DFC) and granular component (GC). The FCs are structures with a low electron density, often circular shape of ~0.1 to 1µm in diameter. The FCs are enriched with rDNA, RNA polymerase I, topoisomerase I and upstream binding factor (UBF). DFCs are a compact fibrillar region containing a high concentration of ribonucleoprotein molecules that confer a high electrodensity. This compo‐ nent entirely or partially surrounds the FCs. DFCs contains important proteins such as fibrillarin and nucleolin as well as small nucleolar RNAs, pre-rRNA and some transcription factors. FCs and DFCs are embedded in the GC, composed mainly of granules of 15 to 20nm in diameter with a loosely organized distribution. In the GC are located B23/nucleophosmin, Nop 52, r-proteins, auxiliary assembly factors, and the 40S and 60S subunits that the GC is itself composed of at least two distintc molecular domains. Considering the species, cell type and physiological state of the cell, there is considerable diversity in the prevalence and

20 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

On the other hand, the current eukaryotic nucleolus is involved in the ribosomal biogenesis but has been described as a multifunctional entity. Extra ribosomal functions include biogen‐ esis and/or maturation of other ribonucleoprotein machines, including the signal recognition particle, the spliceosomal small nuclear RNPs and telomerase, processing or export of some mRNAs and tRNA, cell cycle and cell proliferation control, stress response and apoptosis [116]. The plurifunctional nucleolus hypothesis is reinforced by the description of nucleolar pro‐ teome of several eukaryotes. A proteomic analysis has identified more than 200 nucleolar proteins in Arabidopsis and almost 700 proteins in the nucleolus of HeLa cells. A comparison of nucleolar proteome from humans and budding yeast showed that ~90% of human nucleolar proteins have yeast homologues. Interestingly, only 30% of the human nucleolar proteome is

arrangement of the three nucleolar components.

intended for ribosomal biogenesis [120, 122].

Fundamental to approach the cell at the nanoscale in cell nanobiology are the classical and also remarkably new types of microscopy. Three different epochs characterize microscopy: 1) Light microscopy, developed since ca. 1500, where glass lenses and light as source of illumination are used to get resolution of up to 0.2 µm. Different types such as bright field, phase contrast, differential interference contrast (Nomarsky), dark field, polarization, fluorescence, confocal, and super-resolution, are variants of this type of microscopy. 2) Electron microscopy, devel‐ oped since early 1930s, where electromagnetic lenses and electrons as source of illumination are used to get resolution of up to nm or A°. Transmission and scanning electron microscopy –including the environmental and high resolution modes- are the two forms of this microsco‐ py. 3) Scanning probe microscopy, developed in the early 1980s, where no lenses or illumina‐ tions are used, but instead the microscope consists of a fine tip interacting with the samples to potentially obtaining atomic resolution. Scanning tunneling microscopy and atomic force microscopy are the major variants of this type of modern microscopy. Because atomic force microscopy may produce images at high resolution even under liquid, we have been using such microscopy for imaging the cell components. To test this approach, we used several cell types and generated images at low magnification (Figure 9a). Nuclear particles *i.e*. Lacandonia granules were already visualized using this approach (Figure 9b).

**Figure 9.** a) Atomic force microscopy image of a cell from the tegument of the plant *Lacandonia schismatica*. Cell wall (CW), vacuole (V), cytoplasm (Cy), Within the nucleus (N), compact chromatin (cc), nucleolus (nu), nucleolar organizer (small arrow) and nuclear pore (large arrow). b) Atomic force microscopy of Lacandonia granules within the nucleus of a tegument cell of the plant *Lacandonia schismatica*. Three dimensional displaying shows compact chromatin (cc) and associated particles (arrow).

### *1.1.4. Further research*

Further research in our laboratory will focusing in visualizing the nanoscale cell structures involved in fundamental processes as ribosome biogenesis, at a high resolution *in situ* under liquid conditions to perform quantitative analysis.

[3] Vázquez-nin, G. H, & Echeverría, O. M. The polytene nucleus in morphological, cytochemical, and functional studies of Messenger RNA transcription, processing, and

Cell Nanobiology

23

http://dx.doi.org/10.5772/52003

[4] Jiménez-garcía, J, Agredano-moreno, L. F, Segura-valdez, L. T, Echeverría, M. L, Martínez, O. M, Ramos, E, & Vázquez-nin, C. H. GH. The ultrastructural study of the interphase cell nucleus of Lacandonia schismatica (Lacandoniaceae:Triuridales)

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### **2. Conclusion**

A view of the cell emphasizing vertical resolution obtained by atomic force microscopy may represent a way to understand cell structure and function at the nanoscale, an interphase between molecular biology and cell biology.

### **Acknowledgements**

DGAPA-UNAM PAPIIT IN-227810, PAPIME PE211412, CONACyT 180835.

Rogelio Fragoso-Soriano and Tomás Nepomuceno-Mejía are postdoctoral fellows from ICyTDF and DGAPA-UNAM at Faculty of Sciences-UNAM, respectively. Georgina Alvarez-Fernandez is on-a leave-of-absence from the Department of Biochemistry, Faculty of Medicine, UNAM. Luis and Teresa Jiménez Segura for SRP and ATP synthase figures.

### **Author details**

María de Lourdes Segura-Valdez1 , Lourdes T. Agredano-Moreno1 , Tomás Nepomuceno-Mejía2 , Rogelio Fragoso-Soriano2 , Georgina Álvarez-Fernández2 , Alma Zamora-Cura1 , Reyna Lara-Martínez1 and Luis F. Jiménez-García1

1 Laboratory of Cell Nanobiology and Electron Microscopy Laboratory (Tlahuizcalpan), De‐ partment of Cell Biology, Faculty of Sciences, National Autonomous University of Mexico (UNAM), Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, México D.F, México

2 Visiting from the Department of Biochemistry, Faculty of Medicine, UNAM, México

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*1.1.4. Further research*

**2. Conclusion**

**Acknowledgements**

**Author details**

Alma Zamora-Cura1

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22 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

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Further research in our laboratory will focusing in visualizing the nanoscale cell structures involved in fundamental processes as ribosome biogenesis, at a high resolution *in situ* under

A view of the cell emphasizing vertical resolution obtained by atomic force microscopy may represent a way to understand cell structure and function at the nanoscale, an interphase

Rogelio Fragoso-Soriano and Tomás Nepomuceno-Mejía are postdoctoral fellows from ICyTDF and DGAPA-UNAM at Faculty of Sciences-UNAM, respectively. Georgina Alvarez-Fernandez is on-a leave-of-absence from the Department of Biochemistry, Faculty of Medicine,

, Lourdes T. Agredano-Moreno1

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**Chapter 2**

**The Exogenous Antioxidants**

Elena G. Olvera Hernández, Telma Flores Cerón and

One theory that was initially questioned was the proposal of Dr. Denhan Harman (1956) of the University of Nebraska. He was the first researcher to propose Free radicals (FR) as an important cause of cellular aging. Today this theory enjoys wide approval. FR are "disequili‐ brated" molecules that travel through our organism attempting to capture an electron of the

FR perform many useful functions in the organism (in fact, our own bodies manufacture these in moderate amounts to combat, for example, infections). When the increase of the in‐ tracellular contents of FR exceeds the cells' antioxidant defenses and are not efficient for in‐ hibiting them, this causes organic damage known as Oxidative stress (OS), which leads to a variety of physiological and biochemical changes that induce damage to biological mole‐ cules such as nucleic acids, proteins, lipids, etc., which consequently cause deterioration and cell death. An FR comprises any atom or group that possesses one or more unpaired elec‐

OS traditionally has been considered a static cell-damage process that derives from the aero‐ bic metabolism, and its clinical importance has been recognized to the point of currently be‐ ing considered a central component of any pathological process. OS in diverse pathological states affects a wide variety of physiological functions, contributing to or providing biofeed‐ back on the development of a great number of human degenerative diseases, such as athero‐ sclerosis, diabetes, cardiomyopathies, chronic inflammatory diseases (rheumatoid arthritis, intestinal inflammatory intestinal disease, and pancreatitis), neurological diseases, high blood pressure, ocular diseases, and pulmonary and hematological disease, cancer, and im‐

> © 2013 Romero et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

stable molecules to obtain its electrochemical stability [1,2].

Alejandro Chehue Romero,

Angelina Álvarez Chávez

http://dx.doi.org/10.5772/52490

trons; thus, FR are very reactive[3].

munodepression, asthma, among others [4].

**1. Introduction**

**Chapter 2**

### **The Exogenous Antioxidants**

Alejandro Chehue Romero, Elena G. Olvera Hernández, Telma Flores Cerón and Angelina Álvarez Chávez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52490

### **1. Introduction**

One theory that was initially questioned was the proposal of Dr. Denhan Harman (1956) of the University of Nebraska. He was the first researcher to propose Free radicals (FR) as an important cause of cellular aging. Today this theory enjoys wide approval. FR are "disequili‐ brated" molecules that travel through our organism attempting to capture an electron of the stable molecules to obtain its electrochemical stability [1,2].

FR perform many useful functions in the organism (in fact, our own bodies manufacture these in moderate amounts to combat, for example, infections). When the increase of the in‐ tracellular contents of FR exceeds the cells' antioxidant defenses and are not efficient for in‐ hibiting them, this causes organic damage known as Oxidative stress (OS), which leads to a variety of physiological and biochemical changes that induce damage to biological mole‐ cules such as nucleic acids, proteins, lipids, etc., which consequently cause deterioration and cell death. An FR comprises any atom or group that possesses one or more unpaired elec‐ trons; thus, FR are very reactive[3].

OS traditionally has been considered a static cell-damage process that derives from the aero‐ bic metabolism, and its clinical importance has been recognized to the point of currently be‐ ing considered a central component of any pathological process. OS in diverse pathological states affects a wide variety of physiological functions, contributing to or providing biofeed‐ back on the development of a great number of human degenerative diseases, such as athero‐ sclerosis, diabetes, cardiomyopathies, chronic inflammatory diseases (rheumatoid arthritis, intestinal inflammatory intestinal disease, and pancreatitis), neurological diseases, high blood pressure, ocular diseases, and pulmonary and hematological disease, cancer, and im‐ munodepression, asthma, among others [4].

© 2013 Romero et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This implication does not mean that Reactive oxygen species (ROS) always play a di‐ rect role in the development of the disease. In fact, reactive species predispose the or‐ ganism to diseases caused by other agents. In many cases, oxidative damage is to a greater degree the consequence of the tissue damage that the disease produces than a cause of the disease itself and therefore can contribute to worsening of the tissue dam‐ age generated [3].

Many benefits are conferred on antioxidants against diverse pathological states; in adition to this, an unequaled richness in natural foods is exhibited as well as our obligation to take ad‐

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 35

In the present work, the description is performed of the characteristics of the exogenous an‐

Vitamins are organic micronutrients that possess no energetic value, are biologically active, and with diverse molecular structure, which are necessary for humans in very small quanti‐ ties (micronutrients) and which should be supplied by the diet because humans are unable

The majority of vitamins are not synthesized by the organism, some can be formed in varia‐ ble amounts in the organism (vitamin D and niacin are synthesized endogenously; the for‐ mer forms in the skin by exposure to the sun, niacin can be obtained from tryptophan, and vitamins K2, B1, B2, and biotin are synthesized by bacteria). However, this synthesis is gen‐

The functions of the vitamins and the need of the organism for these are highly varied. Per‐ sons always need vitamins and at all life stages. However, during specific periods such as

The majority of vitamins have a basic function in the maintenance of health (doing honor to their name: "vita" means life. The term vitamin, proposed for the first time (in 1912) by Pol‐ ish Chemist Casimir Funk, is demonstrated by the appearance of deficiency or deficiencyrelated diseases that were caused by the lack of vitamins in the diet; for example, lack of vitamin A can produce blindness and the lack of vitamin D can retard bone growth; vita‐ mins also facilitate the metabolic reactions necessary for utilization of proteins, fats, and car‐

In addition, today we know that their nutritional role extends beyond that of the preven‐ tion of deficiency or deficiency-associated diseases. They can also aid in preventing some of the most prevalent chronic diseases in developed societies. Vitamin C, for example, prevents scurvy and also appears to prevent certain types of cancer. Vitamin E, a potent antioxidant, is a protector factor in cardiovascular disease and folates help in preventing

Traditionally, vitamins have been classified into two large groups in terms of their solubility

**Liposoluble vitamins:** A (retinol); D (ergocalciferol); E (tocopherol), and K (filoquinone and menadione), which are soluble in lipids but not in water; thus, they are generally vehicu‐ lized in the fat found in foods. These vitamins can accumulate and cause toxicity when in‐

tioxidants with regard to their employment in human health [6].

to synthesize and which are essential for maintaining health [7].

growth, pregnancy, lactancy, and disease, the needs are increased [8].

erally not sufficient to cover the organism's needs. [8,9].

vantage of and assess these.

**2. Vitamins**

bohydrates.

as follows:

fetal neural tube defects [9].

gested in large amounts [9].

While our own body produces FR in moderate amounts, amounts that decrease when we age, we must also bear in mind ROS-generated exogenous sources in organisms, such as an‐ tibiotics, drugs, alcohol, tobacco, stress, contaminants, chemotherapy, and exposure to Ultra‐ violet (UV) and ionizing radiation.

On the other hand, numerous epidemiological studies suggest that more persons could avoid the appearance of pathological processes if they consumed antioxidant-rich diets (fruits and vegetables). Thus, it would be possible to protect the organism more efficiently against OS, with the presentation of lesser risk of developing human degenerative diseases.

This has led to conducting experiments to identify the specific components responsible for the positive effects on health by the consumption of foods of plant origin. One explanation that has found great acceptance is that this is due to the presence of antioxidant nutrients such as vitamins C and E, carotenoids, flavonoids, selenium, etc., which would interfere with oxidative damage to the DNA, proteins, and lipids [3].

Antioxidants are synthetic or natural substances that present in low concentrations com‐ pared with the biomolecules that they should protect. Antioxidants protect by retarding or inhibiting the harmful effects of FR. They are classified as follows: endogens (glutathione, co-enzyme Q, etc.), which are manufactured by the cell itself; exogens, which enter the or‐ ganism through the diet (existing in determined foods) or through supplements with antiox‐ idant formulations, and co-factors (copper, zinc, manganese, iron, and selenium). The consumption of antioxidant exogens can increase protection of the body and aid antioxidant endogens in combating diseases [5].

Fortunately, numerous foods and supplements that we ingest are rich in the antioxidants that protect against damage to the cells. Vitamin C, which is found in abundance in citrics and vegetables, is perhaps the best known antioxidant. Vitamin E, which is liposoluble, can be found in nuts, unrefined vegetable oils such as corn, cotton seed, and wheat germ, and in whole grains. Beta carotene, which is converted into vitamin A in the organism, can be found in dark-leafed vegetables, carrots, and sweet potatoes.

In recent years, plant-derived natural antioxidants have been used frequently, given that they present activity that is comparable with the most frequently employed synthetic antiox‐ idants. Antioxidants are also found in a variety of herbs and foods that are to a great extent unknown in and not easily available in our environment, such as green tea, cardo mariano, ginkgo biloba, pine bark, and red wine; however, we do have dulcamara, dragon's blood, cat's claw, anamu/guinea hen weed), garlic, onion, aloe vera, and others that are very rich in antioxidants.

Many benefits are conferred on antioxidants against diverse pathological states; in adition to this, an unequaled richness in natural foods is exhibited as well as our obligation to take ad‐ vantage of and assess these.

In the present work, the description is performed of the characteristics of the exogenous an‐ tioxidants with regard to their employment in human health [6].

### **2. Vitamins**

This implication does not mean that Reactive oxygen species (ROS) always play a di‐ rect role in the development of the disease. In fact, reactive species predispose the or‐ ganism to diseases caused by other agents. In many cases, oxidative damage is to a greater degree the consequence of the tissue damage that the disease produces than a cause of the disease itself and therefore can contribute to worsening of the tissue dam‐

While our own body produces FR in moderate amounts, amounts that decrease when we age, we must also bear in mind ROS-generated exogenous sources in organisms, such as an‐ tibiotics, drugs, alcohol, tobacco, stress, contaminants, chemotherapy, and exposure to Ultra‐

On the other hand, numerous epidemiological studies suggest that more persons could avoid the appearance of pathological processes if they consumed antioxidant-rich diets (fruits and vegetables). Thus, it would be possible to protect the organism more efficiently against OS, with the presentation of lesser risk of developing human degenerative diseases.

This has led to conducting experiments to identify the specific components responsible for the positive effects on health by the consumption of foods of plant origin. One explanation that has found great acceptance is that this is due to the presence of antioxidant nutrients such as vitamins C and E, carotenoids, flavonoids, selenium, etc., which would interfere

Antioxidants are synthetic or natural substances that present in low concentrations com‐ pared with the biomolecules that they should protect. Antioxidants protect by retarding or inhibiting the harmful effects of FR. They are classified as follows: endogens (glutathione, co-enzyme Q, etc.), which are manufactured by the cell itself; exogens, which enter the or‐ ganism through the diet (existing in determined foods) or through supplements with antiox‐ idant formulations, and co-factors (copper, zinc, manganese, iron, and selenium). The consumption of antioxidant exogens can increase protection of the body and aid antioxidant

Fortunately, numerous foods and supplements that we ingest are rich in the antioxidants that protect against damage to the cells. Vitamin C, which is found in abundance in citrics and vegetables, is perhaps the best known antioxidant. Vitamin E, which is liposoluble, can be found in nuts, unrefined vegetable oils such as corn, cotton seed, and wheat germ, and in whole grains. Beta carotene, which is converted into vitamin A in the organism, can be

In recent years, plant-derived natural antioxidants have been used frequently, given that they present activity that is comparable with the most frequently employed synthetic antiox‐ idants. Antioxidants are also found in a variety of herbs and foods that are to a great extent unknown in and not easily available in our environment, such as green tea, cardo mariano, ginkgo biloba, pine bark, and red wine; however, we do have dulcamara, dragon's blood, cat's claw, anamu/guinea hen weed), garlic, onion, aloe vera, and others that are very rich in

age generated [3].

violet (UV) and ionizing radiation.

endogens in combating diseases [5].

antioxidants.

with oxidative damage to the DNA, proteins, and lipids [3].

34 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

found in dark-leafed vegetables, carrots, and sweet potatoes.

Vitamins are organic micronutrients that possess no energetic value, are biologically active, and with diverse molecular structure, which are necessary for humans in very small quanti‐ ties (micronutrients) and which should be supplied by the diet because humans are unable to synthesize and which are essential for maintaining health [7].

The majority of vitamins are not synthesized by the organism, some can be formed in varia‐ ble amounts in the organism (vitamin D and niacin are synthesized endogenously; the for‐ mer forms in the skin by exposure to the sun, niacin can be obtained from tryptophan, and vitamins K2, B1, B2, and biotin are synthesized by bacteria). However, this synthesis is gen‐ erally not sufficient to cover the organism's needs. [8,9].

The functions of the vitamins and the need of the organism for these are highly varied. Per‐ sons always need vitamins and at all life stages. However, during specific periods such as growth, pregnancy, lactancy, and disease, the needs are increased [8].

The majority of vitamins have a basic function in the maintenance of health (doing honor to their name: "vita" means life. The term vitamin, proposed for the first time (in 1912) by Pol‐ ish Chemist Casimir Funk, is demonstrated by the appearance of deficiency or deficiencyrelated diseases that were caused by the lack of vitamins in the diet; for example, lack of vitamin A can produce blindness and the lack of vitamin D can retard bone growth; vita‐ mins also facilitate the metabolic reactions necessary for utilization of proteins, fats, and car‐ bohydrates.

In addition, today we know that their nutritional role extends beyond that of the preven‐ tion of deficiency or deficiency-associated diseases. They can also aid in preventing some of the most prevalent chronic diseases in developed societies. Vitamin C, for example, prevents scurvy and also appears to prevent certain types of cancer. Vitamin E, a potent antioxidant, is a protector factor in cardiovascular disease and folates help in preventing fetal neural tube defects [9].

Traditionally, vitamins have been classified into two large groups in terms of their solubility as follows:

**Liposoluble vitamins:** A (retinol); D (ergocalciferol); E (tocopherol), and K (filoquinone and menadione), which are soluble in lipids but not in water; thus, they are generally vehicu‐ lized in the fat found in foods. These vitamins can accumulate and cause toxicity when in‐ gested in large amounts [9].

These are fat-soluble compounds and are found associated in foods with fats, mainly of ani‐ mal origin, and are absorbed with them. Therefore, any problem with respect to the absorp‐ tion of fats will be an obstacle to the absorption of liposoluble vitamins. The latter are stored in moderate amounts in the vital organs, especially in the liver [8].

as well as *in vivo*. Health benefits have been attributed to vitamin C, such as the anticancer‐ igenous, immunoregulator, antiinflammatory, and neuroprotector effect. Vitamin C rapidly eliminates Reactive oxygen species (ROS), Reactive nitrogen species (ROS), or both, and re‐ duces the transitional metallic ions of specific biosynthetic enzymes; thus, it can prevent bio‐ logical oxidation (García G.A., et al., 2006). The damage exercised by electronically disequilibrated and instable Reactive oxygen-derived species (ROS) (Free radicals, FR), ni‐ trogen-derived FR, NOS), and sulfa-derived or mixed FR harm through oxidation any of the cellular macromolecular components. If these are not neutralized, so-called "propagation" or "amplification" is produced and, in the case of oxidation, the peroxides are again oxi‐

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 37

**Clinical Uses:** Vascular diseases, cancer, cataracts, High blood pressure, acute pancreatitis, the common cold, iron fixation in blood hemoglobin, dermatological uses (photochronoag‐ ing, photoprotection, prevention of contact dermatitis, non-scarring of wounds, and hyper‐

Foods are substances or products of any nature that due to their characteristics and compo‐ nents are utilized for human nutrition. Ascorbic acid, commonly known as vitamin C, pro‐ motes resistance to infections by means of the immunological activity of leukocytes; it is useful for preventing and curing the common cold, as well as improving iron absorption in the human body and diminishing the incidence of anemia caused by lack of this mineral,

**Chemical structure:** Ascorbic acid is a 6-carbon ketolactone that has a structural relationship with glucose; it is a white substance, stable in its dry form, but in solution it oxidizes easily, even more so if exposed to heat. An alkaline pH (>7), copper, and iron also accelerate its oxi‐ dation. Its chemical structure is reminiscent of that of glucose (in many mammals and

Vitamin C is found mainly in foods of plant origin and can present in two chemically inter‐ changeable forms: ascorbic acid (the reduced form), and dihydroascorbic acid (the oxidated form) (See Figure 1), with both forms biologically functional and maintaining themselves in physiological equilibrium. If dihydroascorbic acid is hydrated, it is transformed into diketo‐ gluconic acid, which is not biologically active, and with this an irreversible transformation.

**Deficit:** It is well known that a deficiency of vitamin C causes scurvy in humans, thus the origin of the name "ascorbic" given to the acid [10].Scurvy was recognized for the first time in the XV and XVI Centuries as a serious disease contracted by sailors on long sea journeys (it appeared in adults after a nutritional need had existed for >6 months, because sailors had no access to fresh foods, including fruits and vegetables). Prior to the era of research on vita‐ mins, the British Navy established the practice of supplying lemons and other citric fruits to their sailors to avoid scurvy [13]. Scurvy is related with defective collagen synthesis, which manifests itself as the lack of scarring, progressive asthenia, gum inflammation, falling out of the teeth, joint inflammation and pain, capillary fragility, and esquimosis, thus the impor‐

which presents a high incidence in Mexican population.

plants, this vitamin is synthesized by glucose and galactose).

tance of the ingestion of vitamin C in the diet [11].

This hydration occurs spontaneously on neutral or alkaline dissolution.

dized into peroxyls [12].

pigmentation) [9].

**Hydrosoluble vitamins:** The following are vitamins of the B group: [B1 (thiamin); B2 (ribofla‐ vin; B3 (niacin); pantothenic acid; B6 (pyridoxine); biotin; folic acid, and B12 (cyanocobala‐ min)], and vitamin C (ascorbic acid), contained in the aqueous compartments of foods. [9].

These are water-soluble compounds that are found in foods of animal and plant origin. Dif‐ ferent from liposoluble vitamins, water-soluble vitamins are not stored in the body; thus, they should be ingested daily with food to avoid their supply becoming exhausted [8]. The hydrosoluble vitamins participate as co-enzymes in processes linked with the metabolism of organic foods: carbohydrates; lipids, and proteins.

One important difference between these two vitamin groups lies in their final destiny in the organism. An excess of water-soluble vitamins is rapidly excreted in the urine; on the other hand, liposoluble vitamins cannot be eliminated in this manner; they accumulate in tissues and organs. This characteristic is associated with a greater risk of toxicity, which means the ingestion of excessive amounts of liposoluble vitamins, especially vitamins A and E. Vita‐ min B12 constitutes an exception because it is stored in the liver in important quantities.

### **2.1. Vitamin C**

Vitamin C, also known as ascorbic acid (enantiomer, L-ascorbic acid) is an antioxidant hy‐ drosoluble vitamin, this due to that it is an electron donor, which explains its being a reduc‐ er that directly neutralizes or reduces the damage exercised by electronically disequilibrated and instable reactive species, denominated Free radicals (FR).

**Action:** The presence of this vitamin is required for a certain number of metabolic reactions in all animals and plants and is created internally by nearly all organisms, humans compris‐ ing a notable exception [10].Vitamin C is essential for the biosynthesis of collagen proteins, carnitine (which is a pro-catabolic transporter of fatty acids in the mitochondria), neuro‐ transmitters (mediators of cell communications, primarily of nerve expression), neuroendo‐ crine peptides, and in the control of angiogenesis; it aids in the development of teeth and gums, bone, cartilage, iron absorption, the growth and repair of normal connective tissue, the metabolism of fats, and the scarring of wounds; it promotes resistance to infections by means of the immunological activity of the leukocytes [11].

In addition to the biological functions mentioned, there are an infinite number of scientific and pseudoscientific reports that qualify this vitamin as an immunomodulator, an antiviral influenza protector, an antiatherogenic, an antiangiogenic, and as an anti-inflammatory, and debate continues on its activity in cancer and its antioxidant properties, given that there is information that lends support to its procancerigenenous and to its role as a pro-oxidant. Currently, this vitamin is the most widely employed vitamin in drugs, premedication, and nutritional supplements worldwide [11]. Various lines of experimental and epidemiological evidence suggest that vitamin C is a powerful antioxidant in biological systems, both *in vitro* as well as *in vivo*. Health benefits have been attributed to vitamin C, such as the anticancer‐ igenous, immunoregulator, antiinflammatory, and neuroprotector effect. Vitamin C rapidly eliminates Reactive oxygen species (ROS), Reactive nitrogen species (ROS), or both, and re‐ duces the transitional metallic ions of specific biosynthetic enzymes; thus, it can prevent bio‐ logical oxidation (García G.A., et al., 2006). The damage exercised by electronically disequilibrated and instable Reactive oxygen-derived species (ROS) (Free radicals, FR), ni‐ trogen-derived FR, NOS), and sulfa-derived or mixed FR harm through oxidation any of the cellular macromolecular components. If these are not neutralized, so-called "propagation" or "amplification" is produced and, in the case of oxidation, the peroxides are again oxi‐ dized into peroxyls [12].

These are fat-soluble compounds and are found associated in foods with fats, mainly of ani‐ mal origin, and are absorbed with them. Therefore, any problem with respect to the absorp‐ tion of fats will be an obstacle to the absorption of liposoluble vitamins. The latter are stored

**Hydrosoluble vitamins:** The following are vitamins of the B group: [B1 (thiamin); B2 (ribofla‐ vin; B3 (niacin); pantothenic acid; B6 (pyridoxine); biotin; folic acid, and B12 (cyanocobala‐ min)], and vitamin C (ascorbic acid), contained in the aqueous compartments of foods. [9]. These are water-soluble compounds that are found in foods of animal and plant origin. Dif‐ ferent from liposoluble vitamins, water-soluble vitamins are not stored in the body; thus, they should be ingested daily with food to avoid their supply becoming exhausted [8]. The hydrosoluble vitamins participate as co-enzymes in processes linked with the metabolism of

One important difference between these two vitamin groups lies in their final destiny in the organism. An excess of water-soluble vitamins is rapidly excreted in the urine; on the other hand, liposoluble vitamins cannot be eliminated in this manner; they accumulate in tissues and organs. This characteristic is associated with a greater risk of toxicity, which means the ingestion of excessive amounts of liposoluble vitamins, especially vitamins A and E. Vita‐ min B12 constitutes an exception because it is stored in the liver in important quantities.

Vitamin C, also known as ascorbic acid (enantiomer, L-ascorbic acid) is an antioxidant hy‐ drosoluble vitamin, this due to that it is an electron donor, which explains its being a reduc‐ er that directly neutralizes or reduces the damage exercised by electronically disequilibrated

**Action:** The presence of this vitamin is required for a certain number of metabolic reactions in all animals and plants and is created internally by nearly all organisms, humans compris‐ ing a notable exception [10].Vitamin C is essential for the biosynthesis of collagen proteins, carnitine (which is a pro-catabolic transporter of fatty acids in the mitochondria), neuro‐ transmitters (mediators of cell communications, primarily of nerve expression), neuroendo‐ crine peptides, and in the control of angiogenesis; it aids in the development of teeth and gums, bone, cartilage, iron absorption, the growth and repair of normal connective tissue, the metabolism of fats, and the scarring of wounds; it promotes resistance to infections by

In addition to the biological functions mentioned, there are an infinite number of scientific and pseudoscientific reports that qualify this vitamin as an immunomodulator, an antiviral influenza protector, an antiatherogenic, an antiangiogenic, and as an anti-inflammatory, and debate continues on its activity in cancer and its antioxidant properties, given that there is information that lends support to its procancerigenenous and to its role as a pro-oxidant. Currently, this vitamin is the most widely employed vitamin in drugs, premedication, and nutritional supplements worldwide [11]. Various lines of experimental and epidemiological evidence suggest that vitamin C is a powerful antioxidant in biological systems, both *in vitro*

in moderate amounts in the vital organs, especially in the liver [8].

36 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

organic foods: carbohydrates; lipids, and proteins.

and instable reactive species, denominated Free radicals (FR).

means of the immunological activity of the leukocytes [11].

**2.1. Vitamin C**

**Clinical Uses:** Vascular diseases, cancer, cataracts, High blood pressure, acute pancreatitis, the common cold, iron fixation in blood hemoglobin, dermatological uses (photochronoag‐ ing, photoprotection, prevention of contact dermatitis, non-scarring of wounds, and hyper‐ pigmentation) [9].

Foods are substances or products of any nature that due to their characteristics and compo‐ nents are utilized for human nutrition. Ascorbic acid, commonly known as vitamin C, pro‐ motes resistance to infections by means of the immunological activity of leukocytes; it is useful for preventing and curing the common cold, as well as improving iron absorption in the human body and diminishing the incidence of anemia caused by lack of this mineral, which presents a high incidence in Mexican population.

**Chemical structure:** Ascorbic acid is a 6-carbon ketolactone that has a structural relationship with glucose; it is a white substance, stable in its dry form, but in solution it oxidizes easily, even more so if exposed to heat. An alkaline pH (>7), copper, and iron also accelerate its oxi‐ dation. Its chemical structure is reminiscent of that of glucose (in many mammals and plants, this vitamin is synthesized by glucose and galactose).

Vitamin C is found mainly in foods of plant origin and can present in two chemically inter‐ changeable forms: ascorbic acid (the reduced form), and dihydroascorbic acid (the oxidated form) (See Figure 1), with both forms biologically functional and maintaining themselves in physiological equilibrium. If dihydroascorbic acid is hydrated, it is transformed into diketo‐ gluconic acid, which is not biologically active, and with this an irreversible transformation. This hydration occurs spontaneously on neutral or alkaline dissolution.

**Deficit:** It is well known that a deficiency of vitamin C causes scurvy in humans, thus the origin of the name "ascorbic" given to the acid [10].Scurvy was recognized for the first time in the XV and XVI Centuries as a serious disease contracted by sailors on long sea journeys (it appeared in adults after a nutritional need had existed for >6 months, because sailors had no access to fresh foods, including fruits and vegetables). Prior to the era of research on vita‐ mins, the British Navy established the practice of supplying lemons and other citric fruits to their sailors to avoid scurvy [13]. Scurvy is related with defective collagen synthesis, which manifests itself as the lack of scarring, progressive asthenia, gum inflammation, falling out of the teeth, joint inflammation and pain, capillary fragility, and esquimosis, thus the impor‐ tance of the ingestion of vitamin C in the diet [11].

**Obtaining Vitamin C:** This is a nutrient that is localized, above all, in citric fruits and vege‐ tables. All fruits and vegetables contain a certain amount of vitamin C. Foods that tend to be greater sources of vitamin C are, among others, the following: citrics (oranges, limes, lem‐ ons, grapefruit); guavas; pineapple; strawberries; kiwis; mangoes; melon; watermelon, and cantaloupe and, as examples of vegetables, green peppers, tomatoes, broccoli, cabbage, cau‐ liflower, green peas, asparagus, parsley, turnips, green tea, and other green-leafed vegeta‐ bles (spinach), potatoes or sweet potatoes, and yams.

is eliminated at a high percentage by the urine in the form of oxalic acid (catabolite) or, if it is ingested in very high amounts, as ascorbic acid. If there are deficiencies, absorption is very high and there is no elimination by urine. Ascorbic acid is found at high concentrations in various tissues, for example, suprarenal, liver, spleen, and kidneys.Alcohol consumption diminishes absorption of the vitamin, and the smoking habit depletes the levels of the vita‐ min in the organism; thus, it is recommended that smokers and regular alcohol consumers

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 39

The half-life of ascorbic acid in the organism is approximately16 days. Thus, the symptoms

**Figure 2.** Mechanism of absorption of vitamin C. The L-dihydroascorbic acid molecule is better absorbed than that of L-ascorbic acid. Passive absorption is dependent on a glucose transporter and active absorption is dependent on Na+.

**Toxicity:** It is scarcely probable for vitamin C intoxication (megadose) to occur because it is a hydrosoluble vitamin and excesses are eliminated through the urine. But if the daily dose

of vitamin C exceeds 2,000 mg/day, the following can appear [16]:

**•** Important alterations of glucose in persons with diabetes

**•** Diarrhea

**•** Insomnia

**•** Smarting on urinating

**•** Excessive iron absorption

**•** Prickling and irritation of the skin

**•** Formation of oxalate and uric kidney stones.

of scurvy do not appear for months in subjects with a diet deficient in vitamin C [7].

supplement their diet with vitamin C.

**Figure 1.** The oxidation-reduction (redox) reaction of vitamin C, molecular forms in equilibrium. L-dihydroascorbic acid also possesses biological activity, due to that in the body it is reduced to form ascorbic acid.

However, it is noteworthy that vitamin C diminishes on boiling, drying, or soaking foods; thus, it is convenient to consume these raw.

Daily recommended doses of ascorbic acid are 75 mg/day (for women) and 90 mg/day (for men). There are between 1.2 and 2 g (20 mg/kg body weight) of ascorbic acid available in the entire organism and its half-life ranges from 10‒20 days [11‒15].

**Absorption:** Vitamin C is easily absorbed in the small intestine, more precisely, in the duo‐ denum. It enters the blood by active transport and perhaps also by diffusion. It would ap‐ pear that the mechanism of absorption is saturable, due to that when large amounts of the vitamin are ingested, the percentage absorbed is much lower (Figure 2). In normal inges‐ tions (30‒180 mg), vitamin C is absorbed (bioavailability) at 70‒90% vs. a 16% ingestion of 12 g. Its concentrations in plasma are 10‒20 mcg/ml.

The vitamin C concentration in the leukocytes is in relation to the concentration of the vita‐ min in the tissues: therefore, by measuring the concentration of vitamin C in the leukocytes, we can know the real level of the vitamin in the tissues. The pool of vitamin C that humans possess under normal conditions is approximately 1,500 g. When this pool is full, vitamin C is eliminated at a high percentage by the urine in the form of oxalic acid (catabolite) or, if it is ingested in very high amounts, as ascorbic acid. If there are deficiencies, absorption is very high and there is no elimination by urine. Ascorbic acid is found at high concentrations in various tissues, for example, suprarenal, liver, spleen, and kidneys.Alcohol consumption diminishes absorption of the vitamin, and the smoking habit depletes the levels of the vita‐ min in the organism; thus, it is recommended that smokers and regular alcohol consumers supplement their diet with vitamin C.

The half-life of ascorbic acid in the organism is approximately16 days. Thus, the symptoms of scurvy do not appear for months in subjects with a diet deficient in vitamin C [7].

**Figure 2.** Mechanism of absorption of vitamin C. The L-dihydroascorbic acid molecule is better absorbed than that of L-ascorbic acid. Passive absorption is dependent on a glucose transporter and active absorption is dependent on Na+.

**Toxicity:** It is scarcely probable for vitamin C intoxication (megadose) to occur because it is a hydrosoluble vitamin and excesses are eliminated through the urine. But if the daily dose of vitamin C exceeds 2,000 mg/day, the following can appear [16]:

**•** Diarrhea

**Obtaining Vitamin C:** This is a nutrient that is localized, above all, in citric fruits and vege‐ tables. All fruits and vegetables contain a certain amount of vitamin C. Foods that tend to be greater sources of vitamin C are, among others, the following: citrics (oranges, limes, lem‐ ons, grapefruit); guavas; pineapple; strawberries; kiwis; mangoes; melon; watermelon, and cantaloupe and, as examples of vegetables, green peppers, tomatoes, broccoli, cabbage, cau‐ liflower, green peas, asparagus, parsley, turnips, green tea, and other green-leafed vegeta‐

**Figure 1.** The oxidation-reduction (redox) reaction of vitamin C, molecular forms in equilibrium. L-dihydroascorbic

However, it is noteworthy that vitamin C diminishes on boiling, drying, or soaking foods;

Daily recommended doses of ascorbic acid are 75 mg/day (for women) and 90 mg/day (for men). There are between 1.2 and 2 g (20 mg/kg body weight) of ascorbic acid available in the

**Absorption:** Vitamin C is easily absorbed in the small intestine, more precisely, in the duo‐ denum. It enters the blood by active transport and perhaps also by diffusion. It would ap‐ pear that the mechanism of absorption is saturable, due to that when large amounts of the vitamin are ingested, the percentage absorbed is much lower (Figure 2). In normal inges‐ tions (30‒180 mg), vitamin C is absorbed (bioavailability) at 70‒90% vs. a 16% ingestion of 12

The vitamin C concentration in the leukocytes is in relation to the concentration of the vita‐ min in the tissues: therefore, by measuring the concentration of vitamin C in the leukocytes, we can know the real level of the vitamin in the tissues. The pool of vitamin C that humans possess under normal conditions is approximately 1,500 g. When this pool is full, vitamin C

acid also possesses biological activity, due to that in the body it is reduced to form ascorbic acid.

entire organism and its half-life ranges from 10‒20 days [11‒15].

thus, it is convenient to consume these raw.

g. Its concentrations in plasma are 10‒20 mcg/ml.

bles (spinach), potatoes or sweet potatoes, and yams.

38 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants


Great controversy on the theme of Free radicals (FR) and antioxidants such as vitamin C continues, although there is conceptual dispute on whether these are the cause or conse‐ quence of the pathology. Biochemically, L-ascorbic acid donates two of its electrons from a double loop between the carbons in positions 2 and 3 (See Figure 1), and this donation is sequential, because the first molecular species generated after the loss of an electron is an FR denominated ascorbile acid. Similar to other FR with an unpaired electron, ascorbile is rela‐ tively stable and regularly non-reactive, with half-life of 10‒15 seconds.

It can be easily destroyed by the action of heat and of oxygen in the air. Vitamin E is one of

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 41

**Action:** It has been proposed that in addition to its antioxidant function, vitamin E can per‐ form a specific physicochemical function in the ordering of the lipic membranes, especially

*In vivo*, vitamin E acts when it breaks the chain of antioxidants, thus preventing the propa‐ gation of damage to the biological membranes that give rise to FR, something akin to a pro‐ tective shield of the cells' membranes that allows them not to age or to deteriorate due to oxygen-containing FR, retarding cellular catabolism, impeding the chain reaction that can produce peroxides from ensuing. It participates in the hemo group and in vitamin E defi‐ ciency; hemolytic anemia appears as a result of damage by FR. It also exercises an antitoxic function, a protector in the face of various chemical agents, especially preventing the forma‐ tion of peroxides from PFA, thus favoring the maintenance and stability of the biological

Tocopherols act as intra- and extracellular liposoluble antioxidants within the body. In par‐ ticular, the tocopherols protect the highly stored fatty acids (PFA) that are present in cellular and subcellular membranes, maintaining the integrity of the biological membranes, as well as other reactive compounds (e.g., vitamins A and C) from the oxidative damage that they could undergo on acting as FR traps. It has also been suggested that the tocopherols play an

Tocopherols favor normal growth and development, act as an anticoagulant agent, stimulate the formation of red globules, stimulate the recycling of vitamin C, reduce the risk of the first mortal heart attack in males, protect against prostate cancer, improve immunity, and is a potent antioxidant against cancer in general, cardiac diseases, and FR, thus possessing a

Vitamin E can reduce circulatory problems in the lower limbs, prevent coronary diseases, increase strength and muscular resistance (fostering achievement in sports), drive the sexual metabolism, and relieve menopausal symptoms. It can reduce the formation of scars (stimu‐ lating the curing of burns and wounds), could help in the treatment of acne, and is a poten‐

**Chemical structure:** The chemical formula for vitamin E (C29H50O2) is utilized for designat‐ ing a group of eight natural species (vitamers) of tocopherols and tocotrienols (α, β, γ, and δ). Together with vitamins A, D, and K, these constitute the group of liposoluble vitamins, characterized by deriving from the isoprenoid nucleus, soluble in lipids and organic sol‐ vents. They are essentials, given that the organism cannot synthesize them; therefore, their contribution is carried out through the diet in small amounts. For efficient absorption by the organism, these require the presence of fatty acids, bile, and lipolytic enzymes of the pan‐

Their structure comprises two primary parts: they contain a substitute aromatic ring de‐ nominated chromate and a long side chain (See Figure 3). These eight vitamers are divided

of phospholipids rich in arachidonic acid (thus acting as a membrane stabilizer) [1].

membranes and of the lisosomes in erythrocytes, liver, and muscle [19].

important role in cell respiration and in DNA and co-enzyme Q biosynthesis.

the least toxic liposoluble vitamins [9].

potent anti-aging function.

creas and intestinal mucosa [20].

tial treatment for diaper dermatitis and bee stings.

A great diversity of scientific works has allowed increasing the knowledge of the biological function of vitamin C, but this has also generated doubts, given that controversies have sur‐ faced. One of these controversial points comprises the pro-oxidant activity of vitamin C [12,17]. In the meanwhile, there are starting points, such as that its pro-oxidant activity de‐ pends as much on the dose in the diet as on the presence of trace metals, such as iron and free copper, in order for these to produce Fenton-type reactions, and this is amplified by the additional presence of certain FR in the circulating medium [16,18]. This would also depend on the vitamin C-directed reaction.

### **2.2. Vitamin E**

Discovered at the beginning of the 1920s in vegetable oils such as that of wheat germ by Herbert Evans and Katherine Bishop, vitamin E is also denominated tocopherol or the anti‐ sterile vitamin, due to its activity. Vitamin E is present in small amounts in all of the cells.

Vitamin E is a group of methylated phenolic compounds known as tocopherols and toco‐ trienols (a combination of the Greek words "τόκος" [birth] and "φέρειν" [possess or car‐ ry], which together mean "to carry a pregnancy"). Alpha-tocopherol is the most common of these and biologically that with the greatest vitaminic action. It is a lipophilic antioxi‐ dant that is localized in the cell membranes whose absorption and transport are found to be very highly linked with that of lipids. It is considered the most important lipid mole‐ cule protector because its action consists of protecting the polyunsaturated fatty acids of cell membrane phospholipids from cellular peroxidation, and also inhibiting the peroxi‐ dation of Low-density lipoproteins (LDL). It oxidizes the oxygen singlet, takes up hy‐ droxyl FR, neutralizes peroxides, and captures the superoxide anion in order to convert it into less reactive forms [1].

Fortunately, the foods with the greatest amounts of Polyunsaturated fatty acids (PFA) also tend to have a high content of this vitamin. Sunflower seed oil, one of the foods richest in PFA, also has the highest content of vitamin E among all of the foods that we habitually con‐ sume. It is also found in other vegetable oils, in dry fruit, and in eggs. In the mean diet of Spaniards, vegetable oils furnish 79% of the vitamin E that they consume [9].

Ingestion that adequately covers the recommended allowance appears to behave as a factor of protection in cardiovascular disease, on protecting LDL from oxidation, one of the main risk factors of this pathology. Vitamin E acts jointly and synergically with the mineral seleni‐ um, another of the organism's antioxidants.

It can be easily destroyed by the action of heat and of oxygen in the air. Vitamin E is one of the least toxic liposoluble vitamins [9].

Great controversy on the theme of Free radicals (FR) and antioxidants such as vitamin C continues, although there is conceptual dispute on whether these are the cause or conse‐ quence of the pathology. Biochemically, L-ascorbic acid donates two of its electrons from a double loop between the carbons in positions 2 and 3 (See Figure 1), and this donation is sequential, because the first molecular species generated after the loss of an electron is an FR denominated ascorbile acid. Similar to other FR with an unpaired electron, ascorbile is rela‐

A great diversity of scientific works has allowed increasing the knowledge of the biological function of vitamin C, but this has also generated doubts, given that controversies have sur‐ faced. One of these controversial points comprises the pro-oxidant activity of vitamin C [12,17]. In the meanwhile, there are starting points, such as that its pro-oxidant activity de‐ pends as much on the dose in the diet as on the presence of trace metals, such as iron and free copper, in order for these to produce Fenton-type reactions, and this is amplified by the additional presence of certain FR in the circulating medium [16,18]. This would also depend

Discovered at the beginning of the 1920s in vegetable oils such as that of wheat germ by Herbert Evans and Katherine Bishop, vitamin E is also denominated tocopherol or the anti‐ sterile vitamin, due to its activity. Vitamin E is present in small amounts in all of the cells.

Vitamin E is a group of methylated phenolic compounds known as tocopherols and toco‐ trienols (a combination of the Greek words "τόκος" [birth] and "φέρειν" [possess or car‐ ry], which together mean "to carry a pregnancy"). Alpha-tocopherol is the most common of these and biologically that with the greatest vitaminic action. It is a lipophilic antioxi‐ dant that is localized in the cell membranes whose absorption and transport are found to be very highly linked with that of lipids. It is considered the most important lipid mole‐ cule protector because its action consists of protecting the polyunsaturated fatty acids of cell membrane phospholipids from cellular peroxidation, and also inhibiting the peroxi‐ dation of Low-density lipoproteins (LDL). It oxidizes the oxygen singlet, takes up hy‐ droxyl FR, neutralizes peroxides, and captures the superoxide anion in order to convert

Fortunately, the foods with the greatest amounts of Polyunsaturated fatty acids (PFA) also tend to have a high content of this vitamin. Sunflower seed oil, one of the foods richest in PFA, also has the highest content of vitamin E among all of the foods that we habitually con‐ sume. It is also found in other vegetable oils, in dry fruit, and in eggs. In the mean diet of

Ingestion that adequately covers the recommended allowance appears to behave as a factor of protection in cardiovascular disease, on protecting LDL from oxidation, one of the main risk factors of this pathology. Vitamin E acts jointly and synergically with the mineral seleni‐

Spaniards, vegetable oils furnish 79% of the vitamin E that they consume [9].

tively stable and regularly non-reactive, with half-life of 10‒15 seconds.

40 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

on the vitamin C-directed reaction.

it into less reactive forms [1].

um, another of the organism's antioxidants.

**2.2. Vitamin E**

**Action:** It has been proposed that in addition to its antioxidant function, vitamin E can per‐ form a specific physicochemical function in the ordering of the lipic membranes, especially of phospholipids rich in arachidonic acid (thus acting as a membrane stabilizer) [1].

*In vivo*, vitamin E acts when it breaks the chain of antioxidants, thus preventing the propa‐ gation of damage to the biological membranes that give rise to FR, something akin to a pro‐ tective shield of the cells' membranes that allows them not to age or to deteriorate due to oxygen-containing FR, retarding cellular catabolism, impeding the chain reaction that can produce peroxides from ensuing. It participates in the hemo group and in vitamin E defi‐ ciency; hemolytic anemia appears as a result of damage by FR. It also exercises an antitoxic function, a protector in the face of various chemical agents, especially preventing the forma‐ tion of peroxides from PFA, thus favoring the maintenance and stability of the biological membranes and of the lisosomes in erythrocytes, liver, and muscle [19].

Tocopherols act as intra- and extracellular liposoluble antioxidants within the body. In par‐ ticular, the tocopherols protect the highly stored fatty acids (PFA) that are present in cellular and subcellular membranes, maintaining the integrity of the biological membranes, as well as other reactive compounds (e.g., vitamins A and C) from the oxidative damage that they could undergo on acting as FR traps. It has also been suggested that the tocopherols play an important role in cell respiration and in DNA and co-enzyme Q biosynthesis.

Tocopherols favor normal growth and development, act as an anticoagulant agent, stimulate the formation of red globules, stimulate the recycling of vitamin C, reduce the risk of the first mortal heart attack in males, protect against prostate cancer, improve immunity, and is a potent antioxidant against cancer in general, cardiac diseases, and FR, thus possessing a potent anti-aging function.

Vitamin E can reduce circulatory problems in the lower limbs, prevent coronary diseases, increase strength and muscular resistance (fostering achievement in sports), drive the sexual metabolism, and relieve menopausal symptoms. It can reduce the formation of scars (stimu‐ lating the curing of burns and wounds), could help in the treatment of acne, and is a poten‐ tial treatment for diaper dermatitis and bee stings.

**Chemical structure:** The chemical formula for vitamin E (C29H50O2) is utilized for designat‐ ing a group of eight natural species (vitamers) of tocopherols and tocotrienols (α, β, γ, and δ). Together with vitamins A, D, and K, these constitute the group of liposoluble vitamins, characterized by deriving from the isoprenoid nucleus, soluble in lipids and organic sol‐ vents. They are essentials, given that the organism cannot synthesize them; therefore, their contribution is carried out through the diet in small amounts. For efficient absorption by the organism, these require the presence of fatty acids, bile, and lipolytic enzymes of the pan‐ creas and intestinal mucosa [20].

Their structure comprises two primary parts: they contain a substitute aromatic ring de‐ nominated chromate and a long side chain (See Figure 3). These eight vitamers are divided into two basic groups: four tocopherols, and four tocotrienols, which are differentiated in the side-chain saturation; the tocopherols possess a saturated chain, and the tocotrienols, an unsaturated one with three double loops on carbons 3, 7, and 11 (Figure 4).

WIthin each group, the vitamers differ in the number and position of the methyl groups in the chromate ring, designating these as α, β, and δ (Figures 4 and 5) [19,20].

**Figure 3.** Components of the tocotrienol structure.

**Figure 4.** Chemical structure of the possible stereoisomers of the tocopherols and tocotrienols that make up the natu‐ ral vitamin E. The presence of the -CH3 or -H groups in the chromate ring define that these substances as α, β, γ, and δ.

**Figure 5.** Chemical structure of the tocopherols.

**Deficit:** The deficiency of vitamin E can be due to two causes: not consuming a certain food that contains it, or poor fat absorption, due to that vitamin E is a liposoluble vitamin, that is,

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 43

Vitamin E is essential for humans. Its deficiency is not frequent even with persons who con‐ sume diets that are relatively poor in this vitamin, and could develop in cases of intense malabsorption of fats, cystic fibrosis, some forms of chronic liver disease, and congenital

it is diluted in fats for its absorption in the intestine in the micelles.

The presence of three chiral centers (position C2 of the chromate ring, positions C4 and C8 of the phytyl chain) allow there to be a total of eight configurations depending on the R or S orientation of the methyl group in each of the chiral centers (Figures 3 and 5) [19].

During vitamin E synthesis, equimolar amounts of these isomers (vitamers) are produced.

**Figure 5.** Chemical structure of the tocopherols.

into two basic groups: four tocopherols, and four tocotrienols, which are differentiated in the side-chain saturation; the tocopherols possess a saturated chain, and the tocotrienols, an

WIthin each group, the vitamers differ in the number and position of the methyl groups in

**Figure 4.** Chemical structure of the possible stereoisomers of the tocopherols and tocotrienols that make up the natu‐ ral vitamin E. The presence of the -CH3 or -H groups in the chromate ring define that these substances as α, β, γ, and δ.

The presence of three chiral centers (position C2 of the chromate ring, positions C4 and C8 of the phytyl chain) allow there to be a total of eight configurations depending on the R or S

During vitamin E synthesis, equimolar amounts of these isomers (vitamers) are produced.

orientation of the methyl group in each of the chiral centers (Figures 3 and 5) [19].

unsaturated one with three double loops on carbons 3, 7, and 11 (Figure 4).

42 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

the chromate ring, designating these as α, β, and δ (Figures 4 and 5) [19,20].

**Figure 3.** Components of the tocotrienol structure.

**Deficit:** The deficiency of vitamin E can be due to two causes: not consuming a certain food that contains it, or poor fat absorption, due to that vitamin E is a liposoluble vitamin, that is, it is diluted in fats for its absorption in the intestine in the micelles.

Vitamin E is essential for humans. Its deficiency is not frequent even with persons who con‐ sume diets that are relatively poor in this vitamin, and could develop in cases of intense malabsorption of fats, cystic fibrosis, some forms of chronic liver disease, and congenital abetalipoproteinemia. The newborn, fundamentally the premature infant, is particularly vulnerable to vitamin E deficiency because of its deficient body reserves. The majority of vi‐ tamin E deficiency-associated sequelae are subclinical. Neuropathological alterations have been described in at-risk patients and the most frequent manifestations comprise diverse grades of areflexia, walk proprioception disorders, diminution of vibratory sensations, and ophthalmoplegia [1].

Erythrocytic hemolysis. The FR attack membrane and erythrocyte integrity; thus, these are

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 45

This produces sterility in some animals and certain disorders associated with reproduction,

**Obtaining Vitamin E:** Tocopherol-rich dietary sources include the following: alfalfa flour; wheat germ flour (125‒100 mg/kg); hen's egg (egg yolk); polished rice (100‒75 mg/kg); rice bran; mediator wheat (75‒50 mg/kg); dry yeast; dry distillery solubles; barley grains; whole soy flour; corn grains; ground wheat residues (50‒25 mg/kg); corn gluten flour; wheat bran; rye grains; sorghum; fish flour; oatmeal; sunflower seed flour; cotton seed flour (25‒10 mg/ kg); almonds; hazelnuts; sunflower seeds; nuts, and peanuts. Other sources include all vege‐ table oils and green vegetable harvests, above all those with green leaves, sweet chile pep‐

pers, avocado, fresh potatoes, celery, cabbage, fruits, chicken, fish, and butter [19, 20].

1 International unit (IU) of vitamin E = 1 mg αlpha-tocopherol, and 1 IU of vitamin E = 0.67 mg of vitamin E. In adults, the Minimum daily requirement (MDR) for vitamin E is 15 mg/

The principal sources are vegetable oils and wheat germ. Hydrogenation of the oils does not produce a very important loss of tocopherols in terms of their content in the original oil;

One hundred percent of the MDR of vitamin E can be covered with two tablespoons of sun‐

**Absorption:** The absorption of vitamin E in the intestinal lumen depends on the process necessary for the digestion of fats and uptake by the erythrocytes. In order to liberate the free fatty acids from the triglycerides the diet requires pancreatic esterases. Bile acids, mono‐ glycerides, and free fatty acids are important components of mixed micelles. Esterases are required for the hydrolytic unfolding of tocopherol esters, a common form of vitamin E in dietary supplements. Bile acids, necessary for the formation of mixed micelles, are indispen‐ sable for the absorption of vitamin E, and its secretion in the lymphatic system is deficient. In patients with biliary obstruction, cholestasic disease of the liver, pancreatitis, or cystic fib‐ rosis, a vitamin E deficiency presents as the result of malabsorption. Vitamin E is transport‐ ed by means of plasma lipoproteins in an unspecific manner. The greater part of vitamin E

The four forms of tocopherol are similarly absorbed in the diet and are transported to the peripheral cells by the kilomicrons. After hydrolysis by the lipoprotein lipases, part of the

Vitamin E accumulates in the liver as the other liposoluble vitamins (A and D) do, but dif‐

**Toxicity:** High doses of vitamin E can interfere with the action of vitamin K and also inter‐

death, and fetal reabsorption in females and testicular degeneration in males.

The excess of vitamin E does not appear to produce noxious toxic effects.

day, and up to 200‒600 mg/day would not cause any disorder.

present in the body is localized in adipose tissue [19, 20].

fere with the effect of anticoagulants: hemorrhages.

tocopherols is liberated by the kilomicrons of the peripheral tissues [19].

ferent from these, it also accumulates in muscle and adipose tissue.

thus, margarine and mayonnaise contain this vitamin, in lesser amounts.

also hemolysis-sensitive.

flower seed or corn oil.

With regard to the relationship of vitamin E deficiency and the development of cardiovascu‐ lar disease and cancer, there are no conclusive results to date [1,19].

The existence of a lack of vitamin E is rare. If this occurs, it is manifested in specific cases, that is, in the following three situations:


Likewise, vitamin E levels can fall due to a zinc deficiency.

Lipid-absorption disorders can present in adults. From 3 years on, lack of absorption presents neurological conditions. The deficiency appears in less time due to the infants' not possessing so great a vitamin-E reserve.

#### *2.2.1. Symptoms of vitamin E deficiency*

Irritability, Fluid retention, Hemolytic anemia (destruction of red globules), Ocular alteration

Damage to the nervous system, Difficulty in maintaining equilibrium, Tiredness, apathy

Inability to concentrate, Alterations in the walk and Diminished immune response.

#### *2.2.2. Vitamin E deficiency-related diseases*

Encephalomalacia. This is due to the lack of vitamin E, which does not avoid PFA oxida‐ tion of the ration of the vitamin; consequently, hemorrhages and edema are produced in the cerebellum.

Exudative diathesis. This is due to deficient rations of vitamin E and selenium. The disease can be prevented with the administration of selenium, which acts on vitamin E as an agent that favors the storage of selenium in the organism.

Nutritional white muscle or muscular dystrophy. Rations with a scarcity of vitamin E, sele‐ nium, and azo-containing amino acids and a high content of polyunsaturated fats cause muscle degeneration in chest and thighs.

Ceroid pigmentation. This corresponds to the yellowish-brown coloration of adipose tissue in the liver due to the oxidation *in vivo* of lipids.

Erythrocytic hemolysis. The FR attack membrane and erythrocyte integrity; thus, these are also hemolysis-sensitive.

This produces sterility in some animals and certain disorders associated with reproduction, death, and fetal reabsorption in females and testicular degeneration in males.

The excess of vitamin E does not appear to produce noxious toxic effects.

abetalipoproteinemia. The newborn, fundamentally the premature infant, is particularly vulnerable to vitamin E deficiency because of its deficient body reserves. The majority of vi‐ tamin E deficiency-associated sequelae are subclinical. Neuropathological alterations have been described in at-risk patients and the most frequent manifestations comprise diverse grades of areflexia, walk proprioception disorders, diminution of vibratory sensations, and

With regard to the relationship of vitamin E deficiency and the development of cardiovascu‐

The existence of a lack of vitamin E is rare. If this occurs, it is manifested in specific cases,

**a.** Persons with a difficulty of absorbing or secreting bile or who suffer from fat metabo‐

**b.** Premature infants (with Very low birth weight, VLB) who weigh <1,500 grams at birth

Lipid-absorption disorders can present in adults. From 3 years on, lack of absorption presents neurological conditions. The deficiency appears in less time due to the infants' not

Irritability, Fluid retention, Hemolytic anemia (destruction of red globules), Ocular alteration

Encephalomalacia. This is due to the lack of vitamin E, which does not avoid PFA oxida‐ tion of the ration of the vitamin; consequently, hemorrhages and edema are produced in

Exudative diathesis. This is due to deficient rations of vitamin E and selenium. The disease can be prevented with the administration of selenium, which acts on vitamin E as an agent

Nutritional white muscle or muscular dystrophy. Rations with a scarcity of vitamin E, sele‐ nium, and azo-containing amino acids and a high content of polyunsaturated fats cause

Ceroid pigmentation. This corresponds to the yellowish-brown coloration of adipose tissue

Damage to the nervous system, Difficulty in maintaining equilibrium, Tiredness, apathy

Inability to concentrate, Alterations in the walk and Diminished immune response.

**c.** Persons with genetic abnormalities in alpha-tocopherol transporter proteins.

lar disease and cancer, there are no conclusive results to date [1,19].

lism-related disease (celiac disease or cystic fibrosis)

44 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Likewise, vitamin E levels can fall due to a zinc deficiency.

ophthalmoplegia [1].

that is, in the following three situations:

possessing so great a vitamin-E reserve.

*2.2.1. Symptoms of vitamin E deficiency*

*2.2.2. Vitamin E deficiency-related diseases*

muscle degeneration in chest and thighs.

in the liver due to the oxidation *in vivo* of lipids.

that favors the storage of selenium in the organism.

the cerebellum.

**Obtaining Vitamin E:** Tocopherol-rich dietary sources include the following: alfalfa flour; wheat germ flour (125‒100 mg/kg); hen's egg (egg yolk); polished rice (100‒75 mg/kg); rice bran; mediator wheat (75‒50 mg/kg); dry yeast; dry distillery solubles; barley grains; whole soy flour; corn grains; ground wheat residues (50‒25 mg/kg); corn gluten flour; wheat bran; rye grains; sorghum; fish flour; oatmeal; sunflower seed flour; cotton seed flour (25‒10 mg/ kg); almonds; hazelnuts; sunflower seeds; nuts, and peanuts. Other sources include all vege‐ table oils and green vegetable harvests, above all those with green leaves, sweet chile pep‐ pers, avocado, fresh potatoes, celery, cabbage, fruits, chicken, fish, and butter [19, 20].

1 International unit (IU) of vitamin E = 1 mg αlpha-tocopherol, and 1 IU of vitamin E = 0.67 mg of vitamin E. In adults, the Minimum daily requirement (MDR) for vitamin E is 15 mg/ day, and up to 200‒600 mg/day would not cause any disorder.

The principal sources are vegetable oils and wheat germ. Hydrogenation of the oils does not produce a very important loss of tocopherols in terms of their content in the original oil; thus, margarine and mayonnaise contain this vitamin, in lesser amounts.

One hundred percent of the MDR of vitamin E can be covered with two tablespoons of sun‐ flower seed or corn oil.

**Absorption:** The absorption of vitamin E in the intestinal lumen depends on the process necessary for the digestion of fats and uptake by the erythrocytes. In order to liberate the free fatty acids from the triglycerides the diet requires pancreatic esterases. Bile acids, mono‐ glycerides, and free fatty acids are important components of mixed micelles. Esterases are required for the hydrolytic unfolding of tocopherol esters, a common form of vitamin E in dietary supplements. Bile acids, necessary for the formation of mixed micelles, are indispen‐ sable for the absorption of vitamin E, and its secretion in the lymphatic system is deficient. In patients with biliary obstruction, cholestasic disease of the liver, pancreatitis, or cystic fib‐ rosis, a vitamin E deficiency presents as the result of malabsorption. Vitamin E is transport‐ ed by means of plasma lipoproteins in an unspecific manner. The greater part of vitamin E present in the body is localized in adipose tissue [19, 20].

The four forms of tocopherol are similarly absorbed in the diet and are transported to the peripheral cells by the kilomicrons. After hydrolysis by the lipoprotein lipases, part of the tocopherols is liberated by the kilomicrons of the peripheral tissues [19].

Vitamin E accumulates in the liver as the other liposoluble vitamins (A and D) do, but dif‐ ferent from these, it also accumulates in muscle and adipose tissue.

**Toxicity:** High doses of vitamin E can interfere with the action of vitamin K and also inter‐ fere with the effect of anticoagulants: hemorrhages.

Since 2001, it was calculated that 70% of the U.S. population occasionally consumes dietary supplements and that 40% do so on a regular basis. In 2002, Montuiler and collaborators in‐ formed, in a population of physicians, that 64% consumed doses of >400 IU/day of vitamin E and that the average obtained from food sources is 9.3 mg of αlpha-tocopherol per day (ap‐ proximately 14 IU/day). In 2005, Ford and coworkers found that 11.3% of the U.S. popula‐ tion consumes at least 400 IU/day of vitamin E and that median daily ingestion is 8.8 IU/day.

**Sources:** Retinol is only found in the lipidic part of foods of animal origin as follows; whole milk; lard; cream; cheese; egg yolk; eels, and fatty fish, due to their self-storage in the liver and in the oils extracted from the liver. The latter, as well as the oils extracted from the liver (veal and pork), comprise an important source of vitamin A. Cod liver oil constitutes source richest in vitamin A, although this cannot be considered a food in the strictest sense. In the case of skim/low-fat milk, this vitamin would be eliminated, but by law it is restituted to its

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 47

Vegetables contain only provitamins or carotenes (all of these coloring pigments, such as al‐ pha, beta, and gamma carotene). Garden vegetables (spinach and similar vegetables), car‐ rots, sweet chile peppers, potatoes, tomatoes, and red and yellow fruits are the main suppliers. We must bear on mind that there are numerous carotenes that do not possess any provitamnic A activity, such as lycopene from the tomato, although it does act as a neutral‐

**Structure of Vitamin A:** This vitamin is a diterpene (C20H32) that can present in the follow‐

Retinyl-palmitate, in the case of lengthening of a side chain from esterification with palmitic

**Absorption:** The metabolism of the vitamin responds to the same general mechanisms of di‐ gestion and absorption as those of other lipidic substances. Absorption is carried out in the form of carotenes or similar substances at the intestinal level within the interior of the mi‐

Retinol esters are absorbed from 80‒90%, while the beta-carotenes are absorbed at only 40‒ 50%. Factors in the diet that affect carotene absorption include the origin and the concentration of the fat in the diet, the amount of carotenoids, and the digestibility of the foods. Vitamin A is first processed in the intestine, and afterward it arrives at the liver via portal, the liver being the main storage organ. In addition, the liver is responsible for regulating the secretion of the reti‐ nol bound to the retinoid-binding protein. Carotene absorption in particular is very inefficient in raw foods, and its content in lipids in the diet is low. The efficiency of conversion into retinol, which is quite variable and, in general, low, depends not only on the structure of the carote‐ noids, but also on their proteinic ingestion. Thus, when carotene ingestion is very high, those which have not been transformed into retinol in the retinal mucosa are absorbed unaltered, bind with the lipoproteins, and are deposited in the skin and the mucosa, on which they confer

**Toxicity:** Both the deficiency as well as the excess of vitamin A causes fetal malformations. Ingestion of large amounts of this vitamin can give rise to skin alterations (scaling), hair fall, weakness, choking, vomiting, etc. In extreme cases, great amounts accumulate in the liver,

Retinol (See Figure 6), when the side chain terminal is an alcohol group (–CH2OH)

original content; examples of these include manchego cheese, margarine, and butter.

izer of FR [21, 22].

ing various molecular forms:

acid (–CH2O-CO-(CH2)14-CH3).

Retinal, the carbon terminal, is an aldehyde (–CHO)

celles and quilomicrons, together with other fats.

Retinoic acid, when the terminal group is acidic (–COOH)

a typical yellowish color, constituting hypercarotenosis [21].

producing hepatic disorders that end up as fatty liver.

Part of the potential danger of consuming high doses of vitamin E could be attributed to its effect on displacing other soluble antioxidants in fats and breaking up the natural balance of the antioxidant system. This can also inhibit the Glutathione S-transferase (GST) cytosolic enzymes, which contribute to the detoxification of drugs and endogenous toxins. In fact, one study on αlpha-tocopherol and β-carotene demonstrated a significant increase in the risk of hemorrhagic shock among study participants treated with vitamin E. Other data suggest that vitamin E could also affect the conversion of β-carotene into vitamin E and the distribu‐ tion of the latter in animal tissues. Vitamin E possesses anticoagulant properties, possibly on interfering with the mechanisms mediated by vitamin K. In recent studies conducted *in vi‐ tro*, it was demonstrated that vitamin E potentiates the antiplatelet effects of acetylsalicylic acid; therefore, one should be alert to this effect when both substances are consumed [19].

### **2.3. Vitamin A**

This is a term that is employed to describe a family of liposoluble compounds that are essen‐ tial in the diet and that have a structural relationship and share their biological activity. It is an antioxidant vitamin that eliminates Free radicals (FR) and protects the DNA from their mutagenic action, thus continuing to halt cellular aging. Their oxygen sensitivity is due to the large amount of double loops present in their structure. Their biological activity is attrib‐ uted to all-*trans* retinol, but from the nutritional viewpoint, they should be included in un‐ der the denomination of A provitamins, certain carotenoids, and similar compounds, the carotenals, which have the capacity to give rise to retinol from the organism.

Vitamin A is a hydrosoluble alcohol that is soluble in fats and organic solvents. It is stable when exposed to heat and light, but is destroyed by oxidation; thus, cooking in contact with the air can diminish the vitamin A content in foods. Its bioavailability increases with the presence of vitamin E and other antioxidants [21].

**Function:** In its different forms, vitamin A, also known as an antixerophthalmic, is necessary in vision, normal growth (its deficiency causes bone growth delay), reproduction, cellular proliferation, differentiation (which confers upon it a role in processes such as spermatogen‐ esis, fetal development, immunological response, etc.), fetal development, and the integrity of the immune system. Others of these include its being an antioxidant, amino acid metabo‐ lism, the structure and function of other cells, reproduction, and epithelial tissues.

Vitamin A participates in the synthesis of glycoproteins, which contributes to maintaining the integrity of epithelial tissue in all of the body's cavities. Epithelial dissection especially affects the conjunctivae of the eye (xerophthalmia), which renders the cornea opaque and causes crevices, producing blindness and facilitating eye infections.

**Sources:** Retinol is only found in the lipidic part of foods of animal origin as follows; whole milk; lard; cream; cheese; egg yolk; eels, and fatty fish, due to their self-storage in the liver and in the oils extracted from the liver. The latter, as well as the oils extracted from the liver (veal and pork), comprise an important source of vitamin A. Cod liver oil constitutes source richest in vitamin A, although this cannot be considered a food in the strictest sense. In the case of skim/low-fat milk, this vitamin would be eliminated, but by law it is restituted to its original content; examples of these include manchego cheese, margarine, and butter.

Vegetables contain only provitamins or carotenes (all of these coloring pigments, such as al‐ pha, beta, and gamma carotene). Garden vegetables (spinach and similar vegetables), car‐ rots, sweet chile peppers, potatoes, tomatoes, and red and yellow fruits are the main suppliers. We must bear on mind that there are numerous carotenes that do not possess any provitamnic A activity, such as lycopene from the tomato, although it does act as a neutral‐ izer of FR [21, 22].

**Structure of Vitamin A:** This vitamin is a diterpene (C20H32) that can present in the follow‐ ing various molecular forms:

Retinol (See Figure 6), when the side chain terminal is an alcohol group (–CH2OH)

Retinal, the carbon terminal, is an aldehyde (–CHO)

Since 2001, it was calculated that 70% of the U.S. population occasionally consumes dietary supplements and that 40% do so on a regular basis. In 2002, Montuiler and collaborators in‐ formed, in a population of physicians, that 64% consumed doses of >400 IU/day of vitamin E and that the average obtained from food sources is 9.3 mg of αlpha-tocopherol per day (ap‐ proximately 14 IU/day). In 2005, Ford and coworkers found that 11.3% of the U.S. popula‐ tion consumes at least 400 IU/day of vitamin E and that median daily ingestion is 8.8 IU/day.

46 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Part of the potential danger of consuming high doses of vitamin E could be attributed to its effect on displacing other soluble antioxidants in fats and breaking up the natural balance of the antioxidant system. This can also inhibit the Glutathione S-transferase (GST) cytosolic enzymes, which contribute to the detoxification of drugs and endogenous toxins. In fact, one study on αlpha-tocopherol and β-carotene demonstrated a significant increase in the risk of hemorrhagic shock among study participants treated with vitamin E. Other data suggest that vitamin E could also affect the conversion of β-carotene into vitamin E and the distribu‐ tion of the latter in animal tissues. Vitamin E possesses anticoagulant properties, possibly on interfering with the mechanisms mediated by vitamin K. In recent studies conducted *in vi‐ tro*, it was demonstrated that vitamin E potentiates the antiplatelet effects of acetylsalicylic acid; therefore, one should be alert to this effect when both substances are consumed [19].

This is a term that is employed to describe a family of liposoluble compounds that are essen‐ tial in the diet and that have a structural relationship and share their biological activity. It is an antioxidant vitamin that eliminates Free radicals (FR) and protects the DNA from their mutagenic action, thus continuing to halt cellular aging. Their oxygen sensitivity is due to the large amount of double loops present in their structure. Their biological activity is attrib‐ uted to all-*trans* retinol, but from the nutritional viewpoint, they should be included in un‐ der the denomination of A provitamins, certain carotenoids, and similar compounds, the

Vitamin A is a hydrosoluble alcohol that is soluble in fats and organic solvents. It is stable when exposed to heat and light, but is destroyed by oxidation; thus, cooking in contact with the air can diminish the vitamin A content in foods. Its bioavailability increases with the

**Function:** In its different forms, vitamin A, also known as an antixerophthalmic, is necessary in vision, normal growth (its deficiency causes bone growth delay), reproduction, cellular proliferation, differentiation (which confers upon it a role in processes such as spermatogen‐ esis, fetal development, immunological response, etc.), fetal development, and the integrity of the immune system. Others of these include its being an antioxidant, amino acid metabo‐

Vitamin A participates in the synthesis of glycoproteins, which contributes to maintaining the integrity of epithelial tissue in all of the body's cavities. Epithelial dissection especially affects the conjunctivae of the eye (xerophthalmia), which renders the cornea opaque and

lism, the structure and function of other cells, reproduction, and epithelial tissues.

causes crevices, producing blindness and facilitating eye infections.

carotenals, which have the capacity to give rise to retinol from the organism.

presence of vitamin E and other antioxidants [21].

**2.3. Vitamin A**

Retinoic acid, when the terminal group is acidic (–COOH)

Retinyl-palmitate, in the case of lengthening of a side chain from esterification with palmitic acid (–CH2O-CO-(CH2)14-CH3).

**Absorption:** The metabolism of the vitamin responds to the same general mechanisms of di‐ gestion and absorption as those of other lipidic substances. Absorption is carried out in the form of carotenes or similar substances at the intestinal level within the interior of the mi‐ celles and quilomicrons, together with other fats.

Retinol esters are absorbed from 80‒90%, while the beta-carotenes are absorbed at only 40‒ 50%. Factors in the diet that affect carotene absorption include the origin and the concentration of the fat in the diet, the amount of carotenoids, and the digestibility of the foods. Vitamin A is first processed in the intestine, and afterward it arrives at the liver via portal, the liver being the main storage organ. In addition, the liver is responsible for regulating the secretion of the reti‐ nol bound to the retinoid-binding protein. Carotene absorption in particular is very inefficient in raw foods, and its content in lipids in the diet is low. The efficiency of conversion into retinol, which is quite variable and, in general, low, depends not only on the structure of the carote‐ noids, but also on their proteinic ingestion. Thus, when carotene ingestion is very high, those which have not been transformed into retinol in the retinal mucosa are absorbed unaltered, bind with the lipoproteins, and are deposited in the skin and the mucosa, on which they confer a typical yellowish color, constituting hypercarotenosis [21].

**Toxicity:** Both the deficiency as well as the excess of vitamin A causes fetal malformations. Ingestion of large amounts of this vitamin can give rise to skin alterations (scaling), hair fall, weakness, choking, vomiting, etc. In extreme cases, great amounts accumulate in the liver, producing hepatic disorders that end up as fatty liver.

It is noteworthy that the administration of vitamin A in chronic form and at doses high‐ er than the recommended doses those can produce a clinical condition of toxicity charac‐ terized by fatigue, irritability, cephalea, febricula, hemorrhages in different tissues, and cutaneous alterations.

**2.4. Flavonoids and their antioxidant actions**

rodegenerative diseases [22].

their chemical structure[22].

**Figure 7.** Base structure of the flavonoids

health, 2010) [24].

Flavo comes from the Latin *flavus* and means the color found between yellow and red, such as that of honey or of gold, and flavonoid refers to an aromatic group, with heterocyclic pig‐ ments that contain oxygen, which are widely distributed among plants, constituting the ma‐ jority of yellow, red, and blue fruits. Consequently, flavonoids are found in abundance in grapes, apples, onions, cherries, and cabbage, in addition to forming part of the ginkgo bilo‐ ba tree and *Camellia sinensis* (green tea). On consuming these, we obtain the anti-inflamma‐ tory, antimicrobial, antithrombotic, antiallergic, antitumor, anticancerigenous, and antioxidant properties. With regard to the latter properties, these lie within its function in the nervous system, because a protector relationship has been observed with regard to neu‐

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 49

Flavonoids are Low-molecular-weight (LMW) compounds that share a common skeleton with diphenylpyrenes (C6-C3-C6); a flavonoid is a 2-phenyl-ring (A and B) compound linked through the pyrene C ring (heterocyclic). The carbon atoms in the C and A rings are numbered from 2‒8, while those of the B ring are numbered from 2'‒6'12 (Figure 7). The activity of flavonoids as antioxidants depends on the redox properties of their hy‐ droxy phenolic groups and on the structural relationship among the different parts of

Thanks to the variations of pyrene, the flavonoids achieved classification, as shown in Table 1 (Antiatherogenic properties of flavonoids: Implications for cardiovascular

In children, this can trigger the early closing of the long bones, which causes the height to descend. Megadoses of vitamin A can produce acute intoxication that will be characterized by clinical features of sedation, dizziness, nausea, vomiting, erythema, pruritis, and general‐ ized desquamation of the skin. We should also point out that in the elderly, the safety mar‐ gin when we administer this vitamin is small; thus, we must be especially cautious and adjust the dose well [21].

**Figure 6.** Molecular forms of vitamin A.

### **2.4. Flavonoids and their antioxidant actions**

It is noteworthy that the administration of vitamin A in chronic form and at doses high‐ er than the recommended doses those can produce a clinical condition of toxicity charac‐ terized by fatigue, irritability, cephalea, febricula, hemorrhages in different tissues, and

48 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

In children, this can trigger the early closing of the long bones, which causes the height to descend. Megadoses of vitamin A can produce acute intoxication that will be characterized by clinical features of sedation, dizziness, nausea, vomiting, erythema, pruritis, and general‐ ized desquamation of the skin. We should also point out that in the elderly, the safety mar‐ gin when we administer this vitamin is small; thus, we must be especially cautious and

cutaneous alterations.

adjust the dose well [21].

**Figure 6.** Molecular forms of vitamin A.

Flavo comes from the Latin *flavus* and means the color found between yellow and red, such as that of honey or of gold, and flavonoid refers to an aromatic group, with heterocyclic pig‐ ments that contain oxygen, which are widely distributed among plants, constituting the ma‐ jority of yellow, red, and blue fruits. Consequently, flavonoids are found in abundance in grapes, apples, onions, cherries, and cabbage, in addition to forming part of the ginkgo bilo‐ ba tree and *Camellia sinensis* (green tea). On consuming these, we obtain the anti-inflamma‐ tory, antimicrobial, antithrombotic, antiallergic, antitumor, anticancerigenous, and antioxidant properties. With regard to the latter properties, these lie within its function in the nervous system, because a protector relationship has been observed with regard to neu‐ rodegenerative diseases [22].

Flavonoids are Low-molecular-weight (LMW) compounds that share a common skeleton with diphenylpyrenes (C6-C3-C6); a flavonoid is a 2-phenyl-ring (A and B) compound linked through the pyrene C ring (heterocyclic). The carbon atoms in the C and A rings are numbered from 2‒8, while those of the B ring are numbered from 2'‒6'12 (Figure 7). The activity of flavonoids as antioxidants depends on the redox properties of their hy‐ droxy phenolic groups and on the structural relationship among the different parts of their chemical structure[22].

**Figure 7.** Base structure of the flavonoids

Thanks to the variations of pyrene, the flavonoids achieved classification, as shown in Table 1 (Antiatherogenic properties of flavonoids: Implications for cardiovascular health, 2010) [24].


**e.** Isoflavonoids: such as genestein and daidzein, present in soy foods such as tofu, soy milk, soybeans, soy vegetable protein, tempeh/fermented soybeans, miso/soybean

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 51

**g.** Proanthocyanidines: these appear in grape seeds, sea pine bark extract, and in red wine. These merit incorporation into the group of essential nutrients. The mean value of the inges‐ tion of flavonoids is 23 mg/day. The main flavonoid consumed is quercetin, tea being its

**Properties:** The flavonoids are white or yellowish solid, crystallized substances. Their here‐ tosides are soluble in hot water, alcohol, and polar organic dissolvents, being insoluble in apolar organic dissolvents. However, when they are in their free state, they are scarcely hy‐ drosoluble, but are soluble in more or less oxygenated organic substances, depending on

On the other hand, these are easily oxidizable substances; thus, they exert an antioxidant ef‐

**Pharmacological activity:** Pharmacologically, flavonoids are prominent due to their low tox‐ icity, presenting in general activity on the vascular system with P vitaminic action (protector effect of the vascular wall due to the diminution of permeability and to the increase of capil‐ lary resistance). Likewise, they possess an antioxidant effect, can inhibit lipid peroxidation, have antimutagenic effects, and possess the capacity to inhibit diverse enzymes [23, 24].

**Antioxidant functions:** The flavonoids' antioxidant action depends mainly on their se‐ questering capacity of FR and on their chelant properties of metals such as iron, imped‐ ing the catalytic actions of FR, and they also act by inhibiting the enzyme systems related with vascular functionality, such as the following: Catechol-O-methyl transferase (COMT), with which it increases the duration of the action of the catecholamines, thus inciding in vascular resistance; histidine decarboxylase, thus affecting the histamine's ac‐ tion, and the phosphodiesterases, thus inhibiting platelet aggregation and adhesiveness, in addition to the following oxidases: lipo-oxygenase; cyclo-oxygenase; myeloperoxidase, and xanthinic oxide, therefore avoiding the formation of Reactive oxygen species (ROS)

In addition to this, it has been observed that they also indirectly inhibit oxidative processes, such as phospholipase A2, at the same time stimulating others with recognized antioxidant

With respect to their structure, flavonoids are their hydroxylic constituents in positions 3´ and 4´; in the B ring, they demonstrate more action as antioxidants and this effect is potenti‐ ated by the presence of a double loop between carbons 2 and 3 and a free OH group in posi‐ tion 4. Additionally, the glycols show to be the most potent in their antilipoperoxidative

fect because they are oxidized more rapidly than other types of substances [23].

**f.** Kaempherol: found in broccoli, leeks, endives, red beets, and radishes

paste, and soy flour

main source [22].

their polarity.

and organic hydroperoxides.

properties, such as catalase and SOD.

actions than in their corresponding glycosidic actions.

**Table 1.** Classification of flavonoids

**Distribution:** The flavonoids are widely distributed among the higher plants, with the ruta‐ ceous, polygonaceous, compound, and umbelliferous plant families the principal ones con‐ taining flavonoids. Flavonoids abound, above all, in young, aerial plant parts and in those most exposed to the sun, such as the leaves, fruits, and flowers, because solar light favors their synthesis, controlling the levels of the auxins (vegetables hormones), which are growth regulators.

These compounds are important for the plant, similar to what occurs with the greater part of secondary metabolites, in addition to being responsible for the coloration of many flowers, fruits, leaves, and seeds, achieving >5,000 distinct flavonoids, because these can be found in the following groups:


**Name Structure Description Family members Dietary sources**

Quercetin, myricetin, isorhamnetin, kaempferol, pachypodol, rhamnazin

Catechins, epigallocatechin gallate, epicatechins, epicatechin gallate

Apigenin, nobiletin, tangeritin, luteolin

Cyanidin, delphinidin, peonidin, malvidin, pelargonidin

Onions, apples, broccoli, cranberries, berries, grapes, parsley, spinach

Tea, red wine, cocoa, grapes, plums, fruits, legumes

Celery, lettuce, parsley, citrus fruits, beets, bell peppers, spinach, Brussels sprouts, thyme

Red wine, blueberries cranberries, black currants, plums, red onions, red potatoes

Carbonyl group at position 4 and an -OH group in position 3 of ring C

position 3 of ring C

Have a carbonyl group in position 4 of ring C and lacking the hydroxyl group at position C3

Carbonyl group at position 4 and an -OH group in position 3 of ring C

**Distribution:** The flavonoids are widely distributed among the higher plants, with the ruta‐ ceous, polygonaceous, compound, and umbelliferous plant families the principal ones con‐ taining flavonoids. Flavonoids abound, above all, in young, aerial plant parts and in those most exposed to the sun, such as the leaves, fruits, and flowers, because solar light favors their synthesis, controlling the levels of the auxins (vegetables hormones), which are growth

These compounds are important for the plant, similar to what occurs with the greater part of secondary metabolites, in addition to being responsible for the coloration of many flowers, fruits, leaves, and seeds, achieving >5,000 distinct flavonoids, because these can be found in

**b.** Anthocyanidines: the pigment responsible for the reddish-blue and red color of cherries

**d.** Citroflavonoids: such as quercetin, lemonene, pyridine, rutin, and orangenine. The bit‐ ter flavor of the orange, lemon, and grapefruit confers orangenine on these fruits, while

**a.** Elegiac acid: found in fruits such as grapes and in vegetables

lemonene has been isolated from the lime and the lemon.

**c.** Catechins: found in black and green tea

Flavan-3-ols With an -OH group in

50 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Flavanones

Flavones

Anthocyanidins

regulators.

the following groups:

**Table 1.** Classification of flavonoids

**g.** Proanthocyanidines: these appear in grape seeds, sea pine bark extract, and in red wine.

These merit incorporation into the group of essential nutrients. The mean value of the inges‐ tion of flavonoids is 23 mg/day. The main flavonoid consumed is quercetin, tea being its main source [22].

**Properties:** The flavonoids are white or yellowish solid, crystallized substances. Their here‐ tosides are soluble in hot water, alcohol, and polar organic dissolvents, being insoluble in apolar organic dissolvents. However, when they are in their free state, they are scarcely hy‐ drosoluble, but are soluble in more or less oxygenated organic substances, depending on their polarity.

On the other hand, these are easily oxidizable substances; thus, they exert an antioxidant ef‐ fect because they are oxidized more rapidly than other types of substances [23].

**Pharmacological activity:** Pharmacologically, flavonoids are prominent due to their low tox‐ icity, presenting in general activity on the vascular system with P vitaminic action (protector effect of the vascular wall due to the diminution of permeability and to the increase of capil‐ lary resistance). Likewise, they possess an antioxidant effect, can inhibit lipid peroxidation, have antimutagenic effects, and possess the capacity to inhibit diverse enzymes [23, 24].

**Antioxidant functions:** The flavonoids' antioxidant action depends mainly on their se‐ questering capacity of FR and on their chelant properties of metals such as iron, imped‐ ing the catalytic actions of FR, and they also act by inhibiting the enzyme systems related with vascular functionality, such as the following: Catechol-O-methyl transferase (COMT), with which it increases the duration of the action of the catecholamines, thus inciding in vascular resistance; histidine decarboxylase, thus affecting the histamine's ac‐ tion, and the phosphodiesterases, thus inhibiting platelet aggregation and adhesiveness, in addition to the following oxidases: lipo-oxygenase; cyclo-oxygenase; myeloperoxidase, and xanthinic oxide, therefore avoiding the formation of Reactive oxygen species (ROS) and organic hydroperoxides.

In addition to this, it has been observed that they also indirectly inhibit oxidative processes, such as phospholipase A2, at the same time stimulating others with recognized antioxidant properties, such as catalase and SOD.

With respect to their structure, flavonoids are their hydroxylic constituents in positions 3´ and 4´; in the B ring, they demonstrate more action as antioxidants and this effect is potenti‐ ated by the presence of a double loop between carbons 2 and 3 and a free OH group in posi‐ tion 4. Additionally, the glycols show to be the most potent in their antilipoperoxidative actions than in their corresponding glycosidic actions.

As previously mentioned, quercetin is the flavonoid that unites the requisites for exercising an effective antioxidant function, because it is five times higher than vitamins A and C and addi‐ tionally possesses a hydrosolubility similar to that of the latter. Therefore, rutin (quercetin-3-b-D-rutinoside) is, to date, the sole flavonoid with a pharmacological presence in Mexico.

The highest concentrations of lycopene are found in prostatic tissue. High consumption of lyco‐ pene has been related with the prevention of some cancer types, precisely that of the prostate.

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 53

Although the tomato is the greatest source of lycopene, there are also other vegetables and fruits that present intense colors, such as watermelons, papayas, apricots, and pink grape‐ fruit. The tomato is the food that concentrates the greatest amount of lycopene, and it should be considered that there are factors that affect its assimilation into the organism, such as its maturity, the distinct varieties, or the manner of cooking, all of which exert an influence on

Of all of these, the fried tomato is that which best assimilates this substance, frying being the best way of cooking because, in addition to the heat, there is a certain amount of fat in‐ volved, which renders better assimilation of lycopene (fat-soluble). In concrete fashion, its presence in the fried tomato is some 25 µg per 100 g, while in the fresh tomato, this is

Other potent antioxidants include minerals such as copper, manganese, selenium, zinc, and iron. These minerals exercise their antioxidant function in diverse processes and metabolic

Zinc intervenes in >200 enzymatic reactions and its deficit increases the production of oxi‐

Copper participates in functions with antioxidant features of the enzyme family denominat‐ ed Superoxide dismutase (SOD), which is responsible for eliminating the superoxide anion. It empowers the immune system, participates in the formation of enzymes, proteins, and brain neurotransmitters (cell renovation and stimulation of the nervous system) and is an

Similarly, it facilitates the synthesis of collagen and elastin (necessary constituents of the

In addition, it acts as an antioxidant, protecting the cells from the toxic effects of FR, and it

Manganese also intervenes in this family of enzymes, concretely, in enzymes localized with‐

the amount and degree of exploitation of lycopene.

around 2 µg per 100 g [6, 26, 27].

steps in the organism [6, 26, 27].

dant species and Oxidative stress (OS) [6, 26, 27].

anti-inflammatory and anti-infectious agent.

good state of the blood vessels, lungs, and the skin).

facilitates calcium and phosphorous fixation [6, 26, 27].

**3. Minerals**

**3.1. Zinc**

**3.2. Copper**

**3.3. Manganese**

in the mitochondria [6, 26, 27].

There is a synergic effect with all of the vitamins to which we have alluded. This is due to that ascorbic acid reduces the oxidation of quercetin in such a way that combines with it and allows the flavonoids to maintain their functions for a longer time. For its part, quercetin protects vitamin E from oxidation.

The flavonoids remove reactive oxygen, especially in the form of SOD, hydroxyl radicals, hydroperoxides, and lipid peroxides, blocking the harmful effects of these substances on the cell, in which antioxidant protection of flavonoids has been corroborated in the following: queratinocytes; dermal fibroblasts; sensory lymph nodes; the endothelium; nervous tissue, and LDL.

On the other hand, the flavonoids exercise other actions as follows: diuretic; antispasmodic; anti-gastriculcerous, and anti-inflammatory.

In phytotherapy, the flavonoids are mainly employed in cases of capillary fragility as veno‐ tonics, although they are also utilized in proctology, metrorrhages, and retinopathies [22].

#### **2.5. Pro-oxidant mechanisms**

Due to the structural characteristics of some flavonoids, such as the anthocyanidines, these cause low oxidation potentials (EP/2), which permits them to reduce Fe3+ and Cu2+ in order for them to undergo auto-oxidation or even to become involved in the redox recycling proc‐ ess, acting in this manner as pro-oxidant agents, which explains the mutagenic and genotox‐ ic effects of some flavonoids.

Some of these mechanisms include the temporary reduction of Cu (II) to CU (I), auto-oxida‐ tion of the aroxyl radical and generating the superoxide anion (O2‒) that, on following its general sequence, becomes the harmful hydroxyl radical (HO.), as well as the affectation of the functions of the components of the nuclear antioxidant defense system: glutathione, and glutathione-S-transferase.

What determines the antioxidant or pro-oxidant character is the redox stability/lability of the radical compound forming part of the original flavonoid. The pro-oxidant actions only appear to be produced when the flavonoid doses are excessively high [25].

Under this heading, we will present a brief review of the remaining antioxidants present in our diet, their activity, and the foods that supply them.

#### **2.6. Lycopene**

Lycopene is the carotenoid that imparts the red color to the tomato and watermelon and that it not converted into vitamin A in the human organism, which does not impede it from pos‐ sessing very high antioxidant properties.

The highest concentrations of lycopene are found in prostatic tissue. High consumption of lyco‐ pene has been related with the prevention of some cancer types, precisely that of the prostate.

Although the tomato is the greatest source of lycopene, there are also other vegetables and fruits that present intense colors, such as watermelons, papayas, apricots, and pink grape‐ fruit. The tomato is the food that concentrates the greatest amount of lycopene, and it should be considered that there are factors that affect its assimilation into the organism, such as its maturity, the distinct varieties, or the manner of cooking, all of which exert an influence on the amount and degree of exploitation of lycopene.

Of all of these, the fried tomato is that which best assimilates this substance, frying being the best way of cooking because, in addition to the heat, there is a certain amount of fat in‐ volved, which renders better assimilation of lycopene (fat-soluble). In concrete fashion, its presence in the fried tomato is some 25 µg per 100 g, while in the fresh tomato, this is around 2 µg per 100 g [6, 26, 27].

### **3. Minerals**

As previously mentioned, quercetin is the flavonoid that unites the requisites for exercising an effective antioxidant function, because it is five times higher than vitamins A and C and addi‐ tionally possesses a hydrosolubility similar to that of the latter. Therefore, rutin (quercetin-3-b-

There is a synergic effect with all of the vitamins to which we have alluded. This is due to that ascorbic acid reduces the oxidation of quercetin in such a way that combines with it and allows the flavonoids to maintain their functions for a longer time. For its part, quercetin

The flavonoids remove reactive oxygen, especially in the form of SOD, hydroxyl radicals, hydroperoxides, and lipid peroxides, blocking the harmful effects of these substances on the cell, in which antioxidant protection of flavonoids has been corroborated in the following: queratinocytes; dermal fibroblasts; sensory lymph nodes; the endothelium; nervous tissue,

On the other hand, the flavonoids exercise other actions as follows: diuretic; antispasmodic;

In phytotherapy, the flavonoids are mainly employed in cases of capillary fragility as veno‐ tonics, although they are also utilized in proctology, metrorrhages, and retinopathies [22].

Due to the structural characteristics of some flavonoids, such as the anthocyanidines, these cause low oxidation potentials (EP/2), which permits them to reduce Fe3+ and Cu2+ in order for them to undergo auto-oxidation or even to become involved in the redox recycling proc‐ ess, acting in this manner as pro-oxidant agents, which explains the mutagenic and genotox‐

Some of these mechanisms include the temporary reduction of Cu (II) to CU (I), auto-oxida‐ tion of the aroxyl radical and generating the superoxide anion (O2‒) that, on following its general sequence, becomes the harmful hydroxyl radical (HO.), as well as the affectation of the functions of the components of the nuclear antioxidant defense system: glutathione, and

What determines the antioxidant or pro-oxidant character is the redox stability/lability of the radical compound forming part of the original flavonoid. The pro-oxidant actions only

Under this heading, we will present a brief review of the remaining antioxidants present in

Lycopene is the carotenoid that imparts the red color to the tomato and watermelon and that it not converted into vitamin A in the human organism, which does not impede it from pos‐

appear to be produced when the flavonoid doses are excessively high [25].

our diet, their activity, and the foods that supply them.

sessing very high antioxidant properties.

D-rutinoside) is, to date, the sole flavonoid with a pharmacological presence in Mexico.

protects vitamin E from oxidation.

**2.5. Pro-oxidant mechanisms**

ic effects of some flavonoids.

glutathione-S-transferase.

**2.6. Lycopene**

anti-gastriculcerous, and anti-inflammatory.

52 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

and LDL.

Other potent antioxidants include minerals such as copper, manganese, selenium, zinc, and iron. These minerals exercise their antioxidant function in diverse processes and metabolic steps in the organism [6, 26, 27].

### **3.1. Zinc**

Zinc intervenes in >200 enzymatic reactions and its deficit increases the production of oxi‐ dant species and Oxidative stress (OS) [6, 26, 27].

### **3.2. Copper**

Copper participates in functions with antioxidant features of the enzyme family denominat‐ ed Superoxide dismutase (SOD), which is responsible for eliminating the superoxide anion.

It empowers the immune system, participates in the formation of enzymes, proteins, and brain neurotransmitters (cell renovation and stimulation of the nervous system) and is an anti-inflammatory and anti-infectious agent.

Similarly, it facilitates the synthesis of collagen and elastin (necessary constituents of the good state of the blood vessels, lungs, and the skin).

In addition, it acts as an antioxidant, protecting the cells from the toxic effects of FR, and it facilitates calcium and phosphorous fixation [6, 26, 27].

### **3.3. Manganese**

Manganese also intervenes in this family of enzymes, concretely, in enzymes localized with‐ in the mitochondria [6, 26, 27].

### **3.4. Selenium**

Selenium intervenes in the synthesis of enzymes related with the oxidative function, such as glutathione peroxidase, which, as its name indicates, eliminates peroxide groups, including oxygen peroxide.

Numerous studies have shown the protector effect of red globules and of the fatty acids of oxidative damage typical of intense exercise and excessive exposure to the sun's UV rays. It is synthesized by plants and animals, as well as by the human organism, although in the latter case, in very small amounts. Lipoic acid is considered a very good regenerator of po‐ tent antioxidants such as vitamin C, vitamin E, glycation, and co-enzyme Q10. It is liposolu‐

The Exogenous Antioxidants http://dx.doi.org/10.5772/52490 55

It is found in spinach and similar green-leafed vegetables, broccoli, meat, yeast, and in cer‐

The hypolipidemic and anti-inflammatory activities *in vivo* as well as *in vitro* of the flavonoids of citric fruits have been widely demonstrated. Among the flavonoids, naringenine, one of the compounds that causes the bitter taste of grapefruit, has been studied extensively in recent years. In a recently conducted clinical assay, it was found that naringenine reduced Low-densi‐ ty-lipoprotein (LDL) levels in the circulation of 17% of patients with hypercholesterolemia. Ad‐ ditionally, the reducer effects of cholesterol in rabbits and rats were demonstrated, in addition to the reducer effects of Very-low-density-lipoprotein (VLDL) levels through the inhibition of key proteins for their assembly. Other studies reported that naringenine activates enzymes

A good diet influences the development and treatment of diseases, it is increasingly evident. After that epidemiological studies have shown the association between moderate consump‐ tion of certain foods and reduced incidence of various diseases at the rate of these observa‐ tions has attracted considerable interest in studying the properties of substances inherent in the chemical composition of food. Among the characteristics of these substances is the anti‐ oxidant activity, associated with the elimination of free radicals and therefore to the preven‐ tion of early stages which can trigger degenerative diseases. In this regard it is important to continue the study of dietary antioxidants on the activity may have on human diseases, pay‐ ing attention to the substances primarily natural antioxidants of food and synthetic way to

, Elena G. Olvera Hernández, Telma Flores Cerón and

\*Address all correspondence to: chehue\_alex@yahoo.com, chehuea@uaeh.edu.mx

ble and hydrosoluble, which means that it can act on any part of the organism.

that are important for the oxidation of fatty acids, such as CYP4A1 [28].

tain organs (such as kidney and heart) [6, 26, 27].

**4.1. Naringenine**

**5. Conclusion**

**Author details**

Alejandro Chehue Romero\*

Angelina Álvarez Chávez

assess its protective effect on the body.

Autonomous University of Hidalgo State, Mexico

This mineral is incorporated into proteins in the form of selenoproteins and, in this manner, aids in the prevention of cell damage. Epidemiological studies related the lack of selenium in the diet with the incidence of lung, colorectal, and prostate cancer.

The selenium content in the diet is directly related with the selenium content of the soil in which the food was grown. Thus, selenium-deficient soils give rise to a deficit of this ele‐ ment in the population, as in the case of China.

In this specific latter case, the method-of-choice comprises supplementing the diet with con‐ tributions of selenium, preferably in the form of selenomethionide, which is the analog, or‐ ganic form of selenium and which easily increases selenium levels in the blood [6, 26, 27].

### **3.5. Iron**

Iron forms part of the organism's antioxidant system because it contributes to eliminat‐ ing the peroxide groups. However, its capacity to change valence with ease (2+/3+) renders that it can also intervene, depending on the environment, in the formation of Free radicals (FR) [6, 26, 27].

### **3.6. Co-enzyme Q**

Co-enzyme Q10 or ubiquinone is a liposoluble compound that can be carried in many foods, although it can also be synthesized in the human organism. Co-enzyme Q10 diminishes with age; thus, the metabolic processes in which it has been found implicated are also coenzyme Q10-sensitive.

Given its liposolubility, its absorption is very los, especially when the diet is poor in fats.

Its principal antioxidant activity resides in that, in its reduced form, it is a liposoluble antiox‐ idant that inhibits lipid peroxidation in LDL. It is also found in the mitochondria, where it could protect protein membranes and the DNA from the oxidative damage that accompa‐ nies lipid peroxidation in these membranes.

It additionally acts as an immune system stimulant and through this stimulation also functions as an anticancerigen. In addition, it is capable of directly regenerating alphatocopherol [6, 26, 27].

### **4. Lipoic acid**

Lipoic acid or thioctic acid is also a compound that forms part of the antioxidant capital of the organism.

Numerous studies have shown the protector effect of red globules and of the fatty acids of oxidative damage typical of intense exercise and excessive exposure to the sun's UV rays.

It is synthesized by plants and animals, as well as by the human organism, although in the latter case, in very small amounts. Lipoic acid is considered a very good regenerator of po‐ tent antioxidants such as vitamin C, vitamin E, glycation, and co-enzyme Q10. It is liposolu‐ ble and hydrosoluble, which means that it can act on any part of the organism.

It is found in spinach and similar green-leafed vegetables, broccoli, meat, yeast, and in cer‐ tain organs (such as kidney and heart) [6, 26, 27].

### **4.1. Naringenine**

**3.4. Selenium**

oxygen peroxide.

**3.5. Iron**

Free radicals (FR) [6, 26, 27].

**3.6. Co-enzyme Q**

enzyme Q10-sensitive.

tocopherol [6, 26, 27].

**4. Lipoic acid**

the organism.

nies lipid peroxidation in these membranes.

Selenium intervenes in the synthesis of enzymes related with the oxidative function, such as glutathione peroxidase, which, as its name indicates, eliminates peroxide groups, including

This mineral is incorporated into proteins in the form of selenoproteins and, in this manner, aids in the prevention of cell damage. Epidemiological studies related the lack of selenium

The selenium content in the diet is directly related with the selenium content of the soil in which the food was grown. Thus, selenium-deficient soils give rise to a deficit of this ele‐

In this specific latter case, the method-of-choice comprises supplementing the diet with con‐ tributions of selenium, preferably in the form of selenomethionide, which is the analog, or‐ ganic form of selenium and which easily increases selenium levels in the blood [6, 26, 27].

Iron forms part of the organism's antioxidant system because it contributes to eliminat‐ ing the peroxide groups. However, its capacity to change valence with ease (2+/3+) renders that it can also intervene, depending on the environment, in the formation of

Co-enzyme Q10 or ubiquinone is a liposoluble compound that can be carried in many foods, although it can also be synthesized in the human organism. Co-enzyme Q10 diminishes with age; thus, the metabolic processes in which it has been found implicated are also co-

Given its liposolubility, its absorption is very los, especially when the diet is poor in fats.

Its principal antioxidant activity resides in that, in its reduced form, it is a liposoluble antiox‐ idant that inhibits lipid peroxidation in LDL. It is also found in the mitochondria, where it could protect protein membranes and the DNA from the oxidative damage that accompa‐

It additionally acts as an immune system stimulant and through this stimulation also functions as an anticancerigen. In addition, it is capable of directly regenerating alpha-

Lipoic acid or thioctic acid is also a compound that forms part of the antioxidant capital of

in the diet with the incidence of lung, colorectal, and prostate cancer.

54 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ment in the population, as in the case of China.

The hypolipidemic and anti-inflammatory activities *in vivo* as well as *in vitro* of the flavonoids of citric fruits have been widely demonstrated. Among the flavonoids, naringenine, one of the compounds that causes the bitter taste of grapefruit, has been studied extensively in recent years. In a recently conducted clinical assay, it was found that naringenine reduced Low-densi‐ ty-lipoprotein (LDL) levels in the circulation of 17% of patients with hypercholesterolemia. Ad‐ ditionally, the reducer effects of cholesterol in rabbits and rats were demonstrated, in addition to the reducer effects of Very-low-density-lipoprotein (VLDL) levels through the inhibition of key proteins for their assembly. Other studies reported that naringenine activates enzymes that are important for the oxidation of fatty acids, such as CYP4A1 [28].

### **5. Conclusion**

A good diet influences the development and treatment of diseases, it is increasingly evident. After that epidemiological studies have shown the association between moderate consump‐ tion of certain foods and reduced incidence of various diseases at the rate of these observa‐ tions has attracted considerable interest in studying the properties of substances inherent in the chemical composition of food. Among the characteristics of these substances is the anti‐ oxidant activity, associated with the elimination of free radicals and therefore to the preven‐ tion of early stages which can trigger degenerative diseases. In this regard it is important to continue the study of dietary antioxidants on the activity may have on human diseases, pay‐ ing attention to the substances primarily natural antioxidants of food and synthetic way to assess its protective effect on the body.

### **Author details**

Alejandro Chehue Romero\* , Elena G. Olvera Hernández, Telma Flores Cerón and Angelina Álvarez Chávez

\*Address all correspondence to: chehue\_alex@yahoo.com, chehuea@uaeh.edu.mx

Autonomous University of Hidalgo State, Mexico

### **References**

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[7] Goodman, L.S; Gilman, A. Las Bases Farmacológicas da Terapéutica. 10ª ed., Editora

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**Chapter 3**

**Chemistry of Natural Antioxidants and Studies**

Jorge Alberto Mendoza Pérez and Tomás Alejandro Fregoso Aguilar

http://dx.doi.org/10.5772/52247

molecules. For example:

properly cited.

(H2O2)

**1. Introduction**

**1.1. Antioxidants**

Additional information is available at the end of the chapter

**Performed with Different Plants Collected in Mexico**

Oxidation is the transfer of electrons from one atom to another and represents an essential part of both aerobic life and our metabolism, since oxygen is the ultimate electron acceptor in the electron flow system that produces energy in the form of ATP. However, problems may arise when the electron flow becomes uncoupled (transfer of unpaired single electrons), generating free radicals [1]. Antioxidants are important in living organisms as well as in food because they may delay or stop formation of free radical by giving hydrogen atoms or scavenging them. Oxidative stress is involved in the pathology of cancer, atherosclerosis, malaria and rheumatoid arthritis. An antioxidant can be defined in the broadest sense of the word, as any molecule capable of preventing or delaying oxidation (loss of one or more elec‐ trons) from other molecules, usually biological substrates such as lipids, proteins or nucleic acids. The oxidation of such substrates may be initiated by two types of reactive species: free radicals and those species without free radicals are reactive enough to induce the oxidation

of substrates such as those mentioned. There are three main types of antioxidants:

**1.** Primary: Prevent the formation of new free radicals, converting them into less harmful molecules before they can react or preventing the formation of free radicals from other

**•** Enzyme superoxide dismutase (SOD) which converts O2 • - to hydrogen peroxide

© 2013 Mendoza Pérez and Fregoso Aguilar; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

### **Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico**

Jorge Alberto Mendoza Pérez and Tomás Alejandro Fregoso Aguilar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52247

### **1. Introduction**

#### **1.1. Antioxidants**

Oxidation is the transfer of electrons from one atom to another and represents an essential part of both aerobic life and our metabolism, since oxygen is the ultimate electron acceptor in the electron flow system that produces energy in the form of ATP. However, problems may arise when the electron flow becomes uncoupled (transfer of unpaired single electrons), generating free radicals [1]. Antioxidants are important in living organisms as well as in food because they may delay or stop formation of free radical by giving hydrogen atoms or scavenging them. Oxidative stress is involved in the pathology of cancer, atherosclerosis, malaria and rheumatoid arthritis. An antioxidant can be defined in the broadest sense of the word, as any molecule capable of preventing or delaying oxidation (loss of one or more elec‐ trons) from other molecules, usually biological substrates such as lipids, proteins or nucleic acids. The oxidation of such substrates may be initiated by two types of reactive species: free radicals and those species without free radicals are reactive enough to induce the oxidation of substrates such as those mentioned. There are three main types of antioxidants:

	- **•** Enzyme superoxide dismutase (SOD) which converts O2 - to hydrogen peroxide (H2O2)

© 2013 Mendoza Pérez and Fregoso Aguilar; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**•** Enzyme glutathione peroxidase (GPx), which converts H2O2 and lipid peroxides to harmless molecules before they form free radicals.

hibition, which involves these antioxidants reacting with over-reduced forms of the photo‐ synthetic reaction centers and thereby prevent the production of superoxide. Another process which produces reactive oxygen species is lipid oxidation that takes place following the production of eicosanoids. However, the cells are provided with mechanisms that pre‐ vent unnecessary oxidation. Oxidative enzymes of these biosynthetic pathways are coordi‐

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico

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61

A free radical from the chemical viewpoint, is any species (atom, molecule or ion) contain‐ ing at least one unpaired electron and its outermost orbital, and which is in turn able to exist

The atoms arrange their electrons in regions called "atomic orbitals" in the form of pairs of electrons. The latter confers stability atom, or low chemical reactivity towards its environ‐ ment. However, under certain circumstances, it may lose its parity orbital, either giving or capturing an electron. When this occurs, the resulting orbit exhibits an unpaired electron, making the atom in a free radical. The presence of an unpaired electron in an orbital outer‐ most atom latter confers an increased ability to react with other atoms and / or molecules present in the environment, usually, lipids, proteins and nucleic acids (Figure 2). The inter‐ action between free radicals and such substrates results in eventually structural and func‐

Free radicals cause damage to different levels in the cell: Attack lipids and proteins in the cell membrane so the cell cannot perform its vital functions (transport of nutrients, waste

The superoxide radical, O2, which is normally in the metabolism cause a chain reaction of lipid peroxidation of the fatty acids of phospholipids of the cell membrane. Free radicals at‐

tack DNA avoiding cell replication and contributing to cellular aging.

nated and highly regulated [3].

independently (Figure 1).

**Figure 1.** Atomic orbitals

tional alterations [4].

disposal, cell division, etc.).

**1.2. Free radicals**


It also handles the classification based according to where they perform their activities, their background and their biochemical characteristics. So, antioxidants are also classified into two broad groups, depending on whether they are water soluble (hydrophilic) or lipid (hy‐ drophobic). In general, water soluble antioxidants react with oxidants in the cell cytoplasm and blood plasma, whereas the liposoluble antioxidants protecting cell membranes against lipid peroxidation. In the metabolism it is a contradiction that while the vast majority of life requires oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by producing reactive oxygen species. Therefore, organisms possess a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids. Usually antioxidant sys‐ tems prevent these reactive species are formed or removed before they can damage vital components of the cell. Reactive oxygen species produced in cells include hydrogen perox‐ ide (H2O2), hypochlorous acid (HClO), and free radicals such as hydroxyl radical (•OH) and superoxide radical (O2•-). The hydroxyl radical is particularly unstable and reacts rapidly and non-specifically with most biological molecules. This species produces hydrogen perox‐ ide redox reactions catalyzed by metals such as the Fenton reaction. These oxidants can damage cells starting chemical chain reactions such as lipid peroxidation or by oxidizing DNA or DNA damage proteins. These effects can cause mutations and possibly cancer if not reversed by DNA repair mechanisms, while damage proteins will cause enzyme inhibition, denaturation and degradation of proteins. The use of oxygen as part of the process for gen‐ erating metabolic energy produces reactive oxygen species. In this process, the superoxide anion is produced as a byproduct of several steps in the electron transport chain. Particular‐ ly important is the reduction of coenzyme Q in the compound III as a highly reactive free radical is formed as intermediate (Q•-). This unstable intermediate can lead to loss of elec‐ trons when these jump directly to molecular oxygen to form superoxide anion instead of moving with well controlled series of reactions of electron transport chain. In a similar set of reactions in plants reactive oxygen species are also produced during photosynthesis under high light intensity. This effect is partly offset by the involvement of carotenoids in photoin‐ hibition, which involves these antioxidants reacting with over-reduced forms of the photo‐ synthetic reaction centers and thereby prevent the production of superoxide. Another process which produces reactive oxygen species is lipid oxidation that takes place following the production of eicosanoids. However, the cells are provided with mechanisms that pre‐ vent unnecessary oxidation. Oxidative enzymes of these biosynthetic pathways are coordi‐ nated and highly regulated [3].

### **1.2. Free radicals**

**•** Enzyme glutathione peroxidase (GPx), which converts H2O2 and lipid peroxides to

**•** Proteins that bind to metals (ferritin, transferrin and ceruloplasmin) limit the availa‐

**2.** Secondary: Capture free radicals, preventing the chain reaction (eg vitamin E or alphatocopherol, vitamin C or ascorbic acid, beta-carotene, uric acid, bilirubin, albumin, ubiq‐

**3.** Tertiary: They repair damaged biomolecules by free radicals (eg DNA repair enzymes

It also handles the classification based according to where they perform their activities, their background and their biochemical characteristics. So, antioxidants are also classified into two broad groups, depending on whether they are water soluble (hydrophilic) or lipid (hy‐ drophobic). In general, water soluble antioxidants react with oxidants in the cell cytoplasm and blood plasma, whereas the liposoluble antioxidants protecting cell membranes against lipid peroxidation. In the metabolism it is a contradiction that while the vast majority of life requires oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by producing reactive oxygen species. Therefore, organisms possess a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids. Usually antioxidant sys‐ tems prevent these reactive species are formed or removed before they can damage vital components of the cell. Reactive oxygen species produced in cells include hydrogen perox‐ ide (H2O2), hypochlorous acid (HClO), and free radicals such as hydroxyl radical (•OH) and superoxide radical (O2•-). The hydroxyl radical is particularly unstable and reacts rapidly and non-specifically with most biological molecules. This species produces hydrogen perox‐ ide redox reactions catalyzed by metals such as the Fenton reaction. These oxidants can damage cells starting chemical chain reactions such as lipid peroxidation or by oxidizing DNA or DNA damage proteins. These effects can cause mutations and possibly cancer if not reversed by DNA repair mechanisms, while damage proteins will cause enzyme inhibition, denaturation and degradation of proteins. The use of oxygen as part of the process for gen‐ erating metabolic energy produces reactive oxygen species. In this process, the superoxide anion is produced as a byproduct of several steps in the electron transport chain. Particular‐ ly important is the reduction of coenzyme Q in the compound III as a highly reactive free radical is formed as intermediate (Q•-). This unstable intermediate can lead to loss of elec‐ trons when these jump directly to molecular oxygen to form superoxide anion instead of moving with well controlled series of reactions of electron transport chain. In a similar set of reactions in plants reactive oxygen species are also produced during photosynthesis under high light intensity. This effect is partly offset by the involvement of carotenoids in photoin‐

harmless molecules before they form free radicals.

60 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

bility of iron necessary to form the radical OH

and methionine sulfoxide reductase) [2].

**•** Catalases

**•** Glutathione reductase.

uinol-10, methionine)

**•** Glutathione S transferase.

A free radical from the chemical viewpoint, is any species (atom, molecule or ion) contain‐ ing at least one unpaired electron and its outermost orbital, and which is in turn able to exist independently (Figure 1).

**Figure 1.** Atomic orbitals

The atoms arrange their electrons in regions called "atomic orbitals" in the form of pairs of electrons. The latter confers stability atom, or low chemical reactivity towards its environ‐ ment. However, under certain circumstances, it may lose its parity orbital, either giving or capturing an electron. When this occurs, the resulting orbit exhibits an unpaired electron, making the atom in a free radical. The presence of an unpaired electron in an orbital outer‐ most atom latter confers an increased ability to react with other atoms and / or molecules present in the environment, usually, lipids, proteins and nucleic acids (Figure 2). The inter‐ action between free radicals and such substrates results in eventually structural and func‐ tional alterations [4].

Free radicals cause damage to different levels in the cell: Attack lipids and proteins in the cell membrane so the cell cannot perform its vital functions (transport of nutrients, waste disposal, cell division, etc.).

The superoxide radical, O2, which is normally in the metabolism cause a chain reaction of lipid peroxidation of the fatty acids of phospholipids of the cell membrane. Free radicals at‐ tack DNA avoiding cell replication and contributing to cellular aging.

**Antioxidant metabolite Solubility**

**Table 1.** Biochemical properties of antioxidant metabolites

**Figure 3.** Different kinds of antioxidant metaboilites produced by plants

**2. Enzyme systems**

α-tocopherol

**Concentration in human serum (M)**

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico

Ascorbic acid (vitamin C) Water 50 to 60 260 (male)

Glutathione Water from 325 to 650 6,400 (male) Lipoic acid Water from 0.1 to 0.7 4- 5 (rat) Uric acid Water from 200 to 400 1,600 (male)

(vitamin E) Lipid 10 to 40 50 (male)

As with chemical antioxidants, cells are protected against oxidative stress by a network of antioxidant enzymes. Superoxide released by processes such as oxidative phosphorylation,

Ubiquinol (coenzyme Q) Lipid 5 200 (male)

Carotene Lipid β-carotene: 0.5 to 1 retinol (vitamin A): 1 - 3 5

**Liver tissue concentration (mol / kg)**

http://dx.doi.org/10.5772/52247

63

(male, total carotenoids)

**Figure 2.** Unpaired electron in a free radical

The normal body processes produce free radicals that involve food metabolism, breathing and exercise. We are also exposed to environmental elements that create free radicals such as industrial pollution, snuff, radiation, drugs, chemical additives in processed foods and pesticides. Not all free radicals are dangerous because, for example, immune cells create free radicals to kill bacteria and viruses, but if there is sufficient control by antioxidants, healthy cell can be damaged.

The reactive oxygen species (ROS) is a collective term, widely used, comprising all the reac‐ tive species, whether or not free radicals, focus their reactivity in an oxygen atom. However, often under the designation ROS include other chemical species whose reactivity is focused on other than oxygen atoms [5].

#### **1.3. Metabolites**

In general, water soluble antioxidants react with oxidants in the cell cytoplasm and blood plasma, whereas the liposoluble antioxidants protecting cell membranes against lipid per‐ oxidation. These compounds can be synthesized in the body or obtained from the diet (Table 1) [6].

### **1.4. Antioxidant metabolites in plants**

Plants produce many different secondary metabolites some of them are potent antioxidants, some examples of these compounds is shown in Figure 3 [5,7].

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico http://dx.doi.org/10.5772/52247 63


**Table 1.** Biochemical properties of antioxidant metabolites

**Figure 3.** Different kinds of antioxidant metaboilites produced by plants

#### **2. Enzyme systems**

**Figure 2.** Unpaired electron in a free radical

62 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cell can be damaged.

**1.3. Metabolites**

(Table 1) [6].

on other than oxygen atoms [5].

**1.4. Antioxidant metabolites in plants**

some examples of these compounds is shown in Figure 3 [5,7].

The normal body processes produce free radicals that involve food metabolism, breathing and exercise. We are also exposed to environmental elements that create free radicals such as industrial pollution, snuff, radiation, drugs, chemical additives in processed foods and pesticides. Not all free radicals are dangerous because, for example, immune cells create free radicals to kill bacteria and viruses, but if there is sufficient control by antioxidants, healthy

The reactive oxygen species (ROS) is a collective term, widely used, comprising all the reac‐ tive species, whether or not free radicals, focus their reactivity in an oxygen atom. However, often under the designation ROS include other chemical species whose reactivity is focused

In general, water soluble antioxidants react with oxidants in the cell cytoplasm and blood plasma, whereas the liposoluble antioxidants protecting cell membranes against lipid per‐ oxidation. These compounds can be synthesized in the body or obtained from the diet

Plants produce many different secondary metabolites some of them are potent antioxidants,

As with chemical antioxidants, cells are protected against oxidative stress by a network of antioxidant enzymes. Superoxide released by processes such as oxidative phosphorylation, is first converted into hydrogen peroxide and immediately reduced to give water. This route of detoxification is the result of multiple enzymes with superoxide dismutase catalyzing the first step and then catalases and peroxidases that eliminate several hydrogen peroxide [8].

lion diabetics worldwide and likely to double this number for 2030 is quite high. Countries like China, India, United States of America and Mexico are at the top of this pathology [16]. In Mexico, this condition is a major cause of mortality and morbidity are estimated to be ap‐ proximately 10 million individuals with diabetes, of whom 22.7% did not know they are sick, while 55% do not have good control netheir condition. This pathology is multifactorial, presenting various metabolic problems (polyuria, polyphagia, polydipsia, weight changes). The disorder is characterized by the inadequate use of glucose, due to insufficient produc‐ tion, insulin resistance and some without production of the hormone, resulting in unfavora‐ ble a high index of this monosaccharide in the blood. This causes abnormal function of some organs, tissues and systems that can cause kidney failure, vision loss, and amputation of a

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico

http://dx.doi.org/10.5772/52247

65

Different factors increase the likelihood of the individual to develop diabetes as are smok‐ ing, sedentary lifestyle, lack of exercise coupled with unbalanced diet causes both over‐ weight and obesity. Naturally the body causes the formation of free radicals (highly unstable molecules), these chemical species are responsible for cellular aging, but when there is a greater concentration of these molecules may contribute to the development of various diseases and chronic degenerative neuro Parkinson's, Alzheimer's and diabetes. Obesity increases oxygen consumption and thus the production of free radicals, thus creat‐ ing the phenomenon known as oxidative stress. Excess fat naturally stored in fat cells, caus‐ es the more than normal synthesis of substances called adiposines IL6 or leptines. These

Due to the current problem in the health issue we propose the use of herbs as an option to improve the style of living of the people, not only for the adjuvant treatment, but because the use of plants offers great nutritional benefits somehow reducing the incidence of such chronic degenerative diseases. This is not intended to impair the option of preventive diag‐ nosis by the health sector does not provide such benefits, but rather the use of plants known to have medicinal activity coupled with the clinical - pharmacology, could present better re‐ sults, for the treatment of the various degenerative chronic diseases. Given the increasing scientific evidence that the etiology of several chronic degenerative diseases such as diabetes is influenced by factors such as metabolic redox imbalance. Is currently booming studying the formation of metabolites against free radicals that diverse plant species presents. An ex‐ ample of this has been widely documented, is the cranberry, a plant used for treating vari‐ ous diseases and, as has been discovered, is due to its potential antioxidant that has these

Similarly, Mexico has focused attention on other plants with potential antioxidant properties and for some years and was used in the treatment of diabetes. In this regard, since 2006, our research work focused on the task of describing the effects of plants such as Noni *(Morinda citrifolia*), Moringa (*Moringa oleifera*), the Guarumbo (*Cecropia obtusifolia Bertolt*), the Musaro

substances in higher concentrations also cause insulin resistance [17].

limb, diabetic coma and even death.

**3. Alternative medicine**

properties beneficial to health [18, 19].

#### **2.1. Oxidative stress**

Free radicals oxidize many biological structures, damaging them. This is known as oxidative damage, a major cause of aging, cancer, atherosclerosis, chronic inflammatory processes and cataracts, which are the most characteristic.

In certain circumstances, production of free radicals can increase uncontrollably, a situation known as oxidative stress. This means an imbalance between the speeds of production and destruction of toxic molecules, leading to an increase in cellular concentration of free radi‐ cals. Cells have mechanisms to protect against the harmful effects of free radicals based on a complex defense mechanism consisting of the antioxidants. Oxidative stress has been impli‐ cated in over one hundred human disease conditions, such as cancer, cardiovascular dis‐ ease, aging and neurodegenerative diseases [9]. However, the innate defense in the human body may not be enough for severe oxidative stress. Hence, certain amounts of exogenous antioxidants are constantly required to maintain an adequate level of antioxidants in order to balance the ROS. As an example, epidemiological evidence indicates that the consump‐ tion of grapes reduces the incidence of coronary heart disease (CHD), atherosclerosis and platelet aggregation [10]. This greater protection may be due to the phenolic components of grapes, which are particularly abundant since they behave as reactive oxygen species-scav‐ engers and metal-chelators. Polyphenolic substances in grapes and other red fruits are usu‐ ally subdivided into two groups: flavonoids and nonflavonoids. The most common flavonoids are flavonols (quercetin, kaempferol, and myricetin), flavan-3-ols (catechin, epi‐ catechin, and tannins), and anthocyanins (cyanin). Nonflavonoids comprise stilbenes, hy‐ droxycinnamic acids and benzoic acids. Numerous papers have been published red fruits and their antioxidant properties have been correlated with their polyphenol contents [11,12,13,14,15].

#### **2.2. Oxidative stress and disease**

It is thought that oxidative stress contributes to the development of a wide range of diseases including Alzheimer's disease, Parkinson's disease, the pathologies caused by diabetes, rheumatoid arthritis, and neurodegeneration in motor neuron diseases. In many cases, it is unclear if oxidants trigger the disease, or occur as a result of this and cause the symptoms of the disease as a plausible alternative, a neurodegenerative disease may result from defective axonal transport of mitochondria that perform oxidation reactions. A case in which it fits is particularly well understood in the role of oxidative stress in cardiovascular disease. Here, the oxidation of low density lipoprotein (LDL) seems to trigger the process of atherogenesis, which leads to atherosclerosis, and ultimately to cardiovascular disease.

In diseases that have a high impact on the health sector Diabetes Mellitus is one of the most known. The World Health Organization (WHO) estimates that there are just over 180 mil‐ lion diabetics worldwide and likely to double this number for 2030 is quite high. Countries like China, India, United States of America and Mexico are at the top of this pathology [16]. In Mexico, this condition is a major cause of mortality and morbidity are estimated to be ap‐ proximately 10 million individuals with diabetes, of whom 22.7% did not know they are sick, while 55% do not have good control netheir condition. This pathology is multifactorial, presenting various metabolic problems (polyuria, polyphagia, polydipsia, weight changes). The disorder is characterized by the inadequate use of glucose, due to insufficient produc‐ tion, insulin resistance and some without production of the hormone, resulting in unfavora‐ ble a high index of this monosaccharide in the blood. This causes abnormal function of some organs, tissues and systems that can cause kidney failure, vision loss, and amputation of a limb, diabetic coma and even death.

Different factors increase the likelihood of the individual to develop diabetes as are smok‐ ing, sedentary lifestyle, lack of exercise coupled with unbalanced diet causes both over‐ weight and obesity. Naturally the body causes the formation of free radicals (highly unstable molecules), these chemical species are responsible for cellular aging, but when there is a greater concentration of these molecules may contribute to the development of various diseases and chronic degenerative neuro Parkinson's, Alzheimer's and diabetes. Obesity increases oxygen consumption and thus the production of free radicals, thus creat‐ ing the phenomenon known as oxidative stress. Excess fat naturally stored in fat cells, caus‐ es the more than normal synthesis of substances called adiposines IL6 or leptines. These substances in higher concentrations also cause insulin resistance [17].

### **3. Alternative medicine**

is first converted into hydrogen peroxide and immediately reduced to give water. This route of detoxification is the result of multiple enzymes with superoxide dismutase catalyzing the first step and then catalases and peroxidases that eliminate several hydrogen peroxide [8].

Free radicals oxidize many biological structures, damaging them. This is known as oxidative damage, a major cause of aging, cancer, atherosclerosis, chronic inflammatory processes and

In certain circumstances, production of free radicals can increase uncontrollably, a situation known as oxidative stress. This means an imbalance between the speeds of production and destruction of toxic molecules, leading to an increase in cellular concentration of free radi‐ cals. Cells have mechanisms to protect against the harmful effects of free radicals based on a complex defense mechanism consisting of the antioxidants. Oxidative stress has been impli‐ cated in over one hundred human disease conditions, such as cancer, cardiovascular dis‐ ease, aging and neurodegenerative diseases [9]. However, the innate defense in the human body may not be enough for severe oxidative stress. Hence, certain amounts of exogenous antioxidants are constantly required to maintain an adequate level of antioxidants in order to balance the ROS. As an example, epidemiological evidence indicates that the consump‐ tion of grapes reduces the incidence of coronary heart disease (CHD), atherosclerosis and platelet aggregation [10]. This greater protection may be due to the phenolic components of grapes, which are particularly abundant since they behave as reactive oxygen species-scav‐ engers and metal-chelators. Polyphenolic substances in grapes and other red fruits are usu‐ ally subdivided into two groups: flavonoids and nonflavonoids. The most common flavonoids are flavonols (quercetin, kaempferol, and myricetin), flavan-3-ols (catechin, epi‐ catechin, and tannins), and anthocyanins (cyanin). Nonflavonoids comprise stilbenes, hy‐ droxycinnamic acids and benzoic acids. Numerous papers have been published red fruits and their antioxidant properties have been correlated with their polyphenol contents

It is thought that oxidative stress contributes to the development of a wide range of diseases including Alzheimer's disease, Parkinson's disease, the pathologies caused by diabetes, rheumatoid arthritis, and neurodegeneration in motor neuron diseases. In many cases, it is unclear if oxidants trigger the disease, or occur as a result of this and cause the symptoms of the disease as a plausible alternative, a neurodegenerative disease may result from defective axonal transport of mitochondria that perform oxidation reactions. A case in which it fits is particularly well understood in the role of oxidative stress in cardiovascular disease. Here, the oxidation of low density lipoprotein (LDL) seems to trigger the process of atherogenesis,

In diseases that have a high impact on the health sector Diabetes Mellitus is one of the most known. The World Health Organization (WHO) estimates that there are just over 180 mil‐

which leads to atherosclerosis, and ultimately to cardiovascular disease.

**2.1. Oxidative stress**

[11,12,13,14,15].

**2.2. Oxidative stress and disease**

cataracts, which are the most characteristic.

64 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Due to the current problem in the health issue we propose the use of herbs as an option to improve the style of living of the people, not only for the adjuvant treatment, but because the use of plants offers great nutritional benefits somehow reducing the incidence of such chronic degenerative diseases. This is not intended to impair the option of preventive diag‐ nosis by the health sector does not provide such benefits, but rather the use of plants known to have medicinal activity coupled with the clinical - pharmacology, could present better re‐ sults, for the treatment of the various degenerative chronic diseases. Given the increasing scientific evidence that the etiology of several chronic degenerative diseases such as diabetes is influenced by factors such as metabolic redox imbalance. Is currently booming studying the formation of metabolites against free radicals that diverse plant species presents. An ex‐ ample of this has been widely documented, is the cranberry, a plant used for treating vari‐ ous diseases and, as has been discovered, is due to its potential antioxidant that has these properties beneficial to health [18, 19].

Similarly, Mexico has focused attention on other plants with potential antioxidant properties and for some years and was used in the treatment of diabetes. In this regard, since 2006, our research work focused on the task of describing the effects of plants such as Noni *(Morinda citrifolia*), Moringa (*Moringa oleifera*), the Guarumbo (*Cecropia obtusifolia Bertolt*), the Musaro (*Lophocereus sp.*) and Neem (*Azadirachta indica*) in murine models of chemically induced dia‐ betes with streptozotocine. More recently, we began to evaluate the antioxidant properties of some of these plants through *in vitro* techniques [20].

Polyphenolic compounds are commonly found in both edible and inedible plants, and they have been reported to have multiple biological effects, including antioxidant activity [25]. Herbs are used in many domains, including medicine, nutrition, flavouring, beverages, dye‐ ing, repellents, fragrances, cosmetics [26]. Many species have been recognized to have me‐ dicinal properties and beneficial impact on health, e.g. antioxidant activity, digestive stimulation action, antiinflammatory, antimicrobial, hypolipidemic, antimutagenic effects and anticarcinogenic potential [27,28]. Crude extracts of herbs and spices, and other plant materials rich in phenolics are of increasing interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food. The basic flavonoids structure is the flavan nucleus, which consists of 15 carbon atoms arranged in three rings (C6–C3–C6), labelled A, B, and C (Figure 3). Various clases of flavo‐ noid differ in the level of oxidation and saturation of ring C, while individual compounds within a class differ in the substitution pattern of rings A and B. The differences in the struc‐ ture and substitution will influence the phenoxyl radical stability and thereby the antioxi‐ dant properties of the flavonoids. Plant species belong to several botanical families, such as Labiatae, Compositae, Umbelliferae, Asteracae, Polygonacae and Myrtacae. Many spices

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico

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67

have been investigated for their antioxidant properties for at least 50 years [29,30].

**4. DPPH experiments with different plants collected in México**

*oleifera*) and the other in the Neem tree (*Azadirachta indica*).

to which they evaluated the antioxidant capacity.

antioxidant capacity [31].

Herein it is presented a brief description of two experiments to evaluate the antioxidant properties of some plants collected in México, one conducted in the Moringa tree (*Moringa*

**1.** Antioxidant properties of *Moringa oleifera*: In this experiment were collected fresh leaves of *M. oleifera* in the Municipality of Apatzingan in the state of Michoacan, Mexico, the leaves are brought to the facilities of the National School of Biological Sciences, IPN where allowed to air dry and then were macerated and placed in containers containing methanol. After one week the solvent was decanted and concentrated under reduced pressure using a rotary evaporator. The crude methanol extract was stable in distilled water obtained the following experimental concentrations: 50, 25, 12.5 and 6.25 mg / mL

It was used as standard test the unstable radical 2,2-diphenyl-picrylhydrazyl (DPPH), which originally has a purple and when it is placed against a substance having antioxi‐ dant properties in a UV spectrophotometer at 517 nm, changes to yellow colour yellow (Figure 4). It was prepared a calibration curve of DPPH type in methanol at concentra‐ tions of 40, 120, 160 and 200 *u*g. Subsequently, aliquots of the above concentrations of methanol extract of *M. oleifera* and combined with DPPH in methanol for measuring its absorbance in the UV spectrophotometer at 0, 10, 30, 60 45 min., and then self-assess their

#### **3.1. Antioxidant effects in Mexican plants**

The use of traditional medicine is widespread in Mexico and plants are indeed the first source for preparing remedies in this form of alternative medicine. Among the various compounds found in plants, antioxidants are of particular importance because they might serve as leads for the development of novel drugs. Several plants used as anti-inflamma‐ tory, digestive, antinecrotic, neuroprotective, and hepatoprotective properties have recent‐ ly been shown to have and antioxidant and/or antiradical scavenging mechanism as part of their activity [21,22]. The search for natural sources of medicinal products that also have antioxidant and radical scavenging activity is on the rise [23,24]. Among the medici‐ nal properties associated with them are the following: the fruitand bark of *Licania arborea* is used as a soap for hair infections, the latex from *Ficus obtusifolia* is employed as an anti parasitic and also for reducing fever, *Bunchosia cannesens* is prescribed as an antidiarrhoe‐ ic, *Sideroxylon capiri* is used for hiccups, as an antiseptic for cleaning wounds, and wom‐ en use its leaves in a water bath after giving birth. The latex of *Sapium macrocarpum* is used against scorpion stings, fever and some skin problems such as warts; its use as an anti-coagulant is also widespread. The latex of *Ficus cotinifolia* is used in the treatments of urinary infections, vomiting, malaria and against inflammatory pathologies of the spleen. The leaves of *Annona squamosa* are used in cicatrisation of wounds, diarrhoea, ulcers, menstrual disorders, and also to help weight loss. The seeds of this plant are also em‐ ployed as an insecticide. The leaves of *Vitex molli* are used to treat stomach ache, diges‐ tion disorders, nervous alterations, and also scorpion stings. *Piper leucophyllum* is employed for reducing fever and its dried leaves are used for cleaning eyes and as spice in cooking. The leaves and bark of *Gliricidia sepium* are used against high fever, skin infec‐ tions, urine disorders, malaria, and headache. However, its seeds are reported to be toxic. *Hamelia paten is* used to accelerate wound cicatrisation. The Mexican and Central America native species of *Astianthus viminalis* is used for the curing of diabetes and malaria and to reduce hair loss. *Swietenia humilis* is used as anti parasitic, and it is also utilized for hair care as a shampoo. It is also used with other plants in mixed herbal teas, and used as home remedies. *Stemmandenia bella* is employed for curing wounds; *Rupechtia fusca* is used in some stomach disorders; *Bursera grandifolia* is used as a tooth paste and against diges‐ tive disorders; *Ziziphus amole* is prepared as infusion and it is applied for washing wounds and to treat gastric ulcers. The fruit and the latex of *Jacaratia mexicana* are used against ulcers in the mouth and digestive disorders. *Gyrocarpus jathrophifolius* leaves and bark are used as an analgesic. *Pseudobombax ellipticum* is used in respiratory disorders such as cough, and also against fever and as an anti microbial. The stems and flowers of *Comocladia engleriana* are toxic because they produce dermatitis. The flowers and the latex of *Plumeria rubra* can be used for stopping vaginal blood shed, and toothache, and the la‐ tex of the plant is used against earache. Infusions are used as an Eye-cleaning liquid [23, 24, 25 & 26].

Polyphenolic compounds are commonly found in both edible and inedible plants, and they have been reported to have multiple biological effects, including antioxidant activity [25]. Herbs are used in many domains, including medicine, nutrition, flavouring, beverages, dye‐ ing, repellents, fragrances, cosmetics [26]. Many species have been recognized to have me‐ dicinal properties and beneficial impact on health, e.g. antioxidant activity, digestive stimulation action, antiinflammatory, antimicrobial, hypolipidemic, antimutagenic effects and anticarcinogenic potential [27,28]. Crude extracts of herbs and spices, and other plant materials rich in phenolics are of increasing interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food. The basic flavonoids structure is the flavan nucleus, which consists of 15 carbon atoms arranged in three rings (C6–C3–C6), labelled A, B, and C (Figure 3). Various clases of flavo‐ noid differ in the level of oxidation and saturation of ring C, while individual compounds within a class differ in the substitution pattern of rings A and B. The differences in the struc‐ ture and substitution will influence the phenoxyl radical stability and thereby the antioxi‐ dant properties of the flavonoids. Plant species belong to several botanical families, such as Labiatae, Compositae, Umbelliferae, Asteracae, Polygonacae and Myrtacae. Many spices have been investigated for their antioxidant properties for at least 50 years [29,30].

(*Lophocereus sp.*) and Neem (*Azadirachta indica*) in murine models of chemically induced dia‐ betes with streptozotocine. More recently, we began to evaluate the antioxidant properties

The use of traditional medicine is widespread in Mexico and plants are indeed the first source for preparing remedies in this form of alternative medicine. Among the various compounds found in plants, antioxidants are of particular importance because they might serve as leads for the development of novel drugs. Several plants used as anti-inflamma‐ tory, digestive, antinecrotic, neuroprotective, and hepatoprotective properties have recent‐ ly been shown to have and antioxidant and/or antiradical scavenging mechanism as part of their activity [21,22]. The search for natural sources of medicinal products that also have antioxidant and radical scavenging activity is on the rise [23,24]. Among the medici‐ nal properties associated with them are the following: the fruitand bark of *Licania arborea* is used as a soap for hair infections, the latex from *Ficus obtusifolia* is employed as an anti parasitic and also for reducing fever, *Bunchosia cannesens* is prescribed as an antidiarrhoe‐ ic, *Sideroxylon capiri* is used for hiccups, as an antiseptic for cleaning wounds, and wom‐ en use its leaves in a water bath after giving birth. The latex of *Sapium macrocarpum* is used against scorpion stings, fever and some skin problems such as warts; its use as an anti-coagulant is also widespread. The latex of *Ficus cotinifolia* is used in the treatments of urinary infections, vomiting, malaria and against inflammatory pathologies of the spleen. The leaves of *Annona squamosa* are used in cicatrisation of wounds, diarrhoea, ulcers, menstrual disorders, and also to help weight loss. The seeds of this plant are also em‐ ployed as an insecticide. The leaves of *Vitex molli* are used to treat stomach ache, diges‐ tion disorders, nervous alterations, and also scorpion stings. *Piper leucophyllum* is employed for reducing fever and its dried leaves are used for cleaning eyes and as spice in cooking. The leaves and bark of *Gliricidia sepium* are used against high fever, skin infec‐ tions, urine disorders, malaria, and headache. However, its seeds are reported to be toxic. *Hamelia paten is* used to accelerate wound cicatrisation. The Mexican and Central America native species of *Astianthus viminalis* is used for the curing of diabetes and malaria and to reduce hair loss. *Swietenia humilis* is used as anti parasitic, and it is also utilized for hair care as a shampoo. It is also used with other plants in mixed herbal teas, and used as home remedies. *Stemmandenia bella* is employed for curing wounds; *Rupechtia fusca* is used in some stomach disorders; *Bursera grandifolia* is used as a tooth paste and against diges‐ tive disorders; *Ziziphus amole* is prepared as infusion and it is applied for washing wounds and to treat gastric ulcers. The fruit and the latex of *Jacaratia mexicana* are used against ulcers in the mouth and digestive disorders. *Gyrocarpus jathrophifolius* leaves and bark are used as an analgesic. *Pseudobombax ellipticum* is used in respiratory disorders such as cough, and also against fever and as an anti microbial. The stems and flowers of *Comocladia engleriana* are toxic because they produce dermatitis. The flowers and the latex of *Plumeria rubra* can be used for stopping vaginal blood shed, and toothache, and the la‐ tex of the plant is used against earache. Infusions are used as an Eye-cleaning liquid [23,

of some of these plants through *in vitro* techniques [20].

66 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**3.1. Antioxidant effects in Mexican plants**

24, 25 & 26].

### **4. DPPH experiments with different plants collected in México**

Herein it is presented a brief description of two experiments to evaluate the antioxidant properties of some plants collected in México, one conducted in the Moringa tree (*Moringa oleifera*) and the other in the Neem tree (*Azadirachta indica*).

**1.** Antioxidant properties of *Moringa oleifera*: In this experiment were collected fresh leaves of *M. oleifera* in the Municipality of Apatzingan in the state of Michoacan, Mexico, the leaves are brought to the facilities of the National School of Biological Sciences, IPN where allowed to air dry and then were macerated and placed in containers containing methanol. After one week the solvent was decanted and concentrated under reduced pressure using a rotary evaporator. The crude methanol extract was stable in distilled water obtained the following experimental concentrations: 50, 25, 12.5 and 6.25 mg / mL to which they evaluated the antioxidant capacity.

It was used as standard test the unstable radical 2,2-diphenyl-picrylhydrazyl (DPPH), which originally has a purple and when it is placed against a substance having antioxi‐ dant properties in a UV spectrophotometer at 517 nm, changes to yellow colour yellow (Figure 4). It was prepared a calibration curve of DPPH type in methanol at concentra‐ tions of 40, 120, 160 and 200 *u*g. Subsequently, aliquots of the above concentrations of methanol extract of *M. oleifera* and combined with DPPH in methanol for measuring its absorbance in the UV spectrophotometer at 0, 10, 30, 60 45 min., and then self-assess their antioxidant capacity [31].

capacity of this plant and that could explain its therapeutic efficacy in the treatment of dia‐

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**2.** Comparison of the antioxidant properties of *Azadirachta indica* with other species and a commercial product. This experiment followed a similar protocol to that described for *M. oleifera*, with the exception that the samples were evaluated for their antioxidant ca‐ pacity at times 1, 15, 30, 60 and 90 minutes. The protocol is divided into two parts, one of which was evaluated for antioxidant activity from four different extracts from leaves of *Azadirachta indica*: a) methanol, b) infusion, c) ethyl acetate and d) ethanol. This was done by measuring the percent inhibition of loss of the color purple to yellow the re‐ spective front DPPH extract (Figure 6). It was found that infusion of Neem showed the highest antioxidant activity (80% inhibition) than the other extracts even from the first minute after initiating the reaction. This would correspond to the ethnomedical use that people from rural zones done with this tree, and then take it as a tea before the first food of the day. The methanol extract of Neem leaves also exhibited a high antioxidant capacity, as a percentage inhibition of the radical DPPH greater than 50% during the en‐

**Figure 6.** Assessment of% inhibition of DPPH radical from 4 extracts of Neem (*Azadirachta indica*).

In the second part of this protocol, we chose a fraction of the methanol extract of Neem leaves and their antioxidant activity was compared against a commercial preparation, a kind of juice containing: Pomegranate (*Punica granatum*) green tea (*Camel sinensis*), cranberry (*Vaccinum my‐ tillus*), red grape (*Vitis vinifera*) and methanol extract of leaves, seed and fruit peel of passion fruit (*Passiflora edulis*). It was found that the commercial preparation showed the highest anti‐ oxidant activity throughout the reaction time (Figure 7), presented as a% inhibition greater than 50% from the first minute (68% inhibition), reaching end of the reaction with values high‐

betes in mouse models that are currently underway [32].

tire reaction time [33].

**Figure 4.** Graph showing the colour change of DPPH from purple to yellow when it is exposed to an antioxidant sub‐ stance

**Figure 5.** Antioxidant activity of different concentrations of methanol extract of *M. oleifera*

It was found that higher concentrations *of M. oleifera* antioxidant activity showed a concen‐ tration dependance, ie the higher the concentration of the extract metabolic higher antioxi‐ dant capacity (Figure 5). It was noted that the highest concentrations (50 mg / mL and 25 mg / mL) reached 50% of its antioxidant activity (assessed by the percentage of inhibition of purple fading to yellow against DPPH respective concentration of the methanol extract of *M. oleifera*) within 10 min. the reaction is initiated, which is indicative of the high antioxidant capacity of this plant and that could explain its therapeutic efficacy in the treatment of dia‐ betes in mouse models that are currently underway [32].

**2.** Comparison of the antioxidant properties of *Azadirachta indica* with other species and a commercial product. This experiment followed a similar protocol to that described for *M. oleifera*, with the exception that the samples were evaluated for their antioxidant ca‐ pacity at times 1, 15, 30, 60 and 90 minutes. The protocol is divided into two parts, one of which was evaluated for antioxidant activity from four different extracts from leaves of *Azadirachta indica*: a) methanol, b) infusion, c) ethyl acetate and d) ethanol. This was done by measuring the percent inhibition of loss of the color purple to yellow the re‐ spective front DPPH extract (Figure 6). It was found that infusion of Neem showed the highest antioxidant activity (80% inhibition) than the other extracts even from the first minute after initiating the reaction. This would correspond to the ethnomedical use that people from rural zones done with this tree, and then take it as a tea before the first food of the day. The methanol extract of Neem leaves also exhibited a high antioxidant capacity, as a percentage inhibition of the radical DPPH greater than 50% during the en‐ tire reaction time [33].

**Figure 6.** Assessment of% inhibition of DPPH radical from 4 extracts of Neem (*Azadirachta indica*).

**Figure 4.** Graph showing the colour change of DPPH from purple to yellow when it is exposed to an antioxidant sub‐

68 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 5.** Antioxidant activity of different concentrations of methanol extract of *M. oleifera*

It was found that higher concentrations *of M. oleifera* antioxidant activity showed a concen‐ tration dependance, ie the higher the concentration of the extract metabolic higher antioxi‐ dant capacity (Figure 5). It was noted that the highest concentrations (50 mg / mL and 25 mg / mL) reached 50% of its antioxidant activity (assessed by the percentage of inhibition of purple fading to yellow against DPPH respective concentration of the methanol extract of *M. oleifera*) within 10 min. the reaction is initiated, which is indicative of the high antioxidant

stance

In the second part of this protocol, we chose a fraction of the methanol extract of Neem leaves and their antioxidant activity was compared against a commercial preparation, a kind of juice containing: Pomegranate (*Punica granatum*) green tea (*Camel sinensis*), cranberry (*Vaccinum my‐ tillus*), red grape (*Vitis vinifera*) and methanol extract of leaves, seed and fruit peel of passion fruit (*Passiflora edulis*). It was found that the commercial preparation showed the highest anti‐ oxidant activity throughout the reaction time (Figure 7), presented as a% inhibition greater than 50% from the first minute (68% inhibition), reaching end of the reaction with values high‐ er than 85% inhibition of the presence of radical DPPH. Compared to the various organs of *Pas‐ siflora edulis*, the Neem leaf extract showed higher antioxidant capacity from the reaction started, it displayed a% inhibition of DPPH radical by 66% to reach 15 minutes of reaction, and reaching values greater than 85% inhibition at 60 minutes, falling a little activivty (63% approx‐ imately) at 90 min. the reaction is initiated. This would confirm previous data of different au‐ thors and from our laboratory (unpublished data), in the sense of the effectiveness of Neem tree leaves for the treatment of chronic degenerative diseases such as diabetes, it has proven ef‐ fective in significantly reducing blood glucose levels in streptozotocin-treated mice, an effect that may be due to the presence of secondary metabolites of the steroid type saponins, flavo‐ noids and phenols among others that seem to owe much its hypoglycemic action, thanks to its antioxidant properties. In this regard, our laboratory is conducting a more rigorous characteri‐ zation of secondary metabolites found in these plants by using spectroscopic techniques (e.g, Infrared Spectroscopy with Fourier Transform, Proton Nuclear Magnetic Resonance, etc.) try‐ ing to isolate, purify these metabolites and test its therapeutic efficacy in animal models of dia‐ betes. However, we should mention that, although the future is promising with regard to therapy options offered by these plants in Mexico (and other developing countries), yet to be made deeper studies and comparative scale phytochemical, toxicological and drug to confirm everything mentioned in this chapter [34].

where it was reported that the antioxidant effect of protection tends to increase with respect

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71

**Figure 8.** Results from DPPH method for testing the free-radical inhibition effect of different concentrations of metha‐

**Figure 9.** Shows the curve concentration vs. inhibition (%) of the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH ●), in which it is observed that the mean effective concentration (MEC) of noni leaf extract was 4,56%, at this concentration

Furthermore a methanolic solution was prepared with a concentration of 860 mmol / mL of DPPH. From this concentration the experiment was performed with a duplicated calibration curve at different concentrations, which was read at 517nm. An average concentration of

nolic extracts of Noni leaves.

is carried out the 50% inhibition of the unpaired radical.

to higher concentrations in tests performed with lyophilized juice extracts [35].

**Figure 7.** Assessment of% inhibition of DPPH radical of Neem (Azadirachta indica) with respect to a commercial prep‐ aration and different organs of Passion fruit (Passiflora edulis).

Another experiment with Noni (*Morinda citrifolia*) showed that the leaves of this plant also have a high antioxidant effect at low concentrations reaching a maximum effect at a dose of 5mg/mL (Figure 8). These results disagreed with the effect observed with the Noni fruit, where it was reported that the antioxidant effect of protection tends to increase with respect to higher concentrations in tests performed with lyophilized juice extracts [35].

er than 85% inhibition of the presence of radical DPPH. Compared to the various organs of *Pas‐ siflora edulis*, the Neem leaf extract showed higher antioxidant capacity from the reaction started, it displayed a% inhibition of DPPH radical by 66% to reach 15 minutes of reaction, and reaching values greater than 85% inhibition at 60 minutes, falling a little activivty (63% approx‐ imately) at 90 min. the reaction is initiated. This would confirm previous data of different au‐ thors and from our laboratory (unpublished data), in the sense of the effectiveness of Neem tree leaves for the treatment of chronic degenerative diseases such as diabetes, it has proven ef‐ fective in significantly reducing blood glucose levels in streptozotocin-treated mice, an effect that may be due to the presence of secondary metabolites of the steroid type saponins, flavo‐ noids and phenols among others that seem to owe much its hypoglycemic action, thanks to its antioxidant properties. In this regard, our laboratory is conducting a more rigorous characteri‐ zation of secondary metabolites found in these plants by using spectroscopic techniques (e.g, Infrared Spectroscopy with Fourier Transform, Proton Nuclear Magnetic Resonance, etc.) try‐ ing to isolate, purify these metabolites and test its therapeutic efficacy in animal models of dia‐ betes. However, we should mention that, although the future is promising with regard to therapy options offered by these plants in Mexico (and other developing countries), yet to be made deeper studies and comparative scale phytochemical, toxicological and drug to confirm

**Figure 7.** Assessment of% inhibition of DPPH radical of Neem (Azadirachta indica) with respect to a commercial prep‐

Another experiment with Noni (*Morinda citrifolia*) showed that the leaves of this plant also have a high antioxidant effect at low concentrations reaching a maximum effect at a dose of 5mg/mL (Figure 8). These results disagreed with the effect observed with the Noni fruit,

everything mentioned in this chapter [34].

70 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

aration and different organs of Passion fruit (Passiflora edulis).

**Figure 8.** Results from DPPH method for testing the free-radical inhibition effect of different concentrations of metha‐ nolic extracts of Noni leaves.

**Figure 9.** Shows the curve concentration vs. inhibition (%) of the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH ●), in which it is observed that the mean effective concentration (MEC) of noni leaf extract was 4,56%, at this concentration is carried out the 50% inhibition of the unpaired radical.

Furthermore a methanolic solution was prepared with a concentration of 860 mmol / mL of DPPH. From this concentration the experiment was performed with a duplicated calibration curve at different concentrations, which was read at 517nm. An average concentration of those used for standard curve analysis was chosen for the initial concentration of the metha‐ nol extract from leaves of *Morinda citrifolia*. From this methanol extract different percentage dilutions were prepared. After obtaining the extract dilutions 1000µL of each one was mixed with 860µL methanol and 140µL of DDPH and after that it were allowed to stand for 10 mi‐ nutes at 30°C and then absorbance was read (517nm) at time intervals of 10, 30, 45 and 60 minutes in a UV-Vis spectrophotometer HACH DR 5000. Through a mathematical analysis of linear regression curve was obtained concentration vs. inhibition (%), and finally deter‐ mined the mean effective antioxidant concentration (Figure 9) [36].

**Metabolites Reagent**

Potassium

ammonium

Sodium

Libermann

Kedde, Baljet,

Alkaloids

phenols

reducing sugars

Coumarins

Flavonoids

Saponines

cardiotonic Glycosides

cyanogenic

extracts of the leaves.

**i.** Alkaloids

**ii.** Coumarins

**iii.** Phenolics

**iv.** Flavonoids

Sesquiterpenlactones hidroxylamine

*Azadirachta indica*

**Test**

*Bertolt Lophocereus sp*

*Morinda citrifolia* 73

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*Cecropia obtusifolia*

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico

Dragendorf ++ - ++ - Mayer + + + + Wagner + - + + Sonneshain + - - - Silicotungstine + - - -

ferricianide <sup>+</sup> <sup>+</sup> <sup>+</sup> <sup>+</sup>

Fehling - - + + Benedic - - + +

hydroxide - - <sup>+</sup> <sup>+</sup>

Erlich + + + +

Hydroxide <sup>+</sup> <sup>+</sup> - <sup>+</sup>

chlorehydrate - <sup>+</sup> - -

Bouchard <sup>+</sup> <sup>+</sup> <sup>+</sup> <sup>+</sup>

legal - <sup>+</sup> - <sup>+</sup>

Glycosides Guignar - <sup>+</sup> <sup>+</sup> -

**Table 3.** Phytochemical sieve of different plants collected in Mexico. All the tests were performed with methanolic

The results of this phytochemical analysis presented in Table 2, shows qualitatively the sec‐ ondary metabolites found in the methanol extract of leaves of *M. oleifera*. The more abun‐ dant compounds in the leaves of this tree and also the must reported in several articles are:

Tanines ferric chloride - + - -

### **5. Phytochemical results**

#### **5.1. Moringa oleifera**

Extraction by maceration from *M. Oleifera* leaves was carried out in methyl alcohol for the phytochemical sieve, the results are shown in the following table (Table 2), and qualitative tests that were performed for each secondary metabolite are named in the same table.


**Table 2.** Phytochemical sieve of methanolic extract from *M. olerifera* leaves.

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico http://dx.doi.org/10.5772/52247 73


**Table 3.** Phytochemical sieve of different plants collected in Mexico. All the tests were performed with methanolic extracts of the leaves.

The results of this phytochemical analysis presented in Table 2, shows qualitatively the sec‐ ondary metabolites found in the methanol extract of leaves of *M. oleifera*. The more abun‐ dant compounds in the leaves of this tree and also the must reported in several articles are:

**i.** Alkaloids

those used for standard curve analysis was chosen for the initial concentration of the metha‐ nol extract from leaves of *Morinda citrifolia*. From this methanol extract different percentage dilutions were prepared. After obtaining the extract dilutions 1000µL of each one was mixed with 860µL methanol and 140µL of DDPH and after that it were allowed to stand for 10 mi‐ nutes at 30°C and then absorbance was read (517nm) at time intervals of 10, 30, 45 and 60 minutes in a UV-Vis spectrophotometer HACH DR 5000. Through a mathematical analysis of linear regression curve was obtained concentration vs. inhibition (%), and finally deter‐

Extraction by maceration from *M. Oleifera* leaves was carried out in methyl alcohol for the phytochemical sieve, the results are shown in the following table (Table 2), and qualitative

> Tanines ferric chloride phenols Potassium ferricianide +

Flavonoids Sodium Hydroxide + Sesquiterpenlactones hidroxylamine chlorehydrate - Saponines Libermann Bouchard + cardiotonic Glycosides Kedde, Baljet, legal cyanogenic Glycosides Guignar -

**Reagent Test** Dragendorf ++ Mayer + Wagner + Sonneshain + Silicotungstine +

Fehling - Benedic -

Erlich +

ammonium hydroxide -

tests that were performed for each secondary metabolite are named in the same table.

mined the mean effective antioxidant concentration (Figure 9) [36].

72 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**5. Phytochemical results**

**Alkaloids**

reducing sugars

Coumarins

**Table 2.** Phytochemical sieve of methanolic extract from *M. olerifera* leaves.

**5.1. Moringa oleifera**


### **v.** Saponins

In the case of metabolites such as tannins, cardiac glycosides, cyanogenic glycosides, the tests results were negative for the methanol extract of leaves of *M. oleifera* [37, 38].

Phytochemical results for the extracts obtained from leaves of Guarumbo (Cecropia obtusi‐ folia Bertolt), Musaro (Lophocereus sp.), Neem (Azadirachta indica) and Noni (Morinda cit‐ rifolia) are showed in Table 3. Several of those metabolites are highly active antioxidants.

### **6. Description of those plants collected in México and presenting high content of antioxidant compounds**

#### **6.1.** *Azadirachta indica*

Active compounds of Neem have been identified while others have not, and analyzed the most common are: nimbin; nimbidin; ninbidol; gedunin; sodium nimbinate; queceretin; sal‐ annin and azadirachtin.

**Figure 11.** Branch and leaves of Neem or Azadirachta indica.

**Figure 12.** Fruits from Neem Tree

The Neem fruit is bitter, and it is used as a purgative and for hemorrhoids (Figure 12)

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The flowers are astringent and expectorant and also Seed oil is extracted. (Figure 13).

Parts used and their uses:

Neem bark is bitter, astringent, is used to treat diseases of the mouth, teeth, loss of appetite, fever, cough and intestinal parasites (Figure 10)

**Figure 10.** Stem of the Neem tree.

The leaves help to neuromuscular problems, eliminate toxins, purify the blood, are also used to treat snake bites and insect (Figure 11)

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico http://dx.doi.org/10.5772/52247 75

**Figure 11.** Branch and leaves of Neem or Azadirachta indica.

**v.** Saponins

**6.1.** *Azadirachta indica*

annin and azadirachtin.

Parts used and their uses:

**Figure 10.** Stem of the Neem tree.

to treat snake bites and insect (Figure 11)

**content of antioxidant compounds**

74 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

fever, cough and intestinal parasites (Figure 10)

In the case of metabolites such as tannins, cardiac glycosides, cyanogenic glycosides, the

Phytochemical results for the extracts obtained from leaves of Guarumbo (Cecropia obtusi‐ folia Bertolt), Musaro (Lophocereus sp.), Neem (Azadirachta indica) and Noni (Morinda cit‐ rifolia) are showed in Table 3. Several of those metabolites are highly active antioxidants.

**6. Description of those plants collected in México and presenting high**

Active compounds of Neem have been identified while others have not, and analyzed the most common are: nimbin; nimbidin; ninbidol; gedunin; sodium nimbinate; queceretin; sal‐

Neem bark is bitter, astringent, is used to treat diseases of the mouth, teeth, loss of appetite,

The leaves help to neuromuscular problems, eliminate toxins, purify the blood, are also used

tests results were negative for the methanol extract of leaves of *M. oleifera* [37, 38].

The Neem fruit is bitter, and it is used as a purgative and for hemorrhoids (Figure 12)

**Figure 12.** Fruits from Neem Tree

The flowers are astringent and expectorant and also Seed oil is extracted. (Figure 13).

armpit, with serrated margins, the flowers (Figure 16) are solitary and axillary, fragrant and showy, the fruit is a spherical berry, globose or ellipsoid, measuring 10 cm in diameter and

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The ethnopharmacological information reveals that *Passiflora edulis Sims* has been used in traditional medicine around the world. In India, the fresh leaves of this plant are boiled in small amount of water and the extract is drunk to treat dysentery and hypertension, and the fruits are eaten to relieve constipation. In South America, native people drink the tea leaves and flowers as a sedative, infusion of the aerial parts is used in the treatment of tetanus, epi‐

weighs up to 190 g, yellow or purple, with a highly aromatic pulp [43].

**Figure 14.** Passion fruit

**Figure 15.** Leaves and flower of passion fruit

**Figure 13.** Flowers from Neem Tree

Neem kills some infectious organisms, contributes to the immune response at various levels, this increases the possibility that the body fight bacterial infections alone, viral and fungal. Neem increases the production of antibodies, improves the response of immune cells that release mediators white blood cells. For Diabetes: Neem extract orally reduces insulin re‐ quirements by between 30% and 50% to people who are insulin dependent. With Cancer: polysaccharides and limonoids found in the bark, leaves and Neem oil reduces tumors and cancer [39].

#### **6.2.** *Passiflora edulis Sims*

Another plant which has been described with therapeutic applications is the genus Passi‐ flora, whics comprises about 500 species and is the largest in the family Passifloraceae. *Passi‐ flora edulis Sims* is native from the Brazilian Amazon, known by the common name for passion fruit [40, 41]. The word passion comes from the Portuguese- Brazilian passion fruit, which means food prepared in Totuma [41, 42].

*Passiflora edulis* (Figure 14) is a widely cultivated species in tropical and subtropical coun‐ tries, there are two varieties: *Passiflora edulis Sims* var. *flavicarpa*, whose fruits are yellow and *Passiflora edulis Sims* var. purple, with purple fruits and adapts to higher ground [42].

Passion fruit (Figure 15) is a woody perennial, climbing habit and rapid development, which can reach up to 10 m long, the leaves are simple, alternate, and a tendril conestipules in the armpit, with serrated margins, the flowers (Figure 16) are solitary and axillary, fragrant and showy, the fruit is a spherical berry, globose or ellipsoid, measuring 10 cm in diameter and weighs up to 190 g, yellow or purple, with a highly aromatic pulp [43].

**Figure 14.** Passion fruit

**Figure 13.** Flowers from Neem Tree

**6.2.** *Passiflora edulis Sims*

which means food prepared in Totuma [41, 42].

76 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cancer [39].

Neem kills some infectious organisms, contributes to the immune response at various levels, this increases the possibility that the body fight bacterial infections alone, viral and fungal. Neem increases the production of antibodies, improves the response of immune cells that release mediators white blood cells. For Diabetes: Neem extract orally reduces insulin re‐ quirements by between 30% and 50% to people who are insulin dependent. With Cancer: polysaccharides and limonoids found in the bark, leaves and Neem oil reduces tumors and

Another plant which has been described with therapeutic applications is the genus Passi‐ flora, whics comprises about 500 species and is the largest in the family Passifloraceae. *Passi‐ flora edulis Sims* is native from the Brazilian Amazon, known by the common name for passion fruit [40, 41]. The word passion comes from the Portuguese- Brazilian passion fruit,

*Passiflora edulis* (Figure 14) is a widely cultivated species in tropical and subtropical coun‐ tries, there are two varieties: *Passiflora edulis Sims* var. *flavicarpa*, whose fruits are yellow and

Passion fruit (Figure 15) is a woody perennial, climbing habit and rapid development, which can reach up to 10 m long, the leaves are simple, alternate, and a tendril conestipules in the

*Passiflora edulis Sims* var. purple, with purple fruits and adapts to higher ground [42].

**Figure 15.** Leaves and flower of passion fruit

The ethnopharmacological information reveals that *Passiflora edulis Sims* has been used in traditional medicine around the world. In India, the fresh leaves of this plant are boiled in small amount of water and the extract is drunk to treat dysentery and hypertension, and the fruits are eaten to relieve constipation. In South America, native people drink the tea leaves and flowers as a sedative, infusion of the aerial parts is used in the treatment of tetanus, epi‐ lepsy, insomnia and hypertension is also indicated as a muscle relaxant, diuretic, to treat stomach aches, fever and intestinal tumors[44].

branch with age. The flowers are nocturnal, appear only on copies of more than two meters high and are colored green, but in the four seasons can take a pink color, the stem prolifera‐ tion and leads to masses of slender spines long they can get to cover it completely. Multiply by seeds in spring or summer. These plants are drought resistant so as to avoid daily water‐ ing and exposure to damp, besides being very sensitive to frost. But they need full sun and well drained soils. It is endemic to Baja California Sur, Baja California and Sonora in Mexico and Arizona in the United States It is a common species that has spread efficiently over the worldwide. The fruits are edible, but are hard to come by competition with birds and in‐ sects. This plant has curative properties. Southeastern Mexican indians prepare a tea from the pulp and skin of the cactus to relieve arthritis. Some members of this ethnic group say the plants with five ribs are very good for treating cancer. For diabetes: using the Musaro with a seven-pointed cladode, boiled in 1 liter of water, strain and drink three glasses of cooking, one before each meal. It is also widely used to treat ulcers, wounds, and stomach

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79

Musaro or gooseberry is the tea made by slicing sections fifteen or twenty two inches long from the stems of cactus. These cuts are then placed in a container large enough to contain five gallons of water and boiled for eight to ten hours until the liquid is reduced to approximately one gallon. People take this treatment for serious stomach diseases but they should drink tea in large amount. From extracts of *Lophocereus sp* two compounds have been isolated: pylocereine and lophocine a dimeric alkaloid with cytotoxic activity

Common name: The names that are known are "guarumbo", "chancarro", "hormiguillo", "chiflón" and "koochlle" among others. This plant is widely used by people suffering from

Botanical description: A tree 20 m tall, commonly grows in tropical rain forests. Its trunk is straight and presents a cavity where you can find some ants inside it, with branches that grow along this horizontally. The leaves are placed in a spiral on the branches. They have a

Distribution in Mexico: located along the coasts of Tamaulipas and San Luis Potosi to Ta‐ basco on the side of the Gulf of Mexico and Sinaloa to Chiapas Pacific side. Traditionally the dried leaves (15 g) are heated in water (500 mL) and the result is an infusion which is then filtered and taken as "daily water", cold infusion is often consumed in hot weather. States where is often used as traditional medicine this plant are: Hidalgo, Guerrero, Vera‐ cruz, Yucatan, Campeche, Tabasco, Mexico State, Oaxaca and Chiapas. It is reported that this plant contains β-sitosterol, stigmasterol, 4-ethyl-5-(n-3 valeroil)-6-hexahydrocoumar‐

deep green on the upper surface of the leaf and gray on the back (Figure 18).

ins. From butanol extracts were isolated chlorogenic acid and isorientin [48, 49].

diseases.

[46, 47].

**6.4.** *Cecropia obtusifolia Bertolt*

type 2 diabetes. It is known mainly in rural areas.

Family: Cecropiaceae

The phytochemical study of *Passiflora edulis Sims* (Passifloraceae) shows the presence of gly‐ cosides, including passiflorine, flavonoid glycosides: luteolin-6-Cchinovóside, cyanogenic glycosides, alkaloids harman, triterpenes and saponins, phenols, carotenoids, anthocyanins, L-ascorbic acid, γ-lactones, esters, volatile oils, eugenol, amino acids, carbohydrates and minerals [45].

**Figure 16.** Five ribs cladode from Musaro (*Lophocereus* sp )

#### **6.3.** *Lophocereus* **sp**

Gender *Lophocereus* sp. (Figure 17) develops as a succulent plant which presents a plus size and can reach 7m tall. These cacti have a columnar development right, has ribbed stems that branch with age. The flowers are nocturnal, appear only on copies of more than two meters high and are colored green, but in the four seasons can take a pink color, the stem prolifera‐ tion and leads to masses of slender spines long they can get to cover it completely. Multiply by seeds in spring or summer. These plants are drought resistant so as to avoid daily water‐ ing and exposure to damp, besides being very sensitive to frost. But they need full sun and well drained soils. It is endemic to Baja California Sur, Baja California and Sonora in Mexico and Arizona in the United States It is a common species that has spread efficiently over the worldwide. The fruits are edible, but are hard to come by competition with birds and in‐ sects. This plant has curative properties. Southeastern Mexican indians prepare a tea from the pulp and skin of the cactus to relieve arthritis. Some members of this ethnic group say the plants with five ribs are very good for treating cancer. For diabetes: using the Musaro with a seven-pointed cladode, boiled in 1 liter of water, strain and drink three glasses of cooking, one before each meal. It is also widely used to treat ulcers, wounds, and stomach diseases.

Musaro or gooseberry is the tea made by slicing sections fifteen or twenty two inches long from the stems of cactus. These cuts are then placed in a container large enough to contain five gallons of water and boiled for eight to ten hours until the liquid is reduced to approximately one gallon. People take this treatment for serious stomach diseases but they should drink tea in large amount. From extracts of *Lophocereus sp* two compounds have been isolated: pylocereine and lophocine a dimeric alkaloid with cytotoxic activity [46, 47].

### **6.4.** *Cecropia obtusifolia Bertolt*

### Family: Cecropiaceae

lepsy, insomnia and hypertension is also indicated as a muscle relaxant, diuretic, to treat

The phytochemical study of *Passiflora edulis Sims* (Passifloraceae) shows the presence of gly‐ cosides, including passiflorine, flavonoid glycosides: luteolin-6-Cchinovóside, cyanogenic glycosides, alkaloids harman, triterpenes and saponins, phenols, carotenoids, anthocyanins, L-ascorbic acid, γ-lactones, esters, volatile oils, eugenol, amino acids, carbohydrates and

Gender *Lophocereus* sp. (Figure 17) develops as a succulent plant which presents a plus size and can reach 7m tall. These cacti have a columnar development right, has ribbed stems that

stomach aches, fever and intestinal tumors[44].

78 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 16.** Five ribs cladode from Musaro (*Lophocereus* sp )

**6.3.** *Lophocereus* **sp**

minerals [45].

Common name: The names that are known are "guarumbo", "chancarro", "hormiguillo", "chiflón" and "koochlle" among others. This plant is widely used by people suffering from type 2 diabetes. It is known mainly in rural areas.

Botanical description: A tree 20 m tall, commonly grows in tropical rain forests. Its trunk is straight and presents a cavity where you can find some ants inside it, with branches that grow along this horizontally. The leaves are placed in a spiral on the branches. They have a deep green on the upper surface of the leaf and gray on the back (Figure 18).

Distribution in Mexico: located along the coasts of Tamaulipas and San Luis Potosi to Ta‐ basco on the side of the Gulf of Mexico and Sinaloa to Chiapas Pacific side. Traditionally the dried leaves (15 g) are heated in water (500 mL) and the result is an infusion which is then filtered and taken as "daily water", cold infusion is often consumed in hot weather. States where is often used as traditional medicine this plant are: Hidalgo, Guerrero, Vera‐ cruz, Yucatan, Campeche, Tabasco, Mexico State, Oaxaca and Chiapas. It is reported that this plant contains β-sitosterol, stigmasterol, 4-ethyl-5-(n-3 valeroil)-6-hexahydrocoumar‐ ins. From butanol extracts were isolated chlorogenic acid and isorientin [48, 49].

diseases, which are associated nowadays with stress, fast food diets and lack of daily exer‐

Chemistry of Natural Antioxidants and Studies Performed with Different Plants Collected in Mexico

http://dx.doi.org/10.5772/52247

81

The authors wants to thank the ENCB-IPN for the support received for this work through

1 Department of Environmental Systems Engineering at National School of Biological Scien‐

2 Department of Physiology at National School of Biological Sciences-National Polytechnic

[1] PIETTA, P. G. (2000) Flavonoids as Antioxidants, *Journal Natural Product,* 63: 1035–

[2] Rivera Arce E, Morales González J A, Fernández Sánchez A M, Bautista Ávila M, Vargas Mendoza N, Madrigal Santillán E O. (eds.) Chemistry of Natural Antioxi‐ dants and Studies Performed with Different Plants Collected in Mexico, (2009)

[3] Devasagaya T P, Tilak J C, Boloor K K. Free Radicals and Antioxidants in Human Health: Currens Status and Future Prospects. J. Assoc. Physicians India*.* 2004;

[5] Camacho Luis A, Mendoza Pérez J A, The Ephemeral Nature of Free Radicals: Chem‐ istry and Biochemistry of Free Radicals. In: Morales González J A, Fernández Sán‐ chez A M, Bautista Ávila M, Vargas Mendoza N, Madrigal Santillán E O. (eds.) Antioxidants and Chronic Degenerative Diseases. México: Ciencia al Día; 2009. p.

[6] VanderJagt T J, Ghattas R, VanderJagt D J, Crossey M, Glew R H. Comparison of the Total Antioxidant Content of 30 Widely Used Medicinal Plants of New Mexico. Life

and Tomás Alejandro Fregoso Aguilar2\*

cise, just to name a few factors.

the SIP projects 20120789 and 20120899.

ces-National Polytechnic Institute. Mexico, D.F., Mexico

[4] ACS. Chemistry. España:Reverte; 2010. p.460-65

**Acknowledgments**

**Author details**

**References**

1042.

227-238

52:794-804.

27-76

Sci. 2002; 70: 1035-1040.

Jorge Alberto Mendoza Pérez1

Institute. Mexico, D.F., Mexico

**Figure 17.** Guarumbo (*Cecropia obtusifolia Bertolt)* leaves.

### **7. Conclusion**

The plant kingdom has been the best source of remedies for curing a variety of disease and pain. This is why medicinal plants have played a key role in the worldwide maintenance of health. Traditional herbal medicine is intimately related to the Mexican popular culture; its use has origins based on ancestral knowledge. Natural products of higher plants are an im‐ portant source of therapeutic agents; therefore, many research groups are currently screen‐ ing the different biological activities of plants. Mexico has an extensive variety of plants; it is the fourth richest country worldwide in this aspect. Some 25 000 species are registered, and it is thought that there are almost 30 000 not yet described. Natural antioxidants that are present in herbs and spices are responsible for inhibiting or preventing the deleterious con‐ sequences of oxidative stress. Spices and herbs contain free radical scavengers like polyphe‐ nols, flavonoids and phenolic compounds, having antioxidant activities, Indeed all these plant studied have several biological effects and they could also be used as a source of natu‐ ral antioxidants. Further pharmacological studies are underway to identify the active con‐ stituents of the plant extracts responsible for the showed activities. As a final comment, compounds in plants are of great importance for the treatment of several chronic and degen‐ erative diseases like diabetes and cancer, among others. For that reason the use in traditional medicine is of great interest in order to know the activity and the mechanism of action of these compounds which could be used for the treatment and prevention of that mentioned diseases, which are associated nowadays with stress, fast food diets and lack of daily exer‐ cise, just to name a few factors.

### **Acknowledgments**

The authors wants to thank the ENCB-IPN for the support received for this work through the SIP projects 20120789 and 20120899.

### **Author details**

Jorge Alberto Mendoza Pérez1 and Tomás Alejandro Fregoso Aguilar2\*

1 Department of Environmental Systems Engineering at National School of Biological Scien‐ ces-National Polytechnic Institute. Mexico, D.F., Mexico

2 Department of Physiology at National School of Biological Sciences-National Polytechnic Institute. Mexico, D.F., Mexico

### **References**

**Figure 17.** Guarumbo (*Cecropia obtusifolia Bertolt)* leaves.

80 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

The plant kingdom has been the best source of remedies for curing a variety of disease and pain. This is why medicinal plants have played a key role in the worldwide maintenance of health. Traditional herbal medicine is intimately related to the Mexican popular culture; its use has origins based on ancestral knowledge. Natural products of higher plants are an im‐ portant source of therapeutic agents; therefore, many research groups are currently screen‐ ing the different biological activities of plants. Mexico has an extensive variety of plants; it is the fourth richest country worldwide in this aspect. Some 25 000 species are registered, and it is thought that there are almost 30 000 not yet described. Natural antioxidants that are present in herbs and spices are responsible for inhibiting or preventing the deleterious con‐ sequences of oxidative stress. Spices and herbs contain free radical scavengers like polyphe‐ nols, flavonoids and phenolic compounds, having antioxidant activities, Indeed all these plant studied have several biological effects and they could also be used as a source of natu‐ ral antioxidants. Further pharmacological studies are underway to identify the active con‐ stituents of the plant extracts responsible for the showed activities. As a final comment, compounds in plants are of great importance for the treatment of several chronic and degen‐ erative diseases like diabetes and cancer, among others. For that reason the use in traditional medicine is of great interest in order to know the activity and the mechanism of action of these compounds which could be used for the treatment and prevention of that mentioned

**7. Conclusion**


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**Chapter 4**

**Food Phenolic Compounds: Main Classes, Sources and**

The natural phenolic compounds have received increasing interest in the last years, since a great amount of them can be found in plants and consumption of vegetables and beverages with a high level of such compounds may reduce the risk of development of several diseases

It is known that the metabolism of plants is divided in primary and secondary. The substan‐ ces that are common to living things and essential to cells maintenance (lipids, proteins, car‐ bohydrates, and nucleic acids) are originated from the primary metabolism. On the other hand, substances originated from several biosynthetic pathways and that are restricted to determined groups of organisms are results of the secondary metabolism [1]. Phenolic com‐ pounds are constituted in one of the biggest and widely distributed groups of secondary

Figure 1 shows the inter-relationships between the primary and secondary metabolism

Biogenetically, phenolic compounds proceed of two metabolic pathways: the shikimic acid pathway where, mainly, phenylpropanoids are formed and the acetic acid pathway, in which the main products are the simple phenol [3]. Most plants phenolic compounds are synthe‐ sized through the phenylpropanoid pathway [4]. The combination of both pathways leads to the formation of flavonoids, the most plentiful group of phenolic compounds in nature [3]. Additionally, through the biosynthetic pathways to the flavonoids synthesis, among the not well elucidated condensation and polymerization phases, the condensed tannins or non-hy‐ drolysable tannins are formed. Hydrolysable tannins are derivatives of gallic acid or hexa‐

> © 2013 Reis Giada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Their Antioxidant Power**

Additional information is available at the end of the chapter

due to their antioxidant power, among other factors.

Maria de Lourdes Reis Giada

http://dx.doi.org/10.5772/51687

**1. Introduction**

metabolites in plants [2].

hydroxydiphenic acid [5].

in plants.

### **Food Phenolic Compounds: Main Classes, Sources and Their Antioxidant Power**

Maria de Lourdes Reis Giada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51687

### **1. Introduction**

The natural phenolic compounds have received increasing interest in the last years, since a great amount of them can be found in plants and consumption of vegetables and beverages with a high level of such compounds may reduce the risk of development of several diseases due to their antioxidant power, among other factors.

It is known that the metabolism of plants is divided in primary and secondary. The substan‐ ces that are common to living things and essential to cells maintenance (lipids, proteins, car‐ bohydrates, and nucleic acids) are originated from the primary metabolism. On the other hand, substances originated from several biosynthetic pathways and that are restricted to determined groups of organisms are results of the secondary metabolism [1]. Phenolic com‐ pounds are constituted in one of the biggest and widely distributed groups of secondary metabolites in plants [2].

Figure 1 shows the inter-relationships between the primary and secondary metabolism in plants.

Biogenetically, phenolic compounds proceed of two metabolic pathways: the shikimic acid pathway where, mainly, phenylpropanoids are formed and the acetic acid pathway, in which the main products are the simple phenol [3]. Most plants phenolic compounds are synthe‐ sized through the phenylpropanoid pathway [4]. The combination of both pathways leads to the formation of flavonoids, the most plentiful group of phenolic compounds in nature [3].

Additionally, through the biosynthetic pathways to the flavonoids synthesis, among the not well elucidated condensation and polymerization phases, the condensed tannins or non-hy‐ drolysable tannins are formed. Hydrolysable tannins are derivatives of gallic acid or hexa‐ hydroxydiphenic acid [5].

© 2013 Reis Giada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

such as anthocyanin, also lead to the different derivatives that add colors and color grada‐

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This way, phenolic compounds are essential to the physiology and cellular metabolism. They are involved in many functions in plants, such as sensorial properties (color, aroma, taste and astringency), structure, pollination, resistance to pests and predators, germinative processes of seed after harvesting and growth as well as development and reproduction,

Phenolic compounds can be classified in different ways because they are constituted in a large number of heterogeneous structures that range from simple molecules to highly poly‐

According to their carbon chain, phenolic compounds can be divided into 16 major classes [9]. The main classes of phenolic compounds regarding to their carbon chain can be seen in Figure 2. On the other hand, as to their distribution in nature, phenolic compounds can be divided into three classes: *shortly distributed* (as simple phenols, pyrocatechol, hydroquinone, resorci‐ nol, Aldehydes derived from benzoic acids that are components of essential oils, such as va‐ nillin), *widely distributed* (divided in flavonoids and their derivatives, coumarins and phenolic acids, such as benzoic and cinnamic acid and their derivatives) and *polymers* (tan‐

Finally, as to the location in the plant (free in the soluble fraction of cell or bound to com‐ pounds of cell wall), together with the chemical structure of these substances, phenolic compounds may also be classified as: *soluble* (such as simple phenol, flavonoids and tan‐ nins of low and medium molecular weight not bound to membranes compounds) and *insoluble* (essentially constituted by condensed tannins, phenolic acids and other phenolic compounds of low molecular weight bound to cell wall polysaccharides or proteins form‐ ing insoluble stable complexes). This classification is useful from the nutritional view‐ point, to the extent that the metabolic fate in the gastrointestinal tract and the physiological effects of each group will depend largely on their solubility characteristics. Insoluble phe‐ nolic compounds are not digested and may be partially or fully recovered quantitatively in the feces, while a part of the soluble can cross the intestinal barrier and be found in the

The antioxidant activity of food phenolic compounds is of nutritional interest, since it has been associated with the potentiation of the promoting effects of human health through the prevention of several diseases [11]. Additionally, in some cases, these compounds may also be used with therapeutic purposes due to their pharmacological properties [12]. Many phe‐ nolic compounds with low molecular weight, such as thymol, are used in medicine as anti‐

However, the antioxidant activity of phenolic compounds depends largely on the chemi‐ cal structure of these substances [2]. Among the phenolic compounds with known antiox‐ idant activity, flavonoids, tannins chalcones and coumarins as well as phenolic acids are

tion to flowers [7].

among others [8].

merized compounds.

nin and lignin) [10].

blood, unchanged or as metabolites [3].

septic due to its toxicity [7].

highlighted.

**Figure 1.** Inter-relationships between the primary and secondary metabolism in plants.

Therefore, phenolic compounds have, as a common characteristic, the presence of at least one aromatic ring hydroxyl-substituted [6]. Another characteristic of these substances is that they are presented commonly bound to other molecules, frequently to sugars (glycosyl resi‐ due) and proteins. The existence of phenolic compounds in free form also occurs in plant tissues. However, it is less common, possibly because they are toxic when present in the free state and detoxified, at least in part, when bound.

As a result, phenolic compounds play a role of protection against insects and other animals to the plants. The different types of bond between the glycosyl residue and the flavonoids, such as anthocyanin, also lead to the different derivatives that add colors and color grada‐ tion to flowers [7].

This way, phenolic compounds are essential to the physiology and cellular metabolism. They are involved in many functions in plants, such as sensorial properties (color, aroma, taste and astringency), structure, pollination, resistance to pests and predators, germinative processes of seed after harvesting and growth as well as development and reproduction, among others [8].

Phenolic compounds can be classified in different ways because they are constituted in a large number of heterogeneous structures that range from simple molecules to highly poly‐ merized compounds.

According to their carbon chain, phenolic compounds can be divided into 16 major classes [9].

The main classes of phenolic compounds regarding to their carbon chain can be seen in Figure 2.

On the other hand, as to their distribution in nature, phenolic compounds can be divided into three classes: *shortly distributed* (as simple phenols, pyrocatechol, hydroquinone, resorci‐ nol, Aldehydes derived from benzoic acids that are components of essential oils, such as va‐ nillin), *widely distributed* (divided in flavonoids and their derivatives, coumarins and phenolic acids, such as benzoic and cinnamic acid and their derivatives) and *polymers* (tan‐ nin and lignin) [10].

Finally, as to the location in the plant (free in the soluble fraction of cell or bound to com‐ pounds of cell wall), together with the chemical structure of these substances, phenolic compounds may also be classified as: *soluble* (such as simple phenol, flavonoids and tan‐ nins of low and medium molecular weight not bound to membranes compounds) and *insoluble* (essentially constituted by condensed tannins, phenolic acids and other phenolic compounds of low molecular weight bound to cell wall polysaccharides or proteins form‐ ing insoluble stable complexes). This classification is useful from the nutritional view‐ point, to the extent that the metabolic fate in the gastrointestinal tract and the physiological effects of each group will depend largely on their solubility characteristics. Insoluble phe‐ nolic compounds are not digested and may be partially or fully recovered quantitatively in the feces, while a part of the soluble can cross the intestinal barrier and be found in the blood, unchanged or as metabolites [3].

The antioxidant activity of food phenolic compounds is of nutritional interest, since it has been associated with the potentiation of the promoting effects of human health through the prevention of several diseases [11]. Additionally, in some cases, these compounds may also be used with therapeutic purposes due to their pharmacological properties [12]. Many phe‐ nolic compounds with low molecular weight, such as thymol, are used in medicine as anti‐ septic due to its toxicity [7].

**Figure 1.** Inter-relationships between the primary and secondary metabolism in plants.

88 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

state and detoxified, at least in part, when bound.

Therefore, phenolic compounds have, as a common characteristic, the presence of at least one aromatic ring hydroxyl-substituted [6]. Another characteristic of these substances is that they are presented commonly bound to other molecules, frequently to sugars (glycosyl resi‐ due) and proteins. The existence of phenolic compounds in free form also occurs in plant tissues. However, it is less common, possibly because they are toxic when present in the free

As a result, phenolic compounds play a role of protection against insects and other animals to the plants. The different types of bond between the glycosyl residue and the flavonoids, However, the antioxidant activity of phenolic compounds depends largely on the chemi‐ cal structure of these substances [2]. Among the phenolic compounds with known antiox‐ idant activity, flavonoids, tannins chalcones and coumarins as well as phenolic acids are highlighted.


tions according to the degree of hydrogenation and hydroxylation of the three ring sys‐ tems of these compounds. Flavonoids also occur as sulfated and methylated derivatives, conjugated with monosaccharides and disaccharides and forming complexes with oligosac‐ charides, lipids, amines, carboxylic acids and organic acids, being known approximately

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The basic chemical structures of the main classes of flavonoids are presented in Figure 3.

While members of certain classes of flavonoids (eg., flavonones) are colorless, the other (eg,

Flavonoids are important constituents of the human diet [14, 15], and are the most widely

anthocyanins) are always colored, such as flowers pigments and other plant parts [7].

distributed phenolic compounds in plant foods and also the most studied ones [10].

8000 compounds [13].

**Figure 3.** Chemical structures of the main classes of flavonoids.

**Figure 2.** Main classes of phenolic compounds regarding to their carbon chain.

### **2. Main Classes**

#### **2.1. Flavonoids**

According to the degree of hydroxylation and the presence of a C2-C3 double bond in the heterocycling pyrone ring, flavonoids can be divided into 13 classes [3], the most impor‐ tant being represented by the flavonols, flavanols, flavones, isoflavones, anthocyanidins or anthocyanins and flavanones [2]. Within these classes there are many structural varia‐ tions according to the degree of hydrogenation and hydroxylation of the three ring sys‐ tems of these compounds. Flavonoids also occur as sulfated and methylated derivatives, conjugated with monosaccharides and disaccharides and forming complexes with oligosac‐ charides, lipids, amines, carboxylic acids and organic acids, being known approximately 8000 compounds [13].

The basic chemical structures of the main classes of flavonoids are presented in Figure 3.


**Figure 3.** Chemical structures of the main classes of flavonoids.

**Figure 2.** Main classes of phenolic compounds regarding to their carbon chain.

90 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

According to the degree of hydroxylation and the presence of a C2-C3 double bond in the heterocycling pyrone ring, flavonoids can be divided into 13 classes [3], the most impor‐ tant being represented by the flavonols, flavanols, flavones, isoflavones, anthocyanidins or anthocyanins and flavanones [2]. Within these classes there are many structural varia‐

**2. Main Classes**

**2.1. Flavonoids**

While members of certain classes of flavonoids (eg., flavonones) are colorless, the other (eg, anthocyanins) are always colored, such as flowers pigments and other plant parts [7].

Flavonoids are important constituents of the human diet [14, 15], and are the most widely distributed phenolic compounds in plant foods and also the most studied ones [10].

It is known that flavonoids are among the most potent antioxidants from plants. The excel‐ lent antioxidant activity of these substances is related to the presence of hydroxyl groups in positions 3' and 4' of the B ring, which confer high stability to the formed radical by partici‐ pating in the displacement of the electron, and a double bond between carbons C2 and C3 of the ring C together with the carbonyl group at the C4 position, which makes the displace‐ ment of an electron possible from the ring B. Additionally, free hydroxyl groups in position 3 of ring C and in position 5 of ring A, together with the carbonyl group in position 4, are also important for the antioxidant activity of these compounds [16]. However, the effective‐ ness of the flavonoids decreases with the substitution of hydroxyl groups for sugars, being the glycosides less antioxidants than their corresponding aglycons [17].

#### **2.2. Tannins**

Tannins are phenolic compounds of molecular weight from intermediate to high (500-3000 D) [3] and can be classified into two major groups: hydrolysable tannins and non-hydrolysa‐ ble or condensed tannins [18]. There is a third group of tannins, phlorotannins, which are only found in brown seaweeds and are not commonly consumed by humans [19].

**Figure 4.** Chemical structures of hydrolysable tannin (A) and non-hydrolysable or condensed tannins (B).

The chalcones are intermediate in the biosynthesis of flavonoids, being the phloretin and its glucoside phloridzin (phloretin 2'-*o*-glucose), as well as the chalconaringenin and the arbu‐ tin, the most frequently found in foods. The phloretin and phloridzin are characteristics of apples, as well as the chalconaringenin is characteristic of tomatoes and arbutin of pears. However, arbutin is also found in strawberries, wheat and its derivatives, as well as in trace amounts in tea, coffee, red wine and broccoli. In some species of plants, the main pigments

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Like the other phenylpropanoids, coumarins constitute a class of secondary metabolites of plants derivatives from cinnamic acid by cyclization of the side chain of the *o*-coumaric acid [22]. These substances are more common in nature in the form of glycosides, such as umbel‐ liferone, esculetin and scopoletin, and are mainly found in olive oil, oats and spices [3].

The chemical structures of the main coumarins can be seen in Figure 6.

**2.3. Chalcones and Coumarins**

of yellow flowers are chalcones [21].

**Figure 5.** Chemical structures of the main chalcones.

Figure 5 shows the chemical estructures of the main chalcones.

The hydrolysable tannins have a center of glucose or a polyhydric alcohol partially or com‐ pletely esterified with gallic acid or hexahydroxydiphenic acid, forming gallotannin and el‐ lagitannins, respectively [20]. These metabolites are readily hydrolyzed with acids, bases or enzymes. However, they may also be oxidatively condensed to other galoil and hexahydrox‐ ydiphenic molecules and form polymers of high molecular weight. The best known hydro‐ lysable tannin is the tannic acid, which is a gallotannin consisting of a pentagalloyl glucose molecule that can additionally be esterified with another five units of gallic acid [10].

The condensed tannins are polymers of catechin and/or leucoanthocyanidin, not readily hy‐ drolyzed by acid treatment, and constitute the main phenolic fraction responsible for the characteristics of astringency of the vegetables. Although the term condensed tannins is still widely used, the chemically more descriptive term "proanthocyanidins" has gained more ac‐ ceptance. These substances are polymeric flavonoids that form the anthocyanidins pig‐ ments. The proanthocyanidins most widely studied are based on flavan-3-ols (-)-epicatechin and (+)-catechin [5].

The chemical structures of casuarictin (hydrolysable tannin) and proanthocyanidins (nonhydrolysable or condensed tannins) are shown in Figure 4 A and 4B, respectively.

Although the antioxidant activity of tannins has been much less marked than the activity of flavonoids, recent researches have shown that the degree of polymerization of these sub‐ stances is related to their antioxidant activity. In condensed tannins and hydrolysable (ella‐ gitannins) of high molecular weight, this activity can be up to fifteen to thirty times superior to those attributed to simple phenols [16].

Food Phenolic Compounds: Main Classes, Sources and Their Antioxidant Power http://dx.doi.org/10.5772/51687 93

**Figure 4.** Chemical structures of hydrolysable tannin (A) and non-hydrolysable or condensed tannins (B).

#### **2.3. Chalcones and Coumarins**

It is known that flavonoids are among the most potent antioxidants from plants. The excel‐ lent antioxidant activity of these substances is related to the presence of hydroxyl groups in positions 3' and 4' of the B ring, which confer high stability to the formed radical by partici‐ pating in the displacement of the electron, and a double bond between carbons C2 and C3 of the ring C together with the carbonyl group at the C4 position, which makes the displace‐ ment of an electron possible from the ring B. Additionally, free hydroxyl groups in position 3 of ring C and in position 5 of ring A, together with the carbonyl group in position 4, are also important for the antioxidant activity of these compounds [16]. However, the effective‐ ness of the flavonoids decreases with the substitution of hydroxyl groups for sugars, being

Tannins are phenolic compounds of molecular weight from intermediate to high (500-3000 D) [3] and can be classified into two major groups: hydrolysable tannins and non-hydrolysa‐ ble or condensed tannins [18]. There is a third group of tannins, phlorotannins, which are

The hydrolysable tannins have a center of glucose or a polyhydric alcohol partially or com‐ pletely esterified with gallic acid or hexahydroxydiphenic acid, forming gallotannin and el‐ lagitannins, respectively [20]. These metabolites are readily hydrolyzed with acids, bases or enzymes. However, they may also be oxidatively condensed to other galoil and hexahydrox‐ ydiphenic molecules and form polymers of high molecular weight. The best known hydro‐ lysable tannin is the tannic acid, which is a gallotannin consisting of a pentagalloyl glucose

only found in brown seaweeds and are not commonly consumed by humans [19].

molecule that can additionally be esterified with another five units of gallic acid [10].

The condensed tannins are polymers of catechin and/or leucoanthocyanidin, not readily hy‐ drolyzed by acid treatment, and constitute the main phenolic fraction responsible for the characteristics of astringency of the vegetables. Although the term condensed tannins is still widely used, the chemically more descriptive term "proanthocyanidins" has gained more ac‐ ceptance. These substances are polymeric flavonoids that form the anthocyanidins pig‐ ments. The proanthocyanidins most widely studied are based on flavan-3-ols (-)-epicatechin

The chemical structures of casuarictin (hydrolysable tannin) and proanthocyanidins (non-

Although the antioxidant activity of tannins has been much less marked than the activity of flavonoids, recent researches have shown that the degree of polymerization of these sub‐ stances is related to their antioxidant activity. In condensed tannins and hydrolysable (ella‐ gitannins) of high molecular weight, this activity can be up to fifteen to thirty times superior

hydrolysable or condensed tannins) are shown in Figure 4 A and 4B, respectively.

the glycosides less antioxidants than their corresponding aglycons [17].

92 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**2.2. Tannins**

and (+)-catechin [5].

to those attributed to simple phenols [16].

The chalcones are intermediate in the biosynthesis of flavonoids, being the phloretin and its glucoside phloridzin (phloretin 2'-*o*-glucose), as well as the chalconaringenin and the arbu‐ tin, the most frequently found in foods. The phloretin and phloridzin are characteristics of apples, as well as the chalconaringenin is characteristic of tomatoes and arbutin of pears. However, arbutin is also found in strawberries, wheat and its derivatives, as well as in trace amounts in tea, coffee, red wine and broccoli. In some species of plants, the main pigments of yellow flowers are chalcones [21].

Figure 5 shows the chemical estructures of the main chalcones.

**Figure 5.** Chemical structures of the main chalcones.

Like the other phenylpropanoids, coumarins constitute a class of secondary metabolites of plants derivatives from cinnamic acid by cyclization of the side chain of the *o*-coumaric acid [22]. These substances are more common in nature in the form of glycosides, such as umbel‐ liferone, esculetin and scopoletin, and are mainly found in olive oil, oats and spices [3].

The chemical structures of the main coumarins can be seen in Figure 6.

In the group of benzoic acids the ones that stand out are protocatechuic acids, vanillic acids,

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Among the cinnamic acids, *p*-coumaric, ferulic, caffeic and sinapic acids are the most com‐

Cinnamic acids rarely found free in plants. They are generally in the form of esters, along with a cyclic alcohol-acid, such as quinic acid to form the isochlorogenic acid, neochlorogen‐ ic acid, cripto chlorogenic acid and chlorogenic acid, an caffeoyl ester, which is the most im‐

Phenolic acids may be about one-third of the phenolic compounds in the human's diet [24]. It is known that these substances and their esters have a high antioxidant activity, especially hydroxybenzoic acid, hydroxycinnamic acid, caffeic acid and chlorogenic acid, and although other characteristics also contribute to the antioxidant activity of phenolic acids and their es‐ ters, this activity is usually determined by the number of hydroxyl groups found in the mol‐ ecule thereof. In general, the hydroxylated cinnamic acids are more effective than their

Despite the antioxidant activity of phenolic compounds and their possible benefits to human health, until the beginning of the last decade, most studies on these substances occurred in relation to their deleterious effects. Tannins, one of the major components of this group, due to the large number of hydroxyl groups contained therein, among other functional groups (1

syringic acid, gentisic acid, salicylic acid, p-hydroxybenzoic acid and gallic acid [3].

**Figure 8.** The general formulas and names of the main cinnamic acids.

Figure 9 shows the chemical structure of chlorogenic acid.

portant combination [10].

benzoic acids counterparts [16].

mon in nature [24].

**Figure 6.** Chemical structures of the main coumarins.

Although the data are still limited, it is known that chalcones and coumarins have antioxi‐ dant activity [23].

#### **2.4. Phenolic acids**

Phenolic acids can be divided into two groups: benzoic acids and cinnamic acids and deriva‐ tives thereof. The benzoic acids have seven carbon atoms (C6-C1) and are the simplest phe‐ nolic acids found in nature. Cinnamic acids have nine carbon atoms (C6-C3), but the most commonly found in vegetables are with seven. These substances are characterized by hav‐ ing a benzenic ring, a carboxylic group and one or more hydroxyl and/or methoxyl groups in the molecule [24].

The general formulas and names of the main benzoic and cinnamic acids are found in Fig‐ ures 7 and 8, respectively.

**Figure 7.** The general formulas and names of the main benzoic acids.

In the group of benzoic acids the ones that stand out are protocatechuic acids, vanillic acids, syringic acid, gentisic acid, salicylic acid, p-hydroxybenzoic acid and gallic acid [3].

Among the cinnamic acids, *p*-coumaric, ferulic, caffeic and sinapic acids are the most com‐ mon in nature [24].

**Figure 8.** The general formulas and names of the main cinnamic acids.

**Figure 6.** Chemical structures of the main coumarins.

94 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

dant activity [23].

**2.4. Phenolic acids**

in the molecule [24].

ures 7 and 8, respectively.

**Figure 7.** The general formulas and names of the main benzoic acids.

Although the data are still limited, it is known that chalcones and coumarins have antioxi‐

Phenolic acids can be divided into two groups: benzoic acids and cinnamic acids and deriva‐ tives thereof. The benzoic acids have seven carbon atoms (C6-C1) and are the simplest phe‐ nolic acids found in nature. Cinnamic acids have nine carbon atoms (C6-C3), but the most commonly found in vegetables are with seven. These substances are characterized by hav‐ ing a benzenic ring, a carboxylic group and one or more hydroxyl and/or methoxyl groups

The general formulas and names of the main benzoic and cinnamic acids are found in Fig‐

Cinnamic acids rarely found free in plants. They are generally in the form of esters, along with a cyclic alcohol-acid, such as quinic acid to form the isochlorogenic acid, neochlorogen‐ ic acid, cripto chlorogenic acid and chlorogenic acid, an caffeoyl ester, which is the most im‐ portant combination [10].

Figure 9 shows the chemical structure of chlorogenic acid.

Phenolic acids may be about one-third of the phenolic compounds in the human's diet [24]. It is known that these substances and their esters have a high antioxidant activity, especially hydroxybenzoic acid, hydroxycinnamic acid, caffeic acid and chlorogenic acid, and although other characteristics also contribute to the antioxidant activity of phenolic acids and their es‐ ters, this activity is usually determined by the number of hydroxyl groups found in the mol‐ ecule thereof. In general, the hydroxylated cinnamic acids are more effective than their benzoic acids counterparts [16].

Despite the antioxidant activity of phenolic compounds and their possible benefits to human health, until the beginning of the last decade, most studies on these substances occurred in relation to their deleterious effects. Tannins, one of the major components of this group, due to the large number of hydroxyl groups contained therein, among other functional groups (1 to 2 per 100 D), are capable of forming strong complexes with proteins, starch and other molecules, particularly digestive enzymes, reducing the digestibility of the feed. Likewise, by joining with their hydroxyl and carbonyl groups, tannins have the ability to chelate diva‐ lent cations, especially Fe and Zn, reducing the bioavailability of these minerals [10].

**Source Total phenolics (mg%) Reference**

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Cowpea (*V. unguicuata*), brown 100 27 Soyabean 414 28 Oat 352 29 Wheat flour 184 30

Black carrot 68 31 Broccoli 88 31 Brussels sprouts 69 31 Cabbage, white 76 32 Cabbage, red 186 32 Endive 92 32 Kale 136 33 Lettuce 107 32 Potato 150 31 Spinach 112 32 Tomato 68 32 Yam 92 31

Basil 4425 34 Chilli, green 107 32 Chilli, red 277 32 Coriander 374 31 Garlic 145 31 Ginger 221 31 Leek 85 32 Mint 400 31 Onion, white 269 35 Onion, yellow 164 35 Onion, red 428 35 Pepper, black 1600 36 Pepper, white 800 36

**Cereals and legumes**

**Vegetables**

**Herbs and spices**

**Figure 9.** Chemical structure of chlorogenic acid.

Although phenolic compounds are traditionally considered antinutrients, and until the moment as non-nutrients because deficiency states are unknown for them, in recent years they have been seen as a group of micro-nutrients in the vegetable kingdom, which are important part of human and animal diet. The condensed and hydrolysable tannins (el‐ lagitannins) of high molecular weight, since they are not absorbed by the mucosa, they have been regarded as insoluble antioxidants that may have high antioxidant activity in the gastrointestinal tract, protecting proteins, lipids and carbohydrates from oxidative dam‐ age during digestion [25].

Researches have also suggested that regular consumption of phenolic compounds directly from plant foods may be more effective in combating oxidative damage in our body than in the form of dietary supplement [26]. This can be explained by the possible synergistic inter‐ actions among food phenolic compounds, increasing the antioxidant capacity of these sub‐ stances..

This way, the content of phenolic compounds and the antioxidant power of a wide variety of plant foods have been investigated.

### **3. Sources and their antioxidant power**

Table 1 shows the mean content of total phenolic compounds (mg/ 100 g of sample) of some plant foods.


to 2 per 100 D), are capable of forming strong complexes with proteins, starch and other molecules, particularly digestive enzymes, reducing the digestibility of the feed. Likewise, by joining with their hydroxyl and carbonyl groups, tannins have the ability to chelate diva‐

Although phenolic compounds are traditionally considered antinutrients, and until the moment as non-nutrients because deficiency states are unknown for them, in recent years they have been seen as a group of micro-nutrients in the vegetable kingdom, which are important part of human and animal diet. The condensed and hydrolysable tannins (el‐ lagitannins) of high molecular weight, since they are not absorbed by the mucosa, they have been regarded as insoluble antioxidants that may have high antioxidant activity in the gastrointestinal tract, protecting proteins, lipids and carbohydrates from oxidative dam‐

Researches have also suggested that regular consumption of phenolic compounds directly from plant foods may be more effective in combating oxidative damage in our body than in the form of dietary supplement [26]. This can be explained by the possible synergistic inter‐ actions among food phenolic compounds, increasing the antioxidant capacity of these sub‐

This way, the content of phenolic compounds and the antioxidant power of a wide variety

Table 1 shows the mean content of total phenolic compounds (mg/ 100 g of sample) of some

lent cations, especially Fe and Zn, reducing the bioavailability of these minerals [10].

96 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 9.** Chemical structure of chlorogenic acid.

age during digestion [25].

of plant foods have been investigated.

**3. Sources and their antioxidant power**

stances..

plant foods.


**Source Total phenolics (mg%) Reference** Raspberry, red 342 46 Raspberry, yellow 426 46 Strawberry 199 47

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Roasted cocoa bean 1305 48 Cocoa liquor 994 48 Alkalised cocoa powder 896 48 Baking chocolate 349 48 Red wine 242 49 Tea, black 62 50 Tea, green 83 50 Coffee 188 51

As can be seen in Table 1, phenolic compounds are widely distributed in plant foods.

Cocoa, potato, yam, tomato, kale, Brussels sprouts, broccoli and others dark green leafy and brightly-colored vegetables as well as legumes and cereals, in addition to spices and fruits such as cherries and citrus, are particularly rich in phenolic compounds. Red wine also has a high concentration of phenolic compounds. It is known that the abundant phenolic com‐ pounds in red wine are anthocyanin [6, 52]. The green and black teas have been extensively studied, since they may contain up to 30% of their dry weight as phenolic compounds [53]. Coffee is also rich in phenolic compounds, especially chlorogenic acid. It has about 7% of the dry weight of the grains [24] and 15% of the dry instant coffee as phenolic compounds [54].

Although in some studies a few statistically significant correlations were found between the levels of total phenolic compounds and antioxidant power of foods, in others the total phe‐ nolics content of samples was highly correlated with the antioxidant capacity. On the other hand, there are still no standard methods and approved for determining the antioxidant power *in vitro*. The several available tests for this purpose involve different mechanisms of antioxidant defense system, from the chelation of metal ions to the measure of preventing oxidative damage to biomolecules, and offer distinct numerical results that are difficult to compare. Because of this, studies have used different methods to evaluate the antioxidant capacity of the studied sample, such as ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfon‐ ic acid radical assay), DPPH (2,2-diphenyl-picrylhydrazyl radical assay), FRAP (Ferric Re‐ ducing/Antioxidant Power assay) and ORAC (Oxygen Radical Absorbance Capacity assay),

**Table 1.** Total phenolic compounds content of some plant foods.

**Others**

among others tests.


**Table 1.** Total phenolic compounds content of some plant foods.

**Source Total phenolics (mg%) Reference** Shallot 1718 35 Sweet onion 142 35 Thyme 1646 37 Turmeric 176 31

98 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Apple, green 118 38 Apple, red 125 38 Apple, yellow 100 38 Blueberry 362 39 Cherry, sour 156 40 Cherry,sweet 79 38 Grape, black 213 38 Grape, white 184 38 Grapefruit 893 41 Guava, pink flesh 247 42 Guava, white flesh 145 42 Kiwi 791 43 Lemon 843 41 Lime 751 41 Litchi 60 44 Nectarine, white flesh 38 45 Nectarine, yellow flesh 25 45 Orange, sweet 1343 41 Peach, white flesh 53 45 Peach, yellow flesh 35 45 Pear 125 38 Pineapple 94 44 Plum, black 88 44 Plum, red 73 44 Pomegranate 147 44 Pomelo 57 44 Raspberry, black 670 46

**Fruits**

As can be seen in Table 1, phenolic compounds are widely distributed in plant foods.

Cocoa, potato, yam, tomato, kale, Brussels sprouts, broccoli and others dark green leafy and brightly-colored vegetables as well as legumes and cereals, in addition to spices and fruits such as cherries and citrus, are particularly rich in phenolic compounds. Red wine also has a high concentration of phenolic compounds. It is known that the abundant phenolic com‐ pounds in red wine are anthocyanin [6, 52]. The green and black teas have been extensively studied, since they may contain up to 30% of their dry weight as phenolic compounds [53]. Coffee is also rich in phenolic compounds, especially chlorogenic acid. It has about 7% of the dry weight of the grains [24] and 15% of the dry instant coffee as phenolic compounds [54].

Although in some studies a few statistically significant correlations were found between the levels of total phenolic compounds and antioxidant power of foods, in others the total phe‐ nolics content of samples was highly correlated with the antioxidant capacity. On the other hand, there are still no standard methods and approved for determining the antioxidant power *in vitro*. The several available tests for this purpose involve different mechanisms of antioxidant defense system, from the chelation of metal ions to the measure of preventing oxidative damage to biomolecules, and offer distinct numerical results that are difficult to compare. Because of this, studies have used different methods to evaluate the antioxidant capacity of the studied sample, such as ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfon‐ ic acid radical assay), DPPH (2,2-diphenyl-picrylhydrazyl radical assay), FRAP (Ferric Re‐ ducing/Antioxidant Power assay) and ORAC (Oxygen Radical Absorbance Capacity assay), among others tests.

By determining the content of total phenolic compounds and ability to reduce FeCl3 as well as DPPH free radical of some commonly consumed and underutilized tropical legumes [27], it was concluded that one of the commonly consumed cowpea *Vigna unguiculata* (brown) as well as underutilized legumes C. cajan (brown) and *S. sternocarpa* could be considered as functional foods due to their relatively higher antioxidant power, which could be as a result of their relative higher total phenolics content. In a similar way, evaluating the antioxidant capacity of twenty soybean hybrids by DPPH assay and their total phenolics content [28], it was concluded that the two cultivars that showed the highest contents of total phenolics al‐ so showed the highest antioxidant powers.

tract contained significantly more phenolic compounds in comparison with the nonhydro‐ lyzed extract. A dose-dependent effect was observed for all extracts concerning the power of removing free radical and reactive oxygen species, the black pepper extracts being the most effective. This study concluded that the pepper, especially black, which is an important com‐ ponent in the diet of many sub-Saharan and Eastern countries due to its nutritional impor‐ tance, can be considered an antioxidant and radical scavenging. However, evaluating the content of phenolic compounds and antioxidant capacity of 14 herbs and spices [37], al‐ though a significant correlation has been obtained between the phenolics content and anti‐ oxidant capacity of samples, it was found that the trend of the antioxidant capacity was different according to the method applied. The leaves of the species Piper showed the high‐ est antioxidant capacity in both methods studied (Folin-Ciocalteu reagent and FRAP meth‐ od). Yet, the African mango showed the greatest content of free antioxidant by FRAP method, while by Folin method *Piper umbellatum* excelled followed by thyme. This study concluded that the antioxidant power of plant samples should be interpreted with caution when measured by different methods. In spite of that fact, regardless of the method used,

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In addition to the studies already mentioned, the antioxidant capacity of 36 plant extracts was evaluated by the β-carotene and linoleic acid model system [31] and the content of total phenolic compounds of the extracts was determined. Mint, black carrots, and ginger showed high content of total phenolics. The antioxidant capacity calculated as percentage of oxida‐ tion inhibition ranged from a maximum of 92% in turmeric extracts to a minimum of 12.8% in long melon. Other foods which have high antioxidant capacity (> 70%) were ginger, mint, black carrots, Brussels sprouts, broccoli, yam, coriander and tomato. The antioxidant power of the samples significantly and positively correlated with their content of total phenolic compounds, allowing the conclusion that the plant foods with high content of phenolic com‐ pounds can be sources of dietary antioxidants. In another study, 66 types of plant foods were analyzed as to their content of phenolic compounds and their antioxidant capacity in the ORAC assay [32]. The results showed that the antioxidants composition and concentra‐ tion varied significantly among the different vegetables. The coriander, Chinese kale, water spinach and red chili showed high content of total phenolics and high antioxidant power.

Due to the growing recognition of their nutritional and therapeutic value, many fruits have also been investigated as to their content of phenolic compounds and antioxidant capacity. By evaluating the antioxidant capacity and total phenolics content, in addition to flavanol and monomeric anthocyanins, it was found from the flesh and peel of 11 apple cultivars [57] that the concentrations of the parameters investigated differed significantly among the culti‐ vars and were higher in the peel in comparison to the flesh. The content of total phenolics and antioxidant capacity were significantly correlated in both flesh and peel. It was conclud‐ ed that the contribution of phenolics to the antioxidant power in apple peel suggests that peel removal may induce a significant loss of antioxidants. It is also known that one of the most important sources of antioxidants among fruits is small red fruits. By determining the antioxidant capacity of four cultivars of blueberry through three different assays (DPPH, ABTS and FRAP), as well as the content of total phenolic compounds, in addition to flavo‐

the samples were rich in antioxidants.

Among cereals, milled oat groat pearlings, trichomes, flour, and bran were evaluated as to their antioxidant capacity against the oxidation of *R*-phycoerythrin protein in the ORAC assay, as well as against the oxidation of low density lipoproteins (LDL) [55]. In both the methods applied the antioxidant capacity of the fractions of oats was in the following order: pearl‐ ings > flour > trichome = bran. It was concluded through this study that a part of oat antioxi‐ dants, which is rich in phenolic compounds [29], is probably heat-labile because greater antioxidant power was found among the non-steam-treated pearlings. In another study, ten varieties of soft wheat were compared as to their content of total phenolic compounds and antioxidant capacity [30]. Important DPPH, oxygen, hydroxyl and ABTS radical removal capacity was found in all the studied varieties and the content of total phenolics of the samples showed correlation with their antioxidant power in DPPH, ORAC and ABTS assays.

On the other hand, searching the antioxidant capacity of vegetables in the genus Brassica and the best solvent (ethanol, acetone and methanol) for the extraction of their phenolic compounds [56], the results showed that the solvent used significantly affects the phenolics content and the properties of the studied extract. Methanolic extract showed the largest con‐ tent of total phenolics of broccoli, Brussels sprouts, and white cabbage. In this study, the an‐ tioxidant power of the samples was confirmed by different reactive oxygen species and showed to be concentration-dependent. Kale extracts have also been evaluated as to their content of total phenolic compounds and antioxidant capacity [33]. It can be observed that all studied fractions (free and conjugated forms) were able to remove the DPPH radical and that the content of total phenolic compounds of fractions was highly correlated with their antioxidant power.

Herbs and spices are of particular interest, since they have been proved to have high content of phenolic compounds and high antioxidant capacity. The values of Trolox Equivalent An‐ tioxidant Capacity (TEAC) and content of total phenolics were determined for 23 basil acces‐ sions [34]. A positive linear relationship was found between the content of total phenolic compounds and the antioxidant power of samples. This study concluded that basils have valuable antioxidant properties for culinary and possible medical application. The concen‐ tration of phenolic compounds in peppercorn (black and white), as well as the ability of hy‐ drolyzed and nonhydrolyzed pepper extracts to remove DPPH, superoxide, and hydroxyl radicals [36] were also investigated. The results obtained showed that hydrolyzed and non‐ hydrolyzed extracts of black pepper contained significantly more phenolic compounds when compared with those of white pepper. For any of these peppers, the hydrolyzed ex‐ tract contained significantly more phenolic compounds in comparison with the nonhydro‐ lyzed extract. A dose-dependent effect was observed for all extracts concerning the power of removing free radical and reactive oxygen species, the black pepper extracts being the most effective. This study concluded that the pepper, especially black, which is an important com‐ ponent in the diet of many sub-Saharan and Eastern countries due to its nutritional impor‐ tance, can be considered an antioxidant and radical scavenging. However, evaluating the content of phenolic compounds and antioxidant capacity of 14 herbs and spices [37], al‐ though a significant correlation has been obtained between the phenolics content and anti‐ oxidant capacity of samples, it was found that the trend of the antioxidant capacity was different according to the method applied. The leaves of the species Piper showed the high‐ est antioxidant capacity in both methods studied (Folin-Ciocalteu reagent and FRAP meth‐ od). Yet, the African mango showed the greatest content of free antioxidant by FRAP method, while by Folin method *Piper umbellatum* excelled followed by thyme. This study concluded that the antioxidant power of plant samples should be interpreted with caution when measured by different methods. In spite of that fact, regardless of the method used, the samples were rich in antioxidants.

By determining the content of total phenolic compounds and ability to reduce FeCl3 as well as DPPH free radical of some commonly consumed and underutilized tropical legumes [27], it was concluded that one of the commonly consumed cowpea *Vigna unguiculata* (brown) as well as underutilized legumes C. cajan (brown) and *S. sternocarpa* could be considered as functional foods due to their relatively higher antioxidant power, which could be as a result of their relative higher total phenolics content. In a similar way, evaluating the antioxidant capacity of twenty soybean hybrids by DPPH assay and their total phenolics content [28], it was concluded that the two cultivars that showed the highest contents of total phenolics al‐

Among cereals, milled oat groat pearlings, trichomes, flour, and bran were evaluated as to their antioxidant capacity against the oxidation of *R*-phycoerythrin protein in the ORAC assay, as well as against the oxidation of low density lipoproteins (LDL) [55]. In both the methods applied the antioxidant capacity of the fractions of oats was in the following order: pearl‐ ings > flour > trichome = bran. It was concluded through this study that a part of oat antioxi‐ dants, which is rich in phenolic compounds [29], is probably heat-labile because greater antioxidant power was found among the non-steam-treated pearlings. In another study, ten varieties of soft wheat were compared as to their content of total phenolic compounds and antioxidant capacity [30]. Important DPPH, oxygen, hydroxyl and ABTS radical removal capacity was found in all the studied varieties and the content of total phenolics of the samples

showed correlation with their antioxidant power in DPPH, ORAC and ABTS assays.

On the other hand, searching the antioxidant capacity of vegetables in the genus Brassica and the best solvent (ethanol, acetone and methanol) for the extraction of their phenolic compounds [56], the results showed that the solvent used significantly affects the phenolics content and the properties of the studied extract. Methanolic extract showed the largest con‐ tent of total phenolics of broccoli, Brussels sprouts, and white cabbage. In this study, the an‐ tioxidant power of the samples was confirmed by different reactive oxygen species and showed to be concentration-dependent. Kale extracts have also been evaluated as to their content of total phenolic compounds and antioxidant capacity [33]. It can be observed that all studied fractions (free and conjugated forms) were able to remove the DPPH radical and that the content of total phenolic compounds of fractions was highly correlated with their

Herbs and spices are of particular interest, since they have been proved to have high content of phenolic compounds and high antioxidant capacity. The values of Trolox Equivalent An‐ tioxidant Capacity (TEAC) and content of total phenolics were determined for 23 basil acces‐ sions [34]. A positive linear relationship was found between the content of total phenolic compounds and the antioxidant power of samples. This study concluded that basils have valuable antioxidant properties for culinary and possible medical application. The concen‐ tration of phenolic compounds in peppercorn (black and white), as well as the ability of hy‐ drolyzed and nonhydrolyzed pepper extracts to remove DPPH, superoxide, and hydroxyl radicals [36] were also investigated. The results obtained showed that hydrolyzed and non‐ hydrolyzed extracts of black pepper contained significantly more phenolic compounds when compared with those of white pepper. For any of these peppers, the hydrolyzed ex‐

so showed the highest antioxidant powers.

100 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

antioxidant power.

In addition to the studies already mentioned, the antioxidant capacity of 36 plant extracts was evaluated by the β-carotene and linoleic acid model system [31] and the content of total phenolic compounds of the extracts was determined. Mint, black carrots, and ginger showed high content of total phenolics. The antioxidant capacity calculated as percentage of oxida‐ tion inhibition ranged from a maximum of 92% in turmeric extracts to a minimum of 12.8% in long melon. Other foods which have high antioxidant capacity (> 70%) were ginger, mint, black carrots, Brussels sprouts, broccoli, yam, coriander and tomato. The antioxidant power of the samples significantly and positively correlated with their content of total phenolic compounds, allowing the conclusion that the plant foods with high content of phenolic com‐ pounds can be sources of dietary antioxidants. In another study, 66 types of plant foods were analyzed as to their content of phenolic compounds and their antioxidant capacity in the ORAC assay [32]. The results showed that the antioxidants composition and concentra‐ tion varied significantly among the different vegetables. The coriander, Chinese kale, water spinach and red chili showed high content of total phenolics and high antioxidant power.

Due to the growing recognition of their nutritional and therapeutic value, many fruits have also been investigated as to their content of phenolic compounds and antioxidant capacity. By evaluating the antioxidant capacity and total phenolics content, in addition to flavanol and monomeric anthocyanins, it was found from the flesh and peel of 11 apple cultivars [57] that the concentrations of the parameters investigated differed significantly among the culti‐ vars and were higher in the peel in comparison to the flesh. The content of total phenolics and antioxidant capacity were significantly correlated in both flesh and peel. It was conclud‐ ed that the contribution of phenolics to the antioxidant power in apple peel suggests that peel removal may induce a significant loss of antioxidants. It is also known that one of the most important sources of antioxidants among fruits is small red fruits. By determining the antioxidant capacity of four cultivars of blueberry through three different assays (DPPH, ABTS and FRAP), as well as the content of total phenolic compounds, in addition to flavo‐ noids, anthocyanins and flavan-3-ols [39], it was found that all cultivars contained high con‐ tent of total phenolics, flavonoids and anthocyanins and lower content of flavan-3-ols. However, significant differences were found in the total phenolics content among the differ‐ ent cultivars and growing seasons. Despite this, the studied cultivars showed high antioxi‐ dant power, which was highly correlated with the samples phenolic compounds. Similarly, by checking the content of total phenolics, in addition to flavonoids and anthocyanins, as well as the antioxidant capacity of three cultivars of sour cherries [58], a significant differ‐ ence was observed in phenolics content among different cultivars and growing seasons. However, the cultivars analyzed showed high antioxidant capacity, which was correlated with the phenolic compounds found in them. In this study significant increases were also found in the content of total phenolic compounds and antioxidant power during the ripen‐ ing of fruits. Additionally, different solvents were applied for comparing the antioxidant ca‐ pacity and the yield of total phenolic compounds present in the extracts of sour and sweet cherries [40]. It was found that the solubility of phenolic compounds was more effective in extracts of sweet cherries with use of methanol at 50% and in extracts of sour cherries with the use of acetone at 50%. Extracts from lyophilized sour cherries (methanolic and acetone water-mixtures) presented in average twice as high phenolic compounds than ethanolic ex‐ tracts. The DPPH antiradical efficiency values were higher in the extracts of sour cherries when compared with those of sweet cherries. It was concluded in this work that the strong antioxidant power of extracts of sour cherries is due to the substantial amount of total phe‐ nolic compounds present in them and that the fresh sour cherry can be considered as a good dietary source of phenolic compounds. The total phenolics content, total monomeric antho‐ cyanins and antioxidant capacities of 14 wild red raspberry accessions were also examined [59]. In this study, more two cultivars were included in the investigation to determine the variation between wild and cultivated raspberries. Antioxidant capacity of fruits was evalu‐ ated by both FRAP and TEAC assays. Significant variability was found for total phenolics, total monomeric anthocyanins and antioxidant capacity of wild raspberries. Nevertheless, the results indicated that some of the wild accessions of red raspberries have higher antioxi‐ dant power and phytonutrients content than existing domesticated cultivars. Finally, two strawberry cultivars were studied as to their content of total phenolic compounds and anti‐ oxidant capacity in different ripeness stages [47]. It was concluded that despite the berries in general have better taste and be more appreciated at ripe stage, higher contents of total phe‐ nolic compounds and antioxidant power were observed at pink stage for both strawberry cultivars studied. Also with respect to the fruits, a less known snake fruit was compared with better known kiwi fruit regarding to their total phenolics content and four radical scav‐ enging (FRAP, ABTS, DPPH and CUPRAC/Cupric Reducing Antioxidant Capacity) ability [43]. It was observed similarity between snake fruit and kiwi fruit in the contents of phenolic compounds as well as antioxidant power in DPPH assay. By this study, it was able to con‐ clude that the two fruits can be applied as antioxidant supplements to the normal diet. Con‐ sumption of a combination of both fruits could be recommended in order to obtain the best results. In another study, 25 cultivars, 5 each of white-flesh nectarines, yellow-flesh nectar‐ ines, white-flesh peaches, yellow-flesh peaches, and plums at the ripe stage were studied for their total phenolics content and antioxidant capacity by the DPPH and FRAP assays [45]. In

descending order, the cultivars presenting higher contents of total phenolics were: whiteflesh peaches, plums, yellow-flesh peaches and yellow-flesh nectarines. There was a strong correlation between total phenolics and antioxidant power of nectarines, peaches, and plums. By continuing to study the plum fruits, 20 genotypes of plums were investigated for their antioxidant capacity and total phenolics content [60]. Among the 20 genotypes, a strong correlation was observed between the total phenolics and antioxidant power of the samples, which was determined upon the FRAP assay. It was concluded that phenolic com‐ pounds seem to play a significant role in antioxidant value and health benefits of plums. Additionally, Mirabelle plums were examinated for their antioxidant capacity by different assays (DPPH, FRAP, ORAC) and total phenolics content [61]. The antioxidant power of the plum peels, flesh and pits reflected the total phenolics content of the samples with efficacy increasing of the order: peels < flesh < pits across the assays. Peel and flesh of six pear culti‐ vars were also investigated for their antioxidant capacity by DPPH assay and total phenolics content [62]. The results obtained showed that the total phenolics content in the peel can be up to 25 times higher than in the flesh. The peel also showed higher antioxidant power. The pomegranate is another fruit that has been researched. Its peel, mesocarp and juice were evaluated for their antioxidant power by TEAC and FRAP assays as well as total phenolics content [63]. It was found not only high correlation between TEAC and FRAP values, but also with the total phenolics content, which was in the following order: mesocarp > peel > juice. This study demonstrated that selection of raw materials (co-extraction of arils and peel) and pressure, respectively, markedly affected the profile and content of phenolics in the pomegranate juices, underlining the necessity to optimise these parameters for obtaining products with well-defined functional qualities. Studies have also been carried out to quan‐ tify the total phenolics content and antioxidant capacity of citrus fruits. Comparing the anti‐ oxidant properties of peel (flavedo and albedo) and juice of grapefruit, lemon, lime and sweet orange, four different antioxidant assays (DPPH, Reducing Power, β-carotene–lino‐ leate Model System and Thiobarbituric Acid Reactive Substances/TBARS) were applied to the volatile and polar fractions of peels and to crude and polar fraction of juices [41]. Phenol‐ ic compounds were among the two main antioxidant substances found in all extracts. Peels polar fractions showed the highest contents in phenolics, which probably contribute to the highest antioxidant power found in these fractions. However, peels volatile fractions showed the lowest antioxidant power. In another experiment, grapefruit and sour orange were extracted with five different polar solvents. The total phenolics content of the extracts was determined and the dried fractions were screened for their antioxidant capacity by four different assays (DPPH, Phosphomolybdenum method, Nitroblue tetrazolium/NBT Reduc‐ tion and Reducing Power) [64]. All citrus extracts showed good antioxidant capacity. The best correlation between total phenolics and radical scavenging activity was observed by DPPH method. It was concluded that the data obtained clearly established the antioxidant power of the studied citrus fruit extracts. Studying the extraction efficiency of five different solvents on the total phenolics content and antioxidant capacities of pomelo and navel or‐ anges by five antioxidant assays (DPPH, ORAC, ABTS, Phospomolybdenum method and Reducing Power) [65], it was found that the total phenolics content of extracts varied accord‐ ing to the solvent used. Significant differences were also found in antioxidant capacity val‐

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descending order, the cultivars presenting higher contents of total phenolics were: whiteflesh peaches, plums, yellow-flesh peaches and yellow-flesh nectarines. There was a strong correlation between total phenolics and antioxidant power of nectarines, peaches, and plums. By continuing to study the plum fruits, 20 genotypes of plums were investigated for their antioxidant capacity and total phenolics content [60]. Among the 20 genotypes, a strong correlation was observed between the total phenolics and antioxidant power of the samples, which was determined upon the FRAP assay. It was concluded that phenolic com‐ pounds seem to play a significant role in antioxidant value and health benefits of plums. Additionally, Mirabelle plums were examinated for their antioxidant capacity by different assays (DPPH, FRAP, ORAC) and total phenolics content [61]. The antioxidant power of the plum peels, flesh and pits reflected the total phenolics content of the samples with efficacy increasing of the order: peels < flesh < pits across the assays. Peel and flesh of six pear culti‐ vars were also investigated for their antioxidant capacity by DPPH assay and total phenolics content [62]. The results obtained showed that the total phenolics content in the peel can be up to 25 times higher than in the flesh. The peel also showed higher antioxidant power. The pomegranate is another fruit that has been researched. Its peel, mesocarp and juice were evaluated for their antioxidant power by TEAC and FRAP assays as well as total phenolics content [63]. It was found not only high correlation between TEAC and FRAP values, but also with the total phenolics content, which was in the following order: mesocarp > peel > juice. This study demonstrated that selection of raw materials (co-extraction of arils and peel) and pressure, respectively, markedly affected the profile and content of phenolics in the pomegranate juices, underlining the necessity to optimise these parameters for obtaining products with well-defined functional qualities. Studies have also been carried out to quan‐ tify the total phenolics content and antioxidant capacity of citrus fruits. Comparing the anti‐ oxidant properties of peel (flavedo and albedo) and juice of grapefruit, lemon, lime and sweet orange, four different antioxidant assays (DPPH, Reducing Power, β-carotene–lino‐ leate Model System and Thiobarbituric Acid Reactive Substances/TBARS) were applied to the volatile and polar fractions of peels and to crude and polar fraction of juices [41]. Phenol‐ ic compounds were among the two main antioxidant substances found in all extracts. Peels polar fractions showed the highest contents in phenolics, which probably contribute to the highest antioxidant power found in these fractions. However, peels volatile fractions showed the lowest antioxidant power. In another experiment, grapefruit and sour orange were extracted with five different polar solvents. The total phenolics content of the extracts was determined and the dried fractions were screened for their antioxidant capacity by four different assays (DPPH, Phosphomolybdenum method, Nitroblue tetrazolium/NBT Reduc‐ tion and Reducing Power) [64]. All citrus extracts showed good antioxidant capacity. The best correlation between total phenolics and radical scavenging activity was observed by DPPH method. It was concluded that the data obtained clearly established the antioxidant power of the studied citrus fruit extracts. Studying the extraction efficiency of five different solvents on the total phenolics content and antioxidant capacities of pomelo and navel or‐ anges by five antioxidant assays (DPPH, ORAC, ABTS, Phospomolybdenum method and Reducing Power) [65], it was found that the total phenolics content of extracts varied accord‐ ing to the solvent used. Significant differences were also found in antioxidant capacity val‐

noids, anthocyanins and flavan-3-ols [39], it was found that all cultivars contained high con‐ tent of total phenolics, flavonoids and anthocyanins and lower content of flavan-3-ols. However, significant differences were found in the total phenolics content among the differ‐ ent cultivars and growing seasons. Despite this, the studied cultivars showed high antioxi‐ dant power, which was highly correlated with the samples phenolic compounds. Similarly, by checking the content of total phenolics, in addition to flavonoids and anthocyanins, as well as the antioxidant capacity of three cultivars of sour cherries [58], a significant differ‐ ence was observed in phenolics content among different cultivars and growing seasons. However, the cultivars analyzed showed high antioxidant capacity, which was correlated with the phenolic compounds found in them. In this study significant increases were also found in the content of total phenolic compounds and antioxidant power during the ripen‐ ing of fruits. Additionally, different solvents were applied for comparing the antioxidant ca‐ pacity and the yield of total phenolic compounds present in the extracts of sour and sweet cherries [40]. It was found that the solubility of phenolic compounds was more effective in extracts of sweet cherries with use of methanol at 50% and in extracts of sour cherries with the use of acetone at 50%. Extracts from lyophilized sour cherries (methanolic and acetone water-mixtures) presented in average twice as high phenolic compounds than ethanolic ex‐ tracts. The DPPH antiradical efficiency values were higher in the extracts of sour cherries when compared with those of sweet cherries. It was concluded in this work that the strong antioxidant power of extracts of sour cherries is due to the substantial amount of total phe‐ nolic compounds present in them and that the fresh sour cherry can be considered as a good dietary source of phenolic compounds. The total phenolics content, total monomeric antho‐ cyanins and antioxidant capacities of 14 wild red raspberry accessions were also examined [59]. In this study, more two cultivars were included in the investigation to determine the variation between wild and cultivated raspberries. Antioxidant capacity of fruits was evalu‐ ated by both FRAP and TEAC assays. Significant variability was found for total phenolics, total monomeric anthocyanins and antioxidant capacity of wild raspberries. Nevertheless, the results indicated that some of the wild accessions of red raspberries have higher antioxi‐ dant power and phytonutrients content than existing domesticated cultivars. Finally, two strawberry cultivars were studied as to their content of total phenolic compounds and anti‐ oxidant capacity in different ripeness stages [47]. It was concluded that despite the berries in general have better taste and be more appreciated at ripe stage, higher contents of total phe‐ nolic compounds and antioxidant power were observed at pink stage for both strawberry cultivars studied. Also with respect to the fruits, a less known snake fruit was compared with better known kiwi fruit regarding to their total phenolics content and four radical scav‐ enging (FRAP, ABTS, DPPH and CUPRAC/Cupric Reducing Antioxidant Capacity) ability [43]. It was observed similarity between snake fruit and kiwi fruit in the contents of phenolic compounds as well as antioxidant power in DPPH assay. By this study, it was able to con‐ clude that the two fruits can be applied as antioxidant supplements to the normal diet. Con‐ sumption of a combination of both fruits could be recommended in order to obtain the best results. In another study, 25 cultivars, 5 each of white-flesh nectarines, yellow-flesh nectar‐ ines, white-flesh peaches, yellow-flesh peaches, and plums at the ripe stage were studied for their total phenolics content and antioxidant capacity by the DPPH and FRAP assays [45]. In

102 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ues via the same method in different solvents, as well as on the antioxidant capacity of each extract via different methods. Nonetheless, the broad range of activity of the extracts led to the conclusion that multiple mechanisms are responsable for the antioxidant power of the samples and clearly indicated the potential application value of the citrus fruits studied. Fi‐ nally, the study of the content of phenolic compounds and antioxidant power of tropical fruits such as guava has also been conducted. One white-fleshed and three pink-fleshed of guava were analyzed as to their content of total phenolics, in addition to ascorbic acid and total carotenoids, as well as to their antioxidant capacity [42]. The ABTS, DPPH and FRAP assays were used for determining the antioxidant capacity in methanol and dichlorome‐ thane extracts of the samples, while the ORAC assay was used only for determining it in methanol extracts. The results obtained showed that white pulp guava had more total phe‐ nolics and ascorbic acid than pink pulp guava. On the other hand, carotenoids were absent in the white pulp guava. In all antioxidant assays the methanol extracts showed good corre‐ lation with the content of total phenolics and ascorbic acid, as well as between them, but showed negative correlation with total carotenoids.

gation, the antioxidant capacities of three Argentine red wines were evaluated by TEAC and FRAP assays. The correlation between antioxidant capacity and content of phenolic compounds as well as between antioxidant capacity and phenolic profile of samples [49] was determined. It can be noted that the wines showed significant antioxidant capacity. However, no significant correlation was found between their antioxidant capacity and to‐ tal phenolics content. Nevertheless, the canonical correlation and multiple regression anal‐ ysis showed that the antioxidant capacity of the samples was highly correlated with their profile of phenolic compounds. The results obtained in this study showed the importance of analyzing the phenolic profile of the sample rather than total phenolics to help under‐ stand the differences in the antioxidant power of wines, which should be extended to oth‐ er food products. Among the alcoholic beverages, antioxidant power has also been reported for whiskey, sake and sherries. [70]. In addition to alcoholic beverages, the free radicalscavenging activity and total phenolic content of commercial tea [50] were determined, finding that green tea contained higher content of phenolic compounds than black tea. The antioxidant capacity per serving of green tea was also much higher than that of black tea. However, comparing the content of total phenolics, flavonoids and antioxidant capacity of black tea, green tea, red wine and cocoa by ABTS and DPPH assays [71], it was found that cocoa contains much higher levels of total phenolics and flavonoids per serving than black tea, green tea and red wine. In the two methods applied, the antioxidant power of the samples per serving was found in the following descending order: cocoa, red wine, green tea and black tea. The content of total phenolic compounds and DPPH and ABTS radical removal capacity of coffee extracts obtained by continuous (Soxhlet 1 h and 3 h) and discon‐ tinuous (solid-liquid extraction and filter coffeemaker) methods, many solvents (water, meth‐ anol, ethanol and their mixtures), successive extractions and water with different pHs (4.5, 7.0 and 9.5) were also evaluated [72]. The coffee extracts with the highest antioxidant capaci‐ ty were obtained after extraction with water neutral (pH 7.0) in the filter coffeemaker (24 g spent coffee per 400 mL water). In addition, the drink degreasing and lyophilization of the extract permitted to obtain coffee extract powder with high antioxidant power, which can be used as an ingredient or additive in the food industry with potential for preservation

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It is also know that tamarind, canola, sesame, linseed and sunflower seeds are other possible sources of phenolic compounds [73] and have high antioxidant capacity. The antioxidant ca‐ pacity of the striped sunflower seed cotyledon extracts, obtained by sequential extraction with different polarities of solvents, was determined by three in vitro methods: FRAP, DPPH and ORAC [74]. In the three methods applied, the aqueous extract showed higher an‐ tioxidant capacity than the ethanolic. When compared with the synthetic antioxidant Buty‐ lated Hydroxyl Toluene (BHT), the antioxidant power of the aqueous extract varied from 45% to 66%, according to the used method. It was concluded in this study that the high anti‐ oxidant power found for the aqueous extract of the studied sunflower seed suggests that the intake of this seed may prevent *in vivo* oxidative reactions responsible for the development

and functional properties.

of several diseases.

In addition to the aforementioned fruits, in the search for new foods rich in phenolic com‐ pounds and high antioxidant capacity, unconventional tropical fruits have been widely researched. Accordingly, the Antilles cherry, Barbados cherry or acerola (1063 mg/100 g), camu-camu (1176 mg/100 g), puçá-preto (868 mg/100 g), assai or açaí (454 mg/100 g) and jaboticaba (440 mg/100 g) showed to be rich in phenolic compounds. When testing the antioxidant capacity of these fruits fresh and dry matter by DPPH assay, it was found an association between their antioxidant power and total phenolics content [66]. Similarly, banana passion fruit (635-1018mg/100 g), cashew (445 mg/100 g) and guava apple (309 mg/ 100 g) also showed a high total phenolic content when evaluated by FRAP and ABTS assays. The antioxidant power of these fruits showed a strong correlation with their total phenolics content [67].

Other plant-originated foods studied for their content of phenolic compounds and antioxi‐ dant capacities are as follows. The cocoa and chocolate liquor antioxidant capacities as well as monomeric and oligomeric procyanidins were studied [68]. The results obtained showed that the procyanidins content was correlated with the antioxidant capacity, which was de‐ termined by the ORAC assay as an indicator for potential biological activity of the sam‐ ples. However, following the changes in total and individual phenolics content as well as antioxidant capacity during the processing of cocoa beans [48], it can be noted that the loss of phenolic compounds and antioxidant capacity of cocoa vary according to the degree of technological processing. The roasting process and cocoa nib alkalization had the greatest influence on the content of phenolic compounds and antioxidant power. The antioxidant capacity of 107 different Spanish red wines, from different varieties of grapes, aging proc‐ esses and vintages [69] was also investigated by different methods and the results showed that all samples had an important capacity of removing hydroxyl radical and were able to block the superoxide radical, but with 10 times lower intensity. The wines also showed important protective action on biomarkers of oxidative stress. However, few statistically significant correlations were found between the levels of total phenolics and antioxidant power of the wines and the values of these correlations were very low. In another investi‐ gation, the antioxidant capacities of three Argentine red wines were evaluated by TEAC and FRAP assays. The correlation between antioxidant capacity and content of phenolic compounds as well as between antioxidant capacity and phenolic profile of samples [49] was determined. It can be noted that the wines showed significant antioxidant capacity. However, no significant correlation was found between their antioxidant capacity and to‐ tal phenolics content. Nevertheless, the canonical correlation and multiple regression anal‐ ysis showed that the antioxidant capacity of the samples was highly correlated with their profile of phenolic compounds. The results obtained in this study showed the importance of analyzing the phenolic profile of the sample rather than total phenolics to help under‐ stand the differences in the antioxidant power of wines, which should be extended to oth‐ er food products. Among the alcoholic beverages, antioxidant power has also been reported for whiskey, sake and sherries. [70]. In addition to alcoholic beverages, the free radicalscavenging activity and total phenolic content of commercial tea [50] were determined, finding that green tea contained higher content of phenolic compounds than black tea. The antioxidant capacity per serving of green tea was also much higher than that of black tea. However, comparing the content of total phenolics, flavonoids and antioxidant capacity of black tea, green tea, red wine and cocoa by ABTS and DPPH assays [71], it was found that cocoa contains much higher levels of total phenolics and flavonoids per serving than black tea, green tea and red wine. In the two methods applied, the antioxidant power of the samples per serving was found in the following descending order: cocoa, red wine, green tea and black tea. The content of total phenolic compounds and DPPH and ABTS radical removal capacity of coffee extracts obtained by continuous (Soxhlet 1 h and 3 h) and discon‐ tinuous (solid-liquid extraction and filter coffeemaker) methods, many solvents (water, meth‐ anol, ethanol and their mixtures), successive extractions and water with different pHs (4.5, 7.0 and 9.5) were also evaluated [72]. The coffee extracts with the highest antioxidant capaci‐ ty were obtained after extraction with water neutral (pH 7.0) in the filter coffeemaker (24 g spent coffee per 400 mL water). In addition, the drink degreasing and lyophilization of the extract permitted to obtain coffee extract powder with high antioxidant power, which can be used as an ingredient or additive in the food industry with potential for preservation and functional properties.

ues via the same method in different solvents, as well as on the antioxidant capacity of each extract via different methods. Nonetheless, the broad range of activity of the extracts led to the conclusion that multiple mechanisms are responsable for the antioxidant power of the samples and clearly indicated the potential application value of the citrus fruits studied. Fi‐ nally, the study of the content of phenolic compounds and antioxidant power of tropical fruits such as guava has also been conducted. One white-fleshed and three pink-fleshed of guava were analyzed as to their content of total phenolics, in addition to ascorbic acid and total carotenoids, as well as to their antioxidant capacity [42]. The ABTS, DPPH and FRAP assays were used for determining the antioxidant capacity in methanol and dichlorome‐ thane extracts of the samples, while the ORAC assay was used only for determining it in methanol extracts. The results obtained showed that white pulp guava had more total phe‐ nolics and ascorbic acid than pink pulp guava. On the other hand, carotenoids were absent in the white pulp guava. In all antioxidant assays the methanol extracts showed good corre‐ lation with the content of total phenolics and ascorbic acid, as well as between them, but

In addition to the aforementioned fruits, in the search for new foods rich in phenolic com‐ pounds and high antioxidant capacity, unconventional tropical fruits have been widely researched. Accordingly, the Antilles cherry, Barbados cherry or acerola (1063 mg/100 g), camu-camu (1176 mg/100 g), puçá-preto (868 mg/100 g), assai or açaí (454 mg/100 g) and jaboticaba (440 mg/100 g) showed to be rich in phenolic compounds. When testing the antioxidant capacity of these fruits fresh and dry matter by DPPH assay, it was found an association between their antioxidant power and total phenolics content [66]. Similarly, banana passion fruit (635-1018mg/100 g), cashew (445 mg/100 g) and guava apple (309 mg/ 100 g) also showed a high total phenolic content when evaluated by FRAP and ABTS assays. The antioxidant power of these fruits showed a strong correlation with their total

Other plant-originated foods studied for their content of phenolic compounds and antioxi‐ dant capacities are as follows. The cocoa and chocolate liquor antioxidant capacities as well as monomeric and oligomeric procyanidins were studied [68]. The results obtained showed that the procyanidins content was correlated with the antioxidant capacity, which was de‐ termined by the ORAC assay as an indicator for potential biological activity of the sam‐ ples. However, following the changes in total and individual phenolics content as well as antioxidant capacity during the processing of cocoa beans [48], it can be noted that the loss of phenolic compounds and antioxidant capacity of cocoa vary according to the degree of technological processing. The roasting process and cocoa nib alkalization had the greatest influence on the content of phenolic compounds and antioxidant power. The antioxidant capacity of 107 different Spanish red wines, from different varieties of grapes, aging proc‐ esses and vintages [69] was also investigated by different methods and the results showed that all samples had an important capacity of removing hydroxyl radical and were able to block the superoxide radical, but with 10 times lower intensity. The wines also showed important protective action on biomarkers of oxidative stress. However, few statistically significant correlations were found between the levels of total phenolics and antioxidant power of the wines and the values of these correlations were very low. In another investi‐

showed negative correlation with total carotenoids.

104 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

phenolics content [67].

It is also know that tamarind, canola, sesame, linseed and sunflower seeds are other possible sources of phenolic compounds [73] and have high antioxidant capacity. The antioxidant ca‐ pacity of the striped sunflower seed cotyledon extracts, obtained by sequential extraction with different polarities of solvents, was determined by three in vitro methods: FRAP, DPPH and ORAC [74]. In the three methods applied, the aqueous extract showed higher an‐ tioxidant capacity than the ethanolic. When compared with the synthetic antioxidant Buty‐ lated Hydroxyl Toluene (BHT), the antioxidant power of the aqueous extract varied from 45% to 66%, according to the used method. It was concluded in this study that the high anti‐ oxidant power found for the aqueous extract of the studied sunflower seed suggests that the intake of this seed may prevent *in vivo* oxidative reactions responsible for the development of several diseases.

### **4. Conclusion**

Phenolic compounds are widely distributed in plant foods (cereals, vegetables, fruits and others), stressing among them the flavonoids, tannins, chalcones, coumarins and phenolic acids. Although some studies have shown few statistically significant correlations between the levels of total phenolics and antioxidant capacity in foods, in others the content of total phenolic compounds was highly correlated with the antioxidant power of samples. Among the plant foods with a high content of phenolic compounds and antioxidant capacity, we can stand out the dark green leafy and brightly-colored vegetables, in addition to cocoa, soya‐ bean, spices and fruits such as cherries and citrus.

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### **Author details**

Maria de Lourdes Reis Giada\*

Address all correspondence to: mlgiada@nutricao.ufrj.br

Department of Basic and Experimental Nutrition, Institute of Nutrition, Health Sciences Center, Federal University of Rio de Janeiro, Brazil

### **References**


**4. Conclusion**

**Author details**

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329, 19-28.

Maria de Lourdes Reis Giada\*

bean, spices and fruits such as cherries and citrus.

106 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Address all correspondence to: mlgiada@nutricao.ufrj.br

nols. *Journal of nutrition*, 130, 2073S-2085S.

Center, Federal University of Rio de Janeiro, Brazil

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Department of Basic and Experimental Nutrition, Institute of Nutrition, Health Sciences

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**Chapter 5**

*Geranium* **Species as Antioxidants**

Additional information is available at the end of the chapter

Complementary alternative medicine (CAM) has been widely used for a long time for the treatment of multiple diseases, despite the great advances in allopathic medicine. It is esti‐

CAM encompasses empirical knowledge and medical practice in which use is made of herb‐ al medicinal plants, animals, minerals, manual therapy and exercise, alone or in conjunction for the treatment of diseases. In the early 1980's there emerged a strong interest in their study that has significantly influenced the pharmaceutical industry in developing technolo‐ gies to identify new chemical entities and structures that are used for the synthesis of drugs. It has been shown that natural products play an important role in the discovery of com‐

Also, is important to recognize that use plants and their products have provided proven benefits to humanity, which falls into four areas: (i) food, (ii) essences and flavoring agents, (iii) perfumes and cosmetics, and (iv) biological and pharmaceutical agents [1]. Within the pharmaceutical area, the current outlook for natural products in drug discovery takes a cen‐ tral role, since at the beginning of this new millennium, only about 10% of 350,000 known species have been investigated from a phytochemical or pharmacology point of view [1].

> © 2013 Ávila et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

mated that about 80% of the world population use some form of CAM.

pounds for drug development to treat multiple diseases.

Mirandeli Bautista Ávila,

Nancy Vargas Mendoza,

Juan Antonio Gayosso de Lúcio,

Claudia Velázquez González, Minarda De la O Arciniega and

Georgina Almaguer Vargas

http://dx.doi.org/10.5772/52002

**1. Introduction**


**Chapter 5**

### *Geranium* **Species as Antioxidants**

Mirandeli Bautista Ávila, Juan Antonio Gayosso de Lúcio, Nancy Vargas Mendoza, Claudia Velázquez González, Minarda De la O Arciniega and Georgina Almaguer Vargas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52002

### **1. Introduction**

[71] Lee, K. W., Kim, Y. J., Lee, H. J., & Lee, C. Y. (2003). Cocoa has more phenolic phyto‐ chemicals and a higher antioxidant capacity than teas and red wine. *Journal of Agri‐*

[72] Bravo, J., Monente, C., Juániz, I., Peña, M. P., & Cid, C. (2011). Influence of extraction process on antioxidant capacity of spent coffee. *Food Research International*, doi:

[73] Duthie, G. G., Duthie, S. J., & Kyle, J. A. M. (2000). Plant polyphenols in cancer an heart disease: implications as nutritional antioxidants. *Nutrition Research Reviews*,

[74] Giada, M. L. R., & Mancini-Filho, J. (2009). Antioxidant capacity of the striped sun‐ flower (Helianthus annuus L.) seed extracts evaluated by three in vitro methods. *In‐*

*ternational Journal of Food Sciences and Nutrition*, 60(5), 395-401.

*cultural and Food Chemistry*, 51(25), 7292-7295.

112 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

10.1016/j.foodres.2011.04.026.

13(1), 79-106.

Complementary alternative medicine (CAM) has been widely used for a long time for the treatment of multiple diseases, despite the great advances in allopathic medicine. It is esti‐ mated that about 80% of the world population use some form of CAM.

CAM encompasses empirical knowledge and medical practice in which use is made of herb‐ al medicinal plants, animals, minerals, manual therapy and exercise, alone or in conjunction for the treatment of diseases. In the early 1980's there emerged a strong interest in their study that has significantly influenced the pharmaceutical industry in developing technolo‐ gies to identify new chemical entities and structures that are used for the synthesis of drugs. It has been shown that natural products play an important role in the discovery of com‐ pounds for drug development to treat multiple diseases.

Also, is important to recognize that use plants and their products have provided proven benefits to humanity, which falls into four areas: (i) food, (ii) essences and flavoring agents, (iii) perfumes and cosmetics, and (iv) biological and pharmaceutical agents [1]. Within the pharmaceutical area, the current outlook for natural products in drug discovery takes a cen‐ tral role, since at the beginning of this new millennium, only about 10% of 350,000 known species have been investigated from a phytochemical or pharmacology point of view [1].

© 2013 Ávila et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A great examples of molecules that have hit the market as drugs by isolation from natu‐ ral products metabolites are: taxol (1), an antitumor agent isolated from Taxus species [2] and camptothecin (2), isolated from the Chinese plant Camptotheca acuminate Decne (Nyssaceae), used to treat ovarian, breast and colorectal cancer, another example is ephe‐ drine (3), which is isolated from the plant *Ephedra sinica*3 and is used as a flu remedy. In drug discovery, researchers around the world use plants as an essential route in the search for new drugs leaders. One of the main objectives of the research laboratories is the preliminary meeting with isolated bioactive natural products, and its uses as anti‐ cancer, antiviral, antimalarial, antifungal and anti-inflammatory [3]. The search for active compounds in plants is an essential way for the development of new drugs, a process in which there is now more advanced and specific methodologies for the analysis of biolog‐ ical activities in particular.

**Figure 1.** *Geranium* genus.

*Erodioidea*

*Geranium*

*Robertium*

**Table 1.** *Geranium* genus clasification

distribution of this classification.

**Subgenus Section Number of Species**

*Erodioidea* 3 *Aculeolata* 1 *Subacaulia* 15 *Brasiliensia* 3

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 115

*Geranium* 339 *Dissecta* 4 *Tuberosa* 19 *Neurophyllodes* 6 *Paramensia* 2 *Azorelloida* 1 *Polyantha* 7

*Trilopha* 5 *Divaricata* 2 *Batrachioidea* 4 *Ungiculata* 5 *Lucida* 1 *Ruberta* 4 *Anemonifolia* 2

Within the classification of *Geranium* genus are accepted 423 species, distributed in three subgenuses: Erodioidea, Geranium and Robertium. The following table (Table 1) shows the

Documentary research from 1981 to 2006 showed that natural products have been a source of 5.7% of drugs produced in those years. The derivatives of natural products are most of the times, chemical molecules synthetized from natural products and contributed to the 27.6% of the total of the new molecule.

### **2. Characterization of** *Geranium* **genus**

*Geranium* genus is taxonomically classified within the family *Geraniaceae Juss*, which in‐ cludes five to eleven genuses, and in total near to 750 species. The genus best known are *Geranium* genus, as wild plants (Figure 1) and *Pelargonium* genus, as garden plants. The names of these genuses usually cause confusion because "geranium", is the common name for certain species of *Pelargonium*.

The names come from Greek and refer to the form that its fruits acquire, likes beaks. Thus, the word "Geranium" comes from "geranos" meaning crane, and "Pelargonium" derived from "Pelargos" meaning stork [4].

#### **Figure 1.** *Geranium* genus.

A great examples of molecules that have hit the market as drugs by isolation from natu‐ ral products metabolites are: taxol (1), an antitumor agent isolated from Taxus species [2] and camptothecin (2), isolated from the Chinese plant Camptotheca acuminate Decne (Nyssaceae), used to treat ovarian, breast and colorectal cancer, another example is ephe‐ drine (3), which is isolated from the plant *Ephedra sinica*3 and is used as a flu remedy. In drug discovery, researchers around the world use plants as an essential route in the search for new drugs leaders. One of the main objectives of the research laboratories is the preliminary meeting with isolated bioactive natural products, and its uses as anti‐ cancer, antiviral, antimalarial, antifungal and anti-inflammatory [3]. The search for active compounds in plants is an essential way for the development of new drugs, a process in which there is now more advanced and specific methodologies for the analysis of biolog‐

114 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Documentary research from 1981 to 2006 showed that natural products have been a source of 5.7% of drugs produced in those years. The derivatives of natural products are most of the times, chemical molecules synthetized from natural products and contributed to the

*Geranium* genus is taxonomically classified within the family *Geraniaceae Juss*, which in‐ cludes five to eleven genuses, and in total near to 750 species. The genus best known are *Geranium* genus, as wild plants (Figure 1) and *Pelargonium* genus, as garden plants. The names of these genuses usually cause confusion because "geranium", is the common name

The names come from Greek and refer to the form that its fruits acquire, likes beaks. Thus, the word "Geranium" comes from "geranos" meaning crane, and "Pelargonium" derived

ical activities in particular.

27.6% of the total of the new molecule.

for certain species of *Pelargonium*.

from "Pelargos" meaning stork [4].

**2. Characterization of** *Geranium* **genus**


#### **Table 1.** *Geranium* genus clasification

Within the classification of *Geranium* genus are accepted 423 species, distributed in three subgenuses: Erodioidea, Geranium and Robertium. The following table (Table 1) shows the distribution of this classification.

Currently, in Hidalgo state, in Central Mexico, are classified 8 different species [5] and any‐ one has chemical or pharmacological studies.

### **2.1. Biological activities and compounds isolated from** *Geranium* **species**

Some species of *Geranium* that have been studied has shown biological activity like: hy‐ potensive, mild astringents, diuretics, hepatoprotection, antioxidants, anti-inflammatory and antiviral. *Geranium* species also are used as a remedy for tonsillitis, cough, whooping cough, urticarial, dysentery, kidney pain and gastrointestinal disorders [6-8]. It is proba‐ bly that the species of this genus that growing in the State of Hidalgo possess a similar biological activities and metabolites. All phytochemicals studies described for these spe‐ cies, indicates the presence of polyphenolic compounds called tannins, which have been considered as water-soluble compounds of molecular weight between 500 and 30,000 g/mol with special properties such as the ability to precipitate alkaloids, gelatin and oth‐ er proteins [9]. Nowadays tannins are well known because of its antioxidant properties. Tannin-protein complexes in the gastrointestinal tract provide persistent antioxidant ac‐ tivity.

One of the major components in *Geranium* species isgeraniin (4) [10] described by its discov‐ erer as a crystallizable tannin. This substance first isolated from *Geranium thunbergi* Sieg. Et Zucc. by T. Okuda in 1976, has been evaluated showing an antihypertensive activity, gera‐ niin inhibits the angiotensin converting enzyme [11,12] and reverse transcriptase of tumoral viruses RNA [13], inhibit HSV-1 and HSV-2 multiplication at different magnitudes of poten‐ cy and also is an excellent antioxidant [14]. The corilagin (5) [15] is a derivative of geraniin, which has presented antimicrobial activity among other activities [16].

Recently the extracts of *G. sylvaticum* were studied [20] for antioxidant potential and all test‐ ed extracts had strong antioxidant activity and will be subject for further investigations. From flowers of *Geranium sylvaticum* was isolated 3-O-(6-O-acetyl-∙-D-glucopyranoside)-5- O-∙-D-glucopyranoside of malvidin (7) [21].*Geranium sanguineum L.* showed significant in‐ hibitory activity of influenza virus and herpes simplex (8). The methanolic extract of *Geranium pratense* inhibited the action of the amylase enzyme in mouse plasma, isolated for

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 117

*Geranium niveum*, widely used by the Tarahumara Indians of Mexico. Is a specie rich in proanthocyanidins and other phenolics [23]. Previous in vitro assays have demonstrated that proanthocyanidins exhibited antiinflammatory, antiviral, antibacterial, enzyme-inhib‐ iting, antioxidant, and radical-scavenging properties, the roots 25 of this species were iso‐ lated new proanthocyanidins named as geraniins A (9) and B (9a), latterlyin 2001 were found geraniins C (10) and D (10a) [24]. A recently study showed that geraniin A has an‐

*Geranium pusillum*, commonly known as Small-flowered Cranesbill or (in North America) small Geranium, contains1-O-galloyl-3,6-hexahidroxibifenil-D-galactopyranoside (11) (pusi‐ lagin) a polyphenolic compound extracted from aerial parts [26]. The aqueous ethanolic ex‐ tract of *Geranium wallichianum* showed antibacterial activity against *Staphylococcus aureus* [27] and the study of the chemical constituents of the whole plant has resulted in the isola‐ tion and characterization of six compounds. These six compounds were identified as ursolic

first time the 3-O-(2-O-galloyl) -∙-D-glucopyranoside myricetin(9) [22].

tioxidant activity [25].

### **2.2. Different species of geraniums and its relevant compounds**

The specie *Geranium* macrorizum presented a significant hypotensive activity in anesthe‐ tized cats [17], plus antioxidant activity. Of this specie germacrone (6) was isolated which is considered a precursor of pheromones.

*Geranium robertianum L.* well-known specie and one of the most variable in Britain has been used in conditions where increased diuresis is required, such as cystitis, urethritis, pyelonephritis, gout, hypertension and edema. Nowadays the phytochemistry of this geranium is relatively well known and its most studied active compounds are tannins, volatile oils, flavonoids and polyphenols (hyperoside, ellagic acid, isoquercitrin, querci‐ trine, kaempferols, caftaric acid, rutoside).Also infusions and decoctions prepared from leaves of this geranium: Robert herb or red Robin, are described as anti-hyperglycae‐ miant and commonly used in Portuguese herbal medicine [18]. In other hand G. robertia‐ num extract treatment increased the efficiency of coupling between oxidative and phosphorylative systems, since RCR was considerably higher in GK rats consuming this plant extract [19].

Currently, in Hidalgo state, in Central Mexico, are classified 8 different species [5] and any‐

Some species of *Geranium* that have been studied has shown biological activity like: hy‐ potensive, mild astringents, diuretics, hepatoprotection, antioxidants, anti-inflammatory and antiviral. *Geranium* species also are used as a remedy for tonsillitis, cough, whooping cough, urticarial, dysentery, kidney pain and gastrointestinal disorders [6-8]. It is proba‐ bly that the species of this genus that growing in the State of Hidalgo possess a similar biological activities and metabolites. All phytochemicals studies described for these spe‐ cies, indicates the presence of polyphenolic compounds called tannins, which have been considered as water-soluble compounds of molecular weight between 500 and 30,000 g/mol with special properties such as the ability to precipitate alkaloids, gelatin and oth‐ er proteins [9]. Nowadays tannins are well known because of its antioxidant properties. Tannin-protein complexes in the gastrointestinal tract provide persistent antioxidant ac‐

One of the major components in *Geranium* species isgeraniin (4) [10] described by its discov‐ erer as a crystallizable tannin. This substance first isolated from *Geranium thunbergi* Sieg. Et Zucc. by T. Okuda in 1976, has been evaluated showing an antihypertensive activity, gera‐ niin inhibits the angiotensin converting enzyme [11,12] and reverse transcriptase of tumoral viruses RNA [13], inhibit HSV-1 and HSV-2 multiplication at different magnitudes of poten‐ cy and also is an excellent antioxidant [14]. The corilagin (5) [15] is a derivative of geraniin,

The specie *Geranium* macrorizum presented a significant hypotensive activity in anesthe‐ tized cats [17], plus antioxidant activity. Of this specie germacrone (6) was isolated which is

*Geranium robertianum L.* well-known specie and one of the most variable in Britain has been used in conditions where increased diuresis is required, such as cystitis, urethritis, pyelonephritis, gout, hypertension and edema. Nowadays the phytochemistry of this geranium is relatively well known and its most studied active compounds are tannins, volatile oils, flavonoids and polyphenols (hyperoside, ellagic acid, isoquercitrin, querci‐ trine, kaempferols, caftaric acid, rutoside).Also infusions and decoctions prepared from leaves of this geranium: Robert herb or red Robin, are described as anti-hyperglycae‐ miant and commonly used in Portuguese herbal medicine [18]. In other hand G. robertia‐ num extract treatment increased the efficiency of coupling between oxidative and phosphorylative systems, since RCR was considerably higher in GK rats consuming this

which has presented antimicrobial activity among other activities [16].

**2.2. Different species of geraniums and its relevant compounds**

considered a precursor of pheromones.

plant extract [19].

**2.1. Biological activities and compounds isolated from** *Geranium* **species**

one has chemical or pharmacological studies.

116 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

tivity.

Recently the extracts of *G. sylvaticum* were studied [20] for antioxidant potential and all test‐ ed extracts had strong antioxidant activity and will be subject for further investigations. From flowers of *Geranium sylvaticum* was isolated 3-O-(6-O-acetyl-∙-D-glucopyranoside)-5- O-∙-D-glucopyranoside of malvidin (7) [21].*Geranium sanguineum L.* showed significant in‐ hibitory activity of influenza virus and herpes simplex (8). The methanolic extract of *Geranium pratense* inhibited the action of the amylase enzyme in mouse plasma, isolated for first time the 3-O-(2-O-galloyl) -∙-D-glucopyranoside myricetin(9) [22].

*Geranium niveum*, widely used by the Tarahumara Indians of Mexico. Is a specie rich in proanthocyanidins and other phenolics [23]. Previous in vitro assays have demonstrated that proanthocyanidins exhibited antiinflammatory, antiviral, antibacterial, enzyme-inhib‐ iting, antioxidant, and radical-scavenging properties, the roots 25 of this species were iso‐ lated new proanthocyanidins named as geraniins A (9) and B (9a), latterlyin 2001 were found geraniins C (10) and D (10a) [24]. A recently study showed that geraniin A has an‐ tioxidant activity [25].

*Geranium pusillum*, commonly known as Small-flowered Cranesbill or (in North America) small Geranium, contains1-O-galloyl-3,6-hexahidroxibifenil-D-galactopyranoside (11) (pusi‐ lagin) a polyphenolic compound extracted from aerial parts [26]. The aqueous ethanolic ex‐ tract of *Geranium wallichianum* showed antibacterial activity against *Staphylococcus aureus* [27] and the study of the chemical constituents of the whole plant has resulted in the isola‐ tion and characterization of six compounds. These six compounds were identified as ursolic acid, β-sitosterol, stigmasterol, β-sitosterol galactoside, herniarin, and 2,4,6-trihydroxyethyl‐ benzoate which were isolated for the first time from *Geranium wallichianum* [28].

From *Geranium pyrenaicum*, which showed antileishmanial activity [32],a new glycosylate flavonoid: 3-O-(2 ", 3"-di-O-galloyl)- ∙-D-glucopyranoside of kaempferol (13) was isolated, and anuncommon quercetin derivative: 3-O-(2 ", 3"-di-O-galloyl) - ∙-D-glucopyranoside of quercetin (13a) too. In *Geranium mexicanum* an antiprotozoal activity was assayed from its roots, where the most active compound founded was the flavan-3-ol-(-)-epicatechin (14), showing moderate activity (+)-catechin (14a), tyramine (15) and 3-O-β-D-glucopyranoside of

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 119

*Geranium bellum* Rose is a perennial plant with long roots, found in the grassy meadows border‐ ing pine/oak forests in the mountains of Hidalgo State, Mexico, where it has the popular name "pata de león" and has been used as a traditional remedy for treatment of fevers, pain, and gas‐ trointestinal disorders. Radical scavenging assay-guided fractionation of the antioxidant EtOAc and MeOH extracts from the aerial parts of *Geranium bellum* resulted in the isolation of b-

β-sitosterol [33].

*Geranium caespitosum* produces neohesperidoside (12) able to potentiate 10 to 100 times the action of drugs such as ciprofloxacin, norfloxacin, berberine and rhein, against bacterias such as *S. aureus*, *NorA S. aureus, B*. and *B. megaterium subtilis* [29]. Besides, *Geranium caroli‐ nianum* L., isa commonly used traditional Chinese medicine (TCM) with the efficacy of elim‐ inating wind-damp and treating diarrhea. It is clinically used to treat the arthralgia due to wind-dampness, anaesthetization and muscular constriction. It has been reports that *Gerani‐ um carolinianum L.* as well of most of the congeneric plants contain significant amounts of tannins, flavonoids, organic acids, and volatile oils [30].Also, has shown that roots contain a substance that is extracted with water and can be a biological mechanism to control bacteria (*Ralstonia solanacearum*) which attacks potatoes [31].

From *Geranium pyrenaicum*, which showed antileishmanial activity [32],a new glycosylate flavonoid: 3-O-(2 ", 3"-di-O-galloyl)- ∙-D-glucopyranoside of kaempferol (13) was isolated, and anuncommon quercetin derivative: 3-O-(2 ", 3"-di-O-galloyl) - ∙-D-glucopyranoside of quercetin (13a) too. In *Geranium mexicanum* an antiprotozoal activity was assayed from its roots, where the most active compound founded was the flavan-3-ol-(-)-epicatechin (14), showing moderate activity (+)-catechin (14a), tyramine (15) and 3-O-β-D-glucopyranoside of β-sitosterol [33].

acid, β-sitosterol, stigmasterol, β-sitosterol galactoside, herniarin, and 2,4,6-trihydroxyethyl‐

*Geranium caespitosum* produces neohesperidoside (12) able to potentiate 10 to 100 times the action of drugs such as ciprofloxacin, norfloxacin, berberine and rhein, against bacterias such as *S. aureus*, *NorA S. aureus, B*. and *B. megaterium subtilis* [29]. Besides, *Geranium caroli‐ nianum* L., isa commonly used traditional Chinese medicine (TCM) with the efficacy of elim‐ inating wind-damp and treating diarrhea. It is clinically used to treat the arthralgia due to wind-dampness, anaesthetization and muscular constriction. It has been reports that *Gerani‐ um carolinianum L.* as well of most of the congeneric plants contain significant amounts of tannins, flavonoids, organic acids, and volatile oils [30].Also, has shown that roots contain a substance that is extracted with water and can be a biological mechanism to control bacteria

(*Ralstonia solanacearum*) which attacks potatoes [31].

benzoate which were isolated for the first time from *Geranium wallichianum* [28].

118 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

*Geranium bellum* Rose is a perennial plant with long roots, found in the grassy meadows border‐ ing pine/oak forests in the mountains of Hidalgo State, Mexico, where it has the popular name "pata de león" and has been used as a traditional remedy for treatment of fevers, pain, and gas‐ trointestinal disorders. Radical scavenging assay-guided fractionation of the antioxidant EtOAc and MeOH extracts from the aerial parts of *Geranium bellum* resulted in the isolation of b-

sitosterol 3-O-b-D-glucopiranoside, quercetin 3-O-a-L-(2"-O-acetyl)arabinofuranoside (16), quercetin 3-O-a-L-arabinofuranoside, quercetin, methyl gallate, gallic acid, methyl brevifolin carboxylate (17), and dehydrochebulic acid trimethyl ester (18). Compounds 2, 7 and 8 are iso‐ lated for the first time from *Geranium* genus [34]. Antioxidant activity of these extracts (both ini‐ tial fractions and pure compounds), was tested by measuring their capacity to scavenge 2,2' azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals, an assay widely used for screening of antioxidant activity of natural products [15].

**3.1. Plant material**

**3.2. Extraction and purification**

rial.

(Figure 3).

**Figure 3.** Extract fractions scheme.

Specimen of *Geranium schiedeanum* was collected at Epazoyucan Municipality, in Hidalgo State, México, during June 2009. A voucher specimen (J. A. Gayosoo-de-Lucio) is preserved at the Herbarium of Biological Research Centre, Autonomous University of Hidalgo, Pachu‐ ca, Hidalgo, Mexico where Professor Manuel González Ledesma identified the plant mate‐

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 121

Air-dried aerial parts (1 kg) were extracted acetone-H2O 7:3 (20 L) by maceration for 7 days. Vacuum evaporation of dissolvent give a 5 L residue Filtration give a fatty solid residue

Lots of 3 g of acetone-water extract were purified on a Sephadex LH-20 (25 g) column us‐ ing H2O, H2O-MeOH (9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 ) and MeOH, as eluents. Frac‐ tions of 300 mL of each polarity were collected and marked "A–K". They were evaporated and analyzed by TLC and NMR. Fractions "B" gave 75 mg, and were purified over silica gel (10 g), using CHCl3, CHCl3-AcOEt(9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 ) and AcOEt (10 mL of each polarity), as eluents and collecting fractions of 7 mL, fractions 13-16 give I 25 mg. Fractions "C" and "D" gave 56 mg, and were purified over silica gel (10 g), using CHCl3-MeOH (50:7.0, 48:7, 45:7, 40:7, 35:7 and 30:7, 40 mL of each), as eluents and collect‐ ing fractions of 7 mL, fractions 33-66 give II 2 mg, (these procedure was repeated ten times to obtain 18 mg of compound), Fractions "F-I" gave 1.8 g, a portion of 500 mg were puri‐ fied over silica gel C-18 (5 g) using H2O, H2O-MeOH (9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 ) and MeOH (20 mL of each polarity), fractions of 10 mL were collected fractions 2-4 gave 325 mg of III, fraction "K" gave 90 mg were added 5 mL of (40°C) pyridine and were placed a room temperature for 72 h, filtrated of mixture give a yellow needles 60 mg of IV

(12g) and complete evaporation of water give the acetone-water extract (115 g).

Constituents from the aerial parts of *Geranium potentillaefoium* founded in certain studies were geraniin, corilagin, gallic acid, methyl gallate, methyl brevifolincarboxylate, quercetin, quercetin 3-O-β-Dglucopyranoside, quercetin 3-O-β-D- [6"-O-galloyl)glucopyranoside, kaempferol, β-sitosterol 3-O-β-D-glucopyranoside and β-sitosterol [35].

### **3. Study of** *Geranium schiedeanum*

*Geranium schiedeanum* (Gs) (Figure 2), species that grows in Central Mexico, has been used as an antipyretic, anti-inflamatory and antiseptic. The use of other geranium species also has been reported a hypoglycemic, antihypertensive and cholesterol-lowering effect. However, scientific evidence does not exist in any literature to corroborate these targets or any other. In the present study the effect of Gs were studied in reference to postnecrotic liver damage induced by thioacetamide (TA).

**Figure 2.** *Geranium schiedeanum* [5]

### **3.1. Plant material**

sitosterol 3-O-b-D-glucopiranoside, quercetin 3-O-a-L-(2"-O-acetyl)arabinofuranoside (16), quercetin 3-O-a-L-arabinofuranoside, quercetin, methyl gallate, gallic acid, methyl brevifolin carboxylate (17), and dehydrochebulic acid trimethyl ester (18). Compounds 2, 7 and 8 are iso‐ lated for the first time from *Geranium* genus [34]. Antioxidant activity of these extracts (both ini‐ tial fractions and pure compounds), was tested by measuring their capacity to scavenge 2,2' azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals, an assay widely used for

Constituents from the aerial parts of *Geranium potentillaefoium* founded in certain studies were geraniin, corilagin, gallic acid, methyl gallate, methyl brevifolincarboxylate, quercetin, quercetin 3-O-β-Dglucopyranoside, quercetin 3-O-β-D- [6"-O-galloyl)glucopyranoside,

*Geranium schiedeanum* (Gs) (Figure 2), species that grows in Central Mexico, has been used as an antipyretic, anti-inflamatory and antiseptic. The use of other geranium species also has been reported a hypoglycemic, antihypertensive and cholesterol-lowering effect. However, scientific evidence does not exist in any literature to corroborate these targets or any other. In the present study the effect of Gs were studied in reference to postnecrotic liver damage

screening of antioxidant activity of natural products [15].

120 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**3. Study of** *Geranium schiedeanum*

induced by thioacetamide (TA).

**Figure 2.** *Geranium schiedeanum* [5]

kaempferol, β-sitosterol 3-O-β-D-glucopyranoside and β-sitosterol [35].

Specimen of *Geranium schiedeanum* was collected at Epazoyucan Municipality, in Hidalgo State, México, during June 2009. A voucher specimen (J. A. Gayosoo-de-Lucio) is preserved at the Herbarium of Biological Research Centre, Autonomous University of Hidalgo, Pachu‐ ca, Hidalgo, Mexico where Professor Manuel González Ledesma identified the plant mate‐ rial.

### **3.2. Extraction and purification**

Air-dried aerial parts (1 kg) were extracted acetone-H2O 7:3 (20 L) by maceration for 7 days. Vacuum evaporation of dissolvent give a 5 L residue Filtration give a fatty solid residue (12g) and complete evaporation of water give the acetone-water extract (115 g).

Lots of 3 g of acetone-water extract were purified on a Sephadex LH-20 (25 g) column us‐ ing H2O, H2O-MeOH (9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 ) and MeOH, as eluents. Frac‐ tions of 300 mL of each polarity were collected and marked "A–K". They were evaporated and analyzed by TLC and NMR. Fractions "B" gave 75 mg, and were purified over silica gel (10 g), using CHCl3, CHCl3-AcOEt(9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 ) and AcOEt (10 mL of each polarity), as eluents and collecting fractions of 7 mL, fractions 13-16 give I 25 mg. Fractions "C" and "D" gave 56 mg, and were purified over silica gel (10 g), using CHCl3-MeOH (50:7.0, 48:7, 45:7, 40:7, 35:7 and 30:7, 40 mL of each), as eluents and collect‐ ing fractions of 7 mL, fractions 33-66 give II 2 mg, (these procedure was repeated ten times to obtain 18 mg of compound), Fractions "F-I" gave 1.8 g, a portion of 500 mg were puri‐ fied over silica gel C-18 (5 g) using H2O, H2O-MeOH (9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 1:9 ) and MeOH (20 mL of each polarity), fractions of 10 mL were collected fractions 2-4 gave 325 mg of III, fraction "K" gave 90 mg were added 5 mL of (40°C) pyridine and were placed a room temperature for 72 h, filtrated of mixture give a yellow needles 60 mg of IV (Figure 3).

**Figure 3.** Extract fractions scheme.

### **3.3. Animals and treatment**

Male adult Wistar rats 2 months old (200–220g) were obtained from UAEH Bioterio, and acclimated to our animal room for two weeks before use. Throughout these two weeks rats were supplied with food and water *ad libitum,* exposed to a 12 h light-dark cycle and giv‐ en intraperitoneally a single necrogenic dose of thioacetamide (6.6 nmol/Kg body weights) freshly dissolved in 0.9% NaCl. The dose of thioacetamide was chosen as the highest dose with survival above 90% [36,37]. Wistar rats were intragastric pre-treated or not with a sin‐ gle dose of *Gs* extract (300 mg/kg) during 4 days, the fourth day of pretreatment were in‐ traperitoneally injected with a single dose of TA (6.6 mmol/Kg). Samples of blood and liver were obtained from rats at 0, 24, 48,72 and 96 h following TA intoxication. Untreated ani‐ mals received 0.5 ml of 0.9% NaCl. Experiments were performed on two different groups: rats treated with a single dose of thioacetamide (TA) and rats pre-treated with Gs and treated with a single dose of thioacetamide (Gs + TA). Each experiment was performed in duplicate from four different animals and followed the international criteria for the use and care of experimental animals outlined in *The Guiding Principles in the use of Animals in Toxicology* adopted by the Society of Toxicology in 1989.

carried out on Merck silica gel 60 (Aldrich, 230-400 mesh ASTM) and sephadex LH-20

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 123

The results were calculated as the means ± SD of four experimental observations in dupli‐ cate (four animals). Differences between groups were analyzed by an ANOVA following Snedecor F (α = 0.05). Students' test was performed for statistical evaluation as follows: (a) all values against their control; b) differences between two groups Gs + TA versus TA.

One kg of the aerial part of *G. schiedeanum* was extracted by maceration for 7 days with 20 L acetone-water (7:3), concentrated under reduced pressure to a volume of 3 L, which was ex‐ tracted with CHCl3 yielding 12.75 g of CHCl3 phase and 125 g of aqueous phase. The phyto‐ chemical study of *Geranium schiedeanum* led to the isolation of hydrolysable tannins (I) gallic acid, (III) acetonylgeraniin and (IV) ellagic acid and to a lesser proportion of kaempferol gly‐ cosideflavonoid (II) (Figure 4 and 5). Is relevant to notice that is the first time discloses the compound II in the Geranium genus and further that the yield of compound III in the crude

**Figure 4.** Compound (II) 3-O-α-L-arabinofuranoside-7-O-β-L-rhamnopyranoside de Kaempferol

Sigma Aldrich.

**3.8. Results**

extract was 40%.

**3.7. Statistical analysis**

*3.8.1. Active compounds of Geranium schiedeanum*

### **3.4. Processing of samples**

In order to clarify the sequential changes during the different stages of liver injury and the post-necrotic regenerative response, samples were obtained from control and at 24 and 48 h of TA intoxication in both Gs pre-treated or non pre-treated animals. Rats were sacrificed by cervical dislocation and samples of liver were obtained and processed as previously descri‐ bed. Blood was collected from hearts and kept at 4 °C for 24 h, centrifuged at 3000 rpm for 15 min, and serum was obtained as the supernatant.

### **3.5. Determination of AST**

Enzymatic determination were carried out in serum in optimal conditions of temperature and substrate and cofactor concentrations. Aspartate aminotransferase (AST) activity were determined in serum. AST (EC 2.6.2.1) and was assayed following the method of Rej and Horder [38].

The activity of this enzyme was determined spectrophotometrically, by measuring the de‐ crease in absorbance at 340 nm at 37 ° C, produced by the oxidation of NADH to NAD+ in the coupled reaction of reduction of oxaloacetate to malate, catalyzed by malate dehydro‐ genase, according to the following process:

#### **3.6. General**

IR spectra measured in MeOH on a Perkin Elmer 2000 FT-IR spectrophotometer. Optical rotations were determined in MeOH on a Perkin Elmer 341 polarimeter. NMR measure‐ ments performed at 400 MHz for 1H and 100 MHz for 13C on a VARIAN 400 spectrom‐ eter from CDCl3, CD3OH, DMSO-d6 solutions. Column chromatography (CC) was carried out on Merck silica gel 60 (Aldrich, 230-400 mesh ASTM) and sephadex LH-20 Sigma Aldrich.

### **3.7. Statistical analysis**

The results were calculated as the means ± SD of four experimental observations in dupli‐ cate (four animals). Differences between groups were analyzed by an ANOVA following Snedecor F (α = 0.05). Students' test was performed for statistical evaluation as follows: (a) all values against their control; b) differences between two groups Gs + TA versus TA.

### **3.8. Results**

**3.3. Animals and treatment**

**3.4. Processing of samples**

**3.5. Determination of AST**

Horder [38].

**3.6. General**

*Toxicology* adopted by the Society of Toxicology in 1989.

122 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

15 min, and serum was obtained as the supernatant.

genase, according to the following process:

Male adult Wistar rats 2 months old (200–220g) were obtained from UAEH Bioterio, and acclimated to our animal room for two weeks before use. Throughout these two weeks rats were supplied with food and water *ad libitum,* exposed to a 12 h light-dark cycle and giv‐ en intraperitoneally a single necrogenic dose of thioacetamide (6.6 nmol/Kg body weights) freshly dissolved in 0.9% NaCl. The dose of thioacetamide was chosen as the highest dose with survival above 90% [36,37]. Wistar rats were intragastric pre-treated or not with a sin‐ gle dose of *Gs* extract (300 mg/kg) during 4 days, the fourth day of pretreatment were in‐ traperitoneally injected with a single dose of TA (6.6 mmol/Kg). Samples of blood and liver were obtained from rats at 0, 24, 48,72 and 96 h following TA intoxication. Untreated ani‐ mals received 0.5 ml of 0.9% NaCl. Experiments were performed on two different groups: rats treated with a single dose of thioacetamide (TA) and rats pre-treated with Gs and treated with a single dose of thioacetamide (Gs + TA). Each experiment was performed in duplicate from four different animals and followed the international criteria for the use and care of experimental animals outlined in *The Guiding Principles in the use of Animals in*

In order to clarify the sequential changes during the different stages of liver injury and the post-necrotic regenerative response, samples were obtained from control and at 24 and 48 h of TA intoxication in both Gs pre-treated or non pre-treated animals. Rats were sacrificed by cervical dislocation and samples of liver were obtained and processed as previously descri‐ bed. Blood was collected from hearts and kept at 4 °C for 24 h, centrifuged at 3000 rpm for

Enzymatic determination were carried out in serum in optimal conditions of temperature and substrate and cofactor concentrations. Aspartate aminotransferase (AST) activity were determined in serum. AST (EC 2.6.2.1) and was assayed following the method of Rej and

The activity of this enzyme was determined spectrophotometrically, by measuring the de‐ crease in absorbance at 340 nm at 37 ° C, produced by the oxidation of NADH to NAD+ in the coupled reaction of reduction of oxaloacetate to malate, catalyzed by malate dehydro‐

IR spectra measured in MeOH on a Perkin Elmer 2000 FT-IR spectrophotometer. Optical rotations were determined in MeOH on a Perkin Elmer 341 polarimeter. NMR measure‐ ments performed at 400 MHz for 1H and 100 MHz for 13C on a VARIAN 400 spectrom‐ eter from CDCl3, CD3OH, DMSO-d6 solutions. Column chromatography (CC) was

#### *3.8.1. Active compounds of Geranium schiedeanum*

One kg of the aerial part of *G. schiedeanum* was extracted by maceration for 7 days with 20 L acetone-water (7:3), concentrated under reduced pressure to a volume of 3 L, which was ex‐ tracted with CHCl3 yielding 12.75 g of CHCl3 phase and 125 g of aqueous phase. The phyto‐ chemical study of *Geranium schiedeanum* led to the isolation of hydrolysable tannins (I) gallic acid, (III) acetonylgeraniin and (IV) ellagic acid and to a lesser proportion of kaempferol gly‐ cosideflavonoid (II) (Figure 4 and 5). Is relevant to notice that is the first time discloses the compound II in the Geranium genus and further that the yield of compound III in the crude extract was 40%.

**Figure 4.** Compound (II) 3-O-α-L-arabinofuranoside-7-O-β-L-rhamnopyranoside de Kaempferol

**Figure 6.** Effect of Gs pre-treatment on aspartate aminotransferase activity in serum of rats intoxicated with one sub‐ lethal dose of thioacetamide. Samples were obtained at 0, 24, 48, 72 & 96 h following thioacetamide (TA). The results, expressed as nmol per min per ml of serum, are the mean ± SD of four determinations in duplicate from four rats. Differences against the respective control are expressed as (a) and differences due to Gs extract are expressed as (b)

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 125

There is evidence that free radicals play a critical role in certain pathological conditions such as some cancers, multiple sclerosis, inflammation, arthritis and arterosclerosis [43]. For this reason, some research objectives directed toward the development or discovery of these

A large number of plant species, like *G. schiedeanum* contain chemical compounds that ex‐ hibit the ability to trap free radicals. The ability to trap free radicals has been called antioxi‐ dant activity. The phytochemical study of *Geranium shiedeanum* led to the isolation of hydrolysable tannins, well known as potent antioxidants: gallic acid, acetonylgeraniin and ellagic acid and a lesser proportion of kaempferol glycoside flavonoid (3-O-α-L-arabinofura‐ noside-7-O-β-D-rhamnoside de Kaempferol), notably is the first time discloses these com‐ pounds in the genus. Further the yield of acetonyl geraniin in the crude extract was 40%.

Also, in the present study TA-induced hepatotoxicity was used to investigate the effect of the pretreatment of *G. schiedeanum* on the events involved in liver regeneration. The results obtained in the present study provide evidence that *Gs*, when administered intravenously

The pre-treatment with the crude extract in the model of thioacetamide-induced hepatotox‐

p<0.05.

**4. Conclusion**

compounds catchers of these radicals.

prior to TA, significantly reduce liver damage.

icity in rats, decreased and delayed liver injury by 66% at 24 h.

**Figure 5.** HMBC experiment of compound II

#### *3.8.2. Aspartate aminotransferase*

The acute liver injury induced by a necrogenic dose of thioacetamide (TA), a potent hepato‐ toxic agent, is characterized by a severe perivenous necrosis [39]. The necrosis develops as a consequence of the biotransformation of TA through the microsomal flavin-dependent mon‐ ooxygenase [40]. The reactive metabolites responsible for TA hepatotoxicity are the radicals derived from thioacetamide-S-oxide and the reactive oxygen species derived as sub prod‐ ucts in the process of microsomal TA oxidation, both of which can depleted reduced gluta‐ thione leading to oxidative stress [41, 42].

Liver damage induced by xenobiotics is characterized by the release in serum of hepatic en‐ zymes due to necrosis of hepatocytes. AST is randomly distributed in the hepatic acinus, and is the enzyme activity used as marker of necrosis. Our results showed that *Gs* extract significantly reduced the level of liver injury. The levels of AST (Figure 6) were significantly lower in the rats pretreated with *Gs.*

**Figure 6.** Effect of Gs pre-treatment on aspartate aminotransferase activity in serum of rats intoxicated with one sub‐ lethal dose of thioacetamide. Samples were obtained at 0, 24, 48, 72 & 96 h following thioacetamide (TA). The results, expressed as nmol per min per ml of serum, are the mean ± SD of four determinations in duplicate from four rats. Differences against the respective control are expressed as (a) and differences due to Gs extract are expressed as (b) p<0.05.

### **4. Conclusion**

**Figure 5.** HMBC experiment of compound II

124 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

thione leading to oxidative stress [41, 42].

lower in the rats pretreated with *Gs.*

The acute liver injury induced by a necrogenic dose of thioacetamide (TA), a potent hepato‐ toxic agent, is characterized by a severe perivenous necrosis [39]. The necrosis develops as a consequence of the biotransformation of TA through the microsomal flavin-dependent mon‐ ooxygenase [40]. The reactive metabolites responsible for TA hepatotoxicity are the radicals derived from thioacetamide-S-oxide and the reactive oxygen species derived as sub prod‐ ucts in the process of microsomal TA oxidation, both of which can depleted reduced gluta‐

Liver damage induced by xenobiotics is characterized by the release in serum of hepatic en‐ zymes due to necrosis of hepatocytes. AST is randomly distributed in the hepatic acinus, and is the enzyme activity used as marker of necrosis. Our results showed that *Gs* extract significantly reduced the level of liver injury. The levels of AST (Figure 6) were significantly

*3.8.2. Aspartate aminotransferase*

There is evidence that free radicals play a critical role in certain pathological conditions such as some cancers, multiple sclerosis, inflammation, arthritis and arterosclerosis [43]. For this reason, some research objectives directed toward the development or discovery of these compounds catchers of these radicals.

A large number of plant species, like *G. schiedeanum* contain chemical compounds that ex‐ hibit the ability to trap free radicals. The ability to trap free radicals has been called antioxi‐ dant activity. The phytochemical study of *Geranium shiedeanum* led to the isolation of hydrolysable tannins, well known as potent antioxidants: gallic acid, acetonylgeraniin and ellagic acid and a lesser proportion of kaempferol glycoside flavonoid (3-O-α-L-arabinofura‐ noside-7-O-β-D-rhamnoside de Kaempferol), notably is the first time discloses these com‐ pounds in the genus. Further the yield of acetonyl geraniin in the crude extract was 40%.

Also, in the present study TA-induced hepatotoxicity was used to investigate the effect of the pretreatment of *G. schiedeanum* on the events involved in liver regeneration. The results obtained in the present study provide evidence that *Gs*, when administered intravenously prior to TA, significantly reduce liver damage.

The pre-treatment with the crude extract in the model of thioacetamide-induced hepatotox‐ icity in rats, decreased and delayed liver injury by 66% at 24 h.

The data obtained indicate that the crude *Gs* extract pre-treatment has hepatoprotective and antioxidant effect in damage induced by TA. This result suggests that *Gs* extract may be used as an alternative for reduction of liver damage. However further investigation on the acute toxicity and on the mechanism of the hepatoprotective effect of the plant species needs to be carried out.

[8] Küpeli E, Tatl I, Akdemir ZS, Yeflilada E. Estimation of antinociceptive and anti-in‐ flammatory activity on *Geranium pratense* subsp. *finitimum*. *J. Ethnopharmacol.* 2007,

*Geranium* Species as Antioxidants http://dx.doi.org/10.5772/52002 127

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### **Acknowledgements**

The authors would like to thank Teresa Vargas for her valuable technical Assistance. Sup‐ ported by Grant PROMEP-MEXICO UAEHGO-PTC-454.

### **Author details**

Mirandeli Bautista Ávila, Juan Antonio Gayosso de Lúcio, Nancy Vargas Mendoza, Claudia Velázquez González, Minarda De la O Arciniega and Georgina Almaguer Vargas

Universidad Autónoma del Estado de Hidalgo, Mexico

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The data obtained indicate that the crude *Gs* extract pre-treatment has hepatoprotective and antioxidant effect in damage induced by TA. This result suggests that *Gs* extract may be used as an alternative for reduction of liver damage. However further investigation on the acute toxicity and on the mechanism of the hepatoprotective effect of the plant species needs

The authors would like to thank Teresa Vargas for her valuable technical Assistance. Sup‐

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**Chapter 6**

**Foods or Bioactive Constituents of Foods as**

**Gastrointestinal Digestion: A Review**

Antonio Cilla, Amparo Alegría, Reyes Barberá and

molecules with antioxidant and antiinflammatory actions [2].

et, they may have noticeable long-term physiological effects [6].

Additional information is available at the end of the chapter

María Jesús Lagarda

**1. Introduction**

http://dx.doi.org/10.5772/51504

**Chemopreventives in Cell Lines After Simulated**

Epidemiological studies on the relationship between dietary habits and disease risk have shown that food has a direct impact on health. Indeed, our diet plays a significant role in health and well-being, since unbalanced nutrition or an inadequate diet is known to be a key risk factor for chronic age-related diseases [1]. An example that illustrates this fact is the pro‐ tective effect of the so-called Mediterranean diet. The lower occurrence of cancer and cardio‐ vascular disease in the population located around the Mediterranean sea has been linked to the dietary habits of the region, in which the components of the diet contain a wide array of

Many diseases with a strong dietary influence include oxidative damage as an initial event or in an early stage of disease progression [3]. In fact, Western diets (typically dense in fat and energy and low in fiber) are associated with disease risk [4]. Therefore, dietary modifi‐ cation, with a major focus on chronic age-related disease prevention through antioxidant in‐ tervention, could be a good and cost-effective strategy [5]. The intake of whole foods and/or new brand developed functional foods rich in antioxidants would be suitable for this pur‐ pose. In this sense, dietary antioxidants such as polyphenols, carotenoids and peptides, as well as other bioactive chemopreventive components such as fiber and phytosterols have been regarded to have low potency as bioactive compounds when compared to pharmaceut‐ ical drugs, but since they are ingested regularly and in significant amounts as part of the di‐

> © 2013 Cilla et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion: A Review**

Antonio Cilla, Amparo Alegría, Reyes Barberá and María Jesús Lagarda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51504

### **1. Introduction**

Epidemiological studies on the relationship between dietary habits and disease risk have shown that food has a direct impact on health. Indeed, our diet plays a significant role in health and well-being, since unbalanced nutrition or an inadequate diet is known to be a key risk factor for chronic age-related diseases [1]. An example that illustrates this fact is the pro‐ tective effect of the so-called Mediterranean diet. The lower occurrence of cancer and cardio‐ vascular disease in the population located around the Mediterranean sea has been linked to the dietary habits of the region, in which the components of the diet contain a wide array of molecules with antioxidant and antiinflammatory actions [2].

Many diseases with a strong dietary influence include oxidative damage as an initial event or in an early stage of disease progression [3]. In fact, Western diets (typically dense in fat and energy and low in fiber) are associated with disease risk [4]. Therefore, dietary modifi‐ cation, with a major focus on chronic age-related disease prevention through antioxidant in‐ tervention, could be a good and cost-effective strategy [5]. The intake of whole foods and/or new brand developed functional foods rich in antioxidants would be suitable for this pur‐ pose. In this sense, dietary antioxidants such as polyphenols, carotenoids and peptides, as well as other bioactive chemopreventive components such as fiber and phytosterols have been regarded to have low potency as bioactive compounds when compared to pharmaceut‐ ical drugs, but since they are ingested regularly and in significant amounts as part of the di‐ et, they may have noticeable long-term physiological effects [6].

© 2013 Cilla et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

For decades, the beneficial role of antioxidants was related to the reduction of unwanted and uncontrolled production of reactive oxygen species (ROS), leading to a situation refer‐ red to as oxidative stress [7]. Nowadays, the term "antioxidant" has become ambiguous, since it has different connotations for distinct audiences. For instance, for biochemists and nutritionists, the term is related to the scavenging of metabolically generated ROS, while for food scientists the term implies use in retarding food oxidation or for the categorization of foods or substances according to *in vitro* assays of antioxidant capacity, such as the ORAC and TEAC tests [8]. The antioxidant values provided by these assays sometimes have been misinterpreted by both food producers and consumers due to the fact that health claims ad‐ vertised on the package labeling are directly associated with benefits that include slowing of the aging process and decreasing the risk of chronic disease. Nevertheless, contemporary scientific evidence indicates that total antioxidant capacity measured by currently popular chemical assays may not reflect the actual activity *in vivo,* since none of them take biological processes such as bioavailability, uptake and metabolism into account [9]. Therefore, no *in vitro* assay that determines the antioxidant capacity of a nutritional product describes *in vivo* outcomes, and such testing should not be used to suggest such a connection. In this sense, it is currently recognized that the mechanisms of action of antioxidants *in vivo* might be far more complex than mere radical scavenging - involving interactions with specific proteins central to intracellular signaling cascades [10], and in the specific case of cancer cells there might be a direct antioxidant effect, antiproliferation and anti-survival action, the induction of cell cycle arrest, the induction of apoptosis, antiinflammatory effects and the inhibition of angiogenesis and metastasis [11].

crude compounds instead of whole foods impedes the detection of synergistic and/or antag‐

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133

Taking this background together, and in order to obtain a more precise view of the *in vivo* situation, we propose the use of whole foods or related target bioactive constituents subject‐ ed to a human simulated gastrointestinal digestion including or not including colonic fer‐ mentation, depending on the nature of the studied compounds, in order to gain better insight from a nutritional/functional point of view of the chemopreventive action derived

This review introduces the main features of the different *in vitro* gastrointestinal digestion (solubility and dialysis) and colonic fermentation procedures (batch, continuous and contin‐ uous with immobilized feces) for studying the bioaccessibility and further bioavailability and bioactivity of nutrients and bioactive compounds. It also includes a definition of the terms: bioavailability including bioaccessibility and bioactivity. Likewise, the main advan‐ tages and disadvantages of these *in vitro* methods versus *in vivo* approaches, the improve‐ ment of these models with the inclusion of cell lines, and a short comment on the main effects that digestion and/or fermentation have on bioactive compounds are included. On the other hand, a short description is provided of the studies involving the use of human simulated gastrointestinal digestion and/or colonic fermentation procedures, and of the sub‐

Bioavailability is a key concept for nutritional effectiveness, irrespective of the type of food considered (functional or otherwise). Only certain amounts of all nutrients or bioactive com‐

The term bioavailability has several working conditions. From the nutritional point of view, bioavailability is defined as the proportion of a nutrient or bioactive compound can be used for normal physiological functions [16]. This term in turn includes two additional terms: bioacces‐ sibility and bioactivity. Bioaccessibility has been defined as the fraction of a compound that is released from its food matrix in the gastrointestinal tract and thus becomes available for intes‐ tinal absorption. Bioaccessibility includes the sequence of events that take place during food digestion for transformation into potentially bioaccessible material, absorption/assimilation through epithelial tissue and pre-systemic metabolism. Bioactivity in turn includes events linked to how the bioactive compound is transported and reaches the target tissue, how it in‐ teracts with biomolecules, the metabolism or biotransformation it may undergo, and the gen‐ eration of biomarkers and the physiologic responses it causes [12]. Depending on the *in vitro*

*In vitro* methods have been developed to simulate the physiological conditions and the se‐ quence of events that occur during digestion in the human gastrointestinal tract. In a first step, simulated gastrointestinal digestion (gastric and intestinal stages, and in some cases a salivary stage) is applied to homogenized foods or isolated bioactive compounds in a closed

onistic actions among bioactive chemopreventive compounds [14, 15].

from foods and bioactive compounds in cell models of disease.

sequent bioactivity-guided assays with cell line models.

**2. Simulated gastrointestinal digestion assays**

pounds are available for use in physiological functions or for storage.

method used, evaluation is made of bioaccessibility and/or bioactivity.

In order to determine and verify the action of these bioactive compounds, it is clear that data from human intervention studies offer the reference standard and the highest scientific evi‐ dence considering the bioavailability and bioactivity of a food component, while *in vitro* methods are used as surrogates for prediction [12]. From a physiological perspective, food after consumption undergoes a gastrointestinal digestion process that may affect the native antioxidant potential of the complex mixture of bioactive compounds present in the food matrix before reaching the proximal intestine. *In vitro* methods which apply human simulat‐ ed digestion models (including or not including colonic fermentation) are considered valua‐ ble and useful tools for the estimation of pre-absorptive events (i.e., stability, bioaccessibility) of different food components from distinct food sources, and also for deter‐ mining the effect which processing may have upon food components bioavailability [13]. In addition, *in vitro* assays combining a simulated gastrointestinal digestion process and cell cultures as pre-clinical models can be useful for unraveling mechanisms of action and for projecting further *in vivo* assays [9]. Nevertheless, in most cases these *in vitro* studies are un‐ realistic, because they involve single compounds used at high concentrations (pharmacolog‐ ical and not dietary concentrations) far from the low micromolar or nanomolar concentrations detected *in vivo*, or use the bioactive compounds "as they are in food" versus the metabolites or derivatives considered to be the true bioactive compounds, over an ex‐ tended period of time (up to 120 h). As a result, biological activity may be overestimated, since no account is taken of the possible transformation of these compounds during gastro‐ intestinal digestion with or without colonic fermentation [6]. Likewise, the use of single or crude compounds instead of whole foods impedes the detection of synergistic and/or antag‐ onistic actions among bioactive chemopreventive compounds [14, 15].

Taking this background together, and in order to obtain a more precise view of the *in vivo* situation, we propose the use of whole foods or related target bioactive constituents subject‐ ed to a human simulated gastrointestinal digestion including or not including colonic fer‐ mentation, depending on the nature of the studied compounds, in order to gain better insight from a nutritional/functional point of view of the chemopreventive action derived from foods and bioactive compounds in cell models of disease.

This review introduces the main features of the different *in vitro* gastrointestinal digestion (solubility and dialysis) and colonic fermentation procedures (batch, continuous and contin‐ uous with immobilized feces) for studying the bioaccessibility and further bioavailability and bioactivity of nutrients and bioactive compounds. It also includes a definition of the terms: bioavailability including bioaccessibility and bioactivity. Likewise, the main advan‐ tages and disadvantages of these *in vitro* methods versus *in vivo* approaches, the improve‐ ment of these models with the inclusion of cell lines, and a short comment on the main effects that digestion and/or fermentation have on bioactive compounds are included. On the other hand, a short description is provided of the studies involving the use of human simulated gastrointestinal digestion and/or colonic fermentation procedures, and of the sub‐ sequent bioactivity-guided assays with cell line models.

### **2. Simulated gastrointestinal digestion assays**

For decades, the beneficial role of antioxidants was related to the reduction of unwanted and uncontrolled production of reactive oxygen species (ROS), leading to a situation refer‐ red to as oxidative stress [7]. Nowadays, the term "antioxidant" has become ambiguous, since it has different connotations for distinct audiences. For instance, for biochemists and nutritionists, the term is related to the scavenging of metabolically generated ROS, while for food scientists the term implies use in retarding food oxidation or for the categorization of foods or substances according to *in vitro* assays of antioxidant capacity, such as the ORAC and TEAC tests [8]. The antioxidant values provided by these assays sometimes have been misinterpreted by both food producers and consumers due to the fact that health claims ad‐ vertised on the package labeling are directly associated with benefits that include slowing of the aging process and decreasing the risk of chronic disease. Nevertheless, contemporary scientific evidence indicates that total antioxidant capacity measured by currently popular chemical assays may not reflect the actual activity *in vivo,* since none of them take biological processes such as bioavailability, uptake and metabolism into account [9]. Therefore, no *in vitro* assay that determines the antioxidant capacity of a nutritional product describes *in vivo* outcomes, and such testing should not be used to suggest such a connection. In this sense, it is currently recognized that the mechanisms of action of antioxidants *in vivo* might be far more complex than mere radical scavenging - involving interactions with specific proteins central to intracellular signaling cascades [10], and in the specific case of cancer cells there might be a direct antioxidant effect, antiproliferation and anti-survival action, the induction of cell cycle arrest, the induction of apoptosis, antiinflammatory effects and the inhibition of

132 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

In order to determine and verify the action of these bioactive compounds, it is clear that data from human intervention studies offer the reference standard and the highest scientific evi‐ dence considering the bioavailability and bioactivity of a food component, while *in vitro* methods are used as surrogates for prediction [12]. From a physiological perspective, food after consumption undergoes a gastrointestinal digestion process that may affect the native antioxidant potential of the complex mixture of bioactive compounds present in the food matrix before reaching the proximal intestine. *In vitro* methods which apply human simulat‐ ed digestion models (including or not including colonic fermentation) are considered valua‐ ble and useful tools for the estimation of pre-absorptive events (i.e., stability, bioaccessibility) of different food components from distinct food sources, and also for deter‐ mining the effect which processing may have upon food components bioavailability [13]. In addition, *in vitro* assays combining a simulated gastrointestinal digestion process and cell cultures as pre-clinical models can be useful for unraveling mechanisms of action and for projecting further *in vivo* assays [9]. Nevertheless, in most cases these *in vitro* studies are un‐ realistic, because they involve single compounds used at high concentrations (pharmacolog‐ ical and not dietary concentrations) far from the low micromolar or nanomolar concentrations detected *in vivo*, or use the bioactive compounds "as they are in food" versus the metabolites or derivatives considered to be the true bioactive compounds, over an ex‐ tended period of time (up to 120 h). As a result, biological activity may be overestimated, since no account is taken of the possible transformation of these compounds during gastro‐ intestinal digestion with or without colonic fermentation [6]. Likewise, the use of single or

angiogenesis and metastasis [11].

Bioavailability is a key concept for nutritional effectiveness, irrespective of the type of food considered (functional or otherwise). Only certain amounts of all nutrients or bioactive com‐ pounds are available for use in physiological functions or for storage.

The term bioavailability has several working conditions. From the nutritional point of view, bioavailability is defined as the proportion of a nutrient or bioactive compound can be used for normal physiological functions [16]. This term in turn includes two additional terms: bioacces‐ sibility and bioactivity. Bioaccessibility has been defined as the fraction of a compound that is released from its food matrix in the gastrointestinal tract and thus becomes available for intes‐ tinal absorption. Bioaccessibility includes the sequence of events that take place during food digestion for transformation into potentially bioaccessible material, absorption/assimilation through epithelial tissue and pre-systemic metabolism. Bioactivity in turn includes events linked to how the bioactive compound is transported and reaches the target tissue, how it in‐ teracts with biomolecules, the metabolism or biotransformation it may undergo, and the gen‐ eration of biomarkers and the physiologic responses it causes [12]. Depending on the *in vitro* method used, evaluation is made of bioaccessibility and/or bioactivity.

*In vitro* methods have been developed to simulate the physiological conditions and the se‐ quence of events that occur during digestion in the human gastrointestinal tract. In a first step, simulated gastrointestinal digestion (gastric and intestinal stages, and in some cases a salivary stage) is applied to homogenized foods or isolated bioactive compounds in a closed system, with determination of the soluble component fraction obtained by centrifugation or dialysis of soluble components across a semipermeable membrane (bioaccessible fraction). Simulated gastrointestinal digestion can be performed with static models where the prod‐ ucts of digestion remain largely immobile and do not mimic physical processes such as shear, mixing, hydration. Dynamic models can also be used, with gradual modifications in pH and enzymes, and removal of the dialyzed components – thereby better simulating the actual *in vivo* situation. All these systems evaluate the aforementioned term "bioaccessibili‐ ty", and can be used to establish trends in relative bioaccessibility.

fatty acids arising from the active metabolism of dietary compounds by the gut micro‐ biota, and especially for substrate digestion evaluation studies [21, 22]. Several of the pub‐ lications in this field are based on a European interlaboratory study for estimation of

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135

Continuous cultures allow us to control the rate and composition of nutrient feed, bacterial metabolism and the environmental conditions. These models simulate proximal (single-state models) or proximal, transverse and distal colonic regions (multistage models). Continuous cultures are used for performing long-term studies, and substrate replenishment and toxic product removal are facilitated - thereby mimicking the conditions found *in vivo*. The most variable factor in these models is the technique used for fecal inoculation. The use of liquid fecal suspension as inoculum, where the bacterial populations are in the free-cell state, pro‐ duces rapid washout of less competitive bacteria; as a result, the operation time is less than four weeks. The formation of fecal beads from the immobilization of fecal microbiota in a porous polysaccharide matrix allows release of the microbiota into the culture medium,

with better reproduction of the *in vivo* flora and longer fermentation times [21, 22].

laboriousness, the use of large working volumes, and long residence times.

tory control by neuroendocrine cells and through the blood [27].

Artificial continuous models including host functions/human digestive functions have been developed. Models of this kind control peristaltic movement, pH and gastrointestinal secre‐ tions. The SHIME model (Simulated Human Intestinal Microbial Ecosystem) comprises a 5 step multi-chamber reactor simulating the duodenum and jejunum, ileum, cecum and the ascending colon, transverse colon and descending colon [24]. In turn, TIM-1 is an intestinal model of the stomach and small intestine, while TIM-2 is a proximal colon simulator model de‐ veloped by TNO (*Netherlands Organization for Applied Scientific Research)*. These models have been validated based on human and animal data [25]. They incorporate some host functions; however, they do not reproduce immune modulating and neuroendocrine responses. A re‐ maining challenge is the difficulty of establishing a representative human gut microbiota *in vi‐ tro*. Other difficulties are the availability of the system, its cost, the prolonged time involved, its

Combined systems that include the fractions obtained from simulated human digestion (gastrointestinal and/or colonic fermentation) and the incorporation of cell culture-based models allow us to evaluate bioaccessibility (estimate the amount of bioactive compounds assimilated from the bioaccessible fraction by cell culture) and to conduct bioactivity stud‐ ies. The Caco-2 cell model is the most widely used and validated intestinal epithelium or hu‐ man colon carcinoma cell model. Although colonic in origin, Caco-2 cells undergo spontaneous differentiation in cell culture to form a monolayer of well-polarized cells at confluence, showing many of the functional and morphological properties of mature human enterocytes (with the formation of microvilli on the brush border membrane, tight intercel‐ lular junctions and the excretion of brush border-associated enzymes) [26]. However it must be mentioned that this cell line differs in some aspects from *in vivo* conditions. For example, it does not reproduce the different populations of cells in the gut, such as goblet, Paneth and crypt cells, which are less organized and therefore leakier. Likewise, the model lacks regula‐

the fermentability of dietary fiber *in vitro* [23].

The principal requirement for successfully conducting experimental studies of this kind is to achieve conditions which are similar to the *in vivo* conditions. Temperature, shaking or agi‐ tation, and the chemical and enzymatic composition of saliva, gastric juice, duodenal and bile juice are all relevant aspects in these studies. Interactions with other food components must also be taken into account, since they can influence the efficiency of digestion [12, 17]. A recent overview of the different *in vitro* digestion models, sample conditions and enzymes used has been published by Hur et al. [13]. En lipophilic compounds such as carotenoids and phytosterols, it is necessary to form mixed micelles in the duodenal stage through the action of bile salts, phospholipases and colipase. This allows the compounds to form part of the micelles, where they remain until uptake by the enterocytes [18]. In the case of lycopene, during digestion isomerization of trans-lycopene may occur with the disadvantage that trans-isomers are less soluble in bile acid micelles [19]. Salivary and gastric digestion exert no substantial effect on major phenolic compounds. However, polyphenols are highly sensi‐ tivity to the mild alkaline conditions in pancreatic digestion, and a good proportion of these compounds can be transformed into other unknown and/or undetected forms [20].

Bioactive compounds such as dietary fiber, carotenoids, polyphenols and phytosterols un‐ dergo very limited absorption, and may experience important modifications as a result of actions on the part of the intestinal microbiota. Small intestine *in vitro* models are devoid of intestinal microbes, and are designed to only replicate digestion and absorption processes; as a result, they are unable to provide information on intestinal fermentation processes. The incorporation of colonic/large intestine fermentation offers a better approximation to the *in vivo* situation, and allows us to study the effect/interaction between these compounds and the intestinal microbiota.

*In vitro* colonic fermentation models are characterized by the inoculation of single or mul‐ tiple chemostats with fecal microbiota (of rat or human origin) and operated under phys‐ iological temperature, pH and anaerobic conditions. There are two types of colonic fermentation models: batch culture and continuous cultures. Batch culture describes the growth of pure or mixed bacterial suspensions in a carefully selected medium with‐ out the further addition of nutrients in closed systems using sealed bottles or reactors containing suspensions of fecal material under anaerobic conditions. The advantages of batch fermentation are that the technique is inexpensive, easy to set up, and allows large number of substrates of fecal samples to be tested. However, these models have their weakness in microbiological control and the need to be of short duration in or‐ der to avoid the selection of non-representative microbial populations. The technique is useful for fermentation studies, for the investigation of metabolic profiles of short chain fatty acids arising from the active metabolism of dietary compounds by the gut micro‐ biota, and especially for substrate digestion evaluation studies [21, 22]. Several of the pub‐ lications in this field are based on a European interlaboratory study for estimation of the fermentability of dietary fiber *in vitro* [23].

system, with determination of the soluble component fraction obtained by centrifugation or dialysis of soluble components across a semipermeable membrane (bioaccessible fraction). Simulated gastrointestinal digestion can be performed with static models where the prod‐ ucts of digestion remain largely immobile and do not mimic physical processes such as shear, mixing, hydration. Dynamic models can also be used, with gradual modifications in pH and enzymes, and removal of the dialyzed components – thereby better simulating the actual *in vivo* situation. All these systems evaluate the aforementioned term "bioaccessibili‐

The principal requirement for successfully conducting experimental studies of this kind is to achieve conditions which are similar to the *in vivo* conditions. Temperature, shaking or agi‐ tation, and the chemical and enzymatic composition of saliva, gastric juice, duodenal and bile juice are all relevant aspects in these studies. Interactions with other food components must also be taken into account, since they can influence the efficiency of digestion [12, 17]. A recent overview of the different *in vitro* digestion models, sample conditions and enzymes used has been published by Hur et al. [13]. En lipophilic compounds such as carotenoids and phytosterols, it is necessary to form mixed micelles in the duodenal stage through the action of bile salts, phospholipases and colipase. This allows the compounds to form part of the micelles, where they remain until uptake by the enterocytes [18]. In the case of lycopene, during digestion isomerization of trans-lycopene may occur with the disadvantage that trans-isomers are less soluble in bile acid micelles [19]. Salivary and gastric digestion exert no substantial effect on major phenolic compounds. However, polyphenols are highly sensi‐ tivity to the mild alkaline conditions in pancreatic digestion, and a good proportion of these

compounds can be transformed into other unknown and/or undetected forms [20].

the intestinal microbiota.

Bioactive compounds such as dietary fiber, carotenoids, polyphenols and phytosterols un‐ dergo very limited absorption, and may experience important modifications as a result of actions on the part of the intestinal microbiota. Small intestine *in vitro* models are devoid of intestinal microbes, and are designed to only replicate digestion and absorption processes; as a result, they are unable to provide information on intestinal fermentation processes. The incorporation of colonic/large intestine fermentation offers a better approximation to the *in vivo* situation, and allows us to study the effect/interaction between these compounds and

*In vitro* colonic fermentation models are characterized by the inoculation of single or mul‐ tiple chemostats with fecal microbiota (of rat or human origin) and operated under phys‐ iological temperature, pH and anaerobic conditions. There are two types of colonic fermentation models: batch culture and continuous cultures. Batch culture describes the growth of pure or mixed bacterial suspensions in a carefully selected medium with‐ out the further addition of nutrients in closed systems using sealed bottles or reactors containing suspensions of fecal material under anaerobic conditions. The advantages of batch fermentation are that the technique is inexpensive, easy to set up, and allows large number of substrates of fecal samples to be tested. However, these models have their weakness in microbiological control and the need to be of short duration in or‐ der to avoid the selection of non-representative microbial populations. The technique is useful for fermentation studies, for the investigation of metabolic profiles of short chain

ty", and can be used to establish trends in relative bioaccessibility.

134 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Continuous cultures allow us to control the rate and composition of nutrient feed, bacterial metabolism and the environmental conditions. These models simulate proximal (single-state models) or proximal, transverse and distal colonic regions (multistage models). Continuous cultures are used for performing long-term studies, and substrate replenishment and toxic product removal are facilitated - thereby mimicking the conditions found *in vivo*. The most variable factor in these models is the technique used for fecal inoculation. The use of liquid fecal suspension as inoculum, where the bacterial populations are in the free-cell state, pro‐ duces rapid washout of less competitive bacteria; as a result, the operation time is less than four weeks. The formation of fecal beads from the immobilization of fecal microbiota in a porous polysaccharide matrix allows release of the microbiota into the culture medium, with better reproduction of the *in vivo* flora and longer fermentation times [21, 22].

Artificial continuous models including host functions/human digestive functions have been developed. Models of this kind control peristaltic movement, pH and gastrointestinal secre‐ tions. The SHIME model (Simulated Human Intestinal Microbial Ecosystem) comprises a 5 step multi-chamber reactor simulating the duodenum and jejunum, ileum, cecum and the ascending colon, transverse colon and descending colon [24]. In turn, TIM-1 is an intestinal model of the stomach and small intestine, while TIM-2 is a proximal colon simulator model de‐ veloped by TNO (*Netherlands Organization for Applied Scientific Research)*. These models have been validated based on human and animal data [25]. They incorporate some host functions; however, they do not reproduce immune modulating and neuroendocrine responses. A re‐ maining challenge is the difficulty of establishing a representative human gut microbiota *in vi‐ tro*. Other difficulties are the availability of the system, its cost, the prolonged time involved, its laboriousness, the use of large working volumes, and long residence times.

Combined systems that include the fractions obtained from simulated human digestion (gastrointestinal and/or colonic fermentation) and the incorporation of cell culture-based models allow us to evaluate bioaccessibility (estimate the amount of bioactive compounds assimilated from the bioaccessible fraction by cell culture) and to conduct bioactivity stud‐ ies. The Caco-2 cell model is the most widely used and validated intestinal epithelium or hu‐ man colon carcinoma cell model. Although colonic in origin, Caco-2 cells undergo spontaneous differentiation in cell culture to form a monolayer of well-polarized cells at confluence, showing many of the functional and morphological properties of mature human enterocytes (with the formation of microvilli on the brush border membrane, tight intercel‐ lular junctions and the excretion of brush border-associated enzymes) [26]. However it must be mentioned that this cell line differs in some aspects from *in vivo* conditions. For example, it does not reproduce the different populations of cells in the gut, such as goblet, Paneth and crypt cells, which are less organized and therefore leakier. Likewise, the model lacks regula‐ tory control by neuroendocrine cells and through the blood [27].

The advantage of these systems versus those which only evaluate the influence of digestion is their greater similarity to the *in vivo* conditions*.* The combination of *in vitro* human intesti‐ nal cell models with *in vitro* digestion models in turn creates an advanced *in vitro* model sys‐ tem where samples obtained from host responses lacking in *in vitro* digestion models can be directly applied to monolayer cell models for host function studies [21].

ievable in colon cells, since the bioaccessible fractions obtained after digestion are considered to be fractions that can pass through the stomach and small intestine reaching the co‐ lon, where they can exert antioxidant activity *in situ* [33]. In addition, polyphenols are stud‐ ied in neuronal cells, liver-derived cells and lymphocytes. In the case of neuronal cells, the concentrations used (0-6 µM polyphenols) are similar to those reported for dietary pol‐ yphenolic-derived metabolites found in plasma (0-4 µM) [34], but for lymphocytes and liv‐ er, the concentrations are unknown or higher than expected *in vivo*, respectively. Another aspect to bear in mind is the time of cell exposure to the digested food or bioactive con‐ stituents. The range found in these studies is from 30 min to 120 h (this latter time‐

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137

Bioactive compounds of digested foods present four different but in some cases comple‐ mentary modes of action: (1) inhibition of cholesterol absorption (phytosterols), and (2) antiproliferative, (3) cytoprotective and (4) antiinflammatory activities (polyphenols and

**1.** The inhibition of cholesterol absorption has been reported to be mainly due to competi‐ tion between phytosterols and cholesterol for incorporation to the micelles as a previ‐

**2.** Antiproliferative activity has been linked to cell growth inhibition associated to pol‐ yphenols [28, 32, 36-38] and lycopene [39], which is mainly regulated by two mech‐ anisms: cell-cycle arrest and apoptosis induction. The cell cycle can be halted at different phases: G0/G1 with down-regulation of cyclin D1 [39], S with down-regula‐ tion of cyclins D1 and B1 [28, 37] and G2/M [36]. Apoptosis induction in turn oc‐ curs as a result of caspase-3 induction and down-regulation of the anti-apoptotic

**3.** The cytoprotective effect of polyphenols, peptides and antioxidants against induced ox‐ idative stress is related to the preservation of cell viability [40-47], an increase in the ac‐ tivity of antioxidant enzymes (such as catalase, glutathione reductase or glutathione peroxidase) [41, 43, 47, 48], the prevention of reduced glutathione (GSH) depletion [46, 47, 49], a decrease in intracellular ROS content [46, 50, 51], the maintenance of correct cell cycle progression [41, 43, 47, 52], the prevention of apoptosis [43], and the preven‐

**4.** The antiinflammatory action of peptides and polyphenols is derived from the decrease in the release of proinflammatory cytokines such as IL-8 when cells are stimulated with

Studies on the chemopreventive effect of foods or isolated bioactive constituents following colonic fermentation or gastrointestinal digestion plus colonic fermentation in cell lines are shown in Tables 2 and 3, respectively. The colonic fermentation procedure used in these as‐ says has always been a batch model, except for one study combining batch and dynamic fer‐ mentation. In turn, when gastrointestinal digestion is involved, dialysis has been the method used. Foods of plant origin rich in fiber, and short chain fatty acids (mainly buty‐ rate) and polyphenols as the target compounds have been used in such studies. The use of colon-derived cell lines is common in these assays, which have been performed using phys‐

point not being expectable from a physiological standpoint).

ous step before absorption by the intestinal epithelial cells [35].

general antioxidants).

proteins Bcl-2 and Bcl-xL [39].

tion of DNA damage [42, 51, 52].

stressors such as H202 or TNFα [53, 54].

### **3. Bioactivity of digested/fermented foods or related target bioactive compounds in cell lines**

The chemopreventive properties of bioactive compounds have been investigated in cultured cells exposed to individual compounds. However, gut epithelial cells are more likely to be exposed to complex food matrixes containing mixtures of bioactive and antioxidant *in vivo* compounds [6]. In addition, food matrixes undergo a digestion process that may affect the structure and properties of the bioactive compounds. Therefore, the *in vitro* protective ef‐ fects of antioxidant bioactive compounds do not necessarily reflect *in vivo* chemoprotection, which is more likely due to the combined effects of all the bioactive components present in the food [28].

A potential cell culture model for cancer or cardiovascular chemoprevention research in‐ volving dietary antioxidants (polyphenols, carotenoids and peptides) and other bioactive chemopreventive components such as phytosterols, should include some of the proposed mechanisms of action: inhibition of cell proliferation, induction of tumor suppressor gene expression, induction of cell cycle arrest, induction of apoptosis, antioxidant enzyme induc‐ tion, and enhanced detoxification, antiinflammatory activities and the inhibition of choles‐ terol absorption [9, 15, 29, 30]. In addition, other mechanisms of chemoprevention could involve protection against genotoxic compounds or reactive oxygen species [31].

It recently has been stated that the measurement of cellular bioactivity of food samples cou‐ pled to *in vitro* digestion can provide information close to the real-life physiological situation [32]. In this sense, we surveyed more than 30 studies conducted in the past 10 years, involv‐ ing human simulated gastrointestinal digestion and/or colonic fermentation procedures and subsequent bioactivity-guided assays with cell line models. These studies are presented in Tables 1, 2 and 3, which correspond to the mechanism of action related to chemoprevention of digested, fermented or digested plus fermented foods or bioactive constituents in cell lines, respectively.

The chemopreventive effect of digested foods or bioactive constituents in cell lines is sum‐ marized in Table 1. From the 22 studies surveyed, and according to the digestion meth‐ od used, it can be seen that most of them involve solubility (n = 17) versus dialysis (n = 5). Samples used are preferably of vegetal origin (n = 15), the target compounds respon‐ sible for the chemopreventive action being polyphenols, antioxidants (in general), antiox‐ idant peptides, lycopene and phytosterols. Furthermore, these compounds are mainly studied in colon-derived cells (as a cancer model when not differentiated, or as an intes‐ tinal epithelial model when differentiated). Concentrations tested are physiologically ach‐ ievable in colon cells, since the bioaccessible fractions obtained after digestion are considered to be fractions that can pass through the stomach and small intestine reaching the co‐ lon, where they can exert antioxidant activity *in situ* [33]. In addition, polyphenols are stud‐ ied in neuronal cells, liver-derived cells and lymphocytes. In the case of neuronal cells, the concentrations used (0-6 µM polyphenols) are similar to those reported for dietary pol‐ yphenolic-derived metabolites found in plasma (0-4 µM) [34], but for lymphocytes and liv‐ er, the concentrations are unknown or higher than expected *in vivo*, respectively. Another aspect to bear in mind is the time of cell exposure to the digested food or bioactive con‐ stituents. The range found in these studies is from 30 min to 120 h (this latter time‐ point not being expectable from a physiological standpoint).

The advantage of these systems versus those which only evaluate the influence of digestion is their greater similarity to the *in vivo* conditions*.* The combination of *in vitro* human intesti‐ nal cell models with *in vitro* digestion models in turn creates an advanced *in vitro* model sys‐ tem where samples obtained from host responses lacking in *in vitro* digestion models can be

**3. Bioactivity of digested/fermented foods or related target bioactive**

The chemopreventive properties of bioactive compounds have been investigated in cultured cells exposed to individual compounds. However, gut epithelial cells are more likely to be exposed to complex food matrixes containing mixtures of bioactive and antioxidant *in vivo* compounds [6]. In addition, food matrixes undergo a digestion process that may affect the structure and properties of the bioactive compounds. Therefore, the *in vitro* protective ef‐ fects of antioxidant bioactive compounds do not necessarily reflect *in vivo* chemoprotection, which is more likely due to the combined effects of all the bioactive components present in

A potential cell culture model for cancer or cardiovascular chemoprevention research in‐ volving dietary antioxidants (polyphenols, carotenoids and peptides) and other bioactive chemopreventive components such as phytosterols, should include some of the proposed mechanisms of action: inhibition of cell proliferation, induction of tumor suppressor gene expression, induction of cell cycle arrest, induction of apoptosis, antioxidant enzyme induc‐ tion, and enhanced detoxification, antiinflammatory activities and the inhibition of choles‐ terol absorption [9, 15, 29, 30]. In addition, other mechanisms of chemoprevention could

It recently has been stated that the measurement of cellular bioactivity of food samples cou‐ pled to *in vitro* digestion can provide information close to the real-life physiological situation [32]. In this sense, we surveyed more than 30 studies conducted in the past 10 years, involv‐ ing human simulated gastrointestinal digestion and/or colonic fermentation procedures and subsequent bioactivity-guided assays with cell line models. These studies are presented in Tables 1, 2 and 3, which correspond to the mechanism of action related to chemoprevention of digested, fermented or digested plus fermented foods or bioactive constituents in cell

The chemopreventive effect of digested foods or bioactive constituents in cell lines is sum‐ marized in Table 1. From the 22 studies surveyed, and according to the digestion meth‐ od used, it can be seen that most of them involve solubility (n = 17) versus dialysis (n = 5). Samples used are preferably of vegetal origin (n = 15), the target compounds respon‐ sible for the chemopreventive action being polyphenols, antioxidants (in general), antiox‐ idant peptides, lycopene and phytosterols. Furthermore, these compounds are mainly studied in colon-derived cells (as a cancer model when not differentiated, or as an intes‐ tinal epithelial model when differentiated). Concentrations tested are physiologically ach‐

involve protection against genotoxic compounds or reactive oxygen species [31].

directly applied to monolayer cell models for host function studies [21].

136 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**compounds in cell lines**

the food [28].

lines, respectively.

Bioactive compounds of digested foods present four different but in some cases comple‐ mentary modes of action: (1) inhibition of cholesterol absorption (phytosterols), and (2) antiproliferative, (3) cytoprotective and (4) antiinflammatory activities (polyphenols and general antioxidants).


Studies on the chemopreventive effect of foods or isolated bioactive constituents following colonic fermentation or gastrointestinal digestion plus colonic fermentation in cell lines are shown in Tables 2 and 3, respectively. The colonic fermentation procedure used in these as‐ says has always been a batch model, except for one study combining batch and dynamic fer‐ mentation. In turn, when gastrointestinal digestion is involved, dialysis has been the method used. Foods of plant origin rich in fiber, and short chain fatty acids (mainly buty‐ rate) and polyphenols as the target compounds have been used in such studies. The use of colon-derived cell lines is common in these assays, which have been performed using phys‐ iologically relevant concentrations and time periods of exposure of samples to cells ranging between 24 h and 72 h.

and for allowing comparisons of results among laboratories. This task is currently being car‐ ried out at European level within the project "Improving health properties of food by shar‐ ing our knowledge on the digestive process (INFOGEST) (2011-2015) (FAO COST Action FA

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion

*Gastrointestinal digestion (dialysis)*

Cell growth inhibition Viability decrease Cell cycle arrest at G2/M phase Up-regulation of tumor suppression gene *CEACAM1*

Prevention of H2O2 (75µM/5min)-induced DNA damage and decrease in G1 phase of cell cycle (HT29 cells) No effect on epithelial integrity (Caco-2 cells) Inhibition of colon cancer cell invasion (HT115 cells)

Protection against H2O2 and Aβ(1-42) induced cytotoxicity (only at low concentrations)

Preservation of cell viability against H2O2 (300 µM- 24 h) –induced oxidative stress (not related to modulation of ROS nor GSH levels)

CECAM1: Carcinoembryonic antigen-related cell adhesion molecule 1. Aβ(1-42): β-amyloid peptide 1-42. ROS: reac‐

**Table 1.** Mechanisms involved in the chemopreventive effect of *in vitro* digested foods or bioactive constituents in cell

The *in vitro* simulation of the conditions of gastrointestinal digestion represents an alterna‐ tive to *in vivo* studies for evaluating the bioavailability and/or functionality of bioactive com‐ ponents of foods. *In vitro* studies do not replace *in vivo* studies; rather, both complement

**Cellular mechanism References**

Bermúdez-Soto et al. (2007) [36]

http://dx.doi.org/10.5772/51504

139

Coates et al. (2007) [52]

Okello et al. (2011) [44]

Tavares et al. (2012a) [45]

1005) (http://www.cost-infogest.eu/ABOUT-Infogest)".

**Cell treatment (Concentrations and time)**

85 to 220 (µM total polyphenols) 2 h a day for a 4 day period

> 3.125 to 50 (µg/mL) 24 h

0.3-10 µg/mL (for H2O2) and 0.03-0.125 µg/mL (for Aβ(1-42)) Pretreatment 24 h and stressed 24 h

> 1.5-6 µM total polyphenols 24 h

**Cell type**

Caco-2 (human colon carcinoma)

HT29, Caco-2 and HT115 (human colon carcinoma)

Differentiate d PC12 (model of neuronal cells)

SK-N-MC (neuroblast oma cells)

tive oxygen species. GSH: reduced glutathione.

**Sample (Target compound/s)**

**(Polyphenols)**

Chokeberry juice

Raspberries

Green tea

Blackberry (*Rubus sp.*)

lines.

The mechanism of action underlying the treatment of cells with colonic fermented foods or isolated bioactive constituents (see Table 2) mainly comprises antiproliferative activity (i) and/or cytoprotective action (ii). In the first case, antiproliferative activity (i) has been attrib‐ uted to cell growth inhibition [55-59], mainly due to apoptosis induction [58-59] and/or the up-regulation of genes involved in cell cycle arrest (*p21*) and apoptosis (*WNT2B*) [59]. Stud‐ ies referred to a cytoprotective effect against oxidative damage (ii) in turn have been linked to the prevention of DNA damage [55, 56] and to the induction of antioxidant enzymes such as glutathione-S-transferase (GST) [56].

The bioactivity observed with the incubation of cells lines with foods or isolated bioactive constituents following gastrointestinal digestion plus colonic fermentation (see Table 3) is derived from antiproliferative activity (i) regulated by cell growth inhibition [60-62], cell cy‐ cle arrest [60] and/or apoptosis induction [60, 62], or by a cytoprotective effect against in‐ duced oxidative stress (ii) as a result of preservation of cell viability [63], protection against DNA damage [31, 61, 63] and/or induction of antioxidant enzymes such as CAT, GST and sulfotransferase (SULT2B1) [31].

### **4. Conclusions and future perspectives**

From the data here reviewed in disease cell models, it can be concluded that gastrointestinal digestion/colonic fermentation applied to whole foods or isolated bioactive constituents may have potential health benefits derived from cell growth inhibition through the induction of cell-cycle arrest and/or apoptosis, cytoprotection against induced oxidative stress, antiin‐ flammatory activity and the reduction of cholesterol absorption.

Studies conducted with single bioactive compounds are unrealistic from a nutritional and physiological point of view, since they do not take into account physicochemical changes during digestion and possible synergistic activities. Thus, a combined model of human si‐ mulated digestion including or not including colonic fermentation (depending on the nature of the studied compounds) with cell lines should be carried out if *in vitro* bioactivity assays with whole foods or bioactive chemopreventive compounds for the prevention of oxidative stress-related diseases are planned.

Although digested/fermented bioactive compounds appear as promising chemopreventive agents, our understanding of the molecular and biochemical pathways behind their mecha‐ nism of action is still limited, and further studies are warranted. In addition, the need for harmonization of the *in vitro* methods: (i) conditions of the gastrointestinal procedure, (ii) cell line used, (iii) concentrations of bioactive compounds used (usually much higher than those achievable in the human body when the digestion process is not considered), and (iv) time of cell exposure to the bioactive compounds (more than 24 h is unlikely to occur *in vivo*), should be considered for improved study designs more similar to the *in vivo* situation and for allowing comparisons of results among laboratories. This task is currently being car‐ ried out at European level within the project "Improving health properties of food by shar‐ ing our knowledge on the digestive process (INFOGEST) (2011-2015) (FAO COST Action FA 1005) (http://www.cost-infogest.eu/ABOUT-Infogest)".

iologically relevant concentrations and time periods of exposure of samples to cells ranging

The mechanism of action underlying the treatment of cells with colonic fermented foods or isolated bioactive constituents (see Table 2) mainly comprises antiproliferative activity (i) and/or cytoprotective action (ii). In the first case, antiproliferative activity (i) has been attrib‐ uted to cell growth inhibition [55-59], mainly due to apoptosis induction [58-59] and/or the up-regulation of genes involved in cell cycle arrest (*p21*) and apoptosis (*WNT2B*) [59]. Stud‐ ies referred to a cytoprotective effect against oxidative damage (ii) in turn have been linked to the prevention of DNA damage [55, 56] and to the induction of antioxidant enzymes such

The bioactivity observed with the incubation of cells lines with foods or isolated bioactive constituents following gastrointestinal digestion plus colonic fermentation (see Table 3) is derived from antiproliferative activity (i) regulated by cell growth inhibition [60-62], cell cy‐ cle arrest [60] and/or apoptosis induction [60, 62], or by a cytoprotective effect against in‐ duced oxidative stress (ii) as a result of preservation of cell viability [63], protection against DNA damage [31, 61, 63] and/or induction of antioxidant enzymes such as CAT, GST and

From the data here reviewed in disease cell models, it can be concluded that gastrointestinal digestion/colonic fermentation applied to whole foods or isolated bioactive constituents may have potential health benefits derived from cell growth inhibition through the induction of cell-cycle arrest and/or apoptosis, cytoprotection against induced oxidative stress, antiin‐

Studies conducted with single bioactive compounds are unrealistic from a nutritional and physiological point of view, since they do not take into account physicochemical changes during digestion and possible synergistic activities. Thus, a combined model of human si‐ mulated digestion including or not including colonic fermentation (depending on the nature of the studied compounds) with cell lines should be carried out if *in vitro* bioactivity assays with whole foods or bioactive chemopreventive compounds for the prevention of oxidative

Although digested/fermented bioactive compounds appear as promising chemopreventive agents, our understanding of the molecular and biochemical pathways behind their mecha‐ nism of action is still limited, and further studies are warranted. In addition, the need for harmonization of the *in vitro* methods: (i) conditions of the gastrointestinal procedure, (ii) cell line used, (iii) concentrations of bioactive compounds used (usually much higher than those achievable in the human body when the digestion process is not considered), and (iv) time of cell exposure to the bioactive compounds (more than 24 h is unlikely to occur *in vivo*), should be considered for improved study designs more similar to the *in vivo* situation

between 24 h and 72 h.

as glutathione-S-transferase (GST) [56].

138 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

sulfotransferase (SULT2B1) [31].

stress-related diseases are planned.

**4. Conclusions and future perspectives**

flammatory activity and the reduction of cholesterol absorption.


CECAM1: Carcinoembryonic antigen-related cell adhesion molecule 1. Aβ(1-42): β-amyloid peptide 1-42. ROS: reac‐ tive oxygen species. GSH: reduced glutathione.

**Table 1.** Mechanisms involved in the chemopreventive effect of *in vitro* digested foods or bioactive constituents in cell lines.

The *in vitro* simulation of the conditions of gastrointestinal digestion represents an alterna‐ tive to *in vivo* studies for evaluating the bioavailability and/or functionality of bioactive com‐ ponents of foods. *In vitro* studies do not replace *in vivo* studies; rather, both complement each other. *In vitro* methods need to be improved and validated with more *in vivo* studies. Thus, caution is mandatory when attempting to extrapolate observations obtained *in vitro* in cell line studies to humans.

**Sample (Target compound/s)**

Feijoadatraditional Brazilian meal

Culinary herbs: rosemary, sage and thyme

**(Antioxidants)**

Fruit beverages with/without milk and/or iron/zinc

Fruit beverages with/without milk or CPPs

Beef patties enriched with sage and oregano

**Table 1.** (continued-II).

**Cell type**

HepG2 (human liver cancer cells)

PBL (peripheral blood lymphocytes ) and Differentitat ed Caco-2 (model of intestinal epithelium)

Differentiate d Caco-2 (model of intestinal epithelia)

Differentiate d Caco-2 (model of intestinal epithelia)

Caco-2 (human colon carcinoma)

**Cell treatment (Concentration s and time)**

10-100 mg/mL 72 h (antiproliferatio n) and 1 h (antioxidant)

1:10 (v/v) in culture medium. Stressors (H2O2 2 mM and TNFα 100 µg/mL) Co-incubation 24 h or preincubation 3h then stress 24 h

1:1 (v/v) in culture medium

1:1 (v/v) in culture medium or CPPs (1.4 mg/mL)

10-100% (v/v) 24 h

phopeptides. GSH-Rd. glutathione reductase. GSH: reduced glutathione.

**Cellular mechanism References**

Kremer-Faller et al. (2012) [32]

http://dx.doi.org/10.5772/51504

141

Chohan et al. (2012) [54]

> Cilla et al. (2008) [40]

Laparra et al. (2008) [41]

> Ryan et al. (2009) [49]

Antiproliferative activity ("/ 80 mg/mL) Increase in cellular antioxidant activity (0.6 µM quercetin equivalents)

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion

PBL: significant decrease in IL-8 release when co-incubation with H2O2 and preincubation prior H202 and TNFα Caco-2: significant decrease in IL-8 release only when co-incubation with TNFα

> Preservation of cell viability No alteration of SOD

Preservation of cell viability (only fruit beverages)

Increase in cell viability at low concentrations (20-40%) but slight decrease at high concentrations (80-100%) Increase in GSH (only sage-enriched samples at 10%) Protection against H202 (200 µM/1h) induced GSH depletion (at 10%)

IL-8: Proinflammatory interleukin-8. TNFα: tumor necrosis factor α. SOD: Superoxide dismutase. CPPs: caseinophos‐


**Table 1.** (continued-I).

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion http://dx.doi.org/10.5772/51504 141


IL-8: Proinflammatory interleukin-8. TNFα: tumor necrosis factor α. SOD: Superoxide dismutase. CPPs: caseinophos‐ phopeptides. GSH-Rd. glutathione reductase. GSH: reduced glutathione.

**Table 1.** (continued-II).

each other. *In vitro* methods need to be improved and validated with more *in vivo* studies. Thus, caution is mandatory when attempting to extrapolate observations obtained *in vitro* in

> Preservation of cell viability and mitochondrial membrane potential against H2O2 (300 µM -24 h)-induced oxidative stress Decrease of intracellular ROS against H2O2 (200 µM -1 h)-induced oxidative stress (only *R. brigantines*) Prevention of GSH depletion against H2O2 (300 µM -24 h)-induced oxidative stress Induction of caspase 3/7 activity against H2O2 (300 µM -24 h)-induced oxidative stress (preconditioning effect)

*Gastrointestinal digestion (solubility)*

Cell growth inhibition (no clear doseresponse) Cell cycle arrest at S phase (7.5%) Down-regulation of cyclins D1 and B1 No apoptosis (cytostatic effect)

Cell growth inhibition (without citotoxicity) Cell cycle arrest at S phase No apoptosis and resumption of cell cycle after digest removal (cytostatic effect)

Cell growth inhibition

**Cellular mechanism References**

Tavares et al. (2012b) [46]

> Cilla et al. (2009) [28]

> Cilla et al. (2010) [37]

Frontela-Saseta et al. (2011) [38]

cell line studies to humans.

**Cell type**

SK-N-MC (neuroblast oma cells)

Caco-2 (human colon carcinoma)

Caco-2 and HT-29 (human colon carcinoma)

Caco-2 (human colon carcinoma)

ROS: reactive oxygen species. GSH: Reduced glutathione.

**Cell treatment (Concentration s and time)**

140 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

0-6 µM total polyphenols 24 h

2%, 5% and 7.5% (v/v) in culture medium (3.4-22.7 mg/mL total polyphenols) 4 hours-4 days or 24 h

7.5% (v/v) in culture medium (~50 µM total polyphenols) 24 h

4% (v/v) in culture medium 24-120 h

**Sample (Target compound/s)**

Wild blackberry species

**(Polyphenols)**

Fruit beverages with/without milk and/or iron

Zinc-fortified fruit beverages with/without iron and/or milk

Fruit juices enriched with pine bark extract

**Table 1.** (continued-I).


**Sample (Target compound/s)**

CPPs from digested cow's skimmed milk

Purified hen egg yolkderived phosvitin phosphopeptid es

**(Lycopene)**

Tomatoes

**(Phytosterols)**

Orange juice enriched with fat-free phytosterols

**Table 1.** (continued-IV).

**Sample (Target compound/s)**

Fibre sources: linseed, watercress, kale, tomato.soya

**(SCFA)**

**Cell type**

Differentiate d Caco-2 (model of intestinal epithelia)

Differentiate d Caco-2 (model of intestinal epithelia)

HT29 and HCT-116 (human colon carcinoma)

Differentiate d Caco-2 (model of intestinal epithelia)

**Cell type**

HT29 (human colon carcinoma)

**Cell treatment (Concentration s and time)**

1, 2 and 3 mg/mL Pre-incubation 24 h then stressed 2h with H202 5 mM

> 0.05-0.5 mg/mL 2 h

20-100 mL/L 24 h

2 mL test medium/well 4 h

GSH: reduced glutathione. CAT: catalase. IL-8: proinflammatory interleukin-8.

**Cell treatment (Concentration s and time)**

2.5-25% (v/v) in culture medium 72 h

**Cellular mechanism References**

García-Nebot et al. (2011) [47]

http://dx.doi.org/10.5772/51504

143

Young et al. (2011) [53]

Palozza et al. (2011) [39]

Bohn et al. (2007) [35]

Beyer-Sehlmeyer et al. (2003) [55]

Preservation of cell viability Increase in GSH content and induction of CAT activity Decrease in lipid peroxidation Maintenance of correct cell cycle progression

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion

Reduced IL-8 secretion in H202 (1 mM/6 h) induced oxidative stress

Cell growth inhibition Cell cycle arrest at G0-G1 phase and apoptosis induction (caspase-3) Down-regulation of cyclin D1 and antiapoptotic proteins Bcl-2 and Bcl-xL

Reduced micellarization of cholesterol Decrease in cholesterol accumulation by Caco-2 cells

Cell growth inhibition (all samples except watercress) Prevention of HNE (150µM/30 min)-induced DNA damage (only soya flour)

**Cellular mechanism References**

AAPH: 2,2'-azobis (2-amidinopropane) dihydrochloride. ROS: reactive oxygen species. GSH-Rd: glutathione reduc‐ tase. Nrf-2: nuclear response factor 2. GSH-Px2: glutathione peroxidase.

**Table 1.** (continued-III).

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion http://dx.doi.org/10.5772/51504 143


#### **Table 1.** (continued-IV).

**Sample (Target compound/s)**

Ellagic acid-, lutein- or sesamolenriched meat patties

Pacific hake fish protein hydrolysates

Human breast milk

Fruit beverages with/without milk and/or iron/zinc

Purified milk hydrolysate peptide fraction from digested human milk

**Table 1.** (continued-III).

**Cell type**

Caco-2 (human colon carcinoma)

Caco-2 (human colon carcinoma)

Co-culture of Caco-2 BBE and HT29-MTX (model of human intestinal mucosa)

Differentiate d Caco-2 (model of intestinal epithelia)

Caco-2 and FHs 74 int (human colon carcinoma and primary fetal enterocytes)

**Cell treatment (Concentration s and time)**

142 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

0-20% (v/v) in culture medium 24 h

0.625-5 mg/mL 2 h

1:3 (v/v) in culture medium 30 min

1:1 (v/v) in culture medium Pre-incubation 24 h then stressed 2h with H202 5 mM

0.31-1.25 g/L (peptide) and 150 µM (tryptophan) 2 h (peptide) and 1-12 h (tryptophan)

tase. Nrf-2: nuclear response factor 2. GSH-Px2: glutathione peroxidase.

**Cellular mechanism References**

Daly et al. (2010) [42]

Samaranayaka et al. (2010) [50]

> Yao et al. (2010) [51]

Cilla et al. (2011) [43]

Elisia et al. (2011) [48]

Viability maintenance against H202 (500 µM/ 1h)-induced stress Prevention of H202 (50 µM/30 min)-induced DNA damage

Inhibition (at non cytotoxic doses) of intracellular oxidation induced by AAPH (50 µM/1-2 h)

Decrease of H202 (1 mM/30 min)-induced ROS Prevention of H202 (500 µM/30 min) induced DNA damage

Preservation of cell viability Increase in GSH-Rd activity (only Fe or Zn with/without milk samples) Prevention of G1 cell cycle phase decrease induced by H202 Prevention of apoptosis (caspase-3) induced by H202

Exacerbation of AAPH (50 µM/1-2 h) induced oxidative stress (peptide) Up-regulation of Nrf-2 and subsequent upregulation of GSH-Px2 gene as adaptive response to stress (tryptophan)

AAPH: 2,2'-azobis (2-amidinopropane) dihydrochloride. ROS: reactive oxygen species. GSH-Rd: glutathione reduc‐



**Sample (Target compound/s)**

> Resistant starches

> Wheat aleurone

> Wheat aleurone

> > Bread

Bread

**(butyrate)**

**(SCFA and polyphenols)**

**(SCFA)**

**Cell type**

Differentiate d Caco-2 (model of intestinal epithelia)

HT29 (human colon carcinoma)

HT29 (human colon carcinoma)

HT29 (human colon carcinoma)

LT97 (human colon adenoma)

(batch) foods or bioactive constituents in cell lines.

**Cell treatment (Concentration s and time)**

10% (v/v) in culture medium 24 h

10% (v/v) in culture medium 24-72 h

5-10% (v/v) in culture medium 24-72 h

2.5-5% (v/v) in culture medium 24-72 h

5-20% (v/v) in culture medium 24-72 h

thione. CAT: catalase. SULT: Sulfotransferase. AP: Alkaline phosphatase.

**Cellular mechanism References**

Fässler et al. (2007) [63]

http://dx.doi.org/10.5772/51504

145

Borowicki et al. (2010b) [60]

> Stein et al. (2010) [31]

Lux et al. (2011) [61]

Schölrmann et al. (2011) [62]

Preservation of cell viability Prevention of H202 (75 µM/5 min)-induced DNA damage Maintenance of barrier function integrity (TEER)

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion

Cell growth inhibition Cell cycle arrest in G0-G1 phase Apoptosis induction (caspase-3)

Induction of antioxidant enzymes (CAT and GST) Up-regulation of genes *CAT*, *GSTP1* and *SULT2B1* Prevention of H202 (75 µM/5 min)-induced DNA damage

Cell growth inhibition Prevention of H202 (75 µM/5 min)-induced DNA damage

Up-regulation of genes from DNA repair, biotransformation, differentiation and apoptosis Increase in GST activity, GSH content and AP activity (differentiation) Cell growth inhibition Apoptosis induction (caspase-3)

SCFA: Shot chain fatty acids. TEER: Trans Epithelial Electrical Resistance. GST: Glutathione-S-Transferase. GSH: Gluta‐

**Table 3.** Mechanisms involved in the chemopreventive effect of *in vitro* digested (dialysis) plus colonic fermented

SCFA: short chain fatty acids. GST: Glutathione-S-Transferase. HNE: 4-Hydroxynonenal. PARP: Poly (ADP-ribose) pol‐ ymerase. WNT2B: Wingless-type MMTV integration site family member 2.

**Table 2.** Mechanisms involved in the chemopreventive effect of *in vitro* colonic fermented (in batch) of foods or bioactive constituents in cell lines.

Foods or Bioactive Constituents of Foods as Chemopreventives in Cell Lines After Simulated Gastrointestinal Digestion http://dx.doi.org/10.5772/51504 145

**Sample (Target compound/s)**

flour, chicory inulin and wheat

Wheat branderived arabinoxylans

> Inulin-type fructans

Wheat aleurone

**(polyphenols)**

Apples

bioactive constituents in cell lines.

**Cell type**

HT29 (human colon carcinoma)

LT97 and HT29 (human colon adenoma and carcinoma)

LT97 and HT29 (human colon adenoma and carcinoma)

LT97 and HT29 (human colon adenoma and carcinoma)

**Cell treatment (Concentration s and time)**

144 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

0.01-50% (v/v) in culture medium 24-72 h

1.25-20% (v/v) in culture medium 24-72 h

5-10% (v/v) in culture medium 24-72 h

100-900 µg/mL 24-48 h

ymerase. WNT2B: Wingless-type MMTV integration site family member 2.

**Cellular mechanism References**

Glei et al. (2006) [56]

Munjal et al. (2009) [58]

Borowicki et al. (2010a) [59]

Veeriah et al. (2007) [57]

Cell growth inhibition Prevention of HNE (200µM/30 min)-induced DNA damage (at 25-50%) Induction of GST activity (at 10%)

Cell growth inhibition (at 5-10%) Apoptosis induction (cleavage of PARP) only in LT97 cells (at 5-10%)

Cell growth inhibition Apoptosis induction (caspase-3) Up-regulation of genes *p21* (cell cycle arrest) and *WNT2B* (apoptosis)

Cell growth inhibition (LT97 more sensitive than HT29 cells)

SCFA: short chain fatty acids. GST: Glutathione-S-Transferase. HNE: 4-Hydroxynonenal. PARP: Poly (ADP-ribose) pol‐

**Table 2.** Mechanisms involved in the chemopreventive effect of *in vitro* colonic fermented (in batch) of foods or


SCFA: Shot chain fatty acids. TEER: Trans Epithelial Electrical Resistance. GST: Glutathione-S-Transferase. GSH: Gluta‐ thione. CAT: catalase. SULT: Sulfotransferase. AP: Alkaline phosphatase.

**Table 3.** Mechanisms involved in the chemopreventive effect of *in vitro* digested (dialysis) plus colonic fermented (batch) foods or bioactive constituents in cell lines.

### **Acknowledgements**

This work was partially supported by Consolider Fun-C-Food CSD2007-00063 and the Gen‐ eralitat Valenciana (ACOMP 2011/195).

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pancreatic digestion. *Food Chem.*, 102, 865-874.

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751-760.

51-59.

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and signaling pathways. *Mol. Nutr. Food Res.*, 52, 507-526.

gestion models for food applications. *Food Chem.*, 125, 1-12.

### **Author details**

Antonio Cilla\* , Amparo Alegría, Reyes Barberá and María Jesús Lagarda

\*Address all correspondence to: antonio.cilla@uv.es

Nutrition and Food Science Area. Faculty of Pharmacy, University of Valencia, Avda. Vice‐ nte Andrés Estellés s/n, 46100 - Burjassot, Valencia, Spain

### **References**


[9] Liu, R. H., & Finley, J. (2005). Potential cell culture models for antioxidant research. *J. Agric. Food Chem.*, 53, 4311-4314.

**Acknowledgements**

**Author details**

Antonio Cilla\*

**References**

eralitat Valenciana (ACOMP 2011/195).

\*Address all correspondence to: antonio.cilla@uv.es

146 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

nte Andrés Estellés s/n, 46100 - Burjassot, Valencia, Spain

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, Amparo Alegría, Reyes Barberá and María Jesús Lagarda

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**Section 2**

**Cell Damage by Free Radicals - Oxidative Stress**

**in Disease**

**Cell Damage by Free Radicals - Oxidative Stress in Disease**

**Chapter 7**

**The Chemoprevention of Chronic Degenerative Disease**

**Through Dietary Antioxidants: Progress, Promise and**

**Evidences**

Eduardo Madrigal-Santillán,

http://dx.doi.org/10.5772/52162

duction of powerful antibiotics [1].

erative diseases has increased [1].

**1. Introduction**

María del Carmen Valadez-Vega, María Teresa Sumaya-Martínez, Karla Guadalupe Pérez-Ávila and José Antonio Morales-González

Eduardo Madrigal-Bujaidar, Sandra Cruz-Jaime,

Additional information is available at the end of the chapter

**1.1. Epidemiology of chronic degenerative diseases in Mexico and the world**

During the last 30 years relevant changes in the public health field have arisen worldwide, among which the most representative are observed in developed countries where a big deal of infectious diseases have been reduced and controlled as a result of the creation and intro‐

In countries such as Australia, Austria, Belgium, Canada, Denmark, Finland, France, Ger‐ many, Greece, Ireland, Italy, Japan, Luxembourg, Netherlands, New Zealand, Norway, Portugal, Spain, Sweden, Switzerland, the United Kingdom, and the United States, incor‐ porated to the OECD (Organization for Economic Cooperation and Development), mortal‐ ity due to those diseases has diminished up to 38% in people between 35 and 69 years old. Likewise, the risk of mortality before age 70 has diminished up to the 23%. Those re‐ ductions have been the result of social changes and of the improvement of preventive methods of infectious diseases. However, in recent years the prevalence of chronic degen‐

> © 2013 Madrigal-Santillán et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **The Chemoprevention of Chronic Degenerative Disease Through Dietary Antioxidants: Progress, Promise and Evidences**

Eduardo Madrigal-Santillán, Eduardo Madrigal-Bujaidar, Sandra Cruz-Jaime, María del Carmen Valadez-Vega, María Teresa Sumaya-Martínez, Karla Guadalupe Pérez-Ávila and José Antonio Morales-González

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52162

### **1. Introduction**

### **1.1. Epidemiology of chronic degenerative diseases in Mexico and the world**

During the last 30 years relevant changes in the public health field have arisen worldwide, among which the most representative are observed in developed countries where a big deal of infectious diseases have been reduced and controlled as a result of the creation and intro‐ duction of powerful antibiotics [1].

In countries such as Australia, Austria, Belgium, Canada, Denmark, Finland, France, Ger‐ many, Greece, Ireland, Italy, Japan, Luxembourg, Netherlands, New Zealand, Norway, Portugal, Spain, Sweden, Switzerland, the United Kingdom, and the United States, incor‐ porated to the OECD (Organization for Economic Cooperation and Development), mortal‐ ity due to those diseases has diminished up to 38% in people between 35 and 69 years old. Likewise, the risk of mortality before age 70 has diminished up to the 23%. Those re‐ ductions have been the result of social changes and of the improvement of preventive methods of infectious diseases. However, in recent years the prevalence of chronic degen‐ erative diseases has increased [1].

© 2013 Madrigal-Santillán et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chronic degenerative diseases (CDDs) represent a problem of public health for they have become the cause of death worldwide both in adolescents and adults. Among the most prevalent CDDs worldwide is obesity, the cardiovascular diseases (such as hypertension, atherosclerosis), heart diseases, diabetes, chronic respiratory diseases, and cancer; which have caused the 60% of the 58 million yearly deaths, which are approximately 35 million people death for these diseases between 2005 and 2007 [2].

and 2050 the proportion of elderly people in Mexico will grow from 7% to 28% and with it

The Chemoprevention of Chronic Degenerative Disease Through Dietary Antioxidants: Progress, Promise and

In the case of the epidemiological transition, this is characterized by a reduction of morbidi‐ ty and mortality from transmissible diseases and an increase in chronic degenerative diseas‐ es. In recent years, this parameter has shown that in both developed and developing countries, the proportion of infectious diseases in individuals over age 15 is stable, but un‐ fortunately the CDDs are increasing, showing that they occupy almost half of value of mor‐ bility globally. A relevant fact is observed in developed countries (like France, Germany, Japan, United Kingdom, and United States) where the greatest impact of transmissible dis‐ eases remains the HIV/AIDS; but the cerebrovascular diseases and the ischemic heart dis‐ ease are among the main causes of morbidity and mortality (Table 1) in individuals over age

In the specific case of Mexico, it is well-known that infectious diseases made up the profile of mortality in the fifties, since half of the deaths were caused by diarrhea and respiratory infections, for reproductive problems and associated malnutrition conditions. Nowadays, these diseases (classified as lag diseases) are concentrated in less than 15% of deaths [2].

In the last 10 years, there has been an overlap between lag diseases and the so-called emerg‐ ing diseases. Thus, the epidemiological transition has ranked the chronic degenerative dis‐ eases among the 10 leading causes of death, highlighting the type 2 diabetes, obesity,

> Mortality (over 60 years)

> > disease

343 10 Colorectal cancer 477

Cause Deaths

(thousands)

Evidences

157

http://dx.doi.org/10.5772/52162

2399

cardiovascular diseases, malignant neoplasms and cerebrovascular diseases [4].

(thousands)

3 Tuberculosis 1036 3 Chronic obstructive pulmonary

**Table 1.** Leading causes of death in people over 15 years in the world (as a function of AVAD index)

1 HIV / AIDS 2279 1 Ischemic heart disease 5825 2 Ischemic heart disease 1332 2 Cerebrovascular diseases 4689

 Injuries from traffic accidents 814 4 Infections of lower respiratory 1396 Cerebrovascular diseases 783 5 Trachea and lung cancer 928 Self-harm 672 6 Diabetes 754 Violence 473 7 Hypertensive heart disease 735 Liver cirrhosis 382 8 Stomach Cancer 605 Infections of lower respiratory 352 9 Tuberculosis 495

the possibility of an chronic degenerative disease is greater [2,3].

15, both diseases represent more than 36% of deaths worldwide [2].

Mortality

(individuals between 15 and 50 years)

10 Chronic obstructive pulmonary

disease

Cause Deaths

Prevalence in chronic degenerative diseases results from different factors, among which the technologic advance and modernization affect life styles where an increase in process‐ ed foods consumption with a high level of fat content, a sedentary lifestyle since child‐ hood, alcohol and tobacco, stress and a lack of culture in terms of damage prevention and health risks [1,2].

Mexico does not escape this situation as a result of specific factors to our country such as economic development, concentration of population in urban areas, lack of support to im‐ prove the health services and the limitations in preventive programs, particularly in the population under 10 years. Besides, there is a transformation of the population pyramid due to a reduction in mortality and a decrease in birth rate; both phenomena are identi‐ fied as epidemiologic and demographic transitions [2]. In México, the morbidity data pro‐ duced by the CDDs are taken from the statistics of the healthcare sector and published by the healthcare ministry. Although in those reports not all the existing cases are included (not all patients request healthcare services), they are a good help to understand the dam‐ age behavior along with other indicators of prevalence that estimate the number of cases in the population within a specific period of time. Such indicators are obtained from the national healthcare survey and from the national healthcare and nutrition survey 2006 [2]. On the other hand, the mortality statistics are considered as more reliable due to the per‐ manent job in updating the database. The information is obtained from the records of the national institute of statistics, geography and informatics (INEGI) and the general bureau of health information, in conjunction with the epidemiological AVAD index, which is a measure that combines years of healthy life lost due to premature mortality and years of life lost due to disability [3].

As mentioned above, the epidemiological and demographic transitions are important factors for the prevalence of chronic degenerative diseases and indicate changes in the behavior of population dynamics, as well as damage to health which are the result of the low socioeco‐ nomic development and the impact of government policies on public health. The demo‐ graphic transition shows the change in a steady state population with high fertility and mortality associated with the low socioeconomic development process and/or moderniza‐ tion. This process is irreversible and was constructed from the first countries reaching socioeconomic development in Europe such as France and England. In recent years it has made rapid changes affecting the world population [2].

According to data from INEGI and the national population council (CONAPO), Mexico has experienced an accelerated process of demographic transition, which has influenced the eco‐ nomic development and migration, leading to a reduction in mortality and a parallel high birth rates, as well as the consequent population growth, so it is estimated that between 2010 and 2050 the proportion of elderly people in Mexico will grow from 7% to 28% and with it the possibility of an chronic degenerative disease is greater [2,3].

Chronic degenerative diseases (CDDs) represent a problem of public health for they have become the cause of death worldwide both in adolescents and adults. Among the most prevalent CDDs worldwide is obesity, the cardiovascular diseases (such as hypertension, atherosclerosis), heart diseases, diabetes, chronic respiratory diseases, and cancer; which have caused the 60% of the 58 million yearly deaths, which are approximately 35 million

Prevalence in chronic degenerative diseases results from different factors, among which the technologic advance and modernization affect life styles where an increase in process‐ ed foods consumption with a high level of fat content, a sedentary lifestyle since child‐ hood, alcohol and tobacco, stress and a lack of culture in terms of damage prevention and

Mexico does not escape this situation as a result of specific factors to our country such as economic development, concentration of population in urban areas, lack of support to im‐ prove the health services and the limitations in preventive programs, particularly in the population under 10 years. Besides, there is a transformation of the population pyramid due to a reduction in mortality and a decrease in birth rate; both phenomena are identi‐ fied as epidemiologic and demographic transitions [2]. In México, the morbidity data pro‐ duced by the CDDs are taken from the statistics of the healthcare sector and published by the healthcare ministry. Although in those reports not all the existing cases are included (not all patients request healthcare services), they are a good help to understand the dam‐ age behavior along with other indicators of prevalence that estimate the number of cases in the population within a specific period of time. Such indicators are obtained from the national healthcare survey and from the national healthcare and nutrition survey 2006 [2]. On the other hand, the mortality statistics are considered as more reliable due to the per‐ manent job in updating the database. The information is obtained from the records of the national institute of statistics, geography and informatics (INEGI) and the general bureau of health information, in conjunction with the epidemiological AVAD index, which is a measure that combines years of healthy life lost due to premature mortality and years of

As mentioned above, the epidemiological and demographic transitions are important factors for the prevalence of chronic degenerative diseases and indicate changes in the behavior of population dynamics, as well as damage to health which are the result of the low socioeco‐ nomic development and the impact of government policies on public health. The demo‐ graphic transition shows the change in a steady state population with high fertility and mortality associated with the low socioeconomic development process and/or moderniza‐ tion. This process is irreversible and was constructed from the first countries reaching socioeconomic development in Europe such as France and England. In recent years it has made

According to data from INEGI and the national population council (CONAPO), Mexico has experienced an accelerated process of demographic transition, which has influenced the eco‐ nomic development and migration, leading to a reduction in mortality and a parallel high birth rates, as well as the consequent population growth, so it is estimated that between 2010

people death for these diseases between 2005 and 2007 [2].

156 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

health risks [1,2].

life lost due to disability [3].

rapid changes affecting the world population [2].

In the case of the epidemiological transition, this is characterized by a reduction of morbidi‐ ty and mortality from transmissible diseases and an increase in chronic degenerative diseas‐ es. In recent years, this parameter has shown that in both developed and developing countries, the proportion of infectious diseases in individuals over age 15 is stable, but un‐ fortunately the CDDs are increasing, showing that they occupy almost half of value of mor‐ bility globally. A relevant fact is observed in developed countries (like France, Germany, Japan, United Kingdom, and United States) where the greatest impact of transmissible dis‐ eases remains the HIV/AIDS; but the cerebrovascular diseases and the ischemic heart dis‐ ease are among the main causes of morbidity and mortality (Table 1) in individuals over age 15, both diseases represent more than 36% of deaths worldwide [2].

In the specific case of Mexico, it is well-known that infectious diseases made up the profile of mortality in the fifties, since half of the deaths were caused by diarrhea and respiratory infections, for reproductive problems and associated malnutrition conditions. Nowadays, these diseases (classified as lag diseases) are concentrated in less than 15% of deaths [2].

In the last 10 years, there has been an overlap between lag diseases and the so-called emerg‐ ing diseases. Thus, the epidemiological transition has ranked the chronic degenerative dis‐ eases among the 10 leading causes of death, highlighting the type 2 diabetes, obesity, cardiovascular diseases, malignant neoplasms and cerebrovascular diseases [4].


**Table 1.** Leading causes of death in people over 15 years in the world (as a function of AVAD index)

### **2. Definition, importance and control of oxidative stress**

The term "oxidative stress" was first introduced in the eighties by Helmut Sies (1985), defin‐ ing it as a disturbance in the prooxidant-oxidant balance in favor of the first. From that time, a great number of researchers have studied this phenomenon; so, the concept has evolved and now, has been defined as "A situation when steady-state ROS concentration is transi‐ ently or chronically enhanced, disturbing cellular metabolism and its regulation and damag‐ ing cellular constituents" [5,6].

ieve this by using the NADPH produced in different metabolic pathways [14]. A prolonged exposure to ROS can result in diminished NADPH concentration, which is needed in other important physiologic processes, even though some enzymatic AO do not consume cofac‐ tors. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) be‐

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Non-enzymatic antioxidants constitute a heterogeneous group of hydrophobic and hydro‐ philic molecules that trap FR and create chemical species that are less noxious to cell integri‐ ty [18]. Essentially, they give an electron to a FR to stabilize it. Hydrophilic non-enzymatic antioxidants are located mainly in the cytosol, mitochondrial and nuclear matrixes and in extracellular fluids. They are vitamin C, glutathione, uric acid, ergothioneine and polyphe‐

**3. Role of oxidative stress in the development and pathogenesis of the**

Currently, studies related to reactive oxygen species (ROS) and reactive nitrogen species (RNS) have become a relevant issue in research with the main purpose of understanding their functions and effects in the organism. Studies developed throughout the 20th century have explained the action mechanisms of ROS and the operation of the systems responsible for their elimination. These evidences have shown the existence of enzyme systems that pro‐ duce ROS (cytochrome P450, xanthine oxidase, respiratory chain) of the Fenton reaction, cat‐

All researches lead to the same conclusions so far: a) the evidence that the cells have special‐ ized systems to convert ROS into less reactive compounds, and b) if those systems would fail, the ROS could be preexisting compounds for the development of diseases. Thus, several researchers agree in the relevance of "oxidative stress" in medical problems, specifically in

Different observations suggest that these pathologies could be originated when reactive spe‐ cies are formed and suffer alterations, or when they are eliminated, or both. However, the situation is real and much more complicated, for it is difficult to determine the crucial event that originates this disease due to the diversity of forms of oxidative stress (Figure 1). Differ‐ ent researches indicate that mutations produced in genes are responsible of the metabolic unbalance of ROS, while others suggest that environmental changes and common habits weigh on human metabolic processes. However, doubt remains, if oxidative stress is the pri‐ mary event that leads to the disease or the oxidative phenomenon is developed throughout

Whatever the means by which oxidative stress is induced and pathology is developed, the majority of evidences coincide in the relevance of alterations or enzyme deficiencies. These

pathogenesis and/or complications of chronic degenerative diseases [9,12,20,21].

long to this group [12,14,17].

nolic flavonoids [9,18].

the disease [22].

**2.2. Non-enzymatic antioxidants**

**chronic degenerative diseases**

alase, peroxidase, and superoxide dismutase [19].

However, oxidative stress is a phenomenon not entirely detrimental for the organism; also, free radicals (FR) have an important function in several homeostatic processes. They act as intermediate agents in essential oxidation-reduction (redox) reactions. Some examples are the destruction of microorganisms through phagocytosis, synthesis of inflammatory media‐ tors and detoxification. Therefore, FR in low concentrations are useful and even essential [7].

FR represents any chemical species that exists independently and has one or more unmatch‐ ed (odd) electrons rotating in its external atomic orbits. This highly unstable configuration causes this chemical species to be very aggressive and to have a short life span. Once gener‐ ated, FR interact with other molecules through redox reactions to obtain a stable electronic configuration [8-10].

Several authors have classified FR according to the functional group in their molecule, being the most frequent the reactive oxygen species (ROS) and reactive nitrogen species (RNS). Thiol radicals are less important, their reactive group contains sulfur; well as those contain‐ ing carbon or phosphorus in their reactive center. ROS are constituted by superoxide anion (O2•−), hydroxyl radical (•OH), hydrogen peroxide (H2O2), and singlet oxygen. While, that the RNS are nitric oxide (NO), nitrogen dioxide (NO2•−) and peroxynitrite (OONO−) [11,12].

Due to the constant production of ROS and RNS during metabolic processes, the organism has developed a powerful, complex defense system that limits its exposure to these agents these are the so-called antioxidants (AO). Several antioxidants are enzymes or essential nu‐ trients, or include these in their molecular structure. An essential nutrient is a compound that must be eaten because the organism is unable to synthesize it. Based on this characteris‐ tic, some authors classify AO as non-enzymatic and enzymatic [12-14].

#### **2.1. Enzimatic antioxidants**

Some researchers state that the AO function performed by enzymes has advantages com‐ pared to AO compounds, for this activity is regulated according to cellular requirements: they can be induced, inhibited or activated by endogenous effectors [15]. Ho and colleagues (1998) showed evidence of the importance of AO enzymes in protection against oxidant agents. When using transgenic mice designed to overexpress the activity of some AO en‐ zymes, it was noticed that there is a notorious tolerance of certain tissues when they are ex‐ posed to toxics and pathologic conditions that would promote ROS action [9,16].

Enzymatic AO catalyze electron transference from a substrate towards FR. Later, the sub‐ strates or reducing agents used in these reactions are regenerated to be used again, they ach‐ ieve this by using the NADPH produced in different metabolic pathways [14]. A prolonged exposure to ROS can result in diminished NADPH concentration, which is needed in other important physiologic processes, even though some enzymatic AO do not consume cofac‐ tors. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) be‐ long to this group [12,14,17].

### **2.2. Non-enzymatic antioxidants**

**2. Definition, importance and control of oxidative stress**

158 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ing cellular constituents" [5,6].

configuration [8-10].

**2.1. Enzimatic antioxidants**

The term "oxidative stress" was first introduced in the eighties by Helmut Sies (1985), defin‐ ing it as a disturbance in the prooxidant-oxidant balance in favor of the first. From that time, a great number of researchers have studied this phenomenon; so, the concept has evolved and now, has been defined as "A situation when steady-state ROS concentration is transi‐ ently or chronically enhanced, disturbing cellular metabolism and its regulation and damag‐

However, oxidative stress is a phenomenon not entirely detrimental for the organism; also, free radicals (FR) have an important function in several homeostatic processes. They act as intermediate agents in essential oxidation-reduction (redox) reactions. Some examples are the destruction of microorganisms through phagocytosis, synthesis of inflammatory media‐ tors and detoxification. Therefore, FR in low concentrations are useful and even essential [7]. FR represents any chemical species that exists independently and has one or more unmatch‐ ed (odd) electrons rotating in its external atomic orbits. This highly unstable configuration causes this chemical species to be very aggressive and to have a short life span. Once gener‐ ated, FR interact with other molecules through redox reactions to obtain a stable electronic

Several authors have classified FR according to the functional group in their molecule, being the most frequent the reactive oxygen species (ROS) and reactive nitrogen species (RNS). Thiol radicals are less important, their reactive group contains sulfur; well as those contain‐ ing carbon or phosphorus in their reactive center. ROS are constituted by superoxide anion (O2•−), hydroxyl radical (•OH), hydrogen peroxide (H2O2), and singlet oxygen. While, that the RNS are nitric oxide (NO), nitrogen dioxide (NO2•−) and peroxynitrite (OONO−) [11,12]. Due to the constant production of ROS and RNS during metabolic processes, the organism has developed a powerful, complex defense system that limits its exposure to these agents these are the so-called antioxidants (AO). Several antioxidants are enzymes or essential nu‐ trients, or include these in their molecular structure. An essential nutrient is a compound that must be eaten because the organism is unable to synthesize it. Based on this characteris‐

Some researchers state that the AO function performed by enzymes has advantages com‐ pared to AO compounds, for this activity is regulated according to cellular requirements: they can be induced, inhibited or activated by endogenous effectors [15]. Ho and colleagues (1998) showed evidence of the importance of AO enzymes in protection against oxidant agents. When using transgenic mice designed to overexpress the activity of some AO en‐ zymes, it was noticed that there is a notorious tolerance of certain tissues when they are ex‐

Enzymatic AO catalyze electron transference from a substrate towards FR. Later, the sub‐ strates or reducing agents used in these reactions are regenerated to be used again, they ach‐

posed to toxics and pathologic conditions that would promote ROS action [9,16].

tic, some authors classify AO as non-enzymatic and enzymatic [12-14].

Non-enzymatic antioxidants constitute a heterogeneous group of hydrophobic and hydro‐ philic molecules that trap FR and create chemical species that are less noxious to cell integri‐ ty [18]. Essentially, they give an electron to a FR to stabilize it. Hydrophilic non-enzymatic antioxidants are located mainly in the cytosol, mitochondrial and nuclear matrixes and in extracellular fluids. They are vitamin C, glutathione, uric acid, ergothioneine and polyphe‐ nolic flavonoids [9,18].

### **3. Role of oxidative stress in the development and pathogenesis of the chronic degenerative diseases**

Currently, studies related to reactive oxygen species (ROS) and reactive nitrogen species (RNS) have become a relevant issue in research with the main purpose of understanding their functions and effects in the organism. Studies developed throughout the 20th century have explained the action mechanisms of ROS and the operation of the systems responsible for their elimination. These evidences have shown the existence of enzyme systems that pro‐ duce ROS (cytochrome P450, xanthine oxidase, respiratory chain) of the Fenton reaction, cat‐ alase, peroxidase, and superoxide dismutase [19].

All researches lead to the same conclusions so far: a) the evidence that the cells have special‐ ized systems to convert ROS into less reactive compounds, and b) if those systems would fail, the ROS could be preexisting compounds for the development of diseases. Thus, several researchers agree in the relevance of "oxidative stress" in medical problems, specifically in pathogenesis and/or complications of chronic degenerative diseases [9,12,20,21].

Different observations suggest that these pathologies could be originated when reactive spe‐ cies are formed and suffer alterations, or when they are eliminated, or both. However, the situation is real and much more complicated, for it is difficult to determine the crucial event that originates this disease due to the diversity of forms of oxidative stress (Figure 1). Differ‐ ent researches indicate that mutations produced in genes are responsible of the metabolic unbalance of ROS, while others suggest that environmental changes and common habits weigh on human metabolic processes. However, doubt remains, if oxidative stress is the pri‐ mary event that leads to the disease or the oxidative phenomenon is developed throughout the disease [22].

Whatever the means by which oxidative stress is induced and pathology is developed, the majority of evidences coincide in the relevance of alterations or enzyme deficiencies. These deficiencies are often caused by mutations in genes coding antioxidant or related enzymes, for example, by genetic polymorphism.

This concept is frequently related to large number of pathologies. Enzymes involved in de‐ fence against ROS are not an exception. All enzymes contributing to antioxidant defence can be classified to really antioxidant ones, dealing directly with ROS as substrates, and auxili‐ ary ones. The latter enzymes respond for reparation or degradation of oxidatively modified molecules, maturation and posttranslational modification of antioxidant enzymes and me‐ tabolism of low molecular mass antioxidants. As a rule, genetic polymorphisms of enzymes may lead to oxidative stress and consequent diseases, among which cancer, neurodegenera‐ tion, cardiovascular disorders, and diabetes are most frequently mentioned. Among the most studied enzymes with genetic polymorphism is the glucose-6-phosphate dehydrogen‐ ase, catalase, superoxide dismutase, glutathione peroxidase and those involved in repara‐ tion of oxidized molecules and the disease progression [22].

### **3.1. Glucose-6-phosphate dehydrogenase deficiency**

The most striking example among polymorphisms of genes coding enzymes related to anti‐ oxidant defence is well-known deficiency in glucose-6-phosphate dehydrogenase (G6PDH) which leads to favism; genetic disease characterized by the lysis of erythrocytes when con‐ sumed broad beans and other substances which are harmless to the general population [23].

Other pathologies which are related to the same deficiency are diabetes [24,25], vascular dis‐ eases [24], and cancer [26]. In these cases, oxidative stress is induced in specific cells; it was shown that GSH may react with superoxide anion radical providing partial defence against this ROS [27]. When decreasing GSH concentration in G6PDH-deficient individuals enhan‐ ces their sensitivity to redox-active compounds, producing superoxide. Superoxide is able to react also with nitric oxide, leading to the formation of rather harmful oxidant peroxynitrite. However, relation of this reaction to diabetes and vascular diseases is not because of peroxy‐ nitrite production and subsequent oxidative damage, but rather because of decrease in nitric oxide level [28].

The latter is an important second messenger in certain signalling pathways particularly re‐ lated to vasodilation [29]. There is some probability also that individuals with G6PDH-defi‐ ciency may fail to regulate properly blood pressure [30]. Despite possible impairment in nitric oxide production, there is also other way to connect G6PDH deficiency with vascular diseases. It is known, that development of vascular diseases depends on the levels of homo‐ cysteine and folate, intermediates in metabolism of sulfur-containing amino acids [31]. Pro‐ duction of two these metabolites depends on GSH and NADPH levels in cells [32].

**Figure 1.** Scheme of the different ways that produce oxidative stress and stimulate the development of chronic de‐ generative diseases. *Abbreviations*: ROS – reactive oxygen species, RNS – reactive nitrogen species, SOD – superoxide dismutase, CAT - catalase, GSH-Px – glutathione-peroxidase, Prx - peroxyredoxin, GSH – reduced glutathione, CoQH2 –

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On the other hand, NADPH supply at certain conditions may be even harmful leading to enhanced oxidative damage and cancer development. Indeed, it was shown that G6PDH was particularly responsible for cell growth and frequently correlated with cell growth [26]. Tian and colleagues (1998) found that cancer cells possessed several times higher G6PDH activity. The positive correlation between tumour progression and G6PDH activity was

Increased NADPH supply resulting from G6PDH overexpression can lead to so-called "re‐ ductive stress" [37]. Enhanced activity of G6PDH, a lipogenic enzyme, was found at diabe‐ tes and obesity [38]. In humans, G6PDH is regulated by many transcription factors, in

ubiquinol.

found also for humans [35,36].

Data regarding association of G6PDH deficiency with cancer are controversial, because some studies demonstrated that G6PDH-deficient patients may additionally suffer from cancer [33], while others state opposite [34]. Nevertheless, both situations are possible. In particular, there is a large data body indicating that different cancer types are developed at increased DNA damage. It often happens under polymorphism in enzymes contributing to DNA repair, what will be discussed below.

The Chemoprevention of Chronic Degenerative Disease Through Dietary Antioxidants: Progress, Promise and Evidences http://dx.doi.org/10.5772/52162 161

deficiencies are often caused by mutations in genes coding antioxidant or related enzymes,

This concept is frequently related to large number of pathologies. Enzymes involved in de‐ fence against ROS are not an exception. All enzymes contributing to antioxidant defence can be classified to really antioxidant ones, dealing directly with ROS as substrates, and auxili‐ ary ones. The latter enzymes respond for reparation or degradation of oxidatively modified molecules, maturation and posttranslational modification of antioxidant enzymes and me‐ tabolism of low molecular mass antioxidants. As a rule, genetic polymorphisms of enzymes may lead to oxidative stress and consequent diseases, among which cancer, neurodegenera‐ tion, cardiovascular disorders, and diabetes are most frequently mentioned. Among the most studied enzymes with genetic polymorphism is the glucose-6-phosphate dehydrogen‐ ase, catalase, superoxide dismutase, glutathione peroxidase and those involved in repara‐

The most striking example among polymorphisms of genes coding enzymes related to anti‐ oxidant defence is well-known deficiency in glucose-6-phosphate dehydrogenase (G6PDH) which leads to favism; genetic disease characterized by the lysis of erythrocytes when con‐ sumed broad beans and other substances which are harmless to the general population [23].

Other pathologies which are related to the same deficiency are diabetes [24,25], vascular dis‐ eases [24], and cancer [26]. In these cases, oxidative stress is induced in specific cells; it was shown that GSH may react with superoxide anion radical providing partial defence against this ROS [27]. When decreasing GSH concentration in G6PDH-deficient individuals enhan‐ ces their sensitivity to redox-active compounds, producing superoxide. Superoxide is able to react also with nitric oxide, leading to the formation of rather harmful oxidant peroxynitrite. However, relation of this reaction to diabetes and vascular diseases is not because of peroxy‐ nitrite production and subsequent oxidative damage, but rather because of decrease in nitric

The latter is an important second messenger in certain signalling pathways particularly re‐ lated to vasodilation [29]. There is some probability also that individuals with G6PDH-defi‐ ciency may fail to regulate properly blood pressure [30]. Despite possible impairment in nitric oxide production, there is also other way to connect G6PDH deficiency with vascular diseases. It is known, that development of vascular diseases depends on the levels of homo‐ cysteine and folate, intermediates in metabolism of sulfur-containing amino acids [31]. Pro‐

Data regarding association of G6PDH deficiency with cancer are controversial, because some studies demonstrated that G6PDH-deficient patients may additionally suffer from cancer [33], while others state opposite [34]. Nevertheless, both situations are possible. In particular, there is a large data body indicating that different cancer types are developed at increased DNA damage. It often happens under polymorphism in enzymes contributing to

duction of two these metabolites depends on GSH and NADPH levels in cells [32].

for example, by genetic polymorphism.

160 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

tion of oxidized molecules and the disease progression [22].

**3.1. Glucose-6-phosphate dehydrogenase deficiency**

oxide level [28].

DNA repair, what will be discussed below.

**Figure 1.** Scheme of the different ways that produce oxidative stress and stimulate the development of chronic de‐ generative diseases. *Abbreviations*: ROS – reactive oxygen species, RNS – reactive nitrogen species, SOD – superoxide dismutase, CAT - catalase, GSH-Px – glutathione-peroxidase, Prx - peroxyredoxin, GSH – reduced glutathione, CoQH2 – ubiquinol.

On the other hand, NADPH supply at certain conditions may be even harmful leading to enhanced oxidative damage and cancer development. Indeed, it was shown that G6PDH was particularly responsible for cell growth and frequently correlated with cell growth [26]. Tian and colleagues (1998) found that cancer cells possessed several times higher G6PDH activity. The positive correlation between tumour progression and G6PDH activity was found also for humans [35,36].

Increased NADPH supply resulting from G6PDH overexpression can lead to so-called "re‐ ductive stress" [37]. Enhanced activity of G6PDH, a lipogenic enzyme, was found at diabe‐ tes and obesity [38]. In humans, G6PDH is regulated by many transcription factors, in particular, SREBP-1a (sterol regulatory element binding protein) [39], AP-1 [40] and Sp1 [41]. It was shown that elevation of G6PDH activity might lead to enhanced lipid synthesis [42] and to possible reductive stress [43].

SOD) encoded in humans by gene SOD3. The enzyme is a homotetramer presenting in plasma, lymph, and synovial fluid [59]. Extracellular SOD is abundant particularly in the lung, blood vessels, and the heart. Consequently, polymorphism of SOD3 gene is associated

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Polymorphism of glutathione peroxidase (GSH-Px) was found to be associated with some cancers. Four GSH-Px isoforms have been described in humans. It was found that mutations in exon 1 of human GSH-Px-1 gene lead to appearance of polyalanine tract at N-terminus of the protein [59]. These tracts themselves are not connected with diminished enzyme activity. Another polymorphism, substitution of proline-198 to leucine, was found in Japanese dia‐ betic patients and associated with intima-media thickness of carotid arteries [61]. The same substitution for adjacent proline-197 was associated with lung and breast cancers, as well as

Mutations may also affect enzymes involved in DNA reparation. The enzyme 8-hydroxy-2′ deoxyguanosine glycosylase (hOGG) encoded in human genome by the gene hOGG1 is probably the most known example. Recent studies associate mutations in hOGG1 with dif‐ ferent cancer types, such as lung, stomach and bladder cancers [62]. Most of the mutations in this gene affect exon 7 and cause serine-to-cysteine substitution. It was demonstrated that substitution S326C in hOGG1 protein confers susceptibility to oxidation and makes the en‐

Hydrolase MTH1 is other important enzyme preventing incorporation of oxidized purine nucleotide triphosphates in DNA [64]. Knockout of this enzyme in mice resulted in in‐

Other important antioxidant enzymes are glutathione S-transferases (GSTs). Its main function is to conjugation of different electrophilic compounds with glutathione [66]. Oxidatively modi‐ fied compounds as well as lipid oxidation products, like 4-hydroxy-2-nonenal, are subjected to conjugation with glutathione. In general, GSTs are belong to xenobiotic-elimitating system. Some of them, namely GSTs of µ class, are known well by their ability to eliminate polycyclic ar‐ omatic hydrocarbons, oxidized previously by cytochrome P450 monooxygenases. To date, eight classes of GSTs have been described: α, *κ*, µ,σ, ξ, π, θ, and ω. Cytosolic enzymes belong to classes α, µ, π and θ [67]. The gene coding GSTM1 (GST of µ class, isoform 1) is appeared to be

Some studies associate polymorphism of GSTM1 with lung cancer [59,68], although reports are controversial. For example, meta-analysis conducted by [69] found no association of GSTM1 null genotype with lung cancers as well as with smoking. Other authors found such association and reported increased susceptibility to cancerogens among Caucasian and Afri‐ can-American populations [70]. Polymorphism of GSTM1 was also found to be associated with head and neck carcinomas [67]. The need in GSTM1 and its role in prevention of lung cancer are explained by the ability of the enzyme to detoxify constituents of cigarette smoke, such as mentioned above polycyclic aromatic hydrocarbons. Some studies also associate

**3.5. Polymorphism of enzymes involved in reparation of oxidized molecules**

zyme prone to form disulfide bond between different polypeptide chains [63].

creased frequency of lung, stomach and liver tumours with age [65].

highly polymorphic and found inactivated in half of human population.

with pulmonary and cardiovascular diseases [60].

with cardiovascular diseases [59].

#### **3.2. Catalase deficiency**

The first case of catalase deficiency was described by Shigeo Takahara (1947) in a child with cold sores and called acatalasemia to the patology [44]. The cause of this patology is related to ability of oral Streptococci to produce hydrogen peroxide which may promote death of mouth mucosa cells in acatalasemic patients [45]. Catalase deficiency is also associated with diabetes mellitus [46]. This association is attributed for Hungarian hypocatalasemic patients. They were shown to possess higher levels of homocysteine and lower levels of folate [32]. It hints, on one hand, to abnormalities of sulfur metabolism, but on the other hand, it is com‐ monly known that higher homocysteine levels are related to cardiovascular diseases [47], the fact we mentioned above in the context of G6PDH deficiency.

### **3.3. Polymorphism of Cu,Zn-SOD and protein aggregation**

In recent years, the main attention has focused on the polymorphism of genes coding the enzyme superoxide dismutase. More than 100 nucleotide substitutions for the gene SOD1 coding human cytosolic copper- and zinc containing SOD (Cu,Zn-SOD) were described [48]. It is known that several mutations in SOD1 gene are associated with cases of familial amyo‐ trophic lateral sclerosis (ALS), a neurodegenerative disease which is characterized by para‐ lysis and subsequent death [49]. Mechanisms of the disease development are still unknown, but there are many evidences that oxidative stress, developed in neurons, is rather caused by unexpected pro-oxidative activity of SOD than by the loss of the activity at all [50]. It was found that the aggregates cause harm to the cells not only via oxidative stress, but also via inhibition of glutamate receptors [51] and induction of apoptosis [52].

Irwin Fridovich presented some examples of unusual activities of SOD, such as oxidase-like or reductase-like ones [53]. His works and data of other authors suggest that SOD, being mutated or placed in specific conditions, may produce more harmful ROS tan hydrogen per‐ oxide, i.e. hydroxyl radical [54, 55]. Some studies suggested that SOD aggregation can be triggered by higher susceptibility to oxidation of mutated protein [56,57]. Indeed, Cu,Zn-SOD is considered to be rather stable, resistant to many, deleterious to other proteins, com‐ pounds [48]. These evidences suggest that Alzheimer, Huntington, and Parkinson diseases are other pathologies related to this enzymatic alteration [22].

#### **3.4. Polymorphism of Mn-SOD, extracellular SOD and glutathione peroxidase**

Unlike Cu,Zn-SOD, less mutations were found in the gene coding human manganese con‐ taining superoxide dismutase (SOD2). Substitution of alanine-16 to valine (so called "Ala variant") is the most known mutation [58]. This mutation has recently been associated with cancers of breast, prostate, ovaries and bladder, as well as non-Hodgkin lymphoma, meso‐ thelioma and hepatic carcinoma [58]. Mammals possess also extracellular Cu,Zn-SOD (EC- SOD) encoded in humans by gene SOD3. The enzyme is a homotetramer presenting in plasma, lymph, and synovial fluid [59]. Extracellular SOD is abundant particularly in the lung, blood vessels, and the heart. Consequently, polymorphism of SOD3 gene is associated with pulmonary and cardiovascular diseases [60].

particular, SREBP-1a (sterol regulatory element binding protein) [39], AP-1 [40] and Sp1 [41]. It was shown that elevation of G6PDH activity might lead to enhanced lipid synthesis

The first case of catalase deficiency was described by Shigeo Takahara (1947) in a child with cold sores and called acatalasemia to the patology [44]. The cause of this patology is related to ability of oral Streptococci to produce hydrogen peroxide which may promote death of mouth mucosa cells in acatalasemic patients [45]. Catalase deficiency is also associated with diabetes mellitus [46]. This association is attributed for Hungarian hypocatalasemic patients. They were shown to possess higher levels of homocysteine and lower levels of folate [32]. It hints, on one hand, to abnormalities of sulfur metabolism, but on the other hand, it is com‐ monly known that higher homocysteine levels are related to cardiovascular diseases [47],

In recent years, the main attention has focused on the polymorphism of genes coding the enzyme superoxide dismutase. More than 100 nucleotide substitutions for the gene SOD1 coding human cytosolic copper- and zinc containing SOD (Cu,Zn-SOD) were described [48]. It is known that several mutations in SOD1 gene are associated with cases of familial amyo‐ trophic lateral sclerosis (ALS), a neurodegenerative disease which is characterized by para‐ lysis and subsequent death [49]. Mechanisms of the disease development are still unknown, but there are many evidences that oxidative stress, developed in neurons, is rather caused by unexpected pro-oxidative activity of SOD than by the loss of the activity at all [50]. It was found that the aggregates cause harm to the cells not only via oxidative stress, but also via

Irwin Fridovich presented some examples of unusual activities of SOD, such as oxidase-like or reductase-like ones [53]. His works and data of other authors suggest that SOD, being mutated or placed in specific conditions, may produce more harmful ROS tan hydrogen per‐ oxide, i.e. hydroxyl radical [54, 55]. Some studies suggested that SOD aggregation can be triggered by higher susceptibility to oxidation of mutated protein [56,57]. Indeed, Cu,Zn-SOD is considered to be rather stable, resistant to many, deleterious to other proteins, com‐ pounds [48]. These evidences suggest that Alzheimer, Huntington, and Parkinson diseases

Unlike Cu,Zn-SOD, less mutations were found in the gene coding human manganese con‐ taining superoxide dismutase (SOD2). Substitution of alanine-16 to valine (so called "Ala variant") is the most known mutation [58]. This mutation has recently been associated with cancers of breast, prostate, ovaries and bladder, as well as non-Hodgkin lymphoma, meso‐ thelioma and hepatic carcinoma [58]. Mammals possess also extracellular Cu,Zn-SOD (EC-

the fact we mentioned above in the context of G6PDH deficiency.

inhibition of glutamate receptors [51] and induction of apoptosis [52].

are other pathologies related to this enzymatic alteration [22].

**3.4. Polymorphism of Mn-SOD, extracellular SOD and glutathione peroxidase**

**3.3. Polymorphism of Cu,Zn-SOD and protein aggregation**

[42] and to possible reductive stress [43].

162 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**3.2. Catalase deficiency**

Polymorphism of glutathione peroxidase (GSH-Px) was found to be associated with some cancers. Four GSH-Px isoforms have been described in humans. It was found that mutations in exon 1 of human GSH-Px-1 gene lead to appearance of polyalanine tract at N-terminus of the protein [59]. These tracts themselves are not connected with diminished enzyme activity. Another polymorphism, substitution of proline-198 to leucine, was found in Japanese dia‐ betic patients and associated with intima-media thickness of carotid arteries [61]. The same substitution for adjacent proline-197 was associated with lung and breast cancers, as well as with cardiovascular diseases [59].

### **3.5. Polymorphism of enzymes involved in reparation of oxidized molecules**

Mutations may also affect enzymes involved in DNA reparation. The enzyme 8-hydroxy-2′ deoxyguanosine glycosylase (hOGG) encoded in human genome by the gene hOGG1 is probably the most known example. Recent studies associate mutations in hOGG1 with dif‐ ferent cancer types, such as lung, stomach and bladder cancers [62]. Most of the mutations in this gene affect exon 7 and cause serine-to-cysteine substitution. It was demonstrated that substitution S326C in hOGG1 protein confers susceptibility to oxidation and makes the en‐ zyme prone to form disulfide bond between different polypeptide chains [63].

Hydrolase MTH1 is other important enzyme preventing incorporation of oxidized purine nucleotide triphosphates in DNA [64]. Knockout of this enzyme in mice resulted in in‐ creased frequency of lung, stomach and liver tumours with age [65].

Other important antioxidant enzymes are glutathione S-transferases (GSTs). Its main function is to conjugation of different electrophilic compounds with glutathione [66]. Oxidatively modi‐ fied compounds as well as lipid oxidation products, like 4-hydroxy-2-nonenal, are subjected to conjugation with glutathione. In general, GSTs are belong to xenobiotic-elimitating system. Some of them, namely GSTs of µ class, are known well by their ability to eliminate polycyclic ar‐ omatic hydrocarbons, oxidized previously by cytochrome P450 monooxygenases. To date, eight classes of GSTs have been described: α, *κ*, µ,σ, ξ, π, θ, and ω. Cytosolic enzymes belong to classes α, µ, π and θ [67]. The gene coding GSTM1 (GST of µ class, isoform 1) is appeared to be highly polymorphic and found inactivated in half of human population.

Some studies associate polymorphism of GSTM1 with lung cancer [59,68], although reports are controversial. For example, meta-analysis conducted by [69] found no association of GSTM1 null genotype with lung cancers as well as with smoking. Other authors found such association and reported increased susceptibility to cancerogens among Caucasian and Afri‐ can-American populations [70]. Polymorphism of GSTM1 was also found to be associated with head and neck carcinomas [67]. The need in GSTM1 and its role in prevention of lung cancer are explained by the ability of the enzyme to detoxify constituents of cigarette smoke, such as mentioned above polycyclic aromatic hydrocarbons. Some studies also associate lung cancer with polymorphism of GSTT1 (GST of θ class) which participates in catabolism of tobacco smoke constituents, such as halomethanes and butadione [70].

It has been known for some time that dietary factors play a role in the development of some human cancers [73,74] and that some foods contain mutagens and carcinogens [74,75]. In‐ vestigations of last decades, has focused on the existence of a number of non nutritional components in our regular diet that possess antimutagenic and anticarcinogenic properties,

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The chemopreventers are classified as food entities that can prevent the appearance of some long-term diseases like cancer or cardiovascular disorders. It has been suggested that che‐ moprevention should be considered as an inexpensive, easily applicable approach to cancer control and "may become a major weapon in the anticancer arsenal" [76,78,79]. These com‐ pounds can be found in all food categories, but mainly in fruits, vegetables, grains and tea [78,79]. Chemopreventers belong to different classes of chemicals but the most recognized are some vitamins, food polyphenols, flavonoids, catechins, and some components in spices

The mechanisms of action of the chemopreventers are complex and can be categorized ac‐ cording to the site of action or by the specific type of action. It appears that most chemo‐ preventers act primarily as antioxidants. As such, they may scavenge free radicals formed during the preparation of food or as a normal biological process in the body. Recall, that the free radicals can react with DNA, lipids, or cell membranes, leading to aging, injuries of the organ, and greater susceptibility to develop the chronic degenerative disease. Therefore, any event that removes free radicals in the human body is considered beneficial for human health. In addition to their antioxidative activities, there are other mechanisms that show in

**5. Chemopreventive evidence of some fruits and food supplements**

Plants from the genus *Opuntia* are the most abundant of the Cactaceae family, grown throughout the Americas as well as the central area of the Mediterranean, Europe, Asia, Af‐ rica, and Australia. *Opuntia* species display flattened stems called "pencas" or cladodes. The cactus pear (also called prickly pears) is the fruit of this plant (*Opuntia* spp.). The fruit is an oval berry with a large number of seeds and a semi-hard rind with thorns, which may be grouped by fruit colors: red, purple, orange-yellow and white. The fruits with white pulp and green rind are preferred for consumption as food, and their domestic production corre‐ sponds to almost 95% of the total production. Mexico is the main producer of cactus pears and accounts for more than 45% of the worldwide production; however, only 1.5% of this

these compounds have been called as chemopreventers [76,77].

[78, 79].

the Table 2 [80-82].

**5.1. Cactus pears**

**evaluated by our research group**

production is exported [83,84].

#### **3.6. Role of oxidative modifications of antioxidant and related enzymes in disease progression**

Many disorders related to the metabolism of transition metals, amino acids or low molecular mass reductants are known to be connected with activities of antioxidant enzymes. Particu‐ larly, impairement in selenium uptake or synthesis of selenocysteine needed for glutathione peroxidase may lead to GSH-Px deficiency and subsequent disorders such as cardiovascular ones [47]. Disruption of iron-sulfur clusters by superoxide anion radicals or peroxynitrite leads frequently to impairment of many metabolic pathways. Indeed, aconitase, NADHubiquinone-oxidoreductase (complex I of mitochondrial electron transport chain), ubiqui‐ nol-cytochrome c oxidoreductase (complex III), ribonucleotide reductase, ferredoxins possess iron-sulfur clusters, susceptible to oxidation. Owing to this, aconitase is used as one of oxidative stress markers [71]. On the other hand, iron is a component of haem, a prosthet‐ ic group in catalase holoenzyme. Susceptibility to oxidative modification is described for catalase, glutathione peroxidase, Cu,Zn-SOD, and G6PDH. The latter is believed to be one of the most susceptible to oxidation enzymes [22]. Thus, oxidative stress induced by exogenous factors, like carcinogens, certain drugs, ions of transition metals, etc., or by metabolic disor‐ ders, like diabetes, can be exacerbated by oxidative modification of antioxidant enzymes. These assumptions demonstrate the potential of antioxidant therapy in particular cases. At some pathological states, whatever the cause of the disease, oxidative stress is seen to be a powerful exacerbating factor. Type II diabetes, cardiovascular diseases and neurodegenera‐ tive diseases, associated with protein aggregation are among such pathologies. Indeed, en‐ hanced level of glucose results in higher probability of protein glycation [72].

### **4. Impact of chemopreventive agents in the chronic degenerative diseases**

The available evidences indicates that individuals with chronic degenerative diseases are more susceptible to oxidative stress and damage because they have elevated levels of oxi‐ dants and/or reduced antioxidants. Therefore, it has been posited that antioxidant supple‐ mentation in such individuals may be beneficial. Different research has confirmed that many common foods contain nonnutritive components that may provide protection against chronic degenerative diseases, however, the most studies have had impact on the cancer [20,21].

The "chemoprevention" seeks to eliminate precancerous cells in order to avoid the necessity of chemotherapy. It can be further classified as primary, secondary, or tertiary prevention. Primary chemoprevention focuses on preventing the development of precancerous lesions, secondary chemoprevention focuses on preventing the progression of these lesions to can‐ cer, and tertiary chemoprevention aims to prevent the recurrence or spread of a primary cancer [73].

It has been known for some time that dietary factors play a role in the development of some human cancers [73,74] and that some foods contain mutagens and carcinogens [74,75]. In‐ vestigations of last decades, has focused on the existence of a number of non nutritional components in our regular diet that possess antimutagenic and anticarcinogenic properties, these compounds have been called as chemopreventers [76,77].

The chemopreventers are classified as food entities that can prevent the appearance of some long-term diseases like cancer or cardiovascular disorders. It has been suggested that che‐ moprevention should be considered as an inexpensive, easily applicable approach to cancer control and "may become a major weapon in the anticancer arsenal" [76,78,79]. These com‐ pounds can be found in all food categories, but mainly in fruits, vegetables, grains and tea [78,79]. Chemopreventers belong to different classes of chemicals but the most recognized are some vitamins, food polyphenols, flavonoids, catechins, and some components in spices [78, 79].

The mechanisms of action of the chemopreventers are complex and can be categorized ac‐ cording to the site of action or by the specific type of action. It appears that most chemo‐ preventers act primarily as antioxidants. As such, they may scavenge free radicals formed during the preparation of food or as a normal biological process in the body. Recall, that the free radicals can react with DNA, lipids, or cell membranes, leading to aging, injuries of the organ, and greater susceptibility to develop the chronic degenerative disease. Therefore, any event that removes free radicals in the human body is considered beneficial for human health. In addition to their antioxidative activities, there are other mechanisms that show in the Table 2 [80-82].

### **5. Chemopreventive evidence of some fruits and food supplements evaluated by our research group**

### **5.1. Cactus pears**

lung cancer with polymorphism of GSTT1 (GST of θ class) which participates in catabolism

Many disorders related to the metabolism of transition metals, amino acids or low molecular mass reductants are known to be connected with activities of antioxidant enzymes. Particu‐ larly, impairement in selenium uptake or synthesis of selenocysteine needed for glutathione peroxidase may lead to GSH-Px deficiency and subsequent disorders such as cardiovascular ones [47]. Disruption of iron-sulfur clusters by superoxide anion radicals or peroxynitrite leads frequently to impairment of many metabolic pathways. Indeed, aconitase, NADHubiquinone-oxidoreductase (complex I of mitochondrial electron transport chain), ubiqui‐ nol-cytochrome c oxidoreductase (complex III), ribonucleotide reductase, ferredoxins possess iron-sulfur clusters, susceptible to oxidation. Owing to this, aconitase is used as one of oxidative stress markers [71]. On the other hand, iron is a component of haem, a prosthet‐ ic group in catalase holoenzyme. Susceptibility to oxidative modification is described for catalase, glutathione peroxidase, Cu,Zn-SOD, and G6PDH. The latter is believed to be one of the most susceptible to oxidation enzymes [22]. Thus, oxidative stress induced by exogenous factors, like carcinogens, certain drugs, ions of transition metals, etc., or by metabolic disor‐ ders, like diabetes, can be exacerbated by oxidative modification of antioxidant enzymes. These assumptions demonstrate the potential of antioxidant therapy in particular cases. At some pathological states, whatever the cause of the disease, oxidative stress is seen to be a powerful exacerbating factor. Type II diabetes, cardiovascular diseases and neurodegenera‐ tive diseases, associated with protein aggregation are among such pathologies. Indeed, en‐

**3.6. Role of oxidative modifications of antioxidant and related enzymes in disease**

of tobacco smoke constituents, such as halomethanes and butadione [70].

164 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

hanced level of glucose results in higher probability of protein glycation [72].

**4. Impact of chemopreventive agents in the chronic degenerative diseases**

The available evidences indicates that individuals with chronic degenerative diseases are more susceptible to oxidative stress and damage because they have elevated levels of oxi‐ dants and/or reduced antioxidants. Therefore, it has been posited that antioxidant supple‐ mentation in such individuals may be beneficial. Different research has confirmed that many common foods contain nonnutritive components that may provide protection against chronic degenerative diseases, however, the most studies have had impact on the

The "chemoprevention" seeks to eliminate precancerous cells in order to avoid the necessity of chemotherapy. It can be further classified as primary, secondary, or tertiary prevention. Primary chemoprevention focuses on preventing the development of precancerous lesions, secondary chemoprevention focuses on preventing the progression of these lesions to can‐ cer, and tertiary chemoprevention aims to prevent the recurrence or spread of a primary

**progression**

cancer [20,21].

cancer [73].

Plants from the genus *Opuntia* are the most abundant of the Cactaceae family, grown throughout the Americas as well as the central area of the Mediterranean, Europe, Asia, Af‐ rica, and Australia. *Opuntia* species display flattened stems called "pencas" or cladodes. The cactus pear (also called prickly pears) is the fruit of this plant (*Opuntia* spp.). The fruit is an oval berry with a large number of seeds and a semi-hard rind with thorns, which may be grouped by fruit colors: red, purple, orange-yellow and white. The fruits with white pulp and green rind are preferred for consumption as food, and their domestic production corre‐ sponds to almost 95% of the total production. Mexico is the main producer of cactus pears and accounts for more than 45% of the worldwide production; however, only 1.5% of this production is exported [83,84].


color. These pigments have shown beneficial effects on the redox-regulated pathways in‐ volved in cell growth and inflammation, and have not shown toxic effects in humans [90,91].

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In addition, a neuroprotector activity against oxidative damage induced in cultures of rat cortical cells has been attributed to the cactus pear flavonoids [92]. Another beneficial effect of the fruit was observed in the prevention of stomach ulcers through the stimulation of prostaglandin production: cactus pear promoted mucous secretion of bicarbonate, involved in the protection of gastric mucosa [93]. On the other hand, their contents of natural antioxi‐ dants has raised interest in the use of cactus pears as substitute for synthetic antioxidants,

In the Institute of Health Sciences (Autonomous University of Hidalgo State) have been per‐ formed studies to demostrate the chemopreventive capacity of the cactus pear. The first studies were developed by Hernández-Ceruelos et al. (2009) with the main objective to eval‐ uate the antioxidant effect of three varieties of prickly pear juice (red-purple, white-green and yellow-orange) in four different concentrations (25, 50, 75 and 100%) by the technique of DPPH (1,1-diphenyl-2-picrylhydrazyl). Their results indicated that the juice of princkly pear variety red-purple (PPRP) had the highest antioxidant capacity in all concentrations in com‐ parison with the positive control (vitamin E). Subsequently, researchers evaluated the anti‐ clastogenic potential of PPRP by micronucleus assay against of methyl methane sulfonate (MMS) in mice. This experiment had a duration of 2 weeks, was included a negative control (animals treated with water), a positive control of MMS (40 mg/kg), a group of mice treated with princkly pear variety red-purple (25mL/Kg), and three groups with PPRP (in doses of 25, 16.5 and 8.3 mL/Kg) plus the mutagen. The PPRP was administered by oral gavage and the mutagen was injected intraperitoneally 5 days before the end of the experiment (single dose). Finally, blood samples were obtained in four times (0, 24, 48 and 72 hours) to deter‐ mine the frequency of micronucleated polychromatic erythrocytes (MNPE). The result indi‐ cated that PPRP is not a genotoxic agent, on the contrary, may reduce the number of micronucleated polychromatic erythrocytes. In this regard, the princkly pear variety redpurple showed an anticlastogenic effect directly proportional to the concentrations. The highest protection was obtained with the concentration of 25 mL/Kg (approximately, 80%)

In the second study was evaluated the antioxidant activities [with three assays: a)1,1-di‐ phenyl-2-picrylhydrazyl radical-scavenging, b) protection against oxidation of a β-carotenelinoleic acid emulsion, and c) iron(II) chelation], the content of total phenolic compounds, ascorbic acid, betacyanin, betaxanthins and the stability of betacyanin pigments in presence of Cu(II)-dependent hydroxyl radicals (OH•), in 18 cultivars of purple, red, yellow and white cactus pear from six Mexican states (Hidalgo, Puebla, Guanajuato, Jalisco, Zacatecas and the State of Mexico). The results indicated that the antiradical activities from yellow and white cactus pear cultivars were not significantly different and were lower than the average antiradical activities in red and purple cultivars. The red cactus pear from the state of Zaca‐ tecas showed the highest antioxidant activity. The free radical scavenging activity for red cactus pears was significantly correlated to the concentration of total phenolic compounds (*R*2 = 0.90) and ascorbic acid (*R*2 = 0.86). All 18 cultivars of cactus pears studied showed sig‐

such as butylhydroxytoluene (BHT), butylhydroxyanisole (BHA) [88].

after 48 hours of treatment [94].

**Table 2.** Other mechanisms of chemoprevention

A viable strategy to increase the competitiveness of the Mexican cactus pear in national and international markets is the innovation and creation of new high value-added products. This could be achieved by determining the nutritional and functional properties that differentiate the Mexican cactus pear from analogous products. In addition, providing functional prod‐ ucts for a market in constant growth would offer a key competitive advantage and would allow the producers to diversify its commercialization, not as fresh fruit only, but also as an ingredient or high-value additive for the food industry. A commercialization of the cactuspear based on its antioxidant properties could generate competitive advantages that may turn into business opportunities and the development of new products [85].

Different studies with the varieties of European and Asian cactus pears have shown notable antioxidant activities that significantly reduce oxidative stress in patients and may help in preventing chronic pathologies (as diabetes and cancer) [85-87]. For this reason, the cactus pear is considered a functional food; this feature is attributed to its bioactive compounds such as vitamin C and vitamin E, polyphenols, carotenoids, flavonoid compounds (e.g., kaempferol, quercetin, and isorhamnetin), taurine and pigments [88,89].

Betalains are water-soluble pigments. Two betalain derivatives are present in cactus-pears: betacyanin, which gives the red-purple color, and betaxanthin, which gives a yellow-orange color. These pigments have shown beneficial effects on the redox-regulated pathways in‐ volved in cell growth and inflammation, and have not shown toxic effects in humans [90,91].

**Mechanism Action Examples**

Inducing agents Agents that induce or enhance enzyme activity (e.g.,

166 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Suppressing agents Agents that may react on different processes (e.g., inhibition of

or have effects on frank malignancies

apoptosis.

**Table 2.** Other mechanisms of chemoprevention

Agents that block or inhibit to the enzymes responsible for the biotransformation of procarcinogens to carcinogen form

glutathione S-transferase, GST) for detoxify and reduce the level of mutagenic/carcinogenic species of the body

arachidonic acid metabolism, activity of protease or protein kinase C) involved in tumor promotion/progression

Since the immune system can influence on growth either via effects on the inflammation status or by causing apoptosis. Some chemopreventers can act on the early stages in neoplasia

Some chemopreventers may alter signaling pathways of receptors for hormones and others factors responsible for cell regulation and can be modified the potential for growth, either by acting to increase mitosis or alter the level of

turn into business opportunities and the development of new products [85].

kaempferol, quercetin, and isorhamnetin), taurine and pigments [88,89].

A viable strategy to increase the competitiveness of the Mexican cactus pear in national and international markets is the innovation and creation of new high value-added products. This could be achieved by determining the nutritional and functional properties that differentiate the Mexican cactus pear from analogous products. In addition, providing functional prod‐ ucts for a market in constant growth would offer a key competitive advantage and would allow the producers to diversify its commercialization, not as fresh fruit only, but also as an ingredient or high-value additive for the food industry. A commercialization of the cactuspear based on its antioxidant properties could generate competitive advantages that may

Different studies with the varieties of European and Asian cactus pears have shown notable antioxidant activities that significantly reduce oxidative stress in patients and may help in preventing chronic pathologies (as diabetes and cancer) [85-87]. For this reason, the cactus pear is considered a functional food; this feature is attributed to its bioactive compounds such as vitamin C and vitamin E, polyphenols, carotenoids, flavonoid compounds (e.g.,

Betalains are water-soluble pigments. Two betalain derivatives are present in cactus-pears: betacyanin, which gives the red-purple color, and betaxanthin, which gives a yellow-orange

Dithiocarbamates, isothiocyanates, diallyl sulfide, and ellagic acid

Isothiocyanates,

curcurains

selenium

lactoferrin

retinoids

sulfaraphane, d-limonene, terpinoids, turmeric, and

isoflavones, phytoestrogens,

Carotenoid, flavonoids,

d-limonene, sulfur compounds, lactoferrin,

Inhibition of carcinogen formation

Immune activity and modulation

Signal transduction pathways and their regulation

In addition, a neuroprotector activity against oxidative damage induced in cultures of rat cortical cells has been attributed to the cactus pear flavonoids [92]. Another beneficial effect of the fruit was observed in the prevention of stomach ulcers through the stimulation of prostaglandin production: cactus pear promoted mucous secretion of bicarbonate, involved in the protection of gastric mucosa [93]. On the other hand, their contents of natural antioxi‐ dants has raised interest in the use of cactus pears as substitute for synthetic antioxidants, such as butylhydroxytoluene (BHT), butylhydroxyanisole (BHA) [88].

In the Institute of Health Sciences (Autonomous University of Hidalgo State) have been per‐ formed studies to demostrate the chemopreventive capacity of the cactus pear. The first studies were developed by Hernández-Ceruelos et al. (2009) with the main objective to eval‐ uate the antioxidant effect of three varieties of prickly pear juice (red-purple, white-green and yellow-orange) in four different concentrations (25, 50, 75 and 100%) by the technique of DPPH (1,1-diphenyl-2-picrylhydrazyl). Their results indicated that the juice of princkly pear variety red-purple (PPRP) had the highest antioxidant capacity in all concentrations in com‐ parison with the positive control (vitamin E). Subsequently, researchers evaluated the anti‐ clastogenic potential of PPRP by micronucleus assay against of methyl methane sulfonate (MMS) in mice. This experiment had a duration of 2 weeks, was included a negative control (animals treated with water), a positive control of MMS (40 mg/kg), a group of mice treated with princkly pear variety red-purple (25mL/Kg), and three groups with PPRP (in doses of 25, 16.5 and 8.3 mL/Kg) plus the mutagen. The PPRP was administered by oral gavage and the mutagen was injected intraperitoneally 5 days before the end of the experiment (single dose). Finally, blood samples were obtained in four times (0, 24, 48 and 72 hours) to deter‐ mine the frequency of micronucleated polychromatic erythrocytes (MNPE). The result indi‐ cated that PPRP is not a genotoxic agent, on the contrary, may reduce the number of micronucleated polychromatic erythrocytes. In this regard, the princkly pear variety redpurple showed an anticlastogenic effect directly proportional to the concentrations. The highest protection was obtained with the concentration of 25 mL/Kg (approximately, 80%) after 48 hours of treatment [94].

In the second study was evaluated the antioxidant activities [with three assays: a)1,1-di‐ phenyl-2-picrylhydrazyl radical-scavenging, b) protection against oxidation of a β-carotenelinoleic acid emulsion, and c) iron(II) chelation], the content of total phenolic compounds, ascorbic acid, betacyanin, betaxanthins and the stability of betacyanin pigments in presence of Cu(II)-dependent hydroxyl radicals (OH•), in 18 cultivars of purple, red, yellow and white cactus pear from six Mexican states (Hidalgo, Puebla, Guanajuato, Jalisco, Zacatecas and the State of Mexico). The results indicated that the antiradical activities from yellow and white cactus pear cultivars were not significantly different and were lower than the average antiradical activities in red and purple cultivars. The red cactus pear from the state of Zaca‐ tecas showed the highest antioxidant activity. The free radical scavenging activity for red cactus pears was significantly correlated to the concentration of total phenolic compounds (*R*2 = 0.90) and ascorbic acid (*R*2 = 0.86). All 18 cultivars of cactus pears studied showed sig‐ nificant chelating activity of ferrous ions. The red and purple cactus pears showed a great stability when exposed to OH• [88].

Although the types of berry fruits consumed worldwide are many, the experiment executed in our laboratory is focuses on cranberries that are commonly consumed in Mexico, especial‐ ly in the states of Tlaxcala, Hidalgo, and Puebla. The purpose of our study was to determine whether cranberry ethanolic extract (CEE) can prevent the DNA damage produced by ben‐ zo[a]pyrene (B[a]P) using an in vivo mouse peripheral blood micronucleus assay. The exper‐ imental groups were organized as follows: a negative control group (without treatment), a positive group treated with B[a]P (200 mg/kg), a group administered with 800 mg/kg of cranberry ethanolic extract, and three groups treated with B[a]P and cranberry ethanolic ex‐ tract (200, 400, and 800 mg/kg) respectively. The CEE and benzo[a]pyrene were adminis‐ tered orally for a week, on a daily basis. During this period the body weight, the feed intake, and the determination of antigenotoxic potential were quantified. At the end of this period, we continued with the same determinations for one week more (recovery period) but any‐ more administration of the substances. The animals treated with B[a]P showed a weight in‐ crease after the first week of administration. The same phenomenon was observed in the lots combined with B[a]P and CEE (low and medium doses). The dose of 800 mg/kg of CEE showed similar values to the control group at the end of the treatment period. In the second part of the assay, when the substances were not administered, these experimental groups re‐ gained their normal weight. The dose of CEE (800 mg/kg) was not genotoxic nor cytotoxic. On the contrary, the B[a]P increases the frequency of micronucleated normochromatic eryth‐ rocytes (MNNE) and reduces the rate of polychromatic erythrocytes (PE) at the end of the treatment period. With respect to the combined lots, a significant decrease in the MN rate was observed from the sixth to the eighth day of treatment with the two high doses applied; the highest protection (60%) was obtained with 800 mg/kg of CEE. The same dose showed an anticytotoxic effect which corresponded to an improvement of 62.5% in relation to the an‐ imals administered with the B[a]P. In the second period, all groups reached values that have been seen in the control group animals. Our results suggest that the inhibition of clastoge‐ nicity of the cranberry ethanolic extract against B[a]P is related to the antioxidant capacity of

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the combination of phytochemicals present in its chemical composition [107].

and largely an oblate spheroid and generally, is consumed in form of juice [108].

The grapefruit is a subtropical citrus tree known for its bitter fruit. These evergreen trees usually grow around 6 meters tall. The leaves are dark green, long and thin. His fruit (called toronja in Spanish) has become popular since the late 19th century, is yellow-orange skinned

The grapefruit juice is an excellent source of many nutrients and phytochemicals that con‐ tribute to a healthy diet. Is a good source of vitamin C, contains the fiber pectin, and the va‐ rieties pink and red contain the beneficial antioxidant lycopene [108]. But, the main flavonoid, existing in highest concentration in grapefruit juice is naringin, which in humans

Since grapefruit juice is known to inhibit enzymes necessary for the clearance of some drugs and hormones, some researchers have hypothesized that grapefruit juice and the naringin may play an indirect role in the development of hormone-dependent cancers. A study found

**5.3. Grapefruit juice and naringin**

is metabolized to naringenin [109].

### **5.2. Cranberries**

Among small soft-fleshed colorful fruits, berries make up the largest proportion consumed in our diet. Berry fruits are popularly consumed not only in fresh and frozen forms, but also as processed and derived products including canned fruits, yogurts, beverages, jams, and jellies. In addition, there has been a growing trend in the intake of berry extracts as ingredi‐ ents in functional foods and dietary supplements, which may or may not be combined with other colorful fruits, vegetables, and herbal extracts [95]. Berry fruits commonly consumed in America include blackberries, black raspberries, red raspberries and strawberries, blue‐ berries, and cranberries.

Other "niche-cultivated" berries and forest/wild berries, for example, bilberries, black cur‐ rant, lingonberry, and cloudberry, are also popularly consumed in other regions of the World [95]. The North American cranberry (*Vaccinium macrocarpon*) is of a growing public interest as a functional food because of potential health benefits linked to phytochemicals of the fruit. Cranberry juice has long been consumed for the prevention of urinary tract infec‐ tions, and research linked this property to the ability of cranberry proanthocyanidins to in‐ hibit the adhesion of *Escherichia coli* bacteria responsible for these infections [96]. These studies, which brought to light the unique structural features of cranberry proanthocyani‐ dins [97], have sparked numerous clinical studies probing a cranberry´s role in the preven‐ tion of urinary tract infections and targeted the nature of the active metabolites. Further antibacterial adhesion studies demonstrated that cranberry constituents also inhibit the ad‐ hesion of *Helicobacter pylori*, a major cause of gastric cancer, to human gastric mucus [98]. The earliest report of potential anti-carcinogenic activity appeared in 1996 in the University of Illinois [99].

Extracts of cranberry and bilberry were observed to inhibit ornithine decarboxylase (ODC) expression and induce the xenobiotic detoxification enzyme quinonereductase *in vitro* [99]. Subsequent studies with cranberry and other berries in cellular models have focused on some cancers such as breast, colon, liver, prostate and lung [100-102]. This biological activity of berries are partially attributed to their high content of a diverse range of phytochemicals such as flavonoids (anthocyanins, flavonols, and flavanols), tannins (proanthocyanidins, el‐ lagitannins, and gallotannins), quercetin, phenolic acids, lignans, and stilbenoids (e.g., re‐ sveratrol) [100]. With respect to his genotoxic and/or antigenotoxic potential, there are few reports in the literature that demonstrate this effect and the majority of studies were per‐ formed *in vitro* cell culture models [101,103,104]. Boateng *et al*. demonstrated that consump‐ tion of some juices of berries (as blueberries, blackberries, and cranberry) can reduce the formation of aberrant crypt foci (ACF) induced by azoxymethane in Fisher male rats [105]. Another study, in which it was administrated a lyophilized extract of *Vaccinium ashei* berries in male Swiss mice during 30 days, showed to have improved the performance on memory tasks and has a protective effect on the DNA damage in brain tissue evaluated with the com‐ et assay [106].

Although the types of berry fruits consumed worldwide are many, the experiment executed in our laboratory is focuses on cranberries that are commonly consumed in Mexico, especial‐ ly in the states of Tlaxcala, Hidalgo, and Puebla. The purpose of our study was to determine whether cranberry ethanolic extract (CEE) can prevent the DNA damage produced by ben‐ zo[a]pyrene (B[a]P) using an in vivo mouse peripheral blood micronucleus assay. The exper‐ imental groups were organized as follows: a negative control group (without treatment), a positive group treated with B[a]P (200 mg/kg), a group administered with 800 mg/kg of cranberry ethanolic extract, and three groups treated with B[a]P and cranberry ethanolic ex‐ tract (200, 400, and 800 mg/kg) respectively. The CEE and benzo[a]pyrene were adminis‐ tered orally for a week, on a daily basis. During this period the body weight, the feed intake, and the determination of antigenotoxic potential were quantified. At the end of this period, we continued with the same determinations for one week more (recovery period) but any‐ more administration of the substances. The animals treated with B[a]P showed a weight in‐ crease after the first week of administration. The same phenomenon was observed in the lots combined with B[a]P and CEE (low and medium doses). The dose of 800 mg/kg of CEE showed similar values to the control group at the end of the treatment period. In the second part of the assay, when the substances were not administered, these experimental groups re‐ gained their normal weight. The dose of CEE (800 mg/kg) was not genotoxic nor cytotoxic. On the contrary, the B[a]P increases the frequency of micronucleated normochromatic eryth‐ rocytes (MNNE) and reduces the rate of polychromatic erythrocytes (PE) at the end of the treatment period. With respect to the combined lots, a significant decrease in the MN rate was observed from the sixth to the eighth day of treatment with the two high doses applied; the highest protection (60%) was obtained with 800 mg/kg of CEE. The same dose showed an anticytotoxic effect which corresponded to an improvement of 62.5% in relation to the an‐ imals administered with the B[a]P. In the second period, all groups reached values that have been seen in the control group animals. Our results suggest that the inhibition of clastoge‐ nicity of the cranberry ethanolic extract against B[a]P is related to the antioxidant capacity of the combination of phytochemicals present in its chemical composition [107].

### **5.3. Grapefruit juice and naringin**

nificant chelating activity of ferrous ions. The red and purple cactus pears showed a great

Among small soft-fleshed colorful fruits, berries make up the largest proportion consumed in our diet. Berry fruits are popularly consumed not only in fresh and frozen forms, but also as processed and derived products including canned fruits, yogurts, beverages, jams, and jellies. In addition, there has been a growing trend in the intake of berry extracts as ingredi‐ ents in functional foods and dietary supplements, which may or may not be combined with other colorful fruits, vegetables, and herbal extracts [95]. Berry fruits commonly consumed in America include blackberries, black raspberries, red raspberries and strawberries, blue‐

Other "niche-cultivated" berries and forest/wild berries, for example, bilberries, black cur‐ rant, lingonberry, and cloudberry, are also popularly consumed in other regions of the World [95]. The North American cranberry (*Vaccinium macrocarpon*) is of a growing public interest as a functional food because of potential health benefits linked to phytochemicals of the fruit. Cranberry juice has long been consumed for the prevention of urinary tract infec‐ tions, and research linked this property to the ability of cranberry proanthocyanidins to in‐ hibit the adhesion of *Escherichia coli* bacteria responsible for these infections [96]. These studies, which brought to light the unique structural features of cranberry proanthocyani‐ dins [97], have sparked numerous clinical studies probing a cranberry´s role in the preven‐ tion of urinary tract infections and targeted the nature of the active metabolites. Further antibacterial adhesion studies demonstrated that cranberry constituents also inhibit the ad‐ hesion of *Helicobacter pylori*, a major cause of gastric cancer, to human gastric mucus [98]. The earliest report of potential anti-carcinogenic activity appeared in 1996 in the University

Extracts of cranberry and bilberry were observed to inhibit ornithine decarboxylase (ODC) expression and induce the xenobiotic detoxification enzyme quinonereductase *in vitro* [99]. Subsequent studies with cranberry and other berries in cellular models have focused on some cancers such as breast, colon, liver, prostate and lung [100-102]. This biological activity of berries are partially attributed to their high content of a diverse range of phytochemicals such as flavonoids (anthocyanins, flavonols, and flavanols), tannins (proanthocyanidins, el‐ lagitannins, and gallotannins), quercetin, phenolic acids, lignans, and stilbenoids (e.g., re‐ sveratrol) [100]. With respect to his genotoxic and/or antigenotoxic potential, there are few reports in the literature that demonstrate this effect and the majority of studies were per‐ formed *in vitro* cell culture models [101,103,104]. Boateng *et al*. demonstrated that consump‐ tion of some juices of berries (as blueberries, blackberries, and cranberry) can reduce the formation of aberrant crypt foci (ACF) induced by azoxymethane in Fisher male rats [105]. Another study, in which it was administrated a lyophilized extract of *Vaccinium ashei* berries in male Swiss mice during 30 days, showed to have improved the performance on memory tasks and has a protective effect on the DNA damage in brain tissue evaluated with the com‐

stability when exposed to OH• [88].

168 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**5.2. Cranberries**

berries, and cranberries.

of Illinois [99].

et assay [106].

The grapefruit is a subtropical citrus tree known for its bitter fruit. These evergreen trees usually grow around 6 meters tall. The leaves are dark green, long and thin. His fruit (called toronja in Spanish) has become popular since the late 19th century, is yellow-orange skinned and largely an oblate spheroid and generally, is consumed in form of juice [108].

The grapefruit juice is an excellent source of many nutrients and phytochemicals that con‐ tribute to a healthy diet. Is a good source of vitamin C, contains the fiber pectin, and the va‐ rieties pink and red contain the beneficial antioxidant lycopene [108]. But, the main flavonoid, existing in highest concentration in grapefruit juice is naringin, which in humans is metabolized to naringenin [109].

Since grapefruit juice is known to inhibit enzymes necessary for the clearance of some drugs and hormones, some researchers have hypothesized that grapefruit juice and the naringin may play an indirect role in the development of hormone-dependent cancers. A study found

a correlation between eating a quarter of grapefruit daily and a 30% increase in risk for breast cancer in post-menopausal women. The study points to the inhibition of CYP3A4 en‐ zyme by grapefruit, which metabolizes estrogen [110]. However, an investigation conducted in 2008 has shown that grapefruit consumption does not increase breast cancer risk and found a significant decrease in breast cancer risk with greater intake of grapefruit in women who never used hormone therapy [111].

With regard to grapefruit juice (GJ), we performed two experiments which are summarized below. The first evaluated the capacity of GJ to inhibit the micronucleated polychromatic er‐ ythrocytes (MNPE) produced by daunorubicin in an acute assay in mice, as well as to deter‐ mine its antioxidant potential in mouse hepatic microsomes, and its capacity to trap free

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The results showed that GJ is not toxic or genotoxic damage; on the contrary, it generated a significant reduction of the MNPE formed by daunorubicin. The effect was found through‐ out the examined schedule (from 24 to 96 h). The two high doses produced inhibition of about 60% at 48 h, 86% at 72 h and 100% at 96 h after the treatment. With respect to the grapefruit juice antioxidant potential, a 50% decrease in liver microsomal lipid peroxidation produced by daunorubicin was found by quantifying malondialdehyde formation. Finally, a strong GJ scavenging activity evaluated with the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was observed, giving rise to a concentration-dependent curve with a correlation coefficient of 0.98. Overall, our results established an efficient anticlastogenic potential of grapefruit juice, probably related to its antioxidant capacity, or to alterations of daunorubicin metabo‐

Based on this background; recently, we finished another study in which using the comet as‐ say was demonstrated a strong effect by hydrogen peroxide (HP) and no damage by grape‐ fruit juice (GJ) in human lymphocytes. Cells exposed to HP and treated with GJ was shown an increase of DNA damage by HP over the control level, and a decrease of such damage by GJ. With the comet assay plus formamidopyrimidine-DNA-glycosylase we found the stron‐ gest increase of DNA damage by HP over the control level, and the strongest reduction of such damage by GJ. By applying the comet/FISH method we determined 98% of the p53 gene signals in the comet head of control cells along the experiment, in contrast with about 90% signals in the comet tail of cells exposed to HP. Cells treated with both agents showed a significant, concentration/time dependent return of p53 signals to the head, suggesting en‐ hancement of the gene repair. Finally, with the annexin V assay we found an increase in apoptosis and necrosis by HP, and no effect by GJ; when GJ was added to HP treated cells no modification was observed in regard to apoptosis, although a decrease of necrosis was

Chamomile (*Matricaria chamomilla* or *Chamomilla recutita*) is an asteraceae plant native to Eu‐ rope and distributed around the world, except in tropical and polar regions. This plant has been used for its curative properties since ancient Egyptian and Greek times, and at present is frequently used as an antiseptic, antiflogistic, diuretic, expectorant, febrifuge, sedative, an‐ ti-inflammatory and anticarcinogen [116]. Pharmacological activities of various components of the plant have been reported, for example, the anti-inflammatory capacity and the modu‐ lating effects of the heat shock protein on apigenin and quercetin flavonoids, as well as the anti-inflammatory, antioxidant, and antiseptic activities detected on α-bisabolol, guargazu‐ lene, and chamazulene [117, 118]. The essential oil extracted from the chamomile flower var‐

radicals in vitro.

lism [114].

observed [115].

**5.4. Chamomile**

In the case of naringin, this compound exerts a variety of pharmacological effects such as antioxidant activity, blood lipid lowering, anticancer activity, and inhibition of selected drug-metabolizing cytochrome P450 enzymes, including CYP3A4 and CYP1A2, which may result in drug-drug interactions in vivo. Ingestion of naringin and related flavonoids can al‐ so affect the intestinal absorption of certain drugs, leading to either an increase or decrease in circulating drug levels [112].

This evidence has motivated to our research group to develop various studies with grape‐ fruit juice (GJ) and the naringin (Nar) to assess his chemoprotective ability.

Our first experience was with naringin in 2001. On that occasion, the study was designed for three main purposes: (1) to determine whether Nar has a genotoxic effect in mouse in vivo. This was evaluated by measuring the rate of micronucleated polychromatic erythrocytes (MNPE); (2) to determine its antigenotoxic and its anticytotoxic potential on the damage produced by ifosfamide. The first study was done by scoring the rate of MNPE, and the sec‐ ond one by establishing the index polychromatic erythrocytes/normochromatic erythrocytes (PE/NE); and (3) to explore whether its antigenotoxic mechanism of action is related to an inhibitory effect of Nar on the expression of the CYP3A enzyme, an effect which could avoid the biotransformation of ifosfamide.

A single oral administration was used for all groups in the experiment: three groups were given different doses of Nar (50, 250, and 500 mg/kg), other groups received the same doses of Nar plus an administration of ifosfamide (60 mg/kg), another group treated with distilled water and another with ifosfamide (60 mg/kg) were used as negative and positive controls, respectively. The micronuclei and the cell scoring were made in blood samples taken from the tail of the animals at 0, 24, 48, 72, and 96 h. The results showed that Nar was neither genotoxic nor cytotoxic with the doses tested, but ifosfamide produced an increase in the rate of MNPE at 24 and 48 h. The highest value was 24+/-1.57 MNPE per thousand cells at 48 h. The index PE/NE was significantly reduced by ifosfamide at 24 and 48 h. Concerning the antigenotoxic capacity of Nar, a significant decrease was observed in the MNPE produced by ifosfamide at the three tested doses. This effect was dose-dependent, showing the highest reduction in MNPE frequency (54.2%) at 48 h with 500 mg/kg of Nar. However, no protec‐ tion on the cytotoxicity produced by ifosfamide was observed. Immunoblot analysis was used to assess the CYP3A expression in liver and intestinal microsomes from mouse ex‐ posed orally to Nar. An induction in the CYP3A protein was observed in both intestinal and hepatic microsomes from treated mice. This induction correlated with an increase in eryth‐ romycin N-demethylase activity. These data suggest that other mechanism(s) are involved in the antigenotoxic action of naringin [113].

With regard to grapefruit juice (GJ), we performed two experiments which are summarized below. The first evaluated the capacity of GJ to inhibit the micronucleated polychromatic er‐ ythrocytes (MNPE) produced by daunorubicin in an acute assay in mice, as well as to deter‐ mine its antioxidant potential in mouse hepatic microsomes, and its capacity to trap free radicals in vitro.

The results showed that GJ is not toxic or genotoxic damage; on the contrary, it generated a significant reduction of the MNPE formed by daunorubicin. The effect was found through‐ out the examined schedule (from 24 to 96 h). The two high doses produced inhibition of about 60% at 48 h, 86% at 72 h and 100% at 96 h after the treatment. With respect to the grapefruit juice antioxidant potential, a 50% decrease in liver microsomal lipid peroxidation produced by daunorubicin was found by quantifying malondialdehyde formation. Finally, a strong GJ scavenging activity evaluated with the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was observed, giving rise to a concentration-dependent curve with a correlation coefficient of 0.98. Overall, our results established an efficient anticlastogenic potential of grapefruit juice, probably related to its antioxidant capacity, or to alterations of daunorubicin metabo‐ lism [114].

Based on this background; recently, we finished another study in which using the comet as‐ say was demonstrated a strong effect by hydrogen peroxide (HP) and no damage by grape‐ fruit juice (GJ) in human lymphocytes. Cells exposed to HP and treated with GJ was shown an increase of DNA damage by HP over the control level, and a decrease of such damage by GJ. With the comet assay plus formamidopyrimidine-DNA-glycosylase we found the stron‐ gest increase of DNA damage by HP over the control level, and the strongest reduction of such damage by GJ. By applying the comet/FISH method we determined 98% of the p53 gene signals in the comet head of control cells along the experiment, in contrast with about 90% signals in the comet tail of cells exposed to HP. Cells treated with both agents showed a significant, concentration/time dependent return of p53 signals to the head, suggesting en‐ hancement of the gene repair. Finally, with the annexin V assay we found an increase in apoptosis and necrosis by HP, and no effect by GJ; when GJ was added to HP treated cells no modification was observed in regard to apoptosis, although a decrease of necrosis was observed [115].

### **5.4. Chamomile**

a correlation between eating a quarter of grapefruit daily and a 30% increase in risk for breast cancer in post-menopausal women. The study points to the inhibition of CYP3A4 en‐ zyme by grapefruit, which metabolizes estrogen [110]. However, an investigation conducted in 2008 has shown that grapefruit consumption does not increase breast cancer risk and found a significant decrease in breast cancer risk with greater intake of grapefruit in women

In the case of naringin, this compound exerts a variety of pharmacological effects such as antioxidant activity, blood lipid lowering, anticancer activity, and inhibition of selected drug-metabolizing cytochrome P450 enzymes, including CYP3A4 and CYP1A2, which may result in drug-drug interactions in vivo. Ingestion of naringin and related flavonoids can al‐ so affect the intestinal absorption of certain drugs, leading to either an increase or decrease

This evidence has motivated to our research group to develop various studies with grape‐

Our first experience was with naringin in 2001. On that occasion, the study was designed for three main purposes: (1) to determine whether Nar has a genotoxic effect in mouse in vivo. This was evaluated by measuring the rate of micronucleated polychromatic erythrocytes (MNPE); (2) to determine its antigenotoxic and its anticytotoxic potential on the damage produced by ifosfamide. The first study was done by scoring the rate of MNPE, and the sec‐ ond one by establishing the index polychromatic erythrocytes/normochromatic erythrocytes (PE/NE); and (3) to explore whether its antigenotoxic mechanism of action is related to an inhibitory effect of Nar on the expression of the CYP3A enzyme, an effect which could avoid

A single oral administration was used for all groups in the experiment: three groups were given different doses of Nar (50, 250, and 500 mg/kg), other groups received the same doses of Nar plus an administration of ifosfamide (60 mg/kg), another group treated with distilled water and another with ifosfamide (60 mg/kg) were used as negative and positive controls, respectively. The micronuclei and the cell scoring were made in blood samples taken from the tail of the animals at 0, 24, 48, 72, and 96 h. The results showed that Nar was neither genotoxic nor cytotoxic with the doses tested, but ifosfamide produced an increase in the rate of MNPE at 24 and 48 h. The highest value was 24+/-1.57 MNPE per thousand cells at 48 h. The index PE/NE was significantly reduced by ifosfamide at 24 and 48 h. Concerning the antigenotoxic capacity of Nar, a significant decrease was observed in the MNPE produced by ifosfamide at the three tested doses. This effect was dose-dependent, showing the highest reduction in MNPE frequency (54.2%) at 48 h with 500 mg/kg of Nar. However, no protec‐ tion on the cytotoxicity produced by ifosfamide was observed. Immunoblot analysis was used to assess the CYP3A expression in liver and intestinal microsomes from mouse ex‐ posed orally to Nar. An induction in the CYP3A protein was observed in both intestinal and hepatic microsomes from treated mice. This induction correlated with an increase in eryth‐ romycin N-demethylase activity. These data suggest that other mechanism(s) are involved

fruit juice (GJ) and the naringin (Nar) to assess his chemoprotective ability.

who never used hormone therapy [111].

170 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

in circulating drug levels [112].

the biotransformation of ifosfamide.

in the antigenotoxic action of naringin [113].

Chamomile (*Matricaria chamomilla* or *Chamomilla recutita*) is an asteraceae plant native to Eu‐ rope and distributed around the world, except in tropical and polar regions. This plant has been used for its curative properties since ancient Egyptian and Greek times, and at present is frequently used as an antiseptic, antiflogistic, diuretic, expectorant, febrifuge, sedative, an‐ ti-inflammatory and anticarcinogen [116]. Pharmacological activities of various components of the plant have been reported, for example, the anti-inflammatory capacity and the modu‐ lating effects of the heat shock protein on apigenin and quercetin flavonoids, as well as the anti-inflammatory, antioxidant, and antiseptic activities detected on α-bisabolol, guargazu‐ lene, and chamazulene [117, 118]. The essential oil extracted from the chamomile flower var‐ ies from 0.42 to 2%, and consists of compounds such as bisabolol, chamazulene, cyclic sesquiterpenes, bisabolol oxides, and other azulenes and terpenes [119].

a group treated with 1000 mg/kg of CEO, and three groups treated first with CEO (250, 500 and 1000 mg/kg) and then with MMS (25 mg/kg). The results indicated a dose-dependent inhibitory effect on the SCEs formed by both mutagens. In the case of daunorubicin, a statis‐ tically significant result was observed in the three tested doses: from the lowest to the high‐ est dose, the inhibitory values corresponded to 25.7, 63.1 and 75.5%. No alterations were found with respect to the cellular proliferation kinetics, but a reduction in the mitotic index was detected. As regards MMS, the inhibitory values were 24.8, 45.8 and 60.6%; no altera‐ tions were found in either the cellular proliferation kinetics or in the mitotic indices [120]. This results suggested that CEO may be an effective antimutagen and was the reason for de‐

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The aim of the second investigation was to determine the inhibitory potential of CEO on the genotoxic damage produced by daunorubicin (DAU) in mice germ cells. We evaluated the effect of 5, 50, and 500 mg/kg of essential oil on the rate of sister chromatid exchange (SCE) induced in spermatogonia by 10 mg/kg of the mutagen. We found no genotoxicity of CEO, but detected an inhibition of SCE after the damage induced by DAU; from the lowest to the highest dose of CEO we found an inhibition of 47.5%, 61.9%, and 93.5%, respectively. As a possible mechanism of action, the antioxidant capacity of CEO was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method and ferric thiocya‐ nate assays. In the first test we observed a moderate scavenging potential of the oil; never‐ theless, the second assay showed an antioxidant capacity similar to that observed with vitamin E. In conclusion, we found that CEO is an efficient chemoprotective agent against the damage induced by DAU in the precursor cells of the germinal line of mice, and that its

*Silybum marianum* is the scientific name of milk thistle or St. Mary's thistle. It is a Mediterra‐ nean native plant belonging to the Asteraceae family. It is characterized by thorny branches, a milky sap, with oval leaves that reach up to 30 centimeters, its flowers are bright pink and

Milk thistle (Mt) grows of wild form in the southern Europe, the northern Africa and the Middle East but is cultivated in Hungary, China and South American countries as Argentina, Venezuela and Ecuador. In México, is consumed as supplement food for

In the sixties years, German scientists performed a chemical investigation of his fruits, isolating a crude extract formed by active compounds with hepatoprotective capacity; this group of compounds was called silymarin. In 1975, it was found that the principal components of silymarin are silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B and silydianin [123]. Currently it is known that the chemical constituents of silymarin are flavonolignans, ie, a combination conformed by flavonoids and lignins

velop the second study.

**5.5. Silymarin**

many years ago [122].

structures [124].

antioxidant capacity may induce this effect [116].

can measure up to 8 cm to diameter [121].

With respect to his genotoxic and/or antigenotoxic potential, there are few reports in the lit‐ erature that demonstrate this effect. Therefore, our laboratory performed two investigations with the main purpose to evaluate the chemoprotection capacity of chamomile. Initially, we obtained the chamomile essential oil (CEO) from flowers of *Chamomilla recutita* by steam dis‐ tillation, and then it was analyzed by gas chromatography to identify the chemical species. Thirteen compounds were determined with this assay, including bisabolol and its oxides, βfarnecene, chamazulene, germacrene, and sesquiterpenes (Table 3).


**Table 3.** Components of the tested chamomile essential oil

The first work was to determine the inhibitory effect of the CEO, on the sister chromatid ex‐ changes (SCEs) produced by daunorubicin and methyl methanesulfonate (MMS) in mouse bone marrow cells.

The authors performed a toxic and genotoxic assay of chamomile essential oil; both showed negative results. To determine whether CEO can inhibit the mutagenic effects induced by daunorubicin, one group of mice was administered corn oil, another group was treated with the mutagen (10 mg/kg), a third group was treated with 500 mg/kg of CEO; three other groups were treated first with CEO (5, 50 and 500 mg/kg) and then with 10 mg/kg of dau‐ norubicin. In the case of MMS, the experimental groups consisted of the following: the nega‐ tive control group which was administered corn oil, a group treated with 25 mg/kg of MMS, a group treated with 1000 mg/kg of CEO, and three groups treated first with CEO (250, 500 and 1000 mg/kg) and then with MMS (25 mg/kg). The results indicated a dose-dependent inhibitory effect on the SCEs formed by both mutagens. In the case of daunorubicin, a statis‐ tically significant result was observed in the three tested doses: from the lowest to the high‐ est dose, the inhibitory values corresponded to 25.7, 63.1 and 75.5%. No alterations were found with respect to the cellular proliferation kinetics, but a reduction in the mitotic index was detected. As regards MMS, the inhibitory values were 24.8, 45.8 and 60.6%; no altera‐ tions were found in either the cellular proliferation kinetics or in the mitotic indices [120]. This results suggested that CEO may be an effective antimutagen and was the reason for de‐ velop the second study.

The aim of the second investigation was to determine the inhibitory potential of CEO on the genotoxic damage produced by daunorubicin (DAU) in mice germ cells. We evaluated the effect of 5, 50, and 500 mg/kg of essential oil on the rate of sister chromatid exchange (SCE) induced in spermatogonia by 10 mg/kg of the mutagen. We found no genotoxicity of CEO, but detected an inhibition of SCE after the damage induced by DAU; from the lowest to the highest dose of CEO we found an inhibition of 47.5%, 61.9%, and 93.5%, respectively. As a possible mechanism of action, the antioxidant capacity of CEO was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method and ferric thiocya‐ nate assays. In the first test we observed a moderate scavenging potential of the oil; never‐ theless, the second assay showed an antioxidant capacity similar to that observed with vitamin E. In conclusion, we found that CEO is an efficient chemoprotective agent against the damage induced by DAU in the precursor cells of the germinal line of mice, and that its antioxidant capacity may induce this effect [116].

### **5.5. Silymarin**

ies from 0.42 to 2%, and consists of compounds such as bisabolol, chamazulene, cyclic

With respect to his genotoxic and/or antigenotoxic potential, there are few reports in the lit‐ erature that demonstrate this effect. Therefore, our laboratory performed two investigations with the main purpose to evaluate the chemoprotection capacity of chamomile. Initially, we obtained the chamomile essential oil (CEO) from flowers of *Chamomilla recutita* by steam dis‐ tillation, and then it was analyzed by gas chromatography to identify the chemical species. Thirteen compounds were determined with this assay, including bisabolol and its oxides, β-

**Compound RTa Area (%)** (*E*)-β-Farnecene 38.46 28.17 Germacrene-D 39.23 2.19 Unidentified sesquiterpene 40.07 1.40 Unidentified sesquiterpene 41.17 0.78 (Ζ,Ε)−α−Farnecene 41.35 1.59 Unidentifiedsesquiterpene 48.52 0.71 α−Bisabolol oxide A 54.46 41.77 α−Bisabolol oxide B 49.28 4.31 α-Bisabolol oxide 50.65 5.30 α−Bisabolol 51.18 2.31 Chamazulene 52.80 2.39 1,6-Dioxaspiro[4,4]non-3-ene,2-(2,4hexadyn-1-ylidene) 60.73 2.19 Hexatriacontane 67.49 0.50

The first work was to determine the inhibitory effect of the CEO, on the sister chromatid ex‐ changes (SCEs) produced by daunorubicin and methyl methanesulfonate (MMS) in mouse

The authors performed a toxic and genotoxic assay of chamomile essential oil; both showed negative results. To determine whether CEO can inhibit the mutagenic effects induced by daunorubicin, one group of mice was administered corn oil, another group was treated with the mutagen (10 mg/kg), a third group was treated with 500 mg/kg of CEO; three other groups were treated first with CEO (5, 50 and 500 mg/kg) and then with 10 mg/kg of dau‐ norubicin. In the case of MMS, the experimental groups consisted of the following: the nega‐ tive control group which was administered corn oil, a group treated with 25 mg/kg of MMS,

sesquiterpenes, bisabolol oxides, and other azulenes and terpenes [119].

172 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

farnecene, chamazulene, germacrene, and sesquiterpenes (Table 3).

RT<sup>a</sup>

bone marrow cells.

, Retention time obtained with gas chromatography.

**Table 3.** Components of the tested chamomile essential oil

*Silybum marianum* is the scientific name of milk thistle or St. Mary's thistle. It is a Mediterra‐ nean native plant belonging to the Asteraceae family. It is characterized by thorny branches, a milky sap, with oval leaves that reach up to 30 centimeters, its flowers are bright pink and can measure up to 8 cm to diameter [121].

Milk thistle (Mt) grows of wild form in the southern Europe, the northern Africa and the Middle East but is cultivated in Hungary, China and South American countries as Argentina, Venezuela and Ecuador. In México, is consumed as supplement food for many years ago [122].

In the sixties years, German scientists performed a chemical investigation of his fruits, isolating a crude extract formed by active compounds with hepatoprotective capacity; this group of compounds was called silymarin. In 1975, it was found that the principal components of silymarin are silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B and silydianin [123]. Currently it is known that the chemical constituents of silymarin are flavonolignans, ie, a combination conformed by flavonoids and lignins structures [124].

Mt is one of the most investigated plant extracts with known mechanisms of action for oral treatment of toxic liver damage. Silymarin is used as a protective treatment in acute and chronic liver diseases [125]. His protective capacity is related with different mechanisms as suppress toxin penetration into the hepatic cells, increasing superoxide dismutase activity, increasing glutathione tissue level, inhibition of lipid peroxidation and enhancing hepato‐ cyte protein synthesis. The hepatoprotective activity of silymarin can be explained based on antioxidant properties due to the phenolic nature of flavonolignans. It also acts through stimulating liver cells regeneration and cell membrane stabilization to prevent hepatotoxic agents from entering hepatocytes [126].

Silymarin is also beneficial for reducing the chances for developing certain cancers [127]. The molecular targets of silymarin for cancer prevention have been studied. Milk thistle in‐ terfere with the expressions of cell cycle regulators and proteins involved in apoptosis to modulate the imbalance between cell survival and apoptosis. Sy-Cordero et al. (2010) isolat‐ ed four key flavonolign and diastereoisomers (silybin A, silybin B, isosilybin A and isosily‐ bin B) from *S*. *marianum* in gram scale. These compounds and other two related analogues, present in extremely minute quantities, were evaluated for antiproliferative/cytotoxic activi‐ ty against human prostate cancer cell lines. Isosilybin B showed the most potent activity [126]. The isolation of six isomers afforded a preliminary analysis of structure-activity rela‐ tionship toward prostate cancer prevention. The results suggested that an *ortho* relationship for the hydroxyl and methoxy substituents in silybin A, silybin B, isosilybin A and isosilybin B was more favorable than the *meta* relationship for the same substituents in the minor fla‐ vonolignans. Silymarin suppressed UVA-induced oxidative stress that can induce skin dam‐ age. Therefore, topical application of silymarin can be a useful strategy for protecting against skin cancer [128].

**Figure 2.** Antigenotoxic effect of silymarin (Sly) against the DNA damage induced by the chronic consumption of eta‐

The Chemoprevention of Chronic Degenerative Disease Through Dietary Antioxidants: Progress, Promise and

with respect to the value of the control group and, b with respect to the value obtained in mice treated with Et-OH

, Eduardo Madrigal-Bujaidar2

, María Teresa Sumaya-Martínez3

and José Antonio Morales-González1

[1] Doll R. Chronic and degenerative disease: Major causes of morbidity and death. The‐

[2] Nava-Chapa G., Ortiz-Espinosa RM., Reyes-Gómez D. Epidemiologia de las enfer‐ medades crónico degenerativas. In: Morales-González JA., Fernández-Sánchez AM.,

American Journal of ClinicalNutrition.1995; 62(6) 1301S-1305S.

statistically significant difference

Evidences

175

http://dx.doi.org/10.5772/52162

,

, Sandra Cruz-Jaime1

,

nol (Et-OH). Results are the mean ± SD of 5 mice per group (100 nuclei per doses) a

only. ANOVA and Student-Newman Keuls tests, p ≤ 0.05.

1 Instituto de Ciencias de la Salud, UAEH, México

2 Escuela Nacional de Ciencias Biológicas, IPN, México

3 Universidad Autónoma de Nayarit, Tepic, México

**Author details**

**References**

Eduardo Madrigal-Santillán1

Karla Guadalupe Pérez-Ávila1

María del Carmen Valadez-Vega1

In our laboratory, we evaluated the antigenotoxic effect of two doses of silymarin (200 and 400 mg/Kg) administered by oral gavage against the chronic consumption of ethanol (solu‐ tion: 92 mL of water/8 mL of ethanol) during a week with alkaline single cell electrophoresis (comet) assay.

Figure 2 shows the comet measurements obtained in our assay. To summarize, at the 24 hours of the schedule we found no significant DNA damage induced in the control group (only water) and the silymarin group (400 mg/kg), both groups had a mean T/N index of 1.1. On the contrary, the mice (strain CD-1) that consumed the solution of etha‐ nol showed a slight comet increase during this same time. But at 48, 72 and 96 hours, this group showed a T/N index increase of about four times as much. During the last times (120, 144, 168 and 192) there is a decrease of DNA damage, suggesting that hepa‐ tocytes are in the process of cell regeneration. With respect to the groups treated with the combination of chemicals, a clear antigenotoxic effect was found with the two doses of silymarin; particularly with 400 mg/kg, the prevention of DNA damage was about 70% during the 48, 72, 96 and 120 hours of treatment. At the end of the experiment, these groups reached similar values to the negative control [129].

The Chemoprevention of Chronic Degenerative Disease Through Dietary Antioxidants: Progress, Promise and Evidences http://dx.doi.org/10.5772/52162 175

**Figure 2.** Antigenotoxic effect of silymarin (Sly) against the DNA damage induced by the chronic consumption of eta‐ nol (Et-OH). Results are the mean ± SD of 5 mice per group (100 nuclei per doses) a statistically significant difference with respect to the value of the control group and, b with respect to the value obtained in mice treated with Et-OH only. ANOVA and Student-Newman Keuls tests, p ≤ 0.05.

### **Author details**

Mt is one of the most investigated plant extracts with known mechanisms of action for oral treatment of toxic liver damage. Silymarin is used as a protective treatment in acute and chronic liver diseases [125]. His protective capacity is related with different mechanisms as suppress toxin penetration into the hepatic cells, increasing superoxide dismutase activity, increasing glutathione tissue level, inhibition of lipid peroxidation and enhancing hepato‐ cyte protein synthesis. The hepatoprotective activity of silymarin can be explained based on antioxidant properties due to the phenolic nature of flavonolignans. It also acts through stimulating liver cells regeneration and cell membrane stabilization to prevent hepatotoxic

Silymarin is also beneficial for reducing the chances for developing certain cancers [127]. The molecular targets of silymarin for cancer prevention have been studied. Milk thistle in‐ terfere with the expressions of cell cycle regulators and proteins involved in apoptosis to modulate the imbalance between cell survival and apoptosis. Sy-Cordero et al. (2010) isolat‐ ed four key flavonolign and diastereoisomers (silybin A, silybin B, isosilybin A and isosily‐ bin B) from *S*. *marianum* in gram scale. These compounds and other two related analogues, present in extremely minute quantities, were evaluated for antiproliferative/cytotoxic activi‐ ty against human prostate cancer cell lines. Isosilybin B showed the most potent activity [126]. The isolation of six isomers afforded a preliminary analysis of structure-activity rela‐ tionship toward prostate cancer prevention. The results suggested that an *ortho* relationship for the hydroxyl and methoxy substituents in silybin A, silybin B, isosilybin A and isosilybin B was more favorable than the *meta* relationship for the same substituents in the minor fla‐ vonolignans. Silymarin suppressed UVA-induced oxidative stress that can induce skin dam‐ age. Therefore, topical application of silymarin can be a useful strategy for protecting

In our laboratory, we evaluated the antigenotoxic effect of two doses of silymarin (200 and 400 mg/Kg) administered by oral gavage against the chronic consumption of ethanol (solu‐ tion: 92 mL of water/8 mL of ethanol) during a week with alkaline single cell electrophoresis

Figure 2 shows the comet measurements obtained in our assay. To summarize, at the 24 hours of the schedule we found no significant DNA damage induced in the control group (only water) and the silymarin group (400 mg/kg), both groups had a mean T/N index of 1.1. On the contrary, the mice (strain CD-1) that consumed the solution of etha‐ nol showed a slight comet increase during this same time. But at 48, 72 and 96 hours, this group showed a T/N index increase of about four times as much. During the last times (120, 144, 168 and 192) there is a decrease of DNA damage, suggesting that hepa‐ tocytes are in the process of cell regeneration. With respect to the groups treated with the combination of chemicals, a clear antigenotoxic effect was found with the two doses of silymarin; particularly with 400 mg/kg, the prevention of DNA damage was about 70% during the 48, 72, 96 and 120 hours of treatment. At the end of the experiment,

these groups reached similar values to the negative control [129].

agents from entering hepatocytes [126].

174 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

against skin cancer [128].

(comet) assay.

Eduardo Madrigal-Santillán1 , Eduardo Madrigal-Bujaidar2 , Sandra Cruz-Jaime1 , María del Carmen Valadez-Vega1 , María Teresa Sumaya-Martínez3 , Karla Guadalupe Pérez-Ávila1 and José Antonio Morales-González1

1 Instituto de Ciencias de la Salud, UAEH, México

2 Escuela Nacional de Ciencias Biológicas, IPN, México

3 Universidad Autónoma de Nayarit, Tepic, México

### **References**


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184 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

lar Sciences. 2010; 11(10) 3793-3802.


**Chapter 8**

**Inflammatory Environmental, Oxidative Stress in**

The incidence and prevalence of cancer has been increasing in such as degree that it has be‐ come the second or third leading cause of death worldwide, depending on ethnicity or country in question and is consequently a major public health, cancer is a leading cause of death in many countries, accounting for 7.6 million deaths (around 13% of all deaths) in 2008. Deaths from cancer worldwide are projected to continue rising, with an estimated 13.1 million deaths in 2030. About 30% of cancer deaths are due to the five leading behavioral and dietary risks: high body mass index, low fruit and vegetable intake, lack of physical ac‐

Cancer is a generic term for a large group of diseases that can affect any part of the body, cancer cells are significantly influenced by the surrounding stromal tissues for the initiation, proliferation, and distant colony formation. When a tumour successfully spreads to other parts of the body and grows, invading and destroying other healthy tissues, this process is referred to as metastasis, and the result is a serious condition that is very difficult to treat, because the progression to metastases is the leading cause of death associated to cancer. Metastatic cells in this process must interact with the endothelium in three stages of tumor progression. In recent years, the interaction between these cell populations has been seen as part of a complex microenvironment tumor-associated. Mantovani et al. have even postulat‐ ed that this tumor microenvironment inflammatory plays an important role in tumorigene‐

> © 2013 Esquivel-Chirino et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Tumoral Progression**

José Antonio Morales-González,

Luis Enrique Hernández-Mora and

Alejandro Zentella-Dehesa

http://dx.doi.org/10.5772/51789

tivity, tobacco use, alcohol use [1-8].

**1. Introduction**

César Esquivel-Chirino, Jaime Esquivel-Soto,

Additional information is available at the end of the chapter

Delina Montes Sánchez, Jose Luis Ventura-Gallegos,

### **Inflammatory Environmental, Oxidative Stress in Tumoral Progression**

César Esquivel-Chirino, Jaime Esquivel-Soto, José Antonio Morales-González, Delina Montes Sánchez, Jose Luis Ventura-Gallegos, Luis Enrique Hernández-Mora and Alejandro Zentella-Dehesa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51789

**1. Introduction**

The incidence and prevalence of cancer has been increasing in such as degree that it has be‐ come the second or third leading cause of death worldwide, depending on ethnicity or country in question and is consequently a major public health, cancer is a leading cause of death in many countries, accounting for 7.6 million deaths (around 13% of all deaths) in 2008. Deaths from cancer worldwide are projected to continue rising, with an estimated 13.1 million deaths in 2030. About 30% of cancer deaths are due to the five leading behavioral and dietary risks: high body mass index, low fruit and vegetable intake, lack of physical ac‐ tivity, tobacco use, alcohol use [1-8].

Cancer is a generic term for a large group of diseases that can affect any part of the body, cancer cells are significantly influenced by the surrounding stromal tissues for the initiation, proliferation, and distant colony formation. When a tumour successfully spreads to other parts of the body and grows, invading and destroying other healthy tissues, this process is referred to as metastasis, and the result is a serious condition that is very difficult to treat, because the progression to metastases is the leading cause of death associated to cancer. Metastatic cells in this process must interact with the endothelium in three stages of tumor progression. In recent years, the interaction between these cell populations has been seen as part of a complex microenvironment tumor-associated. Mantovani et al. have even postulat‐ ed that this tumor microenvironment inflammatory plays an important role in tumorigene‐

© 2013 Esquivel-Chirino et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. sis and tumor progression [18]. Tumor and normal surrounding cells such as endothelial cells, soluble factors derived from this two cell populations and extracellular matrix [9-12], compose the tumor microenvironment.

and the rest of the vessel wall. The cells that form the endothelium are called endothelial cells, these cells have very distinct and unique functions that are paramount to vascular biol‐ ogy. These functions include fluid filtration, formation of new blood vessels in the angiogen‐ esis, neutrophil recruitment. The endothelium acts as a semi-selective barrier between the vessel lumen and surrounding tissue, controlling the passage of materials and the transit of white blood cells, hormones into and out of the bloodstream. Excessive or prolonged in‐ creases in permeability of the endothelial monolayer, as in cases of chronic inflammation, may lead to tissue edema. It is also important in controlling blood pressure, blood coagula‐ tion, vascular tone, degradation of lipoproteins an in the secretion of growth factors and cy‐ tokines [24-25]. In recent decades, it has become clear that the endothelium of venules and smaller capillaries, and lymphatic vessels play a central role in the process of tumor growth, dissemination of metastatic cells, which is accompanied by the development of a characteris‐

Inflammatory Environmental, Oxidative Stress in Tumoral Progression

http://dx.doi.org/10.5772/51789

189

Quiescent, resting endothelial cells in the adult form a highly heterogeneous cell population that varies not only in different organs but also in different vessel calibers within an organ. Endothelium in the normal adult male, although being metabolically active, considered qui‐ escent because the turnover of these cells is very low and this called: constitutive phenotype

In this condition, the apical membrane of endothelial cells exhibits a very low amount of in‐ tercellular adhesion molecules, so that no adhesion of cellular blood components to the ves‐

Endothelial cell activation is associated with a number of distinct phenotype changes that, much like differentiation processes of the constitutive phenotype of endothelial cells, serve their need to adapt to functional requirements. [28] The cytokine-induced phenotype of en‐ dothelial cells during inflammation has been characterized most extensively in the last few years. When endothelial cells are activated by these cytokines are functional disorders in‐ volving immediate responses, for example, some pathological conditions such as sepsis, are associated with endothelial conversion to a phenotype activated [29-30]. The activated phe‐ notype characterized by activation of constitutive nitric oxide synthase (NOS), also accom‐ panied by changes such as increased expression of cell adhesion molecules (CAMs) and Eselectin (CD62E), ICAM-1(CD54), VCAM-1(CD106), P-selectin (CD62P) Fig (1). These changes allow the endothelium to participate in pathological conditions including inflam‐ mation, coagulation, cell proliferation, metastasis, tumor angiogenesis. All these cellular in‐ teractions are regulated by temporal and spatial presentation of various cell adhesion molecules and chemotactical molecules displaying appropriate specificity and affinity for

tic tumor vasculature and tumors formed by endothelial cells [26].

There are two phenotypes endothelial (constitutive and activated);

*2.1.1. The constitutive phenotype of endothelial cells*

*2.1.2. The activated phenotype of endothelial cells*

Fig (1).

sel walls [27].

There are other factors in the development of cancer such as genetic, environmental, as well as the role of oxidative stress and free radicals in response to damage caused by chemicals, radiation, cellular aging, ischemic lesions and cells immune system [13]. Substances such as antioxidants can protect cells against free radical damage. The damage caused by free radi‐ cals can lead to cancer. Antioxidants interact with free radicals to stabilize them so that, be‐ ing able to avoid some of the damage that free radicals can cause. It is important to analyze the role of antioxidants as an alternative that contributes to cancer treatment and to promote their use and consumption in cancer prevention

### **2. Tumoral progression**

Tumors often become more aggressive in their behavior in more aggressive and their char‐ acteristics, although the time course may be quite variable, this phenomenon has been termed tumor progression by Foulds [15].

In the early stages of the tumor progression, there is a detachment of cancer cells from the primary tumor, followed by tumor cell adhesion to endothelial cells of venules in the target organs. After the extravasations occurs extracellular matrix invasion by tumor cells, these cells of primary lesions enter the lymphatics or the bloodstream depending on their anatom‐ ical location. In the circulation, many tumor cells are eradicated by physical forces exerted on them to pass through the microvasculature of secondary organs, and immunological mechanisms of action of host defense. Furthermore, once inside the target tissue tumor cells must find favorable conditions for survival and proliferation [16-18]. The biological charac‐ teristic that define tumor progression have been extensively described, although the under‐ lying mechanisms are still not completely defined, however there are two theories have been proposed to explain how tumor cells invade secondary sites where metastasis occurs are the following [18-20]. The first is similar to the inflammatory process by cell adhesion and migration, while the second involves the aggregation of circulating tumor cells, and that these cells blocked blood vessels. In this theory in which a cell stably adhered frequently starts a homotypic aggregation, capturing other circulating tumor cells, followed by the for‐ mation of multicellular aggregates, these aggregates once grow and emerge from the pri‐ mary tumor site, is carried out which triggers tumor progression in metastasis, where it requires a coordinated interaction of tumor cells and vascular endothelial cells, play a criti‐ cal role in most of the events that characterize tumor progression and metastasis [21-23], so it is important to mention the general aspects of endothelial biology:

#### **2.1. Endothelial Biology**

The endothelium is the thin layer of cells that lines the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. The cells that form the endothelium are called endothelial cells, these cells have very distinct and unique functions that are paramount to vascular biol‐ ogy. These functions include fluid filtration, formation of new blood vessels in the angiogen‐ esis, neutrophil recruitment. The endothelium acts as a semi-selective barrier between the vessel lumen and surrounding tissue, controlling the passage of materials and the transit of white blood cells, hormones into and out of the bloodstream. Excessive or prolonged in‐ creases in permeability of the endothelial monolayer, as in cases of chronic inflammation, may lead to tissue edema. It is also important in controlling blood pressure, blood coagula‐ tion, vascular tone, degradation of lipoproteins an in the secretion of growth factors and cy‐ tokines [24-25]. In recent decades, it has become clear that the endothelium of venules and smaller capillaries, and lymphatic vessels play a central role in the process of tumor growth, dissemination of metastatic cells, which is accompanied by the development of a characteris‐ tic tumor vasculature and tumors formed by endothelial cells [26].

There are two phenotypes endothelial (constitutive and activated);

### *2.1.1. The constitutive phenotype of endothelial cells*

sis and tumor progression [18]. Tumor and normal surrounding cells such as endothelial cells, soluble factors derived from this two cell populations and extracellular matrix [9-12],

There are other factors in the development of cancer such as genetic, environmental, as well as the role of oxidative stress and free radicals in response to damage caused by chemicals, radiation, cellular aging, ischemic lesions and cells immune system [13]. Substances such as antioxidants can protect cells against free radical damage. The damage caused by free radi‐ cals can lead to cancer. Antioxidants interact with free radicals to stabilize them so that, be‐ ing able to avoid some of the damage that free radicals can cause. It is important to analyze the role of antioxidants as an alternative that contributes to cancer treatment and to promote

Tumors often become more aggressive in their behavior in more aggressive and their char‐ acteristics, although the time course may be quite variable, this phenomenon has been

In the early stages of the tumor progression, there is a detachment of cancer cells from the primary tumor, followed by tumor cell adhesion to endothelial cells of venules in the target organs. After the extravasations occurs extracellular matrix invasion by tumor cells, these cells of primary lesions enter the lymphatics or the bloodstream depending on their anatom‐ ical location. In the circulation, many tumor cells are eradicated by physical forces exerted on them to pass through the microvasculature of secondary organs, and immunological mechanisms of action of host defense. Furthermore, once inside the target tissue tumor cells must find favorable conditions for survival and proliferation [16-18]. The biological charac‐ teristic that define tumor progression have been extensively described, although the under‐ lying mechanisms are still not completely defined, however there are two theories have been proposed to explain how tumor cells invade secondary sites where metastasis occurs are the following [18-20]. The first is similar to the inflammatory process by cell adhesion and migration, while the second involves the aggregation of circulating tumor cells, and that these cells blocked blood vessels. In this theory in which a cell stably adhered frequently starts a homotypic aggregation, capturing other circulating tumor cells, followed by the for‐ mation of multicellular aggregates, these aggregates once grow and emerge from the pri‐ mary tumor site, is carried out which triggers tumor progression in metastasis, where it requires a coordinated interaction of tumor cells and vascular endothelial cells, play a criti‐ cal role in most of the events that characterize tumor progression and metastasis [21-23], so

The endothelium is the thin layer of cells that lines the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen

it is important to mention the general aspects of endothelial biology:

compose the tumor microenvironment.

188 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

their use and consumption in cancer prevention

termed tumor progression by Foulds [15].

**2. Tumoral progression**

**2.1. Endothelial Biology**

Quiescent, resting endothelial cells in the adult form a highly heterogeneous cell population that varies not only in different organs but also in different vessel calibers within an organ. Endothelium in the normal adult male, although being metabolically active, considered qui‐ escent because the turnover of these cells is very low and this called: constitutive phenotype Fig (1).

In this condition, the apical membrane of endothelial cells exhibits a very low amount of in‐ tercellular adhesion molecules, so that no adhesion of cellular blood components to the ves‐ sel walls [27].

### *2.1.2. The activated phenotype of endothelial cells*

Endothelial cell activation is associated with a number of distinct phenotype changes that, much like differentiation processes of the constitutive phenotype of endothelial cells, serve their need to adapt to functional requirements. [28] The cytokine-induced phenotype of en‐ dothelial cells during inflammation has been characterized most extensively in the last few years. When endothelial cells are activated by these cytokines are functional disorders in‐ volving immediate responses, for example, some pathological conditions such as sepsis, are associated with endothelial conversion to a phenotype activated [29-30]. The activated phe‐ notype characterized by activation of constitutive nitric oxide synthase (NOS), also accom‐ panied by changes such as increased expression of cell adhesion molecules (CAMs) and Eselectin (CD62E), ICAM-1(CD54), VCAM-1(CD106), P-selectin (CD62P) Fig (1). These changes allow the endothelium to participate in pathological conditions including inflam‐ mation, coagulation, cell proliferation, metastasis, tumor angiogenesis. All these cellular in‐ teractions are regulated by temporal and spatial presentation of various cell adhesion molecules and chemotactical molecules displaying appropriate specificity and affinity for proper development and functioning of the organism [31-32]. Has been postulated that this phenotype or variants of it, are involved in the processes of metastasis [33].

**Figure 2.** Steps in the Metastasis process.

**1.** bearing

**3.** adherence

Metastasis is facilitated by cell-cell interactions between tumor cells and the endothelium in distant tissues and determines the spread. Metastatic cells must act with the endothelium in three different stages of tumor progression: initially during the formation of blood vessels that enable tumor growth (vascularization), during the migration process that allows the pas‐ sage from tissue into the bloodstream (intravasation), and finally during the process allow‐ ing extravasation into the target tissue [41-43]. Metastatic cancer cells require properties that allow them not only to adapt to a foreign microenvironment but also to subvert it in a way that is conducive to their continued proliferation and survival [36-38]. In addition, direct tumor cell interactions with platelets, fibroblasts and monocytes/macrophages, polymorphonu‐ clear cells, soluble components, cytokines, chemokines, proteins of the extracellular matrix, growth factors, and other molecules secreted by host cells, significantly contribute to cancer

Inflammatory Environmental, Oxidative Stress in Tumoral Progression

http://dx.doi.org/10.5772/51789

191

cell adhesion, extravasation, and the establishment of metastatic lesions [44-47].

In the early stages of inflammation, neutrophils are cells that migrate to the site of inflam‐ mation under the influence of growth factors, cytokines and chemokines, which are pro‐ duced by macrophages and mast cells residing in the tissue [48]. The process of cell

If the inflammatory response is not regulated, the cellular response will become chronic and will be dominated by lymphocytes, plasma cells, macrophages metastasis, which is favored by the microenvironment of the organ target. The installation of tumor cells in blood vessels

**2.3. Cellular interactions in the inflammatory reaction and spread tumor**

extravasation from the bloodstream can be divided into four stages:

**2.** activation by stimulation chemoattractant

**4.** transendothelial migration.

**Figure 1.** There are two phenotypes endothelial constitutive and activated.

#### **2.2. Metastasis**

Metastasis is the result of cancer cell adaptation to a tissue microenvironment at a distance from the primary tumor, is a complex process involving multiple steps: first, when cancer cells break away from the primary tumor, they invade the host stroma, intravasate into lym‐ phatic or blood vessels, spread to the capillary bed of distant organs, where they invade into new surrounding tissues and proliferate to form secondary tumors [34-35]. When cancer is detected at an early stage, before it has spread, it can often be treated successfully by con‐ ventional cancer therapies such as surgery, chemotherapy, local irradiation, metastatic diffu‐ sion of cancer cells remains the most important clinical problem, because when cancer is detected after known to have metastasized, treatment are much less successful [36]. The metastatic capacity of tumor cells correlates with their ability to exit from the blood circula‐ tion, to colonize distant organs, and to grow in distant organs. Metastasis is a complex proc‐ ess that includes local infiltration of tumor cells into the adjacent tissue, transendothelial migration of cancer cells into vessels known as intravasation, survival in the circulatory sys‐ tem, extravasation and subsequent proliferation in competent organs leading to colonization [36-38]. Initially, tumor cell aggregates detachment from the primary tumor, next the cells actively infiltrate the surrounding stroma and enter into the circulatory system, traveling to distinct sites to establish the secondary tumor growth. In the bloodstream, a very small number of tumor cells survive to reach the target organ, indicating that metastasis formation must be regarded as a very ineffective event. Millions of carcinoma cells enter into the circu‐ latory system, but the majority of them die during transportation, and only 1-5% of viable cells are successful in formation of secondary deposits in distinct sites [37-40]. Fig (2).

**Figure 2.** Steps in the Metastasis process.

proper development and functioning of the organism [31-32]. Has been postulated that this

Metastasis is the result of cancer cell adaptation to a tissue microenvironment at a distance from the primary tumor, is a complex process involving multiple steps: first, when cancer cells break away from the primary tumor, they invade the host stroma, intravasate into lym‐ phatic or blood vessels, spread to the capillary bed of distant organs, where they invade into new surrounding tissues and proliferate to form secondary tumors [34-35]. When cancer is detected at an early stage, before it has spread, it can often be treated successfully by con‐ ventional cancer therapies such as surgery, chemotherapy, local irradiation, metastatic diffu‐ sion of cancer cells remains the most important clinical problem, because when cancer is detected after known to have metastasized, treatment are much less successful [36]. The metastatic capacity of tumor cells correlates with their ability to exit from the blood circula‐ tion, to colonize distant organs, and to grow in distant organs. Metastasis is a complex proc‐ ess that includes local infiltration of tumor cells into the adjacent tissue, transendothelial migration of cancer cells into vessels known as intravasation, survival in the circulatory sys‐ tem, extravasation and subsequent proliferation in competent organs leading to colonization [36-38]. Initially, tumor cell aggregates detachment from the primary tumor, next the cells actively infiltrate the surrounding stroma and enter into the circulatory system, traveling to distinct sites to establish the secondary tumor growth. In the bloodstream, a very small number of tumor cells survive to reach the target organ, indicating that metastasis formation must be regarded as a very ineffective event. Millions of carcinoma cells enter into the circu‐ latory system, but the majority of them die during transportation, and only 1-5% of viable

cells are successful in formation of secondary deposits in distinct sites [37-40]. Fig (2).

phenotype or variants of it, are involved in the processes of metastasis [33].

190 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 1.** There are two phenotypes endothelial constitutive and activated.

**2.2. Metastasis**

Metastasis is facilitated by cell-cell interactions between tumor cells and the endothelium in distant tissues and determines the spread. Metastatic cells must act with the endothelium in three different stages of tumor progression: initially during the formation of blood vessels that enable tumor growth (vascularization), during the migration process that allows the pas‐ sage from tissue into the bloodstream (intravasation), and finally during the process allow‐ ing extravasation into the target tissue [41-43]. Metastatic cancer cells require properties that allow them not only to adapt to a foreign microenvironment but also to subvert it in a way that is conducive to their continued proliferation and survival [36-38]. In addition, direct tumor cell interactions with platelets, fibroblasts and monocytes/macrophages, polymorphonu‐ clear cells, soluble components, cytokines, chemokines, proteins of the extracellular matrix, growth factors, and other molecules secreted by host cells, significantly contribute to cancer cell adhesion, extravasation, and the establishment of metastatic lesions [44-47].

#### **2.3. Cellular interactions in the inflammatory reaction and spread tumor**

In the early stages of inflammation, neutrophils are cells that migrate to the site of inflam‐ mation under the influence of growth factors, cytokines and chemokines, which are pro‐ duced by macrophages and mast cells residing in the tissue [48]. The process of cell extravasation from the bloodstream can be divided into four stages:


If the inflammatory response is not regulated, the cellular response will become chronic and will be dominated by lymphocytes, plasma cells, macrophages metastasis, which is favored by the microenvironment of the organ target. The installation of tumor cells in blood vessels of the organ target to invade, is related to phenotypic changes in the endothelium allowing vascular extravasation of blood circulation of leukocytes in the inflammatory reaction and, as hypothesized current of tumor cells with metastatic capacity. The phenomenon of extravasa‐ tion in response to a tumor cell interaction cell endothelial or not allowing the passage of cells whether there are appropriate conditions for the invasion with varied morphology [53-55].

migration of metastatic cells [60]. Tumor cells are also capable of produce cytokines and che‐ mokines that facilitate evasion of the system immune and help to establishment and devel‐ opment of metastasis (Fig. 4). The increase of tumor-associated macrophages (TAMs) is associated with poor prognosis through various mechanisms: a) release by macrophage IL-10 and prostaglandin E2 which suppress antitumor response, b) easy to release angiogen‐ ic factors as VEGF, EGF, endothelin-2 and plasminogen activator promote tumor growth, c) to facilitate cell invasion metastasis by releasing matrix metalloproteinases and induce TNF

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production and vasodilatation enzyme nitric oxide synthase [61-62].

**Figure 4.** The tumor microenvironment and its role in promoting tumor growth

angiogenesis and vasculogenesis.

Cells grow within defined environmental sites and are subject to microenvironmental con‐ trol. Outside of their sites, normal cells lack appropriate survival signals. During tumor de‐ velopment and progression, malignant cells escape the local tissue control and escape death. Diverse chemoattractant factors promote the recruitment and infiltration of these cells to the tumor microenvironment where they suppress the antitumor immunity or promote tumor

TNF is expressed in low amounts by other cells such as fibroblasts, smooth muscle cells and tumor cells, his target cell are primary endothelial cells, inducing their activation by changing expression levels of some membrane proteins, primarily as adhesion molecules E-selectin, ICAM-1 and VCAM-1 whose expression and synthesis, are regulated by the transcription factor kB nuclear. Activated endothelial phenotype induced by TNF and char‐ acteristics of the inflammatory response, have served to comparing the endothelial pheno‐

Within the process of inflammation, a phenomenon is well-studied cell migration, which is the entrance of polymorphonuclear neutrophils and the vascular system. This involves a se‐ quential mechanism of recognition, contact formation, and migration mediated by adhesion molecules such as (ICAM-1, VCAM-1, E and P Selectins, Integrins) it has been demonstrated that some of these adhesion molecules, such as E-selectin are not only involved in inflamma‐ tion, but also in tumor metastasis and play a significant role in cancer progression and meta‐ stasis, in some cases of colon cancer. [56-58]. The expression of ICAM-1 has also proven to be a marker associated with an invasive phenotype [59].

Hanahanan et al. suggest that diversity of cancer cell genotypes is a manifestation of six ba‐ sics alterations in cell physiology that together indicate development of malignant growth Fig (3), these alterations are shared in common by most all types of tumors [19]. In recent years, it has been demonstrated that metastatic dissemination can be influenced by inflam‐ matory-reparative processes [46]. The interaction between these cell populations has been seen as part of a complex inflammatory microenvironment tumor-associated. Mantovani et al. have even postulated that this tumor microenvironment inflammatory plays an impor‐ tant role in tumorigenesis and tumor progression [60].

**Figure 3.** Capabilities of cancer and inflammation [19,60].

#### **2.4. Inflammatory Microenvironment**

The tumor microenvironment is composed of stromal fibroblasts, myofibroblasts, myoepi‐ thelial cells, macrophages, endothelial cells, leucocytes, and extracellular matrix (ECM) and soluble factors derived from tumor cells. This inflammatory environment surrounding a tu‐ mor promotes the breaking of the basal membrane, a process required for the invasion and migration of metastatic cells [60]. Tumor cells are also capable of produce cytokines and che‐ mokines that facilitate evasion of the system immune and help to establishment and devel‐ opment of metastasis (Fig. 4). The increase of tumor-associated macrophages (TAMs) is associated with poor prognosis through various mechanisms: a) release by macrophage IL-10 and prostaglandin E2 which suppress antitumor response, b) easy to release angiogen‐ ic factors as VEGF, EGF, endothelin-2 and plasminogen activator promote tumor growth, c) to facilitate cell invasion metastasis by releasing matrix metalloproteinases and induce TNF production and vasodilatation enzyme nitric oxide synthase [61-62].

of the organ target to invade, is related to phenotypic changes in the endothelium allowing vascular extravasation of blood circulation of leukocytes in the inflammatory reaction and, as hypothesized current of tumor cells with metastatic capacity. The phenomenon of extravasa‐ tion in response to a tumor cell interaction cell endothelial or not allowing the passage of cells whether there are appropriate conditions for the invasion with varied morphology [53-55]. Within the process of inflammation, a phenomenon is well-studied cell migration, which is the entrance of polymorphonuclear neutrophils and the vascular system. This involves a se‐ quential mechanism of recognition, contact formation, and migration mediated by adhesion molecules such as (ICAM-1, VCAM-1, E and P Selectins, Integrins) it has been demonstrated that some of these adhesion molecules, such as E-selectin are not only involved in inflamma‐ tion, but also in tumor metastasis and play a significant role in cancer progression and meta‐ stasis, in some cases of colon cancer. [56-58]. The expression of ICAM-1 has also proven to

Hanahanan et al. suggest that diversity of cancer cell genotypes is a manifestation of six ba‐ sics alterations in cell physiology that together indicate development of malignant growth Fig (3), these alterations are shared in common by most all types of tumors [19]. In recent years, it has been demonstrated that metastatic dissemination can be influenced by inflam‐ matory-reparative processes [46]. The interaction between these cell populations has been seen as part of a complex inflammatory microenvironment tumor-associated. Mantovani et al. have even postulated that this tumor microenvironment inflammatory plays an impor‐

The tumor microenvironment is composed of stromal fibroblasts, myofibroblasts, myoepi‐ thelial cells, macrophages, endothelial cells, leucocytes, and extracellular matrix (ECM) and soluble factors derived from tumor cells. This inflammatory environment surrounding a tu‐ mor promotes the breaking of the basal membrane, a process required for the invasion and

be a marker associated with an invasive phenotype [59].

192 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

tant role in tumorigenesis and tumor progression [60].

**Figure 3.** Capabilities of cancer and inflammation [19,60].

**2.4. Inflammatory Microenvironment**

**Figure 4.** The tumor microenvironment and its role in promoting tumor growth

Cells grow within defined environmental sites and are subject to microenvironmental con‐ trol. Outside of their sites, normal cells lack appropriate survival signals. During tumor de‐ velopment and progression, malignant cells escape the local tissue control and escape death. Diverse chemoattractant factors promote the recruitment and infiltration of these cells to the tumor microenvironment where they suppress the antitumor immunity or promote tumor angiogenesis and vasculogenesis.

TNF is expressed in low amounts by other cells such as fibroblasts, smooth muscle cells and tumor cells, his target cell are primary endothelial cells, inducing their activation by changing expression levels of some membrane proteins, primarily as adhesion molecules E-selectin, ICAM-1 and VCAM-1 whose expression and synthesis, are regulated by the transcription factor kB nuclear. Activated endothelial phenotype induced by TNF and char‐ acteristics of the inflammatory response, have served to comparing the endothelial pheno‐ type has been observed, is produced in response to contact with soluble tumor factors [63-64].

[67]. In cancer, constitutive secretion of VEGF-independent hypoxic condition favors tumor

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The involvement of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) in can‐ cer is complex, since it seems to require the presence of other cytokines such as IL-4 and IL-6, and there are reports antagonistic to their involvement in tumorigenesis. For example, expression of GM-CSF and interleukin-12, inhibits the immune response and being ex‐ pressed on B16 murine melanoma cells, decreasing completely tumorigenic capacity of syn‐ genic mice C57B/6. In contrast to the line described breast epithelial MCF7-A benign growth factors secreted able to induce expression of IL-6 and GM-CSF in the tumor line R2T1AS breast cancer that is associated with a higher rate of growth and a higher tumorigenic ca‐ pacity in vivo. Moreover, continued exposure of GM-CSF plus IL-4 of mesenchymal cells from human bone marrow, resulted in an increase in the morphological transformation and increased rate of growth both *in vitro* and *in vitro* mesenchymal cells, indicating the induc‐ tion of a transformed phenotype. Whereas previous reports indicate that the involvement of

It is reported that the soluble form of IL-1 receptor could serve as a functional antagonist of IL-1, the presence of this protein is controversial, since IL-1 by trapping an anti-inflammato‐ ry effect would reduce the adhesion of cells added by tumor cells and their extravasation, given this scenario could be proposed that tumor cells not only produce pro-inflammatory factors through the release of soluble factors tumor, but it can regulate the inflammatory

The IL-6 is recognized as a classic inducer of the states of chronic inflammation and that pro‐ mote the activation of vascular endothelium. It has also been reported that IL-6 is produced by tumors that develop metastases to the liver, as the case of colon and mammary gland cancer. The activity of this cytokine in the soluble factors tumor could be further enhanced

Expression of IL-8 in colorectal cancer favors an increase in tumorigenesis and metastasis, this increase is due to IL-8 is associated with expression of MMP-2 and MMP-9, the activity of these metalloproteases have been identified in physiological tissue remodeling processes like normal healing, but also participate in tissue remodeling associated with pathological processes including invasion [74]. Also in melanomas have been identified as IL-8 acts as an angiogenic factor and also promotes mitosis, therefore the IL-8 has been postulated as a po‐ tential therapeutic target. In particular, we have sought to interfere with IL-8 with the pur‐ pose of reducing tumor growth, an alternative that has developed is the use of an anti-IL-8 in nude mice with liver cancer. The results show that administration of neutralizing anti-IL8 significantly decreased tumor growth, even more interestingly this decrease is associated

angiogenesis and growth in a clinical setting and is a marker of poor prognosis [68].

*GM-CSF*

*IL-1ra*

process [71].

*IL-6*

*IL-8*

GM-CSF requires other cytokines [69-70].

by the presence of other co-factors secreted by cells [72-73].

The nuclear factor kappa B (NF-kB) is a transcription factor paramount in regulation of in‐ flammatory response genes Early involved in cell-cell interaction, communication intercellu‐ lar recruitment or transmigration, amplification of signals pathogenic and acceleration of tumorigenesis [52-53,65].

The study of tumor cells to modify their microenvironment has been a growing area of in‐ terest, which has identified the secretion of pro-inflammatory cytokines such as TNF, IL-6) and chemokines such as IL-8, it is interesting to note that these products are known modula‐ tors of endothelial function [53].

In recent years, it has been found that tumor cells secrete soluble factors, which modify the endothelial constitutive phenotype, and that exposure to these factors increase to a greater or less extent the capacity to adhere endothelial human tumor cells. It has been recognized that these soluble factors released by tumor cells or non-tumor cells surrounding the tumor play an important role in tumor progression [66].

Our group has shown that breast cancer cells, lymphomas, with high metastatic potentially induces a change in human endothelial cells (HUVECs) that is characterized by promoting a pro-adhesive endothelial phenotype, the expression of intercellular adhesion molecules (ICAM-1, VCAM-1 and E-Selectin) and the activation of NF-kB [66]. These studies include soluble factor leukemias (EUHE, Eusebia), cervical cancer (HeLa) and mammary gland can‐ cer (MCF-7, ZR), oral cancer. In all cases, we have used primary cultures of human endothe‐ lial cells (HUVECs), which have generated an *in vitro* model to study the tumor microenvironment. This model is based on induction of a pro-adhesive endothelial pheno‐ type that is associated with expression of adhesion molecules E-selectin, ICAM-1, VCAM dependent activation of NF-kB. These effects are considered essential in the process of adhe‐ sion and extravasation during the inflammatory reaction.

Moreover, we have analyzed the biochemical composition of the soluble factors derived from tumor cells. Proteins such as 27, cytokines, chemokines and growth factors associated with the inflammatory reaction; (IL-1 ra, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, TNF, IFN-gamma, IP-10,RANTES), (IL-8, Eotaxina, MCP-1, MIP-1 beta, MIP-1 alpha), (G-CSF, GM-CSF, PDGF, FGF, VEGF). The molecules identified most sig‐ nificant expression in breast carcinomas, were (VEGF, GM-CSF, IL-1ra, IL6, IL8, IP10, RANTES), which play an important role in endothelial cell activation [52-53,65-66].

### *VEGF:*

An unexpected finding was the abundant presence of the expression of vascular endothelial growth factor (VEGF) in breast carcinoma lines. Whereas in normal cells the expression of VEGF is dependent on a condition of hypoxia, surprisingly VEGF production by tumor cell lines and occurred at a concentration of oxygen partial indicating 20% indicative of impaired VEGF expression. In cancer constitutive secretion of VEGF independent of a condition that favors hypoxic tumor angiogenesis and growth in a clinical setting and is a marker of poor prognosis [67]. In cancer, constitutive secretion of VEGF-independent hypoxic condition favors tumor angiogenesis and growth in a clinical setting and is a marker of poor prognosis [68].

### *GM-CSF*

type has been observed, is produced in response to contact with soluble tumor factors

The nuclear factor kappa B (NF-kB) is a transcription factor paramount in regulation of in‐ flammatory response genes Early involved in cell-cell interaction, communication intercellu‐ lar recruitment or transmigration, amplification of signals pathogenic and acceleration of

The study of tumor cells to modify their microenvironment has been a growing area of in‐ terest, which has identified the secretion of pro-inflammatory cytokines such as TNF, IL-6) and chemokines such as IL-8, it is interesting to note that these products are known modula‐

In recent years, it has been found that tumor cells secrete soluble factors, which modify the endothelial constitutive phenotype, and that exposure to these factors increase to a greater or less extent the capacity to adhere endothelial human tumor cells. It has been recognized that these soluble factors released by tumor cells or non-tumor cells surrounding the tumor

Our group has shown that breast cancer cells, lymphomas, with high metastatic potentially induces a change in human endothelial cells (HUVECs) that is characterized by promoting a pro-adhesive endothelial phenotype, the expression of intercellular adhesion molecules (ICAM-1, VCAM-1 and E-Selectin) and the activation of NF-kB [66]. These studies include soluble factor leukemias (EUHE, Eusebia), cervical cancer (HeLa) and mammary gland can‐ cer (MCF-7, ZR), oral cancer. In all cases, we have used primary cultures of human endothe‐ lial cells (HUVECs), which have generated an *in vitro* model to study the tumor microenvironment. This model is based on induction of a pro-adhesive endothelial pheno‐ type that is associated with expression of adhesion molecules E-selectin, ICAM-1, VCAM dependent activation of NF-kB. These effects are considered essential in the process of adhe‐

Moreover, we have analyzed the biochemical composition of the soluble factors derived from tumor cells. Proteins such as 27, cytokines, chemokines and growth factors associated with the inflammatory reaction; (IL-1 ra, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, TNF, IFN-gamma, IP-10,RANTES), (IL-8, Eotaxina, MCP-1, MIP-1 beta, MIP-1 alpha), (G-CSF, GM-CSF, PDGF, FGF, VEGF). The molecules identified most sig‐ nificant expression in breast carcinomas, were (VEGF, GM-CSF, IL-1ra, IL6, IL8, IP10,

An unexpected finding was the abundant presence of the expression of vascular endothelial growth factor (VEGF) in breast carcinoma lines. Whereas in normal cells the expression of VEGF is dependent on a condition of hypoxia, surprisingly VEGF production by tumor cell lines and occurred at a concentration of oxygen partial indicating 20% indicative of impaired VEGF expression. In cancer constitutive secretion of VEGF independent of a condition that favors hypoxic tumor angiogenesis and growth in a clinical setting and is a marker of poor prognosis

RANTES), which play an important role in endothelial cell activation [52-53,65-66].

[63-64].

*VEGF:*

tumorigenesis [52-53,65].

tors of endothelial function [53].

play an important role in tumor progression [66].

194 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

sion and extravasation during the inflammatory reaction.

The involvement of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) in can‐ cer is complex, since it seems to require the presence of other cytokines such as IL-4 and IL-6, and there are reports antagonistic to their involvement in tumorigenesis. For example, expression of GM-CSF and interleukin-12, inhibits the immune response and being ex‐ pressed on B16 murine melanoma cells, decreasing completely tumorigenic capacity of syn‐ genic mice C57B/6. In contrast to the line described breast epithelial MCF7-A benign growth factors secreted able to induce expression of IL-6 and GM-CSF in the tumor line R2T1AS breast cancer that is associated with a higher rate of growth and a higher tumorigenic ca‐ pacity in vivo. Moreover, continued exposure of GM-CSF plus IL-4 of mesenchymal cells from human bone marrow, resulted in an increase in the morphological transformation and increased rate of growth both *in vitro* and *in vitro* mesenchymal cells, indicating the induc‐ tion of a transformed phenotype. Whereas previous reports indicate that the involvement of GM-CSF requires other cytokines [69-70].

### *IL-1ra*

It is reported that the soluble form of IL-1 receptor could serve as a functional antagonist of IL-1, the presence of this protein is controversial, since IL-1 by trapping an anti-inflammato‐ ry effect would reduce the adhesion of cells added by tumor cells and their extravasation, given this scenario could be proposed that tumor cells not only produce pro-inflammatory factors through the release of soluble factors tumor, but it can regulate the inflammatory process [71].

#### *IL-6*

The IL-6 is recognized as a classic inducer of the states of chronic inflammation and that pro‐ mote the activation of vascular endothelium. It has also been reported that IL-6 is produced by tumors that develop metastases to the liver, as the case of colon and mammary gland cancer. The activity of this cytokine in the soluble factors tumor could be further enhanced by the presence of other co-factors secreted by cells [72-73].

### *IL-8*

Expression of IL-8 in colorectal cancer favors an increase in tumorigenesis and metastasis, this increase is due to IL-8 is associated with expression of MMP-2 and MMP-9, the activity of these metalloproteases have been identified in physiological tissue remodeling processes like normal healing, but also participate in tissue remodeling associated with pathological processes including invasion [74]. Also in melanomas have been identified as IL-8 acts as an angiogenic factor and also promotes mitosis, therefore the IL-8 has been postulated as a po‐ tential therapeutic target. In particular, we have sought to interfere with IL-8 with the pur‐ pose of reducing tumor growth, an alternative that has developed is the use of an anti-IL-8 in nude mice with liver cancer. The results show that administration of neutralizing anti-IL8 significantly decreased tumor growth, even more interestingly this decrease is associated with decreased expression of MMP-2 and MMP-9. Something similar is observed using the same experimental treatment of melanoma with a decrease in angiogenesis [75]. In our re‐ sults, we found increases in IL-8 in the soluble factors soluble breast cancer [66]. This indi‐ cates that IL-8 could be used as a marker associated with tumor progression, regardless of tumor type. In patients where they identified the production of IL-8, using neutralizing anti‐ bodies that interfere with their signaling could serve as a therapeutic alternative.

**3.1. Introduction**

In different pathological process the cell injury is induced by free radicals, is an important mechanism of cell damage in many pathological conditions, such as chemical and radiation injury, ischemia injury, cellular aging and some immune system cells such as the phagocytes [82-84]. The free radicals are an example of instability in a biological system; namely, are chemical substances that they have an unpaired electron in its final orbit, this causes that their energy to be unstable and for they become stable they need for molecules which are adjacent to it either organic or inorganic for example: lipids, proteins, carbohydrates and nu‐

Reactive oxygen species (ROS) and reactive nitrogen (RNS) (Table 1) are created in the some cells such as the hepatocytes and in different normal physiologic processes, including, oxi‐ dative respiration, growth, regeneration, apoptosis and microbial killing by phagocytes [83]. The generation of this species chemical types, is normal in a normal cells; however, when these start to produce in excess and the antioxidant system is deficient, oxidative stress oc‐ curs. This causes damage cells: hepatocytes, kupffer cells and endothelial cells, through in‐ duction of inflammation, ischemia, fibrosis, apoptosis, necrosis or other atypical

> Smooth muscle (control or vascular tone) and other cGMP- depended functions. (glycogenolysis, apoptosis, conductance regulator ion channels, vasodilatation

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Oxidation-reduction reactions within the cell, muscle relaxation, control of erythropoietin production, signal transduction from various receptors, enhancement of

and increased blood flow)

immunological functions and oxidative

cleic acids essentials compounds in the cells mainly in the membrane and the core.

These types of chemical species may be either.

**1.** Oxygen derived (reactive oxygen species ROS)

transformation in the cell structure and function.

Nitrogen derived (nitric oxide) Oxygen derived

**3.2. Reactive oxygen species**

(ROS)

**Type of radical Activating enzyme Physiological process**

**Table 1.** *Important physiological process involved with the free radicals.ROS, Reactive oxygen species.*

Reactive oxygen species are produced in normal condition them in a living cell during cellu‐ lar respiration, energy production and various events of growth and cell death, these are de‐ grade by the defensive systems. Therefore, the cells self-regulate their production and degradation of ROS is found transiently in the cell without causing any damage to the cellu‐ lar level, and for that reason the cell maintains an equilibrium constant but as this produc‐ tion increases oxidative stress is generated, this relates whit different pathological process

Nitric oxide synthase NADPH oxidase

**2.** Nitrogen derived (reactive nitrogen species RNS)

### *IP10*

IP10 is a protein induced by interferon, has been reported that this protein inhibits prolifera‐ tion and metastatic tumors, that expression of IP10 in patients with stages II and III colorec‐ tal cancer correlate with the development of metastasis and a poor prognosis. The detection of IP10 could be used as a prognostic marker in stage II and III colorectal cancer patients. This has led to propose that IP10 might be used as a prognostic marker in stage II and III colorectal cancer patients [76-77].

### *RANTES*

Regulated upon activation normal T-cell expressed, and secreted is a chemokine that be‐ longs to the CC class, which distinguishes it from IL8, which belongs to the CXC class. RANTES expression in tumor cells has been associated with tumorigenesis and is consistent with our finding of RANTES the products secreted by breast carcinomas. The presence of RANTES in the tumor microenvironment may be chemoattractant to tumor cells. From this point of view is interesting that the tumor-associated endothelial cells, when stimulated with MIF1-alpha can release RANTES [78]. Study that evaluated the expression of RANTES and its receptor CCR5 in 60 patients with metastatic gastric cancer, were identified elevated expression levels, where it is concluded that RANTES and its receptor may contribute to gastric cancer metastasis by promoting responses TH1 and TH2. By comparing a variety of biological markers in a group of biopsies of mammary gland cancer, RANTES was the only marker present in all biopsies [79].

The reported findings strengthen the idea that soluble factors of tumor microenvironment may be relevant in the final stages of the metastatic spread and that these effects may be mediated by cytokines, chemokines, and growth factors present in the soluble factors secret‐ ed by tumor cell lines. These elements found in high concentrations are known to be capable of inducing the activated phenotype of endothelial cells to a variety of physiological and pathological cellular responses.

### **3. Oxidative stress and free radicals: role in cancer development**

During the inflammatory process macrophages and endothelial cells, generate a large amount of growth factors, cytokines and reactive oxygen species (ROS) and nitrogen (RNS) that can cause DNA damage. If macrophages and remain on the endothelium may allow the tissue damage continues chronic inflammation predisposes to malignancy [56,80].

### **3.1. Introduction**

with decreased expression of MMP-2 and MMP-9. Something similar is observed using the same experimental treatment of melanoma with a decrease in angiogenesis [75]. In our re‐ sults, we found increases in IL-8 in the soluble factors soluble breast cancer [66]. This indi‐ cates that IL-8 could be used as a marker associated with tumor progression, regardless of tumor type. In patients where they identified the production of IL-8, using neutralizing anti‐

IP10 is a protein induced by interferon, has been reported that this protein inhibits prolifera‐ tion and metastatic tumors, that expression of IP10 in patients with stages II and III colorec‐ tal cancer correlate with the development of metastasis and a poor prognosis. The detection of IP10 could be used as a prognostic marker in stage II and III colorectal cancer patients. This has led to propose that IP10 might be used as a prognostic marker in stage II and III

Regulated upon activation normal T-cell expressed, and secreted is a chemokine that be‐ longs to the CC class, which distinguishes it from IL8, which belongs to the CXC class. RANTES expression in tumor cells has been associated with tumorigenesis and is consistent with our finding of RANTES the products secreted by breast carcinomas. The presence of RANTES in the tumor microenvironment may be chemoattractant to tumor cells. From this point of view is interesting that the tumor-associated endothelial cells, when stimulated with MIF1-alpha can release RANTES [78]. Study that evaluated the expression of RANTES and its receptor CCR5 in 60 patients with metastatic gastric cancer, were identified elevated expression levels, where it is concluded that RANTES and its receptor may contribute to gastric cancer metastasis by promoting responses TH1 and TH2. By comparing a variety of biological markers in a group of biopsies of mammary gland cancer, RANTES was the only

The reported findings strengthen the idea that soluble factors of tumor microenvironment may be relevant in the final stages of the metastatic spread and that these effects may be mediated by cytokines, chemokines, and growth factors present in the soluble factors secret‐ ed by tumor cell lines. These elements found in high concentrations are known to be capable of inducing the activated phenotype of endothelial cells to a variety of physiological and

During the inflammatory process macrophages and endothelial cells, generate a large amount of growth factors, cytokines and reactive oxygen species (ROS) and nitrogen (RNS) that can cause DNA damage. If macrophages and remain on the endothelium may allow the

**3. Oxidative stress and free radicals: role in cancer development**

tissue damage continues chronic inflammation predisposes to malignancy [56,80].

bodies that interfere with their signaling could serve as a therapeutic alternative.

196 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

*IP10*

*RANTES*

colorectal cancer patients [76-77].

marker present in all biopsies [79].

pathological cellular responses.

In different pathological process the cell injury is induced by free radicals, is an important mechanism of cell damage in many pathological conditions, such as chemical and radiation injury, ischemia injury, cellular aging and some immune system cells such as the phagocytes [82-84]. The free radicals are an example of instability in a biological system; namely, are chemical substances that they have an unpaired electron in its final orbit, this causes that their energy to be unstable and for they become stable they need for molecules which are adjacent to it either organic or inorganic for example: lipids, proteins, carbohydrates and nu‐ cleic acids essentials compounds in the cells mainly in the membrane and the core.

These types of chemical species may be either.


Reactive oxygen species (ROS) and reactive nitrogen (RNS) (Table 1) are created in the some cells such as the hepatocytes and in different normal physiologic processes, including, oxi‐ dative respiration, growth, regeneration, apoptosis and microbial killing by phagocytes [83]. The generation of this species chemical types, is normal in a normal cells; however, when these start to produce in excess and the antioxidant system is deficient, oxidative stress oc‐ curs. This causes damage cells: hepatocytes, kupffer cells and endothelial cells, through in‐ duction of inflammation, ischemia, fibrosis, apoptosis, necrosis or other atypical transformation in the cell structure and function.


**Table 1.** *Important physiological process involved with the free radicals.ROS, Reactive oxygen species.*

#### **3.2. Reactive oxygen species**

Reactive oxygen species are produced in normal condition them in a living cell during cellu‐ lar respiration, energy production and various events of growth and cell death, these are de‐ grade by the defensive systems. Therefore, the cells self-regulate their production and degradation of ROS is found transiently in the cell without causing any damage to the cellu‐ lar level, and for that reason the cell maintains an equilibrium constant but as this produc‐ tion increases oxidative stress is generated, this relates whit different pathological process such as damage in the cell structure and function, degenerative process and cancer, also in‐ fluence within the inflammation and the immune response, as these are generated by macro‐ phages and neutrophils as mediators for the destruction of pathogens and dead tissue. The generation of free radicals can be made by different pathways [85-86]. (Table 2)

When increased production of reactive species and have a deficiency in the antioxidant sys‐ tem they cause significant neoplastic changes. In some diseases, such as Bloom syndrome develops lymphomas, leukemias and carcinomas, in anemia are implicated the production of these and alterations of antioxidant defense mechanisms at the systemic level [82-83]. Some epidemiological information indicates that tumor incidence is lower in populations

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Tumor cells have a high activity of free radical formation in contrast to healthy cells. It is known that tumor cells not only produce oxygen peroxide (H2O2) but also decreases the pro‐

Increasing the metabolic activity of a neoplastic cell, lead to the increased energy require‐ ments (ATP) produced in the mitochondria this in order to enhance the growth and prolifer‐

**1.** The progression of cancer, primarily because of the damage they cause in to the genetic material of a normal cell. Has been shown that the oxidation of guanine to 8-oxo-dG (oxidation product DNA) induces errors in their replication. By dependent of DNA pol‐ ymerase that generates the nitrogenous base pairing, not complementary, to permit the

**2.** Oncogenic transformation is conditioned by the presence of mutated genes or onco‐ genes that control essential cellular functions in which the redox state within or outside the cellular microenvironment is very important ROS are potential carcinogens because they facilitate mutagenesis, tumor growth, and metastasis and all those process that has

Cancer is a multifactorial disease where endogenous and exogenous factors are involved but the roles played by free radicals in this disease are very important because, these produce

Antioxidants are substances that prevent damage to cells caused by free radicals, it can cause damage to DNA, leading to the possible development of cancer [87]. Antioxidants search for these free radicals and lend them an, this stabilizes the molecule, thus preventing damage to other cells. Antioxidants also turn free radicals into waste by products, and they eventually are eliminated from the body. The inability of our body to neutralize free radicals we are exposed daily forces us to rely on foods with antioxidant properties capable of neu‐

establishment of hydrogen bonds with (A) adenine and (T) thymine [85].

damage in de DNA structure and this produces an important negative effects [86].

**4. Antioxidants and Chemoprevention in cancer**

Some pathways by which cancer cells have high amounts of ROS is for multiple factors:

where the diet is rich in antioxidants like fruits and vegetables [84].

duction of antioxidants such as glutathione peroxidase and SOD.

ation.

been showed [86].

tralizing them [88].


**Table 2.** The generation of free radicals can be made by different pathways.

#### **3.3. Free radical and carcinogenesis**

Free radicals are atoms or groups of atoms that in their atomic structure present one or more unpaired electrons in the outer orbit. These free radicals steal electrons from other molecules in effort to heal themselves, ultimately creating new free radicals in the process by stealing electrons. Free radicals are formed from a number of causes such as cigarette smoke, pollu‐ tion, exposure to sunlight all cause the formation of free radicals. Other factors include nor‐ mal daily processes like food digestion and breathing.

When increased production of reactive species and have a deficiency in the antioxidant sys‐ tem they cause significant neoplastic changes. In some diseases, such as Bloom syndrome develops lymphomas, leukemias and carcinomas, in anemia are implicated the production of these and alterations of antioxidant defense mechanisms at the systemic level [82-83]. Some epidemiological information indicates that tumor incidence is lower in populations where the diet is rich in antioxidants like fruits and vegetables [84].

such as damage in the cell structure and function, degenerative process and cancer, also in‐ fluence within the inflammation and the immune response, as these are generated by macro‐ phages and neutrophils as mediators for the destruction of pathogens and dead tissue. The

Redox reactions in metabolic processes. During cellular respiration O2 is reduced by four

Absorption of radiant energy Electromagnetic radiation (x rays), gamma rays,

Inflammation and immune response. This response induced by activated leukocytes, that

Metabolism of drugs Most chemicals do not show biological activity in its

Metals The transmission metals donate or accept electrons

Nitric oxide Nitric oxide is generated by different cells are an

Free radicals are atoms or groups of atoms that in their atomic structure present one or more unpaired electrons in the outer orbit. These free radicals steal electrons from other molecules in effort to heal themselves, ultimately creating new free radicals in the process by stealing electrons. Free radicals are formed from a number of causes such as cigarette smoke, pollu‐ tion, exposure to sunlight all cause the formation of free radicals. Other factors include nor‐

**Table 2.** The generation of free radicals can be made by different pathways.

mal daily processes like food digestion and breathing.

**3.3. Free radical and carcinogenesis**

superoxide anion.

electrons to the transport of H2 for generating two molecules of water through an oxidative enzyme which results is the formation of superoxide anion (electron), hydrogen peroxide (two electron ) and

infrared, UV, microwave etc. These to hydrolyze the water and generate hydroxyl ions and hydrogen

is caused by a protein complex located at the plasma membrane that employs NADPH oxidase and some intracellular oxidase and this generated a

native form these have to become toxic reactive metabolites to act on their target molecules. This is made by oxidases. These metabolites induce the formation of free radicals, such as acetaminophen.

intracellular free during the reactions thus causing

example of this are the endothelial cells giving rise to reactive species of nitrogen, such as the

) also NO2 and NO3 -

.

the formation of free radicals

peroxynitrite anion (ONOO-

hydroxyl ions (three electrons).

generation of free radicals can be made by different pathways [85-86]. (Table 2)

198 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Tumor cells have a high activity of free radical formation in contrast to healthy cells. It is known that tumor cells not only produce oxygen peroxide (H2O2) but also decreases the pro‐ duction of antioxidants such as glutathione peroxidase and SOD.

Some pathways by which cancer cells have high amounts of ROS is for multiple factors:

Increasing the metabolic activity of a neoplastic cell, lead to the increased energy require‐ ments (ATP) produced in the mitochondria this in order to enhance the growth and prolifer‐ ation.


Cancer is a multifactorial disease where endogenous and exogenous factors are involved but the roles played by free radicals in this disease are very important because, these produce damage in de DNA structure and this produces an important negative effects [86].

### **4. Antioxidants and Chemoprevention in cancer**

Antioxidants are substances that prevent damage to cells caused by free radicals, it can cause damage to DNA, leading to the possible development of cancer [87]. Antioxidants search for these free radicals and lend them an, this stabilizes the molecule, thus preventing damage to other cells. Antioxidants also turn free radicals into waste by products, and they eventually are eliminated from the body. The inability of our body to neutralize free radicals we are exposed daily forces us to rely on foods with antioxidant properties capable of neu‐ tralizing them [88].

### **4.1. Flavonoids**

Flavonoids are found in numerous plants and vegetables, with a wide distribution through the plant kingdom. This class compounds numbers more than 4000 members and can be divided into five subcategories: flavones, monomeric flavanols, flavanones, flavonols and anthocyanidines. Are natural compounds chemically derivate from bezo-y-pirone (phenyl‐ chromone) or flavone. They are considered important constituents of the human diet. It has been reported that they exert multiple biological effects due to their antioxidant and free radical-scavenging abilities [89].

tween tumor cells and endothelial cells or not allowing the passage of cells whether there

Inflammatory Environmental, Oxidative Stress in Tumoral Progression

http://dx.doi.org/10.5772/51789

201

Understanding the molecular basis of these interactions between metastatic cells and endo‐ thelial cells, will enable us to design strategies to interfere with this inter-cellular communi‐ cation. It is important to recognize the tumor-associated inflammatory microenvironment and what is the contribution to tumor progression. The importance of these factors on endo‐ thelial activation being evaluated by reconstituting the mixture with cytokines, chemokines and growth factors recombinant depleted mixtures of tumor soluble factors of each of these

Is important mention that during the inflammatory process macrophages, fibroblasts and endothelial cells generate a large amount of growth factors, cytokines, chemokines and reac‐ tive oxygen species (ROS) and nitrogen (RNS) that can cause DNA damage. These process allow the tissue damage continues chronic inflammation predisposes to malignancy. There‐ fore, it is important to note that people with chronic degenerative diseases, which clearly show chronic inflammatory processes, they may promote or contribute to present or devel‐

The use of antioxidants consumed in a balanced diet can be used as an element in the diet that can become a preventive or contributing to diminish the appearance of a tumor lesion.

and Alejandro Zentella-Dehesa4,5

2 Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo (UAEH),

3 Programa de Genómica Funcional de Procariotes, Centro de Ciencias Genómicas, Univer‐

4 Departamento de Medicina y Toxicología Ambiental, Instituto de Investigaciones Bioméd‐

5 Departamento de Bioquímica. (INNCMSZ) Instituto Nacional de Ciencias Médicas y Nu‐

,

César Esquivel-Chirino1,4,5,6\*, Jaime Esquivel-Soto1,6, José Antonio Morales-González2

1 Facultad de Odontología, Universidad Nacional Autónoma de México, México

Delina Montes Sánchez3,4,5, Jose Luis Ventura-Gallegos4,5,

\*Address all correspondence to: cesquivelch@gmail.com

sidad Nacional Autónoma de México, Campus Morelos, México

6 Facultad de Odontología, Universidad Intercontinental, México

icas. Universidad Nacional Autónoma de México, México

trición "Salvador Zubirán" México D.F., México

are appropriate conditions for the invasion.

proteins by specific monoclonal antibodies.

op a tumor lesion.

**Author details**

México

Luis Enrique Hernández-Mora1

Flavanoids possess a lot of pharmacological and therapeutically properties; antioxidant, an‐ titumor, antiangiogenic (vascular protective), anti-inflammatory, antiallergic, antihepatotox‐ ic, anticancerigenic, antimutagenic, anticancer effects, antiosteoporotic, and antiviral properties [90]. Many studies emphasize take adequate diets that are active allies against cancer. These diets are based on enzymes and antioxidant substances in certain foods that are rich in components that collect above [91].

They also have the ability to repair previous damage to cells, examples of antioxidants in‐ clude (beta-carotene, lycopene, vitamins C, E, and A), and other substances. Nutrients such as; green tea, flavonoids, vitamins C, E, and Beta-carotene in the carcinogen process, has been showed that have function in the elimination of carcinogenic factors, inhibition of precarcinogens and reparation of DNA damage. The mechanisms are diverse and range from inhibition to an active reaction of the immune system in general. This has caused the use of multiple antioxidant micronutrients as preventive agents [90]. Several experimental data have demonstrated the antiproliferative and anti-carcinogenic and the role of chemopreven‐ tive agent of flavonoids [91-92].

Currently investigations are performed to determine the mechanisms by which act flavo‐ noids, because it has been observed that their effects are greater at high doses, which gives them inducing side effects, so it is important to moderate their consumption by a bal‐ anced diet.

### **5. Conclusions**

It is important to analyze the role of tumor-associated inflammatory microenvironment and has been identified that plays an important role in tumor progression. This microenviron‐ ment is composed of molecules that play an important role in inflammatory processes and chronic, and favor the invasion and metastasis process that triggers the death of many peo‐ ple with any cancer.

The installation of tumor cells in blood vessels of the target organ to invade, is related to phenotypic changes in the endothelium allowing vascular extravasation of blood circulation of leukocytes in the inflammatory reaction and, as hypothesized current of tumor cells with metastatic capacity. The phenomenon of extravasation in response to a cell interactions be‐ tween tumor cells and endothelial cells or not allowing the passage of cells whether there are appropriate conditions for the invasion.

Understanding the molecular basis of these interactions between metastatic cells and endo‐ thelial cells, will enable us to design strategies to interfere with this inter-cellular communi‐ cation. It is important to recognize the tumor-associated inflammatory microenvironment and what is the contribution to tumor progression. The importance of these factors on endo‐ thelial activation being evaluated by reconstituting the mixture with cytokines, chemokines and growth factors recombinant depleted mixtures of tumor soluble factors of each of these proteins by specific monoclonal antibodies.

Is important mention that during the inflammatory process macrophages, fibroblasts and endothelial cells generate a large amount of growth factors, cytokines, chemokines and reac‐ tive oxygen species (ROS) and nitrogen (RNS) that can cause DNA damage. These process allow the tissue damage continues chronic inflammation predisposes to malignancy. There‐ fore, it is important to note that people with chronic degenerative diseases, which clearly show chronic inflammatory processes, they may promote or contribute to present or devel‐ op a tumor lesion.

The use of antioxidants consumed in a balanced diet can be used as an element in the diet that can become a preventive or contributing to diminish the appearance of a tumor lesion.

### **Author details**

**4.1. Flavonoids**

radical-scavenging abilities [89].

tive agent of flavonoids [91-92].

anced diet.

**5. Conclusions**

ple with any cancer.

are rich in components that collect above [91].

200 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Flavonoids are found in numerous plants and vegetables, with a wide distribution through the plant kingdom. This class compounds numbers more than 4000 members and can be divided into five subcategories: flavones, monomeric flavanols, flavanones, flavonols and anthocyanidines. Are natural compounds chemically derivate from bezo-y-pirone (phenyl‐ chromone) or flavone. They are considered important constituents of the human diet. It has been reported that they exert multiple biological effects due to their antioxidant and free

Flavanoids possess a lot of pharmacological and therapeutically properties; antioxidant, an‐ titumor, antiangiogenic (vascular protective), anti-inflammatory, antiallergic, antihepatotox‐ ic, anticancerigenic, antimutagenic, anticancer effects, antiosteoporotic, and antiviral properties [90]. Many studies emphasize take adequate diets that are active allies against cancer. These diets are based on enzymes and antioxidant substances in certain foods that

They also have the ability to repair previous damage to cells, examples of antioxidants in‐ clude (beta-carotene, lycopene, vitamins C, E, and A), and other substances. Nutrients such as; green tea, flavonoids, vitamins C, E, and Beta-carotene in the carcinogen process, has been showed that have function in the elimination of carcinogenic factors, inhibition of precarcinogens and reparation of DNA damage. The mechanisms are diverse and range from inhibition to an active reaction of the immune system in general. This has caused the use of multiple antioxidant micronutrients as preventive agents [90]. Several experimental data have demonstrated the antiproliferative and anti-carcinogenic and the role of chemopreven‐

Currently investigations are performed to determine the mechanisms by which act flavo‐ noids, because it has been observed that their effects are greater at high doses, which gives them inducing side effects, so it is important to moderate their consumption by a bal‐

It is important to analyze the role of tumor-associated inflammatory microenvironment and has been identified that plays an important role in tumor progression. This microenviron‐ ment is composed of molecules that play an important role in inflammatory processes and chronic, and favor the invasion and metastasis process that triggers the death of many peo‐

The installation of tumor cells in blood vessels of the target organ to invade, is related to phenotypic changes in the endothelium allowing vascular extravasation of blood circulation of leukocytes in the inflammatory reaction and, as hypothesized current of tumor cells with metastatic capacity. The phenomenon of extravasation in response to a cell interactions be‐

César Esquivel-Chirino1,4,5,6\*, Jaime Esquivel-Soto1,6, José Antonio Morales-González2 , Delina Montes Sánchez3,4,5, Jose Luis Ventura-Gallegos4,5, Luis Enrique Hernández-Mora1 and Alejandro Zentella-Dehesa4,5

\*Address all correspondence to: cesquivelch@gmail.com

1 Facultad de Odontología, Universidad Nacional Autónoma de México, México

2 Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo (UAEH), México

3 Programa de Genómica Funcional de Procariotes, Centro de Ciencias Genómicas, Univer‐ sidad Nacional Autónoma de México, Campus Morelos, México

4 Departamento de Medicina y Toxicología Ambiental, Instituto de Investigaciones Bioméd‐ icas. Universidad Nacional Autónoma de México, México

5 Departamento de Bioquímica. (INNCMSZ) Instituto Nacional de Ciencias Médicas y Nu‐ trición "Salvador Zubirán" México D.F., México

6 Facultad de Odontología, Universidad Intercontinental, México

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[64] Watts, M. E., Parkins, C. S., & Chaplin, D. J. (2002). Influence of hypoxia and tumourconditioned medium on endothelial cell adhesion molecule expression in vitro. *Anti‐*

[65] Estrada-Bernal, A., Mendoza-Milla, C., Ventura-Gallegos, J. L., Lopez-Bojorquez, L. N., Miranda-Peralta, E., Arechavaleta-Velasco, F., Vadillo-Ortega, F., Sanchez-San‐ chez, L., & Zentella-Dehesa, A. (2003). NF-kappaB dependent activation of human endothelial cells treated with soluble products derived from human lymphomas.

[66] Montes-Sanchez, D., Ventura, J. L., Mitre, I., Frias, S., Michan, L., Espejel-Nunez, A., Vadillo-Ortega, F., & Zentella, A. (2009). Glycosylated VCAM-1 isoforms revealed in 2D western blots of HUVECs treated with tumoral soluble factors of breast cancer

[67] Baldewijns , M. M., van Vlodrop, I. J., Vermeulen, P. B., Soetekouw, P. M., van Enge‐ land, M., & de Bruïne, A. P. (2010). VHL and HIF signalling in renal cell carcinogene‐

[68] Kamada, H., Tsutsumi, Y., Kihira, T., Tsunoda, S., Yamamoto, Y., & Mayumi, T. (2000). In vitro remodeling of tumor vascular endothelial cells using conditioned me‐ dium from various tumor cells and their sensitivity to TNF-alpha. *Biochemical and Bi‐*

[69] Edeline, J., Vigneau, C., Patard, J. J., & Rioux-Leclercq, N. (2010). Signalling pathways in renal-cell carcinoma: from the molecular biology to the future therapy]. *Bull Can‐*

[70] Kulbe, H., Chakravarty, P., Leinster, D. A., Charles, K. A., Kwong, J., Thompson, R. G., Gallagher, W. M., Galletta, L., Salako, M. A., Smyth, J. F., Hagemann, T., Brennan, D. J., Bowtell, D. D., & Balkwill, F. R. (2011). A dynamic inflammatory cytokine net‐

[71] Friberg, E., Orsini, N., Mantzoros, C. S., & Wolk, A. (2007). Diabetes mellitus and risk

[72] Mc Lean, M. H., Murray, G. I., Stewart, K. N., Norrie, G., Mayer, C., Hold, G. L., Thomson, J., & El -Omar, E. M. (2011). The inflammatory microenvironment in color‐

[73] Rajkumar, T., Shirley, S., Raja, U. M., & Ramakrishnan, S. A. Identification and vali‐

dation of genes involved in gastric tumorigenesis. *Cancer Cell Int.*, 10, 45.

work in the human ovarian cancer microenvironment. *Cancer Res.*

of endometrial cancer: a meta-analysis. *Diabetologia.*, 50(7), 1365-74.

*Sci.*, 117, 373-380.

*cancer Res.*, 22, 953-958.

*Cancer Lett*, 191, 239-48.

cells. *BMC Chem Biol*, 9, 7.

sis. *J Pathol.*, 221(2), 125-38.

*cer*, 97, 5-15.

*ophysical Research Communications*, 268, 809-813.

ectal neoplasia. PLoS One. Jan 7 , 6(1), e15366.

D1/E. *Clin Hemorheol Microcirc.*, 29, 383-390.

206 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants


[89] Jovanovic, S. V., Steenken, S., Simic, M. G. y., & Hara, Y. (1998). Antioxidant proper‐ ties of flavonoids: reduction potentials and electron transfer reactions of flavonoid radicals. En: Rice Evans C, Parker L (eds.): Flavonoids in health and disease. *Marcel Dekker, Nueva York*, 137-161.

**Chapter 9**

**Oxidative Stress in Diabetes Mellitus and the Role Of**

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects of insulin action, insulin secretion or both [1]. Diabetes has taken place as one of the most important diseases worldwide, reaching epidemic proportions. Global estimates predict that the proportion of adult population with diabetes will increase 69% for the year 2030 [2].

Hyperglycemia in the course of diabetes usually leads to the development of microvascular complications, and diabetic patients are more prone to accelerated atherosclerotic macrovas‐ cular disease. These complications account for premature mortality and most of the social

Increasing evidence suggests that oxidative stress plays a role in the pathogenesis of diabe‐ tes mellitus and its complications [4]. Hyperglycemia increases oxidative stress, which con‐ tributes to the impairment of the main processes that fail during diabetes, insulin action and insulin secretion. In addition, antioxidant mechanisms are diminished in diabetic patients, which may further augment oxidative stress [5, 6]. Several studies have addressed the possi‐ ble participation of dietary antioxidants, such as vitamins, in ameliorating the diabetic state

The aim of this chapter is to revise the current knowledge of the role of oxidative stress in the pathogenesis of diabetes mellitus and its complications, and to discuss the existing evi‐

> © 2013 Lazo-de-la-Vega-Monroy and Fernández-Mejía; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the

**Vitamins with Antioxidant Actions**

Maria-Luisa Lazo-de-la-Vega-Monroy and

Additional information is available at the end of the chapter

and economical burden in the long term of diabetes [3].

and retarding the development of diabetes complications [7, 8].

original work is properly cited.

dence of the effects of vitamins as antioxidant therapy for this disease.

Cristina Fernández-Mejía

http://dx.doi.org/10.5772/51788

**1. Introduction**


### **Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions**

Maria-Luisa Lazo-de-la-Vega-Monroy and Cristina Fernández-Mejía

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51788

### **1. Introduction**

[89] Jovanovic, S. V., Steenken, S., Simic, M. G. y., & Hara, Y. (1998). Antioxidant proper‐ ties of flavonoids: reduction potentials and electron transfer reactions of flavonoid radicals. En: Rice Evans C, Parker L (eds.): Flavonoids in health and disease. *Marcel*

[90] Letan, A. (1966). The relation of structure to antioxidant activity of quercitin and

[91] Stahl, W., Ale-Agha, N. Y., & Polidori, M. C. (2002). Non-antioxidant properties of

[92] Stacvric, B. (1994). Quercitin in our diet: From potent mutagen to probable anticarci‐

[93] Da Silva, J., Herrmann, S. M., Peres, W., Possa, Marroni. N., Gonzalez Gallego, J. Y., & Erdtmann, B. (2002). Evaluation of the genotoxic effect of rutin and quercetin by

comet assay and micronucleus test. *Food Chem Toxicol*, 40, 941-947.

*Dekker, Nueva York*, 137-161.

some of its derivates. *J Food Sci*, 31, 518-523.

208 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

carotenoids. *Biol Chem*, 383, 553-558.

nogen. *Clinical Biochemistry*, 27, 245-248.

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects of insulin action, insulin secretion or both [1]. Diabetes has taken place as one of the most important diseases worldwide, reaching epidemic proportions. Global estimates predict that the proportion of adult population with diabetes will increase 69% for the year 2030 [2].

Hyperglycemia in the course of diabetes usually leads to the development of microvascular complications, and diabetic patients are more prone to accelerated atherosclerotic macrovas‐ cular disease. These complications account for premature mortality and most of the social and economical burden in the long term of diabetes [3].

Increasing evidence suggests that oxidative stress plays a role in the pathogenesis of diabe‐ tes mellitus and its complications [4]. Hyperglycemia increases oxidative stress, which con‐ tributes to the impairment of the main processes that fail during diabetes, insulin action and insulin secretion. In addition, antioxidant mechanisms are diminished in diabetic patients, which may further augment oxidative stress [5, 6]. Several studies have addressed the possi‐ ble participation of dietary antioxidants, such as vitamins, in ameliorating the diabetic state and retarding the development of diabetes complications [7, 8].

The aim of this chapter is to revise the current knowledge of the role of oxidative stress in the pathogenesis of diabetes mellitus and its complications, and to discuss the existing evi‐ dence of the effects of vitamins as antioxidant therapy for this disease.

© 2013 Lazo-de-la-Vega-Monroy and Fernández-Mejía; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. Oxidative stress**

At the beginning of life, the organisms obtained their energy (ATP) by anoxygenic photosin‐ thesis, for which oxygen was toxic. Most of the metabolic pathways were developed during this anaerobic stage of life, in which oxygen came later. Cyanobacteria started producing oxygen from photosynthesis, which raised the atmospheric oxygen, and favored those or‐ ganisms which have evolved into eukaryotic cells with mitochondria, able to use oxygen for a more efficient energy production [9].

and into intracellular and extracellular compartments, such as nucleus, endoplasmic reticu‐ lum, endosome, phagosome, mitochondria and extracellular space. It is the only enzyme whose primary function is generating superoxide and/or hydrogen peroxide, mainly for

Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions

http://dx.doi.org/10.5772/51788

211

Mitochondrion is the site of eukaryotic oxidative metabolism. It contains the enzymes need‐ ed for converting pyruvate into Acetyl-CoA, the citric acid cycle (also known as the Krebs cycle) and for fatty acid oxidation. Additionally, it performs the electron transport and oxi‐ dative phosphorylation. Substrate (amino acid, fatty acid and carbohydrate) oxidation in the citric acid cycle release electrons, which are transferred to the coenzymes NAD+ and FAD to form NADH and FADH2. These electrons then pass into the mitochondrial electron-trans‐ port chain, a system of linked electron carrier proteins comprised by Complexes I, II, III and IV. Complex I, III, and IV drive the exit of protons from the mitochondrial matrix, producing a proton gradient across the inner mitochondrial membrane. The free energy stored in this electrochemical gradient drives the condensation of ADP with inorganic phosphate in order to form ATP by oxidative phosphorylation. Along this electron transport, molecular oxygen is the final electron acceptor, which will be then reduced to H2O [14, 15]. However, between 0.4 and 4% of all oxygen consumed will be converted into superoxide anion [16]. There is also a normal threshold for protonic potential above which electron transfer is inhibited at complex III, causing the electrons to go back to complex II where there are transferred to molecular oxygen prematurely and not to complex IV as it naturally occurs. Therefore, the

Mitochondria play an important role in the maintenance of cellular redox status, acting as a redox sink and limiting NADPH oxidase activity. However, when the proton potential threshold is surpassed, mitochondria is also a significant source of ROS, which may further stimulate NADPH oxidases, creating a vicious cycle of ROS production [18]. When mito‐ chondria cannot further extract oxygen, cell and tissue oxygen levels rise, decreasing the tis‐ sue extraction of oxygen from the blood. This results in tissue vascularity reduction, which may be associated with peripheral vascular disease and, in time, chronic tissue hypoxia and

Nitric oxide •NO is produced by the enzyme nitric oxide synthase (NOS), of which there are three isoforms: neural (nNOS or NOS-I) expressed in neurons, inducible (iNOS or NOS-II) expressed in smooth muscle of bold vessels, hepatocites, macrophagues and neuroendocirne tissue, and endothelial (eNOS or NOS-III) expressed constitutively in endothelial cells. iNOS and eNOS can be stimulated by the redox state in the cell, cytokines, hormones and nu‐ trients [19, 20]. NOS catalyze the oxidation of the terminal guanidine nitrogen of the L–argi‐

nine, in presence of oxygen and NADPH, to yield L-citruline and •NO [21].

preventing the transfer of pathogens and for cellular bactericidal function[12, 13].

*2.1.2. Mitochondrial electron transport chain*

endproduct of this transfer is superoxide [17].

ischemia [9].

*2.1.3. NO and RNS production*

Whenever a cell's internal environment is perturbed by infections, disease, toxins or nutri‐ tional imbalance, mitochondria diverts electron flow away from itself, forming reactive oxy‐ gen species (ROS) and reactive nitrogen species (RNS), thus lowering oxygen consumption. This "oxidative shielding" acts as a defense mechanism for either decreasing cellular uptake of toxic pathogens or chemicals from the environment, or to kill the cell by apoptosis and thus avoid the spreading to neighboring cells [9]. Therefore, ROS formation is a physiologi‐ cal response to stress.

The term "oxidative stress" has been used to define a state in which ROS and RNS reach excessive levels, either by excess production or insufficient removal. Being highly reactive molecules, the pathological consequence of ROS and RNS excess is damage to proteins, lip‐ ids and DNA [10]. Consistent with the primary role of ROS and RNS formation, this oxida‐ tive stress damage may lead to physiological dysfunction, cell death, pathologies such as diabetes and cancer, and aging of the organism [11].

### **2.1. ROS and RNS production**

ROS and RNS are highly reactive molecules, which can be free radicals such as superoxide (•O2 - ), hydroxyl (•OH), peroxyl (•RO2), hydroperoxyl (•HRO2 - ), nitric oxide (•NO) and nitro‐ gen dioxide (•NO2 - ), or nonradicals such as hydrogen peroxide (H2O2), hydrochlorous acid (HOCl), peroxynitrite (ONOO- ), nitrous oxide (HNO2), and alkyl peroxynitrates (RONOO). Most of the studies regarding diabetes and its complications have addressed the role of su‐ peroxide (•O2 - ), nitric oxide (•NO), and peroxynitrite (ONOO- ) in this disease. There are ba‐ sically two pathways for •O2 production: NADPH oxidases and mitochondrial function, while •NO and ONOO are produced by the Nitric Oxide Synthase pathway [10].

### *2.1.1. NADPH oxidases*

Oxidases are enzymes which catalyze redox reactions involving molecular oxygen (O2). Su‐ peroxide is generated by oxidases via one-electron reduction of oxygen and the oxidation of their substrates. Several oxidases exist in the body, such as xantine oxidase, glucose oxidase, monoamine oxidase, cytochrome P450 oxidase, and NADPH oxidases.

NADP in the cell exists in its reduced (NADPH) and oxidized (NADP+) forms. NADPH supplies reducing power in reactions for biosynthesis, and it also serves as electron donor substrate for the NADPH oxidase. This enzyme is a membrane-bound electron transport complex which pumps electrons from NADPH in the cytosol across biological membranes and into intracellular and extracellular compartments, such as nucleus, endoplasmic reticu‐ lum, endosome, phagosome, mitochondria and extracellular space. It is the only enzyme whose primary function is generating superoxide and/or hydrogen peroxide, mainly for preventing the transfer of pathogens and for cellular bactericidal function[12, 13].

### *2.1.2. Mitochondrial electron transport chain*

**2. Oxidative stress**

cal response to stress.

**2.1. ROS and RNS production**

(HOCl), peroxynitrite (ONOO-

sically two pathways for •O2 -

while •NO and ONOO-

*2.1.1. NADPH oxidases*

gen dioxide (•NO2 -

peroxide (•O2 -

(•O2 -

a more efficient energy production [9].

210 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

diabetes and cancer, and aging of the organism [11].

), hydroxyl (•OH), peroxyl (•RO2), hydroperoxyl (•HRO2 -

), nitric oxide (•NO), and peroxynitrite (ONOO-

monoamine oxidase, cytochrome P450 oxidase, and NADPH oxidases.

At the beginning of life, the organisms obtained their energy (ATP) by anoxygenic photosin‐ thesis, for which oxygen was toxic. Most of the metabolic pathways were developed during this anaerobic stage of life, in which oxygen came later. Cyanobacteria started producing oxygen from photosynthesis, which raised the atmospheric oxygen, and favored those or‐ ganisms which have evolved into eukaryotic cells with mitochondria, able to use oxygen for

Whenever a cell's internal environment is perturbed by infections, disease, toxins or nutri‐ tional imbalance, mitochondria diverts electron flow away from itself, forming reactive oxy‐ gen species (ROS) and reactive nitrogen species (RNS), thus lowering oxygen consumption. This "oxidative shielding" acts as a defense mechanism for either decreasing cellular uptake of toxic pathogens or chemicals from the environment, or to kill the cell by apoptosis and thus avoid the spreading to neighboring cells [9]. Therefore, ROS formation is a physiologi‐

The term "oxidative stress" has been used to define a state in which ROS and RNS reach excessive levels, either by excess production or insufficient removal. Being highly reactive molecules, the pathological consequence of ROS and RNS excess is damage to proteins, lip‐ ids and DNA [10]. Consistent with the primary role of ROS and RNS formation, this oxida‐ tive stress damage may lead to physiological dysfunction, cell death, pathologies such as

ROS and RNS are highly reactive molecules, which can be free radicals such as superoxide

Most of the studies regarding diabetes and its complications have addressed the role of su‐

Oxidases are enzymes which catalyze redox reactions involving molecular oxygen (O2). Su‐ peroxide is generated by oxidases via one-electron reduction of oxygen and the oxidation of their substrates. Several oxidases exist in the body, such as xantine oxidase, glucose oxidase,

NADP in the cell exists in its reduced (NADPH) and oxidized (NADP+) forms. NADPH supplies reducing power in reactions for biosynthesis, and it also serves as electron donor substrate for the NADPH oxidase. This enzyme is a membrane-bound electron transport complex which pumps electrons from NADPH in the cytosol across biological membranes

), or nonradicals such as hydrogen peroxide (H2O2), hydrochlorous acid

are produced by the Nitric Oxide Synthase pathway [10].

), nitrous oxide (HNO2), and alkyl peroxynitrates (RONOO).

production: NADPH oxidases and mitochondrial function,

), nitric oxide (•NO) and nitro‐

) in this disease. There are ba‐

Mitochondrion is the site of eukaryotic oxidative metabolism. It contains the enzymes need‐ ed for converting pyruvate into Acetyl-CoA, the citric acid cycle (also known as the Krebs cycle) and for fatty acid oxidation. Additionally, it performs the electron transport and oxi‐ dative phosphorylation. Substrate (amino acid, fatty acid and carbohydrate) oxidation in the citric acid cycle release electrons, which are transferred to the coenzymes NAD+ and FAD to form NADH and FADH2. These electrons then pass into the mitochondrial electron-trans‐ port chain, a system of linked electron carrier proteins comprised by Complexes I, II, III and IV. Complex I, III, and IV drive the exit of protons from the mitochondrial matrix, producing a proton gradient across the inner mitochondrial membrane. The free energy stored in this electrochemical gradient drives the condensation of ADP with inorganic phosphate in order to form ATP by oxidative phosphorylation. Along this electron transport, molecular oxygen is the final electron acceptor, which will be then reduced to H2O [14, 15]. However, between 0.4 and 4% of all oxygen consumed will be converted into superoxide anion [16]. There is also a normal threshold for protonic potential above which electron transfer is inhibited at complex III, causing the electrons to go back to complex II where there are transferred to molecular oxygen prematurely and not to complex IV as it naturally occurs. Therefore, the endproduct of this transfer is superoxide [17].

Mitochondria play an important role in the maintenance of cellular redox status, acting as a redox sink and limiting NADPH oxidase activity. However, when the proton potential threshold is surpassed, mitochondria is also a significant source of ROS, which may further stimulate NADPH oxidases, creating a vicious cycle of ROS production [18]. When mito‐ chondria cannot further extract oxygen, cell and tissue oxygen levels rise, decreasing the tis‐ sue extraction of oxygen from the blood. This results in tissue vascularity reduction, which may be associated with peripheral vascular disease and, in time, chronic tissue hypoxia and ischemia [9].

### *2.1.3. NO and RNS production*

Nitric oxide •NO is produced by the enzyme nitric oxide synthase (NOS), of which there are three isoforms: neural (nNOS or NOS-I) expressed in neurons, inducible (iNOS or NOS-II) expressed in smooth muscle of bold vessels, hepatocites, macrophagues and neuroendocirne tissue, and endothelial (eNOS or NOS-III) expressed constitutively in endothelial cells. iNOS and eNOS can be stimulated by the redox state in the cell, cytokines, hormones and nu‐ trients [19, 20]. NOS catalyze the oxidation of the terminal guanidine nitrogen of the L–argi‐ nine, in presence of oxygen and NADPH, to yield L-citruline and •NO [21].

Once produced and released, •NO can diffuse freely through membranes or act on different cellular targets. •NO participates as mediator of several physiological effects such as vasore‐ laxation, macrophague activation, gene expression and apoptosis. Usually, •NO is consid‐ ered as a vasculoprotective molecule. However, one of its multiple effects is also protein nitrosilation at the thiol groups and RNS generation such as peroxynitrite (ONOO- ), as •NO easily reacts with •O2 - . Therefore, the amount of •O2 determines whether •NO acts as a pro‐ tective or harmful molecule [10, 22].

Regeneration of GSH by GSH-reductase, requires NADPH, which is oxidized to NADP+. Catalase, on the other hand, is localized primarily in peroxisomes, and so it detoxifies the H2O2 that diffuses from the mitochondria to the cytosol, converting it into water and molec‐

Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions

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There are also nonenzymatic antioxidant mechanisms, which mostly help regenerate GSSG back into GSH. Antioxidant vitamins such as A, C, E and alpha-lipoic acid are among these mechanisms. Although all these antioxidant defenses work together to eliminate H2O2 (and thus superoxide) from the cell, in the presence of reduced transition metals (Cu, Fe), H2O2 can be transformed into •OH, which is a highly reactive ROS, by the Fenton reaction [10, 23].

There are several molecular pathways involved in ROS formation and ROS induced dam‐ age. Here we will review the ones that have been related to oxidative stress in diabetes. Not

In order to generate energy, glucose needs to be first oxidized inside the cells by glycolysis. In this process, once glucose enters the cells, it is phosphorylated to form glucose-6-phos‐ phate, a reaction mediated by hexocinases. Glucose-6-P is then converted to Fructose-6-P by phosphoglucoisomerase, which can undergo two fates: the pentose phosphate pathway,

hyde-3-P. Glyceraldehyde-3-P dehydrogenase (GAPDH) phosphorylates this product and glycolysis is further completed until its end product pyruvate, which enters the Krebs cycle

It has been proposed that hyperglycemia-induced mitochondrial superoxide production activates damaging pathways by inhibiting glyderaldehyde-3-phosphate dehydrogenase (GAPDH) [4, 24], an enzyme that normally translocates in and out of the nucleus [25, 26]. ROS inhibit glyderaldehyde-3-phosphate dehydrogenase through a mechanism involv‐ ing the activation of enzyme poly-ADP-ribose polymerase-1 (PARP-1). This enzyme is involved in DNA repair and apoptotic pathways. ROS cause strand breaks in nuclear DNA which activates PARP-1. PARP-1 activation results in inhibition of glyderaldehyde-3 phosphate dehydrogenase by poly-ADP-ribosylation [27]. This results in increased levels of all the glycolytic intermediates upstream of GAPDH. Accumulation of glyceralde‐ hyde 3-phosphate activates two major pathways involved in hyperglycemia-complica‐ tions: a) It activates the AGE pathway deriving glyceraldehyde phosphate and dihydroxyacetone phosphate to the nonenzymatic synthesis of methylglyoxal. b) Increased glyceraldehyde 3-phosphate favors diacylglycerol production which activates PKC pathway. Further up‐ stream, levels of the glycolytic metabolite fructose 6-phosphate increase, which then increas‐ es flux through the hexosamine pathway, where fructose 6-phosphate is converted by the enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) to UDP–N-Acetylglucos‐

to NADPH occurs, or to continue glycolysis to yield Gliceralde‐

**2.3. Metabolic and signaling pathways involved in oxidative stress in diabetes**

surprisingly, most of them are related to glucose and/or lipid metabolism.

ular oxygen [10, 12].

*2.3.1. Glucose oxidation and GAPDH*

and mitochondrial metabolism (Figure 2).

where reduction of NADP+

#### **2.2. Antioxidant defenses in the organism**

As a small part the oxygen consumed for aerobic processes will be converted into superox‐ ide anion [16], which will have to be scavenged or converted into less reactive (and harmful) molecules. The main enzymes that regulate this process are Superoxide dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Catalase (Figure 1). When ROS overproduction or chronic hyperglycemia occurs, the activity of these enzymes is insufficient, leading to more ROS and RNS formation and activation oxidative stress pathways.

**Figure 1.** Antioxidant defenses in the organism.

SOD is considered a first-line defense against ROS. This enzyme is present in nearly all cells, and converts •O2 into H2O2. Mitochondrial and bacterial SOD contain Mn, while cytosolic SOD is a dimer containing Cu and Zn. As the H2O2 may still react with other ROS, it needs to be degraded by either one of the other two antioxidant enzymes, GSH-Px or catalase [10, 12].

GSH peroxidase is located in the mitochondria. It catalyzes degradation of H2O2 by reduc‐ tion, where two gluthathione (GSH) molecules are oxidized to glutathione disulfide (GSSG). Regeneration of GSH by GSH-reductase, requires NADPH, which is oxidized to NADP+. Catalase, on the other hand, is localized primarily in peroxisomes, and so it detoxifies the H2O2 that diffuses from the mitochondria to the cytosol, converting it into water and molec‐ ular oxygen [10, 12].

There are also nonenzymatic antioxidant mechanisms, which mostly help regenerate GSSG back into GSH. Antioxidant vitamins such as A, C, E and alpha-lipoic acid are among these mechanisms. Although all these antioxidant defenses work together to eliminate H2O2 (and thus superoxide) from the cell, in the presence of reduced transition metals (Cu, Fe), H2O2 can be transformed into •OH, which is a highly reactive ROS, by the Fenton reaction [10, 23].

### **2.3. Metabolic and signaling pathways involved in oxidative stress in diabetes**

There are several molecular pathways involved in ROS formation and ROS induced dam‐ age. Here we will review the ones that have been related to oxidative stress in diabetes. Not surprisingly, most of them are related to glucose and/or lipid metabolism.

### *2.3.1. Glucose oxidation and GAPDH*

Once produced and released, •NO can diffuse freely through membranes or act on different cellular targets. •NO participates as mediator of several physiological effects such as vasore‐ laxation, macrophague activation, gene expression and apoptosis. Usually, •NO is consid‐ ered as a vasculoprotective molecule. However, one of its multiple effects is also protein

As a small part the oxygen consumed for aerobic processes will be converted into superox‐ ide anion [16], which will have to be scavenged or converted into less reactive (and harmful) molecules. The main enzymes that regulate this process are Superoxide dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Catalase (Figure 1). When ROS overproduction or chronic hyperglycemia occurs, the activity of these enzymes is insufficient, leading to more

SOD is considered a first-line defense against ROS. This enzyme is present in nearly all cells,

SOD is a dimer containing Cu and Zn. As the H2O2 may still react with other ROS, it needs to be degraded by either one of the other two antioxidant enzymes, GSH-Px or catalase [10, 12].

GSH peroxidase is located in the mitochondria. It catalyzes degradation of H2O2 by reduc‐ tion, where two gluthathione (GSH) molecules are oxidized to glutathione disulfide (GSSG).

into H2O2. Mitochondrial and bacterial SOD contain Mn, while cytosolic

), as •NO

determines whether •NO acts as a pro‐

nitrosilation at the thiol groups and RNS generation such as peroxynitrite (ONOO-

. Therefore, the amount of •O2 -

ROS and RNS formation and activation oxidative stress pathways.

easily reacts with •O2 -

tective or harmful molecule [10, 22].

**2.2. Antioxidant defenses in the organism**

212 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 1.** Antioxidant defenses in the organism.

and converts •O2 -

In order to generate energy, glucose needs to be first oxidized inside the cells by glycolysis. In this process, once glucose enters the cells, it is phosphorylated to form glucose-6-phos‐ phate, a reaction mediated by hexocinases. Glucose-6-P is then converted to Fructose-6-P by phosphoglucoisomerase, which can undergo two fates: the pentose phosphate pathway, where reduction of NADP+ to NADPH occurs, or to continue glycolysis to yield Gliceralde‐ hyde-3-P. Glyceraldehyde-3-P dehydrogenase (GAPDH) phosphorylates this product and glycolysis is further completed until its end product pyruvate, which enters the Krebs cycle and mitochondrial metabolism (Figure 2).

It has been proposed that hyperglycemia-induced mitochondrial superoxide production activates damaging pathways by inhibiting glyderaldehyde-3-phosphate dehydrogenase (GAPDH) [4, 24], an enzyme that normally translocates in and out of the nucleus [25, 26]. ROS inhibit glyderaldehyde-3-phosphate dehydrogenase through a mechanism involv‐ ing the activation of enzyme poly-ADP-ribose polymerase-1 (PARP-1). This enzyme is involved in DNA repair and apoptotic pathways. ROS cause strand breaks in nuclear DNA which activates PARP-1. PARP-1 activation results in inhibition of glyderaldehyde-3 phosphate dehydrogenase by poly-ADP-ribosylation [27]. This results in increased levels of all the glycolytic intermediates upstream of GAPDH. Accumulation of glyceralde‐ hyde 3-phosphate activates two major pathways involved in hyperglycemia-complica‐ tions: a) It activates the AGE pathway deriving glyceraldehyde phosphate and dihydroxyacetone phosphate to the nonenzymatic synthesis of methylglyoxal. b) Increased glyceraldehyde 3-phosphate favors diacylglycerol production which activates PKC pathway. Further up‐ stream, levels of the glycolytic metabolite fructose 6-phosphate increase, which then increas‐ es flux through the hexosamine pathway, where fructose 6-phosphate is converted by the enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) to UDP–N-Acetylglucos‐ amine. Finally, inhibition of GAPDH favors the accumulation of the first glycolytic metab‐ olite, glucose. This increases its flux through the polyol pathway, consuming NADPH in the process [24].

vascular endothelial growth factor (VEGF) mRNA expression and increase leukocyte-endo‐

Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions

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215

Accumulation of glyceraldehyde 3-phosphate, besides activating the AGE formation and the PKC pathway, it can oxidate itself. This autoxidation generates H2O2, which further contrib‐

Intracellular hyperglycaemia is the primary initiating event in the formation of both intracel‐ lular and extracellular AGEs [32]. AGEs can arise from intracellular auto-oxidation of glu‐ cose to glyoxal, decomposition of the Amadori product (glucose-derived 1-amino-1 deoxyfructose lysine adducts) to 3-deoxyglucosone (perhaps accelerated by an amadoriase), and nonenzymatic phosphate elimination from glyceraldehyde phosphate and dihydroxya‐ cetone phosphate to form methylglyoxal. These reactive intracellular dicarbonyl glyoxal, methylglyoxal and 3-deoxyglucosone react with amino groups of intracellular and extracel‐ lular proteins to form AGEs [4]. Intracellular production of AGE precursors can damage cells by three general mechanisms: 1) Intracellular proteins modified by AGEs have altered function, 2) Extracellular matrix components modified by AGE precursors interact abnor‐ mally with other matrix components and with matrix receptors (integrins) that are ex‐ pressed on the surface of cells, and 3) Plasma proteins modified by AGE precursors bind to AGE receptors (such as RAGE and AGE-R1,2 and 3) on cells such as macrophages, vascular endothelial cells and vascular smooth muscle cells. AGE receptors binding induces the pro‐ duction of ROS, which in turn activates PKC. It also activates NF-κB and NADPH oxidase,

In addition to direct damage of biomolecules in the cells, oxidative stress is also involved in activation of several stress-sensitive signaling pathways, which can result in inflamma‐ tion, cytokine release, and even apoptosis. Among these pathways we find the transcrip‐ tion factor NF-κB, which together with PARP acts as a transcriptional coactivator of inflammation molecules such as iNOS, intracellular adhesion molecule-1 (ICAM-I), and histocompatibility complex class II [33]. p38 MAPK pathway and c-Jun Nterminal kinase (JNK) (also known as stress-activated protein kinase (SAPK) participate in cellular respons‐ es to stress due to osmotic shock, cytokines and UV light, playing a role in cellular prolif‐ eration, apoptosis, and inflammatory responses [33]. Jak/STAT is another important signaling pathway, which initiates and mediates cellular responses to cytokines such as interferons

thelium interaction [11].

*2.3.5. Glyceraldehyde autoxidation*

utes to oxidative stress [31].

*2.3.6. Advanced glycation end-products (AGEs)*

and disturbs MAPK signaling [31].

and interleukins [33].

*2.3.7. Stress-sensitive signaling pathways*

### *2.3.2. The polyol pathway*

The family of aldo-keto reductase enzymes catalyzes the reduction of a wide variety of car‐ bonyl compounds to their respective alcohols. These reactions utilize nicotinic acid adenine dinucleotide phosphate (NADPH). Aldo-keto reductase has a low affinity (high Km) for glu‐ cose, and at the normal glucose concentrations, metabolism of glucose by this pathway is a very small percentage of total glucose metabolism. However, in a hyperglycemic environ‐ ment, increased intracellular glucose results in its increased enzymatic conversion to the polyalcohol sorbitol, with concomitant decreases in NADPH [4] (Figure 2). Since NADPH is a cofactor required to regenerate reduced glutathione, an antioxidant mechanism, and this compound is an important scavenger of reactive oxygen species (ROS), this could induce or exacerbate intracellular oxidative stress [24]. Moreover, sorbitol is oxidated to fructose by sorbitol dehydrogenase, which can lead to PKC activation via the increased NADH/NAD+ ratio [4]. Although this mechanism does not produce ROS in a direct way, it takes part in the redox imbalance causing oxidative stress.

#### *2.3.3. Hexosamine pathway*

When glucose levels are within normal range, a relatively low amount of fructose-6-P is drived away from glycolysis. If intracellular glucose rises, excess fructose-6-phosphate is di‐ verted from glycolysis to provide substrate for the rate-limiting enzyme of this pathway, GFAT. This enzyme converts fructose 6-phosphate to glucosamine 6-phosphate, which is then converted to UDP-NAcetylglucosamine, which is essential for making the glycosyl chains of proteins and lipids. Specific O-Glucosamine-N-Acetyl transferases use this metab‐ olite for post-translational modification of specific serine and threonine residues on cyto‐ plasmic and nuclear proteins [24, 28].

### *2.3.4. Diacylglycerol formation and PKC activation*

The Protein Kinase C (PKC) family comprises at least eleven isoforms of serine/threonine kinases, which participate in signaling pathways activated by phosphatidyl serine, Calci‐ um and Diacylglycerol (DAG). DAG levels are elevated chronically in the hyperglycemic or diabetic environment due to an increase in the glycolytic intermediate dihydroxyace‐ tone phosphate (figure 2). This intermediate is reduced to glycerol-3-phosphate, which, conjugated with fatty acids, increases de novo synthesis of DAG [29]. Evidence suggests that the enhanced activity of PKC isoforms could arise from inhibition of the glycolytic enzyme glyceraldehide-3-phosphate dehydrogenase by increased ROS intracellular levels [4, 24]. Other studies suggest that enhanced activity of PKC isoforms could also result from the interaction between AGEs and their extracellular receptors [30]. PKC isoforms consti‐ tute a wide range of cellular signals, including activation of NADPH oxidase and NF-κB, resulting in excessive ROS production. They also increase vascular permeability, stabilize vascular endothelial growth factor (VEGF) mRNA expression and increase leukocyte-endo‐ thelium interaction [11].

#### *2.3.5. Glyceraldehyde autoxidation*

amine. Finally, inhibition of GAPDH favors the accumulation of the first glycolytic metab‐ olite, glucose. This increases its flux through the polyol pathway, consuming NADPH in

The family of aldo-keto reductase enzymes catalyzes the reduction of a wide variety of car‐ bonyl compounds to their respective alcohols. These reactions utilize nicotinic acid adenine dinucleotide phosphate (NADPH). Aldo-keto reductase has a low affinity (high Km) for glu‐ cose, and at the normal glucose concentrations, metabolism of glucose by this pathway is a very small percentage of total glucose metabolism. However, in a hyperglycemic environ‐ ment, increased intracellular glucose results in its increased enzymatic conversion to the polyalcohol sorbitol, with concomitant decreases in NADPH [4] (Figure 2). Since NADPH is a cofactor required to regenerate reduced glutathione, an antioxidant mechanism, and this compound is an important scavenger of reactive oxygen species (ROS), this could induce or exacerbate intracellular oxidative stress [24]. Moreover, sorbitol is oxidated to fructose by sorbitol dehydrogenase, which can lead to PKC activation via the increased NADH/NAD+ ratio [4]. Although this mechanism does not produce ROS in a direct way, it takes part in the

When glucose levels are within normal range, a relatively low amount of fructose-6-P is drived away from glycolysis. If intracellular glucose rises, excess fructose-6-phosphate is di‐ verted from glycolysis to provide substrate for the rate-limiting enzyme of this pathway, GFAT. This enzyme converts fructose 6-phosphate to glucosamine 6-phosphate, which is then converted to UDP-NAcetylglucosamine, which is essential for making the glycosyl chains of proteins and lipids. Specific O-Glucosamine-N-Acetyl transferases use this metab‐ olite for post-translational modification of specific serine and threonine residues on cyto‐

The Protein Kinase C (PKC) family comprises at least eleven isoforms of serine/threonine kinases, which participate in signaling pathways activated by phosphatidyl serine, Calci‐ um and Diacylglycerol (DAG). DAG levels are elevated chronically in the hyperglycemic or diabetic environment due to an increase in the glycolytic intermediate dihydroxyace‐ tone phosphate (figure 2). This intermediate is reduced to glycerol-3-phosphate, which, conjugated with fatty acids, increases de novo synthesis of DAG [29]. Evidence suggests that the enhanced activity of PKC isoforms could arise from inhibition of the glycolytic enzyme glyceraldehide-3-phosphate dehydrogenase by increased ROS intracellular levels [4, 24]. Other studies suggest that enhanced activity of PKC isoforms could also result from the interaction between AGEs and their extracellular receptors [30]. PKC isoforms consti‐ tute a wide range of cellular signals, including activation of NADPH oxidase and NF-κB, resulting in excessive ROS production. They also increase vascular permeability, stabilize

the process [24].

*2.3.2. The polyol pathway*

redox imbalance causing oxidative stress.

214 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

plasmic and nuclear proteins [24, 28].

*2.3.4. Diacylglycerol formation and PKC activation*

*2.3.3. Hexosamine pathway*

Accumulation of glyceraldehyde 3-phosphate, besides activating the AGE formation and the PKC pathway, it can oxidate itself. This autoxidation generates H2O2, which further contrib‐ utes to oxidative stress [31].

### *2.3.6. Advanced glycation end-products (AGEs)*

Intracellular hyperglycaemia is the primary initiating event in the formation of both intracel‐ lular and extracellular AGEs [32]. AGEs can arise from intracellular auto-oxidation of glu‐ cose to glyoxal, decomposition of the Amadori product (glucose-derived 1-amino-1 deoxyfructose lysine adducts) to 3-deoxyglucosone (perhaps accelerated by an amadoriase), and nonenzymatic phosphate elimination from glyceraldehyde phosphate and dihydroxya‐ cetone phosphate to form methylglyoxal. These reactive intracellular dicarbonyl glyoxal, methylglyoxal and 3-deoxyglucosone react with amino groups of intracellular and extracel‐ lular proteins to form AGEs [4]. Intracellular production of AGE precursors can damage cells by three general mechanisms: 1) Intracellular proteins modified by AGEs have altered function, 2) Extracellular matrix components modified by AGE precursors interact abnor‐ mally with other matrix components and with matrix receptors (integrins) that are ex‐ pressed on the surface of cells, and 3) Plasma proteins modified by AGE precursors bind to AGE receptors (such as RAGE and AGE-R1,2 and 3) on cells such as macrophages, vascular endothelial cells and vascular smooth muscle cells. AGE receptors binding induces the pro‐ duction of ROS, which in turn activates PKC. It also activates NF-κB and NADPH oxidase, and disturbs MAPK signaling [31].

#### *2.3.7. Stress-sensitive signaling pathways*

In addition to direct damage of biomolecules in the cells, oxidative stress is also involved in activation of several stress-sensitive signaling pathways, which can result in inflamma‐ tion, cytokine release, and even apoptosis. Among these pathways we find the transcrip‐ tion factor NF-κB, which together with PARP acts as a transcriptional coactivator of inflammation molecules such as iNOS, intracellular adhesion molecule-1 (ICAM-I), and histocompatibility complex class II [33]. p38 MAPK pathway and c-Jun Nterminal kinase (JNK) (also known as stress-activated protein kinase (SAPK) participate in cellular respons‐ es to stress due to osmotic shock, cytokines and UV light, playing a role in cellular prolif‐ eration, apoptosis, and inflammatory responses [33]. Jak/STAT is another important signaling pathway, which initiates and mediates cellular responses to cytokines such as interferons and interleukins [33].

**3. Diabetes mellitus**

betes: type 1 and type 2 diabetes.

insulin resistance leads to hyperglycemia.

**3.1. Insulin action and insulin resistance**

sis and gene expression [40] (Figure 3).

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, caused by a defect on insulin production, insulin action or both [1]. There are two main types of dia‐

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217

Type 1 diabetes is due to an autoimmune destruction of the insulin producing pancreatic betacells, which usually leads to absolute insulin deficiency. Patients with type 1 diabetes re‐ quire insulin for survival. This type of diabetes accounts for 5-10% of the total cases of diabetes worldwide. Type 2 diabetes represents approximately 90% of the total diabetes cases, and it is characterized by impairment in insulin action and/or abnormal insulin secretion [1].

The origins of type 2 diabetes are multifactorial. Obesity, age, ethnic origin and familiar his‐ tory of diabetes are among the factors that contribute to its development. Even though a strong genetic component has been recognized, genotype only establishes the conditions for the individual to be more or less prone to environmental effects and lifestyle factors [34].

Type 2 diabetes develops when insulin secretion or insulin action fails. The impairment of insulin actions is known as insulin resistance, presented as a suppression or retard in meta‐ bolic responses of the muscle, liver and adipose tissue to insulin action. This failure is locat‐ ed at the signaling pathways held after insulin binding to its specific receptor [35]. Chronic

When the beta cells cannot secrete enough insulin in response to the metabolic demand caused by insulin resistance, frank diabetes type 2 occurs. This failure in the beta cell may be due to an acquired secretory dysfunction and/or a decrease in beta-cell mass [36]. All type 2 diabet‐ ic patients have some defect in the ability of beta cells to produce or secrete insulin [37].

Once secreted to the portal circulation, insulin is transported to peripheral tissues, on which it will exert mainly anabolic actions [38]. Insulin starts its action by binding to insulin recep‐ tor, a transmembrane protein belonging to protein tyrosine kinase activity receptors super‐ family, which can autophosphorylate. This initiates a series of events involving protein and membrane lipid phosphorylation, coupling proteins and cytoskeleton activity [39] [40]. The three main signaling pathways activated in response to insulin receptor phosphorylation are 1) PI3K 2)MAPK, and 3) Cb1. These pathways act in a concerted way to translate the signal of insulin receptor into biological actions in target organs, such as glucose transport by transporting GLUT4 vesicles to the membrane, protein, lipid and glycogen synthesis, mito‐

As protein phosphorylation activates these signaling pathways, dephosphorylation inhibits them. Different phosphatases such as protein-tyrosine phosphatase 1B (PTP1B), Phospha‐ tase and tensin homolog (PTEN), SH2-containing tyrosine- protein phosphatase (SHO2), and suppressor of cytokine signaling 3 (SOCS-3) dephosphorylate and shut down insulin signaling [35]. Any alteration in the insulin pathway, being inefficient phosphorylation or

**Figure 2.** Oxidative stress-related pathways derived from glucose metabolism.

#### **2.4. ROS induced damages**

Being highly reactive species, ROS may modify and damage nucleic acids, proteins, lipids and carbohydrates, finally leading to cell damage. Among the motifs that can react with ROS we have the metal ligand from metalloproteases and Fe from oxihemoglobin. •O2 - can also modify and inhibit catalases, while •OH can bind to the histidine residue from SOS causing its inhibition. ROS react mostly with insaturated and sulfur containing molecules, thus, proteins with high contents of tryptophan, tyrosine, phenylalanine, histidine, methio‐ nine and cysteine can suffer ROS modifications. Finally, ROS may also break peptidic bonds after oxidation of proline residues by •O2 or •OH [31].

ROS and RNS may also modify fatty acids, lipoproteins, and phospholipids, a process termed lipid peroxidation, where •OH and •O2 - form hydroperoxide lipids. Hydroperoxyde products cause severe damage to plasma membranes, or they can diffuse to other cells in the organisms and cause vascular permeability and inflammation by binding to (oxidized lowdensity lipoprotein) LOX receptors, and apoptosis [31].

H2O2 in cells can function as a signaling molecule leading to cellular proliferation or can re‐ sult in cell death. At low concentrations, H2O2 serves as a second messenger to activate NF*κ*B and various kinases (p38 MAPK, ERK, PI3K, Akt, JAK2, STAT). H2O2 at slightly higher concentrations can induce the release of cytochrome *c* and apoptosis-inducing factor (AIF) from mitochondria into the cytosol where they trigger the activation of caspase, leading to cell death by apoptosis [12].

### **3. Diabetes mellitus**

**Figure 2.** Oxidative stress-related pathways derived from glucose metabolism.

216 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Being highly reactive species, ROS may modify and damage nucleic acids, proteins, lipids and carbohydrates, finally leading to cell damage. Among the motifs that can react with ROS we have the metal ligand from metalloproteases and Fe from oxihemoglobin. •O2 - can also modify and inhibit catalases, while •OH can bind to the histidine residue from SOS causing its inhibition. ROS react mostly with insaturated and sulfur containing molecules, thus, proteins with high contents of tryptophan, tyrosine, phenylalanine, histidine, methio‐ nine and cysteine can suffer ROS modifications. Finally, ROS may also break peptidic bonds

or •OH [31].

ROS and RNS may also modify fatty acids, lipoproteins, and phospholipids, a process termed lipid peroxidation, where •OH and •O2 - form hydroperoxide lipids. Hydroperoxyde products cause severe damage to plasma membranes, or they can diffuse to other cells in the organisms and cause vascular permeability and inflammation by binding to (oxidized low-

H2O2 in cells can function as a signaling molecule leading to cellular proliferation or can re‐ sult in cell death. At low concentrations, H2O2 serves as a second messenger to activate NF*κ*B and various kinases (p38 MAPK, ERK, PI3K, Akt, JAK2, STAT). H2O2 at slightly higher concentrations can induce the release of cytochrome *c* and apoptosis-inducing factor (AIF) from mitochondria into the cytosol where they trigger the activation of caspase, leading to

**2.4. ROS induced damages**

cell death by apoptosis [12].

after oxidation of proline residues by •O2 -

density lipoprotein) LOX receptors, and apoptosis [31].

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, caused by a defect on insulin production, insulin action or both [1]. There are two main types of dia‐ betes: type 1 and type 2 diabetes.

Type 1 diabetes is due to an autoimmune destruction of the insulin producing pancreatic betacells, which usually leads to absolute insulin deficiency. Patients with type 1 diabetes re‐ quire insulin for survival. This type of diabetes accounts for 5-10% of the total cases of diabetes worldwide. Type 2 diabetes represents approximately 90% of the total diabetes cases, and it is characterized by impairment in insulin action and/or abnormal insulin secretion [1].

The origins of type 2 diabetes are multifactorial. Obesity, age, ethnic origin and familiar his‐ tory of diabetes are among the factors that contribute to its development. Even though a strong genetic component has been recognized, genotype only establishes the conditions for the individual to be more or less prone to environmental effects and lifestyle factors [34].

Type 2 diabetes develops when insulin secretion or insulin action fails. The impairment of insulin actions is known as insulin resistance, presented as a suppression or retard in meta‐ bolic responses of the muscle, liver and adipose tissue to insulin action. This failure is locat‐ ed at the signaling pathways held after insulin binding to its specific receptor [35]. Chronic insulin resistance leads to hyperglycemia.

When the beta cells cannot secrete enough insulin in response to the metabolic demand caused by insulin resistance, frank diabetes type 2 occurs. This failure in the beta cell may be due to an acquired secretory dysfunction and/or a decrease in beta-cell mass [36]. All type 2 diabet‐ ic patients have some defect in the ability of beta cells to produce or secrete insulin [37].

### **3.1. Insulin action and insulin resistance**

Once secreted to the portal circulation, insulin is transported to peripheral tissues, on which it will exert mainly anabolic actions [38]. Insulin starts its action by binding to insulin recep‐ tor, a transmembrane protein belonging to protein tyrosine kinase activity receptors super‐ family, which can autophosphorylate. This initiates a series of events involving protein and membrane lipid phosphorylation, coupling proteins and cytoskeleton activity [39] [40]. The three main signaling pathways activated in response to insulin receptor phosphorylation are 1) PI3K 2)MAPK, and 3) Cb1. These pathways act in a concerted way to translate the signal of insulin receptor into biological actions in target organs, such as glucose transport by transporting GLUT4 vesicles to the membrane, protein, lipid and glycogen synthesis, mito‐ sis and gene expression [40] (Figure 3).

As protein phosphorylation activates these signaling pathways, dephosphorylation inhibits them. Different phosphatases such as protein-tyrosine phosphatase 1B (PTP1B), Phospha‐ tase and tensin homolog (PTEN), SH2-containing tyrosine- protein phosphatase (SHO2), and suppressor of cytokine signaling 3 (SOCS-3) dephosphorylate and shut down insulin signaling [35]. Any alteration in the insulin pathway, being inefficient phosphorylation or increment in phosphatase acticity, causes impairment in insulin action. This is the molecular mechanism leading to insulin resistance.

**Figure 4.** Mechanisms of biphasic glucose-stimulated insulin secretion.

ters, use these pathways to modulate insulin secretion.

**4. Oxidative stress in diabetes mellitus**

**4.1. Oxidative stress processes in insulin resistance**

thus increasing tyrosine phosphorylation [57].

sistance and insulin secretion.

Besides increasing ATP/ADP ratio, glucose metabolism in the beta cell can generate a series of metabolic coupling signals that can initiate and sustain a second insulin secretion phase. Some of these coupling factors participate in mitocondrial metabolism and anaplerosis, con‐ stituting cycles involving NADPH, pyruvate, malate, citrate, isocitrate, Acyl-CoA and gluta‐ mate [46]. Diverse signaling pathways can also contribute to glucose-induced insulin secretion such as CaMKII [47-49], PKA [50, 51], PKC [51, 52] y PKG [53, 54]. Most secretago‐ gues and potentiators of insulin secretion, such as nutrients, hormones and neurotransmit‐

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Hyperglycemia and free fatty acid intake are among the causes for oxidative stress condi‐ tions [23]. Hence, it may not be surprising that diabetic subjects tend to have more oxidative cell and organism environments than healthy subjects, i.e. an increase in ROS generation [5, 55, 56]. Moreover, diabetic patients present a decrease in antioxidant defenses. The antioxi‐ dant enzyme levels are affected by diabetes, which further increase oxidative stress [5, 6]. Oxidative stress has been proposed as a major participant in the patophysiology of diabetic complications [27]. Nevertheless, regarding diabetes onset and development, oxidative stress has also shown to affect the two major mechanisms failing during diabetes: insulin re‐

ROS and RNS affect the insulin signaling cascade [5]. As with other ROS effects, low doses play a physiological role in insulin signaling. After insulin stimulation of its receptor in adi‐ pocytes, H2O2 is produced via NADPH oxidase, which by inhibits PTP1B catalytic activity,

**Figure 3.** Molecular mechanisms of insulin signaling.

#### **3.2. Insulin secretion**

Beta-cells in the endocrine pancreas are responsible for secreting insulin in response to rises in blood nutrient levels during the postprandial state. Glucose is the most important nu‐ trient for insulin secretion. The process by which glucose promotes insulin secretion re‐ quires glucose sensing and metabolism by the beta-cell, a process called glucose-stimulated insulin secretion (Figure 4). In the first phase of insulin secretion, glucose enters the cell by glucose transporters (GLUT2 in rodents, GLUT1 in humans). Glucose is then phosphorylat‐ ed to form glucose-6-phosphate by glucokinase [41]. The generation of ATP by glycolysis, the Krebs cycle and the respiratory chain closes the ATP-sensitive K+ channel (KATP) [42], allowing sodium (Na+) entry without balance. These two events depolarize the membrane and open voltage-dependent T-type calcium (Ca2+) and sodium (Na+) channels. Na+ and Ca2+ entry further depolarizes the membrane and voltage-dependent calcium channels open. This activation increases intracellular Ca2+ ([Ca2+]i) [43], which leads to fusion of in‐ sulin-containing secretory granules with the plasma membrane and the first phase insulin secretion [44, 45].

Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions http://dx.doi.org/10.5772/51788 219

**Figure 4.** Mechanisms of biphasic glucose-stimulated insulin secretion.

increment in phosphatase acticity, causes impairment in insulin action. This is the molecular

Beta-cells in the endocrine pancreas are responsible for secreting insulin in response to rises in blood nutrient levels during the postprandial state. Glucose is the most important nu‐ trient for insulin secretion. The process by which glucose promotes insulin secretion re‐ quires glucose sensing and metabolism by the beta-cell, a process called glucose-stimulated insulin secretion (Figure 4). In the first phase of insulin secretion, glucose enters the cell by glucose transporters (GLUT2 in rodents, GLUT1 in humans). Glucose is then phosphorylat‐ ed to form glucose-6-phosphate by glucokinase [41]. The generation of ATP by glycolysis, the Krebs cycle and the respiratory chain closes the ATP-sensitive K+ channel (KATP) [42], allowing sodium (Na+) entry without balance. These two events depolarize the membrane and open voltage-dependent T-type calcium (Ca2+) and sodium (Na+) channels. Na+ and Ca2+ entry further depolarizes the membrane and voltage-dependent calcium channels open. This activation increases intracellular Ca2+ ([Ca2+]i) [43], which leads to fusion of in‐ sulin-containing secretory granules with the plasma membrane and the first phase insulin

mechanism leading to insulin resistance.

218 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 3.** Molecular mechanisms of insulin signaling.

**3.2. Insulin secretion**

secretion [44, 45].

Besides increasing ATP/ADP ratio, glucose metabolism in the beta cell can generate a series of metabolic coupling signals that can initiate and sustain a second insulin secretion phase. Some of these coupling factors participate in mitocondrial metabolism and anaplerosis, con‐ stituting cycles involving NADPH, pyruvate, malate, citrate, isocitrate, Acyl-CoA and gluta‐ mate [46]. Diverse signaling pathways can also contribute to glucose-induced insulin secretion such as CaMKII [47-49], PKA [50, 51], PKC [51, 52] y PKG [53, 54]. Most secretago‐ gues and potentiators of insulin secretion, such as nutrients, hormones and neurotransmit‐ ters, use these pathways to modulate insulin secretion.

### **4. Oxidative stress in diabetes mellitus**

Hyperglycemia and free fatty acid intake are among the causes for oxidative stress condi‐ tions [23]. Hence, it may not be surprising that diabetic subjects tend to have more oxidative cell and organism environments than healthy subjects, i.e. an increase in ROS generation [5, 55, 56]. Moreover, diabetic patients present a decrease in antioxidant defenses. The antioxi‐ dant enzyme levels are affected by diabetes, which further increase oxidative stress [5, 6].

Oxidative stress has been proposed as a major participant in the patophysiology of diabetic complications [27]. Nevertheless, regarding diabetes onset and development, oxidative stress has also shown to affect the two major mechanisms failing during diabetes: insulin re‐ sistance and insulin secretion.

#### **4.1. Oxidative stress processes in insulin resistance**

ROS and RNS affect the insulin signaling cascade [5]. As with other ROS effects, low doses play a physiological role in insulin signaling. After insulin stimulation of its receptor in adi‐ pocytes, H2O2 is produced via NADPH oxidase, which by inhibits PTP1B catalytic activity, thus increasing tyrosine phosphorylation [57].

However, oxidative stress caused by hyperglycemia in diabetes may impair insulin signal‐ ing, leading to insulin resistance. Although no mechanisms have been completely establish‐ ed, several responses to ROS excess in the insulin signaling have been proposed.

**5. Diabetic complications**

Hyperglycemia, is the responsible of the development of diabetes complications as well. Hy‐ perglycemia damage is produced in cells in which glucose uptake is independent of insulin, which, similarly to what happens in beta-cells, explains that the cause of the complications resides inside the cells [4]. Prolonged exposure to high glucose levels, genetic determinants of susceptibility and accelerating factors such as hypertension and dyslipidemia participate in the development of diabetic complications. Moreover, the development and progression of damage is proportional to hyperglycemia, which makes the lowering of glucose levels the

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The main tissues affected by diabetes complications at the microvasculature levels are reti‐ na, renal glomerulus, and peripheral nerves. Diabetes is also associated with accelerated atherosclerotic disease affecting arteries that supply the heart, brain, and lower extremities.

Oxidative stress plays a pivotal role in the development of diabetes complications, both at the microvascular and macrovascular levels. Results derived from two decades of diabetes complications investigation point towards mitochondrial superoxide overproduction as the main cause of metabolic abnormalities of diabetes. Thus, all of the above reviewed pathways

Diabetic retinopathy: Diabetic retinopathy appears in most patients after 10 to 15 years after diabetes onset. Background retinopathy presents small hemorrhages in the middle layers of the retina, appearing as "dots". Lipid deposition occurs at the margins of the hemorraghe, and microaneurisms (small vascular dilatations) and edema may appear. Proliferative retin‐ opathy occurs when new blood vessels on the surface of the retina cause vitreous hemor‐ rhage, and eventually, blindness. As the cells of the retina contain high amounts of aldoketoreductase, they have high susceptibility to increase the polyol pathway in the presence of excess glucose, with concomitant decreases in NADPH [4]. Sorbitol produced in this proc‐ ess increases osmotic stress, which has been linked to microaneurysm formation, thickening of the basement membranes and loss of pericytes. It is also thought that retina cells are dam‐ aged by glycoproteins, particularly form AGEs. Additionally, ROS by themselves may dam‐ age the cells. Importantly, VEGF, growth hormone and TGF-beta increases during diabetes

Diabetic nephropaty: this complication causes glomerular basement membrane thickness, microaneurism formation, and mesangial nodule formation, all which are reflected in protei‐ nuria and, in the end, renal insufficiency. The mechanisms for injury also involve the in‐ creased polyol pathway and AGE formation. AGE binding to its receptors has been proven to play a role as well in renal damage, fibrosis and inflammation associated with diabetic

are involved in microvasculature and macrovasculature hyperglycemic damage [24].

most important goal for preventing complications and treating diabetes.

In addition, diabetic cardiomyopathy is a major diabetic complication [24].

**5.1. Oxidative stress in diabetic complications**

may be the cause of proliferation of blood vessels [70].

**5.2. Microvascular complications**

Disturbs in cellular redistribution of insulin signaling components may alter the insulin cas‐ cade, a process mediated by NF-κB [58]. A decrease in GLUT4 gene transcription and in‐ crease in GLUT1 (insulin independent glucose transporter) has also been observed, as well as increases in phosphorylation of IRS protein in an insulin receptor-independent fashion (perhaps by the stress kinases). Altogether, hyperglycemia and insulin resistance may also lead to altered mitochondrial function, and insulin action impairment by cytokines in re‐ sponse to metabolic stress [59, 60]. An increase in the hexosamine pathway has also been linked to insulin resistance. Moreover, it has been proposed that this pathway acts as a cellu‐ lar sensor for the glucose excess. From that point of view, insulin resistance may be a protec‐ tive mechanism from the glucose excess entrance [28].

#### **4.2. Oxidative stress processes in insulin secretion**

Pancreatic beta-cells are especially sensitive to ROS and RNS, because their natural enzymat‐ ic antioxidant defenses are lower compared to other tissues such as liver. Moreover, they lack the ability to adapt their low enzyme activity levels in response to stress such as high glu‐ cose or high oxygen [61]. Glucose enters to the beta-cell in an insulin independent fashion, because besides providing energy, glucose sensing in the beta-cell is crucial for insulin secre‐ tion. It has been suggested that hyperglycemia can generate chronic oxidative stress by the glucose oxidation pathway [62], leading to an excess in mitochondrial superoxide produc‐ tion, which further activates uncoupling protein-2 (UCP-2). This protein lowers ATP/ADP relationship through proton leak in the beta-cell, which reduces insulin secretion [63].

ROS also increase the stress signaling pathways in the beta cells, such as NF-κB activity, which potentially leading to beta-cell apoptosis [64], and the JNK pathway which has been related to suppression of insulin gene expression, possibly by reduction of PDX-1 DNA binding activity, a major regulator of insulin expression [65]. It has also been shown that the activation of the hexosamine pathway in beta-cells leads to suppression of PDX-1 binding to the insulin and other genes involved in insulin secretion, perhaps contributing to the betacell dysfunction present in diabetes mellitus [66].

As in other cell types, NO in beta-cells has physiologic roles. NO may regulate glucokinase activity by s-nitrosilation [67] in the beta-cell, and possibly increase insulin secretion. How‐ ever, NO excess and concomitant NRS may cause apoptosis through caspase-3 activation and decrease in ATP levels [68].

Besides ROS hyperproduction, excess mitochondrial metabolism resulting form hypergly‐ cemia in the beta-cell may also alter mitochondrial shape, volume and behavior, uncou‐ pling K-ATP channels from mitochondrial activity and thus altering glucose-induced insulin secretion [69].

### **5. Diabetic complications**

However, oxidative stress caused by hyperglycemia in diabetes may impair insulin signal‐ ing, leading to insulin resistance. Although no mechanisms have been completely establish‐

Disturbs in cellular redistribution of insulin signaling components may alter the insulin cas‐ cade, a process mediated by NF-κB [58]. A decrease in GLUT4 gene transcription and in‐ crease in GLUT1 (insulin independent glucose transporter) has also been observed, as well as increases in phosphorylation of IRS protein in an insulin receptor-independent fashion (perhaps by the stress kinases). Altogether, hyperglycemia and insulin resistance may also lead to altered mitochondrial function, and insulin action impairment by cytokines in re‐ sponse to metabolic stress [59, 60]. An increase in the hexosamine pathway has also been linked to insulin resistance. Moreover, it has been proposed that this pathway acts as a cellu‐ lar sensor for the glucose excess. From that point of view, insulin resistance may be a protec‐

Pancreatic beta-cells are especially sensitive to ROS and RNS, because their natural enzymat‐ ic antioxidant defenses are lower compared to other tissues such as liver. Moreover, they lack the ability to adapt their low enzyme activity levels in response to stress such as high glu‐ cose or high oxygen [61]. Glucose enters to the beta-cell in an insulin independent fashion, because besides providing energy, glucose sensing in the beta-cell is crucial for insulin secre‐ tion. It has been suggested that hyperglycemia can generate chronic oxidative stress by the glucose oxidation pathway [62], leading to an excess in mitochondrial superoxide produc‐ tion, which further activates uncoupling protein-2 (UCP-2). This protein lowers ATP/ADP relationship through proton leak in the beta-cell, which reduces insulin secretion [63].

ROS also increase the stress signaling pathways in the beta cells, such as NF-κB activity, which potentially leading to beta-cell apoptosis [64], and the JNK pathway which has been related to suppression of insulin gene expression, possibly by reduction of PDX-1 DNA binding activity, a major regulator of insulin expression [65]. It has also been shown that the activation of the hexosamine pathway in beta-cells leads to suppression of PDX-1 binding to the insulin and other genes involved in insulin secretion, perhaps contributing to the beta-

As in other cell types, NO in beta-cells has physiologic roles. NO may regulate glucokinase activity by s-nitrosilation [67] in the beta-cell, and possibly increase insulin secretion. How‐ ever, NO excess and concomitant NRS may cause apoptosis through caspase-3 activation

Besides ROS hyperproduction, excess mitochondrial metabolism resulting form hypergly‐ cemia in the beta-cell may also alter mitochondrial shape, volume and behavior, uncou‐ pling K-ATP channels from mitochondrial activity and thus altering glucose-induced

ed, several responses to ROS excess in the insulin signaling have been proposed.

tive mechanism from the glucose excess entrance [28].

220 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**4.2. Oxidative stress processes in insulin secretion**

cell dysfunction present in diabetes mellitus [66].

and decrease in ATP levels [68].

insulin secretion [69].

Hyperglycemia, is the responsible of the development of diabetes complications as well. Hy‐ perglycemia damage is produced in cells in which glucose uptake is independent of insulin, which, similarly to what happens in beta-cells, explains that the cause of the complications resides inside the cells [4]. Prolonged exposure to high glucose levels, genetic determinants of susceptibility and accelerating factors such as hypertension and dyslipidemia participate in the development of diabetic complications. Moreover, the development and progression of damage is proportional to hyperglycemia, which makes the lowering of glucose levels the most important goal for preventing complications and treating diabetes.

The main tissues affected by diabetes complications at the microvasculature levels are reti‐ na, renal glomerulus, and peripheral nerves. Diabetes is also associated with accelerated atherosclerotic disease affecting arteries that supply the heart, brain, and lower extremities. In addition, diabetic cardiomyopathy is a major diabetic complication [24].

### **5.1. Oxidative stress in diabetic complications**

Oxidative stress plays a pivotal role in the development of diabetes complications, both at the microvascular and macrovascular levels. Results derived from two decades of diabetes complications investigation point towards mitochondrial superoxide overproduction as the main cause of metabolic abnormalities of diabetes. Thus, all of the above reviewed pathways are involved in microvasculature and macrovasculature hyperglycemic damage [24].

### **5.2. Microvascular complications**

Diabetic retinopathy: Diabetic retinopathy appears in most patients after 10 to 15 years after diabetes onset. Background retinopathy presents small hemorrhages in the middle layers of the retina, appearing as "dots". Lipid deposition occurs at the margins of the hemorraghe, and microaneurisms (small vascular dilatations) and edema may appear. Proliferative retin‐ opathy occurs when new blood vessels on the surface of the retina cause vitreous hemor‐ rhage, and eventually, blindness. As the cells of the retina contain high amounts of aldoketoreductase, they have high susceptibility to increase the polyol pathway in the presence of excess glucose, with concomitant decreases in NADPH [4]. Sorbitol produced in this proc‐ ess increases osmotic stress, which has been linked to microaneurysm formation, thickening of the basement membranes and loss of pericytes. It is also thought that retina cells are dam‐ aged by glycoproteins, particularly form AGEs. Additionally, ROS by themselves may dam‐ age the cells. Importantly, VEGF, growth hormone and TGF-beta increases during diabetes may be the cause of proliferation of blood vessels [70].

Diabetic nephropaty: this complication causes glomerular basement membrane thickness, microaneurism formation, and mesangial nodule formation, all which are reflected in protei‐ nuria and, in the end, renal insufficiency. The mechanisms for injury also involve the in‐ creased polyol pathway and AGE formation. AGE binding to its receptors has been proven to play a role as well in renal damage, fibrosis and inflammation associated with diabetic nephropaty. This actions of AGE also potentiate oxidative stress, while synergizing with rennin-angiotensin system activation, which leads to a vicious cycle causing kidney failure. As mentioned, diabetic patients, and particularly those with nephropaty, have lowered anti‐ oxidant defenses. Moreover, AGE receptors are significantly increased [71].

sion causing capillary occlusion, in the case of TGF-beta, and decreased fibrinolysis in the case of PAI-1. O-GlcNAcylation impairs cardiomyocyte calcium cycling decreasing sarco‐

Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions

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223

As mentioned above, vitamins C, E, and A constitute the non enzymatic defense against oxidative stress, by regenerating endogenous antioxidants (Figure 1). Vitamin C has a role in scavenging ROS and RNS by becoming oxidated itself. The oxidized products of vita‐ min C, ascorbic radical and dehydroascorbic radical are regenerated by glutathione, NADH or NADPH. In addition, vitamin C can reduce the oxidized forms of vitamin E and glutha‐ tione [81]. Vitamin E is a fat-soluble vitamin which may interact with lipid hydroperox‐ ides and scavenge them. It also participates, together with vitamin C, in gluthatione regeneration by interaction with lipoic acid [23]. Vitamin A has a plethora of cellular ac‐ tions. Besides modulating gene expression, cell growth and differentiation, this vitamin may also act as antioxidant, although the mechanisms of action in this role are not fully deci‐ phered. The antioxidant potential of carotenoids (vitamin A) depends on their distinct mem‐ brane-lipid interactions, while some carotenoids can decrease lipid peroxidation, others can

plasmic reticulum calcium ATPase 2a (Serca 2a) [78-80].

**Figure 5.** Oxidative stress pathways in diabetes mellitus.

stimulate it [82].

**6. Antioxidant vitamins and diabetes mellitus**

Diabetic neuropathy: Diabetic neuropathy is defined as the presence of symptoms and/or signs of peripheral nerve dysfunction in diabetic patients after exclusion of other causes. Pe‐ ripheral neuropathy in diabetes may manifest in several different forms, including sensory, focal/multifocal, and autonomic neuropathies. [72]. Mechanisms of nerve injury are less known but likely related also to the polyol pathway, AGE formation and ROS themselves [70]. Oxidized proteins and lipoproteins also interact with receptors in the membrane of neurons, initiating inflammatory signaling mechanisms which further produce ROS, damag‐ ing cellular components and leading to neuronal injury [73].

### **5.3. Macrovascular complications**

The central pathological mechanism in macrovascular complications is atherosclerotic dis‐ ease. Atherosclerosis occurs as a result of chronic inflammation and injury to the arterial wall in the peripheral or coronary vascular system. This damages cause accumulation of oxi‐ dized lipids from LDL particles in the endothelial wall of arteries, whose rupture leads to acute vascular infarction. Additionally, platelet adhesion and hypercoagulability also occurs in type 2 diabetes, increasing the risk of vascular occlusion [70]. It has been proposed that increased superoxide production is the central and major mediator of endothelial tissue damage, causing direct inactivation of two antiatherosclerotic enzymes, endothelial nitric oxide synthase and prostacyclin synthase and that the activation of oxidative stress path‐ ways is involved in the pathogenesis of complications [24].

Endothelial cells also contain high amounts of aldo-keto reductase, and are thus prone to in‐ creased polyol pathway activation. Moreover, a large body of evidence supports hypothesis that hyperglycemia or diabetes leads to vascular diacylglycerol accumulation and subse‐ quent PKC activation, causing a variety of cardiovascular defects [29]. PKC activation has been associated with vascular alterations such as increases in permeability, contractility, ex‐ tracellular matrix synthesis, cell growth and apoptosis, angiogenesis, leukocyte adhesion, and cytokine activation and inhibition [29]. Hyperglycemia-induced activation of PKC has also been implicated in the overexpression of the fibrinolytic inhibitor, plasminogen activa‐ tor inhibitor-1 (PAI-1) [74]. In smooth muscle PKC hyperactivity is associated with de‐ creased NO production [75] and has been shown to inhibit insulin-stimulated expression of eNOs in endothelial cells.

In arterial endothelial cells O-glucosamine-acylation participates in vascular complications interfering with the action of Akt/PKB, a critical insulin signaling protein, on eNOS [76]. GFAT activity is associated with increased transcription of transforming growth factor (TGF) alpha and beta and PAI-1, factors involved in the proliferation of vascular smoothmuscle and endothelial cells. This effect appears to be mediated by O-glucosamine-acylation of the transcription factor, Sp1 [77]. Increased TGF-beta and PAI-1 are associated with capil‐ lary and vascular occlusion by mechanisms associated with collagen and fibronectin expres‐ sion causing capillary occlusion, in the case of TGF-beta, and decreased fibrinolysis in the case of PAI-1. O-GlcNAcylation impairs cardiomyocyte calcium cycling decreasing sarco‐ plasmic reticulum calcium ATPase 2a (Serca 2a) [78-80].

**Figure 5.** Oxidative stress pathways in diabetes mellitus.

nephropaty. This actions of AGE also potentiate oxidative stress, while synergizing with rennin-angiotensin system activation, which leads to a vicious cycle causing kidney failure. As mentioned, diabetic patients, and particularly those with nephropaty, have lowered anti‐

Diabetic neuropathy: Diabetic neuropathy is defined as the presence of symptoms and/or signs of peripheral nerve dysfunction in diabetic patients after exclusion of other causes. Pe‐ ripheral neuropathy in diabetes may manifest in several different forms, including sensory, focal/multifocal, and autonomic neuropathies. [72]. Mechanisms of nerve injury are less known but likely related also to the polyol pathway, AGE formation and ROS themselves [70]. Oxidized proteins and lipoproteins also interact with receptors in the membrane of neurons, initiating inflammatory signaling mechanisms which further produce ROS, damag‐

The central pathological mechanism in macrovascular complications is atherosclerotic dis‐ ease. Atherosclerosis occurs as a result of chronic inflammation and injury to the arterial wall in the peripheral or coronary vascular system. This damages cause accumulation of oxi‐ dized lipids from LDL particles in the endothelial wall of arteries, whose rupture leads to acute vascular infarction. Additionally, platelet adhesion and hypercoagulability also occurs in type 2 diabetes, increasing the risk of vascular occlusion [70]. It has been proposed that increased superoxide production is the central and major mediator of endothelial tissue damage, causing direct inactivation of two antiatherosclerotic enzymes, endothelial nitric oxide synthase and prostacyclin synthase and that the activation of oxidative stress path‐

Endothelial cells also contain high amounts of aldo-keto reductase, and are thus prone to in‐ creased polyol pathway activation. Moreover, a large body of evidence supports hypothesis that hyperglycemia or diabetes leads to vascular diacylglycerol accumulation and subse‐ quent PKC activation, causing a variety of cardiovascular defects [29]. PKC activation has been associated with vascular alterations such as increases in permeability, contractility, ex‐ tracellular matrix synthesis, cell growth and apoptosis, angiogenesis, leukocyte adhesion, and cytokine activation and inhibition [29]. Hyperglycemia-induced activation of PKC has also been implicated in the overexpression of the fibrinolytic inhibitor, plasminogen activa‐ tor inhibitor-1 (PAI-1) [74]. In smooth muscle PKC hyperactivity is associated with de‐ creased NO production [75] and has been shown to inhibit insulin-stimulated expression of

In arterial endothelial cells O-glucosamine-acylation participates in vascular complications interfering with the action of Akt/PKB, a critical insulin signaling protein, on eNOS [76]. GFAT activity is associated with increased transcription of transforming growth factor (TGF) alpha and beta and PAI-1, factors involved in the proliferation of vascular smoothmuscle and endothelial cells. This effect appears to be mediated by O-glucosamine-acylation of the transcription factor, Sp1 [77]. Increased TGF-beta and PAI-1 are associated with capil‐ lary and vascular occlusion by mechanisms associated with collagen and fibronectin expres‐

oxidant defenses. Moreover, AGE receptors are significantly increased [71].

ing cellular components and leading to neuronal injury [73].

222 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ways is involved in the pathogenesis of complications [24].

**5.3. Macrovascular complications**

eNOs in endothelial cells.

### **6. Antioxidant vitamins and diabetes mellitus**

As mentioned above, vitamins C, E, and A constitute the non enzymatic defense against oxidative stress, by regenerating endogenous antioxidants (Figure 1). Vitamin C has a role in scavenging ROS and RNS by becoming oxidated itself. The oxidized products of vita‐ min C, ascorbic radical and dehydroascorbic radical are regenerated by glutathione, NADH or NADPH. In addition, vitamin C can reduce the oxidized forms of vitamin E and glutha‐ tione [81]. Vitamin E is a fat-soluble vitamin which may interact with lipid hydroperox‐ ides and scavenge them. It also participates, together with vitamin C, in gluthatione regeneration by interaction with lipoic acid [23]. Vitamin A has a plethora of cellular ac‐ tions. Besides modulating gene expression, cell growth and differentiation, this vitamin may also act as antioxidant, although the mechanisms of action in this role are not fully deci‐ phered. The antioxidant potential of carotenoids (vitamin A) depends on their distinct mem‐ brane-lipid interactions, while some carotenoids can decrease lipid peroxidation, others can stimulate it [82].

Since oxidative stress is present during the progression of diabetes and its complications, amelioration of oxidative status, mainly by increasing antioxidant non-enzymatic defenses, has been largely proposed and studied. Several clinical observational trials have particularly studied the correlation between vitamin E status in plasma and/or diet, and markers of oxi‐ dation, inflammation, type 2 diabetes incidence, and diabetic complications. Although in‐ verse association has been found for vitamin E in some studies [7, 83, 84], the association found in other study disappeared after adjustment for cardiovascular risk factors such as obesity, smoking, and hypertension [85], or have observed no beneficial effect at all [7, 8]. Such contrasting results have also been reported for studies looking association of vitamin A and C consumption and amelioration of diabetes status and/or complications [7, 8, 81, 86].

dering diabetic complications. Considering that hyperglycemia is the main cause of oxida‐ tive stress in diabetes, in a similar way, the chronic undesirable effects that occur by ROS production may generate a vicious cycle difficult to break, in which ROS damage exacer‐ bates the diabetic state, increasing glucose levels, which will further induce more oxida‐

Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions

http://dx.doi.org/10.5772/51788

225

Diabetes mellitus has reached epidemic proportions in the last decade, becoming one of the most important diseases worldwide. Several studies indicate oxidative stress is present in the dysfunction of insulin action and secretion that occur during diabetes, as well as in the development of diabetic complications. Nevertheless, oxidative stress is not the primary cause of diabetes, but rather a consequence of nutrient excess, given that oxidative stress is a

Vitamins such as E, C and A with antioxidant properties constitute the physiological nonenzymatic defense against oxidative stress. However, the evidence in favor of the use of vi‐ tamin supplementation as antioxidant therapy remains uncertain. Although some beneficial effects have been proven in observational studies, the results of interventional trials are still ineffective. Perhaps more studies on the physiopathology of oxidative stress and the role of vitamins in it, as well as standardizing vitamin dosage and assessing their undesirable ef‐ fects are needed in order to determine a clear participation of vitamin supplementation in amelioration of the oxidative balance. More studies addressing the possibility of targeting directly at the enzymes and mechanisms involved in ROS production and not by antioxi‐

Given that it is mostly dietary vitamin intake which has shown an association with amelio‐ rating the diabetic state, and that oxidative stress is a response to excess of nutrients, it seems that attending the cause of excessive ROS production represents the best therapeutic option. Thus, adequate dietary interventions that reduce hyperglycemia, and increases in oxygen consumption (i.e. improve mitochondrial function) by exercise remain the primary

This work was supported by grants from CONACyT and from the Dirección General de

natural response to stress, in this case, to glucose and/or lipid overload.

choices for diabetes treatment and prevention of its complications.

tive imbalance [24].

**7. Conclusions**

dants are needed as well.

**Acknowledgements**

Asuntos del Personal Académico, UNAM.

On the other hand, in interventional trials with vitamin supplementation, the effects of vita‐ mins E, C and A, alone or in diverse combinations, have yielded barely any promising re‐ sult. There appears to be no beneficial effect of vitamin supplementation on diabetes or macrovascular complications [7, 8, 81]. Some of these studies have even evidenced associa‐ tions between vitamin supplementation and an increased incidence of stroke [7]. Likewise, supplementation with antioxidant vitamins can even block beneficial ROS production dur‐ ing exercise, inhibiting the health-promoting effects of exercise in humans [87].

Paradoxically, in spite of the solid evidence of increased oxidative stress in diabetes, and the well established actions of vitamins as antioxidants, the association studies between antioxi‐ dant vitamin status and its beneficial effects in diabetes has no consistent results at all. What is more, interventional studies have failed in demonstrating a favorable effect of vitamin supplementation, discouraging its use as antioxidant therapy for diabetes.

Several reasons have been suggested for these contradictory observations. First, as vitamins may be easily oxidized, a vitamin may have antioxidant or oxidant properties, depending on the presence of other vitamins and the oxidative state in the cells i.e., if the oxidized form of a vitamin is not correctly reversed into the reduced form. Additionally, some vitamins may also activate oxidative stress pathways and further increase the oxidative stress, such as the activation of PKC by retinoids [88].

Vitamin doses may also be part of the problem, as the effect of vitamins depends on dietary concentrations and/or supplement intake. The wide variety of doses reached with diet and supplements, and the lack of an established "pharmacological" dose of vitamins, makes it difficult to ascertain the true net effect of vitamin status or supplementation needed to gen‐ erate beneficial effects. As well, the required dose for antioxidant effects versus the required for the vitamin's role in the body may differ, which, together with vitamin's bioavailability and its interaction with other vitamins, are caveats for assessing and finding vitamins' ef‐ fects, if any [7, 88].

Finally, the antioxidant effects of vitamins may not be sufficient to scavenge the great amount of ROS present in diabetes. Certainly, glucose levels have been correlated to the presence and severity of the complications. However once hyperglycemia has establish‐ ed, the incidence of complications after tight glycemic control remains the same. This effect has been termed glycemic memory, and is the cause for accumulative damage ren‐ dering diabetic complications. Considering that hyperglycemia is the main cause of oxida‐ tive stress in diabetes, in a similar way, the chronic undesirable effects that occur by ROS production may generate a vicious cycle difficult to break, in which ROS damage exacer‐ bates the diabetic state, increasing glucose levels, which will further induce more oxida‐ tive imbalance [24].

### **7. Conclusions**

Since oxidative stress is present during the progression of diabetes and its complications, amelioration of oxidative status, mainly by increasing antioxidant non-enzymatic defenses, has been largely proposed and studied. Several clinical observational trials have particularly studied the correlation between vitamin E status in plasma and/or diet, and markers of oxi‐ dation, inflammation, type 2 diabetes incidence, and diabetic complications. Although in‐ verse association has been found for vitamin E in some studies [7, 83, 84], the association found in other study disappeared after adjustment for cardiovascular risk factors such as obesity, smoking, and hypertension [85], or have observed no beneficial effect at all [7, 8]. Such contrasting results have also been reported for studies looking association of vitamin A and C consumption and amelioration of diabetes status and/or complications [7, 8, 81, 86].

224 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

On the other hand, in interventional trials with vitamin supplementation, the effects of vita‐ mins E, C and A, alone or in diverse combinations, have yielded barely any promising re‐ sult. There appears to be no beneficial effect of vitamin supplementation on diabetes or macrovascular complications [7, 8, 81]. Some of these studies have even evidenced associa‐ tions between vitamin supplementation and an increased incidence of stroke [7]. Likewise, supplementation with antioxidant vitamins can even block beneficial ROS production dur‐

Paradoxically, in spite of the solid evidence of increased oxidative stress in diabetes, and the well established actions of vitamins as antioxidants, the association studies between antioxi‐ dant vitamin status and its beneficial effects in diabetes has no consistent results at all. What is more, interventional studies have failed in demonstrating a favorable effect of vitamin

Several reasons have been suggested for these contradictory observations. First, as vitamins may be easily oxidized, a vitamin may have antioxidant or oxidant properties, depending on the presence of other vitamins and the oxidative state in the cells i.e., if the oxidized form of a vitamin is not correctly reversed into the reduced form. Additionally, some vitamins may also activate oxidative stress pathways and further increase the oxidative stress, such as

Vitamin doses may also be part of the problem, as the effect of vitamins depends on dietary concentrations and/or supplement intake. The wide variety of doses reached with diet and supplements, and the lack of an established "pharmacological" dose of vitamins, makes it difficult to ascertain the true net effect of vitamin status or supplementation needed to gen‐ erate beneficial effects. As well, the required dose for antioxidant effects versus the required for the vitamin's role in the body may differ, which, together with vitamin's bioavailability and its interaction with other vitamins, are caveats for assessing and finding vitamins' ef‐

Finally, the antioxidant effects of vitamins may not be sufficient to scavenge the great amount of ROS present in diabetes. Certainly, glucose levels have been correlated to the presence and severity of the complications. However once hyperglycemia has establish‐ ed, the incidence of complications after tight glycemic control remains the same. This effect has been termed glycemic memory, and is the cause for accumulative damage ren‐

ing exercise, inhibiting the health-promoting effects of exercise in humans [87].

supplementation, discouraging its use as antioxidant therapy for diabetes.

the activation of PKC by retinoids [88].

fects, if any [7, 88].

Diabetes mellitus has reached epidemic proportions in the last decade, becoming one of the most important diseases worldwide. Several studies indicate oxidative stress is present in the dysfunction of insulin action and secretion that occur during diabetes, as well as in the development of diabetic complications. Nevertheless, oxidative stress is not the primary cause of diabetes, but rather a consequence of nutrient excess, given that oxidative stress is a natural response to stress, in this case, to glucose and/or lipid overload.

Vitamins such as E, C and A with antioxidant properties constitute the physiological nonenzymatic defense against oxidative stress. However, the evidence in favor of the use of vi‐ tamin supplementation as antioxidant therapy remains uncertain. Although some beneficial effects have been proven in observational studies, the results of interventional trials are still ineffective. Perhaps more studies on the physiopathology of oxidative stress and the role of vitamins in it, as well as standardizing vitamin dosage and assessing their undesirable ef‐ fects are needed in order to determine a clear participation of vitamin supplementation in amelioration of the oxidative balance. More studies addressing the possibility of targeting directly at the enzymes and mechanisms involved in ROS production and not by antioxi‐ dants are needed as well.

Given that it is mostly dietary vitamin intake which has shown an association with amelio‐ rating the diabetic state, and that oxidative stress is a response to excess of nutrients, it seems that attending the cause of excessive ROS production represents the best therapeutic option. Thus, adequate dietary interventions that reduce hyperglycemia, and increases in oxygen consumption (i.e. improve mitochondrial function) by exercise remain the primary choices for diabetes treatment and prevention of its complications.

### **Acknowledgements**

This work was supported by grants from CONACyT and from the Dirección General de Asuntos del Personal Académico, UNAM.

### **Author details**

Maria-Luisa Lazo-de-la-Vega-Monroy\* and Cristina Fernández-Mejía

Unidad de Genética de la Nutrición, Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México/ Instituto Nacional de Pediatría, México

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Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions

http://dx.doi.org/10.5772/51788

227

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**Chapter 10**

**Oxidative Stress and Antioxidant Therapy in Chronic**

Chronic kidney disease (CKD) and cardiovascular disease (CVD) have major impacts upon the health of populations worldwide, especially in Western societies. The progression of CKD or CVD independently exerts synergistic deleterious effects on the other, for example, patients with CKD are more likely to die of CVD than to develop renal failure. This overlap between CKD and CVD, in part, relates to common etiologies such as diabetes mellitus and hypertension, but important dynamic and bidirectional interactions between the cardiovas‐ cular system and kidneys may also explain the occurrence of concurrent organ dysfunction [1]. Cardio-renal syndrome (or reno-cardiac syndrome, the prefix depending on the primary failing organ) is becoming increasingly recognised [2]. Conventional treatment targeted at either syndrome generally reduces the onset or progression of the other [3]. Even though our understanding of various factors and steps involved in the pathogenesis of CKD and CVD and their obvious links has improved, a complete picture of the mechanisms involved is still unclear. Oxidative stress has been identified as one unifying mechanism in the patho‐ genesis of CKD and CVD [4]. This current chapter gives a brief review of recent literature on the relationship between CKD, CVD and oxidative stress and indicates how, by applying knowledge of the molecular controls of oxidative stress, this information may help improve

**2. Pathogenesis of chronic kidney and cardiovascular disease – The links**

It is, in fact, very difficult to separate these chronic diseases, because one is a complication of the other in many situations. The development and progression of CKD are closely linked

> © 2013 Small and Gobe; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Kidney and Cardiovascular Disease**

Additional information is available at the end of the chapter

targeted therapy with antioxidants for these diseases.

David M. Small and Glenda C. Gobe

http://dx.doi.org/10.5772/51923

**1. Introduction**


### **Oxidative Stress and Antioxidant Therapy in Chronic Kidney and Cardiovascular Disease**

David M. Small and Glenda C. Gobe

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51923

### **1. Introduction**

[84] Mayer-Davis, E. J., Costacou, T., King, I., Zaccaro, D. J., & Bell, R. A. (2002). Plasma and dietary vitamin E in relation to incidence of type 2 diabetes: The Insulin Resist‐

[85] Reunanen, A., Knekt, P., Aaran, R. K., & Aromaa, A. (1998). Serum antioxidants and risk of non-insulin dependent diabetes mellitus. *Eur J Clin Nutr*, 52(2), 89-93.

[86] Sinclair, A. J., Taylor, P. B., Lunec, J., Girling, A. J., & Barnett, A. H. (1994). Low plas‐ ma ascorbate levels in patients with type 2 diabetes mellitus consuming adequate di‐

[87] Ristow, M., Zarse, K., Oberbach, A., Kloting, N., Birringer, M., Kiehntopf, M., et al. (2009). Antioxidants prevent health-promoting effects of physical exercise in hu‐

[88] Chertow, B. (2004). Advances in diabetes for the millennium: vitamins and oxidant

ance and Atherosclerosis Study (IRAS). *Diabetes Care*, 25(12), 2172-2177.

etary vitamin C. *Diabet Med*, 11(9), 893-898.

232 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

mans. *Proc Natl Acad Sci USA*, 106(21), 8665-8670.

stress in diabetes and its complications. *MedGenMed*, 6(3), 4.

Chronic kidney disease (CKD) and cardiovascular disease (CVD) have major impacts upon the health of populations worldwide, especially in Western societies. The progression of CKD or CVD independently exerts synergistic deleterious effects on the other, for example, patients with CKD are more likely to die of CVD than to develop renal failure. This overlap between CKD and CVD, in part, relates to common etiologies such as diabetes mellitus and hypertension, but important dynamic and bidirectional interactions between the cardiovas‐ cular system and kidneys may also explain the occurrence of concurrent organ dysfunction [1]. Cardio-renal syndrome (or reno-cardiac syndrome, the prefix depending on the primary failing organ) is becoming increasingly recognised [2]. Conventional treatment targeted at either syndrome generally reduces the onset or progression of the other [3]. Even though our understanding of various factors and steps involved in the pathogenesis of CKD and CVD and their obvious links has improved, a complete picture of the mechanisms involved is still unclear. Oxidative stress has been identified as one unifying mechanism in the patho‐ genesis of CKD and CVD [4]. This current chapter gives a brief review of recent literature on the relationship between CKD, CVD and oxidative stress and indicates how, by applying knowledge of the molecular controls of oxidative stress, this information may help improve targeted therapy with antioxidants for these diseases.

### **2. Pathogenesis of chronic kidney and cardiovascular disease – The links**

It is, in fact, very difficult to separate these chronic diseases, because one is a complication of the other in many situations. The development and progression of CKD are closely linked

© 2013 Small and Gobe; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. with hypertension and dyslipidemia, both causes of renal failure. Diabetic nephropathy is arguably the leading cause of renal failure. CKD, hypertension and diabetes mellitus all in‐ volve endothelial dysfunction, a change well known in the development of atherosclerosis and CVD that includes coronary artery disease, heart failure, stroke and peripheral arterial disease [5]. Vascular calcification occurs in progressive atherosclerosis and CVD, but it is al‐ so an important part of vascular injury in end-stage renal disease (ESRD), where patients need renal replacement therapy to survive. It is paradoxical that approximately 50% of indi‐ viduals with ESRD die from a cardiovascular cause [6]. Thus, CKD and CVD patients have closely-linked diseases with increasing morbidity and mortality. Prevention and treatment of these diseases are major aims in health systems worldwide.

as lipid lowering drugs or RAS modifiers [13], to minimize the progression of CKD. Table 1

1 90mL/Min Normal renal function but abnormal urine findings, or structural

2 60-89mL/min Mildly reduced renal function, and other findings (as for stage 1)

5 <15mL/min or on dialysis Very severe, or end-stage kidney failure (sometimes called

\* Measured using the MDRD formula (MDRD= Modification of Diet in Renal Disease). All GFR values are normalized to

Similar to CKD, the initiating causes for CVD are complex. Although exposure to cardiovas‐ cular risk factors such as hypertension, dyslipidemia and diabetes mellitus contributes to CVD, obesity, lack of physical exercise, smoking, genetics, and even depression, also play a role [14]. Common themes for causality are oxidative stress and inflammation, be they local or systemic. The prevalence of CVD also has a strong positive correlation with age, with more than 80% of cases of coronary artery disease and 75% of cases of congestive heart fail‐ ure observed in geriatric patients [14]. Intrinsic cardiac aging, defined as the development of structural and functional alterations during aging, may render the heart more vulnerable to various stressors, and this ultimately favours the development of CVD. In the early stages of CVD, left ventricular hypertrophy and myocardial fibrosis may be seen in many patients [15]. The processes involved in their development, particularly in association with CKD, can be attributed to hypervolaemia, systemic arterial resistance, elevated blood pressure, large vessel compliance, and activation of pathways related to the parathyroid hormone–vitamin D–phosphate axis. Left ventricular hypertrophy and myocardial fibrosis also predispose to

Heart failure resulting from CVD may be staged in a system similar to CKD. In its 2001 guidelines, the American College of Cardiology (ACC) and the American Heart Association working groups introduced four stages of heart failure [17]: Stage A with patients at high risk for developing heart failure in the future but no functional or structural heart disorder; Stage B with a structural heart disorder but no symptoms at any stage; Stage C with previ‐ ous or current symptoms of heart failure in the context of an underlying structural heart problem, but managed with medical treatment; and Stage D with advanced disease requir‐ ing hospital-based support, a heart transplant or palliative care. The ACC staging system is

indicate kidney disease

established renal failure)

Moderately reduced kidney function

abnormalities, or a genetic trait indicating kidney disease

Oxidative Stress and Antioxidant Therapy in Chronic Kidney and Cardiovascular Disease

http://dx.doi.org/10.5772/51923

235

summarises GFR classification and staging for CKD.

**Stage GFR\* Description**

45-59mL/min 30-44mL/min

**Table 1.** Classification and description of the different stages of CKD

an average surface area (size) of 1.73m2

4 15-29mL/min Severely reduced kidney function

an increase in electric excitability and ventricular arrhythmias [16].

3A 3B

The initiating causes of CKD are highly variable, with previously-mentioned hypertension and diabetes being two of the key ones [7]. Epidemiological studies reveal other strong risk factors for CKD, such as a previous episode of acute kidney damage, exposure to nephrotox‐ ins, obesity, smoking, and increasing age [8, 9]. However, no matter the cause, the progres‐ sive structural changes that occur in the kidney are characteristically unifying [10]. The characteristics of CKD are tubulointerstitial inflammation and fibrosis, tubular atrophy, glo‐ merulosclerosis, renal vasculopathy, and presence of granulation tissue. Alterations in the glomerulus include mesangial cell expansion and contraction of the glomerular tuft, fol‐ lowed by a proliferation of connective tissue which leads to significant damage at this first point of the filtration barrier. Structural changes that occur in the kidney produce a vicious cycle of cause and effect, thereby enhancing kidney damage and giving CKD its progressive nature. Whilst early pathological changes in the kidney can occur without clinical presenta‐ tions, due to the high adaptability of the kidney [10], once the adaptive threshold is reached, the progression of CKD is rapid and the development of ESRD imminent. Vascular patholo‐ gy exacerbates development of CKD, and it is perhaps here that the links with CVD are clos‐ est. Hypertension induces intimal and medial hypertrophy of the intrarenal arteries, leading to hypertensive nephropathy. This is followed by outer cortical glomerulosclerosis with lo‐ cal tubular atrophy and interstitial fibrosis. Compensatory hypertrophy of the inner-cortical glomeruli results, leading to hyperfiltration injury and global glomerulosclerosis. Note, however, that although glomerulopathy is an important characteristic of CKD, the incidence of tubulointerstitial fibrosis has the best correlation with CKD development [11]. As such, kidney tubular cells and renal fibroblasts may be the founding cell types in the progressive development of CKD.

The main clinical manifestation of CKD is a loss of glomerular filtration rate (GFR), allowing for staging of CKD with progressively decreasing (estimated) GFR. CKD staging was facili‐ tated by the National Kidney Foundation (NKF) Kidney Disease Outcomes Quality Initia‐ tive (KDOQI) and the Kidney Disease - Improving Global Outcomes (KDIGO), an outcome that highlighted the condition and facilitated its increased diagnosis [12]. The first two stages have normal, or slightly reduced kidney function but some indication of structural deficit in two samples at least 90 days apart. Stages 3-5 are considered the most concerning, with Stage 3 now being sub-classified into Stages 3a and b because of their diagnostic impor‐ tance. It is thought that stages 2 and 3 should be targeted with prophylactic therapies, such as lipid lowering drugs or RAS modifiers [13], to minimize the progression of CKD. Table 1 summarises GFR classification and staging for CKD.


\* Measured using the MDRD formula (MDRD= Modification of Diet in Renal Disease). All GFR values are normalized to an average surface area (size) of 1.73m2

**Table 1.** Classification and description of the different stages of CKD

with hypertension and dyslipidemia, both causes of renal failure. Diabetic nephropathy is arguably the leading cause of renal failure. CKD, hypertension and diabetes mellitus all in‐ volve endothelial dysfunction, a change well known in the development of atherosclerosis and CVD that includes coronary artery disease, heart failure, stroke and peripheral arterial disease [5]. Vascular calcification occurs in progressive atherosclerosis and CVD, but it is al‐ so an important part of vascular injury in end-stage renal disease (ESRD), where patients need renal replacement therapy to survive. It is paradoxical that approximately 50% of indi‐ viduals with ESRD die from a cardiovascular cause [6]. Thus, CKD and CVD patients have closely-linked diseases with increasing morbidity and mortality. Prevention and treatment

The initiating causes of CKD are highly variable, with previously-mentioned hypertension and diabetes being two of the key ones [7]. Epidemiological studies reveal other strong risk factors for CKD, such as a previous episode of acute kidney damage, exposure to nephrotox‐ ins, obesity, smoking, and increasing age [8, 9]. However, no matter the cause, the progres‐ sive structural changes that occur in the kidney are characteristically unifying [10]. The characteristics of CKD are tubulointerstitial inflammation and fibrosis, tubular atrophy, glo‐ merulosclerosis, renal vasculopathy, and presence of granulation tissue. Alterations in the glomerulus include mesangial cell expansion and contraction of the glomerular tuft, fol‐ lowed by a proliferation of connective tissue which leads to significant damage at this first point of the filtration barrier. Structural changes that occur in the kidney produce a vicious cycle of cause and effect, thereby enhancing kidney damage and giving CKD its progressive nature. Whilst early pathological changes in the kidney can occur without clinical presenta‐ tions, due to the high adaptability of the kidney [10], once the adaptive threshold is reached, the progression of CKD is rapid and the development of ESRD imminent. Vascular patholo‐ gy exacerbates development of CKD, and it is perhaps here that the links with CVD are clos‐ est. Hypertension induces intimal and medial hypertrophy of the intrarenal arteries, leading to hypertensive nephropathy. This is followed by outer cortical glomerulosclerosis with lo‐ cal tubular atrophy and interstitial fibrosis. Compensatory hypertrophy of the inner-cortical glomeruli results, leading to hyperfiltration injury and global glomerulosclerosis. Note, however, that although glomerulopathy is an important characteristic of CKD, the incidence of tubulointerstitial fibrosis has the best correlation with CKD development [11]. As such, kidney tubular cells and renal fibroblasts may be the founding cell types in the progressive

The main clinical manifestation of CKD is a loss of glomerular filtration rate (GFR), allowing for staging of CKD with progressively decreasing (estimated) GFR. CKD staging was facili‐ tated by the National Kidney Foundation (NKF) Kidney Disease Outcomes Quality Initia‐ tive (KDOQI) and the Kidney Disease - Improving Global Outcomes (KDIGO), an outcome that highlighted the condition and facilitated its increased diagnosis [12]. The first two stages have normal, or slightly reduced kidney function but some indication of structural deficit in two samples at least 90 days apart. Stages 3-5 are considered the most concerning, with Stage 3 now being sub-classified into Stages 3a and b because of their diagnostic impor‐ tance. It is thought that stages 2 and 3 should be targeted with prophylactic therapies, such

of these diseases are major aims in health systems worldwide.

234 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

development of CKD.

Similar to CKD, the initiating causes for CVD are complex. Although exposure to cardiovas‐ cular risk factors such as hypertension, dyslipidemia and diabetes mellitus contributes to CVD, obesity, lack of physical exercise, smoking, genetics, and even depression, also play a role [14]. Common themes for causality are oxidative stress and inflammation, be they local or systemic. The prevalence of CVD also has a strong positive correlation with age, with more than 80% of cases of coronary artery disease and 75% of cases of congestive heart fail‐ ure observed in geriatric patients [14]. Intrinsic cardiac aging, defined as the development of structural and functional alterations during aging, may render the heart more vulnerable to various stressors, and this ultimately favours the development of CVD. In the early stages of CVD, left ventricular hypertrophy and myocardial fibrosis may be seen in many patients [15]. The processes involved in their development, particularly in association with CKD, can be attributed to hypervolaemia, systemic arterial resistance, elevated blood pressure, large vessel compliance, and activation of pathways related to the parathyroid hormone–vitamin D–phosphate axis. Left ventricular hypertrophy and myocardial fibrosis also predispose to an increase in electric excitability and ventricular arrhythmias [16].

Heart failure resulting from CVD may be staged in a system similar to CKD. In its 2001 guidelines, the American College of Cardiology (ACC) and the American Heart Association working groups introduced four stages of heart failure [17]: Stage A with patients at high risk for developing heart failure in the future but no functional or structural heart disorder; Stage B with a structural heart disorder but no symptoms at any stage; Stage C with previ‐ ous or current symptoms of heart failure in the context of an underlying structural heart problem, but managed with medical treatment; and Stage D with advanced disease requir‐ ing hospital-based support, a heart transplant or palliative care. The ACC staging system is useful in that Stage A may be considered pre-heart failure where intervention with treat‐ ment may prevent progression to overt symptoms.

mental toxins), and physical factors (eg. lack of physical activity) [19]. The inflammatory cells are then a source of free radicals in the forms of reactive oxygen and nitrogen species, although reactive oxygen species (ROS) are considered the most common. The highly reac‐ tive ROS are capable of damaging various structures and functional pathways in cells. In consequence, the presence of inflammatory cells is stimulated by cell damage caused by ROS, creating a cycle of chronic damage that is difficult to break. Oxidative stress arises from alterations in the oxidation-reduction balance of cells. Normally, ROS are countered by endogenous natural defences known as antioxidants, and it is the imbalance between ROS and antioxidants which favours greater relative levels of ROS, thereby giving rise to a state of oxidative stress [20-22]. The simple oxidant "imbalance" theory has now grown to incor‐ porate the various crucial pathways and cell metabolism that are also controlled by the in‐ terplay between oxidants and antioxidants [23-27]. The rationale for antioxidant therapies

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237

(H2O2). Mitochondria are considered the major source of ROS, however other contributing sites of ROS generation include the endoplasmic reticulum, peroxisomes and lysosomes [28-30]. Estimated levels of ROS within mitochondria are 5-10 fold higher than cystolic and nuclear compartments in cells [31] due to the presence of the electron transport chain (ETC) within the mitochondrial inner membrane. 1-3% of inspired molecular oxygen (O2) is con‐

though cellular H2O2 is stable in this form, it has the potential to interact with a variety of substrates to cause damage, especially in the presence of the ferrous iron (Fe2+), which leads to cleavage and formation of the most reactive and damaging of the ROS, the OH• [34]. In healthy metabolic cells, the production of the potentially harmful H2O2 is countered by the catalizing actions of mitochondrial or cystolic catalase (CAT) or thiol peroxidases into water and O2. The ETC consists of 5 multi-enzyme complexes responsible for maintaining the mi‐ tochondrial membrane potential and ATP generation. Each of these complexes presents a site of ROS generation, however complexes I and III have been identified as primary sites of

•- generation [35-38]. ROS generation from mitochondrial complexes increases with age in mice [39]. In humans, Granata and colleagues [40] have demonstrated that patients with

Agreement on the role of oxidative stress in the pathogenesis of chronic disease is, however, not complete. Oxidants are involved in highly conserved basic physiological processes and are effectors of their downstream pathways [41, 42]. The specific mechanisms for "oxidative stress" are difficult to define because of the rapidity of oxidant signalling [31]. For example, protein tyrosine phosphatases are major targets for oxidant signalling since they contain the amino acid residue cysteine that is highly susceptible to oxidative modification [43]. Meng and colleagues [25] demonstrated the oxidation of the SH2 domain of the platelet-derived growth factor (PDGF) receptor, which contains protein tyrosine phosphatases, in response to PDGF binding. This may indicate the induction of free radicals in response to receptor ac‐ tivation by a cognate ligand in a process that is similar to phosphorylation cascades of intra‐

CKD and haemodialysis patients display impaired mitochondrial respiration.

•-), the hydroxyl radical (OH•) and hydrogen peroxide

•- [32, 33], a powerful precursor of H2O2. Al‐

lies in restoring imbalances in the redox environment of cells.

The main ROS are superoxide (O2

O2

cellular signalling.

verted to the most common of the ROS, O2

The links between CKD and CVD are so close that it is often difficult to tease out individual causes and mechanisms, given their chronic nature. However, children with CKD present as a particular population without pre-existing symptomatic cardiac disease. This population could also receive significant benefit from preventing and treating CKD and thereby mini‐ mising the forthcoming development of CVD which is a major cause of death in children with advanced CKD. Left ventricular hypertrophy and dysfunction, and early markers of atherosclerosis such as increased intimal-medial thickness and stiffness of the carotid artery, and coronary artery calcification, may develop in children with CKD. Early CKD, before needing dialysis, is the optimal time to identify and modify risk factors and intervene in an effort to avert risk of premature cardiac disease and death in these children [18]. These ob‐ servations have sparked added interest in the mechanisms of the chronic diseases, and in ways to target these mechanisms with additional therapies, such as antioxidants.

#### **2.1. Inflammation and chronic kidney and cardiovascular disease**

The circulating nature of many inflammatory mediators such as cytokines, and inflammato‐ ry or immune cells, indicates that the immune system can act as a mediator of kidney-heart cross-talk and may be involved in the reciprocal dysfunction that is encountered commonly in the cardio-renal syndromes. Chronic inflammation may follow acute inflammation, but in many chronic diseases like CKD and CVD, it is likely that it begins as a low-grade response with no initial manifestation of an acute reaction. There are many links with visceral obesity and with increased secretion of inflammatory mediators seen in visceral fat [15]. Proinflam‐ matory cytokines are produced by adipocytes, and also cells in the adipose stroma. The links with oxidative stress as an endogenous driver of the chronic diseases become immedi‐ ately obvious when one admits the close association between oxidative stress and inflamma‐ tion. The characteristics of dyslipidaemia (elevated serum triglycerides, elevated lowdensity lipoprotein cholesterol, and/or low high-density lipoprotein cholesterol) are also often seen in obese patients and these are all recognized as risk factors for atherosclerosis. The links between obesity, inflammation, dyslipidaemia, CKD and CVD also occur through yet another syndrome, metabolic syndrome. An improved understanding of the precise mo‐ lecular mechanisms by which chronic inflammation modifies disease is required before the full implications of its presence, including links with persistent oxidative stress as a cause of chronic disease can be realized.

### **3. Oxidative stress and chronic kidney and cardiovascular disease**

#### **3.1. Understanding oxidative stress**

Oxidative stress has been implicated in various pathological systems that are prevalent in both CKD and CVD, most importantly inflammation and fibrosis. Chronic inflammation is induced by biological (eg. infections, autoimmune disease), chemical (eg. drugs, environ‐ mental toxins), and physical factors (eg. lack of physical activity) [19]. The inflammatory cells are then a source of free radicals in the forms of reactive oxygen and nitrogen species, although reactive oxygen species (ROS) are considered the most common. The highly reac‐ tive ROS are capable of damaging various structures and functional pathways in cells. In consequence, the presence of inflammatory cells is stimulated by cell damage caused by ROS, creating a cycle of chronic damage that is difficult to break. Oxidative stress arises from alterations in the oxidation-reduction balance of cells. Normally, ROS are countered by endogenous natural defences known as antioxidants, and it is the imbalance between ROS and antioxidants which favours greater relative levels of ROS, thereby giving rise to a state of oxidative stress [20-22]. The simple oxidant "imbalance" theory has now grown to incor‐ porate the various crucial pathways and cell metabolism that are also controlled by the in‐ terplay between oxidants and antioxidants [23-27]. The rationale for antioxidant therapies lies in restoring imbalances in the redox environment of cells.

useful in that Stage A may be considered pre-heart failure where intervention with treat‐

The links between CKD and CVD are so close that it is often difficult to tease out individual causes and mechanisms, given their chronic nature. However, children with CKD present as a particular population without pre-existing symptomatic cardiac disease. This population could also receive significant benefit from preventing and treating CKD and thereby mini‐ mising the forthcoming development of CVD which is a major cause of death in children with advanced CKD. Left ventricular hypertrophy and dysfunction, and early markers of atherosclerosis such as increased intimal-medial thickness and stiffness of the carotid artery, and coronary artery calcification, may develop in children with CKD. Early CKD, before needing dialysis, is the optimal time to identify and modify risk factors and intervene in an effort to avert risk of premature cardiac disease and death in these children [18]. These ob‐ servations have sparked added interest in the mechanisms of the chronic diseases, and in

ways to target these mechanisms with additional therapies, such as antioxidants.

**3. Oxidative stress and chronic kidney and cardiovascular disease**

Oxidative stress has been implicated in various pathological systems that are prevalent in both CKD and CVD, most importantly inflammation and fibrosis. Chronic inflammation is induced by biological (eg. infections, autoimmune disease), chemical (eg. drugs, environ‐

The circulating nature of many inflammatory mediators such as cytokines, and inflammato‐ ry or immune cells, indicates that the immune system can act as a mediator of kidney-heart cross-talk and may be involved in the reciprocal dysfunction that is encountered commonly in the cardio-renal syndromes. Chronic inflammation may follow acute inflammation, but in many chronic diseases like CKD and CVD, it is likely that it begins as a low-grade response with no initial manifestation of an acute reaction. There are many links with visceral obesity and with increased secretion of inflammatory mediators seen in visceral fat [15]. Proinflam‐ matory cytokines are produced by adipocytes, and also cells in the adipose stroma. The links with oxidative stress as an endogenous driver of the chronic diseases become immedi‐ ately obvious when one admits the close association between oxidative stress and inflamma‐ tion. The characteristics of dyslipidaemia (elevated serum triglycerides, elevated lowdensity lipoprotein cholesterol, and/or low high-density lipoprotein cholesterol) are also often seen in obese patients and these are all recognized as risk factors for atherosclerosis. The links between obesity, inflammation, dyslipidaemia, CKD and CVD also occur through yet another syndrome, metabolic syndrome. An improved understanding of the precise mo‐ lecular mechanisms by which chronic inflammation modifies disease is required before the full implications of its presence, including links with persistent oxidative stress as a cause of

**2.1. Inflammation and chronic kidney and cardiovascular disease**

chronic disease can be realized.

**3.1. Understanding oxidative stress**

ment may prevent progression to overt symptoms.

236 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

The main ROS are superoxide (O2 •-), the hydroxyl radical (OH•) and hydrogen peroxide (H2O2). Mitochondria are considered the major source of ROS, however other contributing sites of ROS generation include the endoplasmic reticulum, peroxisomes and lysosomes [28-30]. Estimated levels of ROS within mitochondria are 5-10 fold higher than cystolic and nuclear compartments in cells [31] due to the presence of the electron transport chain (ETC) within the mitochondrial inner membrane. 1-3% of inspired molecular oxygen (O2) is con‐ verted to the most common of the ROS, O2 •- [32, 33], a powerful precursor of H2O2. Al‐ though cellular H2O2 is stable in this form, it has the potential to interact with a variety of substrates to cause damage, especially in the presence of the ferrous iron (Fe2+), which leads to cleavage and formation of the most reactive and damaging of the ROS, the OH• [34]. In healthy metabolic cells, the production of the potentially harmful H2O2 is countered by the catalizing actions of mitochondrial or cystolic catalase (CAT) or thiol peroxidases into water and O2. The ETC consists of 5 multi-enzyme complexes responsible for maintaining the mi‐ tochondrial membrane potential and ATP generation. Each of these complexes presents a site of ROS generation, however complexes I and III have been identified as primary sites of O2 •- generation [35-38]. ROS generation from mitochondrial complexes increases with age in mice [39]. In humans, Granata and colleagues [40] have demonstrated that patients with CKD and haemodialysis patients display impaired mitochondrial respiration.

Agreement on the role of oxidative stress in the pathogenesis of chronic disease is, however, not complete. Oxidants are involved in highly conserved basic physiological processes and are effectors of their downstream pathways [41, 42]. The specific mechanisms for "oxidative stress" are difficult to define because of the rapidity of oxidant signalling [31]. For example, protein tyrosine phosphatases are major targets for oxidant signalling since they contain the amino acid residue cysteine that is highly susceptible to oxidative modification [43]. Meng and colleagues [25] demonstrated the oxidation of the SH2 domain of the platelet-derived growth factor (PDGF) receptor, which contains protein tyrosine phosphatases, in response to PDGF binding. This may indicate the induction of free radicals in response to receptor ac‐ tivation by a cognate ligand in a process that is similar to phosphorylation cascades of intra‐ cellular signalling.

### **3.2. Endogenous antioxidants – Metabolism or disease modifiers**

The production of ROS is usually in balance with the availability and cellular localisation of antioxidant enzymes such as superoxide dismutase (SOD), CAT and glutathione peroxidase (Gpx). *In vivo* studies have found accumulated oxidative damage occurs from decreased lev‐ els of these enzymes rather than increased ROS production [44, 45]. However, adequate lev‐ els of both are likely to be vital for normal cell function. Mitochondria possess their own pool of antioxidants to counteract their generation of ROS. Mitochondrial manganese-SOD (Mn-SOD) converts O2 •- to H2O2 which is then decomposed to harmless H2O and O2 by CAT and Gpx [46]. Copper/zinc-SOD (Cu/Zn-SOD) has been implicated in stabilizing O2 •- within other cellular compartments, especially peroxisomes, and must be considered in mainte‐ nance of the redox state of the whole cell [47, 48]. Limited antioxidant actions of Cu/Zn-SOD may also occur within the inter-membrane space [49]. There is no evidence to indicate that glutathione synthesis occurs within mitochondria, however the mitochondria have their own distinct pool of glutathione required for the formation of Gpx [50].

ing mitochondrial biogenesis [57]. PPARγ regulation in chronic disease is increasingly rec‐ ognised, with oxidative stress as the unifying initiating feature. Omega-3 polyunsaturated fatty acids (PUFA) reduce inflammation in kidney tubular epithelial cells by upregulating PPARγ [60]. PPARγ activation by pioglitazone reduced cyclo-oxygenase 2 (COX2) expres‐ sion in smooth muscle cells from hypertensive rats, and upregulated endogenous antioxi‐

Oxidative Stress and Antioxidant Therapy in Chronic Kidney and Cardiovascular Disease

http://dx.doi.org/10.5772/51923

239

Recently, the protective responses of the nuclear factor E2-related factor 2/Kelch-like ECHassociated protein 1 (Nrf2/Keap1)/antioxidant response element (ARE) were noted [62]. Nrf2 is a nuclear transcription factor that is suppressed in the cytoplasm by the physical binding of Keap1 preventing its translocation into the nucleus. Nrf2 is activated by a loss of Keap1 binding by alterations in cellular redox status, such as increased ROS, by-products of oxida‐ tive damage, and reduced antioxidant capacity, thereby promoting its transcriptional re‐ sponse at the ARE [63]. The ARE is a vital component of the promoter regions of genes encoding detoxifying, antioxidant, and glutathione-regulatory enzymes such as quinone-re‐ ductase, glutathione-peroxidases, glutathione-reductase, thioredoxins and thioredoxin-re‐ ductase, peroxiredoxins, gamma-glutamyl cysteine, heme-oxygenase-1 (HO-1), CAT, SOD metallothionein and ferritin [64-67]. Important to note is that by-products of oxidative dam‐ age such a 4-hydroxynoneal and J-isoprostanes act as endogenous activators of Nrf2 [68, 69]. Thus, NRF2/Keap1 and the ARE play a crucial role in cellular defence against ROS. Recent pharmacological protocols have allowed the modulation of this pathway to enhance the ca‐

Chronic diseases of the kidney possess various commonalities to chronic disease of the car‐ diovascular system which can be linked through pathways controlled by oxidative stress, as shown in Figure 1. Vascular, cellular and biochemical factors all contribute. Increased serum uric acid levels (hyperuricaemia) can arise from increased purine metabolism, increasing age and decreased renal excretion, and have harmful systemic effects. Hyperuricaemia is as‐ sociated with an increased risk for development and progression of CKD. Hyperuricemia is also a risk factor associated with coronary artery disease [71], left ventricular hypertrophy [72], atrial fibrillation [73], myocardial infarction [74] and ischemic stroke [75]. A 20.6% prevalence of hyperuricemia was found in a cross-sectional study of 18,020 CKD patients [76], and a positive correlation was found between serum uric acid and serum creatinine with impaired renal function [77]. Retention of uremic toxins promotes inflammation and oxidative stress, by priming the acute inflammatory polymorphonuclear lymphocytes, acti‐ vating interleukin (IL)-1β and IL-8 [78] and stimulating the innate immune response through CD8+ cells [79]. Additionally, uric acid synthesis can promote oxidative stress di‐ rectly through the activity of xanthine oxidoreductase. This enzyme is synthesized as xan‐ thine dehydrogenase, which can be converted to xanthine oxidase by calcium-dependant proteolysis [80] or modification of cysteine residues [81]. In doing so, the enzyme loses its capacity to bind NADH by alterations in its catalytic site and, instead, transfers electrons

[82]. However, the role of uric acid in many conditions asso‐

pabilities of cells to combat oxidative stress and inflammation [70].


**3.4. CKD and CVD are unified by oxidative stress**

from O2, thereby generating O2

dants Mn- and Cu/Zn-SOD [61].

Among the various endogenous defences against ROS, glutathione homeostasis is critical for a cellular redox environment. Glutathione-linked enzymatic defences of this family include Gpx, glutathione-S-transferase (GST), glutaredoxins (Grx), thioredoxins (Trx), and peroxire‐ doxins (Prx) [51]. Many of these proteins are known to interact with each other, forming re‐ dox networks that have come under investigation for their contribution to dysfunctional oxidant pathways. Mitochondrial-specific isoforms of these proteins also exist and include Grx2, Grx5, Trx2 and Prx3 [52-54], which may be more critical for cell survival compared to their cystolic counterparts [50]. Mitochondrial dysfunction, resulting in depleted ATP syn‐ thesis, has the potential to reduce the redox control of glutathione since the rate of gluta‐ thione synthesis is ATP-dependent [55]. Intracellular synthesis of glutathione from amino acid derivatives (glycine, glutamic acid and cysteine) accounts for the majority of cellular glutathione compared with extracellular glutathione uptake [56]. Antioxidant networks in which there is interplay, crosstalk and synergism to efficiently and specifically scavenge ROS, may also exist. If this is the case, these antioxidant networks could be harnessed to de‐ velop poly-therapeutic antioxidant supplements to combat oxidant-related pathologies, like CKD and CVD.

#### **3.3. Oxidative stress and transcriptional control**

The role of oxidative stress in upstream transcriptional gene regulation is becoming increas‐ ingly recognised. Not only does this provide insight into the physiological role of oxidative stress, but presents regulatory systems that are possibly prone to deregulation. Furthermore, these sites present targets for pharmacological intervention. Peroxisome proliferator-activat‐ ed receptors (PPARs) are members of the nuclear hormone receptor superfamily of liganddependant transcription factors which have been shown to alter during CKD and CVD [57-59]. They have important roles in the transcriptional regulation of cell differentiation, lipid metabolism, glucose homeostasis, cell cycle progression, and inflammation. There are three PPAR isoforms – α, β/δ and γ. Peroxisome proliferator gamma coactivator (PGCα), in association with PPARγ activation, leads to a variety of cellular protective responses includ‐ ing mitochondrial biogenesis [57]. PPARγ regulation in chronic disease is increasingly rec‐ ognised, with oxidative stress as the unifying initiating feature. Omega-3 polyunsaturated fatty acids (PUFA) reduce inflammation in kidney tubular epithelial cells by upregulating PPARγ [60]. PPARγ activation by pioglitazone reduced cyclo-oxygenase 2 (COX2) expres‐ sion in smooth muscle cells from hypertensive rats, and upregulated endogenous antioxi‐ dants Mn- and Cu/Zn-SOD [61].

Recently, the protective responses of the nuclear factor E2-related factor 2/Kelch-like ECHassociated protein 1 (Nrf2/Keap1)/antioxidant response element (ARE) were noted [62]. Nrf2 is a nuclear transcription factor that is suppressed in the cytoplasm by the physical binding of Keap1 preventing its translocation into the nucleus. Nrf2 is activated by a loss of Keap1 binding by alterations in cellular redox status, such as increased ROS, by-products of oxida‐ tive damage, and reduced antioxidant capacity, thereby promoting its transcriptional re‐ sponse at the ARE [63]. The ARE is a vital component of the promoter regions of genes encoding detoxifying, antioxidant, and glutathione-regulatory enzymes such as quinone-re‐ ductase, glutathione-peroxidases, glutathione-reductase, thioredoxins and thioredoxin-re‐ ductase, peroxiredoxins, gamma-glutamyl cysteine, heme-oxygenase-1 (HO-1), CAT, SOD metallothionein and ferritin [64-67]. Important to note is that by-products of oxidative dam‐ age such a 4-hydroxynoneal and J-isoprostanes act as endogenous activators of Nrf2 [68, 69]. Thus, NRF2/Keap1 and the ARE play a crucial role in cellular defence against ROS. Recent pharmacological protocols have allowed the modulation of this pathway to enhance the ca‐ pabilities of cells to combat oxidative stress and inflammation [70].

### **3.4. CKD and CVD are unified by oxidative stress**

**3.2. Endogenous antioxidants – Metabolism or disease modifiers**

238 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

(Mn-SOD) converts O2

CKD and CVD.

**3.3. Oxidative stress and transcriptional control**

The production of ROS is usually in balance with the availability and cellular localisation of antioxidant enzymes such as superoxide dismutase (SOD), CAT and glutathione peroxidase (Gpx). *In vivo* studies have found accumulated oxidative damage occurs from decreased lev‐ els of these enzymes rather than increased ROS production [44, 45]. However, adequate lev‐ els of both are likely to be vital for normal cell function. Mitochondria possess their own pool of antioxidants to counteract their generation of ROS. Mitochondrial manganese-SOD

and Gpx [46]. Copper/zinc-SOD (Cu/Zn-SOD) has been implicated in stabilizing O2

own distinct pool of glutathione required for the formation of Gpx [50].

other cellular compartments, especially peroxisomes, and must be considered in mainte‐ nance of the redox state of the whole cell [47, 48]. Limited antioxidant actions of Cu/Zn-SOD may also occur within the inter-membrane space [49]. There is no evidence to indicate that glutathione synthesis occurs within mitochondria, however the mitochondria have their

Among the various endogenous defences against ROS, glutathione homeostasis is critical for a cellular redox environment. Glutathione-linked enzymatic defences of this family include Gpx, glutathione-S-transferase (GST), glutaredoxins (Grx), thioredoxins (Trx), and peroxire‐ doxins (Prx) [51]. Many of these proteins are known to interact with each other, forming re‐ dox networks that have come under investigation for their contribution to dysfunctional oxidant pathways. Mitochondrial-specific isoforms of these proteins also exist and include Grx2, Grx5, Trx2 and Prx3 [52-54], which may be more critical for cell survival compared to their cystolic counterparts [50]. Mitochondrial dysfunction, resulting in depleted ATP syn‐ thesis, has the potential to reduce the redox control of glutathione since the rate of gluta‐ thione synthesis is ATP-dependent [55]. Intracellular synthesis of glutathione from amino acid derivatives (glycine, glutamic acid and cysteine) accounts for the majority of cellular glutathione compared with extracellular glutathione uptake [56]. Antioxidant networks in which there is interplay, crosstalk and synergism to efficiently and specifically scavenge ROS, may also exist. If this is the case, these antioxidant networks could be harnessed to de‐ velop poly-therapeutic antioxidant supplements to combat oxidant-related pathologies, like

The role of oxidative stress in upstream transcriptional gene regulation is becoming increas‐ ingly recognised. Not only does this provide insight into the physiological role of oxidative stress, but presents regulatory systems that are possibly prone to deregulation. Furthermore, these sites present targets for pharmacological intervention. Peroxisome proliferator-activat‐ ed receptors (PPARs) are members of the nuclear hormone receptor superfamily of liganddependant transcription factors which have been shown to alter during CKD and CVD [57-59]. They have important roles in the transcriptional regulation of cell differentiation, lipid metabolism, glucose homeostasis, cell cycle progression, and inflammation. There are three PPAR isoforms – α, β/δ and γ. Peroxisome proliferator gamma coactivator (PGCα), in association with PPARγ activation, leads to a variety of cellular protective responses includ‐

•- to H2O2 which is then decomposed to harmless H2O and O2 by CAT

•- within

Chronic diseases of the kidney possess various commonalities to chronic disease of the car‐ diovascular system which can be linked through pathways controlled by oxidative stress, as shown in Figure 1. Vascular, cellular and biochemical factors all contribute. Increased serum uric acid levels (hyperuricaemia) can arise from increased purine metabolism, increasing age and decreased renal excretion, and have harmful systemic effects. Hyperuricaemia is as‐ sociated with an increased risk for development and progression of CKD. Hyperuricemia is also a risk factor associated with coronary artery disease [71], left ventricular hypertrophy [72], atrial fibrillation [73], myocardial infarction [74] and ischemic stroke [75]. A 20.6% prevalence of hyperuricemia was found in a cross-sectional study of 18,020 CKD patients [76], and a positive correlation was found between serum uric acid and serum creatinine with impaired renal function [77]. Retention of uremic toxins promotes inflammation and oxidative stress, by priming the acute inflammatory polymorphonuclear lymphocytes, acti‐ vating interleukin (IL)-1β and IL-8 [78] and stimulating the innate immune response through CD8+ cells [79]. Additionally, uric acid synthesis can promote oxidative stress di‐ rectly through the activity of xanthine oxidoreductase. This enzyme is synthesized as xan‐ thine dehydrogenase, which can be converted to xanthine oxidase by calcium-dependant proteolysis [80] or modification of cysteine residues [81]. In doing so, the enzyme loses its capacity to bind NADH by alterations in its catalytic site and, instead, transfers electrons from O2, thereby generating O2 - [82]. However, the role of uric acid in many conditions asso‐ ciated with oxidative stress is not clear and there are experimental and clinical data showing that uric acid also has a role *in vivo* as an anti-oxidant [83].

oxidants. However, these studies are criticised for their isolated, non-holistic, nature [87, 88]. It is largely the positive pre-clinical results from *in vivo* studies, usually in rodents, which drive progress for applicability in chronic human disease, but even these show considerable discrepancies in translation into patients. Despite the well-documented dysregulated endog‐ enous oxidant/antioxidant profile in chronic degenerative disorders such as CVD and CKD, there is still evidence that certain antioxidants have no effect [89-92]. It may first be impor‐ tant to identify patients having an altered oxidative stress profile, since this population pro‐ vides an ideal "intention to treat" cohort. The following trials of antioxidants need then to be rigorous, identifying not only any positive patient outcomes, but also the underlying mecha‐ nism, and of course any deleterious outcome. Various approaches have been taken to reduce oxidative stress in models of CKD and accelerated CVD, ranging from reducing oxidant in‐ take in food stuffs [93, 94] to targeted polypharmaceutical compounds. The benefit of rigor‐ ous review of outcome from antioxidant therapies in either CKD or CVD is that the primary and secondary outcomes related to both can be measured. In the following section, some an‐

Oxidative Stress and Antioxidant Therapy in Chronic Kidney and Cardiovascular Disease

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241

N-acetyl cysteine (NAC) acts as an essential precursor to many endogenous antioxidants in‐ volved in the decomposition of peroxides [95]. NAC attenuates oxidative stress from vari‐ ous underlying causes by replenishing intracellular glutathione stores. Glutathione is synthesized in the body by three amino acids by the catalysing of intracellular enzymes gamma-glutamylcysteine synthetase and glutathione synthetase. L-glutamic acid and gly‐ cine are two precursors of glutathione that are biologically and readily available. However, the limiting precursor to glutathione biosynthesis and the third amino acid, L-cysteine, is not readily available in a human diet. Although the primary basis for NAC supplementation is to replenish cellular cysteine levels to maintain intracellular glutathione and thus redox control, the sulfhydral-thiol group of L-cysteine is also able to exert direct antioxidant effects by scavenging free radicals, and NAC may also exert its protective effects against 2,3,5 tris(glutathion-S-yl)-hydroquinone toxicity. This was demonstrated in isolated renal tubular epithelial cells, in part by the activation of extracellular signal regulated protein kinase

The results of NAC supplementation in kidney disease have been variable and largely de‐ pendent on the type and cause of kidney injury and also the timing of treatment. In cultured human proximal tubular epithelial cells, NAC reduced lipid peroxidation and maintained the mitochondrial membrane potential, thereby preventing apoptosis following H2O2 ad‐ ministration [97]. Although NAC had no significant effect on markers of oxidative stress and inflammation in rats following unilateral ureteral obstruction [98], it reduced kidney malondialdehyde (MDA) levels in a diabetic mouse model [99]. The treatment of CKD pa‐ tients with NAC with the aim of improving renal function and preventing ESKD has been largely disappointing, with no evidence of reduction in proteinuria [100, 101]. However, NAC seems to exert the greatest antioxidant and anti-inflammatory properties when used against the greatest injury, such as in ESKD patients receiving either haemodialysis or peri‐

tioxidants used for CKD or CVD are reviewed, as shown in Figure 2.

**4.1. N-acetylcysteine – An antioxidant with promise**

(ERK) 1/2 [96].

**Figure 1.** Chronic kidney disease and cardiovascular disease are unified by oxidative stress. Mutual risk factors influ‐ ence the development and progression of CKD and CVD and can either be modifiable (diabetes, obesity, metabolic syndrome, smoking) or non-modifiable (genetic predisposition, increasing age, acute injury). Oxidative stress has been implicated in the majority of initiating factors. The progression of CKD to CVD, or vice versa, is mediated through: (1) inflammation and the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and IL-8 from activated lymphocytes; (2) endothelial dysfunction due to increased retention of uremic toxins, and decreased L-arginine synthesis which causes alterations in nitric oxide (NO) signalling - dyslipidaemia and associ‐ ated pro-oxidative/inflammatory state lead to increased oxidised-low density lipoproteins (ox-LDL), a major compo‐ nent in the pathogenesis of atherosclerosis; (3) redox perturbations that ultimately underlie oxidative stress due to an imbalance between the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and endogenous antioxidants, leading to mitochondrial dysfunction and alterations in redox sensitive pathways such as Nrf2/keap1/ ARB.

The kidney is a vital source of L-arginine which is a precursor for nitric oxide (NO). A re‐ duction in renal mass can therefore reduce the production of L-arginine and NO activity. NO is vital for regular vascular endothelial cell function, and decreased amounts have the potential to manifest into CVD [84]. Additionally, oxidized low density lipoprotein (ox-LDL), a by-product of oxidative damage in human blood, plays a pivotal role in the patho‐ genesis of atherosclerosis [85]. There is also a possible link between CVD and CKD that is regulated by oxidative stress through a functional mitochondrial angiotensin system [86]. Angiotensin type II receptors were co-localised with angiotensin on the inner mitochondrial membrane of human mononuclear cells and mouse renal tubular cells. This system was found to modulate mitochondrial NO production and respiration.

### **4. Antioxidant therapies in chronic kidney and cardiovascular disease**

The current state of antioxidant therapies for CKD and CVD is one of promise, but not with‐ out controversy. *In vitro* studies commonly identify agents that are able to detoxify harmful oxidants. However, these studies are criticised for their isolated, non-holistic, nature [87, 88]. It is largely the positive pre-clinical results from *in vivo* studies, usually in rodents, which drive progress for applicability in chronic human disease, but even these show considerable discrepancies in translation into patients. Despite the well-documented dysregulated endog‐ enous oxidant/antioxidant profile in chronic degenerative disorders such as CVD and CKD, there is still evidence that certain antioxidants have no effect [89-92]. It may first be impor‐ tant to identify patients having an altered oxidative stress profile, since this population pro‐ vides an ideal "intention to treat" cohort. The following trials of antioxidants need then to be rigorous, identifying not only any positive patient outcomes, but also the underlying mecha‐ nism, and of course any deleterious outcome. Various approaches have been taken to reduce oxidative stress in models of CKD and accelerated CVD, ranging from reducing oxidant in‐ take in food stuffs [93, 94] to targeted polypharmaceutical compounds. The benefit of rigor‐ ous review of outcome from antioxidant therapies in either CKD or CVD is that the primary and secondary outcomes related to both can be measured. In the following section, some an‐ tioxidants used for CKD or CVD are reviewed, as shown in Figure 2.

### **4.1. N-acetylcysteine – An antioxidant with promise**

ciated with oxidative stress is not clear and there are experimental and clinical data showing

**Figure 1.** Chronic kidney disease and cardiovascular disease are unified by oxidative stress. Mutual risk factors influ‐ ence the development and progression of CKD and CVD and can either be modifiable (diabetes, obesity, metabolic syndrome, smoking) or non-modifiable (genetic predisposition, increasing age, acute injury). Oxidative stress has been implicated in the majority of initiating factors. The progression of CKD to CVD, or vice versa, is mediated through: (1) inflammation and the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and IL-8 from activated lymphocytes; (2) endothelial dysfunction due to increased retention of uremic toxins, and decreased L-arginine synthesis which causes alterations in nitric oxide (NO) signalling - dyslipidaemia and associ‐ ated pro-oxidative/inflammatory state lead to increased oxidised-low density lipoproteins (ox-LDL), a major compo‐ nent in the pathogenesis of atherosclerosis; (3) redox perturbations that ultimately underlie oxidative stress due to an imbalance between the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and endogenous antioxidants, leading to mitochondrial dysfunction and alterations in redox sensitive pathways such as Nrf2/keap1/

The kidney is a vital source of L-arginine which is a precursor for nitric oxide (NO). A re‐ duction in renal mass can therefore reduce the production of L-arginine and NO activity. NO is vital for regular vascular endothelial cell function, and decreased amounts have the potential to manifest into CVD [84]. Additionally, oxidized low density lipoprotein (ox-LDL), a by-product of oxidative damage in human blood, plays a pivotal role in the patho‐ genesis of atherosclerosis [85]. There is also a possible link between CVD and CKD that is regulated by oxidative stress through a functional mitochondrial angiotensin system [86]. Angiotensin type II receptors were co-localised with angiotensin on the inner mitochondrial membrane of human mononuclear cells and mouse renal tubular cells. This system was

**4. Antioxidant therapies in chronic kidney and cardiovascular disease**

The current state of antioxidant therapies for CKD and CVD is one of promise, but not with‐ out controversy. *In vitro* studies commonly identify agents that are able to detoxify harmful

found to modulate mitochondrial NO production and respiration.

that uric acid also has a role *in vivo* as an anti-oxidant [83].

240 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ARB.

N-acetyl cysteine (NAC) acts as an essential precursor to many endogenous antioxidants in‐ volved in the decomposition of peroxides [95]. NAC attenuates oxidative stress from vari‐ ous underlying causes by replenishing intracellular glutathione stores. Glutathione is synthesized in the body by three amino acids by the catalysing of intracellular enzymes gamma-glutamylcysteine synthetase and glutathione synthetase. L-glutamic acid and gly‐ cine are two precursors of glutathione that are biologically and readily available. However, the limiting precursor to glutathione biosynthesis and the third amino acid, L-cysteine, is not readily available in a human diet. Although the primary basis for NAC supplementation is to replenish cellular cysteine levels to maintain intracellular glutathione and thus redox control, the sulfhydral-thiol group of L-cysteine is also able to exert direct antioxidant effects by scavenging free radicals, and NAC may also exert its protective effects against 2,3,5 tris(glutathion-S-yl)-hydroquinone toxicity. This was demonstrated in isolated renal tubular epithelial cells, in part by the activation of extracellular signal regulated protein kinase (ERK) 1/2 [96].

The results of NAC supplementation in kidney disease have been variable and largely de‐ pendent on the type and cause of kidney injury and also the timing of treatment. In cultured human proximal tubular epithelial cells, NAC reduced lipid peroxidation and maintained the mitochondrial membrane potential, thereby preventing apoptosis following H2O2 ad‐ ministration [97]. Although NAC had no significant effect on markers of oxidative stress and inflammation in rats following unilateral ureteral obstruction [98], it reduced kidney malondialdehyde (MDA) levels in a diabetic mouse model [99]. The treatment of CKD pa‐ tients with NAC with the aim of improving renal function and preventing ESKD has been largely disappointing, with no evidence of reduction in proteinuria [100, 101]. However, NAC seems to exert the greatest antioxidant and anti-inflammatory properties when used against the greatest injury, such as in ESKD patients receiving either haemodialysis or peri‐ toneal dialysis. In those cases, NAC reduced serum 8-isoprostane and the inflammatory cy‐ tokine IL-6 [102, 103]. A recent systemic review on antioxidant therapy in hemodialysis patients highlighted NAC as the most efficacious agent in decreasing oxidative stress [104].

indirectly by restoring vitamin C [111]. This is a prime example of a cellular antioxidant net‐ work prone to dysregulation. Administration of α-tocopherol to kidney proximal tubular cells in culture decreased cisplatin-induced ROS and increased cell viability [112]. The bene‐ ficial effects of α-tocopherol are not limited to its antioxidant properties, and recently atten‐ tion has focused on its blood oxygenising and endogenous cell signalling functions [113]. Vitamin E foodstuffs primarily consist of α-tocotreinol, an isoform of α-tocopherol which has higher antioxidant efficacy in biological membranes. Despite this, the uptake and distri‐ bution of α-tocotreinol is far less than α-tocopherol. Therefore, the basis of vitamin E supple‐ mentation is to enhance α-tocopherol levels in cell plasma membranes to prevent lipid peroxidation and resultant oxidative stress. One drawback of α-tocopherol is that it takes

Oxidative Stress and Antioxidant Therapy in Chronic Kidney and Cardiovascular Disease

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243

Vitamin E therapy has been extensively researched for renal and cardiovascular benefits in human disease populations. Nevertheless, confounding reports mean there is a lack of con‐ sensus as to whether vitamin E therapy induces an overall benefit. It is known that patients with CKD stage 4 display the largest decrease in serum α-tocopherol levels following a pro‐ gressive decline from stage 1 indicating an increased need for α-tocopherol in the CKD pop‐ ulation [115]. Interestingly, within the same cohort of patients, a positive correlation of serum α-tocopherol levels and GFR was found [115]. A large scale trial concluded that vita‐ min E supplementation to cardiovascular high-risk patients over 4.5 years induced no bene‐ fit to cardiovascular outcome [92]. The results from the Selenium and Vitamin E Cancer Prevention Trial (SELECT) are of greater concern. They suggest that vitamin E supplementa‐ tion significantly increases the risk of prostate cancer for young healthy men [116]. Most studies finding beneficial outcomes of α-tocopherol supplementation have largely focused on the ESKD dialysis populations compared to healthy controls and found a reduced risk of CVD, decreased oxidative stress and increased erythrocyte antioxidants SOD, Gpx and CAT [117-119]. The use of α-tocopherol in CKD patients is not without controversy. Miller and colleagues (2005) concluded that high-dose (≥400 IU/day) vitamin E supplementation may increase all cause mortality which may be due to α-tocopherol displacing gamma-(γ)-toco‐ pherol and delta-(δ)-tocopherol in the body [120]. However, this study was highly criticized owing to a bias in data analysis and numerous methodological flaws [121-130]. The appa‐ rent lack of clarity surrounding vitamin E supplementation and associated renal and cardio‐ vascular outcomes appears to stem largely from differences in trial design and failure to

The heart and kidneys contain the highest endogenous levels of co-enzymes (Co)Q9 and CoQ10 compared to all other organs [131, 132]. This is likely due to the respective reliance on aerobic metabolism and high density of mitochondria in the intrinsic functioning cells from these organs. It is imperative that endogenous CoQ10 levels are maintained to ensure mito‐ chondrial health, and this forms the rationale for CoQ10 therapy. CoQ10 is a fundamental lip‐ id-soluble component of all cell membranes including those enclosing subcellular compartments. The physiological roles of CoQ10 act mostly within the mitochondria where it

several days of pre-treatment to exhibit antioxidant effects [114].

specify the form of tocopherol used.

**4.3. Coenzyme Q10 - Maintaining mitochondrial health**

The effect of NAC on cardiovascular pathologies is less well investigated than CKD. Crespo *et al*., (2011) demonstrated *in vivo* that, although long-term NAC supplementation improved cardiac function, it did not delay progression to cardiomyopathy [105]. Endothelial dysfunc‐ tion caused by uremic toxins such as indoxyl sulphate induced ROS-dependent expression of the pro-inflammatory and pro-oxidant nuclear factor-κB (NF-κB), which was ameliorated by NAC pre-treatment [106].

**Figure 2.** Cellular sites for antioxidant therapy targets in CKD and CVD. Inflammation, lipid peroxidation and reactive oxygen species (ROS) from mitochondrial, cytoplasmic and extracellular sources contribute to oxidative stress. Vitamin E incorporates into the phospholipid bilayer halting lipid peroxidation chain reactions. Omega (ω)-3 fatty acids dis‐ place arachadonic acid in the cell membrane and thus reduce arachadonic acid-derived ROS, but also significantly re‐ duce inflammation and subsequent fibrosis. The cysteine residue of N-acetyl-cysteine (NAC) is a precursor for glutathione (GSH) synthesis, and the thiol group is able to scavenge ROS directly. Bardoxolone exerts transcriptional control by promoting nuclear translocation of Nrf2, facilitating antioxidant response element (ARE) binding that upre‐ gulates endogenous antioxidant enzyme activity. Allopurinol inhibits xanthine oxidase-derived ROS and the damag‐ ing effects of hyperuricemia. Coenzyme Q10 (CoQ10) enhances the efficacy of electron transport in the mitochondria, thereby reducing mitochondrial-derived ROS – it is also able to directly scavenge ROS. L-carnitine enhances mitochon‐ drial fatty acid synthesis and subsequent ATP production and thereby maintains cell health. L-arginine is a precursor for nitric oxide which restores endothelial function.

#### **4.2. Vitamin E – An established antioxidant with controversial outcomes**

Vitamin E, or α-tocopherol, is a lipid-soluble antioxidant that incorporates into the plasma membrane of cells, thereby scavenging free radicals, mainly the peroxyl radical, and halting lipid peroxidation chain reactions [107]. A benefit of α-tocopherol is its ability to restore its antioxidant capacity from its oxidized form following free radical scavenging, and incorpo‐ rate back into the plasma membrane. Vitamin C (ascorbic acid) is able to directly reduce αtocopherol [108-110], and intracellular glutathione and lipoic acid can restore α-tocopherol indirectly by restoring vitamin C [111]. This is a prime example of a cellular antioxidant net‐ work prone to dysregulation. Administration of α-tocopherol to kidney proximal tubular cells in culture decreased cisplatin-induced ROS and increased cell viability [112]. The bene‐ ficial effects of α-tocopherol are not limited to its antioxidant properties, and recently atten‐ tion has focused on its blood oxygenising and endogenous cell signalling functions [113]. Vitamin E foodstuffs primarily consist of α-tocotreinol, an isoform of α-tocopherol which has higher antioxidant efficacy in biological membranes. Despite this, the uptake and distri‐ bution of α-tocotreinol is far less than α-tocopherol. Therefore, the basis of vitamin E supple‐ mentation is to enhance α-tocopherol levels in cell plasma membranes to prevent lipid peroxidation and resultant oxidative stress. One drawback of α-tocopherol is that it takes several days of pre-treatment to exhibit antioxidant effects [114].

Vitamin E therapy has been extensively researched for renal and cardiovascular benefits in human disease populations. Nevertheless, confounding reports mean there is a lack of con‐ sensus as to whether vitamin E therapy induces an overall benefit. It is known that patients with CKD stage 4 display the largest decrease in serum α-tocopherol levels following a pro‐ gressive decline from stage 1 indicating an increased need for α-tocopherol in the CKD pop‐ ulation [115]. Interestingly, within the same cohort of patients, a positive correlation of serum α-tocopherol levels and GFR was found [115]. A large scale trial concluded that vita‐ min E supplementation to cardiovascular high-risk patients over 4.5 years induced no bene‐ fit to cardiovascular outcome [92]. The results from the Selenium and Vitamin E Cancer Prevention Trial (SELECT) are of greater concern. They suggest that vitamin E supplementa‐ tion significantly increases the risk of prostate cancer for young healthy men [116]. Most studies finding beneficial outcomes of α-tocopherol supplementation have largely focused on the ESKD dialysis populations compared to healthy controls and found a reduced risk of CVD, decreased oxidative stress and increased erythrocyte antioxidants SOD, Gpx and CAT [117-119]. The use of α-tocopherol in CKD patients is not without controversy. Miller and colleagues (2005) concluded that high-dose (≥400 IU/day) vitamin E supplementation may increase all cause mortality which may be due to α-tocopherol displacing gamma-(γ)-toco‐ pherol and delta-(δ)-tocopherol in the body [120]. However, this study was highly criticized owing to a bias in data analysis and numerous methodological flaws [121-130]. The appa‐ rent lack of clarity surrounding vitamin E supplementation and associated renal and cardio‐ vascular outcomes appears to stem largely from differences in trial design and failure to specify the form of tocopherol used.

### **4.3. Coenzyme Q10 - Maintaining mitochondrial health**

toneal dialysis. In those cases, NAC reduced serum 8-isoprostane and the inflammatory cy‐ tokine IL-6 [102, 103]. A recent systemic review on antioxidant therapy in hemodialysis patients highlighted NAC as the most efficacious agent in decreasing oxidative stress [104].

The effect of NAC on cardiovascular pathologies is less well investigated than CKD. Crespo *et al*., (2011) demonstrated *in vivo* that, although long-term NAC supplementation improved cardiac function, it did not delay progression to cardiomyopathy [105]. Endothelial dysfunc‐ tion caused by uremic toxins such as indoxyl sulphate induced ROS-dependent expression of the pro-inflammatory and pro-oxidant nuclear factor-κB (NF-κB), which was ameliorated

**Figure 2.** Cellular sites for antioxidant therapy targets in CKD and CVD. Inflammation, lipid peroxidation and reactive oxygen species (ROS) from mitochondrial, cytoplasmic and extracellular sources contribute to oxidative stress. Vitamin E incorporates into the phospholipid bilayer halting lipid peroxidation chain reactions. Omega (ω)-3 fatty acids dis‐ place arachadonic acid in the cell membrane and thus reduce arachadonic acid-derived ROS, but also significantly re‐ duce inflammation and subsequent fibrosis. The cysteine residue of N-acetyl-cysteine (NAC) is a precursor for glutathione (GSH) synthesis, and the thiol group is able to scavenge ROS directly. Bardoxolone exerts transcriptional control by promoting nuclear translocation of Nrf2, facilitating antioxidant response element (ARE) binding that upre‐ gulates endogenous antioxidant enzyme activity. Allopurinol inhibits xanthine oxidase-derived ROS and the damag‐ ing effects of hyperuricemia. Coenzyme Q10 (CoQ10) enhances the efficacy of electron transport in the mitochondria, thereby reducing mitochondrial-derived ROS – it is also able to directly scavenge ROS. L-carnitine enhances mitochon‐ drial fatty acid synthesis and subsequent ATP production and thereby maintains cell health. L-arginine is a precursor

Vitamin E, or α-tocopherol, is a lipid-soluble antioxidant that incorporates into the plasma membrane of cells, thereby scavenging free radicals, mainly the peroxyl radical, and halting lipid peroxidation chain reactions [107]. A benefit of α-tocopherol is its ability to restore its antioxidant capacity from its oxidized form following free radical scavenging, and incorpo‐ rate back into the plasma membrane. Vitamin C (ascorbic acid) is able to directly reduce αtocopherol [108-110], and intracellular glutathione and lipoic acid can restore α-tocopherol

**4.2. Vitamin E – An established antioxidant with controversial outcomes**

by NAC pre-treatment [106].

242 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

for nitric oxide which restores endothelial function.

The heart and kidneys contain the highest endogenous levels of co-enzymes (Co)Q9 and CoQ10 compared to all other organs [131, 132]. This is likely due to the respective reliance on aerobic metabolism and high density of mitochondria in the intrinsic functioning cells from these organs. It is imperative that endogenous CoQ10 levels are maintained to ensure mito‐ chondrial health, and this forms the rationale for CoQ10 therapy. CoQ10 is a fundamental lip‐ id-soluble component of all cell membranes including those enclosing subcellular compartments. The physiological roles of CoQ10 act mostly within the mitochondria where it has three well-characterised functions: (1) the transfer of electrons from complexes I and II to complex III along the ETC of the inner mitochondrial membrane and subsequent mem‐ brane polarisation and ATP generation [133, 134]; (2) the pro-oxidant generation of O2 •- and H2O2 [135, 136]; and (3) the anti-oxidant quenching of free radicals [137]. The continual oxi‐ dation-reduction cycle, and existence of CoQ10 in three different redox states, explains its ac‐ tions as an important cellular redox modulator through its pro-oxidant and antioxidant actions. The fully oxidised form of CoQ10, or ubiquinone, is able to accept electrons, primari‐ ly from NADH, to become fully reduced (ubiquinol - CoQ10-H2). The reduced form of CoQ10 is able to give up electrons, thereby scavenging free radicals. The intermediate of ubiqui‐ none and ubiquinol is the univalently-reduced ubisemiquinone (CoQ10-H+ ) which acts as a pro-oxidant to form O2 •- and, subsequently, H2O2.

chain omega-3 PUFA, including docosahexanoic acid (DHA) and eicosapentanoic acid (EPA), have been investigated in a large range of *in vitro* and *in vivo* models and found to possess anti-inflammatory properties. Recently, omega-3 fatty acid treatment of peripheral blood mononuclear cells from pre-dialysis CKD patients reduced the inflammatory markers IL-6, IL-1β, tumor necrosis factor (TNF)-α and C-reactive protein to levels observed in healthy subjects [147]. Although the beneficial effects of EPA/DHA are attributed to their an‐ ti-inflammatory properties, they are also known to enhance endogenous antioxidant defence systems such as γ-glutamyl-cysteinyl ligase and glutathione reductase [148]. DHA and EPA incorporate into the phospholipid bilayer of cells where they displace arachidonic acid. Arachidonic acid can generate ROS through the COX2 and xanthine oxidase inflammatory pathways. DHA/EPA administration to renal epithelial cells and macrophages suppresses this pro-oxidant pathway [149]. Furthermore, chemoattractants derived from EPA are less potent that those derived from arachidonic acid [150, 151]. Recently, *in vitro* studies deter‐ mined that EPA and DHA attenuated TNF-α-stimulated monocyte chemoattractant protein (MCP)-1 gene expression by interacting with ERK and NF-κB in rat mesangial cells [152]. Earlier evidence had shown that EPA and DHA inhibit NF-κB expression by stimulating PPARs in human kidney-2 cells *in vitro* [60]. *In vivo* studies have now confirmed an im‐ provement in kidney function and structure using EPA/DHA supplementation, with re‐ duced oxidative stress, inflammation and tubulointerstitial fibrosis through the reversal of inflammatory and oxidant pathways [153, 154]. Recently, a highly beneficial outcome of fish oil supplementation was found with heart failure patients with co-morbid diabetes [155]. Clinical studies have found fish oil treatment modulates lipid levels [156, 157], and has antithrombotic [158, 159] and anti-hypertensive effects due to its vascular and endothelial ac‐

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Allopurinol treatment aims is to inhibit xanthine oxidase to decrease serum uric acid and its associated toxic effects. Allopurinol and its metabolite, oxypurinol, act as competitive sub‐ strates for xanthine oxidase. They enhance urinary urate excretion and block uric acid reab‐ sorption by urate transporters in the proximal tubule, thereby facilitating enhanced uric acid excretion [161-163]. Allopurinol treatment of diabetic mice attenuated hyperuricaemia, albu‐ minuria, and tubulointerstitial injury [164]. Allopurinol may also have antioxidant activities in addition to its enzyme inhibitory activities, by scavenging OH• as well as chlorine dioxide and HOCl [165, 166]. Although later *in vivo* studies revealed that rat serum obtained after oral administration of allopurinol did not contain allopurinol levels sufficient to scavenge free radicals [167], inhibition of xanthine oxidase-dependent production of NO• and ROS provides allopurinol an indirect mechanism for decreasing oxidative stress in hyperuricae‐ mic CKD patients. Interventional studies of use of allopurinol in renal disease have shown improved uric acid levels, GFR, cardiovascular outcomes and delayed CKD progression. A

trols which decreased, and a 71% decreased risk of CVD [168]. Interestingly, Kanbay and colleagues (2007) found that allopurinol at 300mg/d over 3 months improved GFR, uric acid

given allopurinol

compared to the con‐

prospective randomised trial of 113 patients with GFR <60 ml/min/1.73m3

100mg/d for 2 years found an increase in GFR of 1.31 ml/min/1.73m3

tions [160].

**4.5. Allopurinol – A xanthine oxidase inhibitor**

The major antioxidant role of CoQ10 is in preventing lipid peroxidation directly, and by in‐ teractions with α-tocopherol [138]. Ubiquinol is able to donate a hydrogen atom and thus quench peroxyl radicals, preventing lipid peroxidation chain reactions. CoQ10 and α-toco‐ pherol co-operate as antioxidants through the actions of CoQ10-H2 restoring α-tocopheroxyl back to α-tocopherol [109, 139]. However, the reactivity of α-tocopherol with peroxy radi‐ cals far exceeds that of ubiquinol with peroxyl radicals, suggesting that, *in vivo*, ubiquinols do not act as antioxidants but regenerate the antioxidant properties of α-tocopherols [140]. This is in accordance with *in vivo* studies investigating the effects of CoQ10 supplementation which have primarily found a limited antioxidant capacity. CoQ10, acting as a pro-oxidant in all biological membranes including the Golgi, endosome/lysosome systems, as well as mito‐ chondria, has led to much criticism regarding the claimed antioxidant power of CoQ10 sup‐ plementation in humans [141]. Nonetheless, many *in vitro* studies demonstrate antioxidant properties of CoQ10 in single cells, and benefits of CoQ10 supplementation in humans are at‐ tributed to its ability to maintain efficient mitochondrial energy metabolism and thus pre‐ vent mitochondrial dysfunction, rather than act as a direct cellular antioxidant. CoQ10 supplementation *in vivo* reduced protein oxidation in skeletal muscle of rats but had no ef‐ fect on mitochondrial H2O2 production in the kidney [142]. However, Ishikawa and collea‐ gues (2011) demonstrated a decrease in kidney O2 •- levels in hemi-nephrectomised rats on a CoQ10 supplemented diet, and increased renal function compared with rats on a control diet [143]. Recently, CoQ10 supplementation improved left ventricular diastolic dysfunction and remodelling and reduced oxidative stress in a mouse model of type 2 diabetes [144]. CoQ10 supplementation in CVD patients also receiving statin therapy is becoming increasingly popular due to the CoQ10-inhibitory actions of statins. CoQ10 levels decrease with age, but there are no studies measuring endogenous CoQ10 levels in CKD or CVD patients and this could prove vital in the identification of population where CoQ10 therapy may have benefi‐ cial outcomes.

#### **4.4. Omega-3 poly-unsaturated fatty acids – Inflammation and oxidative stress**

Inflammation and fibrosis are causes, as well as consequences, of oxidative stress [145, 146]. Direct targeting of inflammatory and fibrotic pathways with more specific modifying com‐ pounds presents a way to indirectly decrease oxidative stress in chronic pathologies. Long chain omega-3 PUFA, including docosahexanoic acid (DHA) and eicosapentanoic acid (EPA), have been investigated in a large range of *in vitro* and *in vivo* models and found to possess anti-inflammatory properties. Recently, omega-3 fatty acid treatment of peripheral blood mononuclear cells from pre-dialysis CKD patients reduced the inflammatory markers IL-6, IL-1β, tumor necrosis factor (TNF)-α and C-reactive protein to levels observed in healthy subjects [147]. Although the beneficial effects of EPA/DHA are attributed to their an‐ ti-inflammatory properties, they are also known to enhance endogenous antioxidant defence systems such as γ-glutamyl-cysteinyl ligase and glutathione reductase [148]. DHA and EPA incorporate into the phospholipid bilayer of cells where they displace arachidonic acid. Arachidonic acid can generate ROS through the COX2 and xanthine oxidase inflammatory pathways. DHA/EPA administration to renal epithelial cells and macrophages suppresses this pro-oxidant pathway [149]. Furthermore, chemoattractants derived from EPA are less potent that those derived from arachidonic acid [150, 151]. Recently, *in vitro* studies deter‐ mined that EPA and DHA attenuated TNF-α-stimulated monocyte chemoattractant protein (MCP)-1 gene expression by interacting with ERK and NF-κB in rat mesangial cells [152]. Earlier evidence had shown that EPA and DHA inhibit NF-κB expression by stimulating PPARs in human kidney-2 cells *in vitro* [60]. *In vivo* studies have now confirmed an im‐ provement in kidney function and structure using EPA/DHA supplementation, with re‐ duced oxidative stress, inflammation and tubulointerstitial fibrosis through the reversal of inflammatory and oxidant pathways [153, 154]. Recently, a highly beneficial outcome of fish oil supplementation was found with heart failure patients with co-morbid diabetes [155]. Clinical studies have found fish oil treatment modulates lipid levels [156, 157], and has antithrombotic [158, 159] and anti-hypertensive effects due to its vascular and endothelial ac‐ tions [160].

#### **4.5. Allopurinol – A xanthine oxidase inhibitor**

has three well-characterised functions: (1) the transfer of electrons from complexes I and II to complex III along the ETC of the inner mitochondrial membrane and subsequent mem‐ brane polarisation and ATP generation [133, 134]; (2) the pro-oxidant generation of O2

H2O2 [135, 136]; and (3) the anti-oxidant quenching of free radicals [137]. The continual oxi‐ dation-reduction cycle, and existence of CoQ10 in three different redox states, explains its ac‐ tions as an important cellular redox modulator through its pro-oxidant and antioxidant actions. The fully oxidised form of CoQ10, or ubiquinone, is able to accept electrons, primari‐ ly from NADH, to become fully reduced (ubiquinol - CoQ10-H2). The reduced form of CoQ10 is able to give up electrons, thereby scavenging free radicals. The intermediate of ubiqui‐

The major antioxidant role of CoQ10 is in preventing lipid peroxidation directly, and by in‐ teractions with α-tocopherol [138]. Ubiquinol is able to donate a hydrogen atom and thus quench peroxyl radicals, preventing lipid peroxidation chain reactions. CoQ10 and α-toco‐ pherol co-operate as antioxidants through the actions of CoQ10-H2 restoring α-tocopheroxyl back to α-tocopherol [109, 139]. However, the reactivity of α-tocopherol with peroxy radi‐ cals far exceeds that of ubiquinol with peroxyl radicals, suggesting that, *in vivo*, ubiquinols do not act as antioxidants but regenerate the antioxidant properties of α-tocopherols [140]. This is in accordance with *in vivo* studies investigating the effects of CoQ10 supplementation which have primarily found a limited antioxidant capacity. CoQ10, acting as a pro-oxidant in all biological membranes including the Golgi, endosome/lysosome systems, as well as mito‐ chondria, has led to much criticism regarding the claimed antioxidant power of CoQ10 sup‐ plementation in humans [141]. Nonetheless, many *in vitro* studies demonstrate antioxidant properties of CoQ10 in single cells, and benefits of CoQ10 supplementation in humans are at‐ tributed to its ability to maintain efficient mitochondrial energy metabolism and thus pre‐ vent mitochondrial dysfunction, rather than act as a direct cellular antioxidant. CoQ10 supplementation *in vivo* reduced protein oxidation in skeletal muscle of rats but had no ef‐ fect on mitochondrial H2O2 production in the kidney [142]. However, Ishikawa and collea‐

CoQ10 supplemented diet, and increased renal function compared with rats on a control diet [143]. Recently, CoQ10 supplementation improved left ventricular diastolic dysfunction and remodelling and reduced oxidative stress in a mouse model of type 2 diabetes [144]. CoQ10 supplementation in CVD patients also receiving statin therapy is becoming increasingly popular due to the CoQ10-inhibitory actions of statins. CoQ10 levels decrease with age, but there are no studies measuring endogenous CoQ10 levels in CKD or CVD patients and this could prove vital in the identification of population where CoQ10 therapy may have benefi‐

Inflammation and fibrosis are causes, as well as consequences, of oxidative stress [145, 146]. Direct targeting of inflammatory and fibrotic pathways with more specific modifying com‐ pounds presents a way to indirectly decrease oxidative stress in chronic pathologies. Long

**4.4. Omega-3 poly-unsaturated fatty acids – Inflammation and oxidative stress**

none and ubiquinol is the univalently-reduced ubisemiquinone (CoQ10-H+

•- and, subsequently, H2O2.

244 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

gues (2011) demonstrated a decrease in kidney O2

pro-oxidant to form O2

cial outcomes.

•- and

) which acts as a

•- levels in hemi-nephrectomised rats on a

Allopurinol treatment aims is to inhibit xanthine oxidase to decrease serum uric acid and its associated toxic effects. Allopurinol and its metabolite, oxypurinol, act as competitive sub‐ strates for xanthine oxidase. They enhance urinary urate excretion and block uric acid reab‐ sorption by urate transporters in the proximal tubule, thereby facilitating enhanced uric acid excretion [161-163]. Allopurinol treatment of diabetic mice attenuated hyperuricaemia, albu‐ minuria, and tubulointerstitial injury [164]. Allopurinol may also have antioxidant activities in addition to its enzyme inhibitory activities, by scavenging OH• as well as chlorine dioxide and HOCl [165, 166]. Although later *in vivo* studies revealed that rat serum obtained after oral administration of allopurinol did not contain allopurinol levels sufficient to scavenge free radicals [167], inhibition of xanthine oxidase-dependent production of NO• and ROS provides allopurinol an indirect mechanism for decreasing oxidative stress in hyperuricae‐ mic CKD patients. Interventional studies of use of allopurinol in renal disease have shown improved uric acid levels, GFR, cardiovascular outcomes and delayed CKD progression. A prospective randomised trial of 113 patients with GFR <60 ml/min/1.73m3 given allopurinol 100mg/d for 2 years found an increase in GFR of 1.31 ml/min/1.73m3 compared to the con‐ trols which decreased, and a 71% decreased risk of CVD [168]. Interestingly, Kanbay and colleagues (2007) found that allopurinol at 300mg/d over 3 months improved GFR, uric acid and C-reactive protein levels but made no change to proteinuria [169]. Allopurinol given to ESKD patients on hemodialysis reduced the risk of CVD by decreasing serum low density lipoproteins, triglycerides and uric acid [170]. Large, long-term interventional studies inves‐ tigating kidney function in the CKD, and CVD, populations are needed to fully determine if allopurinol is cardio- and reno-protective via anti-oxidant mechanisms.

crease in homocysteine levels [182]. Interestingly, oxidative stress is a major characteristic of

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As well as the physiological role of L-carnitine in mitochondrial fatty acid synthesis, oxidant reducing capabilities have also been demonstrated and may underlie the health benefits of L-carnitine therapy in CKD and CVD. L-carnitine infusions significantly improved blood urea nitrogen (BUN) and creatinine levels in a 5/6 nephrectomy model of CKD with a con‐ comitant increase in plasma SOD, Gpx, CAT and GSH, and decrease in the oxidative stress marker malondialdehyde [184]. Ye et al., (2010) suggest that L-carnitine attenuates renal tub‐ ular cell oxidant injury and subsequent apoptosis by reducing mitochondrial-derived ROS [97]. They suggest that this anti-apoptotic mechanism may also explain the demonstrated re‐ duction in morbidity from cardiomyopathies in L-carnitine supplemented hemodialysis pa‐

The premise of L-arginine supplementation is to maintain NO signalling and thereby main‐ tain vascular endothelial cell function. L-arginine is a physiological precursor to NO and its availability and transport determine the rate of NO biosynthesis. CKD patients most often present with atherosclerosis, thromboembolitic complications, and endothelial dysfunction, primarily due to altered endothelium-dependant relaxation factors [185]. It is believed that the impaired NO synthesis, common in CKD individuals, contributes significantly to their disease pathogenesis [186]. L-arginine synthesis occurs in the liver and kidney, with the kid‐ ney functioning to maintain homeostatic plasma levels since the liver processes NO from the diet [187]. The addition of L-aspartic acid or L-glutamic acid with L-citrulline and arginiro‐ succinic acid synthase as the rate determining enzyme forms L-arginine [188]. The proximal tubular cells account for the majority of kidney NO synthesis [189, 190], thus kidney damage and atrophy, a primary corollary of CKD, results in decreased synthesis of L-arginine. The majority of research demonstrates decreased levels of NO production in CKD and CVD pa‐ tients [191-193]. However, some research suggests NO activity increases [194, 195]. These disparate findings highlight the need to measure L-arginine levels in patients before com‐ mencing L-arginine supplementation. Rajapaske *et al*. (2012) demonstrated impaired kidney L-arginine transport and a contributing factor to hypertension in rats, irrespective of an un‐ derlying renal disease [196]. During a state of oxidative stress, L-arginine supplementation was shown to decrease MDA, myeloperoxidase and xanthine oxidase and increase gluta‐ thionine in both heart and kidney tissue from rats [197]. As such, L-arginine supplementa‐ tion represents an approach to restoring a dysregulation of NO signalling and subsequent

Compounds commonly used to alleviate oxidative stress exhibit different antioxidant ac‐ tions, and so there exists the potential for different antioxidants to work together to improve whole cell and organ function through a targeted polypharmaceutical approach to decrease oxidative stress. However, most clinical studies investigating the effects of combination anti‐

hemodialysis patients [183].

**4.8. L-Arginine - Maintaining endothelial function**

endothelial dysfunction in both chronic kidney and heart diseases.

**4.9. Combination antioxidants**

tients.

### **4.6. Bardoxolone methyl - Targeting the Nrf/Keap1/ARE pathway**

A different approach has been investigated by modulating pathways that respond to oxida‐ tive stress, rather than targeting ROS by directly increasing endogenous antioxidants. The Nrf2/keap1/ARE pathway presents an exciting target to enhance the oxidant detoxifying ca‐ pabilities of cells. Bardoxolone methyl [2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me)] is a potent activator of the Nrf2/keap1/ARE pathway and currently shows promise for halting the progressive decline of GFR in type 2 diabetic CKD patients [171, 172]. Bardoxolone methyl is a triterperoid derived from natural plant products that has un‐ dergone oleanolic acid-based modification [173]. Its mechanism of action is largely un‐ known, however, it induces an overall antioxidative protective effect with antiinflammatory and cytoprotective characteristics [174, 175]. Bardoxolone methyl administered to mice ameliorated ischemia-reperfusion induced acute kidney injury by Nrf2-dependant expression of HO-1 and PPARγ [176]. Its mechanism may also reside in regulating mitochondrial biogenesis given the involvement of PPARγ. A large international study evaluating the full scale of bardoxolone methyl's effects on CKD progression is in progress, the results of which could determine if bardoxolone methyl should become a standard treatment in renal disease patients. Concurrent benefits to CVD will undoubtedly also be measured.

### **4.7. L-Carnitine – Improving cardiovascular health in dialysis**

Carnitine is an essential cofactor required for the transformation of free fatty acids into acyl‐ carnitine and its subsequent transport into the mitochondria for β-oxidation [177]. This un‐ derlies its importance in the production of ATP for cellular energy. Acylcarnitine is also essential for the removal of toxic fat metabolism by-products. Carnitine is obtained primari‐ ly from food stuffs, however it can be synthesised endogenously from the amino acid L-ly‐ sine and methionine [177]. L-carnitine supplementation primarily benefits ESRD patients on hemodialysis and their associated cardiovascular complications, especially anemia. This is primarily due to the well-described decrease in serum free carnitine in maintenance hemo‐ dialysis patients compared to non-dialysis CKD and healthy patients [178]. L-carnitine sup‐ plementation offsets renal anemia, lipid abnormalities and cardiac dysfunction in hemodialysis patients [179]. Left ventricular hypertrophy regressed in hemodialysis patients receiving 10mg/kg of L-carnitine immediately following hemodialysis for a 12 month peri‐ od. [180]. Other measures of cardiac morbidity such as reduced left ventricular ejection frac‐ tion and increased left ventricular mass also significantly improved following low dose Lcarnitine supplementation [181]. Benefits to the peripheral vasculature have also been demonstrated by L-carnitine through a mechanism thought to involve an associated de‐ crease in homocysteine levels [182]. Interestingly, oxidative stress is a major characteristic of hemodialysis patients [183].

As well as the physiological role of L-carnitine in mitochondrial fatty acid synthesis, oxidant reducing capabilities have also been demonstrated and may underlie the health benefits of L-carnitine therapy in CKD and CVD. L-carnitine infusions significantly improved blood urea nitrogen (BUN) and creatinine levels in a 5/6 nephrectomy model of CKD with a con‐ comitant increase in plasma SOD, Gpx, CAT and GSH, and decrease in the oxidative stress marker malondialdehyde [184]. Ye et al., (2010) suggest that L-carnitine attenuates renal tub‐ ular cell oxidant injury and subsequent apoptosis by reducing mitochondrial-derived ROS [97]. They suggest that this anti-apoptotic mechanism may also explain the demonstrated re‐ duction in morbidity from cardiomyopathies in L-carnitine supplemented hemodialysis pa‐ tients.

### **4.8. L-Arginine - Maintaining endothelial function**

and C-reactive protein levels but made no change to proteinuria [169]. Allopurinol given to ESKD patients on hemodialysis reduced the risk of CVD by decreasing serum low density lipoproteins, triglycerides and uric acid [170]. Large, long-term interventional studies inves‐ tigating kidney function in the CKD, and CVD, populations are needed to fully determine if

A different approach has been investigated by modulating pathways that respond to oxida‐ tive stress, rather than targeting ROS by directly increasing endogenous antioxidants. The Nrf2/keap1/ARE pathway presents an exciting target to enhance the oxidant detoxifying ca‐ pabilities of cells. Bardoxolone methyl [2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me)] is a potent activator of the Nrf2/keap1/ARE pathway and currently shows promise for halting the progressive decline of GFR in type 2 diabetic CKD patients [171, 172]. Bardoxolone methyl is a triterperoid derived from natural plant products that has un‐ dergone oleanolic acid-based modification [173]. Its mechanism of action is largely un‐ known, however, it induces an overall antioxidative protective effect with antiinflammatory and cytoprotective characteristics [174, 175]. Bardoxolone methyl administered to mice ameliorated ischemia-reperfusion induced acute kidney injury by Nrf2-dependant expression of HO-1 and PPARγ [176]. Its mechanism may also reside in regulating mitochondrial biogenesis given the involvement of PPARγ. A large international study evaluating the full scale of bardoxolone methyl's effects on CKD progression is in progress, the results of which could determine if bardoxolone methyl should become a standard treatment in renal disease patients. Concurrent benefits to CVD will undoubtedly

Carnitine is an essential cofactor required for the transformation of free fatty acids into acyl‐ carnitine and its subsequent transport into the mitochondria for β-oxidation [177]. This un‐ derlies its importance in the production of ATP for cellular energy. Acylcarnitine is also essential for the removal of toxic fat metabolism by-products. Carnitine is obtained primari‐ ly from food stuffs, however it can be synthesised endogenously from the amino acid L-ly‐ sine and methionine [177]. L-carnitine supplementation primarily benefits ESRD patients on hemodialysis and their associated cardiovascular complications, especially anemia. This is primarily due to the well-described decrease in serum free carnitine in maintenance hemo‐ dialysis patients compared to non-dialysis CKD and healthy patients [178]. L-carnitine sup‐ plementation offsets renal anemia, lipid abnormalities and cardiac dysfunction in hemodialysis patients [179]. Left ventricular hypertrophy regressed in hemodialysis patients receiving 10mg/kg of L-carnitine immediately following hemodialysis for a 12 month peri‐ od. [180]. Other measures of cardiac morbidity such as reduced left ventricular ejection frac‐ tion and increased left ventricular mass also significantly improved following low dose Lcarnitine supplementation [181]. Benefits to the peripheral vasculature have also been demonstrated by L-carnitine through a mechanism thought to involve an associated de‐

allopurinol is cardio- and reno-protective via anti-oxidant mechanisms.

**4.6. Bardoxolone methyl - Targeting the Nrf/Keap1/ARE pathway**

246 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**4.7. L-Carnitine – Improving cardiovascular health in dialysis**

also be measured.

The premise of L-arginine supplementation is to maintain NO signalling and thereby main‐ tain vascular endothelial cell function. L-arginine is a physiological precursor to NO and its availability and transport determine the rate of NO biosynthesis. CKD patients most often present with atherosclerosis, thromboembolitic complications, and endothelial dysfunction, primarily due to altered endothelium-dependant relaxation factors [185]. It is believed that the impaired NO synthesis, common in CKD individuals, contributes significantly to their disease pathogenesis [186]. L-arginine synthesis occurs in the liver and kidney, with the kid‐ ney functioning to maintain homeostatic plasma levels since the liver processes NO from the diet [187]. The addition of L-aspartic acid or L-glutamic acid with L-citrulline and arginiro‐ succinic acid synthase as the rate determining enzyme forms L-arginine [188]. The proximal tubular cells account for the majority of kidney NO synthesis [189, 190], thus kidney damage and atrophy, a primary corollary of CKD, results in decreased synthesis of L-arginine. The majority of research demonstrates decreased levels of NO production in CKD and CVD pa‐ tients [191-193]. However, some research suggests NO activity increases [194, 195]. These disparate findings highlight the need to measure L-arginine levels in patients before com‐ mencing L-arginine supplementation. Rajapaske *et al*. (2012) demonstrated impaired kidney L-arginine transport and a contributing factor to hypertension in rats, irrespective of an un‐ derlying renal disease [196]. During a state of oxidative stress, L-arginine supplementation was shown to decrease MDA, myeloperoxidase and xanthine oxidase and increase gluta‐ thionine in both heart and kidney tissue from rats [197]. As such, L-arginine supplementa‐ tion represents an approach to restoring a dysregulation of NO signalling and subsequent endothelial dysfunction in both chronic kidney and heart diseases.

#### **4.9. Combination antioxidants**

Compounds commonly used to alleviate oxidative stress exhibit different antioxidant ac‐ tions, and so there exists the potential for different antioxidants to work together to improve whole cell and organ function through a targeted polypharmaceutical approach to decrease oxidative stress. However, most clinical studies investigating the effects of combination anti‐ oxidants have demonstrated confounding results. Mosca *et al*., (2002) demonstrated that dai‐ ly intake of NAC 100mg, L-carntine 100mg, selenomethionine 0.05mg, α-tocopherol 10mg, CoQ10 100mg and α-lipoic acid 100mg successfully increased plasma CAT, Gpx and total an‐ tioxidant capacity whilst decreasing lipid peroxides and ROS generation by lymphocyte mi‐ tochondria [198]. However, this trial only included healthy participants and cannot be extrapolated to the CKD and CVD populations.

**Author details**

Brisbane, Australia

32(1):70-8.

2007.

**References**

David M. Small and Glenda C. Gobe\*

\*Address all correspondence to: g.gobe@uq.edu.au

Centre for Kidney Disease Research, School of Medicine, The University of Queensland,

Oxidative Stress and Antioxidant Therapy in Chronic Kidney and Cardiovascular Disease

http://dx.doi.org/10.5772/51923

249

[1] Rosner MH, Ronco C, Okusa MD. The role of inflammation in the cardio-renal syn‐ drome: a focus on cytokines and inflammatory mediators. Semin Nephrol. 2012 Jan;

[2] Ronco C, McCullough P, Anker SD, Anand I, Aspromonte N, Bagshaw SM, et al. Cardio-renal syndromes: report from the consensus conference of the acute dialysis

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In a murine model of diabetic nephropathy, a major cause of CKD with associated CVD, the beneficial effects of NAC, L-ascorbic acid (vitamin C) and α-tocopherol were demonstrated [199]. Daily supplementation for 8 weeks decreased lipid peroxidation, BUN, serum creati‐ nine and blood glucose, mainly due to a reduction in the inflammatory response induced by hyperglycemia. In comparison, a prospective trial investigating oral supplementation of mixed tocopherols and α-lipoic acid in stage 3 and 4 CKD patients has revealed disappoint‐ ing results. Over 2 months, supplementation did not reduce biomarkers of oxidative stress (F2-isoprostanes and protein thiol concentration) or inflammation (CRP and IL-6). The short period of time (2 months) of the intervention may explain this result and longer trials need to be carried out. The inclusion of vitamin E in these interventions has polarized discussion on the outcomes, because of its negligible benefits when cardiovascular outcomes were measured [91, 92, 200] and also because of contraindications, discussed previously. Despite this, long-term treatment in with the antioxidants vitamin C, vitamin E, CoQ10 and selenium has been shown to reduce multiple cardiovascular risk factors [201]. Recently, multiple anti‐ oxidants in combination with L-arginine have shown promise in animal models of CKD and associated CVD. Korish (2010) has demonstrated in a 5/6 nephrectomy CKD model that Larginine improved the effects of L-carnitine, catechin and vitamins E and C on blood pres‐ sure, dyslipidemia, inflammation and kidney function [84].

### **5. Conclusion**

CKD is a progressive disease with increasing incidence, having very little success in current conventional therapies once CKD reaches stage 4. Stages 2 and 3 are best to target to slow or stop further development of the disease. There is an almost inseparable connection between CKD and CVD, with many patients with CKD dying of the cardiovascular complications be‐ fore renal failure reaches its fullest extent. Oxidative stress and inflammation are closely in‐ terrelated with development of CKD and CVD, and involve a spiralling cycle that leads to progressive patient deterioration. Given the complex nature of oxidative stress and its mo‐ lecular pathways, antioxidants may need to be given as a polypharmacotherapy to target each aberrant pathway, with the aim of reducing the burden of these chronic diseases. It is vital for the progression of antioxidant therapy research in CKD and CVD that measures of oxidative stress are compared with pathophysiological outcome in the diseases, especially in connection with antioxidant therapies that may be delivered with or without more conven‐ tional CKD therapies.

### **Author details**

oxidants have demonstrated confounding results. Mosca *et al*., (2002) demonstrated that dai‐ ly intake of NAC 100mg, L-carntine 100mg, selenomethionine 0.05mg, α-tocopherol 10mg, CoQ10 100mg and α-lipoic acid 100mg successfully increased plasma CAT, Gpx and total an‐ tioxidant capacity whilst decreasing lipid peroxides and ROS generation by lymphocyte mi‐ tochondria [198]. However, this trial only included healthy participants and cannot be

In a murine model of diabetic nephropathy, a major cause of CKD with associated CVD, the beneficial effects of NAC, L-ascorbic acid (vitamin C) and α-tocopherol were demonstrated [199]. Daily supplementation for 8 weeks decreased lipid peroxidation, BUN, serum creati‐ nine and blood glucose, mainly due to a reduction in the inflammatory response induced by hyperglycemia. In comparison, a prospective trial investigating oral supplementation of mixed tocopherols and α-lipoic acid in stage 3 and 4 CKD patients has revealed disappoint‐ ing results. Over 2 months, supplementation did not reduce biomarkers of oxidative stress (F2-isoprostanes and protein thiol concentration) or inflammation (CRP and IL-6). The short period of time (2 months) of the intervention may explain this result and longer trials need to be carried out. The inclusion of vitamin E in these interventions has polarized discussion on the outcomes, because of its negligible benefits when cardiovascular outcomes were measured [91, 92, 200] and also because of contraindications, discussed previously. Despite this, long-term treatment in with the antioxidants vitamin C, vitamin E, CoQ10 and selenium has been shown to reduce multiple cardiovascular risk factors [201]. Recently, multiple anti‐ oxidants in combination with L-arginine have shown promise in animal models of CKD and associated CVD. Korish (2010) has demonstrated in a 5/6 nephrectomy CKD model that Larginine improved the effects of L-carnitine, catechin and vitamins E and C on blood pres‐

CKD is a progressive disease with increasing incidence, having very little success in current conventional therapies once CKD reaches stage 4. Stages 2 and 3 are best to target to slow or stop further development of the disease. There is an almost inseparable connection between CKD and CVD, with many patients with CKD dying of the cardiovascular complications be‐ fore renal failure reaches its fullest extent. Oxidative stress and inflammation are closely in‐ terrelated with development of CKD and CVD, and involve a spiralling cycle that leads to progressive patient deterioration. Given the complex nature of oxidative stress and its mo‐ lecular pathways, antioxidants may need to be given as a polypharmacotherapy to target each aberrant pathway, with the aim of reducing the burden of these chronic diseases. It is vital for the progression of antioxidant therapy research in CKD and CVD that measures of oxidative stress are compared with pathophysiological outcome in the diseases, especially in connection with antioxidant therapies that may be delivered with or without more conven‐

extrapolated to the CKD and CVD populations.

248 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

sure, dyslipidemia, inflammation and kidney function [84].

**5. Conclusion**

tional CKD therapies.

David M. Small and Glenda C. Gobe\*

\*Address all correspondence to: g.gobe@uq.edu.au

Centre for Kidney Disease Research, School of Medicine, The University of Queensland, Brisbane, Australia

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[186] Brunini TM, da SCD, Siqueira MA, Moss MB, Santos SF, Mendes-Ribeiro AC. Ure‐ mia, atherothrombosis and malnutrition: the role of L-arginine-nitric oxide pathway.

[187] Reyes AA, Karl IE, Klahr S. Role of arginine in health and in renal disease. Am J

[188] Morris SM, Jr. Enzymes of arginine metabolism. J Nutr. 2004 Oct;134(10 Suppl):

[189] Stuehr DJ. Enzymes of the L-arginine to nitric oxide pathway. J Nutr. 2004 Oct;134(10

[190] Morel F, Hus-Citharel A, Levillain O. Biochemical heterogeneity of arginine metabo‐

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[192] Brunini TM, Roberts NB, Yaqoob MM, Ellory JC, Mann GE, Mendes RAC. Activation of L-arginine transport in undialysed chronic renal failure and continuous ambulato‐ ry peritoneal dialysis patients. Clin Exp Pharmacol Physiol. 2006 Jan-Feb;33(1-2):

[193] Mendes RAC, Hanssen H, Kiessling K, Roberts NB, Mann GE, Ellory JC. Transport of L-arginine and the nitric oxide inhibitor NG-monomethyl-L-arginine in human er‐

ythrocytes in chronic renal failure. Clin Sci (Lond). 1997 Jul;93(1):57-64.

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[181] Matsumoto Y, Sato M, Ohashi H, Araki H, Tadokoro M, Osumi Y, et al. Effects of Lcarnitine supplementation on cardiac morbidity in hemodialyzed patients. Am J Nephrol. 2000 May-Jun;20(3):201-7.

[169] Kanbay M, Ozkara A, Selcoki Y, Isik B, Turgut F, Bavbek N, et al. Effect of treatment of hyperuricemia with allopurinol on blood pressure, creatinine clearence, and pro‐ teinuria in patients with normal renal functions. Int Urol Nephrol. 2007;39(4):1227-33.

[170] Shelmadine B, Bowden RG, Wilson RL, Beavers D, Hartman J. The effects of lower‐ ing uric acid levels using allopurinol on markers of metabolic syndrome in end-stage renal disease patients: a pilot study. Anadolu Kardiyol Derg. 2009 Oct;9(5):385-9.

[171] Pergola PE, Krauth M, Huff JW, Ferguson DA, Ruiz S, Meyer CJ, et al. Effect of bar‐ doxolone methyl on kidney function in patients with T2D and Stage 3b-4 CKD. Am J

[172] Pergola PE, Raskin P, Toto RD, Meyer CJ, Huff JW, Grossman EB, et al. Bardoxolone methyl and kidney function in CKD with type 2 diabetes. N Engl J Med. 2011 Jul

[173] Honda T, Yoshizawa H, Sundararajan C, David E, Lajoie MJ, Favaloro FG, Jr., et al. Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents. J Med Chem. 2011 Mar 24;54(6):

[174] Eskiocak U, Kim SB, Roig AI, Kitten E, Batten K, Cornelius C, et al. CDDO-Me pro‐ tects against space radiation-induced transformation of human colon epithelial cells.

[175] Nagaraj S, Youn JI, Weber H, Iclozan C, Lu L, Cotter MJ, et al. Anti-inflammatory tri‐ terpenoid blocks immune suppressive function of MDSCs and improves immune re‐

[176] Wu QQ, Wang Y, Senitko M, Meyer C, Wigley WC, Ferguson DA, et al. Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPARgamma, and HO-1. Am J Physiol Renal Physiol. 2011 May;

[177] Kelly GS. L-Carnitine: therapeutic applications of a conditionally-essential amino

[178] Fouque D, Holt S, Guebre-Egziabher F, Nakamura K, Vianey-Saban C, Hadj-Aissa A, et al. Relationship between serum carnitine, acylcarnitines, and renal function in pa‐

[179] Eknoyan G, Latos DL, Lindberg J. Practice recommendations for the use of L-carni‐ tine in dialysis-related carnitine disorder. National Kidney Foundation Carnitine

[180] Sakurabayashi T, Miyazaki S, Yuasa Y, Sakai S, Suzuki M, Takahashi S, et al. L-carni‐ tine supplementation decreases the left ventricular mass in patients undergoing he‐

tients with chronic renal disease. J Ren Nutr. 2006 Apr;16(2):125-31.

Consensus Conference. Am J Kidney Dis. 2003 Apr;41(4):868-76.

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Nephrol. 2011;33(5):469-76.

262 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Radiat Res. 2010 Jul;174(1):27-36.

acid. Altern Med Rev. 1998 Oct;3(5):345-60.

modialysis. Circ J. 2008 Jun;72(6):926-31.

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[194] Noris M, Benigni A, Boccardo P, Aiello S, Gaspari F, Todeschini M, et al. Enhanced nitric oxide synthesis in uremia: implications for platelet dysfunction and dialysis hypotension. Kidney Int. 1993 Aug;44(2):445-50.

**Chapter 11**

**Role of Oxidative Stress in**

Nalini Rajamannan

**1. Introduction**

aortic valve disease.

http://dx.doi.org/10.5772/52275

**Calcific Aortic Valve Disease:**

Additional information is available at the end of the chapter

**From Bench to Bedside - The Role of a Stem Cell Niche**

Calcific Aortic Stenosis is the most common cause of aortic valve disease in developed coun‐ tries. This condition increases in prevalence with the advancing age of the U.S. population, afflicting 2-3 % by age 65 [1]. Aortic valve replacement is the number one indication for sur‐ gical valve replacement in the United States and in Europe. The natural history of severe symptomatic aortic stenosis is associated with 50% mortality within 5 years [2]. Bicuspid aortic valve disease is the most common congenital heart abnormality and it is the most common phenotype of calcific aortic stenosis. The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly, having a prevalence of 0.9 to 1.37% in the general pop‐ ulation [3]. Understanding the cellular mechanisms of tricuspid versus bicuspid aortic valve lesions will provide further understanding the mechanisms of this disease. Currently, there are three fundamental mechanisms defined in the development of aortic valve disease: 1) oxidative stress via traditional cardiovascular risk factors [4-8,6, 7, 9-12], 2) cellular prolifera‐ tion [13] and 3) osteoblastogenesis in the end stage disease process [14, 15]. Previously, the Wnt/Lrp5 signaling pathway has been identified as a signaling mechanism for cardiovascu‐ lar calcification [5, 16, 17]. The corollaries necessary to define a tissue stem cell niche: 1) physical architecture of the endothelial cells signaling to the adjacent subendothelial cells: the valve interstitial cell along the valve fibrosa. 2) defining the oxidative-mechanical stress gradient necessary to activate Wnt3a/Lrp5 in this tissue stem niche to induce disease. Re‐ cently, the mechanisms of oxidative stress have been identified in the development of calcif‐ ic aortic valve disease. This chapter will outline the factors important in the role of calcific

> © 2013 Rajamannan; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.


## **Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche**

Nalini Rajamannan

[194] Noris M, Benigni A, Boccardo P, Aiello S, Gaspari F, Todeschini M, et al. Enhanced nitric oxide synthesis in uremia: implications for platelet dysfunction and dialysis

[195] Aiello S, Noris M, Remuzzi G. Nitric oxide synthesis and L-arginine in uremia. Miner

[196] Rajapakse NW, Kuruppu S, Hanchapola I, Venardos K, Mattson DL, Smith AI, et al. Evidence that renal arginine transport is impaired in spontaneously hypertensive

[197] Huang CC, Tsai SC, Lin WT. Potential ergogenic effects of L-arginine against oxida‐ tive and inflammatory stress induced by acute exercise in aging rats. Exp Gerontol.

[198] Mosca L, Marcellini S, Perluigi M, Mastroiacovo P, Moretti S, Famularo G, et al. Mod‐ ulation of apoptosis and improved redox metabolism with the use of a new antioxi‐

[199] Park NY, Park SK, Lim Y. Long-term dietary antioxidant cocktail supplementation effectively reduces renal inflammation in diabetic mice. Br J Nutr. 2011 Nov;106(10):

[200] Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Lancet. 1999 Aug 7;354(9177):

[201] Shargorodsky M, Debby O, Matas Z, Zimlichman R. Effect of long-term treatment with antioxidants (vitamin C, vitamin E, coenzyme Q10 and selenium) on arterial compliance, humoral factors and inflammatory markers in patients with multiple

hypotension. Kidney Int. 1993 Aug;44(2):445-50.

rats. Am J Physiol Renal Physiol. 2012 Jun;302(12):F1554-62.

dant formula. Biochem Pharmacol. 2002 Apr 1;63(7):1305-14.

cardiovascular risk factors. Nutr Metab (Lond). 2010;7:55.

Electrolyte Metab. 1997;23(3-6):151-6.

264 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

2008 Jun;43(6):571-7.

1514-21.

447-55.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52275

### **1. Introduction**

Calcific Aortic Stenosis is the most common cause of aortic valve disease in developed coun‐ tries. This condition increases in prevalence with the advancing age of the U.S. population, afflicting 2-3 % by age 65 [1]. Aortic valve replacement is the number one indication for sur‐ gical valve replacement in the United States and in Europe. The natural history of severe symptomatic aortic stenosis is associated with 50% mortality within 5 years [2]. Bicuspid aortic valve disease is the most common congenital heart abnormality and it is the most common phenotype of calcific aortic stenosis. The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly, having a prevalence of 0.9 to 1.37% in the general pop‐ ulation [3]. Understanding the cellular mechanisms of tricuspid versus bicuspid aortic valve lesions will provide further understanding the mechanisms of this disease. Currently, there are three fundamental mechanisms defined in the development of aortic valve disease: 1) oxidative stress via traditional cardiovascular risk factors [4-8,6, 7, 9-12], 2) cellular prolifera‐ tion [13] and 3) osteoblastogenesis in the end stage disease process [14, 15]. Previously, the Wnt/Lrp5 signaling pathway has been identified as a signaling mechanism for cardiovascu‐ lar calcification [5, 16, 17]. The corollaries necessary to define a tissue stem cell niche: 1) physical architecture of the endothelial cells signaling to the adjacent subendothelial cells: the valve interstitial cell along the valve fibrosa. 2) defining the oxidative-mechanical stress gradient necessary to activate Wnt3a/Lrp5 in this tissue stem niche to induce disease. Re‐ cently, the mechanisms of oxidative stress have been identified in the development of calcif‐ ic aortic valve disease. This chapter will outline the factors important in the role of calcific aortic valve disease.

© 2013 Rajamannan; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. The role of lipids in vascular and valvular disease**

The role of lipids in vascular atherosclerosis has been defined in the literature for years. Atherosclerosis is a complex multifactorial process which produces a lesion composed of lipids [18, 19], macrophages [20], and proliferating smooth muscle cells [21] apoptosis [22] and extracellular bone matrix production [23] in the vascular wall [24, 25]. The acti‐ vation of these cellular processes is regulated by a number of pathways. Integrins pro‐ vide an important role in the regulation of cellular adhesion in atherosclerosis [26]. Another critical regulator of vascular endothelial biology is nitric oxide (NO) [27, 28]. Cholesterol-rich LDL also has a critical role in the onset and further progression of the atherosclerotic lesion via an inactivation of endothelial nitric oxide synthase (eNOS) [22, 29-31] contributing to an abnormal oxidation state within the vessel. In this inflammato‐ ry environment, growth factors and cytokines are secreted to induce vascular smooth cell proliferation and recruitment of macrophage cells [32-37] which are important in the de‐ velopment of the atherosclerotic plaque lesion.

Until recently the etiology of valvular heart disease has been thought to be a degenerative process related to the passive accumulation of calcium binding to the surface of the valve leaflet. Recent descriptive studies have demonstrated the critical features of aortic valve cal‐ cification, including osteoblast expression, cell proliferation and atherosclerosis [6, 14, 15, 45] and mitral valve degeneration, glycosaminglycan accumulation, proteoglycan expression, and abnormal collagen expression [46-49]. These studies define the biochemical and histo‐ logical characterization of these valve lesions. We and others, have also shown that specific bone cell phenotypes are present in calcifying valve specimens in human specimens [16, 50]. These data provide the evidence that the aortic valve calcification follows the spectrum of

Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche

http://dx.doi.org/10.5772/52275

267

**4. The role of Lrp5/beta-catenin activation in cardiovascular calcification**

Bone and cartilage are major tissues in the vertebrate skeletal system, which is primarily composed of three cell types: osteoblasts, chrondrocytes, and osteoclasts. In the developing embryo, osteoblast and chrondrocytes, both differentiate from common mesenchymal pro‐ genitors in situ, where as osteoclasts are of hematopoietic origin and brought in later by in‐ vading blood vessels. Osteoblast differentiation and maturation lead to bone formation controlled by two distinct mechanisms: intramembranous and endochondral ossification,

To date only two osteoblast-specific transcripts have been identified: 1) Cbfa1 and 2) osteo‐ calcin (OC). The transcription factor Cbfa1 [51] has all the attributes of a 'master gene' differ‐ entiation factor for the osteoblast lineage and bone matrix gene expression. During embryonic development, Cbfa1 expression precedes osteoblast differentiation and is restrict‐ ed to mesenchymal cells destined to become osteoblast. In addition to its critical role in os‐ teoblast commitment and differentiation, Cbfa1 appears to control osteoblast activity, i.e., the rate of bone formation by differentiated osteoblasts [51]. We have shown previously that cholesterol upregulates Cbfa1 gene expression in the aortic valve and atorvastatin decreases the gene expression [6] in an animal model. We have also demonstrated that Sox9 and Cbfa1 are expressed in human degenerative valves removed at the time of surgical valve replace‐ ment [16]. The regulatory mechanism of osteoblast differentiation from osteoblast progeni‐ tor cells into terminally differentiated cells is via a well orchestrated and well studied pathway which involves initial cellular proliferation events and then synthesis of bone ma‐ trix proteins, which requires the actions of specific paracrine/hormonal factors and the acti‐

Genes which code for the bone extracellular matrix proteins in osteoblast cells include alka‐ line phosphatase (AP), osteopontin (OP), osteocalcin (OC), and bone sialoprotein (BSP). This data supports a potential regulatory mechanism that these matrix proteins play a critical role in the development of biomineralization. To date, many of these markers have been shown to be critical in the extracellular mineralization and bone formation that develops in

**and osteoblast bone formation: Connection with the bone axis**

bone formation in calcifying tissues.

both starting from mesenchymal condensations.

vation of the canonical Wntpathway [52].

Recently, similar risk factors for calcific aortic valve disease have recently been described in‐ cluding male gender, hypertension, elevated levels of LDL, and smoking [38, 39] which mimic those that promote the development of vascular atherosclerosis. Surgical pathological studies have demonstrated the presence of LDL and atherosclerosis in calcified valves, dem‐ onstrating similarities between the genesis of valvular and vascular disease and suggesting a common cellular mechanism [40, 41]. Patients who have the diagnosis of familial hyper‐ cholesterolemia develop aggressive peripheral vascular disease, coronary artery disease, as well as aortic valve lesions which calcify with age [10, 42]. Rajamannan et al, have shown that the development of atherosclerosis occurs in the aortic valve in a patient with Familial Hypercholesterolemia with the Low density lipoprotein receptor mutation [10]. The athero‐ sclerosis develops along the aortic surface of the aortic valve and in the lumen of the left cir‐ cumflex artery [10]. This provides the first index case of atherosclerotic aortic valve disease in this patient population. Studies have confirmed in experimental hypercholesterolemia that both atherosclerosis and osteoblast markers are present in the aortic valves [4, 6, 13]. This background provides the foundation for studying valve calcification in an experimental atherosclerotic *in vivo* model.

### **3. Aortic valve calcification**

The presence of calcification in the aortic valve is responsible for valve stenosis. Severe aort‐ ic stenosis can result in symptomatic chest pain, as well as syncope and congestive heart fail‐ ure in patients with severe aortic valve stenosis. For years, aortic valve stenosis was thought to be a degenerative process. However, the pathologic lesion of calcified aortic valves dem‐ onstrate indicate the presence of complex calcification in these tissues. Furthermore, there are a growing number of descriptive studies delineating the presence of bone formation in the aortic valve [15, 43, 44].

Until recently the etiology of valvular heart disease has been thought to be a degenerative process related to the passive accumulation of calcium binding to the surface of the valve leaflet. Recent descriptive studies have demonstrated the critical features of aortic valve cal‐ cification, including osteoblast expression, cell proliferation and atherosclerosis [6, 14, 15, 45] and mitral valve degeneration, glycosaminglycan accumulation, proteoglycan expression, and abnormal collagen expression [46-49]. These studies define the biochemical and histo‐ logical characterization of these valve lesions. We and others, have also shown that specific bone cell phenotypes are present in calcifying valve specimens in human specimens [16, 50]. These data provide the evidence that the aortic valve calcification follows the spectrum of bone formation in calcifying tissues.

**2. The role of lipids in vascular and valvular disease**

266 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

velopment of the atherosclerotic plaque lesion.

atherosclerotic *in vivo* model.

the aortic valve [15, 43, 44].

**3. Aortic valve calcification**

The role of lipids in vascular atherosclerosis has been defined in the literature for years. Atherosclerosis is a complex multifactorial process which produces a lesion composed of lipids [18, 19], macrophages [20], and proliferating smooth muscle cells [21] apoptosis [22] and extracellular bone matrix production [23] in the vascular wall [24, 25]. The acti‐ vation of these cellular processes is regulated by a number of pathways. Integrins pro‐ vide an important role in the regulation of cellular adhesion in atherosclerosis [26]. Another critical regulator of vascular endothelial biology is nitric oxide (NO) [27, 28]. Cholesterol-rich LDL also has a critical role in the onset and further progression of the atherosclerotic lesion via an inactivation of endothelial nitric oxide synthase (eNOS) [22, 29-31] contributing to an abnormal oxidation state within the vessel. In this inflammato‐ ry environment, growth factors and cytokines are secreted to induce vascular smooth cell proliferation and recruitment of macrophage cells [32-37] which are important in the de‐

Recently, similar risk factors for calcific aortic valve disease have recently been described in‐ cluding male gender, hypertension, elevated levels of LDL, and smoking [38, 39] which mimic those that promote the development of vascular atherosclerosis. Surgical pathological studies have demonstrated the presence of LDL and atherosclerosis in calcified valves, dem‐ onstrating similarities between the genesis of valvular and vascular disease and suggesting a common cellular mechanism [40, 41]. Patients who have the diagnosis of familial hyper‐ cholesterolemia develop aggressive peripheral vascular disease, coronary artery disease, as well as aortic valve lesions which calcify with age [10, 42]. Rajamannan et al, have shown that the development of atherosclerosis occurs in the aortic valve in a patient with Familial Hypercholesterolemia with the Low density lipoprotein receptor mutation [10]. The athero‐ sclerosis develops along the aortic surface of the aortic valve and in the lumen of the left cir‐ cumflex artery [10]. This provides the first index case of atherosclerotic aortic valve disease in this patient population. Studies have confirmed in experimental hypercholesterolemia that both atherosclerosis and osteoblast markers are present in the aortic valves [4, 6, 13]. This background provides the foundation for studying valve calcification in an experimental

The presence of calcification in the aortic valve is responsible for valve stenosis. Severe aort‐ ic stenosis can result in symptomatic chest pain, as well as syncope and congestive heart fail‐ ure in patients with severe aortic valve stenosis. For years, aortic valve stenosis was thought to be a degenerative process. However, the pathologic lesion of calcified aortic valves dem‐ onstrate indicate the presence of complex calcification in these tissues. Furthermore, there are a growing number of descriptive studies delineating the presence of bone formation in

### **4. The role of Lrp5/beta-catenin activation in cardiovascular calcification and osteoblast bone formation: Connection with the bone axis**

Bone and cartilage are major tissues in the vertebrate skeletal system, which is primarily composed of three cell types: osteoblasts, chrondrocytes, and osteoclasts. In the developing embryo, osteoblast and chrondrocytes, both differentiate from common mesenchymal pro‐ genitors in situ, where as osteoclasts are of hematopoietic origin and brought in later by in‐ vading blood vessels. Osteoblast differentiation and maturation lead to bone formation controlled by two distinct mechanisms: intramembranous and endochondral ossification, both starting from mesenchymal condensations.

To date only two osteoblast-specific transcripts have been identified: 1) Cbfa1 and 2) osteo‐ calcin (OC). The transcription factor Cbfa1 [51] has all the attributes of a 'master gene' differ‐ entiation factor for the osteoblast lineage and bone matrix gene expression. During embryonic development, Cbfa1 expression precedes osteoblast differentiation and is restrict‐ ed to mesenchymal cells destined to become osteoblast. In addition to its critical role in os‐ teoblast commitment and differentiation, Cbfa1 appears to control osteoblast activity, i.e., the rate of bone formation by differentiated osteoblasts [51]. We have shown previously that cholesterol upregulates Cbfa1 gene expression in the aortic valve and atorvastatin decreases the gene expression [6] in an animal model. We have also demonstrated that Sox9 and Cbfa1 are expressed in human degenerative valves removed at the time of surgical valve replace‐ ment [16]. The regulatory mechanism of osteoblast differentiation from osteoblast progeni‐ tor cells into terminally differentiated cells is via a well orchestrated and well studied pathway which involves initial cellular proliferation events and then synthesis of bone ma‐ trix proteins, which requires the actions of specific paracrine/hormonal factors and the acti‐ vation of the canonical Wntpathway [52].

Genes which code for the bone extracellular matrix proteins in osteoblast cells include alka‐ line phosphatase (AP), osteopontin (OP), osteocalcin (OC), and bone sialoprotein (BSP). This data supports a potential regulatory mechanism that these matrix proteins play a critical role in the development of biomineralization. To date, many of these markers have been shown to be critical in the extracellular mineralization and bone formation that develops in normal osteoblast differentiation (Fig.5). Dr. Spelsberg and Dr. Rajamannan have extensive experience in osteoblast cell biology and will contribute to the translational studies in the aortic valve involving the differentiation and mineralization [53, 54].

experimental hypercholesterolemia to determine how lipids may be regulating Lrp5 in the aortic valve. This background outlines the potential for lipids in the regulation of aortic

Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche

**5. Echocardiography and Computerized Tomography (CT) evaluation of**

Currently the non-invasive "gold standard" for the diagnosis of aortic valve stenosis is 2- Dimensional doppler echocardiography. It is the test of choice to quantify the severity of valve stenosis and pressure differential across the aortic valve. There are a increasing number of studies which have demonstrated the utility of calculating the volume of calci‐ um and the rate of progression of the disease process in the aortic valve [76-80]. Confir‐ mation of hemodynamic valve stenosis by echo will provide the degree of valve stenosis

**6. Development of future medical therapies for calcific aortic stenosis**

The natural history studies of valvular aortic stenosis as defined by clinical and histopatho‐ logic parameters have provided landmark developments towards the understanding of this disease. HMG CoA reductase inhibitors may provide an innovative therapeutic approach by employing both lipid lowering and possibly non-lipid lowering effects to forestall critical stenosis in the aortic valve. Our laboratory has shown that atorvastatin has a number of ef‐ fects in the aortic valve including: 1) inhibition of foam cell accumulation [6], 2) inhibition of Cbfa1 activation [6], 3) eNOS enzymatic activation [11] and 4) attenuation of Lrp5 receptor activation [81]. Statins have potent LDL lowering effects via inhibition of the rate-limiting step in cholesterol synthesis. There are a number of experimental models which demon‐ strate the potential for treating the vasculature with statins to inhibit matrix formation [24, 25], cellular proliferation [6] and vascular aneurysm formation [82]. Although valve replace‐ ment is the current treatment of choice for severe critical aortic stenosis, future insights into the mechanisms of calcification and its progression may indicate a role for lipid lowering

There are a growing number of retrospective studies demonstrating that statins may have benefits in slowing the progression of aortic stenosis [83-85]. A recent clinical trial by Cowell et al, demonstrated that high dose atorvastatin did not slow the progression of aortic steno‐ sis in patients [86]. However, the timing of the initiation of the statin therapy was at a later stage of aortic valve disease. A clinical trial in Portugal called RAAVE- Rosuvastatin Affect‐ ing Aortic Valve Endothelium demonstrated prospectively that statins slow progression in CAVD in an open label study. In the RAAVE study we found a change in aortic valve area (AVA) in the control group was -0.10±0.09 cm2 per year versus -0.05 ±0.12 cm2 per yearin the

. MicroCT will assess the degree of calcification within the

http://dx.doi.org/10.5772/52275

269

valve mineralization via the canonical Wnt pathway.

**the development of calcification and stenosis**

therapy in modifying the rate of progression of stenosis.

using ultrasound techniques4

mineralizing tissues.

A link between lipids and osteoporosis have been studied extensively [55-60]. These groups have shown in *in vitro* and *in vivo* studies that lipids decrease bone formation and increase vascular calcification. Hurska's group from the University of Washington have studied this important hypothesis in the LDLR-/- mice with renal disease [55]. This studied correlated the important understanding of chronic kidney disease with decreased bone formation rates and increase in vascular calcification. This study demonstrates that accelerated vascular cal‐ cification found in patients with end stage renal disease may be related to multifactorial mechanisms including traditional atherosclerotic risk factors and elevated serum phosphate levels. Giachelli has also studied extensively the hypothesis of a sodium phosphate abnor‐ mality in the vascular smooth muscle cell [61]. Her group has also shown that osteopontin expression by vascular smooth muscle cells may have an inhibitory effect in the develop‐ ment of calcification [62] which further defines the complexity of the matrix synthesis phase of bone formation. Demer's laboratory has also studied extensively the correlation of lipids with vascular calcification and osteoporosis via inhibition of Cbfa1 in osteoblast cells [60, 63]. This paradoxical finding between the calcifying vascular aorta and osteoporosis is an important link in the hypercholesterolemia hypothesis. The development of cardiovascular calcification is a multifactorial process which includes a number of mechanisms. Studies in the different laboratories provide important evidence towards the development of therapies depending on the patient population i.e. end stage renal disease versus treatment of the tra‐ ditional risk factors for vascular disease.

Our lab (43) and Towler's laboratory (44) have shown that the Lrp5/Wnt/beta-catenin path‐ way plays an important role in the development of vascular and valvular calcification. Stud‐ ies have shown that different mutations in Lrp5, an LDL receptor related protein; develop a high bone mass phenotype and an osteoporotic phenotype (45, 46). In the presence of the palmitoylation of Wnt an active beta-catenin accumulates in the cytoplasm, presumably in a signaling capacity, and eventually translocates to the nucleus via binding to nucleoporins [64], where it can interacts with LEF-1/TCFs in an inactive transcription complex [65, 66], The Wnt/Lrp5/frizzled complex turns on downstream components such as Dishevelled (Dvl/Dsh) which leads to repression of the glycogen synthase kinase-3 (GSK3) [67]. Inhibi‐ tion of GSK3 allows beta-catenin to accumulate in the nucleus, interacting with members of the LEF/TCF class of architectural HMG box of transcription factors including Cbfa1 in‐ volved in cell differentiation and osteoblast activation [68, 69, 70-72] and Sox 9, a HMG box transcription factor, is required for chondrocyte cell fate determination and marks early chondrocytic differentiation of mesenchymalprogenitors [73].

To determine a potential signaling pathway for the development of aortic valve disease there are numerous pathways which may be implicated in this disease process [50, 74, 75]. Recent evidence suggests that the Wnt pathway regulates the expression of bone mineral markers in cells responsive to the Wnt pathway. Furthermore the Wnt pathway has been shown to be activated by lipids. Therefore we chose to assess this pathway in our model of experimental hypercholesterolemia to determine how lipids may be regulating Lrp5 in the aortic valve. This background outlines the potential for lipids in the regulation of aortic valve mineralization via the canonical Wnt pathway.

normal osteoblast differentiation (Fig.5). Dr. Spelsberg and Dr. Rajamannan have extensive experience in osteoblast cell biology and will contribute to the translational studies in the

A link between lipids and osteoporosis have been studied extensively [55-60]. These groups have shown in *in vitro* and *in vivo* studies that lipids decrease bone formation and increase vascular calcification. Hurska's group from the University of Washington have studied this important hypothesis in the LDLR-/- mice with renal disease [55]. This studied correlated the important understanding of chronic kidney disease with decreased bone formation rates and increase in vascular calcification. This study demonstrates that accelerated vascular cal‐ cification found in patients with end stage renal disease may be related to multifactorial mechanisms including traditional atherosclerotic risk factors and elevated serum phosphate levels. Giachelli has also studied extensively the hypothesis of a sodium phosphate abnor‐ mality in the vascular smooth muscle cell [61]. Her group has also shown that osteopontin expression by vascular smooth muscle cells may have an inhibitory effect in the develop‐ ment of calcification [62] which further defines the complexity of the matrix synthesis phase of bone formation. Demer's laboratory has also studied extensively the correlation of lipids with vascular calcification and osteoporosis via inhibition of Cbfa1 in osteoblast cells [60, 63]. This paradoxical finding between the calcifying vascular aorta and osteoporosis is an important link in the hypercholesterolemia hypothesis. The development of cardiovascular calcification is a multifactorial process which includes a number of mechanisms. Studies in the different laboratories provide important evidence towards the development of therapies depending on the patient population i.e. end stage renal disease versus treatment of the tra‐

Our lab (43) and Towler's laboratory (44) have shown that the Lrp5/Wnt/beta-catenin path‐ way plays an important role in the development of vascular and valvular calcification. Stud‐ ies have shown that different mutations in Lrp5, an LDL receptor related protein; develop a high bone mass phenotype and an osteoporotic phenotype (45, 46). In the presence of the palmitoylation of Wnt an active beta-catenin accumulates in the cytoplasm, presumably in a signaling capacity, and eventually translocates to the nucleus via binding to nucleoporins [64], where it can interacts with LEF-1/TCFs in an inactive transcription complex [65, 66], The Wnt/Lrp5/frizzled complex turns on downstream components such as Dishevelled (Dvl/Dsh) which leads to repression of the glycogen synthase kinase-3 (GSK3) [67]. Inhibi‐ tion of GSK3 allows beta-catenin to accumulate in the nucleus, interacting with members of the LEF/TCF class of architectural HMG box of transcription factors including Cbfa1 in‐ volved in cell differentiation and osteoblast activation [68, 69, 70-72] and Sox 9, a HMG box transcription factor, is required for chondrocyte cell fate determination and marks early

To determine a potential signaling pathway for the development of aortic valve disease there are numerous pathways which may be implicated in this disease process [50, 74, 75]. Recent evidence suggests that the Wnt pathway regulates the expression of bone mineral markers in cells responsive to the Wnt pathway. Furthermore the Wnt pathway has been shown to be activated by lipids. Therefore we chose to assess this pathway in our model of

aortic valve involving the differentiation and mineralization [53, 54].

268 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ditional risk factors for vascular disease.

chondrocytic differentiation of mesenchymalprogenitors [73].

### **5. Echocardiography and Computerized Tomography (CT) evaluation of the development of calcification and stenosis**

Currently the non-invasive "gold standard" for the diagnosis of aortic valve stenosis is 2- Dimensional doppler echocardiography. It is the test of choice to quantify the severity of valve stenosis and pressure differential across the aortic valve. There are a increasing number of studies which have demonstrated the utility of calculating the volume of calci‐ um and the rate of progression of the disease process in the aortic valve [76-80]. Confir‐ mation of hemodynamic valve stenosis by echo will provide the degree of valve stenosis using ultrasound techniques4 . MicroCT will assess the degree of calcification within the mineralizing tissues.

### **6. Development of future medical therapies for calcific aortic stenosis**

The natural history studies of valvular aortic stenosis as defined by clinical and histopatho‐ logic parameters have provided landmark developments towards the understanding of this disease. HMG CoA reductase inhibitors may provide an innovative therapeutic approach by employing both lipid lowering and possibly non-lipid lowering effects to forestall critical stenosis in the aortic valve. Our laboratory has shown that atorvastatin has a number of ef‐ fects in the aortic valve including: 1) inhibition of foam cell accumulation [6], 2) inhibition of Cbfa1 activation [6], 3) eNOS enzymatic activation [11] and 4) attenuation of Lrp5 receptor activation [81]. Statins have potent LDL lowering effects via inhibition of the rate-limiting step in cholesterol synthesis. There are a number of experimental models which demon‐ strate the potential for treating the vasculature with statins to inhibit matrix formation [24, 25], cellular proliferation [6] and vascular aneurysm formation [82]. Although valve replace‐ ment is the current treatment of choice for severe critical aortic stenosis, future insights into the mechanisms of calcification and its progression may indicate a role for lipid lowering therapy in modifying the rate of progression of stenosis.

There are a growing number of retrospective studies demonstrating that statins may have benefits in slowing the progression of aortic stenosis [83-85]. A recent clinical trial by Cowell et al, demonstrated that high dose atorvastatin did not slow the progression of aortic steno‐ sis in patients [86]. However, the timing of the initiation of the statin therapy was at a later stage of aortic valve disease. A clinical trial in Portugal called RAAVE- Rosuvastatin Affect‐ ing Aortic Valve Endothelium demonstrated prospectively that statins slow progression in CAVD in an open label study. In the RAAVE study we found a change in aortic valve area (AVA) in the control group was -0.10±0.09 cm2 per year versus -0.05 ±0.12 cm2 per yearin the Rosuvastatin group (p=0.041). In addition there was an increase in peak aortic valve velocity was +0.24±0.30 m/sec/yr in the control group as compared to the increase in +0.04±0.38 m/sec/yr in the Rosuvastatin group (p=0.007), indicating that in this prospective hypothesis driven study we found by echocardiography a slowing of progression in the aortic valve disease. SALTIRE initiated atorvastatin in patients who had more advanced aortic stenosis as defined by the mean aortic valve area 1.03 cm2 as compared to the average aortic valve area in RAAVE of 1.23 cm2 as the baseline aortic valve area prior to treatment with Rosuvas‐ tatin [87]. The investigators of RAAVE hypothesize that the beneficial effect of the statin was secondary to the early initiation of treatment. Furthermore, the SALTIRE investigators re‐ cently acknowledged the potential of medical therapies may be found if the treatment of this disease is initiated earlier in the disease process [86]. The studies planned in this application should lead to an important understanding of the molecular and cellular mechanisms of aortic valve disease. Furthermore, the experimental approach will also correlate the devel‐ opment of valve calcium by MicroCT and hemodynamic progression by echocardiography in this important disease process.

ing and increased body mass index [38]. The elucidation of these risk factors have provided the experimental basis for hypercholesterolemia as a method to induce aortic valve disease [4-8. Furthermore, studies have shown that the eNOS-/- mouse is a novel mouse model which develops anatomic bicuspid aortic valves (BAV) [91]. To understand if eNOS-/- mice with the BAV phenotype, develops accelerated stenosis earlier than tricuspid aortic valves via the Lrp5 pathway activation, eNOS-/- mice were given a cholesterol diet versus cholesterol and atorvastatin. The Visual Sonics mouse echocardiography machine was used to screen for the BAV phenotype. Echocardiography hemodynamics was also performed to determine the timing of stenosis in bicuspid vs. tricuspid aortic valves eNOS-/- mice on different diets.

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**Figure 1.** Histology of the aortic valves from human bicuspid calcified valves compared to normal aortic valves re‐ moved at the time of surgical valve replacement; **Panel A.** Bicuspid Aortic Valve Removed from patient at the time of surgical valve replacement. **Panel B1.**Notch1 Immunohistochemistry of a Normal Aortic Valve. **Panel B2.**Notch1 Im‐ munohistochemistry of a Bicuspid Aortic Valve. **Panel C.** Notch1 protein expression was decreased in the BAV com‐ pared to controls by immunhistochemistry and Western Blot expression. **Panel D.** Notch1 RNA expression was

decrease in the BAV as compared to Control aortic Valve.

The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly, having a prevalence of 0.9 to 1.37 % in the general population [3]. The natural history of the BAV is progressive stenosis that typically occurs at a faster rate than tricuspid aortic valves requir‐ ing earlier surgical intervention in the BAV patients [2, 3]. With the decline of acute rheu‐ matic fever, calcific aortic stenosis has become the most common indication for surgical valve replacement. Despite the high prevalence of aortic stenosis, few studies have investi‐ gated the mechanisms responsible for aortic valve disease. The cellular mechanism for the development of this disease is not well known. Previously, we and others have demonstrat‐ ed that aortic valve calcification is associated with an osteoblast bone-like phenotype [14, 15]. This bone phenotype is regulated by the canonical Wnt pathway in experimental cardio‐ vascular calcification [5, 17]. We have alsoshown that the canonical Wnt/Lrp5 pathway is upregulated in diseased human valves from patients with valvular heart disease [16]. These studies implicate that inhibition of the canonical Wnt pathway provides a therapeutic ap‐ proach for the treatment of degenerative valvular heart diseases. A recent study [88], discov‐ ered that a loss of function mutation in Notch1 was associated with accelerated aortic valve calcification and a number of congenital heart abnormalities. Normal Notch1 receptor func‐ tions to inhibit osteoblastogenesis [89, 90]. Evaluation of Notch1 gene and protein expres‐ sion in human bicuspid calcified valves compared to normal aortic valves removed at the time of surgical valve replacement is shown in Figure 1, Panel A. Notch1 protein expression was decreased in the BAV compared to controls by immunhistochemistry and Western Blot expression Figure 1, Panel B1 and B2, and C. RNA expression by RTPCR indicates a spliced Notch1 receptor in the diseased valves as compared to controls as shown in Figure 1, Panel D. This Notch1 splicing may be the regulatory switch important for the activation of the Wnt pathway and downstream calcification in these diseased valves [5, 17, 90].

Risk factors for the development of calcific aortic valve disease(CAVD) have been elucidated in a number of epidemiologic databases [38]. The risk factors for CAVD are similar to those of vascular atherosclerosis which include: elevated LDL, hypertension, male gender, smok‐ ing and increased body mass index [38]. The elucidation of these risk factors have provided the experimental basis for hypercholesterolemia as a method to induce aortic valve disease [4-8. Furthermore, studies have shown that the eNOS-/- mouse is a novel mouse model which develops anatomic bicuspid aortic valves (BAV) [91]. To understand if eNOS-/- mice with the BAV phenotype, develops accelerated stenosis earlier than tricuspid aortic valves via the Lrp5 pathway activation, eNOS-/- mice were given a cholesterol diet versus cholesterol and atorvastatin. The Visual Sonics mouse echocardiography machine was used to screen for the BAV phenotype. Echocardiography hemodynamics was also performed to determine the timing of stenosis in bicuspid vs. tricuspid aortic valves eNOS-/- mice on different diets.

Rosuvastatin group (p=0.041). In addition there was an increase in peak aortic valve velocity was +0.24±0.30 m/sec/yr in the control group as compared to the increase in +0.04±0.38 m/sec/yr in the Rosuvastatin group (p=0.007), indicating that in this prospective hypothesis driven study we found by echocardiography a slowing of progression in the aortic valve disease. SALTIRE initiated atorvastatin in patients who had more advanced aortic stenosis

area in RAAVE of 1.23 cm2 as the baseline aortic valve area prior to treatment with Rosuvas‐ tatin [87]. The investigators of RAAVE hypothesize that the beneficial effect of the statin was secondary to the early initiation of treatment. Furthermore, the SALTIRE investigators re‐ cently acknowledged the potential of medical therapies may be found if the treatment of this disease is initiated earlier in the disease process [86]. The studies planned in this application should lead to an important understanding of the molecular and cellular mechanisms of aortic valve disease. Furthermore, the experimental approach will also correlate the devel‐ opment of valve calcium by MicroCT and hemodynamic progression by echocardiography

The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly, having a prevalence of 0.9 to 1.37 % in the general population [3]. The natural history of the BAV is progressive stenosis that typically occurs at a faster rate than tricuspid aortic valves requir‐ ing earlier surgical intervention in the BAV patients [2, 3]. With the decline of acute rheu‐ matic fever, calcific aortic stenosis has become the most common indication for surgical valve replacement. Despite the high prevalence of aortic stenosis, few studies have investi‐ gated the mechanisms responsible for aortic valve disease. The cellular mechanism for the development of this disease is not well known. Previously, we and others have demonstrat‐ ed that aortic valve calcification is associated with an osteoblast bone-like phenotype [14, 15]. This bone phenotype is regulated by the canonical Wnt pathway in experimental cardio‐ vascular calcification [5, 17]. We have alsoshown that the canonical Wnt/Lrp5 pathway is upregulated in diseased human valves from patients with valvular heart disease [16]. These studies implicate that inhibition of the canonical Wnt pathway provides a therapeutic ap‐ proach for the treatment of degenerative valvular heart diseases. A recent study [88], discov‐ ered that a loss of function mutation in Notch1 was associated with accelerated aortic valve calcification and a number of congenital heart abnormalities. Normal Notch1 receptor func‐ tions to inhibit osteoblastogenesis [89, 90]. Evaluation of Notch1 gene and protein expres‐ sion in human bicuspid calcified valves compared to normal aortic valves removed at the time of surgical valve replacement is shown in Figure 1, Panel A. Notch1 protein expression was decreased in the BAV compared to controls by immunhistochemistry and Western Blot expression Figure 1, Panel B1 and B2, and C. RNA expression by RTPCR indicates a spliced Notch1 receptor in the diseased valves as compared to controls as shown in Figure 1, Panel D. This Notch1 splicing may be the regulatory switch important for the activation of the

Wnt pathway and downstream calcification in these diseased valves [5, 17, 90].

Risk factors for the development of calcific aortic valve disease(CAVD) have been elucidated in a number of epidemiologic databases [38]. The risk factors for CAVD are similar to those of vascular atherosclerosis which include: elevated LDL, hypertension, male gender, smok‐

as compared to the average aortic valve

as defined by the mean aortic valve area 1.03 cm2

270 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

in this important disease process.

**Figure 1.** Histology of the aortic valves from human bicuspid calcified valves compared to normal aortic valves re‐ moved at the time of surgical valve replacement; **Panel A.** Bicuspid Aortic Valve Removed from patient at the time of surgical valve replacement. **Panel B1.**Notch1 Immunohistochemistry of a Normal Aortic Valve. **Panel B2.**Notch1 Im‐ munohistochemistry of a Bicuspid Aortic Valve. **Panel C.** Notch1 protein expression was decreased in the BAV com‐ pared to controls by immunhistochemistry and Western Blot expression. **Panel D.** Notch1 RNA expression was decrease in the BAV as compared to Control aortic Valve.

Figure 2 demonstrates the characterization of the eNOS phenotype as defined by histology, RTPCR and echocardiography. In Figure 2, Panel A is the histology for BAV, Figure 2, Panel B is the semi-quantitative RTPCR from the BAV eNOS-/- mice, and echocardiographic data for the bicuspid vs. tricuspid aortic valves Figure 2, Panel C. We measured Notch1, Wnt3a and downstream markers of the canonical Wnt pathway by protein and RNA expression. Notch1 protein was diminished and the RNA expression demonstrates a similar spliced var‐ iant with lipid treatments which was not present with the control and atorvastatin treat‐ ment. Cholesterol diets increased the members of the canonical Wnt pathway and Atorvastatin diminished these markers significantly (p<0.05).

aortic surface is responsible for the secretion of a growth factors [92]. These cells interact with the subendothelial cells that are resident below the endothelial layer of cells. These

Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche

To test the hypothesis that BAV disease develops secondary to a stem cell niche process, the physical cell-cell communication needed to be established [95]. In the aortic valve the com‐ munication for the stem cell niche would be between the aortic valve endothelial cell and the adjacent myofibroblast cell located below the aortic lining endothelial cell. Conditioned media was produced from untreated aortic valve endothelial cells for the microenvironment that activates signaling in the myofibroblast cell. A mitogenic protein (Wnt3a) was isolated from the conditioned media and then tested directly on the responding mesenchymal cell, the cardiac valve myofibroblast [93, 96,95]. This transfer of isolated protein to the adjacent cell was necessary to determine if the cell would proliferate directly in the presence of this protein. This system is appealing because the responding mesenchymal cell is isolated from the anatomic region adjacent and immediately below that of the endothelial cells producing the growth factor activity along the fibrosa surface. Very little is known regarding the char‐ acterization of the endothelial cell conditioned media. These experiments test the corollary that the physical architecture described above is necessary for disease development in the

Figure 3 demonstrates the isolation and characterization of the Wnt3a from the conditioned media microenvironment. Figure 3, Panel A, is light microscopy of aortic valve endothelial cells isolated from the aortic surface of the aortic valve. The results of the mitogen assays for fractions eluting from a DEAE- Sephadex column are shown in Figure 3, Panel B. It can be seen that the mitogenic activity appeared as a single peak eluting at approximately 0.25 M NaCl. The material eluting from DEAE- Sephadex was then applied to Sephadex G-100; the results of mitogen assays on fractions eluting from such a gel filtration column are shown in Figure 3, Panel C. It can be seen that under these native, non-denaturing conditions the bulk of the mitogenic activity eluted as a peak corresponding to standard proteins of 30- 40,000 molecular weight. A SDS denaturing protein gel was run on each sample from the eluted proteins and the bulk of activated protein correlated with the protein peak at 46kd as shown in Figure 3, Panel D. The protein size and charge determination is similar to that previously

characterized as Wnt3a [97]. This material lost all activity when heated to 100o

treatment with trypsin destroyed all activity, implicating a protein structure.

mechanism is important for the development of a gradient in this niche.

nutes; disulfide bond reduction with dithiothreitol also abolished all mitogenic activity; and

The second corollary for identifying a stem cell niche is to define the gradient responsible for the proliferation to differentiation process. The main postulate for this corollary stems from the risk factor hypothesis for the development of aortic valve disease. If traditional atherosclerotic risk factors are necessary for the initiation of disease, then these risk factors are responsible for the gradient necessary for the differentiation of myofibroblast cells to be‐ come an osteoblast calcifying phenotype [5, 17, 62, 75, 94, 95, 98, 99]. If traditional risk fac‐ tors are responsible for the development of valvular heart disease, then an oxidative stress

C for 5 mi‐

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273

cells have been characterized as myofibroblast cells [75, 93, 94].

aortic valve.

**Figure 2.** Characterization of the Bicuspid Aortic valve from the eNOS-/- bicuspid aortic valves. **Panel A**. Immunohisto‐ chemistry stain for Lrp5, Notch1, Proliferating Cell Nuclear Antigen, Osteopontin and Osteocalcin from the eNOS-/ aortic valves on the control, Left column, control diet; middle column, cholesterol diet; right column, cholesterol diet plus atorvastatin. In each panel, the aortic valve leaflet is in the center. (All frames 20X magnification) **Panel B**. RTPCR for Wnt3a, Lrp5, Notch1, cyclin1, Osteopontin and Cbfa1 from the eNOS-/- aortic valves on the control, A1, Cholesterol A2, and the Cholesterol + Atorvastatin diets A3. **Panel C.** Echocardiographic results of the tricuspid versus bicuspid eNOS-/- null mice.

BAV is a complex model to study the mechanisms of calcification. The importance of cellcell communication within a stem cell niche is necessary for the development of valvular heart disease. The two corollaries necessary for an adult stem cell niche is to first define the physical architecture of the stem-cell niche and second is to define the gradient of prolifera‐ tion to differentiation within the stem-cell niche. The endothelial lining cell located along the

aortic surface is responsible for the secretion of a growth factors [92]. These cells interact with the subendothelial cells that are resident below the endothelial layer of cells. These cells have been characterized as myofibroblast cells [75, 93, 94].

Figure 2 demonstrates the characterization of the eNOS phenotype as defined by histology, RTPCR and echocardiography. In Figure 2, Panel A is the histology for BAV, Figure 2, Panel B is the semi-quantitative RTPCR from the BAV eNOS-/- mice, and echocardiographic data for the bicuspid vs. tricuspid aortic valves Figure 2, Panel C. We measured Notch1, Wnt3a and downstream markers of the canonical Wnt pathway by protein and RNA expression. Notch1 protein was diminished and the RNA expression demonstrates a similar spliced var‐ iant with lipid treatments which was not present with the control and atorvastatin treat‐ ment. Cholesterol diets increased the members of the canonical Wnt pathway and

**Figure 2.** Characterization of the Bicuspid Aortic valve from the eNOS-/- bicuspid aortic valves. **Panel A**. Immunohisto‐ chemistry stain for Lrp5, Notch1, Proliferating Cell Nuclear Antigen, Osteopontin and Osteocalcin from the eNOS-/ aortic valves on the control, Left column, control diet; middle column, cholesterol diet; right column, cholesterol diet plus atorvastatin. In each panel, the aortic valve leaflet is in the center. (All frames 20X magnification) **Panel B**. RTPCR for Wnt3a, Lrp5, Notch1, cyclin1, Osteopontin and Cbfa1 from the eNOS-/- aortic valves on the control, A1, Cholesterol A2, and the Cholesterol + Atorvastatin diets A3. **Panel C.** Echocardiographic results of the tricuspid versus bicuspid

BAV is a complex model to study the mechanisms of calcification. The importance of cellcell communication within a stem cell niche is necessary for the development of valvular heart disease. The two corollaries necessary for an adult stem cell niche is to first define the physical architecture of the stem-cell niche and second is to define the gradient of prolifera‐ tion to differentiation within the stem-cell niche. The endothelial lining cell located along the

Atorvastatin diminished these markers significantly (p<0.05).

272 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

eNOS-/- null mice.

To test the hypothesis that BAV disease develops secondary to a stem cell niche process, the physical cell-cell communication needed to be established [95]. In the aortic valve the com‐ munication for the stem cell niche would be between the aortic valve endothelial cell and the adjacent myofibroblast cell located below the aortic lining endothelial cell. Conditioned media was produced from untreated aortic valve endothelial cells for the microenvironment that activates signaling in the myofibroblast cell. A mitogenic protein (Wnt3a) was isolated from the conditioned media and then tested directly on the responding mesenchymal cell, the cardiac valve myofibroblast [93, 96,95]. This transfer of isolated protein to the adjacent cell was necessary to determine if the cell would proliferate directly in the presence of this protein. This system is appealing because the responding mesenchymal cell is isolated from the anatomic region adjacent and immediately below that of the endothelial cells producing the growth factor activity along the fibrosa surface. Very little is known regarding the char‐ acterization of the endothelial cell conditioned media. These experiments test the corollary that the physical architecture described above is necessary for disease development in the aortic valve.

Figure 3 demonstrates the isolation and characterization of the Wnt3a from the conditioned media microenvironment. Figure 3, Panel A, is light microscopy of aortic valve endothelial cells isolated from the aortic surface of the aortic valve. The results of the mitogen assays for fractions eluting from a DEAE- Sephadex column are shown in Figure 3, Panel B. It can be seen that the mitogenic activity appeared as a single peak eluting at approximately 0.25 M NaCl. The material eluting from DEAE- Sephadex was then applied to Sephadex G-100; the results of mitogen assays on fractions eluting from such a gel filtration column are shown in Figure 3, Panel C. It can be seen that under these native, non-denaturing conditions the bulk of the mitogenic activity eluted as a peak corresponding to standard proteins of 30- 40,000 molecular weight. A SDS denaturing protein gel was run on each sample from the eluted proteins and the bulk of activated protein correlated with the protein peak at 46kd as shown in Figure 3, Panel D. The protein size and charge determination is similar to that previously characterized as Wnt3a [97]. This material lost all activity when heated to 100o C for 5 mi‐ nutes; disulfide bond reduction with dithiothreitol also abolished all mitogenic activity; and treatment with trypsin destroyed all activity, implicating a protein structure.

The second corollary for identifying a stem cell niche is to define the gradient responsible for the proliferation to differentiation process. The main postulate for this corollary stems from the risk factor hypothesis for the development of aortic valve disease. If traditional atherosclerotic risk factors are necessary for the initiation of disease, then these risk factors are responsible for the gradient necessary for the differentiation of myofibroblast cells to be‐ come an osteoblast calcifying phenotype [5, 17, 62, 75, 94, 95, 98, 99]. If traditional risk fac‐ tors are responsible for the development of valvular heart disease, then an oxidative stress mechanism is important for the development of a gradient in this niche.

sults for tissue nitrites measured in the endothelial cells providing indirect evidence for the enzyme activity. There was an increase is nitrites with lipid treatments and attenuation with Atorvastatin. This increase in nitrite levels correlates with a decrease in the functional activi‐

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**Figure 4. Evidence for eNOS regulation and Wnt3a Secretion from Aortic Valve Endothelial Cells.** \*p<0.001 for control compared to cholesterol, \*\*p<0.001 for cholesterol compared to cholesterol + Atorvastatin. **Panel A1**. eNOS enzymatic activity in the aortic valve endothelial cells (AEC) in the presence of LDL with and without atorvastatin. **Pan‐ el A2.** Cell Nitrite activity in the aortic valve endothelial cells (AEC) in the presence of LDL with and without atorvasta‐ tin. **Panel A3.** Thymidine incorporation in LDL treated media, compared to AdeNOS treated myofibroblast cells, versus control LacZ virus. **Panel B1**. Caveolin-1 and eNOS protein expression isolated from the lipid with and without atorvas‐ tatin treated cells as shown by Western Blot. **Panel B2**. Electron microscopy immunogold labeling for eNOS and Cav‐ eolin-1 localize in the aortic valve endothelial cells in caveolae. **Panel C.** Wnt3a Immunoprecipitate from Conditioned

Media treated with LDL with and without Atorvastatin.

ty of the eNOS enzyme in the aortic valve endothelium.

**Figure 3.** Protein Isolation and Characterization of Aortic Valve Endothelial Cell Conditioned Media; **Panel A**. Light Microscopy for Aortic Valve Endothelial Cells. **Panel B**. Cell Proliferation for fractions eluting from a DEAE- Sephadex column. (p<0.001) **Panel C**. Fractions from DEAE- Sephadex to characterize weight with Sephadex G-100. (p<0.001) **Panel D.** Southern Blot for Protein Expression of Fractions isolated form the DEAE-Sephadex column.

Nitric oxide is important in terms of the mechanism in adult disease processes and also in the developmental abnormalities such as the bicuspid aortic valve phenotype in the eNOS null mouse. To answer this question of the role of oxidative stress and nitric oxide in the aortic valve, I performed *in vitro* experiments to determine eNOS enzymatic and protein reg‐ ulation in the presence of lipids and attenuation with Atorvastatin. We have previously published that eNOS is regulated in the aortic valve in an experimental hypercholesterole‐ mia model of valvulardisease [11]. Figure 4, demonstrates the eNOS regulation in the endo‐ thelial cells in the presence of lipids with and without Atorvastatin. A number of standard assays were performed to measure eNOS functional activity. Figure 4, Panel A1, tests for eNOS enzymatic activity in the aortic valve endothelial cells (AEC) in the presence of LDL with and without Atorvastatin. ENOS enzymatic activity was decreased in the presence of lipids and Atorvastatin improved functional enzyme activity. Figure 4, Panel A2, shows re‐ sults for tissue nitrites measured in the endothelial cells providing indirect evidence for the enzyme activity. There was an increase is nitrites with lipid treatments and attenuation with Atorvastatin. This increase in nitrite levels correlates with a decrease in the functional activi‐ ty of the eNOS enzyme in the aortic valve endothelium.

**Figure 3.** Protein Isolation and Characterization of Aortic Valve Endothelial Cell Conditioned Media; **Panel A**. Light Microscopy for Aortic Valve Endothelial Cells. **Panel B**. Cell Proliferation for fractions eluting from a DEAE- Sephadex column. (p<0.001) **Panel C**. Fractions from DEAE- Sephadex to characterize weight with Sephadex G-100. (p<0.001)

Nitric oxide is important in terms of the mechanism in adult disease processes and also in the developmental abnormalities such as the bicuspid aortic valve phenotype in the eNOS null mouse. To answer this question of the role of oxidative stress and nitric oxide in the aortic valve, I performed *in vitro* experiments to determine eNOS enzymatic and protein reg‐ ulation in the presence of lipids and attenuation with Atorvastatin. We have previously published that eNOS is regulated in the aortic valve in an experimental hypercholesterole‐ mia model of valvulardisease [11]. Figure 4, demonstrates the eNOS regulation in the endo‐ thelial cells in the presence of lipids with and without Atorvastatin. A number of standard assays were performed to measure eNOS functional activity. Figure 4, Panel A1, tests for eNOS enzymatic activity in the aortic valve endothelial cells (AEC) in the presence of LDL with and without Atorvastatin. ENOS enzymatic activity was decreased in the presence of lipids and Atorvastatin improved functional enzyme activity. Figure 4, Panel A2, shows re‐

**Panel D.** Southern Blot for Protein Expression of Fractions isolated form the DEAE-Sephadex column.

274 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 4. Evidence for eNOS regulation and Wnt3a Secretion from Aortic Valve Endothelial Cells.** \*p<0.001 for control compared to cholesterol, \*\*p<0.001 for cholesterol compared to cholesterol + Atorvastatin. **Panel A1**. eNOS enzymatic activity in the aortic valve endothelial cells (AEC) in the presence of LDL with and without atorvastatin. **Pan‐ el A2.** Cell Nitrite activity in the aortic valve endothelial cells (AEC) in the presence of LDL with and without atorvasta‐ tin. **Panel A3.** Thymidine incorporation in LDL treated media, compared to AdeNOS treated myofibroblast cells, versus control LacZ virus. **Panel B1**. Caveolin-1 and eNOS protein expression isolated from the lipid with and without atorvas‐ tatin treated cells as shown by Western Blot. **Panel B2**. Electron microscopy immunogold labeling for eNOS and Cav‐ eolin-1 localize in the aortic valve endothelial cells in caveolae. **Panel C.** Wnt3a Immunoprecipitate from Conditioned Media treated with LDL with and without Atorvastatin.

The proof of principle experiment to test the importance of eNOS enzymatic activity is an overexpression experiment to determine if eNOS is able to inhibit cell proliferation, an early cellular event in the development of aortic stenosis [13]. Experiments were performed to overexpress eNOS to determine if eNOS overexpression in the aortic valve endothelial cells would regulate cell proliferation. The in vitro myofibroblast cells were directly transduced with an eNOS adenoviral gene construct. Thymidine incorporation was measured to test if overexpressing eNOS can inhibit cellular proliferation. Figure 4, Panel A3, eNOS overex‐ pression inhibits the cell proliferation in the oxidized LDL treated cells induced as com‐ pared to the LacZ control treated cells.

A key regulator of eNOS function is caveolin-1 which is expressed in aortic valve endothe‐ lial cells [9. Caveolin-1 upregulation in the presence of lipids inactivates eNOS enzymatic function and further promotes oxidative stress [100, 101]. Experiments were performed to localize the expression of Caveolin1 and eNOS in the aortic valve endothelial cell caveolae. A well defined mechanism to inactivate eNOS enzymatic activity is functional binding of eNOS with caveolin1 in the presence of lipids [29, 95, 102]. Figure 4, Panel B1, demonstrates that Caveolin-1 is upregulated in the lipid treated cells and decreases with atorvastatin treat‐ ment with no change in the eNOS protein expression as shown by Western Blot. Figure 4, Panel B2, demonstrates the ultrastructural evidence by immunogold labeling for eNOS and Caveolin-1 present in the aortic valve endothelial cells in caveolae, similar to previously re‐ ported data [9, 11]. This caveolin1 upregulation is indirect evidence in addition to the direct data of a decrease in the enzyme activity, that caveolin-1 may play a similar role in AEC found in the aortic valve similar to the vascular endothelium.

**Figure 5.** Schematic Modeling for Calcification in the Aortic Valve Stem Cell Niche.

stem cells into differentiation [105].

Adult tissues stem cells are a population of functionally undifferentiated cells, capable of (i) homing (ii) proliferation, (iii) producing differentiated progeny, (iv) self-renewing, (v) re‐ generation, and (vi) reversibility in the use of these options. Within this definition, stem cells are defined by virtue of their functional potential and not by a specific observable character‐ istic. This data is the first to implicate a cell-cell communication between the aortic valve en‐ dothelial cell and the myofibroblast cell to activate the canonical Wnt pathway. Lrp5 is important in normal valve development [103], in this stem cell niche, reactivation of latent Lrp5 expression [5, 16], regulates osteoblastogenesis in these mesenchymal cells. The two corollary requirements necessary for an adult stem cell niche is to first define the physical architecture of the stem-cell niche and second is to define the gradient of proliferation to dif‐ ferentiation within the stem-cell niche. The aortic valve endothelial cell communicates with the myofibroblast cell to activate the myofibroblast to differentiate to form an osteoblast-like phenotype [14]. This concept is similar to the endothelial/mesenchymal transition critical in normal valve development [104]. This data fulfills these main corollaries of the plausibility of a stem cell niche responsible for the development of valvular heart disease. Within a stem cell niche there is a delicate balance between proliferation and differentiation. Cells near the stem-cell zone are more proliferative, and Wnt likely plays a role in directing cell differentia‐ tion. Stem cell behavior is determined by the number of its stem cell neighbors, which in the valve is the endothelial cell. This assumption is aimed at simply describing the fact that cy‐ tokines, secreted by cells into the micro-environment are capable of activating quiescent

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Experiments were performed to determine if Wnt3a secretion changes in the microenviron‐ ment of the aortic valve endothelial cells with and without lipids. Figure 4, Panel C, demon‐ strates that Wnt3a protein concentration in the conditioned media in the presence of LDL with and without Atorvastatin. There is a significant increase in the protein with the lipids and attenuation of this protein secretion with the Atorvastatin treatments. This experiment tests the effects of lipids regulating the development of a "Wnt3a" gradient in the microen‐ vironment. If LDL increases Wnt3a secretion into the conditioned media or the microenvir‐ onment of the diseased aortic valve, this further contributes to the activation of the canonical Wnt pathway in the subendothelial space of the aortic valve.

The final experiment to test the importance of a stem cell niche to activate the cellular osteo‐ blast gene program in the subendothelial layer cells was to test for the gene expression of the Wnt/Lrp5 pathway in the myofibroblast cells. The stem cell niche is a unique model for the development of an oxidative stress communication within the aortic valve endothelium. As shown in Figure 5, oxidative stress contributes to the release of Wnt3a into the subendo‐ thelial space to activate Lrp5/Frizzeled receptor complex on the extracellular membrane of the myofibroblast. This trimeric complex then induces glycogen synthase kinase to be phos‐ phorylated. This phosphorylation event causes -catenin translocation to the nucleus. catenin acts as a coactivator of osteoblast specific transcription factor Cbfa1 to induce mesenchymalosteoblastogenesis in the aortic valve myofibroblast cell.

Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche http://dx.doi.org/10.5772/52275 277

**Figure 5.** Schematic Modeling for Calcification in the Aortic Valve Stem Cell Niche.

The proof of principle experiment to test the importance of eNOS enzymatic activity is an overexpression experiment to determine if eNOS is able to inhibit cell proliferation, an early cellular event in the development of aortic stenosis [13]. Experiments were performed to overexpress eNOS to determine if eNOS overexpression in the aortic valve endothelial cells would regulate cell proliferation. The in vitro myofibroblast cells were directly transduced with an eNOS adenoviral gene construct. Thymidine incorporation was measured to test if overexpressing eNOS can inhibit cellular proliferation. Figure 4, Panel A3, eNOS overex‐ pression inhibits the cell proliferation in the oxidized LDL treated cells induced as com‐

A key regulator of eNOS function is caveolin-1 which is expressed in aortic valve endothe‐ lial cells [9. Caveolin-1 upregulation in the presence of lipids inactivates eNOS enzymatic function and further promotes oxidative stress [100, 101]. Experiments were performed to localize the expression of Caveolin1 and eNOS in the aortic valve endothelial cell caveolae. A well defined mechanism to inactivate eNOS enzymatic activity is functional binding of eNOS with caveolin1 in the presence of lipids [29, 95, 102]. Figure 4, Panel B1, demonstrates that Caveolin-1 is upregulated in the lipid treated cells and decreases with atorvastatin treat‐ ment with no change in the eNOS protein expression as shown by Western Blot. Figure 4, Panel B2, demonstrates the ultrastructural evidence by immunogold labeling for eNOS and Caveolin-1 present in the aortic valve endothelial cells in caveolae, similar to previously re‐ ported data [9, 11]. This caveolin1 upregulation is indirect evidence in addition to the direct data of a decrease in the enzyme activity, that caveolin-1 may play a similar role in AEC

Experiments were performed to determine if Wnt3a secretion changes in the microenviron‐ ment of the aortic valve endothelial cells with and without lipids. Figure 4, Panel C, demon‐ strates that Wnt3a protein concentration in the conditioned media in the presence of LDL with and without Atorvastatin. There is a significant increase in the protein with the lipids and attenuation of this protein secretion with the Atorvastatin treatments. This experiment tests the effects of lipids regulating the development of a "Wnt3a" gradient in the microen‐ vironment. If LDL increases Wnt3a secretion into the conditioned media or the microenvir‐ onment of the diseased aortic valve, this further contributes to the activation of the canonical

The final experiment to test the importance of a stem cell niche to activate the cellular osteo‐ blast gene program in the subendothelial layer cells was to test for the gene expression of the Wnt/Lrp5 pathway in the myofibroblast cells. The stem cell niche is a unique model for the development of an oxidative stress communication within the aortic valve endothelium. As shown in Figure 5, oxidative stress contributes to the release of Wnt3a into the subendo‐ thelial space to activate Lrp5/Frizzeled receptor complex on the extracellular membrane of the myofibroblast. This trimeric complex then induces glycogen synthase kinase to be phos‐ phorylated. This phosphorylation event causes -catenin translocation to the nucleus. catenin acts as a coactivator of osteoblast specific transcription factor Cbfa1 to induce

pared to the LacZ control treated cells.

276 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

found in the aortic valve similar to the vascular endothelium.

Wnt pathway in the subendothelial space of the aortic valve.

mesenchymalosteoblastogenesis in the aortic valve myofibroblast cell.

Adult tissues stem cells are a population of functionally undifferentiated cells, capable of (i) homing (ii) proliferation, (iii) producing differentiated progeny, (iv) self-renewing, (v) re‐ generation, and (vi) reversibility in the use of these options. Within this definition, stem cells are defined by virtue of their functional potential and not by a specific observable character‐ istic. This data is the first to implicate a cell-cell communication between the aortic valve en‐ dothelial cell and the myofibroblast cell to activate the canonical Wnt pathway. Lrp5 is important in normal valve development [103], in this stem cell niche, reactivation of latent Lrp5 expression [5, 16], regulates osteoblastogenesis in these mesenchymal cells. The two corollary requirements necessary for an adult stem cell niche is to first define the physical architecture of the stem-cell niche and second is to define the gradient of proliferation to dif‐ ferentiation within the stem-cell niche. The aortic valve endothelial cell communicates with the myofibroblast cell to activate the myofibroblast to differentiate to form an osteoblast-like phenotype [14]. This concept is similar to the endothelial/mesenchymal transition critical in normal valve development [104]. This data fulfills these main corollaries of the plausibility of a stem cell niche responsible for the development of valvular heart disease. Within a stem cell niche there is a delicate balance between proliferation and differentiation. Cells near the stem-cell zone are more proliferative, and Wnt likely plays a role in directing cell differentia‐ tion. Stem cell behavior is determined by the number of its stem cell neighbors, which in the valve is the endothelial cell. This assumption is aimed at simply describing the fact that cy‐ tokines, secreted by cells into the micro-environment are capable of activating quiescent stem cells into differentiation [105].

The important inhibitor in this model is Notch1. Notch1 plays a roll in cellular differentia‐ tion decisions. In the osteoblast cell, it serves as an inhibitor of osteoblast differentiation [89, 90]. In the aortic valve, it serves to turn off bone formation via the cell-cell crosstalk between the endothelial and the myofibroblast cells. Normal Notch1 receptor functions to maintain normal valve cellular composition and homeostasis. In the presence of lipids, Notch1 is spliced and therefore activates osteoblastogenesis. In turn, the Wnt3a is secreted and binds to Lrp5 and Frizzled on the extracellular membrane to regulate the osteoblast gene program. This developmental disease process follows a parallel signaling pathway that has been ob‐ served in the normal embryonic valve development that has been well delineated by previ‐ ous investigators [104]. A similar cell-cell communication is necessary for the development of valve disease.

**References**

[1] Lindroos, M., Kupari, M., Heikkila, J., & Tilvis, R. Prevalence of aortic valve abnor‐ malities in the elderly: an echocardiographic study of a random population sample.

Role of Oxidative Stress in Calcific Aortic Valve Disease: From Bench to Bedside - The Role of a Stem Cell Niche

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[2] Ross, J., Jr , , Braunwald, E., Aortic, stenosis., & Circulation, . (1968). Suppl):, 61-67. [3] Roberts WC, Ko JM. (2005). Frequency by decades of unicuspid, bicuspid, and tricus‐ pid aortic valves in adults having isolated aortic valve replacement for aortic steno‐ sis, with or without associated aortic regurgitation. *Circulation*, 111(7), 920-925. [4] Drolet, M. C., Arsenault, M., & Couet, J. (2003). Experimental aortic valve stenosis in

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[6] Rajamannan, N. M., Subramaniam, M., Springett, M., Sebo, T. C., Niekrasz, M., Mc Connell, J. P., Singh, R. J., Stone, N. J., Bonow, R. O., & Spelsberg, T. C. (2002). Ator‐ vastatin inhibits hypercholesterolemia-induced cellular proliferation and bone ma‐

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[11] Rajamannan, N. M., Subramaniam, M., Stock, S. R., Stone, N. J., Springett, M., Igna‐ tiev, K. I., Mc Connell, J. P., Singh, R. J., Bonow, R. O., & Spelsberg, T. C. Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercho‐ lesterolaemic aortic valve. Heart (British Cardiac Society). (2005). , 91(6), 806-810. [12] Makkena, B., Salti, H., Subramaniam, M., Thennapan, S., Bonow, R. H., Caira, F., Bo‐ now, R. O., Spelsberg, T. C., & Rajamannan, N. M. (2005). Atorvastatin decreases cel‐ lular proliferation and bone matrix expression in the hypercholesterolemic mitral

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This study provides the correlates described in the mathematical modeling by Agur [106]. This mathematical model has demonstrated the principal that the universal properties of the stem cells can be described in a simple discrete model as derived from hemopoietic stem cell behavior [106]. The transition of hemopoietic stem cells from quiescence into differentiation, is governed by their cell-cycling status, by stimulatory hormones secreted by neighboring cells into the micro-environment and by the level of amplification of stem-cell population [105, 107]. The model of Agur, defines the corollaries necessary to identify a stem cell niche, first the physical architecture of the stem cell niche and second the gradient necessary to reg‐ ulate the niche. In the BAV the gradient is defined by the niche's microenvironment. The ini‐ tiation of event of oxidative stress inhibits normal endothelial nitric oxide synthase function, activates notch1 splicing which in turn induces Wnt3a secretion to activate bone formation within the valve [5, 17], [99].

The model proposed in the study as described in Figure 5, provides the cellular architecture for the development of this disease process. This model does not take into account other cy‐ tokine/growth factor mediated mechanisms that have been shown to also be important in this disease process [108]. However, understanding CAVD from a development disease per‐ spective will provide a foundation for understanding this and other development disease processes. Clinical trials in the field of CAVD are demonstrating variable results [86, 87]. The possible differences in the published trials are secondary to the timing of therapy and the biological targeting of the lipid levels in these patients. Future medical therapies target‐ ing stem cell niche mediated diseases provides a novel model system to test and to translate clinically for patients in the future.

### **Author details**

#### Nalini Rajamannan

Molecular Biology and Biochemistry, Mayo Clinic School of Medicine, Rochester MN, USA

### **References**

The important inhibitor in this model is Notch1. Notch1 plays a roll in cellular differentia‐ tion decisions. In the osteoblast cell, it serves as an inhibitor of osteoblast differentiation [89, 90]. In the aortic valve, it serves to turn off bone formation via the cell-cell crosstalk between the endothelial and the myofibroblast cells. Normal Notch1 receptor functions to maintain normal valve cellular composition and homeostasis. In the presence of lipids, Notch1 is spliced and therefore activates osteoblastogenesis. In turn, the Wnt3a is secreted and binds to Lrp5 and Frizzled on the extracellular membrane to regulate the osteoblast gene program. This developmental disease process follows a parallel signaling pathway that has been ob‐ served in the normal embryonic valve development that has been well delineated by previ‐ ous investigators [104]. A similar cell-cell communication is necessary for the development

278 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

This study provides the correlates described in the mathematical modeling by Agur [106]. This mathematical model has demonstrated the principal that the universal properties of the stem cells can be described in a simple discrete model as derived from hemopoietic stem cell behavior [106]. The transition of hemopoietic stem cells from quiescence into differentiation, is governed by their cell-cycling status, by stimulatory hormones secreted by neighboring cells into the micro-environment and by the level of amplification of stem-cell population [105, 107]. The model of Agur, defines the corollaries necessary to identify a stem cell niche, first the physical architecture of the stem cell niche and second the gradient necessary to reg‐ ulate the niche. In the BAV the gradient is defined by the niche's microenvironment. The ini‐ tiation of event of oxidative stress inhibits normal endothelial nitric oxide synthase function, activates notch1 splicing which in turn induces Wnt3a secretion to activate bone formation

The model proposed in the study as described in Figure 5, provides the cellular architecture for the development of this disease process. This model does not take into account other cy‐ tokine/growth factor mediated mechanisms that have been shown to also be important in this disease process [108]. However, understanding CAVD from a development disease per‐ spective will provide a foundation for understanding this and other development disease processes. Clinical trials in the field of CAVD are demonstrating variable results [86, 87]. The possible differences in the published trials are secondary to the timing of therapy and the biological targeting of the lipid levels in these patients. Future medical therapies target‐ ing stem cell niche mediated diseases provides a novel model system to test and to translate

Molecular Biology and Biochemistry, Mayo Clinic School of Medicine, Rochester MN, USA

of valve disease.

within the valve [5, 17], [99].

clinically for patients in the future.

**Author details**

Nalini Rajamannan


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286 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants


**Chapter 12**

**Menopause Induces Oxidative Stress**

Claudia Camelia Calzada Mendoza and

**1.1. Menopause: endocrinology and symptoms**

Menopause is a physiologic process in women that occurs around 45-55 years old, which is defined as permanent cessation of menstruation by one year in row [1]. The age of meno‐ pause depends on multiple factors such as number of ovules from the female at birth, the frequency of loss of these ovules through her life and the number of ovarian follicles re‐ quired maintaining the menstrual cycle. The diagnosis of menopause is retrospective and is established after a year without menses [2], and their symptoms may have different intensi‐

This process is characterized by gradual decrease of estrogen (E) secretion and changes re‐ lated with sex hormones, so that estradiol levels ranging from 5 to 25 pg/mL, while increas‐ ing titers of gonadotrophins, so that the values of follicle stimulating hormone (FSH) between 40 and 250 mU/mL and luteinizing hormone (LH), from 30 to 150 mU/Ml [4, 5].

Irregular uterine bleeding is a characteristic symptom which is due to both depletion and resistance of ovarian receptors to gonadotropins and increased FSH, leading to alterations in the volume and frequency of bleeding (polymenorrhea, hypo-or menorrhagia, oligomenor‐

Among symptoms are those related to the genitourinary tract by the common embryological origin of vulva, vagina, bladder, and urethra, consequently alterations as dysuria, urinary urgency and incontinence, epithelial atrophy, decreased production of mucus and vaginal dryness (phenomena that can cause dyspareunia), urethritis, vaginitis or cystitis and local

> © 2013 Mendoza and Zamarripa; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Carlos Alberto Jiménez Zamarripa

http://dx.doi.org/10.5772/52082

**1. Introduction**

ty for each woman [3].

rhea) [6, 7].

infections [8, 9, 10] (Figure 1).

**Chapter 12**

### **Menopause Induces Oxidative Stress**

Claudia Camelia Calzada Mendoza and Carlos Alberto Jiménez Zamarripa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52082

**1. Introduction**

### **1.1. Menopause: endocrinology and symptoms**

Menopause is a physiologic process in women that occurs around 45-55 years old, which is defined as permanent cessation of menstruation by one year in row [1]. The age of meno‐ pause depends on multiple factors such as number of ovules from the female at birth, the frequency of loss of these ovules through her life and the number of ovarian follicles re‐ quired maintaining the menstrual cycle. The diagnosis of menopause is retrospective and is established after a year without menses [2], and their symptoms may have different intensi‐ ty for each woman [3].

This process is characterized by gradual decrease of estrogen (E) secretion and changes re‐ lated with sex hormones, so that estradiol levels ranging from 5 to 25 pg/mL, while increas‐ ing titers of gonadotrophins, so that the values of follicle stimulating hormone (FSH) between 40 and 250 mU/mL and luteinizing hormone (LH), from 30 to 150 mU/Ml [4, 5].

Irregular uterine bleeding is a characteristic symptom which is due to both depletion and resistance of ovarian receptors to gonadotropins and increased FSH, leading to alterations in the volume and frequency of bleeding (polymenorrhea, hypo-or menorrhagia, oligomenor‐ rhea) [6, 7].

Among symptoms are those related to the genitourinary tract by the common embryological origin of vulva, vagina, bladder, and urethra, consequently alterations as dysuria, urinary urgency and incontinence, epithelial atrophy, decreased production of mucus and vaginal dryness (phenomena that can cause dyspareunia), urethritis, vaginitis or cystitis and local infections [8, 9, 10] (Figure 1).

© 2013 Mendoza and Zamarripa; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

anxiety and palpitations, and it usually lasts 2-4 minutes and can vary in frequency, in some women may be daily while others may have one episode per month [11, 12]. The mechanism of hot flushes is not clear, however, it is known that hypothalamus, pituitary gonadotropin releasing hormone and gonadotrophins may be involved in hot flushes [13]. Another fre‐ quent symptom is an oral dryness and intense burning sensation that affects mainly the

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 291

On the other hand decreases the content of collagen and elastic fibers of the skin, so that it becomes thinner and brittle losing elasticity and firmness. The epidermis thins, increases water loss and reduces the number of blood vessels, compromising the supply of oxygen and nutrients [15]. Additionally aging is associated with a natural decline in physiological functions, including a loss of muscle mass and strength. Overall, the decline in muscle mass averages 0.4 to 0.8 kg per decade, starting at the age of 20 years, especially around meno‐

Another alteration that occurs is the osteoporosis, which is defined as a skeletal disorder characterized by decreased bone density and an increased risk of fractures [17, 18]. Before reports have confirmed that postmenopausal women have highest incidence of hip fractures

**Figure 3.** Bone mineral density values by age in women. Bone mineral density decreases around menopause [21].

Menopause is a stage that favors weight gain and development or worsening of obesity, and causes of this problem are many; some are clearly related to hypoestrogenism and oth‐ er age-dependent, conditioning increased intake and decreased energy expenditure [22, 23]

During this period there is an abnormal atherogenic lipid profile characterized by increased lipoprotein cholesterol, low density (LDL-C), triglycerides (TG) and small dense LDL parti‐ cles [24] with reduced HDL-C and elevated serum glucose and insulin, perhaps as a direct result of ovarian failure or indirectly as a result of central redistribution of body fat, and this favors the formation of atheromatous plaques and progression of coronary atherosclerosis

tongue and sometimes lips and gums [14].

pause [16] (Figure 2).

[19, 20, 21] (Figure 3).

(Figure 4).

**Figure 1.** Main genitourinary abnormalities according to age in women [10].

**Figure 2.** Changes observed in muscle mass and strength after menopause [16].

Hot flushes are one of the main symptoms associated with menopause and occur in more than 75% of menopausal, consisting of intense episodes of heat that begins on chest and spreads to face, sweating, and flushing of face. Hot flushes are associated with headache, anxiety and palpitations, and it usually lasts 2-4 minutes and can vary in frequency, in some women may be daily while others may have one episode per month [11, 12]. The mechanism of hot flushes is not clear, however, it is known that hypothalamus, pituitary gonadotropin releasing hormone and gonadotrophins may be involved in hot flushes [13]. Another fre‐ quent symptom is an oral dryness and intense burning sensation that affects mainly the tongue and sometimes lips and gums [14].

On the other hand decreases the content of collagen and elastic fibers of the skin, so that it becomes thinner and brittle losing elasticity and firmness. The epidermis thins, increases water loss and reduces the number of blood vessels, compromising the supply of oxygen and nutrients [15]. Additionally aging is associated with a natural decline in physiological functions, including a loss of muscle mass and strength. Overall, the decline in muscle mass averages 0.4 to 0.8 kg per decade, starting at the age of 20 years, especially around meno‐ pause [16] (Figure 2).

Another alteration that occurs is the osteoporosis, which is defined as a skeletal disorder characterized by decreased bone density and an increased risk of fractures [17, 18]. Before reports have confirmed that postmenopausal women have highest incidence of hip fractures [19, 20, 21] (Figure 3).

**Figure 1.** Main genitourinary abnormalities according to age in women [10].

290 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 2.** Changes observed in muscle mass and strength after menopause [16].

Hot flushes are one of the main symptoms associated with menopause and occur in more than 75% of menopausal, consisting of intense episodes of heat that begins on chest and spreads to face, sweating, and flushing of face. Hot flushes are associated with headache,

**Figure 3.** Bone mineral density values by age in women. Bone mineral density decreases around menopause [21].

Menopause is a stage that favors weight gain and development or worsening of obesity, and causes of this problem are many; some are clearly related to hypoestrogenism and oth‐ er age-dependent, conditioning increased intake and decreased energy expenditure [22, 23] (Figure 4).

During this period there is an abnormal atherogenic lipid profile characterized by increased lipoprotein cholesterol, low density (LDL-C), triglycerides (TG) and small dense LDL parti‐ cles [24] with reduced HDL-C and elevated serum glucose and insulin, perhaps as a direct result of ovarian failure or indirectly as a result of central redistribution of body fat, and this favors the formation of atheromatous plaques and progression of coronary atherosclerosis and therefore cardiovascular disease incidence increases substantially in postmenopausal women [25, 26]. Other disorders such as obesity and metabolic syndrome also occurs at menopause, suggesting that menopause may be the trigger of the metabolic syndrome at that stage of life [27, 28].

Depression occurs frequently in postmenopausal women, which is explained by the loss of estrogenic effect in modulating neuronal excitability, synaptic plasticity, neuronal survival induced expression of regenerative responses, regional neurogenesis, regulation of differen‐ tiation and neuronal development [29], in the processes of cognition, modulation of mood and other mental states, as well as improving learning and memory [30], regulate the syn‐ thesis of tryptophan hydroxylase which is the limiting enzyme in serotonin synthesis so this decline in estrogen at menopause may explain the occurrence of psychological symptoms characteristic of depression (fatigue, irritability, sleep problems, abrupt changes of mood, [31]. With respect depressive symptoms in the Multiethnic Study of Atherosclerosis were analyzed testosterone, estradiol, steroid hormone binding globulin (SHBG) and dehydroe‐ piandrosterone; indicating that in early postmenopausal women, sex hormones were associ‐

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 293

Throughout menopause there are factors that predispose women to the development of oxi‐ dative stress, such as estrogen deficiency, as it has been confirmed that they have an antioxi‐ dant capacity independently of its binding to receptors, so for example the 17β-estradiol (E2), estriol, estrone, ethinylestradiol and 2-hidroxiestradiol besides reducing neuronal death with antioxidant activity, due to the presence of an intact hydroxyl group on ring A of

Estrogens are synthesized from different androgen precursors such as androstenedione and testosterone, yielding as products estrone and 17β-estradiol, respectively. The synthesis is catalyzed by aromatase (ARO), the enzyme cytochrome P450 (CYP19) and estrogen synthe‐ sizing different tissue-specific manner, and the major estrogen in adipose tissue is estrone,

The 2-hidroxiestradiol and 2-hydroxyestrone (4-hidroxiestradiol type) (Figure 6) can partici‐ pate in redox cycling to generate free radicals such as superoxide and chemically reactive es‐ trogen semiquinone/quinone, which can damage DNA and other intracellular constituents.

4-hidroxiestradiol participates in a redox cycle to generate free radicals such as superoxide, and intermediate semiquinone/quinone, these intermediaries may induce cell transforma‐

4 - hydroxyestrogens have estrogenic effects and can stimulate the growth of cell lines of breast cancer, with greater intensity than the 4-hydroxyestrone are unstable and can become highly reactive quinone with the formation of semiquinones as intermediary, this reaction produces oxygen free radicals, which can have toxic effects on DNA, such effects include the formation of 8-hydroxy-2-deoxiguanosine a mutagen, resulting from oxidative damage. The toxic effect of 4-hydroxyestrogens probably is prevented under normal conditions intracel‐ lular defense mechanisms. Oxygen free radicals can be removed immediately transformed into water by enzymes such as catalase and superoxide dismutase and antioxidant vitamins

ated with incident depressive symptoms [32].

the molecule [33].

tion and initiate tumoral growth [35].

**2. Pro and antioxidants propierties of estrogens**

the placenta is estriol and in cells granulosa is 17β-estradiol [34].

**Figure 4.** Body fat distribution. Android-type distribution is present in postmenopausal women. DXA= Dual-energy Xray absorptiometry [23].

Postmenopausal women have higher insulin resistance than premenopausal, which could participate to age, the increase in total body fat, central adiposity, estrogen deficiency, alter‐ ations in lipid profile and glucose homeostasis and insulin are more frequent and favor the high cardiovascular morbidity and mortality after menopause. In this sense the transition of menopause is marked by changes in hormonal balance, with increased visceral fat, which are associated with insulin resistance, although it has been found that the change in insulin sensitivity does not alter the lipid profile in early postmenopausal women [24, 26] (Figure 5).

**Figure 5.** Changes lipid during transition from premenopause to Postmenopause [24].

Depression occurs frequently in postmenopausal women, which is explained by the loss of estrogenic effect in modulating neuronal excitability, synaptic plasticity, neuronal survival induced expression of regenerative responses, regional neurogenesis, regulation of differen‐ tiation and neuronal development [29], in the processes of cognition, modulation of mood and other mental states, as well as improving learning and memory [30], regulate the syn‐ thesis of tryptophan hydroxylase which is the limiting enzyme in serotonin synthesis so this decline in estrogen at menopause may explain the occurrence of psychological symptoms characteristic of depression (fatigue, irritability, sleep problems, abrupt changes of mood, [31]. With respect depressive symptoms in the Multiethnic Study of Atherosclerosis were analyzed testosterone, estradiol, steroid hormone binding globulin (SHBG) and dehydroe‐ piandrosterone; indicating that in early postmenopausal women, sex hormones were associ‐ ated with incident depressive symptoms [32].

### **2. Pro and antioxidants propierties of estrogens**

and therefore cardiovascular disease incidence increases substantially in postmenopausal women [25, 26]. Other disorders such as obesity and metabolic syndrome also occurs at menopause, suggesting that menopause may be the trigger of the metabolic syndrome at

**Figure 4.** Body fat distribution. Android-type distribution is present in postmenopausal women. DXA= Dual-energy X-

Postmenopausal women have higher insulin resistance than premenopausal, which could participate to age, the increase in total body fat, central adiposity, estrogen deficiency, alter‐ ations in lipid profile and glucose homeostasis and insulin are more frequent and favor the high cardiovascular morbidity and mortality after menopause. In this sense the transition of menopause is marked by changes in hormonal balance, with increased visceral fat, which are associated with insulin resistance, although it has been found that the change in insulin sensitivity does not alter the lipid profile in early postmenopausal women [24, 26] (Figure 5).

**Figure 5.** Changes lipid during transition from premenopause to Postmenopause [24].

that stage of life [27, 28].

292 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ray absorptiometry [23].

Throughout menopause there are factors that predispose women to the development of oxi‐ dative stress, such as estrogen deficiency, as it has been confirmed that they have an antioxi‐ dant capacity independently of its binding to receptors, so for example the 17β-estradiol (E2), estriol, estrone, ethinylestradiol and 2-hidroxiestradiol besides reducing neuronal death with antioxidant activity, due to the presence of an intact hydroxyl group on ring A of the molecule [33].

Estrogens are synthesized from different androgen precursors such as androstenedione and testosterone, yielding as products estrone and 17β-estradiol, respectively. The synthesis is catalyzed by aromatase (ARO), the enzyme cytochrome P450 (CYP19) and estrogen synthe‐ sizing different tissue-specific manner, and the major estrogen in adipose tissue is estrone, the placenta is estriol and in cells granulosa is 17β-estradiol [34].

The 2-hidroxiestradiol and 2-hydroxyestrone (4-hidroxiestradiol type) (Figure 6) can partici‐ pate in redox cycling to generate free radicals such as superoxide and chemically reactive es‐ trogen semiquinone/quinone, which can damage DNA and other intracellular constituents.

4-hidroxiestradiol participates in a redox cycle to generate free radicals such as superoxide, and intermediate semiquinone/quinone, these intermediaries may induce cell transforma‐ tion and initiate tumoral growth [35].

4 - hydroxyestrogens have estrogenic effects and can stimulate the growth of cell lines of breast cancer, with greater intensity than the 4-hydroxyestrone are unstable and can become highly reactive quinone with the formation of semiquinones as intermediary, this reaction produces oxygen free radicals, which can have toxic effects on DNA, such effects include the formation of 8-hydroxy-2-deoxiguanosine a mutagen, resulting from oxidative damage. The toxic effect of 4-hydroxyestrogens probably is prevented under normal conditions intracel‐ lular defense mechanisms. Oxygen free radicals can be removed immediately transformed into water by enzymes such as catalase and superoxide dismutase and antioxidant vitamins such as ascorbic acid and alpha tocopherol, quinone themselves can be inactivated by sulfo compounds, such as glutathione [36].

17-β-estradiol plays a critical role in neuroprotection through both genomic and non-genom‐ ic mechanisms and recently was discovered that a new G-protein-coupled receptor 30 (GPR30) participates in the neuroprotection against oxidative insult, which is agonist G1. E2 attenuated apoptosis induced by H2O2 exposure, furthermore, G1 or E2 significantly in‐ creased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),

Actually several oxidative stress biomarkers have been studied in menopause, however, each researcher has used different marker, methodologies and women with dissimilar char‐ acteristics (age, ethnic group, postmenopause time), fact does difficult to make a conclusion about the development of oxidative stress during peri, menopause and postmenopause.

Recently has been propose as indicator to γ-glutamyltransferase (GGT) which is an enzyme involved in the transfer of the γ-glutamyl residue from γ-glutamyl peptides to amino acids, H2O, and other small peptides and can be donated by glutathione [41]. On the other hand, GGT is also involved in the production of glutathione [42], which is limited by cysteine availability. GGT participates in the pathway of extracellular GSH in consequence the bio‐ synthesis of cellular glutathione, the most important cell antioxidant, depends of GGT activ‐ ity; hence this enzyme may play an important role in the anti-oxidative defense system of

Abdul et al, founded a highly significant reduction in glutathione levels in the post-menopaus‐ al-group which could be due to the increase in its free radical scavenging property and in‐ creased consumption to counteract the oxidative stress and to inhibit membrane lipid peroxidation which indicates that the increase in serum GGT with enhanced oxidative stress and reduced antioxidant defense system in the post-menopausal women may lead to the spec‐ ulation that GGT could be considered an index or a oxidative stress marker [43] (Table 1).

> GGT (U/L) 5.96±2.99 9.44±2.89 0.025 GSH (mmole/L) 0.62±0.17 0.47±0.11 0.008 MDA (µmole/L) 1.04±0.06 1.32±0.05 0.035

**Postmenopausal Group (n=16)**

**p value**

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 295

**Group (n=17)**

**Table 1.** Serum γ-glutamyltransferase, glutathione and malondialdehyde levels in the pre- and postmenopausal

Supplementary it was found that perimenopausal women have higher total cholesterol val‐ ues and lower paraoxonase-1 (PON1) activity compared to reference values, 8-oxoG levels were unchanged compared with those of healthy control women, lipoperoxide ranks were

Bcl-2 and pro-caspase-3, which is an anti-apoptotic effect [40].

**Serum level Premenopausal**

**3. Oxidative stress and postmenopause**

the cell [43].

women [43].

**Figure 6.** Estrogen metabolism.

Menopause seems to accelerate the development of atherosclerosis and cardiovascular dis‐ eases and in order to identify this correlation, was assessed the correlations between intimamedia thickness, homocysteine serum levels and oxidative stress both in fertile and postmenopausal women and it was founded that were increased levels of homocysteine, ox‐ idative stress and intima-media tickness (IMT) in postmenopausal women having a positive correlation with IMT, which reinforce the idea that a hyperhomocysteinemia may play a role in the progression of atherosclerosisas a result the lack of estrogens [37].

Vasculo protective effects of estrogen are due in part to the modulation of the balance be‐ tween nitric oxide (mainly derived from endothelial vasodilator molecule) [38] and superox‐ ide anion (oxygen-free radical highly reactive), promoting the availability of the first such so the lack of protection induces high levels of oxidative stress and low concentrations of NO, these processes are interacting with hypertension, as seen in menopause. In addition, estro‐ gen induces the expression of oxide reductasesthiol / disulfide, such as disulfide isomerase, thioredoxin, thioredoxinreductase and glutaredoxin in the endothelium and inhibits apopto‐ sis mediated by hydrogen peroxide. On the other hand has been described that genetic fac‐ tors related to dyslipidemia are most important than due to age, for example antioxidant enzymes (SOD, catalase, GR, inflammatory markers CPR, ALT), oxidative stress (O(2)(-), LOO•), hypoxia (HBNO) and all this related to increase vascular resistance, disorders in oxygen supply in tissue and hypoxic competitions of there metabolism may cause, postme‐ nopausal hypertension, hart ischemic disease, impaired hepatic beta-oxidation of fatty acids and hepathosteatosis [39].

17-β-estradiol plays a critical role in neuroprotection through both genomic and non-genom‐ ic mechanisms and recently was discovered that a new G-protein-coupled receptor 30 (GPR30) participates in the neuroprotection against oxidative insult, which is agonist G1. E2 attenuated apoptosis induced by H2O2 exposure, furthermore, G1 or E2 significantly in‐ creased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), Bcl-2 and pro-caspase-3, which is an anti-apoptotic effect [40].

### **3. Oxidative stress and postmenopause**

such as ascorbic acid and alpha tocopherol, quinone themselves can be inactivated by sulfo

Menopause seems to accelerate the development of atherosclerosis and cardiovascular dis‐ eases and in order to identify this correlation, was assessed the correlations between intimamedia thickness, homocysteine serum levels and oxidative stress both in fertile and postmenopausal women and it was founded that were increased levels of homocysteine, ox‐ idative stress and intima-media tickness (IMT) in postmenopausal women having a positive correlation with IMT, which reinforce the idea that a hyperhomocysteinemia may play a role

Vasculo protective effects of estrogen are due in part to the modulation of the balance be‐ tween nitric oxide (mainly derived from endothelial vasodilator molecule) [38] and superox‐ ide anion (oxygen-free radical highly reactive), promoting the availability of the first such so the lack of protection induces high levels of oxidative stress and low concentrations of NO, these processes are interacting with hypertension, as seen in menopause. In addition, estro‐ gen induces the expression of oxide reductasesthiol / disulfide, such as disulfide isomerase, thioredoxin, thioredoxinreductase and glutaredoxin in the endothelium and inhibits apopto‐ sis mediated by hydrogen peroxide. On the other hand has been described that genetic fac‐ tors related to dyslipidemia are most important than due to age, for example antioxidant enzymes (SOD, catalase, GR, inflammatory markers CPR, ALT), oxidative stress (O(2)(-), LOO•), hypoxia (HBNO) and all this related to increase vascular resistance, disorders in oxygen supply in tissue and hypoxic competitions of there metabolism may cause, postme‐ nopausal hypertension, hart ischemic disease, impaired hepatic beta-oxidation of fatty acids

in the progression of atherosclerosisas a result the lack of estrogens [37].

compounds, such as glutathione [36].

294 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 6.** Estrogen metabolism.

and hepathosteatosis [39].

Actually several oxidative stress biomarkers have been studied in menopause, however, each researcher has used different marker, methodologies and women with dissimilar char‐ acteristics (age, ethnic group, postmenopause time), fact does difficult to make a conclusion about the development of oxidative stress during peri, menopause and postmenopause.

Recently has been propose as indicator to γ-glutamyltransferase (GGT) which is an enzyme involved in the transfer of the γ-glutamyl residue from γ-glutamyl peptides to amino acids, H2O, and other small peptides and can be donated by glutathione [41]. On the other hand, GGT is also involved in the production of glutathione [42], which is limited by cysteine availability. GGT participates in the pathway of extracellular GSH in consequence the bio‐ synthesis of cellular glutathione, the most important cell antioxidant, depends of GGT activ‐ ity; hence this enzyme may play an important role in the anti-oxidative defense system of the cell [43].

Abdul et al, founded a highly significant reduction in glutathione levels in the post-menopaus‐ al-group which could be due to the increase in its free radical scavenging property and in‐ creased consumption to counteract the oxidative stress and to inhibit membrane lipid peroxidation which indicates that the increase in serum GGT with enhanced oxidative stress and reduced antioxidant defense system in the post-menopausal women may lead to the spec‐ ulation that GGT could be considered an index or a oxidative stress marker [43] (Table 1).


**Table 1.** Serum γ-glutamyltransferase, glutathione and malondialdehyde levels in the pre- and postmenopausal women [43].

Supplementary it was found that perimenopausal women have higher total cholesterol val‐ ues and lower paraoxonase-1 (PON1) activity compared to reference values, 8-oxoG levels were unchanged compared with those of healthy control women, lipoperoxide ranks were significantly increased compared with those of premenopausal women and an indirect cor‐ relation between PON1 arylesterase (PON1 A) activity and lipoperoxide levels, between PON1 A activity and atherogenic index, between age and TAS, and between age and 8-oxoG levels. Moreover perimenopausal women had higher total cholesterol levels and PON1 A levels were lower than physiological values (table 2) [44].

In another study were determined age, body weight, and superoxide dismutase (SOD), cata‐ lase (CAT) and malondialdehyde (MDA) in disease-free women aged 25-65 years and did found that postmenopausal women had the highest oxidative stress and body weight, also superoxide dismutase, catalase and malondialdehyde were correlated significantly with

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 297

Pansini demonstrated that the total body fat mass increases significantly in postmenopause in comparison with premenopause, with specific increases in fat deposition at the level of trunk (abdominal and visceral) and arms. Concomitantly, the antioxidant status adjusted for age showed that antioxidant status was retained. Also both antioxidant status and hydro‐

Also has been carried out protocols that analyze the connection between menopause and pe‐ riodontal conditions, though to compare serum and gingival crevicular fluid (GCF) total an‐ tioxidant capacity (TAOC) and superoxide dismutase (SOD) concentrations in postmenopausal patients with chronic periodontitis (PMCP) with those of pre-menopausal chronic periodontitis patients (CP). The results showed Serum and GCF TAOC and SOD concentrations were significantly lower in menopause and periodontitis, the lowest values were in the PMCP group, whereas the highest values were in premenopausal. While the ef‐ fect of menopause was more evident in serum antioxidant analysis, the consequence of pe‐ riodontitis was observed to be more apparent in GCF and a decrease in systemic and local

AO defense was observed owing to both menopause and periodontitis [47].

**Risk factor OR 95% CI Pa** Menopause (hypoestrogenism) 2.62 1.35-5.11 0.005 Consumption of alcoholic beverages (≥2 glasses/d) 2.49 0.28-22.50 0.417 Smoking (≥2 cigarettes/d) 1.98 0.58-6.82 0.277 Overweight (˂25 kg/m2) 1.43 0.64-3.18 0.383 Insomnia (AIS score ≥8) 1.13 0.58-2.22 0.715 Age, y 1.04 0.94-1.14 0.466 Physical inactivity (˂30 min/d of physical activity) 0.85 0.43-1.68 0.632

Unsaturated fatty acids have a role in the pathogenesis of atherosclerosis. They are very sen‐ sitive to oxidation caused by excess free oxygen radicals and the consequent oxidative sta‐ tus, and it is well known that lipid and lipoprotein metabolism is markedly altered in postmenopausal women as it was demonstrated by Signorelli who founded that the oxida‐ tive stress is involved in the pathophysiology of atherosclerosis. Malonaldehyde (MDA), 4 hydroxynenal (4-HNE), oxidized lipoproteins (ox LDL) were higher in postmenopausal

while GSH-PX concentrations were significantly higher in fertile women [48].

Logistic regression, R2=0.106, P=0.036.Risk factors for high

body weight [45].

peroxide level increased with trunk fat mass [46].

**Table 4.** OR, odds ratio; AIS, Athens Insomnia Scale.<sup>a</sup>

lipoperoxide levels, as oxidative stress biomarker, in perimenopausal women [49].


**Table 2.** Data showing departures from normality are expressed as median values with the respective lower and upper quartile. The boldfaced entries indicate values beyond the reference range. PON1 A, paraoxonase with arylesterase activity; PON1 L, paroxonase-1 with lactonase activity.Paroxonase-1 levels in perimenonausal women [44].

Another finding is the lipoperoxide level which was significantly increased in perimeno‐ pausal women (Table 3). The levels of the marker of oxidative damage to DNA-8-oxoG were not statistically between pre and perimenopausal. In contrast women in perimenopause had repair ability 4 times higher compared with premenopausal women and significantly in‐ creased plasma total antioxidant capacity (TAS) [44] (Table 3).


**Table 3.** Data showing departures from normality are given as median values with the respective lower and upper quartile. a these values are significantly different (P˂0.005) compared with controls. Profile oxidant and antioxidant between premenopausal and perimenopausal women [44].

In another study were determined age, body weight, and superoxide dismutase (SOD), cata‐ lase (CAT) and malondialdehyde (MDA) in disease-free women aged 25-65 years and did found that postmenopausal women had the highest oxidative stress and body weight, also superoxide dismutase, catalase and malondialdehyde were correlated significantly with body weight [45].

significantly increased compared with those of premenopausal women and an indirect cor‐ relation between PON1 arylesterase (PON1 A) activity and lipoperoxide levels, between PON1 A activity and atherogenic index, between age and TAS, and between age and 8-oxoG levels. Moreover perimenopausal women had higher total cholesterol levels and PON1 A

**Variable Average±SD or median Physiological values**

3.853±1.009 ˂5.2

TCH **5.673±0.856 mmol/L** ˂5.17 mmol/L TG 1.424±0.66 mmol/L ˂1.9 mmol/L LDL 3.103±0.649 mmol/L ˂3.5 mmol/L HDL 1.563±0.445 mmol/L ˂1.4 mmol/L

PON1 A **89.628±14.798 U/mL** 100-200 U/mL Pon1 L **12.213±2.956 U/mL** 13-20 U/mL Homocysteine 8.48±2.97 µmol/L ˂12 µmol/L Glycemia 5.43µ0.65 mmol/L 4.2-6.2 mmol/L Uric acid 246.5 (209.9-296.9) µmol/L ˂339µmol/L

creased plasma total antioxidant capacity (TAS) [44] (Table 3).

between premenopausal and perimenopausal women [44].

**Variable Perimenopausal women Controls (premenopausal)** TAS 1.532±0.095 mmol/La 1.230±0.100 mmol/mL

Lipoperoxides 37.995 (32.035-44.849) nMol/mLa 28.096 (23.103-30.850) nmol/mL 8-oxoG 0.464 (0.283-0.957) per 106 G 0.503 (0.337-0.674) per 106 G Repair ability 36.919% (30.679%-47.046%)a 10.539% (8.665%-11.475%)

**Table 3.** Data showing departures from normality are given as median values with the respective lower and upper

these values are significantly different (P˂0.005) compared with controls. Profile oxidant and antioxidant

**Table 2.** Data showing departures from normality are expressed as median values with the respective lower and upper quartile. The boldfaced entries indicate values beyond the reference range. PON1 A, paraoxonase with arylesterase activity; PON1 L, paroxonase-1 with lactonase activity.Paroxonase-1 levels in perimenonausal women [44].

Another finding is the lipoperoxide level which was significantly increased in perimeno‐ pausal women (Table 3). The levels of the marker of oxidative damage to DNA-8-oxoG were not statistically between pre and perimenopausal. In contrast women in perimenopause had repair ability 4 times higher compared with premenopausal women and significantly in‐

levels were lower than physiological values (table 2) [44].

296 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Atherogenic index (TCH/HDL)

quartile. a

Pansini demonstrated that the total body fat mass increases significantly in postmenopause in comparison with premenopause, with specific increases in fat deposition at the level of trunk (abdominal and visceral) and arms. Concomitantly, the antioxidant status adjusted for age showed that antioxidant status was retained. Also both antioxidant status and hydro‐ peroxide level increased with trunk fat mass [46].

Also has been carried out protocols that analyze the connection between menopause and pe‐ riodontal conditions, though to compare serum and gingival crevicular fluid (GCF) total an‐ tioxidant capacity (TAOC) and superoxide dismutase (SOD) concentrations in postmenopausal patients with chronic periodontitis (PMCP) with those of pre-menopausal chronic periodontitis patients (CP). The results showed Serum and GCF TAOC and SOD concentrations were significantly lower in menopause and periodontitis, the lowest values were in the PMCP group, whereas the highest values were in premenopausal. While the ef‐ fect of menopause was more evident in serum antioxidant analysis, the consequence of pe‐ riodontitis was observed to be more apparent in GCF and a decrease in systemic and local AO defense was observed owing to both menopause and periodontitis [47].


**Table 4.** OR, odds ratio; AIS, Athens Insomnia Scale.<sup>a</sup> Logistic regression, R2=0.106, P=0.036.Risk factors for high lipoperoxide levels, as oxidative stress biomarker, in perimenopausal women [49].

Unsaturated fatty acids have a role in the pathogenesis of atherosclerosis. They are very sen‐ sitive to oxidation caused by excess free oxygen radicals and the consequent oxidative sta‐ tus, and it is well known that lipid and lipoprotein metabolism is markedly altered in postmenopausal women as it was demonstrated by Signorelli who founded that the oxida‐ tive stress is involved in the pathophysiology of atherosclerosis. Malonaldehyde (MDA), 4 hydroxynenal (4-HNE), oxidized lipoproteins (ox LDL) were higher in postmenopausal while GSH-PX concentrations were significantly higher in fertile women [48].

Similar findings were found in pre and postmenopausal Mexican women by Sánchez. Lipo‐ peroxides, erythrocyte superoxide dismutase and glutathione peroxidase activities, the total antioxidant status, pro-oxidant factors, body mass index were evaluated. The lipoperoxide levels were significantly higher in the postmenopausal group than in the premenopausal group, which concluded that menopause is the main risk factor for oxidative stress [49].

stimulates phosphorylated GSK3b [54]; due to in menopause its reduction induces a depres‐

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 299

Oxidative stress participates in decreasing bone formation and stimulating bone resorption. Furthermore, antioxidant enzymes have been observed to have low protective activity in women with osteoporosis, also has been determined higher urine deoxypyridinoline, total Peroxide (TPx), MDA, nitric oxide, also lower TAS and glutathione reductase, compared with postmenopausal women whitout osteoporosis [56, 57]. Likewise has been studied poly‐ morphism associated with enzymes involved in oxidative balance such as of the glutathione S-reductase (GSR), superoxide dismutase (SOD1 and SOD2), and catalase (CAT), of which polymorphisms from GSR were associated to bone mineral density [58]. Both oxidative stress and associated polymorphisms are useful tool to predict which patients might devel‐

Oxidative stress biomarkers have been linked with the presence and severity of the CVD, and to the presence and number of risk factors. It is known that young women during their fertile life are at lower risk of cardiovascular events compared with men, being protected by estrogen action and that oxidative stress is generally higher in men than in premenopausal women. However, after menopause the risk of experiencing cardiovascular events rapidly rises in women, in conjunction with a parallel increase in oxidative stress. Moreover, al‐ though oxidative stress results are lower in females compared to males during the first deca‐ des of life, this difference decreases until the age range which corresponds to the onset of

An analyses of relationship among excess iron, oxidative stress, and centralized fat mass in healthy postmenopausal women showed that almost 14% of the variability in oxLDL was accounted for by centralized fat mass AndGynFM ratio (waistþhip=thigh¼AndGynFM), age, and serum iron. Similarly, 16% the variability in 15-isoprostane F2aα (PGF F2aα) was ac‐ counted for by the AndGynFM ratio, HOMA, and serum iron. Also it was accounted for 33% of the variability in AndGynFM ratio by high-density lipoprotein cholesterol (HDL-C), ferritin, HOMA, oxLDL, and PGF F2aα, all of before suggests that reducing centralized fat mass and maintaining a favorable lipid profile, antioxidant status, and iron status all may be important in protecting postmenopausal women from atherosclerotic CVD [60]. Similar findings has been observed in diabetic postmenopausal women in whom it has been report‐ ed higher levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein choles‐ terol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), catalase (CAT), and malondialdehyde (MDA) and significantly lower levels of HDL-C, reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase

Leg vasoconstriction has been linked to oxidative stress due to the fact that Intravenous ad‐ ministration of a supraphysiological dose of the antioxidant ascorbic acid increased leg

sive disorder [55]. OSTEOPOROSIS

op osteoporosis.

CARDIOVASCULAR DISEASES

menopause for women [59].

(SOD) [61]. Fact means a cardiovascular risk.

LEG VASOCONSTRICTION

However, there are other contrasting studies, in example in a report was found than postme‐ nopausal women had lower levels of lipid hydroperoxide oxidation, the MDA levels did not differ between pre- and postmenopausal women, no differences in advanced oxidation pro‐ tein products (AOPP) and nitrite levels were observed between pre- and postmenopausal women. Postmenopausal women also exhibited a higher total radical antioxidant level [50].

Another study included pre-menopausal, peri-menopausal, and post-menopausal women classified according to the Staging of Reproductive Aging Workshop (STRAW) criteria. No significant correlations between E2 levels and OS markers were detected and consequently, estrogen decline during menopausal transition is not a determinant factor for oxidative stress [51].

### **4. Associated diseases to oxidative stress**

There are several evidences that related to oxidative stress with diseases present in postme‐ nopausal women in example depression, osteoporosis, cardiovascular diseases and leg vaso‐ constriction.

### DEPRESSION

The depression is the most frequent symptom in postmenopausal women, even is a major cause of medical consultation. This disorder has cerebral implications, as showed post-mor‐ tem studies in patients with depressive disorder pointed a significant decrease of neuronal and glial cells in cortico-limbic regions which can be seen as a consequence of alterations in neuronal plasticity. This could be triggered by an increase of free radicals which in its turn eventually leads to cell death and consequently atrophy of vulnerable neuronal and glial cell population in these regions [52]. In addition elevated levels of MDA adversely affected the efficiency of visual-spatial and auditory-verbal working memories; short-term declarative memory and the delayed recall declarative memory were founded. 1. Higher concentration of plasma MDA in recurrent depressive disorder (rDD) patients is associated with the se‐ verity of depressive symptoms 2. Elevated levels of plasma MDA are related to the impair‐ ment of visual-spatial and auditory-verbal working memory and short-term and delayed declarative memory [53]. Actually too is known that estrogen protect neurons against oxida‐ tive damage excitotoxins, and beta-amyloid-induced toxicity in cell culture, reduces the se‐ rum monoamino oxidase levels and might regulate learning and memory. Nitric oxide (NO) is a messenger and in the central nervous system and acts as neurotransmitter/neuromodu‐ lator like serotonin, bradykinin, endothelin, acetylcholine and noradrenaline. Estrogen indu‐ ces activity of constitutive NO synthase, reduces hyperphosphorylated of Tau and stimulates phosphorylated GSK3b [54]; due to in menopause its reduction induces a depres‐ sive disorder [55].

### OSTEOPOROSIS

Similar findings were found in pre and postmenopausal Mexican women by Sánchez. Lipo‐ peroxides, erythrocyte superoxide dismutase and glutathione peroxidase activities, the total antioxidant status, pro-oxidant factors, body mass index were evaluated. The lipoperoxide levels were significantly higher in the postmenopausal group than in the premenopausal group, which concluded that menopause is the main risk factor for oxidative stress [49].

However, there are other contrasting studies, in example in a report was found than postme‐ nopausal women had lower levels of lipid hydroperoxide oxidation, the MDA levels did not differ between pre- and postmenopausal women, no differences in advanced oxidation pro‐ tein products (AOPP) and nitrite levels were observed between pre- and postmenopausal women. Postmenopausal women also exhibited a higher total radical antioxidant level [50].

Another study included pre-menopausal, peri-menopausal, and post-menopausal women classified according to the Staging of Reproductive Aging Workshop (STRAW) criteria. No significant correlations between E2 levels and OS markers were detected and consequently, estrogen decline during menopausal transition is not a determinant factor for oxidative

There are several evidences that related to oxidative stress with diseases present in postme‐ nopausal women in example depression, osteoporosis, cardiovascular diseases and leg vaso‐

The depression is the most frequent symptom in postmenopausal women, even is a major cause of medical consultation. This disorder has cerebral implications, as showed post-mor‐ tem studies in patients with depressive disorder pointed a significant decrease of neuronal and glial cells in cortico-limbic regions which can be seen as a consequence of alterations in neuronal plasticity. This could be triggered by an increase of free radicals which in its turn eventually leads to cell death and consequently atrophy of vulnerable neuronal and glial cell population in these regions [52]. In addition elevated levels of MDA adversely affected the efficiency of visual-spatial and auditory-verbal working memories; short-term declarative memory and the delayed recall declarative memory were founded. 1. Higher concentration of plasma MDA in recurrent depressive disorder (rDD) patients is associated with the se‐ verity of depressive symptoms 2. Elevated levels of plasma MDA are related to the impair‐ ment of visual-spatial and auditory-verbal working memory and short-term and delayed declarative memory [53]. Actually too is known that estrogen protect neurons against oxida‐ tive damage excitotoxins, and beta-amyloid-induced toxicity in cell culture, reduces the se‐ rum monoamino oxidase levels and might regulate learning and memory. Nitric oxide (NO) is a messenger and in the central nervous system and acts as neurotransmitter/neuromodu‐ lator like serotonin, bradykinin, endothelin, acetylcholine and noradrenaline. Estrogen indu‐ ces activity of constitutive NO synthase, reduces hyperphosphorylated of Tau and

stress [51].

constriction. DEPRESSION

**4. Associated diseases to oxidative stress**

298 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Oxidative stress participates in decreasing bone formation and stimulating bone resorption. Furthermore, antioxidant enzymes have been observed to have low protective activity in women with osteoporosis, also has been determined higher urine deoxypyridinoline, total Peroxide (TPx), MDA, nitric oxide, also lower TAS and glutathione reductase, compared with postmenopausal women whitout osteoporosis [56, 57]. Likewise has been studied poly‐ morphism associated with enzymes involved in oxidative balance such as of the glutathione S-reductase (GSR), superoxide dismutase (SOD1 and SOD2), and catalase (CAT), of which polymorphisms from GSR were associated to bone mineral density [58]. Both oxidative stress and associated polymorphisms are useful tool to predict which patients might devel‐ op osteoporosis.

### CARDIOVASCULAR DISEASES

Oxidative stress biomarkers have been linked with the presence and severity of the CVD, and to the presence and number of risk factors. It is known that young women during their fertile life are at lower risk of cardiovascular events compared with men, being protected by estrogen action and that oxidative stress is generally higher in men than in premenopausal women. However, after menopause the risk of experiencing cardiovascular events rapidly rises in women, in conjunction with a parallel increase in oxidative stress. Moreover, al‐ though oxidative stress results are lower in females compared to males during the first deca‐ des of life, this difference decreases until the age range which corresponds to the onset of menopause for women [59].

An analyses of relationship among excess iron, oxidative stress, and centralized fat mass in healthy postmenopausal women showed that almost 14% of the variability in oxLDL was accounted for by centralized fat mass AndGynFM ratio (waistþhip=thigh¼AndGynFM), age, and serum iron. Similarly, 16% the variability in 15-isoprostane F2aα (PGF F2aα) was ac‐ counted for by the AndGynFM ratio, HOMA, and serum iron. Also it was accounted for 33% of the variability in AndGynFM ratio by high-density lipoprotein cholesterol (HDL-C), ferritin, HOMA, oxLDL, and PGF F2aα, all of before suggests that reducing centralized fat mass and maintaining a favorable lipid profile, antioxidant status, and iron status all may be important in protecting postmenopausal women from atherosclerotic CVD [60]. Similar findings has been observed in diabetic postmenopausal women in whom it has been report‐ ed higher levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein choles‐ terol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), catalase (CAT), and malondialdehyde (MDA) and significantly lower levels of HDL-C, reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) [61]. Fact means a cardiovascular risk.

### LEG VASOCONSTRICTION

Leg vasoconstriction has been linked to oxidative stress due to the fact that Intravenous ad‐ ministration of a supraphysiological dose of the antioxidant ascorbic acid increased leg blood flow in the postmenopausal women as a result of an increase in leg vascular conduc‐ tance, but it did not affect leg blood flow in premenopausal controls or mean arterial pres‐ sure, also changes in leg blood flow and leg vascular conductance with ascorbic acid were related to high plasma oxidized LDL an low antioxidant status [62] (Table 5, Figure 7).

demias, mood among others, is also important to note that hormone replacement therapy is

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 301

**•** 1.- Estrogens. They may be natural or synthetic. Estrogens (17β-Estradiol and Estriol) and conjugated equine estrogens, and these are administered orally. Estrogens may adminis‐ trated by oral, subcutaneous routes, also intravaginal estrogen (tablets, creams, ovules), alone or combined with progestin, are suitable for vaginal symptoms, with no significant

**•** 2 - Progestogens. They are administered in combination with estrogen to reduce the risk of endometrial hyperplasia and cancer. Currently most used active ingredients TH are: oral micronized progesterone, medroxyprogesterone and norethisterone. Progestins are mainly used orally, although there are preparations to be administered in combination

**•** 3 - Another group of drugs called STEAR (Selective Tissue Estrogenic Activity Regulator) is widely used because it has tissue-specific metabolism, and a main representative is Ti‐ bolone, this is a synthetic steroid with weak estrogenic, androgenic and gestagenic activi‐ ties, which controls vasomotor symptoms, prevents bone demineralization and improves mood [66]. Tibolone improves vaginal symptoms and no significant differences when compared to estrogen, decreases menopausal symptoms, although moderately increases bone density and inhibit bone resorption. In the cardiovascular system there is no evi‐ dence of efficacy for the primary or secondary prevention of diseases associated with

As mentioned above, there are different pathologies in the menopause that improve after ad‐ ministrating of hormone replacement therapy, fact that aroused the interest in evaluating their effects on biomarkers of oxidative stress, which has been recognized its participation in illness‐ es as cancer, atherogenesis, Alzheimer's and aging among others. Below are described the find‐ ings on changes in oxidative stress biomarkers after administrating HRT by periods time.

In African American and Caucasian posmenopausal women the HRT reduced plasma levels of free 8-isoprostane after 6 weeks of HT, at the same time nitrite increased, principally in Caucasian women. Both ethnics groups have reduced levels of oxidative stress but the dif‐ ferences were not statistically significant [68]. Even the combined therapy for 3 months had an antioxidant effect in posmenopausal hemodialysis women, who showed reduced levels

Oxidized low-density lipoprotein (oxLDL)/β2-glycoprotein I (β2GPI) complexes are etiologi‐ cally important in the development of atherosclerosis. Combined HRT led to a significant

**6. Effects of hormonal replacement therapy on oxidative stress**

of MDA although TAC, uric acid and C- reactive protein were not changed [69].

There are three Hormonal Replacement Therapies (HRT) treatment regimens:

increase in endometrial hyperplasia or proliferation.

with estrogen transdermal route [65].

menopause at this level [67].

LESS THAN THREE MONTHS

not without risks.


**Table 5.** Values are means ±SE, ACE angiotensin-converting enzyme. \*P˂0.05 vs premenopausal.Serum biomarkers associated to leg vasoconstriction [62].

**Figure 7.** Relationship between plasma oxidized low-density lipoprotein (LDL) and the change in femoral artery BF (top) and vascular conductance VC (bottom) with ascorbic acid in premenopusal ( ˂) and postmenopausal (●) women [62].

In addition, long-term studies indicate that total cholesterol (TC), LDL cholesterol (LDL-C), triglycerides (TG), MDA and common carotid artery wall intima-media thickness (IMT) are higher in women with hormonal depletion over 5 years, reveling a close temporal correla‐ tion between plasma oxidative and carotid wall IMT as postmenopause proceeds [63].

Further investigations are needed to examine the roll of oxidative stress as an endogenous bioactive agent related to disease in post-menopausal women. Since oxidative stress is the imbalance between total oxidants and antioxidants in the body, any single oxidant/ antioxi‐ dant parameter may not reflect oxidative stress. Further studies are needed to understand the underlying mechanisms of before findings.

### **5. Hormonal replacement therapy**

Hormone replacement therapy (HRT) is defined as treatment that estrogen provides women to improve the characteristic symptoms of menopause [64], especially osteoporosis, dyslipi‐ demias, mood among others, is also important to note that hormone replacement therapy is not without risks.

There are three Hormonal Replacement Therapies (HRT) treatment regimens:


### **6. Effects of hormonal replacement therapy on oxidative stress**

As mentioned above, there are different pathologies in the menopause that improve after ad‐ ministrating of hormone replacement therapy, fact that aroused the interest in evaluating their effects on biomarkers of oxidative stress, which has been recognized its participation in illness‐ es as cancer, atherogenesis, Alzheimer's and aging among others. Below are described the find‐ ings on changes in oxidative stress biomarkers after administrating HRT by periods time.

#### LESS THAN THREE MONTHS

blood flow in the postmenopausal women as a result of an increase in leg vascular conduc‐ tance, but it did not affect leg blood flow in premenopausal controls or mean arterial pres‐ sure, also changes in leg blood flow and leg vascular conductance with ascorbic acid were related to high plasma oxidized LDL an low antioxidant status [62] (Table 5, Figure 7).

**Table 5.** Values are means ±SE, ACE angiotensin-converting enzyme. \*P˂0.05 vs premenopausal.Serum biomarkers

**Figure 7.** Relationship between plasma oxidized low-density lipoprotein (LDL) and the change in femoral artery BF (top) and vascular conductance VC (bottom) with ascorbic acid in premenopusal ( ˂) and postmenopausal (●) women

In addition, long-term studies indicate that total cholesterol (TC), LDL cholesterol (LDL-C), triglycerides (TG), MDA and common carotid artery wall intima-media thickness (IMT) are higher in women with hormonal depletion over 5 years, reveling a close temporal correla‐ tion between plasma oxidative and carotid wall IMT as postmenopause proceeds [63].

Further investigations are needed to examine the roll of oxidative stress as an endogenous bioactive agent related to disease in post-menopausal women. Since oxidative stress is the imbalance between total oxidants and antioxidants in the body, any single oxidant/ antioxi‐ dant parameter may not reflect oxidative stress. Further studies are needed to understand

Hormone replacement therapy (HRT) is defined as treatment that estrogen provides women to improve the characteristic symptoms of menopause [64], especially osteoporosis, dyslipi‐

**Variable Premenopausal Postmenopausal**

Oxidized LDL, U/l 36.9±4.9 55.6±3.3\* TAS, mmol/l 1.4±0.1 1.1±0.1\* ACE, U/l 26.2±3.9 30.3±2.6 Endothelin-1, pg/ml 4.8±0.5 5.3±0.3 Norepinephrine, pg/ml 149±39 343±28\* Epinephrine, pg/ml 20±3 27±2

300 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

associated to leg vasoconstriction [62].

the underlying mechanisms of before findings.

**5. Hormonal replacement therapy**

[62].

In African American and Caucasian posmenopausal women the HRT reduced plasma levels of free 8-isoprostane after 6 weeks of HT, at the same time nitrite increased, principally in Caucasian women. Both ethnics groups have reduced levels of oxidative stress but the dif‐ ferences were not statistically significant [68]. Even the combined therapy for 3 months had an antioxidant effect in posmenopausal hemodialysis women, who showed reduced levels of MDA although TAC, uric acid and C- reactive protein were not changed [69].

Oxidized low-density lipoprotein (oxLDL)/β2-glycoprotein I (β2GPI) complexes are etiologi‐ cally important in the development of atherosclerosis. Combined HRT led to a significant increase in TAC and a minor but statistically nonsignificant decrease of oxLDL/β2GPI com‐ plexes when compared with the baseline control levels. There was also no significant associ‐ ation between TAC and oxLDL/β2GPI complexes changes related to HRT. This study indicates that, HRT in postmenopausal women leads to an increase in TAC without an equivalent change in serum levels of oxLDL/β2GPI complexes. It is concluded that beneficial effects of HRT could be explained, at least in part, by improving antioxidant status, but may not be directly associated with a change in oxidized lipoprotein production [70] (Figure 8).

stress [77]. Although another study reports that conjugated estrogens alone (EHRT) or con‐ jugated estrogen with medroxyprogesterone acetate can reduce lipoprotein lipase (LPL), hepatic lipase (HL), oxidized apolipoprotein B in LDL [78] also platelet MDA, glutathione-Stransferase (GST) and SOD levels were lower and total thiol (t-SH) content was higher than pre-treatment levels. These results indicate that hormone replacement therapy may affect platelet membrane fatty acid content and oxidant-antioxidant balance in postmenopausal

> 204.1±30.0/ 178.6±10.7\*

45.7±10.6/ 53.2±7.6\*

131.7±24.9/ 102.8±13.6\*

133.5±62.0/ 115.2±35.2

4.7±0.4/ 4.0±0.4\*

54.9±8.5/ 47.9±4.0\*

51.5±6.7/ 73.9±11.2\*

malondialdehyde compared to those who had no treatment [84, 85].

**Table 6.** Comparinson of parameters before and after HRT in postmenopausal women p˂0.005Serum lipid parameters, MDA, oxLDL and PON1 levels in postmenopausal women before and after HRT [74].

Similarly the effect of DNA damage by oxidative stress has been evaluated. The 8-hydroxy‐ deoxyguanosine (8-OHdG) is widely used for determination of DNA damage since it is ex‐ cised from oxidative damaged DNA with endonuclease repair enzymes coded (OGG1). After HT, mean blood 8-OHdG (DNA damage marker) level significantly decreased com‐ pared to those before HT, while urinary 8-OHdG level did not show any difference, this

Tibolone acts as an antioxidant upon increase the concentration of reduced sulfhydryl [81], however, the exact mechanism has not been elucidated, but it could participate in a direct mechanism, ie through the structure tibolone and its metabolites as it is similar to the struc‐ ture of 17β-estradiol, considered as an antioxidant for its phenol ring, which can act neutral‐ izing to free radical [82, 83]. Moreover tibolone reduces the concentration of

**MPA n=25 NETA n=20 Total n=45**

225.7±30.1/ 191.6±21.1\*

49.1±11.0/ 56.6±8.2

147.5±25.2/ 112.7±21.2\*

138.4±74.4/ 113.5±37.0

4.9±0.4/ 3.6±0.3\*

53.1±8.3/ 44.8±4.4\*

51.4±9.0/ 67.4±10,0\* 213.7±31.7/ 184.4±17.2\*

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 303

47.2±10.8/ 54.8±8.0\*

138.71±26.08/ 107.2±17.9\*

135.67±67.1/ 114.4±35.6

4.82±0.4/ 3.8±0.4\*

54.13±8.34/ 46.6±4.4\*

51.47±7.7/ 71.0±11.1\*

women [79].

Total-C

HDL-C

LDL-C

MDA

OxLDL

PON 1

Triglycerides

Pre/postreatment (mg/100ml)

Pre/postreatment (mg/100ml)

Pre/postreatment (mg/100ml)

Pre/postreatment (mg/100ml)

Pre/postreatment (mg/100ml)

Pre/postreatment (mg/100ml)

Pre/postreatment (mg/100ml)

without relation with S326C polymorphism [80].

**Figure 8.** Effects of hormone replacement therapy (HRT) on oxLDL/β2GPI (a), total antioxidant capacity (TAC) (b), oxLDL/β2GPI to HDL-C ratio (c) in the studied subjects. Values are means ± SE. P < 0.05 (paired sample t-test). total serum antioxidant capacity (TAC) as ferric reducing ability of plasma (FRAP) and related these to HRT and oxLDL/β2GPI complexes level [70].

#### SIX MONTHS

The main studies about effect of HRT has been carried out with combination estrogen plus gestagen, and in them has been founded in example that carbonils groups determined by ELISA showed a reduction of serum levels after six months oral or transdermal treat when compared with control group, and there was not difference between oral and transdermal, which indicates that hormonal therapy reduces the of carbonyl protein, a marker of oxida‐ tive stress, suggesting potential protective effect [71]. Similar result were founded with the serum level of malondialdehyde, superoxide dismutase and sulfhydryl groups without changes on plasma total homocysteine (tHcy) (used as atherogenic indicator) [72]. Another study with equal number of months of follow-up showed that carbonyls, MDA and oxLDL were reduced, while erythrocyte glutathione (GSH) were increased, and nitrotyrosine (NT) levels were not changed [73, 74] (Table 6).

On the other hand Tibolone treatment leads to a decrease in concentrations of plasma lipid peroxide, increase plasma concentrations of vitamin E and alpha-tocopherol and significant decrease in lipid peroxide concentrations [75, 76].

#### ONE YEAR

However, the combined treatment by one year significantly reduced the levels of catechola‐ mines, mean blood pressure and LDL cholesterol while it increased levels of nitrite/nitrate, indicating cardiovascular benefit in healthy recent postmenopausal women. Levels of 8-epi PGF2alpha did not change, suggesting no evident relationship between HRT and oxidative stress [77]. Although another study reports that conjugated estrogens alone (EHRT) or con‐ jugated estrogen with medroxyprogesterone acetate can reduce lipoprotein lipase (LPL), hepatic lipase (HL), oxidized apolipoprotein B in LDL [78] also platelet MDA, glutathione-Stransferase (GST) and SOD levels were lower and total thiol (t-SH) content was higher than pre-treatment levels. These results indicate that hormone replacement therapy may affect platelet membrane fatty acid content and oxidant-antioxidant balance in postmenopausal women [79].

increase in TAC and a minor but statistically nonsignificant decrease of oxLDL/β2GPI com‐ plexes when compared with the baseline control levels. There was also no significant associ‐ ation between TAC and oxLDL/β2GPI complexes changes related to HRT. This study indicates that, HRT in postmenopausal women leads to an increase in TAC without an equivalent change in serum levels of oxLDL/β2GPI complexes. It is concluded that beneficial effects of HRT could be explained, at least in part, by improving antioxidant status, but may not be directly associated with a change in oxidized lipoprotein production [70] (Figure 8).

302 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 8.** Effects of hormone replacement therapy (HRT) on oxLDL/β2GPI (a), total antioxidant capacity (TAC) (b), oxLDL/β2GPI to HDL-C ratio (c) in the studied subjects. Values are means ± SE. P < 0.05 (paired sample t-test). total serum antioxidant capacity (TAC) as ferric reducing ability of plasma (FRAP) and related these to HRT and oxLDL/β2GPI

The main studies about effect of HRT has been carried out with combination estrogen plus gestagen, and in them has been founded in example that carbonils groups determined by ELISA showed a reduction of serum levels after six months oral or transdermal treat when compared with control group, and there was not difference between oral and transdermal, which indicates that hormonal therapy reduces the of carbonyl protein, a marker of oxida‐ tive stress, suggesting potential protective effect [71]. Similar result were founded with the serum level of malondialdehyde, superoxide dismutase and sulfhydryl groups without changes on plasma total homocysteine (tHcy) (used as atherogenic indicator) [72]. Another study with equal number of months of follow-up showed that carbonyls, MDA and oxLDL were reduced, while erythrocyte glutathione (GSH) were increased, and nitrotyrosine (NT)

On the other hand Tibolone treatment leads to a decrease in concentrations of plasma lipid peroxide, increase plasma concentrations of vitamin E and alpha-tocopherol and significant

However, the combined treatment by one year significantly reduced the levels of catechola‐ mines, mean blood pressure and LDL cholesterol while it increased levels of nitrite/nitrate, indicating cardiovascular benefit in healthy recent postmenopausal women. Levels of 8-epi PGF2alpha did not change, suggesting no evident relationship between HRT and oxidative

complexes level [70].

SIX MONTHS

ONE YEAR

levels were not changed [73, 74] (Table 6).

decrease in lipid peroxide concentrations [75, 76].


**Table 6.** Comparinson of parameters before and after HRT in postmenopausal women p˂0.005Serum lipid parameters, MDA, oxLDL and PON1 levels in postmenopausal women before and after HRT [74].

Similarly the effect of DNA damage by oxidative stress has been evaluated. The 8-hydroxy‐ deoxyguanosine (8-OHdG) is widely used for determination of DNA damage since it is ex‐ cised from oxidative damaged DNA with endonuclease repair enzymes coded (OGG1). After HT, mean blood 8-OHdG (DNA damage marker) level significantly decreased com‐ pared to those before HT, while urinary 8-OHdG level did not show any difference, this without relation with S326C polymorphism [80].

Tibolone acts as an antioxidant upon increase the concentration of reduced sulfhydryl [81], however, the exact mechanism has not been elucidated, but it could participate in a direct mechanism, ie through the structure tibolone and its metabolites as it is similar to the struc‐ ture of 17β-estradiol, considered as an antioxidant for its phenol ring, which can act neutral‐ izing to free radical [82, 83]. Moreover tibolone reduces the concentration of malondialdehyde compared to those who had no treatment [84, 85].

Although there are reports that indicate the antioxidant effect of HRT, there are also studies that indicate otherwise; in example in another study combined HRT led to decreased plasma total and LDL cholesterol, but did not affect oxidizability and oxidation of LDL. Circulating levels of antioxidant vitamins (beta-carotene, vitamin C, vitamin E/triglycerides) and total antioxidant capacity of plasma and lipid peroxidation, assessed by plasma TBARs, were not different from controls in postmenopausal women receiving HRT, which indicates that com‐ bined HRT modifies the blood lipid profile, however it does not appear to influence oxida‐ tive status [86]. Additionaly DNA damage, GPx activity and nitrite level as well as a decreased GSH level were observed after oral administrating of estrogens alone or combi‐ nated [87].

[IL]-1beta, IL-6) and oxidative stress (superoxide dismutase [SOD], glutathione peroxidase [GPx], cyclooxygenase-2 [COX-2]) [95]. In this sense has also been found that de-alcoholised wine (DAW) with different polyphenol content by one month does not exert a protective ac‐ tivity towards oxidative DNA damage by comet assay, nor modifies significantly the gene expression profile of peripheral lymphocytes, whereas it shows blood-fluidifying actions, expressed as a significant decrease in blood viscosity. However, this effect does not correlate

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 305

In contrast, the intake for 8 weeks of soya protein diet and soya nut diet decreased MDA and increased the total antioxidant capacity in postmenopausal women with metabolic syn‐ drome [97]. Extra-virgin olive oils (EVOO) is another example of food which reduces DNA damage by oxidative stress when is administrated for 8 weeks in healthy postmenopausal,

Nevertheless there are also reports of foods that raise oxidative stress as with American gin‐ seng (AG) and wine. The AG supplementation at 500 mg every day for 4 months causes oxi‐ dative stress in postmenopausal women, due to reduced total antioxidant capacity, elevated plasma malondialdehyde and urine 8-hydroxydeoxyguanosine concentrations and in‐ creased erythrocyte antioxidant enzyme activity such as erythrocyte superoxide dismutase

Previous studies do not allow a conclusion on the effect of foods rich in antioxidant com‐ pounds, because were used different markers and administration time, and still more the age range of the postmenopausal differs considerably. This shows the need for more studies. Before shows the importance of further research with other foods with antioxidant proper‐

Vitamin C - Citrus fruits and their juices, berries, dark green vegetables (spinach, asparagus, green peppers, brussel sprouts, broccoli, watercress, other greens), red and yellow peppers,

Vitamin E - Vegetable oils such as olive, soybean, corn, cottonseed and safflower, nuts and nut butters, seeds, whole grains, wheat, wheat germ, brown rice, oatmeal, soybeans, sweet potatoes, legumes (beans, lentils, split peas) and dark leafy green vegetables. Selenium - Bra‐ zil nuts, brewer's yeast, oatmeal, brown rice, chicken, eggs, dairy products, garlic, molasses,

Beta Carotene - Variety of dark orange, red, yellow and green vegetables and fruits such as broccoli, kale, spinach, sweet potatoes, carrots, red and yellow peppers, apricots, cantaloupe

With respect to the lycopene, the following mechanism of the role of lycopene in chronic diseases has been mentioned by Agarwal and Rao [101] and Waliszewski and Blasco [102].

This highlights the importance of promote healthy lifestyles (balanced diet and moderate in‐ tensity exercise) in vulnerable populations, such as menopausal women, in order to prevent

tomatoes and tomato juice, pineapple, cantaloupe, mangos, papaya and guava.

onions, salmon, seafood, tuna, wheat germ, whole grains and most vegetables.

which was evaluated by the comet assay in peripheral blood lymphocytes [98].

with the dosage of polyphenols from (DAW) [96].

and GSH reductase [99].

ties known such as:

and mangos [100].

aging induced oxidative stress-related diseases.

With respect to hot flushes, they have been associated to smaller level of total antioxidant activity in plasma, without differences in nitrite-nitrate concentrations, and after HRT there is an increase in total antioxidant activity level and nitrite-nitrate concentrations in meno‐ pausal women, with and without hot flushes [88].

On the other side estrogen increases vasodilatation and inhibits the response of blood ves‐ sels to injury and the development of atherosclerosis, it has been related to hormone´s effect on serum lipid concentration, that is reducing MDA and oxLDL levels and increasing activi‐ ty of paroxonase PON1, which a calcium-dependent enzyme and in serum is exclusively lo‐ cated on HDL. PON is synthesized and secreted by liver and tightly binds to HDL subfractions that also contain apoA-1 and apoJ or clusterin and it has the capacity to protect LDL against oxidation [89, 90, 91].

### **7. Effect of nutrition and exercise on oxidative stress biomarkers**

Adequate nutrition and physical exercise are two factors of health promotion and its effect on oxidative stress has been investigated in postmenopausal women, which has given con‐ troversial data. With respect to foods, they contain large amount of antioxidant molecules from there arouse the interest to check if their use can reduce the oxidative stress observed in postmenopause.

For example it was reported that the intake of fresh, greenhouse-grown vegetables for 3-wk did not induced changes in the urine concentrations of 8-isoprostane F2α, hexanoyl lysine, and serum high sensitivity C-reactive protein despite that plasma carotenoids were elevated in overweight postmenopausal women [92]. Something similar was established with a 2 month supplementation period with the Klamath algae extract, which is an extract naturally rich in powerful algal antioxidant molecules (AFA-phycocyanins) and concentrated with Klamath algae's natural neuromodulators (phenylethylamine as well as natural selective MAO-B inhibitors), whose effect was to increase in the plasma levels of carotenoids, toco‐ pherols and retinol, however in this study oxidative stress was not measured [93, 94].

Otherwise is soy milk consumption for four weeks, which did not reduced markers of in‐ flammation and oxidative stress as (tumor necrosis factor alpha [TNF-alpha], interleukin [IL]-1beta, IL-6) and oxidative stress (superoxide dismutase [SOD], glutathione peroxidase [GPx], cyclooxygenase-2 [COX-2]) [95]. In this sense has also been found that de-alcoholised wine (DAW) with different polyphenol content by one month does not exert a protective ac‐ tivity towards oxidative DNA damage by comet assay, nor modifies significantly the gene expression profile of peripheral lymphocytes, whereas it shows blood-fluidifying actions, expressed as a significant decrease in blood viscosity. However, this effect does not correlate with the dosage of polyphenols from (DAW) [96].

Although there are reports that indicate the antioxidant effect of HRT, there are also studies that indicate otherwise; in example in another study combined HRT led to decreased plasma total and LDL cholesterol, but did not affect oxidizability and oxidation of LDL. Circulating levels of antioxidant vitamins (beta-carotene, vitamin C, vitamin E/triglycerides) and total antioxidant capacity of plasma and lipid peroxidation, assessed by plasma TBARs, were not different from controls in postmenopausal women receiving HRT, which indicates that com‐ bined HRT modifies the blood lipid profile, however it does not appear to influence oxida‐ tive status [86]. Additionaly DNA damage, GPx activity and nitrite level as well as a decreased GSH level were observed after oral administrating of estrogens alone or combi‐

With respect to hot flushes, they have been associated to smaller level of total antioxidant activity in plasma, without differences in nitrite-nitrate concentrations, and after HRT there is an increase in total antioxidant activity level and nitrite-nitrate concentrations in meno‐

On the other side estrogen increases vasodilatation and inhibits the response of blood ves‐ sels to injury and the development of atherosclerosis, it has been related to hormone´s effect on serum lipid concentration, that is reducing MDA and oxLDL levels and increasing activi‐ ty of paroxonase PON1, which a calcium-dependent enzyme and in serum is exclusively lo‐ cated on HDL. PON is synthesized and secreted by liver and tightly binds to HDL subfractions that also contain apoA-1 and apoJ or clusterin and it has the capacity to protect

Adequate nutrition and physical exercise are two factors of health promotion and its effect on oxidative stress has been investigated in postmenopausal women, which has given con‐ troversial data. With respect to foods, they contain large amount of antioxidant molecules from there arouse the interest to check if their use can reduce the oxidative stress observed

For example it was reported that the intake of fresh, greenhouse-grown vegetables for 3-wk did not induced changes in the urine concentrations of 8-isoprostane F2α, hexanoyl lysine, and serum high sensitivity C-reactive protein despite that plasma carotenoids were elevated in overweight postmenopausal women [92]. Something similar was established with a 2 month supplementation period with the Klamath algae extract, which is an extract naturally rich in powerful algal antioxidant molecules (AFA-phycocyanins) and concentrated with Klamath algae's natural neuromodulators (phenylethylamine as well as natural selective MAO-B inhibitors), whose effect was to increase in the plasma levels of carotenoids, toco‐

pherols and retinol, however in this study oxidative stress was not measured [93, 94].

Otherwise is soy milk consumption for four weeks, which did not reduced markers of in‐ flammation and oxidative stress as (tumor necrosis factor alpha [TNF-alpha], interleukin

**7. Effect of nutrition and exercise on oxidative stress biomarkers**

nated [87].

pausal women, with and without hot flushes [88].

304 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

LDL against oxidation [89, 90, 91].

in postmenopause.

In contrast, the intake for 8 weeks of soya protein diet and soya nut diet decreased MDA and increased the total antioxidant capacity in postmenopausal women with metabolic syn‐ drome [97]. Extra-virgin olive oils (EVOO) is another example of food which reduces DNA damage by oxidative stress when is administrated for 8 weeks in healthy postmenopausal, which was evaluated by the comet assay in peripheral blood lymphocytes [98].

Nevertheless there are also reports of foods that raise oxidative stress as with American gin‐ seng (AG) and wine. The AG supplementation at 500 mg every day for 4 months causes oxi‐ dative stress in postmenopausal women, due to reduced total antioxidant capacity, elevated plasma malondialdehyde and urine 8-hydroxydeoxyguanosine concentrations and in‐ creased erythrocyte antioxidant enzyme activity such as erythrocyte superoxide dismutase and GSH reductase [99].

Previous studies do not allow a conclusion on the effect of foods rich in antioxidant com‐ pounds, because were used different markers and administration time, and still more the age range of the postmenopausal differs considerably. This shows the need for more studies. Before shows the importance of further research with other foods with antioxidant proper‐ ties known such as:

Vitamin C - Citrus fruits and their juices, berries, dark green vegetables (spinach, asparagus, green peppers, brussel sprouts, broccoli, watercress, other greens), red and yellow peppers, tomatoes and tomato juice, pineapple, cantaloupe, mangos, papaya and guava.

Vitamin E - Vegetable oils such as olive, soybean, corn, cottonseed and safflower, nuts and nut butters, seeds, whole grains, wheat, wheat germ, brown rice, oatmeal, soybeans, sweet potatoes, legumes (beans, lentils, split peas) and dark leafy green vegetables. Selenium - Bra‐ zil nuts, brewer's yeast, oatmeal, brown rice, chicken, eggs, dairy products, garlic, molasses, onions, salmon, seafood, tuna, wheat germ, whole grains and most vegetables.

Beta Carotene - Variety of dark orange, red, yellow and green vegetables and fruits such as broccoli, kale, spinach, sweet potatoes, carrots, red and yellow peppers, apricots, cantaloupe and mangos [100].

With respect to the lycopene, the following mechanism of the role of lycopene in chronic diseases has been mentioned by Agarwal and Rao [101] and Waliszewski and Blasco [102].

This highlights the importance of promote healthy lifestyles (balanced diet and moderate in‐ tensity exercise) in vulnerable populations, such as menopausal women, in order to prevent aging induced oxidative stress-related diseases.

Whit respect to the exercise has been reported that who practice yoga or tai chi (TC) for a year have same BMI, and even more no effects were shown on erythrocyte superoxide dis‐ mutase activity, plasma lipid peroxidation (TBARS) or total homocysteine concentrations, but the activity of erythrocyte glutathione peroxidase - an aerobic training-responsive en‐ zyme - was higher in TC practitioners [103]. Although short term aerobic physical activity program (8 or 15 weeks) exhibited similar results, namely a reduction of serum glucose, LDL-cholesterol (LDL-C), plasma TBARS concentrations, decreasing of HOMA(IR) as well as an increased of total antioxidant status (TAS) of plasma and reduced glutathione (GSH) concentrations in red blood cells (RBC) increased significantly [104, 105].

This highlights the importance of promote healthy lifestyles (balanced diet and moderate in‐ tensity exercise) in vulnerable populations, such as menopausal women, in order to prevent

Menopause Induces Oxidative Stress http://dx.doi.org/10.5772/52082 307

The studies presented here were performed with different number of patients, methodolo‐ gies and biomarkers, but most of them indicate that estrogen depletion induces oxidative stress and hormone replacement therapy seems to reduce it. With respect to the modifica‐ tion of biomarkers of oxidative stress damage by food and exercise needs more research be‐

1 Section of Post graduate Studies and Research of Escuela Superior de Medicina- Instituto Politécnico Nacional. Street Salvador Díaz Mirón S/N, Colony Casco de Santo Tomás, Dele‐

2 Hospital psychiatry "Dr. Samuel Ramírez Moreno"-psychiatric careservices- Secretaria de Salud, highway México-Puebla Km 5.5, Colony Santa Catarina, Tláhuac, C.P. 13100, México

[1] Reddish, S. (2011). Menopausal transition- assessment in general practice. *Australian*

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[4] Guthrie, J. R., Dennerstein, L., Hopper, J. L., & Burger, H. G. (1996). Hot flushes, menstrual status and hormone levels in a population-based sample of midlife wom‐

Claudia Camelia Calzada Mendoza1\* and Carlos Alberto Jiménez Zamarripa2

aging induced oxidative stress-related diseases.

cause so far no conclusive data have been obtained.

\*Address all correspondence to: cccalzadam@yahoo.com.mx

gation Miguel Hidalgo, C.P. 11340, México D.F.

*Family Physician*, 40(5), 266-72.

*y Nutrición*, 14(3), 141-148.

en. *Obstetrics and Gynecology*, 88, 437-441.

**8. Conclusion**

**Author details**

D.F.

**References**

**Figure 9.** Lycopene and its mechanism in preventing of chronic diseases (Adapted from 101 and 102).

Regular physical training has been shown to upregulate antioxidant enzymatic systems, which may slow down the usual increase of oxidative stress in postmenopausal women, since it has been identified significant negative associations between oxidative stress and in‐ dices of physical fitness-activity (malondialdehyde, 8-iso-prostaglandin F2alpha, 8-hy‐ droxy-2'-deoxyguanosine). Conversely, glutathione peroxidase is positively correlated with fitness level, furthermore mean arterial blood pressure (MABP) and cerebrovascular con‐ ductance (CVC) are directly associated with 8-hydroxy-2'-deoxyguanosine, nitrotyrosine and nitric oxide (NO) These findings demonstrate that, after menopause, fitness level and regular physical activity mediate against oxidative stress by maintaining antioxidant en‐ zyme efficiency. Furthermore, these results suggest that oxidative stress and NO production modulate MABP and CVC [106].

Contrary to the above has also been reported, that the exercise does not modify the antioxi‐ dant status (although this is lower in metabolic healthy obese postmenopausal women than non-metabolic healthy obese postmenopausal women) and worse increases serum levels of thiobarbituric acid-reactive substances [107].

This highlights the importance of promote healthy lifestyles (balanced diet and moderate in‐ tensity exercise) in vulnerable populations, such as menopausal women, in order to prevent aging induced oxidative stress-related diseases.

### **8. Conclusion**

Whit respect to the exercise has been reported that who practice yoga or tai chi (TC) for a year have same BMI, and even more no effects were shown on erythrocyte superoxide dis‐ mutase activity, plasma lipid peroxidation (TBARS) or total homocysteine concentrations, but the activity of erythrocyte glutathione peroxidase - an aerobic training-responsive en‐ zyme - was higher in TC practitioners [103]. Although short term aerobic physical activity program (8 or 15 weeks) exhibited similar results, namely a reduction of serum glucose, LDL-cholesterol (LDL-C), plasma TBARS concentrations, decreasing of HOMA(IR) as well as an increased of total antioxidant status (TAS) of plasma and reduced glutathione (GSH)

concentrations in red blood cells (RBC) increased significantly [104, 105].

306 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 9.** Lycopene and its mechanism in preventing of chronic diseases (Adapted from 101 and 102).

modulate MABP and CVC [106].

thiobarbituric acid-reactive substances [107].

Regular physical training has been shown to upregulate antioxidant enzymatic systems, which may slow down the usual increase of oxidative stress in postmenopausal women, since it has been identified significant negative associations between oxidative stress and in‐ dices of physical fitness-activity (malondialdehyde, 8-iso-prostaglandin F2alpha, 8-hy‐ droxy-2'-deoxyguanosine). Conversely, glutathione peroxidase is positively correlated with fitness level, furthermore mean arterial blood pressure (MABP) and cerebrovascular con‐ ductance (CVC) are directly associated with 8-hydroxy-2'-deoxyguanosine, nitrotyrosine and nitric oxide (NO) These findings demonstrate that, after menopause, fitness level and regular physical activity mediate against oxidative stress by maintaining antioxidant en‐ zyme efficiency. Furthermore, these results suggest that oxidative stress and NO production

Contrary to the above has also been reported, that the exercise does not modify the antioxi‐ dant status (although this is lower in metabolic healthy obese postmenopausal women than non-metabolic healthy obese postmenopausal women) and worse increases serum levels of

The studies presented here were performed with different number of patients, methodolo‐ gies and biomarkers, but most of them indicate that estrogen depletion induces oxidative stress and hormone replacement therapy seems to reduce it. With respect to the modifica‐ tion of biomarkers of oxidative stress damage by food and exercise needs more research be‐ cause so far no conclusive data have been obtained.

### **Author details**

Claudia Camelia Calzada Mendoza1\* and Carlos Alberto Jiménez Zamarripa2

\*Address all correspondence to: cccalzadam@yahoo.com.mx

1 Section of Post graduate Studies and Research of Escuela Superior de Medicina- Instituto Politécnico Nacional. Street Salvador Díaz Mirón S/N, Colony Casco de Santo Tomás, Dele‐ gation Miguel Hidalgo, C.P. 11340, México D.F.

2 Hospital psychiatry "Dr. Samuel Ramírez Moreno"-psychiatric careservices- Secretaria de Salud, highway México-Puebla Km 5.5, Colony Santa Catarina, Tláhuac, C.P. 13100, México D.F.

### **References**


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314 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants


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**Chapter 13**

**Oxidative Stress in Periodontal Disease and Oral Cancer**

The oral cavity is a region interconnected with other systems of the body; it should not be viewed as an isolated area. Diseases that it lays down can have systemic scope and signifi‐ cantly affect the quality of life of individuals who suffer them. Periodontal disease is one of the oral health problems that most often affect the global population, lack of treatment leads to loss of tooth organs and consequently alters the digestion and nutrition, without consid‐ ering other relevant aspects as phonation, aesthetics and social or emotional impact. The im‐ portance of periodontal disease has raised possible bidirectional relationships with systemic diseases such as diabetes, metabolic syndrome and cardiovascular disease. We address here‐ in the role of oxidative stress in the etiopathogeny of periodontal disease. In the same con‐ text, another disease that has become relevant in our days is the oral cancer. Epidemiological data show that the incidence of this neoplasm has been increasing in several countries. The impact of oral cancer on patients, who suffer it, is devastating. The role of oxidative stress in the development of this disease and some alternatives for its treatment, are topics addressed in this brief review. These two oral diseases are a sample of the plethora of effects that oxi‐

Periodontitis is the second world health problem since it affects between 10 to 15% of the

destruction and inflammation present, the American Dental Association classifies according

Although the various states in this disease depend on the degree of

© 2013 Nava-Villalba et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Mario Nava-Villalba, German González-Pérez,

Additional information is available at the end of the chapter

dative stress may have at local and systemic level.

**2. Periodontal disease**

world population [1].

Maribel Liñan-Fernández and

Torres-Carmona Marco

http://dx.doi.org/10.5772/52492

**1. Introduction**


### **Oxidative Stress in Periodontal Disease and Oral Cancer**

Mario Nava-Villalba, German González-Pérez, Maribel Liñan-Fernández and Torres-Carmona Marco

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52492

### **1. Introduction**

[99] Dickman, J. R., Koenig, R. T., & Ji, L. L. (2009). American ginseng supplementation induces an oxidative stress in postmenopausal women. *Journal of the American of Col‐*

[100] Garcia P.M.C. (2008). Antioxidantes en la dietamediterranea. *Nutrición Clínica en Me‐*

[101] Agarwal, S., & Rao, A. V. (2000). Tomato lycopene and its role in human health and chronic diseases. *Canadian Medical Association or its licensors*, 163(6), 739-44.

[102] Waliszewski, K. N., & Blasco, G. (2010). Propiedadesnutraceúticasdellicopeno. *Salud‐*

[103] Palasuwan, A., Margaritis, I., Soogarun, S., & Rousseau, A. S. (2011). Dietary intakes and antioxidant status in mind-body exercising pre- and postmenopausal women.

[104] Karolkiewicz, J., Michalak, E., Pospieszna, B., Deskur-Smielecka, E., Nowak, A., & Pi‐ laczyńska-Szcześniak, Ł. (2009). Response of oxidative stress markers and antioxi‐ dant parameters to an 8week aerobic physical activity program in healthy,

[105] Schmitz, K. H., Warren, M., Rundle, A. G., Williams, N. I., Gross, M. D., & Kurzer, M. S. (2008). Exercise effect on oxidative stress is independent of change in estrogen me‐

[106] Pialoux, V., Brown, A. D., Leigh, R., Friedenreich, C. M., & Poulin, M. J. (2009). Effect of cardiorespiratory fitness on vascular regulation and oxidative stress in postmeno‐

[107] Lwow, F., Dunajska, K., Milewicz, A., Jedrzejuk, D., Kik, K., & Szmigiero, L. (2011). Effect of moderate-intensity exercise on oxidative stress indices in metabolically healthy obese and metabolically unhealthy obese phenotypes in postmenopausal

postmenopausal women. *Archives of Gerontology and Geriatrics* 49(1)e67-71.

tabolism. *Cancer Epidemiology Biomarkers and Prevention*, 17(1), 220-3.

*The Journal of Nutrition Health and Aging*, 15(7), 577-84.

pausal women. *Hypertension*, 54(5), 1014-20.

women: a pilot study. *Menopause*, 18(6), 646-53.

*lege of Nutrition*, 28(2), 219-28.

316 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

*Publica Mexicana*, 52, 254-265.

*dicina*, 2(3), 129-140.

The oral cavity is a region interconnected with other systems of the body; it should not be viewed as an isolated area. Diseases that it lays down can have systemic scope and signifi‐ cantly affect the quality of life of individuals who suffer them. Periodontal disease is one of the oral health problems that most often affect the global population, lack of treatment leads to loss of tooth organs and consequently alters the digestion and nutrition, without consid‐ ering other relevant aspects as phonation, aesthetics and social or emotional impact. The im‐ portance of periodontal disease has raised possible bidirectional relationships with systemic diseases such as diabetes, metabolic syndrome and cardiovascular disease. We address here‐ in the role of oxidative stress in the etiopathogeny of periodontal disease. In the same con‐ text, another disease that has become relevant in our days is the oral cancer. Epidemiological data show that the incidence of this neoplasm has been increasing in several countries. The impact of oral cancer on patients, who suffer it, is devastating. The role of oxidative stress in the development of this disease and some alternatives for its treatment, are topics addressed in this brief review. These two oral diseases are a sample of the plethora of effects that oxi‐ dative stress may have at local and systemic level.

### **2. Periodontal disease**

Periodontitis is the second world health problem since it affects between 10 to 15% of the world population [1]. Although the various states in this disease depend on the degree of destruction and inflammation present, the American Dental Association classifies according

© 2013 Nava-Villalba et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. to a system development based on the severity of the loss of periodontal insertion. The in‐ formation obtained in clinical and radiographic examination classifies the patient in four typical cases that are:

there are acid phosphatase and alkaline, lipopolysaccharides, aminopeptidase, epithelium

Oxidative Stress in Periodontal Disease and Oral Cancer

http://dx.doi.org/10.5772/52492

319

Bacteria causes tissue destruction with its deletion, this is a feature of marginal periodontitis products. Destruction of tissues within a radius of 1.5 to 2.5 mm around the plaque has been ob‐ served (the so-called *influence radioplate)* in periodontitis. The hydrolysis of the connective tis‐ sue associated with the inflammation is due to the reactive oxygen species and the elastase/ lysosomic-like enzymes. Collagenase and gelatinase are segregated to the microenvironment. Prostaglandin E, Interleukin 1-/ J and the lipopolysaccharide activates osteoclasts and induce a resorption of alveolar bone. Cellular and humoral components of the immune system, mainly involved in the periodontal immune response are leukocytes, immunoglobulins, complement system and lysozyme. If the immune defenses are working properly, the periodontium is pro‐ tected from the harmful effect of pathogenic substances secreted by the microorganisms. The immunocompetent host is able to defend itself against microbial attacks that occur every day. Thus prevents *infections,* i.e. the multiplication of microorganisms within the periodontium. We can say that the periodontal inflammation is a local reaction to a tissue injury whose purpose is

The human immune system can be classified according to their function within the perio‐

The system formed by neutrophils, antibodies and complement is crucial to the immune de‐ fense against periodontal infections. When functional defects of neutrophils occur, it increas‐

A phenomenon that occurs within the periodontal disease is called oxidative stress. In order to understand the phenomenon of oxidative stress it is important to know what the free rad‐ icals (FR) are, where they come from and how to act. A FR is considered that molecule pre‐ sented an electron unpaired or odd in the orbital external, in its atomic structure giving it a spatial configuration that generates a high instability. In the molecule of oxygen (O2) know the following FR or also called oxygen reactive species: anion superoxide (O2˙ˉ), hydrogen

FR but by its ability to generate the OH˙ in the presence of metals such as iron, it incorpo‐ rates it as such. A fundamental characteristic of the reactions of free radicals is that act of

O2). The H2O2 is not strictly a

toxin, inhibitor of fibroblasts and a toxin that induces a bone resorption.

the destruction of the causal factor, dilution or its encapsulation.

**•** Neutrophils, antibodies and complement system

es the frequency of serious marginal periodontitis [4].

peroxide (H2O2), hydroxyl radical (OH˙) and singlet oxygen (<sup>1</sup>

chain reactions, where a radical reaction generates another consecutively.

dontium, follows: **•** Secretory system

**•** Leukocytes and macrophages

**•** Immune regulation system.

**3. Oxidative stress**


There are other classifications of the inflammatory process [2]:


Periodontal disease is an inflammatory process involving a set of changes that directly affect tissues that hold the teeth. The etiology plays a role which is essential within the bacterial infection. In fact, within the 300 to 400 species of bacteria located in the oral cavity consider that some of them are exclusive to the periodontal tissues. However in recent years it has been determined that the evolution and spread of the disease will play a decisive role in the host response to bacterial attack. This is reflected in the model of the critical path in the pathogenesis of this disease. Through this one can understand that there are diseases and systemic conditions that have risk factors for periodontal disease, because they are going to modify the host response and favor the development of damage [3].

When it is lost in the inclusion of periodontal fibers, usually after puberty, the cases that are reported before this stage are only 5%. Previously it has reported that there was a ratio of two to one in the frequency of periodontal disease, women being the most affected in this order. Currently known, the presence by gender of this involvement is very similar.

In adults with more than 1 mm of affected dental faces periodontal insertion loss increases with age. An epidemiological report in United States mentions that approximately 80-92% of the population between the ages of 35 and 64 years performed, lost more than 1 mm inser‐ tion in 20 to 47% of teeth. From 18 to 22% of the population of 35 to 64 years were more 2 mm deep in the probing of the periodontal bags in 11 to 13% of tooth surfaces. Periodontitis occurs when tissue destruction due to the direct effect of bacterial toxins and removal prod‐ ucts, in addition, the effects caused indirectly by the harmful organic defense mechanisms. Microorganisms as *p. gingivalis, a. actinomycetemcomitans* and *Capnocytophaga*sp. produce col‐ lagenase (substances similar to trypsin) and phospholipase, among others. Extracellularly there are acid phosphatase and alkaline, lipopolysaccharides, aminopeptidase, epithelium toxin, inhibitor of fibroblasts and a toxin that induces a bone resorption.

Bacteria causes tissue destruction with its deletion, this is a feature of marginal periodontitis products. Destruction of tissues within a radius of 1.5 to 2.5 mm around the plaque has been ob‐ served (the so-called *influence radioplate)* in periodontitis. The hydrolysis of the connective tis‐ sue associated with the inflammation is due to the reactive oxygen species and the elastase/ lysosomic-like enzymes. Collagenase and gelatinase are segregated to the microenvironment. Prostaglandin E, Interleukin 1-/ J and the lipopolysaccharide activates osteoclasts and induce a resorption of alveolar bone. Cellular and humoral components of the immune system, mainly involved in the periodontal immune response are leukocytes, immunoglobulins, complement system and lysozyme. If the immune defenses are working properly, the periodontium is pro‐ tected from the harmful effect of pathogenic substances secreted by the microorganisms. The immunocompetent host is able to defend itself against microbial attacks that occur every day. Thus prevents *infections,* i.e. the multiplication of microorganisms within the periodontium. We can say that the periodontal inflammation is a local reaction to a tissue injury whose purpose is the destruction of the causal factor, dilution or its encapsulation.

The human immune system can be classified according to their function within the perio‐ dontium, follows:

**•** Secretory system

to a system development based on the severity of the loss of periodontal insertion. The in‐ formation obtained in clinical and radiographic examination classifies the patient in four

Periodontal disease is an inflammatory process involving a set of changes that directly affect tissues that hold the teeth. The etiology plays a role which is essential within the bacterial infection. In fact, within the 300 to 400 species of bacteria located in the oral cavity consider that some of them are exclusive to the periodontal tissues. However in recent years it has been determined that the evolution and spread of the disease will play a decisive role in the host response to bacterial attack. This is reflected in the model of the critical path in the pathogenesis of this disease. Through this one can understand that there are diseases and systemic conditions that have risk factors for periodontal disease, because they are going to

When it is lost in the inclusion of periodontal fibers, usually after puberty, the cases that are reported before this stage are only 5%. Previously it has reported that there was a ratio of two to one in the frequency of periodontal disease, women being the most affected in this

In adults with more than 1 mm of affected dental faces periodontal insertion loss increases with age. An epidemiological report in United States mentions that approximately 80-92% of the population between the ages of 35 and 64 years performed, lost more than 1 mm inser‐ tion in 20 to 47% of teeth. From 18 to 22% of the population of 35 to 64 years were more 2 mm deep in the probing of the periodontal bags in 11 to 13% of tooth surfaces. Periodontitis occurs when tissue destruction due to the direct effect of bacterial toxins and removal prod‐ ucts, in addition, the effects caused indirectly by the harmful organic defense mechanisms. Microorganisms as *p. gingivalis, a. actinomycetemcomitans* and *Capnocytophaga*sp. produce col‐ lagenase (substances similar to trypsin) and phospholipase, among others. Extracellularly

order. Currently known, the presence by gender of this involvement is very similar.

typical cases that are: **•** Type I: Gingivitis

**•** Acute gingivitis

**•** Chronic gingivitis

**•** Marginal periodontitis

**•** Type II: Mild Periodontitis

**•** Type III: Moderate Periodontitis

**•** Type IV: Advanced Periodontitis

**•** Ulceronecrotic acute gingivitis

**•** Superficial marginal periodontitis

**•** Deep marginal periodontitis

There are other classifications of the inflammatory process [2]:

318 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

modify the host response and favor the development of damage [3].


The system formed by neutrophils, antibodies and complement is crucial to the immune de‐ fense against periodontal infections. When functional defects of neutrophils occur, it increas‐ es the frequency of serious marginal periodontitis [4].

### **3. Oxidative stress**

A phenomenon that occurs within the periodontal disease is called oxidative stress. In order to understand the phenomenon of oxidative stress it is important to know what the free rad‐ icals (FR) are, where they come from and how to act. A FR is considered that molecule pre‐ sented an electron unpaired or odd in the orbital external, in its atomic structure giving it a spatial configuration that generates a high instability. In the molecule of oxygen (O2) know the following FR or also called oxygen reactive species: anion superoxide (O2˙ˉ), hydrogen peroxide (H2O2), hydroxyl radical (OH˙) and singlet oxygen (<sup>1</sup> O2). The H2O2 is not strictly a FR but by its ability to generate the OH˙ in the presence of metals such as iron, it incorpo‐ rates it as such. A fundamental characteristic of the reactions of free radicals is that act of chain reactions, where a radical reaction generates another consecutively.

Oxygen (O2) that this in the air is fundamental to life, many reactions in which participates the O2 generates reactive oxygen species (ROS), of which some have the chemical character of being free radical (FR), whose biochemical entities in its atomic structure presented an odd or unpaired electron in the outer orbital, giving it a spatial configuration that generates a high instability with an enormous capacity for combined with the diversity of molecules members of the cell structure: carbohydrates, lipids, proteins, nucleic acids, and derivatives of each of them, causing important functional alterations. In this sense, the body has an anti‐ oxidant system to counteract the generation of ROS, which maintains a homeostatic balance. However there are pro-oxidant factors that favor the generation of FR, causing an imbalance in favor of the latter, generating so-called oxidative stress (OS) [5].

ozone. The living organism has adapted to an existence under a continuous output of radi‐ cal free flow. Between the different antioxidant defense mechanism adaptation mechanism is of great importance. Antioxidants are "those substances that when they are present in lower concentrations compared to the substrate of an oxidizable, significantly delay or in‐ hibit the oxidation of the substrate". The various possible mechanisms that antioxidants can

Oxidative Stress in Periodontal Disease and Oral Cancer

http://dx.doi.org/10.5772/52492

321

**•** Interception of the radical free to eliminate reactive metabolites and their conversion to

Antioxidant defense system is very dynamic and responsive to any disturbance that occurs in the body redox balance. Antioxidants can be regulated and neutralize the formation of radical free that can occur due to oxidative stress, such as the factor transcription factors Ac‐ tivator protein 1 and nuclear-kb are redox sensitive. Redox potential is a measure of the af‐

The presence of inflammatory infiltrate is a constant feature in periodontal disease. It is known that these cells release lots of free radicals; it is suspected that these metabolites are involved in the pathogenesis of the disease. The presence of a dense inflammatory infiltrate in periodontal disease leads to the suspicion that the relationship of periodontal leukocytetissue has a double aspect. The role of these cells in the containment of the gingival bacteria and their products must be analyzed according to a balance with the destruction of tissue due to the release of the products of its action (FR and proteases). In this way, a defensive mechanism, under the interaction of various factors, can be harmful to periodontal tissues, and they are therefore involved in the pathogenesis of inflammatory periodontal disease. There is numerous evidence pointing to the involvement of FR in periodontal disease. It has been reported in patients with rapidly progressive periodontitis, that the polymorphonu‐ clear neutrophils (PMN) are functionally activated, produce high levels of O2 and have a high response the luminol-dependent (QL) chemiluminescent. There is an increase of the PMN oxidative response peripherals in patients with localized and generalized juvenile pe‐ riodontitis, as well as in adult patients with periodontitis (AP). This increase is related to

It has also compared the generation O2 by the activated PMN in the gingival crevicular fluid (GCF) of patients with AP. The PMN activation with phorbolmyristateacetate causes a marked increase in the release of O2 in patients with AP, while the antioxidant activity of the gum is similar to the controls. The effect of the PMN in crevicular fluid of patients is de‐ pendent on variations in the rate of formation of O2, relative to the intrinsic antioxidant ca‐

In gingival epithelial cells in culture studies have shown the PMN may cause lysis of these through the action of the free myeloperoxidase(MPO), a leukocyte enzyme generating radi‐

**•** Create a favourable environment for the effective functioning of other antioxidants.

offer protection against damage from free radicals are:

**•** Facilitate the repair of the damage caused by free radicals.

clinical periodontal status and is reversed by therapy.

pacity of the gingival tissue.

**•** The prevention of the formation of radical free.

less reactive molecules.

finity of a substance for electrons [8].

The tetravalent reduction of oxygen to produce water through the electron transport chain in mitochondria is relatively safe. However, the univalent reduction of oxygen generates ROS. The human organism also has antioxidant system to counteract the generation of ROS, which maintains a homeostatic balance. However, there are pro-oxidant factors that favor the generation of FR, causing an imbalance in favor of the latter, generating OS. The antioxi‐ dant enzyme superoxide dismutase (SOD), Glutathione peroxidase (GP), glutathione reduc‐ tase (GR) and catalase (CAT), as well as proteins carriers of metals (ceruplasmina, transferrin, lactoferrin, etc.), and another micronutrients as vitamins A, C and E, bilirubin, uric acid and selenium, constitute the most important elements of the antioxidant system. Also, between the most important pro-oxidant factors we can highlight the process of aging, ionizing radiation, ultraviolet rays, environmental pollution, cigarette smoke, excess of exer‐ cise, intake of alcoholic beverages and inadequate diet [6].

The role of Coenzyme Q10 is the mitochondrial energy coupling. It is an essential part of the cel‐ lular machinery used to produce ATP that provides the energy for muscle contraction and oth‐ er vital cellular functions. Most of the ATP production occurs in the inner membrane of the mitochondria, where the Coenzyme Q10 is located. The most important function is serving as a suppressor of primary free radicals, located in the membranes in the vicinity of unsaturated lipid chains. There are less established functions that include the oxidation/reduction of the control of the origin and transmission of signals in cells that induce the expression of gender, the control of membrane channels, the structure and solubility in lipids [7].

Free radicals cause damage to periodontal tissues by a variety of different mechanisms in‐ cluding:


ROS covers other reactive species that are not true radicals, but are however capable of react in intra and extracellular environment: peroxide of hydrogen, hypochlorous acid, oxygen, ozone. The living organism has adapted to an existence under a continuous output of radi‐ cal free flow. Between the different antioxidant defense mechanism adaptation mechanism is of great importance. Antioxidants are "those substances that when they are present in lower concentrations compared to the substrate of an oxidizable, significantly delay or in‐ hibit the oxidation of the substrate". The various possible mechanisms that antioxidants can offer protection against damage from free radicals are:

**•** The prevention of the formation of radical free.

Oxygen (O2) that this in the air is fundamental to life, many reactions in which participates the O2 generates reactive oxygen species (ROS), of which some have the chemical character of being free radical (FR), whose biochemical entities in its atomic structure presented an odd or unpaired electron in the outer orbital, giving it a spatial configuration that generates a high instability with an enormous capacity for combined with the diversity of molecules members of the cell structure: carbohydrates, lipids, proteins, nucleic acids, and derivatives of each of them, causing important functional alterations. In this sense, the body has an anti‐ oxidant system to counteract the generation of ROS, which maintains a homeostatic balance. However there are pro-oxidant factors that favor the generation of FR, causing an imbalance

The tetravalent reduction of oxygen to produce water through the electron transport chain in mitochondria is relatively safe. However, the univalent reduction of oxygen generates ROS. The human organism also has antioxidant system to counteract the generation of ROS, which maintains a homeostatic balance. However, there are pro-oxidant factors that favor the generation of FR, causing an imbalance in favor of the latter, generating OS. The antioxi‐ dant enzyme superoxide dismutase (SOD), Glutathione peroxidase (GP), glutathione reduc‐ tase (GR) and catalase (CAT), as well as proteins carriers of metals (ceruplasmina, transferrin, lactoferrin, etc.), and another micronutrients as vitamins A, C and E, bilirubin, uric acid and selenium, constitute the most important elements of the antioxidant system. Also, between the most important pro-oxidant factors we can highlight the process of aging, ionizing radiation, ultraviolet rays, environmental pollution, cigarette smoke, excess of exer‐

The role of Coenzyme Q10 is the mitochondrial energy coupling. It is an essential part of the cel‐ lular machinery used to produce ATP that provides the energy for muscle contraction and oth‐ er vital cellular functions. Most of the ATP production occurs in the inner membrane of the mitochondria, where the Coenzyme Q10 is located. The most important function is serving as a suppressor of primary free radicals, located in the membranes in the vicinity of unsaturated lipid chains. There are less established functions that include the oxidation/reduction of the control of the origin and transmission of signals in cells that induce the expression of gender,

Free radicals cause damage to periodontal tissues by a variety of different mechanisms in‐

ROS covers other reactive species that are not true radicals, but are however capable of react in intra and extracellular environment: peroxide of hydrogen, hypochlorous acid, oxygen,

in favor of the latter, generating so-called oxidative stress (OS) [5].

320 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cise, intake of alcoholic beverages and inadequate diet [6].

**•** The oxidation of important enzymes (anti proteases)

**•** Stimulation and release of pro-inflammatory cytokines

cluding:

**•** DNA damage

**•** Lipid peroxidation

**•** Protein damage

the control of membrane channels, the structure and solubility in lipids [7].


Antioxidant defense system is very dynamic and responsive to any disturbance that occurs in the body redox balance. Antioxidants can be regulated and neutralize the formation of radical free that can occur due to oxidative stress, such as the factor transcription factors Ac‐ tivator protein 1 and nuclear-kb are redox sensitive. Redox potential is a measure of the af‐ finity of a substance for electrons [8].

The presence of inflammatory infiltrate is a constant feature in periodontal disease. It is known that these cells release lots of free radicals; it is suspected that these metabolites are involved in the pathogenesis of the disease. The presence of a dense inflammatory infiltrate in periodontal disease leads to the suspicion that the relationship of periodontal leukocytetissue has a double aspect. The role of these cells in the containment of the gingival bacteria and their products must be analyzed according to a balance with the destruction of tissue due to the release of the products of its action (FR and proteases). In this way, a defensive mechanism, under the interaction of various factors, can be harmful to periodontal tissues, and they are therefore involved in the pathogenesis of inflammatory periodontal disease.

There is numerous evidence pointing to the involvement of FR in periodontal disease. It has been reported in patients with rapidly progressive periodontitis, that the polymorphonu‐ clear neutrophils (PMN) are functionally activated, produce high levels of O2 and have a high response the luminol-dependent (QL) chemiluminescent. There is an increase of the PMN oxidative response peripherals in patients with localized and generalized juvenile pe‐ riodontitis, as well as in adult patients with periodontitis (AP). This increase is related to clinical periodontal status and is reversed by therapy.

It has also compared the generation O2 by the activated PMN in the gingival crevicular fluid (GCF) of patients with AP. The PMN activation with phorbolmyristateacetate causes a marked increase in the release of O2 in patients with AP, while the antioxidant activity of the gum is similar to the controls. The effect of the PMN in crevicular fluid of patients is de‐ pendent on variations in the rate of formation of O2, relative to the intrinsic antioxidant ca‐ pacity of the gingival tissue.

In gingival epithelial cells in culture studies have shown the PMN may cause lysis of these through the action of the free myeloperoxidase(MPO), a leukocyte enzyme generating radi‐ cals. Its activity has been increased in the crevicular fluid of sites with gingivitis and perio‐ dontitis with respect to healthy sites.

highest frequency (Figure 3).Since oral squamous cell carcinoma (OSCC) is the main malig‐

Oxidative Stress in Periodontal Disease and Oral Cancer

http://dx.doi.org/10.5772/52492

323

**Figure 1.** Squamous cell cancer of the posterior lateral border of the tongue in a 28-year-old woman.She smoked a

**Figure 2.** Squamous cell cancer of floor of mouth in a 58-year-old woman.She had a history of poorly controlled dia‐ betes type 2 from 42 years. She also has used ill-fitting dentures since age 50. Note the linear lesion with presence of

nant neoplasia we focus in it.

cigarette per day for 15 years.

necrosis in the centre of the fissure.

There is a close relationship between free radical production by leukocytes and activation of proteases. Altogether these actions could have profound effects on the function and integri‐ ty of the gingival epithelium.

The above evidence leads to consider that in the inflammatory periodontal disease, the gen‐ eral etiological factors causing the breakup of physiological systems of inhibition of lipid peroxidation, creates a low level of antioxidant protection of periodontal tissues. In these cir‐ cumstances, the local factors lead to the migration of neutrophils to the gingiva and gingival fluid. The activation of these leukocytes in phagocytosis, causes the release of ROS, which leads to the outbreak of the lipid peroxidation of the soft tissues of the periodontium and activation of protease. This lipid peroxidation is the mechanism that triggers the develop‐ ment of morphofunctionalchangesin periodontium and their vessels, which results in de‐ struction of collagen and bone resorption.

Due to numberless evidences that suggest a participation of the ROS in the pathogenesis of the periodontal disease, it has been raised that the factors that promote a rupture of the anti‐ oxidant physiological system, contribute to the development of oxidative mechanisms that initiate the periodontitis. The main cause of lipid peroxidation in the periodontal disease seems to lie in the liberation of ROS by leukocytes in phagocytosis. These concepts empha‐ size the utility of antioxidants in the prophylaxis and treatment of periodontal disease and therefore justify the search of new antioxidant preparations for this purpose. For example the *p. gingivalis* is a major cause of periodontitis, and their presence is a risk factor for sys‐ temic inflammatory syndromes, such as atherosclerosis and cardiac dysfunction. The capaci‐ ty of the virulence factors such as proteases and LPS to induce inflammation has been studied intensely. In some cases, however, the inflammation occurs regardless of these fac‐ tors, suggesting the existence of other stimulating immune. It was found that the cell death induced by *p. gingivalis*in the tissues is through the production of ROS [9].

### **4. Oral Cancer**

The oral cancer occupies 2-5 % of all whole body cancers. This percentage places this neopla‐ sia within the ten most common cancers [10]. Although its magnitude is relatively low, its impact on affected patients and their costs in health systems is high.There is a considerable variation in the incidence and mortality rates around the world. The incidence is greater in south of India, Australia, North of America, many European countries, Brazil, certain coun‐ tries of Africa and some of central Asia [11]. 90% of oral cancer is of epithelial origin and the rest 10% are distributing in adenocarcinomas, sarcomas, lymphoproliferative disorders, metastasis, melanomas and malignant odontogenic tumors. The intraoral main site of oral squamous cell cancer (OSCC) is the posterior lateral border of tongue (Figure 1) and floor of mouth (Figure 2). If the lips are considered within the oral territory, then this site has the highest frequency (Figure 3).Since oral squamous cell carcinoma (OSCC) is the main malig‐ nant neoplasia we focus in it.

cals. Its activity has been increased in the crevicular fluid of sites with gingivitis and perio‐

There is a close relationship between free radical production by leukocytes and activation of proteases. Altogether these actions could have profound effects on the function and integri‐

The above evidence leads to consider that in the inflammatory periodontal disease, the gen‐ eral etiological factors causing the breakup of physiological systems of inhibition of lipid peroxidation, creates a low level of antioxidant protection of periodontal tissues. In these cir‐ cumstances, the local factors lead to the migration of neutrophils to the gingiva and gingival fluid. The activation of these leukocytes in phagocytosis, causes the release of ROS, which leads to the outbreak of the lipid peroxidation of the soft tissues of the periodontium and activation of protease. This lipid peroxidation is the mechanism that triggers the develop‐ ment of morphofunctionalchangesin periodontium and their vessels, which results in de‐

Due to numberless evidences that suggest a participation of the ROS in the pathogenesis of the periodontal disease, it has been raised that the factors that promote a rupture of the anti‐ oxidant physiological system, contribute to the development of oxidative mechanisms that initiate the periodontitis. The main cause of lipid peroxidation in the periodontal disease seems to lie in the liberation of ROS by leukocytes in phagocytosis. These concepts empha‐ size the utility of antioxidants in the prophylaxis and treatment of periodontal disease and therefore justify the search of new antioxidant preparations for this purpose. For example the *p. gingivalis* is a major cause of periodontitis, and their presence is a risk factor for sys‐ temic inflammatory syndromes, such as atherosclerosis and cardiac dysfunction. The capaci‐ ty of the virulence factors such as proteases and LPS to induce inflammation has been studied intensely. In some cases, however, the inflammation occurs regardless of these fac‐ tors, suggesting the existence of other stimulating immune. It was found that the cell death

The oral cancer occupies 2-5 % of all whole body cancers. This percentage places this neopla‐ sia within the ten most common cancers [10]. Although its magnitude is relatively low, its impact on affected patients and their costs in health systems is high.There is a considerable variation in the incidence and mortality rates around the world. The incidence is greater in south of India, Australia, North of America, many European countries, Brazil, certain coun‐ tries of Africa and some of central Asia [11]. 90% of oral cancer is of epithelial origin and the rest 10% are distributing in adenocarcinomas, sarcomas, lymphoproliferative disorders, metastasis, melanomas and malignant odontogenic tumors. The intraoral main site of oral squamous cell cancer (OSCC) is the posterior lateral border of tongue (Figure 1) and floor of mouth (Figure 2). If the lips are considered within the oral territory, then this site has the

induced by *p. gingivalis*in the tissues is through the production of ROS [9].

dontitis with respect to healthy sites.

322 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

struction of collagen and bone resorption.

ty of the gingival epithelium.

**4. Oral Cancer**

**Figure 1.** Squamous cell cancer of the posterior lateral border of the tongue in a 28-year-old woman.She smoked a cigarette per day for 15 years.

**Figure 2.** Squamous cell cancer of floor of mouth in a 58-year-old woman.She had a history of poorly controlled dia‐ betes type 2 from 42 years. She also has used ill-fitting dentures since age 50. Note the linear lesion with presence of necrosis in the centre of the fissure.

There are premalignant lesions recognized like: leukoplakia, erythroplakia, oralsubmucosal fibrosis, palatal lesions of reverse cigar smoking, oral liken planus, discoid lupus erythema‐ tosus, and hereditary disorders likecongenital dyskeratosis and epidermolysisbullosa, but beyond of a clinical standpoint, diverse carcinogenic molecular mechanisms have been postulated.The main target is the DNA, since mutations that occur in it generates a wide range of deleterious effects in the cell. In a very general overview, the balance between tu‐ mor suppressor genes and those genes that induce cell cycle is altered.Allowing cells to es‐ cape cell cycle control and developing an unpredictable biological behavior. Subsequently, the cells express molecules that allow them to acquire an invasive phenotype, a phenomen‐ on known as epithelial-mesenchymal transition. Why malignant cells colonize distant sites? Is not yet fully understood, but it is the feature that makes it lethal.

Several works explore the levels of oxidative stress in patients with oral cancer [13-15] most of them quantified the products of lipid-peroxidation(mainly malonilaldehyde) and contrast them with the activity of antioxidant enzymes or exogenous antioxidants levels in blood or even saliva. The results agree that there is an imbalance between the high amount of free radicals and insufficient antioxidant system activity.Added to this, some researchers have observed that high levels of lipid-peroxidation combined with low levels of thiols and anti‐

Oxidative Stress in Periodontal Disease and Oral Cancer

http://dx.doi.org/10.5772/52492

325

The OSCC is a multifactorial disease, however, a factor strongly associated, is smoking. 90% of individuals with oral cancer are smokers. It is considered that the smoke from cigarettes have 4000 chemicals, 40 of which have carcinogenic potential. It has been shown that ciga‐ rette smoke contains pro-oxidants that are capable of initiating the process of lipid-peroxida‐

In contrast, there is epidemiological evidence that demonstrates the protective effect of diet on some populations [19-21].For example in Greece, which has the lowest rates of oral can‐ cer among European countries,its population is exposed to latent risk factors such as alcohol intake and smoking; micronutrients consume such as riboflavin, magnesium and iron corre‐

Consequently, several authors have proposed the ingestion of diverse exogenous antioxi‐ dants; supporting in those epidemiological studies, where the diet offers protection for the development of cancer, and taking into account that the endogenous antioxidant systems

For example, vitamin C is one of the most extensively evaluated antioxidants in oral cancer alternative co-therapies. Low or even undetectable levels of vitamin C correlate with the presence of oral cancer [17, 22]; in contrast, is one of the micronutrients that have a consis‐ tent inverse correlation in different studies [23].Vitamin C acts as a scavenger of free radicals and impedes the detrimental chain reactions triggered by the free radicals.The l-glutamine is another antioxidant that has shown a beneficial modulating effect in patients with oral can‐ cer in stages III and IV. The l-glutamine is administered in the diet as a complementary ther‐ apy; the proposal is that restores glutathione cascade system [15].In addition, other antioxidants such as carotene, vitamin E, thiamine, vitamin B6, folic acid, niacin and potassi‐ um have shown a convincing protective effect [24]. Even more,when them are administered

, Maribel Liñan-Fernández3

and

oxidant status, correlate with poor survival rate in patients with oral cancer [16].

tion and deplete levels of antioxidants from the diet [17,18].

lated inversely with oral cancer [19].

have been overwhelmed by oxidative stress.

together during the cycles of radiotherapy [25].

Mario Nava-Villalba, German González-Pérez2

\*Address all correspondence to: marionava23@gmail.com

**Author details**

Torres-Carmona Marco4

**Figure 3.** Squamous cell cancer of the lip in a 74-year-old man. He was a farmer and consumed alcohol chronically.

Free radicals are products of the oxidation-reduction systems of the cell and its participation in cellular metabolic functions is essential for cell survival. A classic example is the electron transport chain in mitochondria. However, in whatpathologicalconditions, free radicals can become deleterious? In fact, what are the results of its harmful effects? The involvement of free radicals in cancer development has been studied for 3 decades, and there is sufficient evidence that implicates theirs in the multistage theory of carcinogenesis. They are proposed to cause diverse DNA alterations like: punctual mutations, DNA base oxidations, strand breaks, mutation of tumor suppressor genes and can induce overexpression of proto-onco‐ genes [12].It should be added that oxidative protein damage participates in facilitating the development of cancer.

Several works explore the levels of oxidative stress in patients with oral cancer [13-15] most of them quantified the products of lipid-peroxidation(mainly malonilaldehyde) and contrast them with the activity of antioxidant enzymes or exogenous antioxidants levels in blood or even saliva. The results agree that there is an imbalance between the high amount of free radicals and insufficient antioxidant system activity.Added to this, some researchers have observed that high levels of lipid-peroxidation combined with low levels of thiols and anti‐ oxidant status, correlate with poor survival rate in patients with oral cancer [16].

The OSCC is a multifactorial disease, however, a factor strongly associated, is smoking. 90% of individuals with oral cancer are smokers. It is considered that the smoke from cigarettes have 4000 chemicals, 40 of which have carcinogenic potential. It has been shown that ciga‐ rette smoke contains pro-oxidants that are capable of initiating the process of lipid-peroxida‐ tion and deplete levels of antioxidants from the diet [17,18].

In contrast, there is epidemiological evidence that demonstrates the protective effect of diet on some populations [19-21].For example in Greece, which has the lowest rates of oral can‐ cer among European countries,its population is exposed to latent risk factors such as alcohol intake and smoking; micronutrients consume such as riboflavin, magnesium and iron corre‐ lated inversely with oral cancer [19].

Consequently, several authors have proposed the ingestion of diverse exogenous antioxi‐ dants; supporting in those epidemiological studies, where the diet offers protection for the development of cancer, and taking into account that the endogenous antioxidant systems have been overwhelmed by oxidative stress.

For example, vitamin C is one of the most extensively evaluated antioxidants in oral cancer alternative co-therapies. Low or even undetectable levels of vitamin C correlate with the presence of oral cancer [17, 22]; in contrast, is one of the micronutrients that have a consis‐ tent inverse correlation in different studies [23].Vitamin C acts as a scavenger of free radicals and impedes the detrimental chain reactions triggered by the free radicals.The l-glutamine is another antioxidant that has shown a beneficial modulating effect in patients with oral can‐ cer in stages III and IV. The l-glutamine is administered in the diet as a complementary ther‐ apy; the proposal is that restores glutathione cascade system [15].In addition, other antioxidants such as carotene, vitamin E, thiamine, vitamin B6, folic acid, niacin and potassi‐ um have shown a convincing protective effect [24]. Even more,when them are administered together during the cycles of radiotherapy [25].

### **Author details**

There are premalignant lesions recognized like: leukoplakia, erythroplakia, oralsubmucosal fibrosis, palatal lesions of reverse cigar smoking, oral liken planus, discoid lupus erythema‐ tosus, and hereditary disorders likecongenital dyskeratosis and epidermolysisbullosa, but beyond of a clinical standpoint, diverse carcinogenic molecular mechanisms have been postulated.The main target is the DNA, since mutations that occur in it generates a wide range of deleterious effects in the cell. In a very general overview, the balance between tu‐ mor suppressor genes and those genes that induce cell cycle is altered.Allowing cells to es‐ cape cell cycle control and developing an unpredictable biological behavior. Subsequently, the cells express molecules that allow them to acquire an invasive phenotype, a phenomen‐ on known as epithelial-mesenchymal transition. Why malignant cells colonize distant sites?

**Figure 3.** Squamous cell cancer of the lip in a 74-year-old man. He was a farmer and consumed alcohol chronically.

development of cancer.

Free radicals are products of the oxidation-reduction systems of the cell and its participation in cellular metabolic functions is essential for cell survival. A classic example is the electron transport chain in mitochondria. However, in whatpathologicalconditions, free radicals can become deleterious? In fact, what are the results of its harmful effects? The involvement of free radicals in cancer development has been studied for 3 decades, and there is sufficient evidence that implicates theirs in the multistage theory of carcinogenesis. They are proposed to cause diverse DNA alterations like: punctual mutations, DNA base oxidations, strand breaks, mutation of tumor suppressor genes and can induce overexpression of proto-onco‐ genes [12].It should be added that oxidative protein damage participates in facilitating the

Is not yet fully understood, but it is the feature that makes it lethal.

324 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Mario Nava-Villalba, German González-Pérez2 , Maribel Liñan-Fernández3 and Torres-Carmona Marco4

\*Address all correspondence to: marionava23@gmail.com

1 Dentistry Department, School of Medicine.Autonomous University of Querétaro.andDen‐ tistry Department, Health Science Division,University of the Valley of México, Campus QuerétaroQuerétaro, México

[11] (2009). Petersen P.E. Oral cancer prevention and control- The approach of the World

Oxidative Stress in Periodontal Disease and Oral Cancer

http://dx.doi.org/10.5772/52492

327

[12] Halliwell, B. (2007). Oxidative stress and cancer: have we moved forward?, *Biochemi‐*

[13] Sultan-Beevi, S. S., Hassanal-Rasheed, A. M., & Geetha, A. (2004). Evaluation of oxi‐ dative stress and nitric oxide levels in patients with oral cavity cancer., *Japan Journal*

[14] Khanna, R., Thapa, P. B., Khanna, H. D., Khanna, S., Khanna, A. K., & Shukla, H. S. (2005). Lipid peroxidation and antioxidant enzyme status in oral carcinoma patients.,

[15] Das, S., Mahapatra, S. K., Gautam, N., Das, A., & Roy, S. (2007). Oxidative stress in lymphocytes, neutrophils, and serum of oral cavity cancer patients: modulatory ar‐

[16] (2007). Patel B.P, Rawal U.M, Dave T.K, Rawal R.M, Shukla S.N, Shah P.M, Patel P.S. Lipid peroxidation, total antioxidant status, and total thiol levels predict overall sur‐ vival in patients with oral squamous cell carcinoma., *Integrative Cancer Therapies*, 6(4),

[17] Hedge, N. D., Kumari, S., Hedge, M. N., Bekal, M., & Rajaram, P. (2012). Status of serum vitamin C level and peroxidation in smokers and non-smokers with oral can‐ cer., *Research Journal of Pharmaceutical, Biological and Chemical Sciences*, 3(1), 170-175. [18] Raghavendra, U. D., Souza, V. D., & Souza, B. (2012). Erythrocyte malonilaldheyde and antioxidant status in oral squamous cell carcinoma patients and tobacco chew‐

[19] Petridou, E., et al. (2002). The role of diet and specific micronutrients in the etiology

[20] Winn, D. M., Ziegler, R. G., Pickle, L. W., Gridley, G., Blot, W. J., & Hoover, R. N. (1984). Diet in the etiology of oral and pharyngeal cancer among women from the

[21] Franceschi S, Favero A, Conti E, Talamini R, Volpe R, Negri E, Barzan L, La Vecchia C. Food groups, oils and butter, and cancer of the oral cavity and pharynx. *British*

[22] Aravindh, L., Jagathesh, P., Shanmugam, S., Sarkar, S., Kumar, P. M., & Ramasubra‐ manian, S. (2012). Estimation of plasma antioxidants beta carotene, vitamin C and vi‐ tamin E levels in patients with OSMF and oral Cancer- Indian population.,

[23] World Cancer Research Fund & American Institute for CancerResearch. (1997). Food, nutrition and the prevention of cancer.Mouth and pharynx., *Cancers, nutrition, and food. Menasha,WI: World Cancer Research Fund & American Institutefor Cancer Research*.

*International Journal of Biological and Medical Research*, 3(2), 1655-1657.

Health Organization., *Oral Oncology*.

*of Clinical Oncology*, 34(7), 379-385.

*Kathmandu University Medical Journal*, 3(4), 334-339.

ers/smokers., *Biomedical Research*, 21(4), 441-444.

southern United States., *Cancer Research*, 44(3), 1216-1222.

of oral carcinoma., *Cancer*, 94(11), 2981-2988.

*Journal of Cancer* 1999; 80 (3/4) 614-620.

ray of l-glutamine., *Supportive Care in Cancer*, 15(12), 1399-1405.

*cal Journal*, 401(1), 1-11.

365-372.

2 Dentistry Department, School of Medicine.AutonomousUniversity of Querétaro, Queré‐ taro, México

3 Dentistry Department, School of Medicine.AutonomousUniversity of Querétaro, Queré‐ taro, México

4 Dentistry Department, School of Medicine.Autonomous University of Querétaro.andGe‐ netics Department,Comprehensive Rehabilitation Center of Querétaro.Querétaro, México

### **References**


[11] (2009). Petersen P.E. Oral cancer prevention and control- The approach of the World Health Organization., *Oral Oncology*.

1 Dentistry Department, School of Medicine.Autonomous University of Querétaro.andDen‐ tistry Department, Health Science Division,University of the Valley of México, Campus

2 Dentistry Department, School of Medicine.AutonomousUniversity of Querétaro, Queré‐

3 Dentistry Department, School of Medicine.AutonomousUniversity of Querétaro, Queré‐

4 Dentistry Department, School of Medicine.Autonomous University of Querétaro.andGe‐ netics Department,Comprehensive Rehabilitation Center of Querétaro.Querétaro, México

[1] (2010). Mendes S.A, A.M Vargas Duarte Ferreira faith, Nogueira GH.Periodontitis in individuals with diabetes treated in the public health system of Belo Horizonte, Bra‐

[2] Hee-Kyung, L., Sang-Hee, C., Kyu, C. W., Anwar, T. M., Keun-Bae, S., & Seong-Hwa, J. (2009). The effect of intensive oral hygiene care on gingivitis and periodontal de‐

[3] (2002). Garcia T.B, Saldaña B.A, Soto F.C.Oxidative stress in systemic effects of in‐ flammatory periodontal disease., *Revista Cubana de Investigación Biomédica*, 21(3),

[5] Valdez P.A, Mendoza N.V.Relationship of oxidative stress with periodontal disease in older adults with type 2 diabetes mellitus. Revista ADM (2006). , 63(5), 189-94.

[6] (2004). García M.A.Influence of oxidative stress in periodontal disease., *Revista Cuba‐ na de Ciencias Médicas*, http://www.cpicmha.sld.cu/hab/10 2\_04/hab07204.htm.

[7] Shobha, P., Sunitha, J., & Mayank, H. (2010). Role of coenzyme Q10 as an antioxidant and bioenergizer in periodontal diseases., *Indian journal Pharmacology*, 42(6), 334-37.

[8] Gowri, P., Biju, T., & Suchetha, K. (2008). The challenge of antioxidants to free radi‐

[9] Kenichi, I., Hiroshi, H., Katsutoshi, I., Tatsuo, A., Mikio, S., Koji, N., et al. (2010). Por‐ phyromonasgingivalis Peptidoglycans induce excessive activation of the innate im‐ mune system in silkworm slrvae., *Journal Biological Chemistry*, 285(43), 33338-33347.

[10] Stewart B.W, Kleihues P, editors. World Cancer Report. Lyon:, *WHO International*

cals in periodontitis., *Journan Indian Society Periodontology*, 12(3), 79-83.

*Agency for Research on Cancer; 2003.*

struction in Type 2 diabetic patients., *Yonsei Medical Journal*, 50(4), 529-36.

[4] (2010). Newman T.C. Clinical Periodontology. McGraw-Hill, 10thEdition,.

zil., *Revista Brasileira de Epidemiología*, 13(1), 118-25.

QuerétaroQuerétaro, México

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taro, México

taro, México

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[24] Negri, E., Franceschi, S., Bosetti, C., Levi, F., Conti, E., Parpinel, M., & La Vecchia, C. (2002). Selected micronutrients and oral and pharyngeal cancer., *International Journal of Cancer*, 86(1), 122-127.

**Aging**

**Section 3**

[25] (2009). Shariff A.K, Patil S.R, Shukla P.S, Sontakke A.V, Hendre A.S, Gudur A.K. Ef‐ fect of oral antioxidant supplementation on lipid peroxidation during radiotherapy in head and neck malignancies., *Indian Journal of Clinical Biochemistry*, 24(3), 307-311.

**Section 3**

### **Aging**

[24] Negri, E., Franceschi, S., Bosetti, C., Levi, F., Conti, E., Parpinel, M., & La Vecchia, C. (2002). Selected micronutrients and oral and pharyngeal cancer., *International Journal*

[25] (2009). Shariff A.K, Patil S.R, Shukla P.S, Sontakke A.V, Hendre A.S, Gudur A.K. Ef‐ fect of oral antioxidant supplementation on lipid peroxidation during radiotherapy in head and neck malignancies., *Indian Journal of Clinical Biochemistry*, 24(3), 307-311.

*of Cancer*, 86(1), 122-127.

328 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Chapter 14**

**Aging, Oxidative Stress and Antioxidants**

Aging is an extremely complex and multifactorial process that proceeds to the gradual dete‐ rioration in functions. It usually manifest after maturity and leads to disability and death. Traditionally researchers focused primarily on understanding how physiological functions decline with the increasing age; almost no research was dedicated to investigation of causes or methods of aging intervention. If scientists would discover a drug for healing all major chronic degenerative diseases, the average lifetime would be increased for just 12 years. Peo‐

Defects formed in human body as a consequence of the aging process start to arise very ear‐ ly in life, probably *in utero*. In the early years, both the fraction of affected cells and the aver‐ age burden of damage per affected cell are low [1]. The signs of aging start to appear after maturity, when optimal health, strength and appearance are at the peak. After puberty, all physiological functions gradually start to decline (e.g. the maximum lung, heart and kidney capacities are decreased, the secretion of sexual hormones is lowered, arthritic changes, skin wrinkling, etc). The precise biological and cellular mechanisms responsible for the aging are not known, but according to Fontana and Klein [2], "they are likely to involve a constella‐ tion of complex and interrelated factors, including [1] oxidative stress–induced protein and DNA damage in conjunction with insufficient DNA damage repair, as well as genetic insta‐ bility of mitochondrial and nuclear genomes; [2] noninfectious chronic inflammation caused by increased adipokine and cytokine production; [3] alterations in fatty acid metabolism, in‐ cluding excessive free fatty acid release into plasma with subsequent tissue insulin resist‐ ance; [4] accumulation of end products of metabolism, such as advanced glycation end products, amyloid, and proteins that interfere with normal cell function; [5] sympathetic nerve system and angiotensin system activation as well as alterations in neuroendocrine sys‐ tems; and [6] loss of post-mitotic cells, resulting in a decreased number of neurons and mus‐ cle cells as well as deterioration in structure and function of cells in all tissues and organs".

> © 2013 Poljsak and Milisav; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ple would still die from complications connected with the aging process.

Additional information is available at the end of the chapter

B. Poljsak and I. Milisav

http://dx.doi.org/10.5772/51609

**1. Introduction**

### **Chapter 14**

### **Aging, Oxidative Stress and Antioxidants**

B. Poljsak and I. Milisav

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51609

### **1. Introduction**

Aging is an extremely complex and multifactorial process that proceeds to the gradual dete‐ rioration in functions. It usually manifest after maturity and leads to disability and death. Traditionally researchers focused primarily on understanding how physiological functions decline with the increasing age; almost no research was dedicated to investigation of causes or methods of aging intervention. If scientists would discover a drug for healing all major chronic degenerative diseases, the average lifetime would be increased for just 12 years. Peo‐ ple would still die from complications connected with the aging process.

Defects formed in human body as a consequence of the aging process start to arise very ear‐ ly in life, probably *in utero*. In the early years, both the fraction of affected cells and the aver‐ age burden of damage per affected cell are low [1]. The signs of aging start to appear after maturity, when optimal health, strength and appearance are at the peak. After puberty, all physiological functions gradually start to decline (e.g. the maximum lung, heart and kidney capacities are decreased, the secretion of sexual hormones is lowered, arthritic changes, skin wrinkling, etc). The precise biological and cellular mechanisms responsible for the aging are not known, but according to Fontana and Klein [2], "they are likely to involve a constella‐ tion of complex and interrelated factors, including [1] oxidative stress–induced protein and DNA damage in conjunction with insufficient DNA damage repair, as well as genetic insta‐ bility of mitochondrial and nuclear genomes; [2] noninfectious chronic inflammation caused by increased adipokine and cytokine production; [3] alterations in fatty acid metabolism, in‐ cluding excessive free fatty acid release into plasma with subsequent tissue insulin resist‐ ance; [4] accumulation of end products of metabolism, such as advanced glycation end products, amyloid, and proteins that interfere with normal cell function; [5] sympathetic nerve system and angiotensin system activation as well as alterations in neuroendocrine sys‐ tems; and [6] loss of post-mitotic cells, resulting in a decreased number of neurons and mus‐ cle cells as well as deterioration in structure and function of cells in all tissues and organs".

© 2013 Poljsak and Milisav; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In recent years, oxidative stress has been implicated in a wide variety of degenerative proc‐ esses, diseases and syndromes, including the following: mutagenesis, cell transformation and cancer; heart attacks, strokes, atherosclerosis, and ischemia/reperfusion injury; chronic inflam‐ matory diseases, like rheumatoid arthritis, lupus erythematosus and psoriatic arthritis; acute inflammatory problems; photooxidative stresses to the eye, e.g. cataract; neurological disor‐ ders, such as certain forms of familial amyotrophic lateral sclerosis, certain glutathione per‐ oxidase-linked adolescent seizures, Parkinson's and Alzheimer's diseases; and other agerelated disorders, perhaps even including factors underlying the aging process itself [3].

unpaired electron in their outer shells [15, 16]. Consistently, aged mammals contain high quantities of oxidized lipids and proteins as well as damaged/mutated DNA, particularly in the mitochondrial genome [13, 14]. In support of a mitochondrial theory of aging, the mito‐ chondrial DNA damage increases with aging [17, 18]. Thus, a modern version of this tenet is the "oxidative stress theory" of aging, which holds that increases in ROS accompany aging

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 333

The oxygen consumption, production of ATP by mitochondria and free-radical production are linked processes [20, 21]. Harman first proposed that normal aging results from random deleterious damage to tissues by free radicals [14] and subsequently focused on mitochon‐ dria as generators of free radicals [13]. Halliwell and Gutteridge later suggested to rename this free radical theory of aging as the "oxidative damage theory of aging" [22], since aging and diseases are caused not only by free radicals, but also by other reactive oxygen and ni‐

Increases in mitochondrial energy production at the cellular level might have beneficial and/or deleterious effects [23]. Increased regeneration of reducing agents (NADH, NADPH and FADH2) and ATP can improve the recycling of antioxidants and assist the antioxidant defence system. On the other hand, enhanced mitochondrial activity may increase the pro‐ duction of superoxide, thereby aggravating the oxidative stress and further burdening the antioxidant defence system. The mitochondria are the major source of toxic oxidants, which have the potential of reacting with and destroying cell constituents and which accumulate with age. The result of this destructive activity is lowererd energy production and a body that more readily displays signs of age (e.g., wrinkled skin, production of lower energy lev‐ els). There is now a considerable evidence that mitochondria are altered in the tissues of ag‐ ing individuals and that the damage to mitochondrial DNA (mtDNA) increases 1,000-fold

The mutation rate of mitochondrial DNA is ten-times higher than that of nuclear DNA. Mi‐ tochondrial DNA (mtDNA) is a naked, mostly double-stranded, circular, and is continuous‐ ly exposed to ROS. It is replicated much faster than nuclear DNA with less proofreading and efficient DNA repair mechanisms [25]. Thus, mtDNA is more vulnerable to attack by ROS. Damaged mitochondria can cause the energy crisis in the cell, leading to senescence and aging of tissue. Accumulation of damage decreases the cell's ability to generate ATP, so that cells, tissues, and individuals function less well. The gradual loss of energy experienced with age is paralleled by a decrease in a number of mitochondria per cell, as well as energy-

A major effect of mitochondrial dysfunction is an unappropriately high generation of ROS and proton leakage, resulting in lowering of ATP production in relation to electron input from metabolism. Leaked ROS and protons cause damage to a wide range of macromole‐ cules, including enzymes, nucleic acids and membrane lipids within and beyond mitochon‐ dria, and thus are consistent with the inflammation theory of aging as being proximal events triggering the production of pro-inflammatory cytokines. The age-related increases in the levels of both oxidative damage and mutational load of mtDNA predicted by the mitochon‐ drial theory of aging have been described in multiple species and organ systems [26]. How‐

and lead to functional alterations, pathological conditions, and even death [19].

trogen species.

with age [24].

producing efficiency of remaining mitochondria.

### **2. Aging theories**

Scientists estimated that the allelic variation or mutations in up to 7,000 relevant genes might modulate their expression patterns and/or induce senescence in an aging person, even in the absence of aging specific genes [4, 5]. As these are complex processes they may result from different mechanisms and causes. Consequently, there are many theories trying to ex‐ plain the aging process, each from its own perspective, and none of the theories can explain all details of aging. The aging theories are not mutually exclusive, especially, when oxida‐ tive stress is considered [6].

Mild oxidative stress is the result of normal metabolism; the resulting biomolecular damage cannot be totally repaired or removed by cellular degradation systems, like lysosomes, pro‐ teasomes, and cytosolic and mitochondrial proteases. About 1% to 4% of the mitochondrial‐ ly metabolized oxygen is converted to the superoxide ion that can be converted subsequently to hydrogen peroxide, hydroxyl radical and eventually other reactive species, including other peroxides and singlet oxygen that can in turn, generate free radicals capable of damaging structural proteins and DNA [7, 8, 9, 10, 11]. Since extensive research on the relation between polymorphisms likely to accelerate/decelerate the common mechanisms of aging and resistance to the oxidative stress has been neglected in almost all scientific stud‐ ies, the data do not allow us to conclude that the oxidative theory supports the theory of programmed aging so far [7]. However, the most recent studies support the idea that oxida‐ tive stress is a significant marker of senescence in different species. Resistance to oxidative stress is a common trait of long-lived genetic variations in mammals and lower organisms [5, 12]. Theories on aging process can be divided into programmed and stochastic.

#### **2.1. Free radical theory, oxidative stress theory and mitochondrial theory of aging**

Denham Harman was first to propose the free radical theory of aging in the 1950s, and ex‐ tended the idea to implicate mitochondrial production of reactive oxygen species in 1970s, [13]. According to this theory, enhanced and unopposed metabolism-driven oxidative stress has a major role in diverse chronic age-related diseases [13, 14, 7]. Organisms age because of accumulation of free radical damage in the cells. It was subsequently discovered that reac‐ tive oxygen species (ROS) generally contribute to the accumulation of oxidative damage to cellular constituents, eventhough some of them are not free radicals, as they do not have an unpaired electron in their outer shells [15, 16]. Consistently, aged mammals contain high quantities of oxidized lipids and proteins as well as damaged/mutated DNA, particularly in the mitochondrial genome [13, 14]. In support of a mitochondrial theory of aging, the mito‐ chondrial DNA damage increases with aging [17, 18]. Thus, a modern version of this tenet is the "oxidative stress theory" of aging, which holds that increases in ROS accompany aging and lead to functional alterations, pathological conditions, and even death [19].

In recent years, oxidative stress has been implicated in a wide variety of degenerative proc‐ esses, diseases and syndromes, including the following: mutagenesis, cell transformation and cancer; heart attacks, strokes, atherosclerosis, and ischemia/reperfusion injury; chronic inflam‐ matory diseases, like rheumatoid arthritis, lupus erythematosus and psoriatic arthritis; acute inflammatory problems; photooxidative stresses to the eye, e.g. cataract; neurological disor‐ ders, such as certain forms of familial amyotrophic lateral sclerosis, certain glutathione per‐ oxidase-linked adolescent seizures, Parkinson's and Alzheimer's diseases; and other agerelated disorders, perhaps even including factors underlying the aging process itself [3].

332 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Scientists estimated that the allelic variation or mutations in up to 7,000 relevant genes might modulate their expression patterns and/or induce senescence in an aging person, even in the absence of aging specific genes [4, 5]. As these are complex processes they may result from different mechanisms and causes. Consequently, there are many theories trying to ex‐ plain the aging process, each from its own perspective, and none of the theories can explain all details of aging. The aging theories are not mutually exclusive, especially, when oxida‐

Mild oxidative stress is the result of normal metabolism; the resulting biomolecular damage cannot be totally repaired or removed by cellular degradation systems, like lysosomes, pro‐ teasomes, and cytosolic and mitochondrial proteases. About 1% to 4% of the mitochondrial‐ ly metabolized oxygen is converted to the superoxide ion that can be converted subsequently to hydrogen peroxide, hydroxyl radical and eventually other reactive species, including other peroxides and singlet oxygen that can in turn, generate free radicals capable of damaging structural proteins and DNA [7, 8, 9, 10, 11]. Since extensive research on the relation between polymorphisms likely to accelerate/decelerate the common mechanisms of aging and resistance to the oxidative stress has been neglected in almost all scientific stud‐ ies, the data do not allow us to conclude that the oxidative theory supports the theory of programmed aging so far [7]. However, the most recent studies support the idea that oxida‐ tive stress is a significant marker of senescence in different species. Resistance to oxidative stress is a common trait of long-lived genetic variations in mammals and lower organisms

[5, 12]. Theories on aging process can be divided into programmed and stochastic.

**2.1. Free radical theory, oxidative stress theory and mitochondrial theory of aging**

Denham Harman was first to propose the free radical theory of aging in the 1950s, and ex‐ tended the idea to implicate mitochondrial production of reactive oxygen species in 1970s, [13]. According to this theory, enhanced and unopposed metabolism-driven oxidative stress has a major role in diverse chronic age-related diseases [13, 14, 7]. Organisms age because of accumulation of free radical damage in the cells. It was subsequently discovered that reac‐ tive oxygen species (ROS) generally contribute to the accumulation of oxidative damage to cellular constituents, eventhough some of them are not free radicals, as they do not have an

**2. Aging theories**

tive stress is considered [6].

The oxygen consumption, production of ATP by mitochondria and free-radical production are linked processes [20, 21]. Harman first proposed that normal aging results from random deleterious damage to tissues by free radicals [14] and subsequently focused on mitochon‐ dria as generators of free radicals [13]. Halliwell and Gutteridge later suggested to rename this free radical theory of aging as the "oxidative damage theory of aging" [22], since aging and diseases are caused not only by free radicals, but also by other reactive oxygen and ni‐ trogen species.

Increases in mitochondrial energy production at the cellular level might have beneficial and/or deleterious effects [23]. Increased regeneration of reducing agents (NADH, NADPH and FADH2) and ATP can improve the recycling of antioxidants and assist the antioxidant defence system. On the other hand, enhanced mitochondrial activity may increase the pro‐ duction of superoxide, thereby aggravating the oxidative stress and further burdening the antioxidant defence system. The mitochondria are the major source of toxic oxidants, which have the potential of reacting with and destroying cell constituents and which accumulate with age. The result of this destructive activity is lowererd energy production and a body that more readily displays signs of age (e.g., wrinkled skin, production of lower energy lev‐ els). There is now a considerable evidence that mitochondria are altered in the tissues of ag‐ ing individuals and that the damage to mitochondrial DNA (mtDNA) increases 1,000-fold with age [24].

The mutation rate of mitochondrial DNA is ten-times higher than that of nuclear DNA. Mi‐ tochondrial DNA (mtDNA) is a naked, mostly double-stranded, circular, and is continuous‐ ly exposed to ROS. It is replicated much faster than nuclear DNA with less proofreading and efficient DNA repair mechanisms [25]. Thus, mtDNA is more vulnerable to attack by ROS. Damaged mitochondria can cause the energy crisis in the cell, leading to senescence and aging of tissue. Accumulation of damage decreases the cell's ability to generate ATP, so that cells, tissues, and individuals function less well. The gradual loss of energy experienced with age is paralleled by a decrease in a number of mitochondria per cell, as well as energyproducing efficiency of remaining mitochondria.

A major effect of mitochondrial dysfunction is an unappropriately high generation of ROS and proton leakage, resulting in lowering of ATP production in relation to electron input from metabolism. Leaked ROS and protons cause damage to a wide range of macromole‐ cules, including enzymes, nucleic acids and membrane lipids within and beyond mitochon‐ dria, and thus are consistent with the inflammation theory of aging as being proximal events triggering the production of pro-inflammatory cytokines. The age-related increases in the levels of both oxidative damage and mutational load of mtDNA predicted by the mitochon‐ drial theory of aging have been described in multiple species and organ systems [26]. How‐ ever, whether this damage affects mitochondrial function or significantly modulates the physiology of aging has remained controversial [27, 28]. As already mentioned, free radicals can damage the mitochondrial inner membrane, creating a positive feedback-loop for in‐ creased free-radical creation. Induction of ROS generates mtDNA mutations, in turn leading to a defective respiratory chain. Defective respiratory chain generates even more ROS and generates a vicious cycle. The result is even more damage.

**2.2. Other theories of aging**

an organism.

senescence [32].

bodies that attack the body's tissues [32].

Apart from the free radical theory, the aging is explained by many other theories:

The Telomere shortening hypothesis (also described as "replicative senescence," the "Hay‐ flick phenomenon" or Hayflick limit) is based on the fact that telomeres shorten with each successive cell division. Shortened telomeres activate a mechanism that prevents cell divi‐ sion [32]. The telomere shortening hypothesis cannot explain the aging of the non-dividing cells, e.g. neurons and muscle cells, thus cannot explain the aging process in all the cells of

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 335

The Reproductive-cell cycle theory states that aging is regulated by reproductive hor‐ mones, which act in an antagonistic pleiotropic manner through cell cycle signaling. This promotes growth and development early in life in order to achieve reproduction, howev‐ er later in life, in a futile attempt to maintain reproduction, become dysregulated and drive

The Wear and tear theory of aging is based on the idea that changes associated with aging result from damage by chance that accumulates over time [32]. The wear-and-tear theories describe aging as an accumulation of damage and garbage that eventually overwhelms our ability to function. Similar are Error accumulation and Accumulative waste theories; Error accumulation theory explains aging as the results from chance events that escape proofread‐ ing mechanisms of genetic code [32], according to Accumulative waste theory the aging re‐ sults from build-up of cell waste products in time because of defective repair-removal processes. Terman, [33] believes that the process of aging derives from imperfect clearance of oxidatively damaged, relatively indigestible material, the accumulation of which further hinders cellular catabolic and anabolic functions (e.g. accumulation of lipofuscin in lyso‐ somes). The programmed theories (e.g. aging clock theory) propose a time-switch in our bodies that controls not only our process of development but also triggers our self-destruc‐ tion. The shortening of telomeres would provide such a clock in rapidly dividing cells. The Autoimmune theory of aging is based on the idea that aging results from an increase in anti‐

Mitohormesis theory of aging is based on the "hormesis effects". It describes beneficial ac‐ tions resulting from the response of an organism to a low-intensity stressor. It has been known since the 1930s that restricting calories while maintaining adequate amounts of other nutrients can extend the lifespan in laboratory animals. Michael Ristow's group has provid‐ ed evidence for the theory that this effect is due to increased formation of free radicals with‐ in the mitochondria causing a secondary induction of increased antioxidant defense capacity [34]. Finkel et al., [35] stated that the best strategy to enhance endogenous antioxidant levels may actually be oxidative stress itself, based on the classical physiological concept of horme‐ sis (for detailed information on hormesis see paragraph Adaptive responses and hormesis). Additionally, the Disposable soma theory was proposed [36, 37], which postulated a special class of gene mutations with the following antagonistic pleiotropic effects: these hypotheti‐ cal mutations save energy for reproduction (positive effect) by partially disabling molecular proofreading and other accuracy promoting devices in somatic cells (negative effect). The

**Figure 1.** Oxidative stress from endogenous or exogenous sources can trigger the chain reaction, which leads to accel‐ erated aging process of cells and organisms.

On the other hand, the "vicious cycle" theory, which states that free radical damage to mito‐ chondrial DNA leads to mitochondria that produce more superoxide, has been questioned by some scientists since the most damaged mitochondria are degraded by autophagy, whereas the less defective mitochondria (which produce less ATP as well as less superox‐ ide) remain to reproduce themselves [29]. But the efficiency of autophagy to consume mal‐ functioning mitochondria also declines with age, resulting in more mitochondria producing higher levels of superoxide [30]. Mitochondria of older organisms are fewer in number, larg‐ er in size and less efficient (produce less energy and more superoxide).

Free radicals could also be involved in signalling responses, which subsequently stimu‐ late pathways related to cell senescence and death, and in pro-inflammatory gene expres‐ sion. This inflammatory cascade is more active during aging and has been linked with ageassociated pathologies, like cancer, cardiovascular diseases, arthritis, and neurodegenerative diseases [31].

### **2.2. Other theories of aging**

ever, whether this damage affects mitochondrial function or significantly modulates the physiology of aging has remained controversial [27, 28]. As already mentioned, free radicals can damage the mitochondrial inner membrane, creating a positive feedback-loop for in‐ creased free-radical creation. Induction of ROS generates mtDNA mutations, in turn leading to a defective respiratory chain. Defective respiratory chain generates even more ROS and

**Figure 1.** Oxidative stress from endogenous or exogenous sources can trigger the chain reaction, which leads to accel‐

On the other hand, the "vicious cycle" theory, which states that free radical damage to mito‐ chondrial DNA leads to mitochondria that produce more superoxide, has been questioned by some scientists since the most damaged mitochondria are degraded by autophagy, whereas the less defective mitochondria (which produce less ATP as well as less superox‐ ide) remain to reproduce themselves [29]. But the efficiency of autophagy to consume mal‐ functioning mitochondria also declines with age, resulting in more mitochondria producing higher levels of superoxide [30]. Mitochondria of older organisms are fewer in number, larg‐

Free radicals could also be involved in signalling responses, which subsequently stimu‐ late pathways related to cell senescence and death, and in pro-inflammatory gene expres‐ sion. This inflammatory cascade is more active during aging and has been linked with ageassociated pathologies, like cancer, cardiovascular diseases, arthritis, and neurodegenerative

er in size and less efficient (produce less energy and more superoxide).

generates a vicious cycle. The result is even more damage.

334 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

erated aging process of cells and organisms.

diseases [31].

Apart from the free radical theory, the aging is explained by many other theories:

The Telomere shortening hypothesis (also described as "replicative senescence," the "Hay‐ flick phenomenon" or Hayflick limit) is based on the fact that telomeres shorten with each successive cell division. Shortened telomeres activate a mechanism that prevents cell divi‐ sion [32]. The telomere shortening hypothesis cannot explain the aging of the non-dividing cells, e.g. neurons and muscle cells, thus cannot explain the aging process in all the cells of an organism.

The Reproductive-cell cycle theory states that aging is regulated by reproductive hor‐ mones, which act in an antagonistic pleiotropic manner through cell cycle signaling. This promotes growth and development early in life in order to achieve reproduction, howev‐ er later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence [32].

The Wear and tear theory of aging is based on the idea that changes associated with aging result from damage by chance that accumulates over time [32]. The wear-and-tear theories describe aging as an accumulation of damage and garbage that eventually overwhelms our ability to function. Similar are Error accumulation and Accumulative waste theories; Error accumulation theory explains aging as the results from chance events that escape proofread‐ ing mechanisms of genetic code [32], according to Accumulative waste theory the aging re‐ sults from build-up of cell waste products in time because of defective repair-removal processes. Terman, [33] believes that the process of aging derives from imperfect clearance of oxidatively damaged, relatively indigestible material, the accumulation of which further hinders cellular catabolic and anabolic functions (e.g. accumulation of lipofuscin in lyso‐ somes). The programmed theories (e.g. aging clock theory) propose a time-switch in our bodies that controls not only our process of development but also triggers our self-destruc‐ tion. The shortening of telomeres would provide such a clock in rapidly dividing cells. The Autoimmune theory of aging is based on the idea that aging results from an increase in anti‐ bodies that attack the body's tissues [32].

Mitohormesis theory of aging is based on the "hormesis effects". It describes beneficial ac‐ tions resulting from the response of an organism to a low-intensity stressor. It has been known since the 1930s that restricting calories while maintaining adequate amounts of other nutrients can extend the lifespan in laboratory animals. Michael Ristow's group has provid‐ ed evidence for the theory that this effect is due to increased formation of free radicals with‐ in the mitochondria causing a secondary induction of increased antioxidant defense capacity [34]. Finkel et al., [35] stated that the best strategy to enhance endogenous antioxidant levels may actually be oxidative stress itself, based on the classical physiological concept of horme‐ sis (for detailed information on hormesis see paragraph Adaptive responses and hormesis).

Additionally, the Disposable soma theory was proposed [36, 37], which postulated a special class of gene mutations with the following antagonistic pleiotropic effects: these hypotheti‐ cal mutations save energy for reproduction (positive effect) by partially disabling molecular proofreading and other accuracy promoting devices in somatic cells (negative effect). The Evolutionary theory of aging is based on life history theory and is constituted of a set of ideas that themselves require further elaboration and validation [38].

**3. The Role of Oxidative Stress on the General Aging Process**

which depends on their formation and quenching.

Endogenous

**•** inflammation

**•** others

Exogenous

**•** smoking

**•** lifestyle

**•** others

**•** poor nutrition

**•** increased respiration

**•** elevation in O2 concentration

**•** increased mitochondrial leakage

**•** disorders and chronic diseases

**•** psychological and emotional stress

**•** chronic inflammation

**•** strenuous excercise

Causes of increased free-radical production include [43]:

**•** environment (pollution, pesticides, radiation, etc.)

In order to understand strategies to reduce oxidative stress and aging, it is first important to briefly explain reasons for oxidative stress formation. Oxidative damage is a result of the in‐ trinsic and extrinsic ROS formation factors. The most important endogenous sources of oxi‐ dants are mitochondrial electron transport chain and nitric oxide synthase reaction, and the non-mitochondrial soruces: Fenton reaction, reactions involving cytochromes P450 in micro‐ somes, peroxisomal beta - oxidation and respiratory burst of phagocytic cells [6]. Free radi‐ cal reactions have been implicated also as the consequence of exposure to many environmental pollutants, e.g. cigarette smoke, alcohol, ionizing and UV radiations, pesti‐ cides, ozone, etc. Oxidative stress is the direct consequence of an increased generation of free radicals and/or reduced physiological activity of antioxidant defenses against free radi‐ cals. The degree of oxidative stress is proportional to the concentration of free radicals,

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 337

**Figure 2.** Oxidative stress as the common denominator of majority of aging theories.

Evidence implies that an important theme linking several different kinds of cellular damage is the consequence of exposure to reactive oxygen species [5, 39].

Many of the theories overlap, e.g., ROS can cause DNA damage (free radical theory) and al‐ so accelerate telomere shortening (telomere theory), since telomere shortening is accelerated by oxidative stress in vascular endothelial cells [40, 41]. None of the theories explain the ag‐ ing process, as it may be too complex to be covered by only one theory. Perhaps there is no single mechanism responsible for aging in all living organisms [42]. The definitive mecha‐ nisms of aging across species remain equivocal. Diminished capacity for protein synthesis and DNA repair, decline in immune functions, loss of muscle mass and strength, a decrease in bone mineral density as well as a decrease in enzymatic and non-enzymatic antioxidative protections are well established. In essence, aging is progressive accumulation through life of many random molecular defects that build up within the cells and tissues. For this reason, only one "magic bullet" will never be able to prevent or reverse the complex and multicaus‐ al process of aging.

### **3. The Role of Oxidative Stress on the General Aging Process**

In order to understand strategies to reduce oxidative stress and aging, it is first important to briefly explain reasons for oxidative stress formation. Oxidative damage is a result of the in‐ trinsic and extrinsic ROS formation factors. The most important endogenous sources of oxi‐ dants are mitochondrial electron transport chain and nitric oxide synthase reaction, and the non-mitochondrial soruces: Fenton reaction, reactions involving cytochromes P450 in micro‐ somes, peroxisomal beta - oxidation and respiratory burst of phagocytic cells [6]. Free radi‐ cal reactions have been implicated also as the consequence of exposure to many environmental pollutants, e.g. cigarette smoke, alcohol, ionizing and UV radiations, pesti‐ cides, ozone, etc. Oxidative stress is the direct consequence of an increased generation of free radicals and/or reduced physiological activity of antioxidant defenses against free radi‐ cals. The degree of oxidative stress is proportional to the concentration of free radicals, which depends on their formation and quenching.

Causes of increased free-radical production include [43]:

#### Endogenous

Evolutionary theory of aging is based on life history theory and is constituted of a set of

ideas that themselves require further elaboration and validation [38].

336 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 2.** Oxidative stress as the common denominator of majority of aging theories.

is the consequence of exposure to reactive oxygen species [5, 39].

al process of aging.

Evidence implies that an important theme linking several different kinds of cellular damage

Many of the theories overlap, e.g., ROS can cause DNA damage (free radical theory) and al‐ so accelerate telomere shortening (telomere theory), since telomere shortening is accelerated by oxidative stress in vascular endothelial cells [40, 41]. None of the theories explain the ag‐ ing process, as it may be too complex to be covered by only one theory. Perhaps there is no single mechanism responsible for aging in all living organisms [42]. The definitive mecha‐ nisms of aging across species remain equivocal. Diminished capacity for protein synthesis and DNA repair, decline in immune functions, loss of muscle mass and strength, a decrease in bone mineral density as well as a decrease in enzymatic and non-enzymatic antioxidative protections are well established. In essence, aging is progressive accumulation through life of many random molecular defects that build up within the cells and tissues. For this reason, only one "magic bullet" will never be able to prevent or reverse the complex and multicaus‐


#### Exogenous


Causes of decreased antioxidant defense include:


Oxidative stress is caused mainly by:

**•** mutation or reduced activity of enzymes (catalase, SOD, glutathione peroxidase)

**4.1. Primary Antioxidant Defenses**

SODs are a group of metalloenzymes, which catalyze the conversion of superoxide anion to hydrogen peroxide and dioxygen [46]. This reaction is a source of cellular hydrogen

Hydrogen peroxide formed by SOD, from other metabolic reactions or from the non-enzymat‐ ic reaction of the hydroperoxyl radical, is scavenged by a ubiquitous heme protein catalase. It catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen [47].

One antioxidative role of catalases is to lower the risk of hydroxyl radical formation from

All glutathione peroxidases may catalyze the reduction of H2O2 using glutathione (GSH) as a substrate. They can also reduce other peroxides (e.g., lipid peroxides in cell membranes) to

GPx is responsible for detoxification of low H2O2 amounts, while in higher H2O2 amounts,

In addition to enzymatic defenses described above, there is an intracellular non-enzymatic defense system to protect cellular constituents against ROS and for maintaining the redox state. Glutathione (GSH) is the most abundant intracellular thiol-based antioxidant, present in millimolar concentrations in all aerobic cells, eukaryotic and prokaryotic [48]. It is a sulf‐ hydryl buffer, detoxifies compounds through conjugation reactions catalyzed by glutathione S-transferases, directly, as in the case with peroxide in the GPx-catalyzed reaction [47] or with Cr(VI) [49]. GSH is capable of reacting with Cr(VI) to yield Cr(V), Cr(IV), GSH thiyl radicals and Cr(III)-GSH complexes [50, 51]. The ratios of reduced-to-oxidized glutathione (GSH/GSSG) in normal cells are high (> 10 : 1), as the enzyme, glutathione reductase, help to

˙− <sup>+</sup> 2H+→H2O2 <sup>+</sup> O2 (1)

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 339

2 H2O2→O2 + 2H2O (2)

ROOH + 2 GSH→ROH + GSSG + H2O (3)

2O2

H2O2 via Fenton reaction catalyzed by chromium or ferrous ions.

catalase takes the leading part in cellular detoxification [15].

reduce oxidized glutathione in the following reaction:

*Superoxide Dismutase (SOD)*

*Glutathione Peroxidase (GPx)*

*Glutathione-Related Systems*

peroxide.

*Catalase*

alcohols.


Excessive generation of free radicals may overwhelm natural cellular antioxidant defenses, leading to oxidation and further functional impairment. There is an oxidative damage po‐ tential, as there is a constant free radical formation in small amounts, which escape the cell defense.

The reduction of oxidative stress can be achieved on three levels [44]: i) by lowering expo‐ sure to environmental pollutants ii) by increasing the levels of endogenous and exogenous antioxidants in order to scavenge ROS before they can cause any damage; or iii) lowering the generation of oxidative stress by stabilizing mitochondrial energy production and effi‐ ciency - reducing the amount of ROS formed per amount of O2 consumed.

### **4. Defenses against ROS and strategies to reduce oxidative stress**

Generation of ROS and the activity of antioxidant defenses are balanced *in vivo.* In fact, the balance may be slightly tipped in favor of ROS so that there is continuous low-level oxida‐ tive damage in the human body.

Besides the endogenous and exogenous antioxidative protection, the second category of de‐ fence are repair processes, which remove the damaged biomolecules before they accumulate to cause altered cell metabolism or viability [45].

### **4.1. Primary Antioxidant Defenses**

### *Superoxide Dismutase (SOD)*

SODs are a group of metalloenzymes, which catalyze the conversion of superoxide anion to hydrogen peroxide and dioxygen [46]. This reaction is a source of cellular hydrogen peroxide.

$$\text{2O}\_2\text{'} + \text{2H}^+ \rightarrow \text{H}\_2\text{O}\_2 + \text{O}\_2 \tag{1}$$

#### *Catalase*

Causes of decreased antioxidant defense include:

**•** reduced biokinetics of antioxidant metabolism

**•** reduced intake of antioxidants

**•** others

**•** reduced bioabsorption of antioxidants

Oxidative stress is caused mainly by:

**•** increased triplet oxigen (3

**•** chronic inflammation

tive damage in the human body.

to cause altered cell metabolism or viability [45].

defense.

**•** reduced activity of endogenous antioxidative enzymes

338 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**•** decreased intake of exogenous antioxidants from food

**•** increased physical activity of an untrained individual

**•** ROS from ionizing radiation, air pollution, smoking

**•** increased metal ion intake (e.g., Fe, Cu, Cr)

**•** easiliy peroxidized amino acids (e.g., lysine)

**•** mutation or reduced activity of enzymes (catalase, SOD, glutathione peroxidase)

Excessive generation of free radicals may overwhelm natural cellular antioxidant defenses, leading to oxidation and further functional impairment. There is an oxidative damage po‐ tential, as there is a constant free radical formation in small amounts, which escape the cell

The reduction of oxidative stress can be achieved on three levels [44]: i) by lowering expo‐ sure to environmental pollutants ii) by increasing the levels of endogenous and exogenous antioxidants in order to scavenge ROS before they can cause any damage; or iii) lowering the generation of oxidative stress by stabilizing mitochondrial energy production and effi‐

Generation of ROS and the activity of antioxidant defenses are balanced *in vivo.* In fact, the balance may be slightly tipped in favor of ROS so that there is continuous low-level oxida‐

Besides the endogenous and exogenous antioxidative protection, the second category of de‐ fence are repair processes, which remove the damaged biomolecules before they accumulate

O2) concentration

ciency - reducing the amount of ROS formed per amount of O2 consumed.

**4. Defenses against ROS and strategies to reduce oxidative stress**

Hydrogen peroxide formed by SOD, from other metabolic reactions or from the non-enzymat‐ ic reaction of the hydroperoxyl radical, is scavenged by a ubiquitous heme protein catalase. It catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen [47].

$$2\,\mathrm{H}\_{2}\mathrm{O}\_{2}\to\mathrm{O}\_{2} + 2\,\mathrm{H}\_{2}\mathrm{O}\tag{2}$$

One antioxidative role of catalases is to lower the risk of hydroxyl radical formation from H2O2 via Fenton reaction catalyzed by chromium or ferrous ions.

#### *Glutathione Peroxidase (GPx)*

All glutathione peroxidases may catalyze the reduction of H2O2 using glutathione (GSH) as a substrate. They can also reduce other peroxides (e.g., lipid peroxides in cell membranes) to alcohols.

$$\text{ROOH} + 2\,\text{GSH} \rightarrow \text{ROH} + \text{GSSG} + \text{H}\_2\text{O} \tag{3}$$

GPx is responsible for detoxification of low H2O2 amounts, while in higher H2O2 amounts, catalase takes the leading part in cellular detoxification [15].

#### *Glutathione-Related Systems*

In addition to enzymatic defenses described above, there is an intracellular non-enzymatic defense system to protect cellular constituents against ROS and for maintaining the redox state. Glutathione (GSH) is the most abundant intracellular thiol-based antioxidant, present in millimolar concentrations in all aerobic cells, eukaryotic and prokaryotic [48]. It is a sulf‐ hydryl buffer, detoxifies compounds through conjugation reactions catalyzed by glutathione S-transferases, directly, as in the case with peroxide in the GPx-catalyzed reaction [47] or with Cr(VI) [49]. GSH is capable of reacting with Cr(VI) to yield Cr(V), Cr(IV), GSH thiyl radicals and Cr(III)-GSH complexes [50, 51]. The ratios of reduced-to-oxidized glutathione (GSH/GSSG) in normal cells are high (> 10 : 1), as the enzyme, glutathione reductase, help to reduce oxidized glutathione in the following reaction:

$$\text{GSSG} + \text{NADPH} + \text{H}^\* \rightarrow \text{2GSH} + \text{NADP}^\* \tag{4}$$

DNA glycosylases, Ogg1p and Ntg2p to remove the damaged bases, like 7,8-dihydro-8-oxo‐ guanine, 2,6-diamino-4-hydroxy-5-n-methylformamidopyrimidine, thymine glycol, and 5 hydroxycytosine (reviewed in 65). Lipid peroxides or damaged lipids are metabolized by peroxidases or lipases. Overall, antioxidant defenses seems to be approximately balanced with the generation of ROS *in vivo*. There appears to be no great reserve of antioxidant de‐ fenses in mammals, but as previously mentioned, some oxygen-derived species perform useful metabolic roles [66]. The production of H2O2 by activated phagocytes is the classic ex‐

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 341

The intake of exogenous antioxidants from fruit and vegetables is important in preventing the oxidative stress and cellular damage. Natural antioxidants like vitamin C and E, carote‐ noids and polyphenols are generally considered as beneficial components of fruits and vege‐ tables. Their antioxidative properties are often claimed to be responsible for the protective effects of these food components against cardiovascular diseases, certain forms of cancers, photosensitivity diseases and aging [68]. However, many of the reported health claims are based on epidemiological studies in which specific diets were associated with reduced risks for specific forms of cancer and cardiovascular diseases.The identification of the actual in‐ gredient in a specific diet responsible for the beneficial health effect remains an important bottleneck for translating observational epidemiology to the development of functional food ingredients. When ingesting high amounts of synthetic antoxidants, toxic pro-oxidant ac‐

The adaptive response is a phenomenon in which exposure to minimal stress results in in‐ creased resistance to higher levels of the same stressor or other stressors. Stressors can in‐ duce cell repair mechanisms, temporary adaptation to the same or other stressor, induce autophagy or trigger cell death [69]. The molecular mechanisms of adaptation to stress is the least investigated of the stress responses described above. It may inactivate the activation of apoptosis through caspase-9, i.e. through the intrinsic pathway, one of the main apoptotic pathways [70, 117]. Early stress responses result also in the post-translational activation of pre-existing defenses, as well as activation of signal transduction pathways that initiate late responses, namely the *de novo* synthesis of stress proteins and antioxidant defenses [65]. Hormesis is characterized by dose-response relationships displaying low-dose stimulation and high-dose inhibition [71]. Hormesis is observed also upon the exposure to low dose of a toxin, which may increase cell's tolerance for greater toxicity [35]. Reactive oxygen species (ROS) can be thought of as hormetic compounds. They are beneficial in moderate amounts and harmful in the amounts that cause the oxidative stress. Many studies investigated the

ample of the deliberate metabolic generation of ROS for organism's advantage [67].

**4.3. Exogenous Antioxidant Defenses: Compounds Derived from the Diet**

tions may be important to consider [68].

**4.4. Adaptive responses and hormesis**

The NADPH required is from several reactions, the best known from the oxidative phase of pentose phosphate pathway [15]. Both, glutathione reductase and glucose-6-phosphate de‐ hydrogenase are involved in the glutathione recycling system [52].

#### **4.2. Secondary Antioxidant Defenses**

Although efficient, the antioxidant enzymes and compounds do not prevent the oxidative damage completely. A series of damage removal and repair enzymes deal with this damage. Many of these essential maintenance and repair systems become deficient in senescent cells, thus a high amount of biological "garbage" is accumulated (e.g., intralysosomal accumula‐ tion of lipofuscin) [53, 54]. Age-related oxidative changes are most common in non-prolifer‐ ating cells, like the neurons and cardiac myocites, as there is no "dilution effect" of damaged structures through cell division [33]. The ability to repair DNA correlates with species-spe‐ cific lifespan, and is necessary, but not sufficient for longevity [55]. There is an age-related decline in proteasome activity and proteasome content in different tissues (e.g. rat liver, hu‐ man epidermis); this leads to accumulation of oxidatively modified proteins [56]. Protea‐ somes are a part of the protein-removal system in eukaryotic cells. Proteasome activity and function may be decreased upon replicative senescence. On the other hand, proteasome acti‐ vation was shown to enhance the survival during oxidative stress, lifespan extension and maintenance of the juvenile morphology longer in specific cells, e.g. human primary fibro‐ blasts [57]. The total amount of oxidatively modified proteins of an 80-year-old man may be up to 50% [58]. Besides, elevated levels of oxidized proteins, oxidized lipids, advanced DNA oxidation and glycoxidation end products are found in aged organisms [7, 59, 60]. Torres and Perez [61] have shown that proteasome inhibition is a mediator of oxidative stress and ROS production and is affecting mitochondrial function. These authors propose that a pro‐ gressive decrease in proteasome function during aging can promote mitochondrial damage and ROS accumulation. It is likely that changes in proteasome dynamics could generate a prooxidative conditions that could cause tissue injury during aging, *in vivo* [61].

Numerous studies have reported age-related increases in somatic mutation and other forms of DNA damage, indicating that the capacity for DNA repair is an important determinant of the rate of aging at the cellular and molecular levels [62, 63]. An important player in the im‐ mediate cellular response to ROS-induced DNA damage is the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). It recognizes DNA lesions and flags them for repair. Grube and Burkle [64] discovered a strong positive correlation of PARP activity with the lifespan of species: cells from long-lived species had higher levels of PARP activity than cells from short-lived species.

The DNA-repair enzymes, excision-repair enzymes, operate on the basis of damage or muti‐ lation occurring to only one of the two strands of the DNA. The undamaged strand is used as a template to repair the damaged one. The excision repair of oxidized bases involves two DNA glycosylases, Ogg1p and Ntg2p to remove the damaged bases, like 7,8-dihydro-8-oxo‐ guanine, 2,6-diamino-4-hydroxy-5-n-methylformamidopyrimidine, thymine glycol, and 5 hydroxycytosine (reviewed in 65). Lipid peroxides or damaged lipids are metabolized by peroxidases or lipases. Overall, antioxidant defenses seems to be approximately balanced with the generation of ROS *in vivo*. There appears to be no great reserve of antioxidant de‐ fenses in mammals, but as previously mentioned, some oxygen-derived species perform useful metabolic roles [66]. The production of H2O2 by activated phagocytes is the classic ex‐ ample of the deliberate metabolic generation of ROS for organism's advantage [67].

#### **4.3. Exogenous Antioxidant Defenses: Compounds Derived from the Diet**

The intake of exogenous antioxidants from fruit and vegetables is important in preventing the oxidative stress and cellular damage. Natural antioxidants like vitamin C and E, carote‐ noids and polyphenols are generally considered as beneficial components of fruits and vege‐ tables. Their antioxidative properties are often claimed to be responsible for the protective effects of these food components against cardiovascular diseases, certain forms of cancers, photosensitivity diseases and aging [68]. However, many of the reported health claims are based on epidemiological studies in which specific diets were associated with reduced risks for specific forms of cancer and cardiovascular diseases.The identification of the actual in‐ gredient in a specific diet responsible for the beneficial health effect remains an important bottleneck for translating observational epidemiology to the development of functional food ingredients. When ingesting high amounts of synthetic antoxidants, toxic pro-oxidant ac‐ tions may be important to consider [68].

#### **4.4. Adaptive responses and hormesis**

GSSG + NADPH + H+→2GSH + NADP<sup>+</sup> (4)

The NADPH required is from several reactions, the best known from the oxidative phase of pentose phosphate pathway [15]. Both, glutathione reductase and glucose-6-phosphate de‐

Although efficient, the antioxidant enzymes and compounds do not prevent the oxidative damage completely. A series of damage removal and repair enzymes deal with this damage. Many of these essential maintenance and repair systems become deficient in senescent cells, thus a high amount of biological "garbage" is accumulated (e.g., intralysosomal accumula‐ tion of lipofuscin) [53, 54]. Age-related oxidative changes are most common in non-prolifer‐ ating cells, like the neurons and cardiac myocites, as there is no "dilution effect" of damaged structures through cell division [33]. The ability to repair DNA correlates with species-spe‐ cific lifespan, and is necessary, but not sufficient for longevity [55]. There is an age-related decline in proteasome activity and proteasome content in different tissues (e.g. rat liver, hu‐ man epidermis); this leads to accumulation of oxidatively modified proteins [56]. Protea‐ somes are a part of the protein-removal system in eukaryotic cells. Proteasome activity and function may be decreased upon replicative senescence. On the other hand, proteasome acti‐ vation was shown to enhance the survival during oxidative stress, lifespan extension and maintenance of the juvenile morphology longer in specific cells, e.g. human primary fibro‐ blasts [57]. The total amount of oxidatively modified proteins of an 80-year-old man may be up to 50% [58]. Besides, elevated levels of oxidized proteins, oxidized lipids, advanced DNA oxidation and glycoxidation end products are found in aged organisms [7, 59, 60]. Torres and Perez [61] have shown that proteasome inhibition is a mediator of oxidative stress and ROS production and is affecting mitochondrial function. These authors propose that a pro‐ gressive decrease in proteasome function during aging can promote mitochondrial damage and ROS accumulation. It is likely that changes in proteasome dynamics could generate a

prooxidative conditions that could cause tissue injury during aging, *in vivo* [61].

Numerous studies have reported age-related increases in somatic mutation and other forms of DNA damage, indicating that the capacity for DNA repair is an important determinant of the rate of aging at the cellular and molecular levels [62, 63]. An important player in the im‐ mediate cellular response to ROS-induced DNA damage is the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). It recognizes DNA lesions and flags them for repair. Grube and Burkle [64] discovered a strong positive correlation of PARP activity with the lifespan of species: cells from long-lived species had higher levels of PARP activity than cells from

The DNA-repair enzymes, excision-repair enzymes, operate on the basis of damage or muti‐ lation occurring to only one of the two strands of the DNA. The undamaged strand is used as a template to repair the damaged one. The excision repair of oxidized bases involves two

hydrogenase are involved in the glutathione recycling system [52].

340 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**4.2. Secondary Antioxidant Defenses**

short-lived species.

The adaptive response is a phenomenon in which exposure to minimal stress results in in‐ creased resistance to higher levels of the same stressor or other stressors. Stressors can in‐ duce cell repair mechanisms, temporary adaptation to the same or other stressor, induce autophagy or trigger cell death [69]. The molecular mechanisms of adaptation to stress is the least investigated of the stress responses described above. It may inactivate the activation of apoptosis through caspase-9, i.e. through the intrinsic pathway, one of the main apoptotic pathways [70, 117]. Early stress responses result also in the post-translational activation of pre-existing defenses, as well as activation of signal transduction pathways that initiate late responses, namely the *de novo* synthesis of stress proteins and antioxidant defenses [65]. Hormesis is characterized by dose-response relationships displaying low-dose stimulation and high-dose inhibition [71]. Hormesis is observed also upon the exposure to low dose of a toxin, which may increase cell's tolerance for greater toxicity [35]. Reactive oxygen species (ROS) can be thought of as hormetic compounds. They are beneficial in moderate amounts and harmful in the amounts that cause the oxidative stress. Many studies investigated the induction of adaptive response by oxidative stress [72, 73, 74, 75]. An oxidative stress re‐ sponse is triggered when cells sense an increase of ROS, which may result from exposure of cells to low concentrations of oxidants, increased production of ROS or a decrease in antioxi‐ dant defenses. In order to survive, the cells induce the antioxidant defenses and other pro‐ tective factors, such as stress proteins. Finkel and Holbrook [35] stated that the best strategy to enhance endogenous antioxidant levels may be the oxidative stress itself, based on the classical physiological concept of hormesis.

ions are likely to lead to the formation of high levels of hydroxyl radical by the chemical mechanisms listed above. Therefore, the valence state and bioavailability of redox active

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 343


A chelator is a molecule that has the ability to bind to metal ions, e.g. iron molecules, in or‐ der to remove heavy metals from the body. According to Halliwell and Gutteridge [22] che‐ lators act by multiple mechanisms; mainly to i) alter the reduction potential or accessibility of metal ions to stop them catalysing OH˙production (e.g. transferrin or lactoferrin) ii) pre‐ vent the escape of the free radical into solution (e.g. albumin). In this case the free radicals are formed at the biding site of the metal ions to chelating agent. Chelators can be manmade or be produced naturally, e.g. plant phenols. Because the iron catalyzes ROS genera‐ tion, sequestering iron by chelating agents is thought to be an effective approach toward preventing intracellular oxidative damage. Many chelating agents have been used to inhibit iron- or copper-mediated ROS formation, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepenta-acetic acid (DETAPAC), N,n'-Bis- (2-Hydroxybenzyl)ethylenedia‐ mine-N,n'-diacetic acid (HBED), 2-3-Dihydroxybenzoate, Desferrioxamine B (DFO), defera‐ sirox (ICL 670), N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N,-diacetic acid

dihydrochloride (OR10141), phytic acid, PYSer and others (for details see 22).

Desferrioxamine can react directly with several ROS and is used as iron(III) chelator for pre‐ vention and treatment of iron overload in patients who ingested toxic oral doses of iron [22]. Also, the intracellular protein ferritin plays a role in cellular antioxidant defense. It binds nonmetabolized intracellular iron, therefore, aids to regulation of iron availability. In this way it can decrease the availability of iron for participation in Fenton reaction and lipid per‐ oxidations. Body iron burden can be assessed by using a variety of measurements, such as serum ferritin levels and liver iron concentration by liver biopsies [for detailed information

metal ions contribute significantly to the generation of reactive oxygen species.

ed animals [86, 87].

*Iron Chelators*

see 88, 89, 90].

The enzymatic, non-enzymatic and indirect antioxidant defense systems could be involved in the induction of adaptive response to oxidative stress [76, 77, 78, 79, 80, 81]. It was ob‐ served, that a wide variety of stressors, such as pro-oxidants, aldehydes, caloric restriction, irradiation, UV-radiation, osmotic stress, heat shock, hypergravity, etc. can have a life-pro‐ longing effect.The effects of these stresses are linked also to changes in intracellular redox potential, which are transmitted to changes in activity of numerous enzymes and pathways. The main physiological benefit of adaptive response is to protect the cells and organisms from moderate doses of a toxic agent [82, 69]. As such, the stress responses that result in en‐ hanced defense and repair and even cross protection against multiple stressors could have clinical or public-health use.

#### **4.5. Sequestration of metal ions; Fenton-like reactions**

Many metal ions are necessary for normal metabolism, however they may represent a health risk when present in higher concentrations. Increased ROS generation has been implicated as a consequence of exposure to high levels of metal ions, like, iron, copper, lead, cobalt, mercury, nickel, chromium, selenium and arsenic, but not to manganese and zinc. The above mentioned transition metal ions are redox active: reduced forms of redox active metal ions participate in already discussed Fenton reaction where hydroxyl radical is generated from hydrogen peroxide [83]. Furthermore, the Haber-Weiss reaction, which involves the oxidized forms of redox active metal ions and superoxide anion, generates the reduced form of metal ion, which can be coupled to Fenton reaction to generate hydroxyl radical [15].

Fenton reaction

$$\text{Metal}^{\text{(n+1)}} + \text{H}\_2\text{O}\_2 \rightarrow \text{Metal}^{\text{(n+1)\*}} + \text{HO}^\cdot + \text{OH} \cdot \text{H} \tag{5}$$

Haber-Weiss reaction

$$\text{Metal}^{\text{(n+1)+}} + 2\text{O}\_2^{-\text{-}} \rightarrow \text{Metal}^{\text{(n+1)}} + \text{O}\_2 \tag{6}$$

Redox cycling is a characteristic of transition metals [84], and Fenton-like production of ROS appear to be involved in iron-, copper-, chromium-, and vanadium-mediated tissue damage [85]. Increases in levels of superoxide anion, hydrogen peroxide or the redox active metal ions are likely to lead to the formation of high levels of hydroxyl radical by the chemical mechanisms listed above. Therefore, the valence state and bioavailability of redox active metal ions contribute significantly to the generation of reactive oxygen species.


#### *Iron Chelators*

induction of adaptive response by oxidative stress [72, 73, 74, 75]. An oxidative stress re‐ sponse is triggered when cells sense an increase of ROS, which may result from exposure of cells to low concentrations of oxidants, increased production of ROS or a decrease in antioxi‐ dant defenses. In order to survive, the cells induce the antioxidant defenses and other pro‐ tective factors, such as stress proteins. Finkel and Holbrook [35] stated that the best strategy to enhance endogenous antioxidant levels may be the oxidative stress itself, based on the

The enzymatic, non-enzymatic and indirect antioxidant defense systems could be involved in the induction of adaptive response to oxidative stress [76, 77, 78, 79, 80, 81]. It was ob‐ served, that a wide variety of stressors, such as pro-oxidants, aldehydes, caloric restriction, irradiation, UV-radiation, osmotic stress, heat shock, hypergravity, etc. can have a life-pro‐ longing effect.The effects of these stresses are linked also to changes in intracellular redox potential, which are transmitted to changes in activity of numerous enzymes and pathways. The main physiological benefit of adaptive response is to protect the cells and organisms from moderate doses of a toxic agent [82, 69]. As such, the stress responses that result in en‐ hanced defense and repair and even cross protection against multiple stressors could have

Many metal ions are necessary for normal metabolism, however they may represent a health risk when present in higher concentrations. Increased ROS generation has been implicated as a consequence of exposure to high levels of metal ions, like, iron, copper, lead, cobalt, mercury, nickel, chromium, selenium and arsenic, but not to manganese and zinc. The above mentioned transition metal ions are redox active: reduced forms of redox active metal ions participate in already discussed Fenton reaction where hydroxyl radical is generated from hydrogen peroxide [83]. Furthermore, the Haber-Weiss reaction, which involves the oxidized forms of redox active metal ions and superoxide anion, generates the reduced form of metal ion, which can be coupled to Fenton reaction to generate hydroxyl radical [15].

Redox cycling is a characteristic of transition metals [84], and Fenton-like production of ROS appear to be involved in iron-, copper-, chromium-, and vanadium-mediated tissue damage [85]. Increases in levels of superoxide anion, hydrogen peroxide or the redox active metal

<sup>+</sup> H2O2<sup>→</sup> Metal(n+1)+ <sup>+</sup> HO˙ <sup>+</sup> OH- (5)

<sup>−</sup> ⋅→Metal(n+1) <sup>+</sup> O2 (6)

classical physiological concept of hormesis.

342 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**4.5. Sequestration of metal ions; Fenton-like reactions**

Metal(n+1)

Metal(n+1)+ <sup>+</sup> 2O2

clinical or public-health use.

Fenton reaction

Haber-Weiss reaction

A chelator is a molecule that has the ability to bind to metal ions, e.g. iron molecules, in or‐ der to remove heavy metals from the body. According to Halliwell and Gutteridge [22] che‐ lators act by multiple mechanisms; mainly to i) alter the reduction potential or accessibility of metal ions to stop them catalysing OH˙production (e.g. transferrin or lactoferrin) ii) pre‐ vent the escape of the free radical into solution (e.g. albumin). In this case the free radicals are formed at the biding site of the metal ions to chelating agent. Chelators can be manmade or be produced naturally, e.g. plant phenols. Because the iron catalyzes ROS genera‐ tion, sequestering iron by chelating agents is thought to be an effective approach toward preventing intracellular oxidative damage. Many chelating agents have been used to inhibit iron- or copper-mediated ROS formation, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepenta-acetic acid (DETAPAC), N,n'-Bis- (2-Hydroxybenzyl)ethylenedia‐ mine-N,n'-diacetic acid (HBED), 2-3-Dihydroxybenzoate, Desferrioxamine B (DFO), defera‐ sirox (ICL 670), N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N,-diacetic acid dihydrochloride (OR10141), phytic acid, PYSer and others (for details see 22).

Desferrioxamine can react directly with several ROS and is used as iron(III) chelator for pre‐ vention and treatment of iron overload in patients who ingested toxic oral doses of iron [22]. Also, the intracellular protein ferritin plays a role in cellular antioxidant defense. It binds nonmetabolized intracellular iron, therefore, aids to regulation of iron availability. In this way it can decrease the availability of iron for participation in Fenton reaction and lipid per‐ oxidations. Body iron burden can be assessed by using a variety of measurements, such as serum ferritin levels and liver iron concentration by liver biopsies [for detailed information see 88, 89, 90].

#### **4.6. Stabilizing mitochondrial ROS production**

Oxidative stress and oxidative damage accumulation could be decreased by regulating the electron leakage from electron transport chain and the resultant ROS production [44]. Nutri‐ tional and lifestyle modifications may decrease mitochondrial ROS formation, e.g. by caloric restriction (CR), sport activities and healthy eating habits. The anti-aging action of caloric re‐ striction is an example of hormesis [91, 92, 93]. The works of Yu and Lee [94], Koizumi et al. [95] and Chen and Lovry [96] imply that food restriction (energetic stress) increases the overall antioxidant capacity to maintain the optimal status of intracellular environment by balancing ROS in CR thus promotes the metabolic shift to result in more efficient electron transport at the mitochondrial respiratory chain [97]. In this way, the leakage of electrons from the respiratory chain is reduced [98, 99]. There are reports of slower aging by intermit‐ tent fasting without the overall reduction of caloric intake [100, 101]. Since it is extremely hard to maintain the long-term CR, the search is on for CR mimetics. These are the agents or strategies that can mimic the beneficial health-promoting and anti-aging effects of CR. Sev‐ eral compounds have been tested for a potential to act as CR mimetic; such as plant-derived polyphenols (e.g., resveratrol, quercetin, butein, piceatannol), insulin-action enhancers (e.g., metformin), or pharmacological agents that inhibit glycolysis (e.g., 2-deoxyglucose) [102].

**5. Conclusion and perspectives**

In conclusion, excessive production of ROS and reduced antioxidant defence with age sig‐ nificantly contribute to aging. It seems that oxidative damage is the major cause and the most important contributor to human aging. Antioxidant defense seems to be approximatly balanced with the generation of oxygen-derived species in young individuals, however, there is an increase of oxidative stress later in life. Then the approaches to lower the in‐ creased ROS formation in our bodies could be implemented by avoiding the exposure to exogenous free radicals, by intake of adequate amounts of antioxidants and/or by stimulat‐

Aging, Oxidative Stress and Antioxidants http://dx.doi.org/10.5772/51609 345

Developing natural or pharmacological agents capable of increasing the antioxidative pro‐ tection and/or modulating the endogenous defense and repair mechanisms may potentially improve health, increase longevity and contribute to treatment of degenerative age-related diseases, such as cardiovascular and neurodegenerative disorders and cancer. The lifestyle changes, e.g. regular physical activity, increased intake of fruits and vegetables, and reduced calorie intake may improve health and increase cellular resistance to stress. Synthetic antiox‐ idant supplements may help to correct the high levels of oxidative stress that cannot be con‐

1 University of Ljubljana, Laboratory of oxidative stress research, Faculty of Health Sciences,

2 University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Ljubljana, Slov‐

[1] Kirkwood, B., & Mathers, J. C. (2009). The basic biology of aging. In: Stanner S., Thompson R., Buttriss J. [eds.], *Healthy aging-The role of nutrition and lifestyle*, NY:Wi‐

[2] Fontana, L., & Klein, S. (2007). Aging, Adiposity, and Calorie Restriction. *Jama*, 297,

[3] Davies, K. J. (1995). Oxidative stress: the paradox of aerobic life. *Biochem. Soc. Symp.*,

ing the damage-repair systems of the cells [44 and references within].

trolled by the sinergy of endogenous antioxidant systems.

\*Address all correspondence to: borut.poljsak@zf.uni-lj.si

**Author details**

Ljubljana, Slovenia

**References**

ley-Blackwell.

986-994.

61, 1-31.

enia

B. Poljsak1\* and I. Milisav1,2

Mitochondrial uncoupling has been proposed as a mechanism that reduces the production of reactive oxygen species and may account for the paradox between longevity and activity [103]. Moderate and regular exercise enhances health and longevity relative to sedentary lifestyles. Endurance training adaptation results in increased efficiency in ATP synthesis at the expense of potential increase in oxidative stress that is likely to be compensated by en‐ hanced activities of antioxidant enzymes [104] and proteasome [105]. Exercise requires a large flux of energy and a shift in substrate metabolism in mitochondria from state 4 to state 3. This shift may cause an increase in superoxide production [106]. Indeed, a single bout of exercise was found to increase the metabolism and oxidative stress during and immediately after exercise [107, 108, 109]. While a single bout of exercise of sedentary animals is likely to cause increased detrimental oxidative modification of proteins [110], moderate daily exer‐ cise appears to be beneficial by reducing the damage in rat skeletal muscle [105]. Organisms exposed to oxidative stress often decrease their rate of metabolism [111, 112]. Metabolic un‐ coupling may reduce the mitochondrial oxidant production [113]. It may account for the paradox between longevity and activity [103]. Heat is produced when oxygen consumption is uncoupled from ATP generation. When the mitochondria are uncoupled and membrane potential is low animals might produce less free radicals when expending the most energy [114]. Postprandial oxidative stress is characterized by an increased susceptibility of the or‐ ganism toward oxidative damage after consumption of a meal rich in lipids and/or carbohy‐ drates [115]. The generation of excess superoxide due to abundance of energy substrates after the meal may be a predominate factor resulting in oxidative stress and a decrease in nitric oxide. A mixture of antioxidant compounds is required to provide protection from the oxidative effects of postprandial fats and sugars. No specific antioxidant can be claimed to be the most important, as consumption of food varies enormously in humans. However, a variety of polyphenolic compounds derived from plants appear to be effective dietary anti‐ oxidants, especially when consumed with high-fat meals [116].

### **5. Conclusion and perspectives**

**4.6. Stabilizing mitochondrial ROS production**

344 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Oxidative stress and oxidative damage accumulation could be decreased by regulating the electron leakage from electron transport chain and the resultant ROS production [44]. Nutri‐ tional and lifestyle modifications may decrease mitochondrial ROS formation, e.g. by caloric restriction (CR), sport activities and healthy eating habits. The anti-aging action of caloric re‐ striction is an example of hormesis [91, 92, 93]. The works of Yu and Lee [94], Koizumi et al. [95] and Chen and Lovry [96] imply that food restriction (energetic stress) increases the overall antioxidant capacity to maintain the optimal status of intracellular environment by balancing ROS in CR thus promotes the metabolic shift to result in more efficient electron transport at the mitochondrial respiratory chain [97]. In this way, the leakage of electrons from the respiratory chain is reduced [98, 99]. There are reports of slower aging by intermit‐ tent fasting without the overall reduction of caloric intake [100, 101]. Since it is extremely hard to maintain the long-term CR, the search is on for CR mimetics. These are the agents or strategies that can mimic the beneficial health-promoting and anti-aging effects of CR. Sev‐ eral compounds have been tested for a potential to act as CR mimetic; such as plant-derived polyphenols (e.g., resveratrol, quercetin, butein, piceatannol), insulin-action enhancers (e.g., metformin), or pharmacological agents that inhibit glycolysis (e.g., 2-deoxyglucose) [102].

Mitochondrial uncoupling has been proposed as a mechanism that reduces the production of reactive oxygen species and may account for the paradox between longevity and activity [103]. Moderate and regular exercise enhances health and longevity relative to sedentary lifestyles. Endurance training adaptation results in increased efficiency in ATP synthesis at the expense of potential increase in oxidative stress that is likely to be compensated by en‐ hanced activities of antioxidant enzymes [104] and proteasome [105]. Exercise requires a large flux of energy and a shift in substrate metabolism in mitochondria from state 4 to state 3. This shift may cause an increase in superoxide production [106]. Indeed, a single bout of exercise was found to increase the metabolism and oxidative stress during and immediately after exercise [107, 108, 109]. While a single bout of exercise of sedentary animals is likely to cause increased detrimental oxidative modification of proteins [110], moderate daily exer‐ cise appears to be beneficial by reducing the damage in rat skeletal muscle [105]. Organisms exposed to oxidative stress often decrease their rate of metabolism [111, 112]. Metabolic un‐ coupling may reduce the mitochondrial oxidant production [113]. It may account for the paradox between longevity and activity [103]. Heat is produced when oxygen consumption is uncoupled from ATP generation. When the mitochondria are uncoupled and membrane potential is low animals might produce less free radicals when expending the most energy [114]. Postprandial oxidative stress is characterized by an increased susceptibility of the or‐ ganism toward oxidative damage after consumption of a meal rich in lipids and/or carbohy‐ drates [115]. The generation of excess superoxide due to abundance of energy substrates after the meal may be a predominate factor resulting in oxidative stress and a decrease in nitric oxide. A mixture of antioxidant compounds is required to provide protection from the oxidative effects of postprandial fats and sugars. No specific antioxidant can be claimed to be the most important, as consumption of food varies enormously in humans. However, a variety of polyphenolic compounds derived from plants appear to be effective dietary anti‐

oxidants, especially when consumed with high-fat meals [116].

In conclusion, excessive production of ROS and reduced antioxidant defence with age sig‐ nificantly contribute to aging. It seems that oxidative damage is the major cause and the most important contributor to human aging. Antioxidant defense seems to be approximatly balanced with the generation of oxygen-derived species in young individuals, however, there is an increase of oxidative stress later in life. Then the approaches to lower the in‐ creased ROS formation in our bodies could be implemented by avoiding the exposure to exogenous free radicals, by intake of adequate amounts of antioxidants and/or by stimulat‐ ing the damage-repair systems of the cells [44 and references within].

Developing natural or pharmacological agents capable of increasing the antioxidative pro‐ tection and/or modulating the endogenous defense and repair mechanisms may potentially improve health, increase longevity and contribute to treatment of degenerative age-related diseases, such as cardiovascular and neurodegenerative disorders and cancer. The lifestyle changes, e.g. regular physical activity, increased intake of fruits and vegetables, and reduced calorie intake may improve health and increase cellular resistance to stress. Synthetic antiox‐ idant supplements may help to correct the high levels of oxidative stress that cannot be con‐ trolled by the sinergy of endogenous antioxidant systems.

### **Author details**

B. Poljsak1\* and I. Milisav1,2

\*Address all correspondence to: borut.poljsak@zf.uni-lj.si

1 University of Ljubljana, Laboratory of oxidative stress research, Faculty of Health Sciences, Ljubljana, Slovenia

2 University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Ljubljana, Slov‐ enia

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rie intake. *Proc. Natl. Acad. Sci. U S A., USA, 203*, 100(10), 6216-20.

emerging research field. *Aging Cell.*, 5, 97-108.

production. *Int. J. Biochem. Cell Biol.*, 39(1), 146-60.

tation to endurance training. *Am. J. Physiol.*, 277, R856-R862.

[100] Gredilla, R., Sanz, A., Lopez-Torres, M., & Barja, G. (2001). Caloric restriction de‐ creases mitochondrial free radical generation at complex I and lowers oxidative dam‐

[101] Anson, R. M., Guo, Z., de Cabo, R., Iyun, T., Rios, M., Hagepanos, A., Ingram, D. K., Lane, M. A., & Mattson, M. P. Intermittent fasting dissociates beneficial effects of di‐ etary restriction on glucose metabolism and neuronal resistance to injury from calo‐

[102] Ingram, D. K., Zhu, M., & Mamczarz, J. (2006). Calorie restriction mimetics: an

[103] Cámara, Y., Duval, C., Sibille, B., & Villarroya, F. (2007). Activation of mitochondrialdriven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species

[104] Hollander, J., Fiebig, R., Gore, M., Bejma, J., Ookawara, T., Ohno, H., & Ji, L. L. (1999). Superoxide dismutase gene expression in skeletal muscle: fiber-specific adap‐

[105] Radak, Z., Nakamura, A., & Nakamoto, H. (1998). A period of exercise increases the accumulation of reactive carbonyl derivatives in the lungs of rats. *Pfluger Arch: Eur. J.*

[106] Barja, G. (1999). Mitochondrial oxygen radical generation and leak: sites of produc‐ tion in states 4 and 3, organ specificity, and relation to aging and longevity. *J Bioenerg*

[107] Alessio, H. M., & Goldfarb, A. H. (1988). Lipid peroxidation and scavenger enzymes during exercise. Adaptive response to training. *J Appl Physiol*, 64, 1333-1336.

[108] Ji, L. L. (1993). Antioxidant enzyme response to exercise and aging. *Med Sci Sport Ex‐*

361-7.

*Res*, 287, 247-56.

352 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

*ence*, 273, 59-63.

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*Physiol.*, 435, 439-441.

*Biomembr.*, 31(4), 347-66.

*erc.*, 25, 225-231.


**Section 4**

**Disease and Therapy - A Role for Antioxidants**

**Disease and Therapy - A Role for Antioxidants**

**Chapter 15**

**Disease and Therapy: A Role for Oxidants**

Ana María Téllez López and Marisela López Orozco

Oxidative stress is a large increase reduction potential in cell or a decrease in reducing ca‐ pacity of the cellular redox couples such as glutation. Effects of oxidative stress depend on the magnitude of these changes, if the cell is able to overcome small perturbations and re‐ gain its original state. However, severe oxidative stress can cause cell death and even mod‐

A particularly destructive aspect of oxidative stress is the production of reactive oxygen spe‐ cies, which include free radicals and peroxides. Some of the less reactive species (superox‐ ide) can be converted by a redox reaction with transition metals or other compounds quinines redox cycle, more aggressive radical species which can cause extensive damage cel‐ lular. Most of these species derived from oxygen are produced at a low level in normal aero‐ bic metabolism and the damage they cause to cells is constantly repaired. However, under the severe levels of oxidative stress that causes necrotic damage produces ATP depletion

The antioxidants are substances that may protect your cells against the effects of free radi‐ cals. Free radicals are molecules produced when your body breaks down food, or by envi‐ ronmental exposures like tobacco smoke and radiation. Free radicals can damage cells, and

Antioxidant substances include beta-carotene, lutein, lycopene, selenium, vitamin A; and vi‐ tamin C. Antioxidants are found in many foods. These include fruits and vegetables, nuts,

> © 2013 Molina Trinidad et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

erate oxidation can trigger apoptosis, whereas if it is too intense can cause necrosis.

Additional information is available at the end of the chapter

Eva María Molina Trinidad, Sandra Luz de Ita Gutiérrez,

http://dx.doi.org/10.5772/51994

prevents cell death by apoptosis control.

grains, and some meats, poultry and fish.

may play a role in heart disease, cancer and other diseases.

**1. Introduction**

### **Disease and Therapy: A Role for Oxidants**

Eva María Molina Trinidad, Sandra Luz de Ita Gutiérrez, Ana María Téllez López and Marisela López Orozco

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51994

### **1. Introduction**

Oxidative stress is a large increase reduction potential in cell or a decrease in reducing ca‐ pacity of the cellular redox couples such as glutation. Effects of oxidative stress depend on the magnitude of these changes, if the cell is able to overcome small perturbations and re‐ gain its original state. However, severe oxidative stress can cause cell death and even mod‐ erate oxidation can trigger apoptosis, whereas if it is too intense can cause necrosis.

A particularly destructive aspect of oxidative stress is the production of reactive oxygen spe‐ cies, which include free radicals and peroxides. Some of the less reactive species (superox‐ ide) can be converted by a redox reaction with transition metals or other compounds quinines redox cycle, more aggressive radical species which can cause extensive damage cel‐ lular. Most of these species derived from oxygen are produced at a low level in normal aero‐ bic metabolism and the damage they cause to cells is constantly repaired. However, under the severe levels of oxidative stress that causes necrotic damage produces ATP depletion prevents cell death by apoptosis control.

The antioxidants are substances that may protect your cells against the effects of free radi‐ cals. Free radicals are molecules produced when your body breaks down food, or by envi‐ ronmental exposures like tobacco smoke and radiation. Free radicals can damage cells, and may play a role in heart disease, cancer and other diseases.

Antioxidant substances include beta-carotene, lutein, lycopene, selenium, vitamin A; and vi‐ tamin C. Antioxidants are found in many foods. These include fruits and vegetables, nuts, grains, and some meats, poultry and fish.

© 2013 Molina Trinidad et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Free radicals damage may lead to cancer. Antioxidants interact with and stabilize free radi‐ cals and may prevent some of the damage free radicals might otherwise cause.

cancer, particularly lung cancer. The initial findings of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study) indicated, however, that lung cancer incidence was increased among participants who received beta-carotene as a supplement. Similar results were recently reported by the Beta-Carotene and Retinol Efficacy Trial (CARET), which test‐

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 359

The Physicians' Health Study II (PHS II) is a follow up to the earlier clinical trial by the same name. The study is investigating the effects of vitamin E, C, and multivitamins on prostate cancer and total cancer incidence. In another case the supplementation with alpha-tocopher‐ ol or beta-carotene does not prevent lung cancer in older men who smoke. Beta-Carotene supplementation at pharmacologic levels may modestly increase lung cancer incidence in cigarette smokers, and this effect may be associated with heavier smoking and higher alco‐

Antioxidants neutralize free radicals as the natural by-product of normal cell processes. Free radicals are molecules with incomplete electron shells which make them more chemically re‐ active than those with complete electron shells. Exposure to various environmental factors, including tobacco smoke and radiation, can also lead to free radical formation. In humans, the most common form of free radicals is oxygen. When an oxygen molecule (O2) becomes electrically charged or "radicalized" it tries to steal electrons from other molecules, causing damage to the DNA and other molecules. Over time, such damage may become irreversible and lead to disease including cancer. Antioxidants are often described as "mopping up" free radicals, meaning they neutralize the electrical charge and prevent the free radical from tak‐

Because of the importance that involves using antioxidants as an alternative in the treatment and prevention of chronic degenerative diseases is useful to express the potential in the use

Free radicals are highly reactive chemical species that possess an unpaired electron. Due to it is reactivity, the radicals react readily with other molecules. When free radicals come into contact with the molecules of the human body such as proteins, lipids, carbohydrates, DNA nucleic acids, react with them. These reactions cause changes in the normal functions of these primary metabolites, which cause severe damage that can cause diseases such as can‐ cer and degenerative diseases like Parkinson´s disease or Alzheimer´s disease and athero‐

When any of these afore mentioned diseases, the patient receive the treatment used to treat the particular disease, however, prevention plays a big role. Oxidation in the body tissues caused by free radicals can be prevented with a daily intake of foods that have antioxidants.

The implications of modern life cause changes in eating habits of people, these results in a lack of antioxidants in the body to cope with free radicals that are in contact. The role of an‐ tioxidants is to react with free radicals and thus prevent, to react with the primary metabo‐

ed a combination of beta-carotene and vitamin A.

ing electrons from other molecules.

and development of new drugs that include antioxidants.

sclerosis, coronary heart disease and diabetes [1-4].

lites, thus acting as natural shields against diseases like cancer [5, 6].

hol intake.

Studies in cancer cells *in vitro* and *in vivo* animal's models suggest that the use of free radi‐ cals decreases the growth of malignant cells. However, information from recent clinical tri‐ als is less clear. In recent years, large-scale, randomized clinical trials reached inconsistent conclusions.

Clinical trials published in the 1990s reached differing conclusions about the effect of antiox‐ idants on cancer. The studies examined the effect of beta-carotene and other antioxidants on cancer in different patient groups. However, beta-carotene appeared to have different effects depending upon the patient population, therefore it is important to personalize treatment, and we must take into account the variability to treatment and individualize or personalize therapy. Studies made by Blot WJ et al., in 1993 for the treatment of cancer published in Chi‐ nese Cancer Prevention Study, investigated the effect of a combination of beta-carotene, vi‐ tamin E, and selenium on cancer in healthy Chinese men and women at high risk for gastric cancer. The study showed a combination of beta-carotene, vitamin E, and selenium signifi‐ cantly reduced incidence of both gastric cancer and cancer overall.

A 1994 cancer prevention study entitled the Alpha-Tocopherol (vitamin E)/ Beta-Carotene Cancer Prevention Study (ATBC) demonstrated that lung cancer rates of Finnish male smok‐ ers increased significantly with beta-carotene and were not affected by vitamin E. Epidemio‐ logic evidence indicates that diets high in carotenoid-rich fruits and vegetables, as well as high serum levels of vitamin E (alpha-tocopherol) and beta carotene are associated with a reduced risk of lung cancer. Another study made by Omenn GS in 1994, the Beta-Carotene and Retinol (vitamin A). Efficacy Trial (CARET) also demonstrated a possible increase in lung cancer associated with antioxidants.

The 1996 Physicians' Health Study I (PHS) found no change in cancer rates associated with beta-carotene and aspirin taken by U.S. male physicians.

The 1999 Women's Health Study (WHS) made by Lee IM, tested effects of vitamin E and be‐ ta-carotene in the prevention of cancer and cardiovascular disease among women age 45 years or older. Among apparently healthy women, there was no benefit or harm from betacarotene supplementation. Investigation of the effect of vitamin E is ongoing.

Three large-scale clinical trials continue to investigate the effect of antioxidants on cancer. The Women's Health Study (WHS) is currently evaluating the effect of vitamin E in the pri‐ mary prevention of cancer among U.S. female health professionals age 45 and older.

In 2006, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) is taking place in the United States, Puerto Rico, and Canada. SELECT is trying to find out if taking selenium and/or vitamin E supplements can prevent prostate cancer in men age 50 or older. Also the experimental and epidemiologic investigations suggest that alpha-tocopherol (the most prevalent chemical form of vitamin E found in vegetable oils, seeds, grains, nuts, and other foods) and beta-carotene (a plant pigment and major precursor of vitamin A found in many yellow, orange, and dark-green, leafy vegetables and some fruit) might reduce the risk of cancer, particularly lung cancer. The initial findings of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study) indicated, however, that lung cancer incidence was increased among participants who received beta-carotene as a supplement. Similar results were recently reported by the Beta-Carotene and Retinol Efficacy Trial (CARET), which test‐ ed a combination of beta-carotene and vitamin A.

Free radicals damage may lead to cancer. Antioxidants interact with and stabilize free radi‐

Studies in cancer cells *in vitro* and *in vivo* animal's models suggest that the use of free radi‐ cals decreases the growth of malignant cells. However, information from recent clinical tri‐ als is less clear. In recent years, large-scale, randomized clinical trials reached inconsistent

Clinical trials published in the 1990s reached differing conclusions about the effect of antiox‐ idants on cancer. The studies examined the effect of beta-carotene and other antioxidants on cancer in different patient groups. However, beta-carotene appeared to have different effects depending upon the patient population, therefore it is important to personalize treatment, and we must take into account the variability to treatment and individualize or personalize therapy. Studies made by Blot WJ et al., in 1993 for the treatment of cancer published in Chi‐ nese Cancer Prevention Study, investigated the effect of a combination of beta-carotene, vi‐ tamin E, and selenium on cancer in healthy Chinese men and women at high risk for gastric cancer. The study showed a combination of beta-carotene, vitamin E, and selenium signifi‐

A 1994 cancer prevention study entitled the Alpha-Tocopherol (vitamin E)/ Beta-Carotene Cancer Prevention Study (ATBC) demonstrated that lung cancer rates of Finnish male smok‐ ers increased significantly with beta-carotene and were not affected by vitamin E. Epidemio‐ logic evidence indicates that diets high in carotenoid-rich fruits and vegetables, as well as high serum levels of vitamin E (alpha-tocopherol) and beta carotene are associated with a reduced risk of lung cancer. Another study made by Omenn GS in 1994, the Beta-Carotene and Retinol (vitamin A). Efficacy Trial (CARET) also demonstrated a possible increase in

The 1996 Physicians' Health Study I (PHS) found no change in cancer rates associated with

The 1999 Women's Health Study (WHS) made by Lee IM, tested effects of vitamin E and be‐ ta-carotene in the prevention of cancer and cardiovascular disease among women age 45 years or older. Among apparently healthy women, there was no benefit or harm from beta-

Three large-scale clinical trials continue to investigate the effect of antioxidants on cancer. The Women's Health Study (WHS) is currently evaluating the effect of vitamin E in the pri‐

In 2006, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) is taking place in the United States, Puerto Rico, and Canada. SELECT is trying to find out if taking selenium and/or vitamin E supplements can prevent prostate cancer in men age 50 or older. Also the experimental and epidemiologic investigations suggest that alpha-tocopherol (the most prevalent chemical form of vitamin E found in vegetable oils, seeds, grains, nuts, and other foods) and beta-carotene (a plant pigment and major precursor of vitamin A found in many yellow, orange, and dark-green, leafy vegetables and some fruit) might reduce the risk of

mary prevention of cancer among U.S. female health professionals age 45 and older.

carotene supplementation. Investigation of the effect of vitamin E is ongoing.

cals and may prevent some of the damage free radicals might otherwise cause.

358 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cantly reduced incidence of both gastric cancer and cancer overall.

lung cancer associated with antioxidants.

beta-carotene and aspirin taken by U.S. male physicians.

conclusions.

The Physicians' Health Study II (PHS II) is a follow up to the earlier clinical trial by the same name. The study is investigating the effects of vitamin E, C, and multivitamins on prostate cancer and total cancer incidence. In another case the supplementation with alpha-tocopher‐ ol or beta-carotene does not prevent lung cancer in older men who smoke. Beta-Carotene supplementation at pharmacologic levels may modestly increase lung cancer incidence in cigarette smokers, and this effect may be associated with heavier smoking and higher alco‐ hol intake.

Antioxidants neutralize free radicals as the natural by-product of normal cell processes. Free radicals are molecules with incomplete electron shells which make them more chemically re‐ active than those with complete electron shells. Exposure to various environmental factors, including tobacco smoke and radiation, can also lead to free radical formation. In humans, the most common form of free radicals is oxygen. When an oxygen molecule (O2) becomes electrically charged or "radicalized" it tries to steal electrons from other molecules, causing damage to the DNA and other molecules. Over time, such damage may become irreversible and lead to disease including cancer. Antioxidants are often described as "mopping up" free radicals, meaning they neutralize the electrical charge and prevent the free radical from tak‐ ing electrons from other molecules.

Because of the importance that involves using antioxidants as an alternative in the treatment and prevention of chronic degenerative diseases is useful to express the potential in the use and development of new drugs that include antioxidants.

Free radicals are highly reactive chemical species that possess an unpaired electron. Due to it is reactivity, the radicals react readily with other molecules. When free radicals come into contact with the molecules of the human body such as proteins, lipids, carbohydrates, DNA nucleic acids, react with them. These reactions cause changes in the normal functions of these primary metabolites, which cause severe damage that can cause diseases such as can‐ cer and degenerative diseases like Parkinson´s disease or Alzheimer´s disease and athero‐ sclerosis, coronary heart disease and diabetes [1-4].

When any of these afore mentioned diseases, the patient receive the treatment used to treat the particular disease, however, prevention plays a big role. Oxidation in the body tissues caused by free radicals can be prevented with a daily intake of foods that have antioxidants.

The implications of modern life cause changes in eating habits of people, these results in a lack of antioxidants in the body to cope with free radicals that are in contact. The role of an‐ tioxidants is to react with free radicals and thus prevent, to react with the primary metabo‐ lites, thus acting as natural shields against diseases like cancer [5, 6].

tion of disease is certainly a great alternative to the aggressive use of medications commonly used. The simplest method to prevent cancer and other diseases is undoubtedly add to the diet foods that contain high concentrations of antioxidants, this treatment is easy to perform and causes no adverse side effects. Other organisms containing large amounts of secondary metabolites some of which can act as antioxidants and thereby help prevent cancer and pre‐

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 361

Blocking cancer, in the initial stage protecting cells against oxidative species and enhancing

Suppressing cancer by inhibiting the progressive stages after formation of pre-neoplastic

Studies are underway to help better understand the mechanism of action of antioxidants and test its efficacy against cancer and other diseases. Several studies report that the addition to the diet of foods containing antioxidants may increase the effectiveness of cancer treatment, and help strengthen the body against the side effects associated with treatment [9-11]. The antioxidants found in fruits and vegetables can mention vitamins C and E, carotenoids group and the group of polyphenols. Polyphenols are a group of antioxidant flavonoids to which they belong. There are several types of flavonoids and can be found in foods such as blackberries, blueberries, strawber‐ ries, plum, peach, apple, tomato, cherry, broccoli, onion, soya been, legumes like green gram, lu‐ pine peas, soy beans, white and horse gram, green leafy spices, citrus fruits, tea, red grapes,

The following briefly discuss some results of studies using antioxidants from fruits and veg‐

Research conducted in Canada by Hakimuddin and colleagues [13] showed that the poly‐ phenols found in red wine have selective toxicity against MCF-7 cell type of breast cancer; the authors indicate the importance of a diet that incorporates red wine and feeding grapes to serve as a preventive strategy against cancer, which also can be combined with standard

As mentioned above plums and peaches are fruits that contain phenolic compounds. In a study to test the activity of phenolic species as cancer chemopreventive agents present in ex‐ tracts of plums and peaches, we found that peaches and plums contain a mixture of phenol‐ ic compounds with the ability to inhibit cell lines MCF-7 and MDA -MB435. A very important point to consider is that phenolic acids were isolated chlorogenic and neo-chloro‐ genic which have great potential for use as chemopreventive agents exerting growth inhibi‐ tion of the cell line MDA-MB-435 and low toxicity to the normal cell line MCF-10A [14].

Another recent study [15], focused on the action of terpenes located in the skin of the olives suggests that they may serve as natural potential protective against breast cancer. The triter‐ penes were isolated in significant quantities from the pulp of the olive oil and can act pro‐

vent its development (Fruits, vegetables, plants).

chocolate, cocoa and red wine beverages [12].

etables for the treatment of breast cancer.

phylactically and therapeutically.

Antioxidants can act in two ways:

DNA repair.

cells [8].

therapies.

**Figure 1.** Antioxidants decrement oxidative processes. [Bruce Ames Ph.D., University of California Lecture U.C.T.V. viewed on 08-14-2004].

### **2. Cancer**

#### **2.1. Breast cancer**

Currently breast cancer is a disease of high incidence worldwide and causes millions of deaths annually [7]. In the treatment of various cancers have been used drugs that originate from natural products. To get to the application of the drug as a treatment, it requires years of research. The use of treatment leads to the destruction of cancer cells and normal cells in addition, there are numbers side effects resulting from the application of therapies. Preven‐ tion of disease is certainly a great alternative to the aggressive use of medications commonly used. The simplest method to prevent cancer and other diseases is undoubtedly add to the diet foods that contain high concentrations of antioxidants, this treatment is easy to perform and causes no adverse side effects. Other organisms containing large amounts of secondary metabolites some of which can act as antioxidants and thereby help prevent cancer and pre‐ vent its development (Fruits, vegetables, plants).

Antioxidants can act in two ways:

**Figure 1.** Antioxidants decrement oxidative processes. [Bruce Ames Ph.D., University of California Lecture U.C.T.V.

Currently breast cancer is a disease of high incidence worldwide and causes millions of deaths annually [7]. In the treatment of various cancers have been used drugs that originate from natural products. To get to the application of the drug as a treatment, it requires years of research. The use of treatment leads to the destruction of cancer cells and normal cells in addition, there are numbers side effects resulting from the application of therapies. Preven‐

viewed on 08-14-2004].

360 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**2. Cancer**

**2.1. Breast cancer**

Blocking cancer, in the initial stage protecting cells against oxidative species and enhancing DNA repair.

Suppressing cancer by inhibiting the progressive stages after formation of pre-neoplastic cells [8].

Studies are underway to help better understand the mechanism of action of antioxidants and test its efficacy against cancer and other diseases. Several studies report that the addition to the diet of foods containing antioxidants may increase the effectiveness of cancer treatment, and help strengthen the body against the side effects associated with treatment [9-11]. The antioxidants found in fruits and vegetables can mention vitamins C and E, carotenoids group and the group of polyphenols. Polyphenols are a group of antioxidant flavonoids to which they belong. There are several types of flavonoids and can be found in foods such as blackberries, blueberries, strawber‐ ries, plum, peach, apple, tomato, cherry, broccoli, onion, soya been, legumes like green gram, lu‐ pine peas, soy beans, white and horse gram, green leafy spices, citrus fruits, tea, red grapes, chocolate, cocoa and red wine beverages [12].

The following briefly discuss some results of studies using antioxidants from fruits and veg‐ etables for the treatment of breast cancer.

Research conducted in Canada by Hakimuddin and colleagues [13] showed that the poly‐ phenols found in red wine have selective toxicity against MCF-7 cell type of breast cancer; the authors indicate the importance of a diet that incorporates red wine and feeding grapes to serve as a preventive strategy against cancer, which also can be combined with standard therapies.

As mentioned above plums and peaches are fruits that contain phenolic compounds. In a study to test the activity of phenolic species as cancer chemopreventive agents present in ex‐ tracts of plums and peaches, we found that peaches and plums contain a mixture of phenol‐ ic compounds with the ability to inhibit cell lines MCF-7 and MDA -MB435. A very important point to consider is that phenolic acids were isolated chlorogenic and neo-chloro‐ genic which have great potential for use as chemopreventive agents exerting growth inhibi‐ tion of the cell line MDA-MB-435 and low toxicity to the normal cell line MCF-10A [14].

Another recent study [15], focused on the action of terpenes located in the skin of the olives suggests that they may serve as natural potential protective against breast cancer. The triter‐ penes were isolated in significant quantities from the pulp of the olive oil and can act pro‐ phylactically and therapeutically.

Currently the investigation for the treatment of breast cancer using apigenin, a flavonoid found in celery. The study conducted at the University of Missouri (United States) [16] was performed in mice that were implanted cell line BT-474, of rapid growth. Mice were also treated with medroxyprogesterone acetate (MPA), which is used in postmenopausal wom‐ en. Another group of mice was used as a blank. The group of mice treated with MPA was injected apigenin, found that cancerous tumors grew rapidly in mice that were treated with apigenin. Moreover, in mice treated with apigenin was observed a decrease of the tumor when compared with the group of mice used as a blank. Yet unknown mechanism of action of apigenin chemical, however, although the study was conducted in mice, is very promis‐ ing for future treatment of breast cancer.

teristic red color to the fruit [31, 32]. It has been reported that tomato consumption reduces

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 363

Another study used extracts of potato species Solanum jamesii to test their cytotoxic activity toward antiproliferatva and prostate cancer cells and colon *in vitro*. The extracts were found to inhibit proliferation of cancer cells PC-3 prostate as well as in colon cancer cells LNCaP. Fractions were also tested extract containing anthocyanin and it showed the same activity as

It is a type of cancer that has one of the top female deaths worldwide [37]. Its main cause is due to Human Papilloma Virus, which is a group of more than 150 types of viruses and is transmitted by sexual contact [38]. To the treatment of cervical cancer, chemotherapy and ra‐ diation therapy is performed. As prevention against this type of cancer was recommended not realize sexual contact with infected persons. Another form of prevention is the applica‐ tion of the vaccine that protects against types of HPV high risk of developing cancer. These vaccines Gardasil ® and Cervarix ® were approved by the Federal Drug Administration (FDA) of EU, but these vaccines are only for women, 9 to 26 years of age who are not infect‐

Another recommendation to prevent this cancer is to stimulate the immune system by eat‐ ing foods rich in antioxidants, because if the body is weakened, the virus is an opportunity to attack and develop cancer [38]. Have also been performed *in vitro* studies to observe foods as antioxidants influence on the growth of cervical cancer cells [39]. One study was carried out with extracts of different types of berries and tested for anti-proliferative activity on HeLa cells (cervical carcinoma). The results show that extracts from blueberry and pome‐ granate have little effect inhibiting the growth of HeLa cells. The most effective extracts with increasing concentration were: strawberry extract, arctic bramble, lingonberry and cloudber‐ ry. It has also been reported [40] that glycoalkaloids present in commercial potatoes inhibit

the growth of different types of cancer cell lines, including HeLa cervical cancer cells.

In therapy of cancer selenium doses is 4000 µg in continuous infusion of 1000 µg/9 days, to‐ tal: 13 mg [41] (Forceville et al, 2007), i.v. bolus 1000 µg in 30 minutes for continuous infu‐ sion 1000 µg/d 14 d, total: 15 mg; i.v. bolus 2000 µg in 2 hours continuous infusion 1600

Diabetes is a metabolic disorder associated with defects in secretion and insulin action [43]. Type 1 diabetes also known as insulin dependent and type 2 diabetes called non-insulin de‐ pendent. Both conditions are associated with the formation of free radicals that cause oxida‐ tive stress and disease manifestation. Diabetes is associated with health problems such as neuropathy, retinopathy, erectile dysfunction in men, kidney problems, healing and more

the occurrence of prostate cancer [33-35].

the full extract [36].

**2.3. Cervical cancer**

ed by the virus.

µg/d, 10 d, total 18 mg [42].

**3. Diabetes**

### **2.2. Prostate cancer**

Prostate cancer is a very common type of cancer afflicting men; it is now easy detection by prostate specific antigen test (PSA for its initials in English) [17]. Which are still unknown factors that cause this type of cancer, the disease also takes years in some cases to express symptoms, making it necessary for men to undergo regular medical examinations to detect early. One form of treatment of prostate cancer is surgery, whereby the prostate is removed, but this is a procedure which results in urinary incontinence and impotence, which in some cases is permanent.

Prevention through diet prostate cancer has increased because it is recognized as a way to combat this disease [18, 19]. Among the foods that are recommended for the prevention of prostate cancer are generally fruits and vegetables due to its high content of antioxidants. Fruits like pomegranate containing metabolites such as polyphenols and delphinidin uroliti‐ na A and B chloride, kaempferol, and punicic acid are considered biologically active against prostate cancer [20, 21].

Other fruit that contains a variety of polyphenolic compounds is strawberry [22] has been found that extracts of strawberry juice cell lines tested against prostate cancer proved effec‐ tive as antiproliferative agents, is also noteworthy to mention that were tested individually some of the individual components of the extract (cyanidin-3-glucoside, pelargonidin, pelar‐ gonidin-3-glucoside, pelargonidin-3-rutinoside, kaempferol, quercetin, kaempferol-3-(6' coumaroyl) glucoside, 3,4,5 trihydroxyphenyl-acrylic acid-, glucose ester of (E)-*p*-coumaric acid, and ellagic acid) which also showed efficacy individually [23]. These studies confirm the effectiveness of the cutter to inhibit growth of cancer cells.

The apple is considered the quintessential fruit of health, its daily intake is associated with low risk of chronic diseases and cancer, particularly prostate and colon [24-26]. The block contains a variety of compounds polyphenolic that are responsible for their biological activi‐ ty among these compounds, studies were performed with quercetin which has proven effec‐ tive as an inhibitor *in vitro* cell growth of prostate cancer [23, 24]. Another study showed that the antioxidant activity of apples is correlated [27] with the total concentration of phe‐ nolic compounds present in it clear that this concentration varies according to growing re‐ gion, and other growth period factors [28-30]. The tomato is another fruit with high antioxidant capacity and owes its activity to lycopene, a carotenoid, which gives the charac‐ teristic red color to the fruit [31, 32]. It has been reported that tomato consumption reduces the occurrence of prostate cancer [33-35].

Another study used extracts of potato species Solanum jamesii to test their cytotoxic activity toward antiproliferatva and prostate cancer cells and colon *in vitro*. The extracts were found to inhibit proliferation of cancer cells PC-3 prostate as well as in colon cancer cells LNCaP. Fractions were also tested extract containing anthocyanin and it showed the same activity as the full extract [36].

### **2.3. Cervical cancer**

Currently the investigation for the treatment of breast cancer using apigenin, a flavonoid found in celery. The study conducted at the University of Missouri (United States) [16] was performed in mice that were implanted cell line BT-474, of rapid growth. Mice were also treated with medroxyprogesterone acetate (MPA), which is used in postmenopausal wom‐ en. Another group of mice was used as a blank. The group of mice treated with MPA was injected apigenin, found that cancerous tumors grew rapidly in mice that were treated with apigenin. Moreover, in mice treated with apigenin was observed a decrease of the tumor when compared with the group of mice used as a blank. Yet unknown mechanism of action of apigenin chemical, however, although the study was conducted in mice, is very promis‐

Prostate cancer is a very common type of cancer afflicting men; it is now easy detection by prostate specific antigen test (PSA for its initials in English) [17]. Which are still unknown factors that cause this type of cancer, the disease also takes years in some cases to express symptoms, making it necessary for men to undergo regular medical examinations to detect early. One form of treatment of prostate cancer is surgery, whereby the prostate is removed, but this is a procedure which results in urinary incontinence and impotence, which in some

Prevention through diet prostate cancer has increased because it is recognized as a way to combat this disease [18, 19]. Among the foods that are recommended for the prevention of prostate cancer are generally fruits and vegetables due to its high content of antioxidants. Fruits like pomegranate containing metabolites such as polyphenols and delphinidin uroliti‐ na A and B chloride, kaempferol, and punicic acid are considered biologically active against

Other fruit that contains a variety of polyphenolic compounds is strawberry [22] has been found that extracts of strawberry juice cell lines tested against prostate cancer proved effec‐ tive as antiproliferative agents, is also noteworthy to mention that were tested individually some of the individual components of the extract (cyanidin-3-glucoside, pelargonidin, pelar‐ gonidin-3-glucoside, pelargonidin-3-rutinoside, kaempferol, quercetin, kaempferol-3-(6' coumaroyl) glucoside, 3,4,5 trihydroxyphenyl-acrylic acid-, glucose ester of (E)-*p*-coumaric acid, and ellagic acid) which also showed efficacy individually [23]. These studies confirm

The apple is considered the quintessential fruit of health, its daily intake is associated with low risk of chronic diseases and cancer, particularly prostate and colon [24-26]. The block contains a variety of compounds polyphenolic that are responsible for their biological activi‐ ty among these compounds, studies were performed with quercetin which has proven effec‐ tive as an inhibitor *in vitro* cell growth of prostate cancer [23, 24]. Another study showed that the antioxidant activity of apples is correlated [27] with the total concentration of phe‐ nolic compounds present in it clear that this concentration varies according to growing re‐ gion, and other growth period factors [28-30]. The tomato is another fruit with high antioxidant capacity and owes its activity to lycopene, a carotenoid, which gives the charac‐

the effectiveness of the cutter to inhibit growth of cancer cells.

ing for future treatment of breast cancer.

362 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**2.2. Prostate cancer**

cases is permanent.

prostate cancer [20, 21].

It is a type of cancer that has one of the top female deaths worldwide [37]. Its main cause is due to Human Papilloma Virus, which is a group of more than 150 types of viruses and is transmitted by sexual contact [38]. To the treatment of cervical cancer, chemotherapy and ra‐ diation therapy is performed. As prevention against this type of cancer was recommended not realize sexual contact with infected persons. Another form of prevention is the applica‐ tion of the vaccine that protects against types of HPV high risk of developing cancer. These vaccines Gardasil ® and Cervarix ® were approved by the Federal Drug Administration (FDA) of EU, but these vaccines are only for women, 9 to 26 years of age who are not infect‐ ed by the virus.

Another recommendation to prevent this cancer is to stimulate the immune system by eat‐ ing foods rich in antioxidants, because if the body is weakened, the virus is an opportunity to attack and develop cancer [38]. Have also been performed *in vitro* studies to observe foods as antioxidants influence on the growth of cervical cancer cells [39]. One study was carried out with extracts of different types of berries and tested for anti-proliferative activity on HeLa cells (cervical carcinoma). The results show that extracts from blueberry and pome‐ granate have little effect inhibiting the growth of HeLa cells. The most effective extracts with increasing concentration were: strawberry extract, arctic bramble, lingonberry and cloudber‐ ry. It has also been reported [40] that glycoalkaloids present in commercial potatoes inhibit the growth of different types of cancer cell lines, including HeLa cervical cancer cells.

In therapy of cancer selenium doses is 4000 µg in continuous infusion of 1000 µg/9 days, to‐ tal: 13 mg [41] (Forceville et al, 2007), i.v. bolus 1000 µg in 30 minutes for continuous infu‐ sion 1000 µg/d 14 d, total: 15 mg; i.v. bolus 2000 µg in 2 hours continuous infusion 1600 µg/d, 10 d, total 18 mg [42].

### **3. Diabetes**

Diabetes is a metabolic disorder associated with defects in secretion and insulin action [43]. Type 1 diabetes also known as insulin dependent and type 2 diabetes called non-insulin de‐ pendent. Both conditions are associated with the formation of free radicals that cause oxida‐ tive stress and disease manifestation. Diabetes is associated with health problems such as neuropathy, retinopathy, erectile dysfunction in men, kidney problems, healing and more [44, 45]. Because diabetes is a disease of oxidative stress, it is expected that the antioxidants in fruits, vegetables and plants to help combat it.

In this fatty acid properties are attributed as an antioxidant par excellence also can help reuse of other antioxidants like vitamins C and E, glutathione and coenzyme Q10. Among the many properties that are attributed to reduction of varicose veins, skin moisture, enhances

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 365

Also attributed the reduction in blood glucose levels for type 2 diabetes and help combat the discomforts caused by peripheral neuropathy, and therefore coupled with the effects men‐

Currently sold in different forms under different names, but the diabetic patient can take doses of lipoic acid consuming identified through the diet. No indication that lipoic acid has

Arteriosclerosis is the hardening of the arteries due to fat accumulation; this may lead to a heart attack that can end life [69]. Atherosclerosis is a preventable disease with a balanced diet and exercise. The diet should include variety of fruits and vegetables and be low in fat. Antioxidants play an important role in preventing this disease, it is known that there is a relationship between red wine consumption and the low incidence of cardiovascular dis‐ ease; this is due to the action of the antioxidants present in grapes. We recommend a daily intake of 375 mL of red wine to increase levels of high density lipoprotein HDL proteins, ie proteins responsible for transporting fat [70, 71]. Studies with another fruits can be deter‐

Another fruit that has been investigated for its antioxidant and cardiovascular protec‐ tive effects are blueberries. Studies realized in Arkansas State University, evaluated the effect on two groups of mice for twenty weeks. One group was used as a target, lead‐ ing a normal diet; the other group was fed a blueberry base [72], found that mice with arterial lesions, a significant percentage decreased injuries, compared with the group of mice that did not eat blueberries. The researchers suggest incorporating blueberries to the diet to improve cardiovascular health and recommended as the ideal fruit for the

It is known that fruits such as cranberries have high antioxidant levels and tested their effec‐ tiveness in promoting cardiovascular health [73-75]. This study was supplemented to a group of men for two weeks with cranberry juice. Over time he found an increase in plasma antioxidant capacity and a decrease in LDL (low density lipoprotein) in addition to an in‐ crease in HDL in obese men. Work is to show whether supplementation based cranberry juice may have the same antioxidant capacity and the same protective benefit as red wine, if

In another study conducted at the University of Buffalo studied the effect of resveratrol as an antioxidant and its possible use in treating atherosclerosis. In this investigation were not used fruits or vegetables, but was used an extract of the plant. The extract containing resver‐

contraindications, although high doses can cause episodes of hypoglycemia [68].

energy levels in the body, cancer protection among others.

tioned above, this antioxidant is ideal for diabetics [62-67].

mining its effectiveness in the prevention of arteriosclerosis.

treatment of hypercholesterolemia.

so would avoid alcohol.

**4. Arteriosclerosis**

Several studies report that a proper diet that includes antioxidants is important to reduce the risk of diabetes. We have found that various antioxidants present in some foods and plants as coumarins, some terpenes, flavonoids, lignans, phenylpropanoids, tannins and can help people prevent disease and for helping diabetics [46, 47]. These substances exert their activity by inhibiting the action of R-amylase enzyme. Amylase is an enzyme produced in the pancreas and salivary glands; their function is to help the digestion of carbohydrates [48]. Among the flavonoids that can inhibit R-amylase are the quercetin, myricetin, epigallo‐ catechin gallate, and cyanidin. Tannins, present in green and black teas, grapes, wine, rasp‐ berry, and strawberry, also seem to be good R-amylase inhibitors. Among fruits and vegetables reported with inhibitory capacity toward the R-amylase in vitro are the red grapes, strawberry, raspberry and, green pepper, broccoli, ginger, and carrot [49-54].

Thanks to these findings, it has been proposed the use of some natural metabolites present in these fruits for the control of hyperglycemia following ingestion of food. The advantage of these natural metabolites is that its use can avoid the side effects that occur when drugs are used for this purpose [55, 56].

Consumption of foods rich in antioxidants can also prevent the complications of this dis‐ ease has recently been shown that biotin is a vitamin which is part of the B vitamins, which can be found in foods such as biotin find when we eat certain vegetables: cauli‐ flower, peanut butter, mushrooms, yeast, potatoes, mushrooms, almonds, walnuts, soy‐ beans, chickpeas, grapes, strawberries, watermelon, bananas, wheat, flour, pasta, bread, oats, rice, liver, yolk egg, kidney, fish, poultry and offal in general, can help improve metabolism and insulin sensitivity, leading to decreased levels of blood sugar, also sold capsules containing biotin [57, 58].

Resveratrol is a polyphenol present in red wine. According to research Medical Center, University of Texas Southwestern in the U.S. [60], resveratrol administered directly in‐ to the brain of diabetic mice, can help control type two diabetes by improving blood sugar levels. What makes the resveratrol is to activate a protein called sirtuin which is expressed in parts of the brain that govern the metabolism of glucose. Much remains to be investigated but it is certainly likely that the intake of red wine under medical super‐ vision can help control diabetes.

Also been studied antioxidants in plants and animals such as the following examples show.

A group of researchers at the University of Jaen in Spain isolated a compound called Cin‐ namtannin B-1 of the laurel, which has antioxidant properties that can eliminate free radi‐ cals that cause diseases such as diabetes. The university has signed an agreement with a pharmaceutical for the distribution of this antioxidant [61].

Lipoic acid, also known as alpha lipoic acid or thioctic acid, is produced in small quantities our bodies, it participates in the metabolism significantly. Can also be found in foods like red meat, yeast and some vegetables such as spinach, broccoli.

In this fatty acid properties are attributed as an antioxidant par excellence also can help reuse of other antioxidants like vitamins C and E, glutathione and coenzyme Q10. Among the many properties that are attributed to reduction of varicose veins, skin moisture, enhances energy levels in the body, cancer protection among others.

Also attributed the reduction in blood glucose levels for type 2 diabetes and help combat the discomforts caused by peripheral neuropathy, and therefore coupled with the effects men‐ tioned above, this antioxidant is ideal for diabetics [62-67].

Currently sold in different forms under different names, but the diabetic patient can take doses of lipoic acid consuming identified through the diet. No indication that lipoic acid has contraindications, although high doses can cause episodes of hypoglycemia [68].

### **4. Arteriosclerosis**

[44, 45]. Because diabetes is a disease of oxidative stress, it is expected that the antioxidants

Several studies report that a proper diet that includes antioxidants is important to reduce the risk of diabetes. We have found that various antioxidants present in some foods and plants as coumarins, some terpenes, flavonoids, lignans, phenylpropanoids, tannins and can help people prevent disease and for helping diabetics [46, 47]. These substances exert their activity by inhibiting the action of R-amylase enzyme. Amylase is an enzyme produced in the pancreas and salivary glands; their function is to help the digestion of carbohydrates [48]. Among the flavonoids that can inhibit R-amylase are the quercetin, myricetin, epigallo‐ catechin gallate, and cyanidin. Tannins, present in green and black teas, grapes, wine, rasp‐ berry, and strawberry, also seem to be good R-amylase inhibitors. Among fruits and vegetables reported with inhibitory capacity toward the R-amylase in vitro are the red

grapes, strawberry, raspberry and, green pepper, broccoli, ginger, and carrot [49-54].

Thanks to these findings, it has been proposed the use of some natural metabolites present in these fruits for the control of hyperglycemia following ingestion of food. The advantage of these natural metabolites is that its use can avoid the side effects that occur when drugs

Consumption of foods rich in antioxidants can also prevent the complications of this dis‐ ease has recently been shown that biotin is a vitamin which is part of the B vitamins, which can be found in foods such as biotin find when we eat certain vegetables: cauli‐ flower, peanut butter, mushrooms, yeast, potatoes, mushrooms, almonds, walnuts, soy‐ beans, chickpeas, grapes, strawberries, watermelon, bananas, wheat, flour, pasta, bread, oats, rice, liver, yolk egg, kidney, fish, poultry and offal in general, can help improve metabolism and insulin sensitivity, leading to decreased levels of blood sugar, also sold

Resveratrol is a polyphenol present in red wine. According to research Medical Center, University of Texas Southwestern in the U.S. [60], resveratrol administered directly in‐ to the brain of diabetic mice, can help control type two diabetes by improving blood sugar levels. What makes the resveratrol is to activate a protein called sirtuin which is expressed in parts of the brain that govern the metabolism of glucose. Much remains to be investigated but it is certainly likely that the intake of red wine under medical super‐

Also been studied antioxidants in plants and animals such as the following examples show.

A group of researchers at the University of Jaen in Spain isolated a compound called Cin‐ namtannin B-1 of the laurel, which has antioxidant properties that can eliminate free radi‐ cals that cause diseases such as diabetes. The university has signed an agreement with a

Lipoic acid, also known as alpha lipoic acid or thioctic acid, is produced in small quantities our bodies, it participates in the metabolism significantly. Can also be found in foods like

in fruits, vegetables and plants to help combat it.

364 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

are used for this purpose [55, 56].

capsules containing biotin [57, 58].

vision can help control diabetes.

pharmaceutical for the distribution of this antioxidant [61].

red meat, yeast and some vegetables such as spinach, broccoli.

Arteriosclerosis is the hardening of the arteries due to fat accumulation; this may lead to a heart attack that can end life [69]. Atherosclerosis is a preventable disease with a balanced diet and exercise. The diet should include variety of fruits and vegetables and be low in fat. Antioxidants play an important role in preventing this disease, it is known that there is a relationship between red wine consumption and the low incidence of cardiovascular dis‐ ease; this is due to the action of the antioxidants present in grapes. We recommend a daily intake of 375 mL of red wine to increase levels of high density lipoprotein HDL proteins, ie proteins responsible for transporting fat [70, 71]. Studies with another fruits can be deter‐ mining its effectiveness in the prevention of arteriosclerosis.

Another fruit that has been investigated for its antioxidant and cardiovascular protec‐ tive effects are blueberries. Studies realized in Arkansas State University, evaluated the effect on two groups of mice for twenty weeks. One group was used as a target, lead‐ ing a normal diet; the other group was fed a blueberry base [72], found that mice with arterial lesions, a significant percentage decreased injuries, compared with the group of mice that did not eat blueberries. The researchers suggest incorporating blueberries to the diet to improve cardiovascular health and recommended as the ideal fruit for the treatment of hypercholesterolemia.

It is known that fruits such as cranberries have high antioxidant levels and tested their effec‐ tiveness in promoting cardiovascular health [73-75]. This study was supplemented to a group of men for two weeks with cranberry juice. Over time he found an increase in plasma antioxidant capacity and a decrease in LDL (low density lipoprotein) in addition to an in‐ crease in HDL in obese men. Work is to show whether supplementation based cranberry juice may have the same antioxidant capacity and the same protective benefit as red wine, if so would avoid alcohol.

In another study conducted at the University of Buffalo studied the effect of resveratrol as an antioxidant and its possible use in treating atherosclerosis. In this investigation were not used fruits or vegetables, but was used an extract of the plant. The extract containing resver‐ atrol was administered at doses of 40 mg daily to a group of 10 people, another group of 10 people also served as a target. During the six weeks of the study, blood tests were per‐ formed on the results; researchers concluded that Polygonum cuspidatum extract has a ther‐ apeutic effect against oxidative stress. These results show that resveratrol, as already mentioned above, are effective to counteract the effect of free radicals, and in the case of ar‐ teriosclerosis, can also help prevent it [76].

er, when the ability of the liver to store retinol is exceeded or when liver function is impaired [84]. Thus, the higher retinyl ester concentrations among those who did not have the metabolic syndrome may indicate that they consumed larger amounts of vitamin A com‐ pared with people who have this syndrome. Our findings may have implications for people with the metabolic syndrome, health care professionals who care for them and researchers who study the metabolic syndrome. People with the metabolic syndrome are at increased risk for diabetes and cardiovascular disease, and a role for oxidative stress in the patho‐ physiology of these conditions has been postulated. Free radical species is one of the princi‐ pal mechanisms of action of antioxidants, other mechanisms that affect the pathophysiology of diabetes and cardiovascular disease may be operating as well [83]. The effects of vitamins C and E have received a great deal of interest. Through effects on oxidation of LDL choles‐ terol concentration, leukocyte adhesion, and endothelial function, vitamins C and E may

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 367

Currently the evidence supports the role of nutritional deficiency in Alcoholic Liver Disease (ALD) [88–95]. Lieber and colleagues show that progressive ALD proceeds despite adequate nutrition [96, 97]. The latter hypothesis was based primarily on the observation that baboons fed a nutritionally adequate liquid diet containing ethanol at 50% calories developed nearly the whole spectrum of ALD including cirrhosis. Studies demonstrated profound effects on ethanol-induced liver injury by intake of nutrients such as polyunsaturated fat and iron in quantities that were never thought to be important. The concept of 'sensitization' and 'pri‐ ming' is currently considered fundamental to our pursuit for elucidation of pathogenetic mechanisms of ALD. The sensitization is a conditioning that makes the target cells, hepato‐ cytes, more vulnerable to harmful effects triggered by ethanol and priming as the effect that promotes specific injurious mechanisms. The sensitizing and priming are rendered by the complex interactions of primary mechanistic factors and secondary risk factors. For exam‐ ple, intake of polyunsaturated fat in ethanol-fed rats, but not in pair-fed controls, results in a synergistic priming effect on induction of cytochrome P4502. E1 (CYP2E1) with consequent oxidative injury to the liver [98]. Conversely, saturated fat prevents this priming effect and abrogates depletion of a mitochondrial pool of glutathione (GSH) [99], one of the most cru‐ cial sensitization effects of ethanol on hepatocytes [100]. Iron is another example. Whereas a slight increase in hepatic iron content by dietary iron supplementation is harmless in control rats, it exacerbates alcoholic liver injury via accentuation of oxidative stress [101]. Further, increased iron storage in hepatic macrophages is a potential priming mechanism fore‐ nhanced expression of tumor necrosis factor a (TNF-a) in experimental ALD [102] Besides nutritional factors, female gender, age, concomitant intake of other drugs that can induce CYP2E1, hepatitis virus infection, and genetic predisposition are all considered risk factors. Even among the primary mechanistic factors that include acetaldehyde, oxidative stress, im‐ mune response, hypoxia, and membrane alterations, there are cross-interactive relationships to render sensitization or priming effects. For instance, acetaldehyde, a potent toxic metabo‐

slow atherosclerosis [86, 87].

**6. Liver cirrhosis**

### **5. Obesity and metabolic syndrome**

The metabolic syndrome has been identified as a target for dietary therapies to reduce risk of cardiovascular disease; however, the role of diet in the etiology of the metabolic syn‐ drome is poorly understood. The metabolic syndrome consists of a constellation of factors that increase the risk of cardiovascular disease and type 2 diabetes. The etiology of this syn‐ drome is largely unknown but presumably represents a complex interaction between genet‐ ic, metabolic, and environmental factors including diet [77-79]. The studies endothelial function by assessing the vascular responses to L-arginine, the natural precursor of nitric ox‐ ide it´s characterized for the low-grade inflammatory state of patients with the metabolic syndrome by measuring circulating levels of high-sensitivity C-reactive protein (hs-CRP) as well as of interleukins 6 (IL-6), 7 (IL-7), and 18 (IL-18). These proinflammatory ILs have been prospectively associated with thrombotic cardiovascular events [80, 81] or have been sug‐ gested to be involved in plaque destabilization [82]. The diet designed to increase consump‐ tion of foods rich in phytochemicals, antioxidants, α-linolenic acid, and fiber prevent Metabolic Syndrome.

The diet rich in whole grains, fruits, vegetables, legumes, walnuts, and olive oil might be ef‐ fective in reducing both the prevalence of the metabolic syndrome and its associated cardio‐ vascular risk. One of the mechanisms responsible for the cardioprotective effect of such a diet may be through reduction of the low-grade inflammatory state associated with the met‐ abolic syndrome. Although weight reduction remains a cornerstone of therapy for the meta‐ bolic syndrome, from a public health perspective adoption of a diet rich in phytochemicals, antioxidants, α-linolenic acid, and fiber may provide further benefit on cardiovascular risk, especially in patients who do not lose weight.

If antioxidants play a protective role in the pathophysiology of diabetes and cardiovascular disease, understanding the physiological status of antioxidant concentrations among people at high risk for developing these conditions, such as people with the metabolic syndrome, is of interest. However, little is known about this topic. Because the prevalence of obesity, which is associated with decreased concentrations of antioxidants [83], is high among peo‐ ple with the metabolic syndrome, they are probably more likely to have low antioxidant concentrations. Consequently, our purpose was to examine whether concentrations of sever‐ al antioxidants are lower among those with than those without the metabolic syndrome.

For example a retinol from the liver, the main storage site for retinol is transported to pe‐ ripheral tissues by retinol binding protein. Retinol may be released as a retinyl ester; howev‐ er, when the ability of the liver to store retinol is exceeded or when liver function is impaired [84]. Thus, the higher retinyl ester concentrations among those who did not have the metabolic syndrome may indicate that they consumed larger amounts of vitamin A com‐ pared with people who have this syndrome. Our findings may have implications for people with the metabolic syndrome, health care professionals who care for them and researchers who study the metabolic syndrome. People with the metabolic syndrome are at increased risk for diabetes and cardiovascular disease, and a role for oxidative stress in the patho‐ physiology of these conditions has been postulated. Free radical species is one of the princi‐ pal mechanisms of action of antioxidants, other mechanisms that affect the pathophysiology of diabetes and cardiovascular disease may be operating as well [83]. The effects of vitamins C and E have received a great deal of interest. Through effects on oxidation of LDL choles‐ terol concentration, leukocyte adhesion, and endothelial function, vitamins C and E may slow atherosclerosis [86, 87].

### **6. Liver cirrhosis**

atrol was administered at doses of 40 mg daily to a group of 10 people, another group of 10 people also served as a target. During the six weeks of the study, blood tests were per‐ formed on the results; researchers concluded that Polygonum cuspidatum extract has a ther‐ apeutic effect against oxidative stress. These results show that resveratrol, as already mentioned above, are effective to counteract the effect of free radicals, and in the case of ar‐

The metabolic syndrome has been identified as a target for dietary therapies to reduce risk of cardiovascular disease; however, the role of diet in the etiology of the metabolic syn‐ drome is poorly understood. The metabolic syndrome consists of a constellation of factors that increase the risk of cardiovascular disease and type 2 diabetes. The etiology of this syn‐ drome is largely unknown but presumably represents a complex interaction between genet‐ ic, metabolic, and environmental factors including diet [77-79]. The studies endothelial function by assessing the vascular responses to L-arginine, the natural precursor of nitric ox‐ ide it´s characterized for the low-grade inflammatory state of patients with the metabolic syndrome by measuring circulating levels of high-sensitivity C-reactive protein (hs-CRP) as well as of interleukins 6 (IL-6), 7 (IL-7), and 18 (IL-18). These proinflammatory ILs have been prospectively associated with thrombotic cardiovascular events [80, 81] or have been sug‐ gested to be involved in plaque destabilization [82]. The diet designed to increase consump‐ tion of foods rich in phytochemicals, antioxidants, α-linolenic acid, and fiber prevent

The diet rich in whole grains, fruits, vegetables, legumes, walnuts, and olive oil might be ef‐ fective in reducing both the prevalence of the metabolic syndrome and its associated cardio‐ vascular risk. One of the mechanisms responsible for the cardioprotective effect of such a diet may be through reduction of the low-grade inflammatory state associated with the met‐ abolic syndrome. Although weight reduction remains a cornerstone of therapy for the meta‐ bolic syndrome, from a public health perspective adoption of a diet rich in phytochemicals, antioxidants, α-linolenic acid, and fiber may provide further benefit on cardiovascular risk,

If antioxidants play a protective role in the pathophysiology of diabetes and cardiovascular disease, understanding the physiological status of antioxidant concentrations among people at high risk for developing these conditions, such as people with the metabolic syndrome, is of interest. However, little is known about this topic. Because the prevalence of obesity, which is associated with decreased concentrations of antioxidants [83], is high among peo‐ ple with the metabolic syndrome, they are probably more likely to have low antioxidant concentrations. Consequently, our purpose was to examine whether concentrations of sever‐ al antioxidants are lower among those with than those without the metabolic syndrome.

For example a retinol from the liver, the main storage site for retinol is transported to pe‐ ripheral tissues by retinol binding protein. Retinol may be released as a retinyl ester; howev‐

teriosclerosis, can also help prevent it [76].

366 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**5. Obesity and metabolic syndrome**

especially in patients who do not lose weight.

Metabolic Syndrome.

Currently the evidence supports the role of nutritional deficiency in Alcoholic Liver Disease (ALD) [88–95]. Lieber and colleagues show that progressive ALD proceeds despite adequate nutrition [96, 97]. The latter hypothesis was based primarily on the observation that baboons fed a nutritionally adequate liquid diet containing ethanol at 50% calories developed nearly the whole spectrum of ALD including cirrhosis. Studies demonstrated profound effects on ethanol-induced liver injury by intake of nutrients such as polyunsaturated fat and iron in quantities that were never thought to be important. The concept of 'sensitization' and 'pri‐ ming' is currently considered fundamental to our pursuit for elucidation of pathogenetic mechanisms of ALD. The sensitization is a conditioning that makes the target cells, hepato‐ cytes, more vulnerable to harmful effects triggered by ethanol and priming as the effect that promotes specific injurious mechanisms. The sensitizing and priming are rendered by the complex interactions of primary mechanistic factors and secondary risk factors. For exam‐ ple, intake of polyunsaturated fat in ethanol-fed rats, but not in pair-fed controls, results in a synergistic priming effect on induction of cytochrome P4502. E1 (CYP2E1) with consequent oxidative injury to the liver [98]. Conversely, saturated fat prevents this priming effect and abrogates depletion of a mitochondrial pool of glutathione (GSH) [99], one of the most cru‐ cial sensitization effects of ethanol on hepatocytes [100]. Iron is another example. Whereas a slight increase in hepatic iron content by dietary iron supplementation is harmless in control rats, it exacerbates alcoholic liver injury via accentuation of oxidative stress [101]. Further, increased iron storage in hepatic macrophages is a potential priming mechanism fore‐ nhanced expression of tumor necrosis factor a (TNF-a) in experimental ALD [102] Besides nutritional factors, female gender, age, concomitant intake of other drugs that can induce CYP2E1, hepatitis virus infection, and genetic predisposition are all considered risk factors. Even among the primary mechanistic factors that include acetaldehyde, oxidative stress, im‐ mune response, hypoxia, and membrane alterations, there are cross-interactive relationships to render sensitization or priming effects. For instance, acetaldehyde, a potent toxic metabo‐ lite of ethanol, induces liver injury via its covalent binding to structural or functional pro‐ teins of the cells [103] while promoting oxidative stress via consumption of GSH. In turn, deleterious effects of acetaldehyde-protein adduct formation may be accentuated by oxida‐ tive stress since malondialdehyde, a lipid peroxidation end product, can increase the bind‐ ing affinity of acetaldehyde by 13-fold [104]. The resulting novel hybrid adducts are highly immunogenic and may incite immune response mediated liver injury [105, 106]. Although cellular immune response and inflammation are regarded as independent mechanisms of ALD, they can also lead to oxidative stress via the release of reactive oxygen species (ROS) by NADPH oxidase or action of TNF-a at the electron transport chain in target cells. The multifactorial nature and complex interaction among primary mechanistic factors and be‐ tween primary and secondary factors appear to be the basis for the heterogeneous response that alcoholics exhibit for ALD. Elucidation of the sensitization and priming mechanisms in‐ volving cross-interactions of these factors should allow us to gain insight into the most fun‐ damental question, which is why only a small fraction of alcoholics develop advanced ALD. The experimental models to use for control deletion and addition analyses in order to identi‐ fy what primary and secondary factors are required for the expression of a particular aspect or whole spectrum of experimental ALD. It is need experts in various disciplines need to work together to provide cutting-edge science for elucidating the precise nature and mecha‐ nisms that underlie interactions.

**7. Hypertension**

therapy at this time.

tration of O2

−

these sources is superoxide (O2

Excessive reactive oxygen species (ROS) have emerged as a central common pathway by which disparate influences may induce and exacerbate hypertension. Potential sources of excessive ROS in hypertension include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mitochondria, xanthine oxidase, endothelium-derived NO synthase, cy‐ clooxygenase 1 and 2, cytochrome P450 epoxygenase, and transition metals. While a signifi‐ cant body of epidemiological and clinical data suggests that antioxidant-rich diets reduce blood pressure and cardiovascular risk, randomized trials and population studies using nat‐ ural antioxidants have yielded disappointing results. The reasons behind this lack of efficacy are not completely clear, but likely include a combination of [122] ineffective dosing regi‐ mens, [123] the potential pro-oxidant capacity of some of these agents, [124] selection of sub‐ jects less likely to benefit from antioxidant therapy (too healthy or too sick), and inefficiency of nonspecific quenching of prevalent ROS versus prevention of excessive ROS production. Antioxidants as vitamins A, C and E, L-arginine, flavonoids, and mitochondria-targeted agents (Coenzyme Q10, acetyl-L-carnitine, and alpha-lipoic acid) can be use to treatment hy‐ pertension. Currently exist incomplete knowledge of the mechanisms of action of these agents, lack of target specificity, and potential interindividual differences in therapeutic effi‐ cacy preclude us from recommending any specific natural antioxidant for antihypertensive

Reactive oxygen species (ROS) are generated by multiple cellular sources, including NADPH oxidase, mitochondria, xanthine oxidase, uncoupled endothelium-derived NO syn‐ thase, cycloxygenase, and lipoxygenase. The dominant initial ROS species produced by

undergo enzymatic dismutation to hydrogen peroxide. Superoxide can oxidize proteins and lipids, or react with endothelium-derived nitric oxide (NO) to create the reactive nitrogen species peroxynitrite. Peroxynitrite and other reactive nitrogen species can subsequently ox‐ idize proteins, lipids, and critical enzymatic cofactors that may further increase oxidative

convert to highly reactive hydroxyl radical (via Fenton chemistry) that can cause DNA dam‐ age. The balance between superoxide production and consumption likely keeps the concen‐

These homeostatic levels of reactive oxygen species appear to be important in normal cellu‐

Randomized trials employing non-pharmacological dietary interventions emphasizing fruits, vegetables, whole grains, and nuts have shown impressive blood pressure lowering results in both hypertensive and normotensive subjects [135, 136]. Similar interventions demonstrated to reduce cardiovascular morbidity and mortality continue to maintain inter‐ est in the potential of isolating specific compounds enriched in these diets that may be re‐

The dietary components in these studies are high in compounds known to have antioxidant properties leading many to ascribe the benefits of these diets to their increased content of

in the picomolar range and hydrogen peroxide in the nanomolar range [126].

). Superoxide is short-lived molecule that can subsequently

<sup>−</sup> can be further

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 369

−

lar signaling [127-132] and normal reactions to stressors [133, 134].

sponsible for the overall dietary benefits [137].

stress [125]. Hydrogen peroxide produced by enzymatic dismutation of O2

Antioxidants represent a potential group of therapeutic agents for ALD. They likely provide beneficial effects on hepatocytes via desensitization against oxidant stress while inhibiting priming mechanisms for expression of proinflammatory and cytotoxic mediators via sup‐ pression of NF-kB [107, 108]. Potential approaches may include cell type-specific targeting of antioxidant therapy and development of modalities for more specific and selective regula‐ tion of NF-kB-mediated signaling.

The development of cirrhosis is usually associated with oxidative stress and lipid peroxida‐ tion (LPO). Studies in models of cirrhosis to use carbon tetrachloride (CCl4) inhalation in the rat show several similarities with human cirrhosis. The metabolism of CCl4 into trichloro‐ methyl (CCl3•) and peroxy trichloromethyl (•OOCCl3) free radicals has been reported to cause hepatotoxic effects, like fibrosis, steatosis, necrosis, and hepatocarcinoma [109, 111].

Some compounds that have been studied as possible protectors against liver cirrhosis are known for their anti-inflammatory and antioxidant properties. Plants contain numerous pol‐ yphenols, which have been shown to reduce inflammation and thereby to increase resist‐ ance to disease [112]. Quercetin (Q), a polyphenolic flavonoid compound present in large amounts in vegetables, fruits, and tea, exhibits its therapeutic potential against many diseas‐ es, including hepatoprotection and the inhibition of liver fibrosis [113–114]. It contains a number of phenolic hydroxyl groups, which have strong antioxidant activity [116, 117]. The average intake varies between countries but is approximately 23 mg/day [118].

By increasing the endogenous antioxidant defenses, flavonoids can modulate the redox state of organisms. The major endogenous antioxidant systems include superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx), which is essential for the detoxification of lipid peroxides [119-121].

### **7. Hypertension**

lite of ethanol, induces liver injury via its covalent binding to structural or functional pro‐ teins of the cells [103] while promoting oxidative stress via consumption of GSH. In turn, deleterious effects of acetaldehyde-protein adduct formation may be accentuated by oxida‐ tive stress since malondialdehyde, a lipid peroxidation end product, can increase the bind‐ ing affinity of acetaldehyde by 13-fold [104]. The resulting novel hybrid adducts are highly immunogenic and may incite immune response mediated liver injury [105, 106]. Although cellular immune response and inflammation are regarded as independent mechanisms of ALD, they can also lead to oxidative stress via the release of reactive oxygen species (ROS) by NADPH oxidase or action of TNF-a at the electron transport chain in target cells. The multifactorial nature and complex interaction among primary mechanistic factors and be‐ tween primary and secondary factors appear to be the basis for the heterogeneous response that alcoholics exhibit for ALD. Elucidation of the sensitization and priming mechanisms in‐ volving cross-interactions of these factors should allow us to gain insight into the most fun‐ damental question, which is why only a small fraction of alcoholics develop advanced ALD. The experimental models to use for control deletion and addition analyses in order to identi‐ fy what primary and secondary factors are required for the expression of a particular aspect or whole spectrum of experimental ALD. It is need experts in various disciplines need to work together to provide cutting-edge science for elucidating the precise nature and mecha‐

368 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Antioxidants represent a potential group of therapeutic agents for ALD. They likely provide beneficial effects on hepatocytes via desensitization against oxidant stress while inhibiting priming mechanisms for expression of proinflammatory and cytotoxic mediators via sup‐ pression of NF-kB [107, 108]. Potential approaches may include cell type-specific targeting of antioxidant therapy and development of modalities for more specific and selective regula‐

The development of cirrhosis is usually associated with oxidative stress and lipid peroxida‐ tion (LPO). Studies in models of cirrhosis to use carbon tetrachloride (CCl4) inhalation in the rat show several similarities with human cirrhosis. The metabolism of CCl4 into trichloro‐ methyl (CCl3•) and peroxy trichloromethyl (•OOCCl3) free radicals has been reported to cause hepatotoxic effects, like fibrosis, steatosis, necrosis, and hepatocarcinoma [109, 111]. Some compounds that have been studied as possible protectors against liver cirrhosis are known for their anti-inflammatory and antioxidant properties. Plants contain numerous pol‐ yphenols, which have been shown to reduce inflammation and thereby to increase resist‐ ance to disease [112]. Quercetin (Q), a polyphenolic flavonoid compound present in large amounts in vegetables, fruits, and tea, exhibits its therapeutic potential against many diseas‐ es, including hepatoprotection and the inhibition of liver fibrosis [113–114]. It contains a number of phenolic hydroxyl groups, which have strong antioxidant activity [116, 117]. The

average intake varies between countries but is approximately 23 mg/day [118].

is essential for the detoxification of lipid peroxides [119-121].

By increasing the endogenous antioxidant defenses, flavonoids can modulate the redox state of organisms. The major endogenous antioxidant systems include superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx), which

nisms that underlie interactions.

tion of NF-kB-mediated signaling.

Excessive reactive oxygen species (ROS) have emerged as a central common pathway by which disparate influences may induce and exacerbate hypertension. Potential sources of excessive ROS in hypertension include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mitochondria, xanthine oxidase, endothelium-derived NO synthase, cy‐ clooxygenase 1 and 2, cytochrome P450 epoxygenase, and transition metals. While a signifi‐ cant body of epidemiological and clinical data suggests that antioxidant-rich diets reduce blood pressure and cardiovascular risk, randomized trials and population studies using nat‐ ural antioxidants have yielded disappointing results. The reasons behind this lack of efficacy are not completely clear, but likely include a combination of [122] ineffective dosing regi‐ mens, [123] the potential pro-oxidant capacity of some of these agents, [124] selection of sub‐ jects less likely to benefit from antioxidant therapy (too healthy or too sick), and inefficiency of nonspecific quenching of prevalent ROS versus prevention of excessive ROS production. Antioxidants as vitamins A, C and E, L-arginine, flavonoids, and mitochondria-targeted agents (Coenzyme Q10, acetyl-L-carnitine, and alpha-lipoic acid) can be use to treatment hy‐ pertension. Currently exist incomplete knowledge of the mechanisms of action of these agents, lack of target specificity, and potential interindividual differences in therapeutic effi‐ cacy preclude us from recommending any specific natural antioxidant for antihypertensive therapy at this time.

Reactive oxygen species (ROS) are generated by multiple cellular sources, including NADPH oxidase, mitochondria, xanthine oxidase, uncoupled endothelium-derived NO syn‐ thase, cycloxygenase, and lipoxygenase. The dominant initial ROS species produced by these sources is superoxide (O2 − ). Superoxide is short-lived molecule that can subsequently undergo enzymatic dismutation to hydrogen peroxide. Superoxide can oxidize proteins and lipids, or react with endothelium-derived nitric oxide (NO) to create the reactive nitrogen species peroxynitrite. Peroxynitrite and other reactive nitrogen species can subsequently ox‐ idize proteins, lipids, and critical enzymatic cofactors that may further increase oxidative stress [125]. Hydrogen peroxide produced by enzymatic dismutation of O2 <sup>−</sup> can be further convert to highly reactive hydroxyl radical (via Fenton chemistry) that can cause DNA dam‐ age. The balance between superoxide production and consumption likely keeps the concen‐ tration of O2 − in the picomolar range and hydrogen peroxide in the nanomolar range [126]. These homeostatic levels of reactive oxygen species appear to be important in normal cellu‐ lar signaling [127-132] and normal reactions to stressors [133, 134].

Randomized trials employing non-pharmacological dietary interventions emphasizing fruits, vegetables, whole grains, and nuts have shown impressive blood pressure lowering results in both hypertensive and normotensive subjects [135, 136]. Similar interventions demonstrated to reduce cardiovascular morbidity and mortality continue to maintain inter‐ est in the potential of isolating specific compounds enriched in these diets that may be re‐ sponsible for the overall dietary benefits [137].

The dietary components in these studies are high in compounds known to have antioxidant properties leading many to ascribe the benefits of these diets to their increased content of natural antioxidants. However, prior randomized trials and population studies in healthy populations and patients at high risk for cardiovascular events that have employed combi‐ nations of some of these natural antioxidants as dietary supplements have, for the most part, shown disappointing results [138-145]. The reasons behind these disappointing results are not completely clear, but likely include a combination of 1) ineffective dosing and dosing regimens 2) the potential pro-oxidant capacity and other potentially deleterious effects of these some of these compounds under certain conditions [146-148], 3) selection of subjects less likely to benefit from antioxidant therapy (too healthy or too sick). Populations at inter‐ mediate cardiovascular risk may be better suitable to see effects of antioxidants in shorter term studies [149], 4) inefficiency of non-specific quenching of prevalent ROS versus preven‐ tion of excessive ROS production [150, 151].

**7.1. Antioxidant vitamins**

*7.1.2. Ascorbic acid (Vitamin C)*

grams/day).

*7.1.1. Vitamin A precursors and derivatives*

compound as an effective antihypertensive agent.

Vitamin A precursors and derivatives are retinoids that consist of a beta-ionone ring attach‐ ed to an isoprenoid carbon chain. Foods high in vitamin A include liver, sweet potato, car‐ rot, pumpkin, and broccoli leaf. Initial interest in vitamin A-related compounds focused primarily on beta-carotene, given initial promising epidemiological data with respect to its cardioprotective effects and some correlation with higher plasma levels to lower blood pres‐ sure in men. However, concerns about beta-carotene's pro-oxidative potential came to light with a report suggesting adverse mitochondrial effects of beta-carotene cleavage products. Further, adverse mortality data with respect to beta-carotene has limited interest in this

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 371

Recently, interest in vitamin A derivatives has turned to lycopene, itself a potent antioxidant [152], found concentrated in tomatoes. One small study has shown a reduction in blood pressure with a tomato-extract based intervention (containing a combination of potential an‐ ti-oxidant compounds including lycopene) in patients with stage I hypertension, [153] al‐

L-ascorbic acid is a six-carbon lactone and, for humans, is an essential nutrient. In Western diets, commonly consumed foods that contain high levels of ascorbic acid include broccoli, lemons, limes, oranges, and strawberries. Toxicity potential of this compound is low, al‐ though an increased risk of oxalate renal calculi may exist at higher doses (exceeding 2

The initial purported mechanisms for the potential benefits of ascorbate supplementation were centered on quenching of single-electron free radicals. Subsequent research has dem‐ onstrated that the plasma concentrations of ascorbate required for this mechanism to be physiologically relevant are not attainable by oral supplementation [155]. However, vitamin C can concentrate in local tissues to levels an order of magnitude higher than that of plasma. At this ascorbate may to effectively compete for superoxide and reduce thiols [156]. Recent data also suggest potential suppressive effects of ascorbate on NADPH oxidase activity [157,

Ascorbate's anti-hypertensive efficacy has been evaluated in multiple small studies [160-163] but not all, show modest reductions in blood pressure in both normotensive and hypertensive populations. These data also suggest that supplementation has limited effect on systemic antioxidant markers and little additional blood pressure benefits are seen be‐ yond the 500 mg daily dose. Large scale randomized trial data specific to ascorbate supple‐ mentation and its effects on hypertension are currently lacking. Data from Heart Protection Study (HPS) suggest no significant mortality from supplementation with 250mg/day of as‐ corbate supplementation. However, the relatively low dose of ascorbate, use of combination therapy, and high-risk patient population studied in HPS leave unanswered the key ques‐

though second study showed no effect in pre-hypertensive patients [154].

158]. Ascorbate appears to have limited pro-oxidant ability. [159].

When considering antioxidant therapy for hypertension, lessons from prior disappointing attempts to reduce blood pressure and cardiovascular risk with antioxidant therapy should be considered. The profile of an ideal agent is outlined in The importance of patient selec‐ tion is being increasingly recognized in light of emerging data suggesting that antioxidant supplementation in healthy subjects may blunt the protective benefits of aerobic exercise training, suggesting ROS generation can be beneficial under certain circumstances.


**Table 1.** Antioxidants neutralize the oxidative processes and modify levels in plasma. [150]

### **7.1. Antioxidant vitamins**

natural antioxidants. However, prior randomized trials and population studies in healthy populations and patients at high risk for cardiovascular events that have employed combi‐ nations of some of these natural antioxidants as dietary supplements have, for the most part, shown disappointing results [138-145]. The reasons behind these disappointing results are not completely clear, but likely include a combination of 1) ineffective dosing and dosing regimens 2) the potential pro-oxidant capacity and other potentially deleterious effects of these some of these compounds under certain conditions [146-148], 3) selection of subjects less likely to benefit from antioxidant therapy (too healthy or too sick). Populations at inter‐ mediate cardiovascular risk may be better suitable to see effects of antioxidants in shorter term studies [149], 4) inefficiency of non-specific quenching of prevalent ROS versus preven‐

When considering antioxidant therapy for hypertension, lessons from prior disappointing attempts to reduce blood pressure and cardiovascular risk with antioxidant therapy should be considered. The profile of an ideal agent is outlined in The importance of patient selec‐ tion is being increasingly recognized in light of emerging data suggesting that antioxidant supplementation in healthy subjects may blunt the protective benefits of aerobic exercise

training, suggesting ROS generation can be beneficial under certain circumstances.

**Antioxidants neutralize the oxidative processes and modify levels in plasma**

↑ Lipid peroxidation ↑ MDA (TBAR), F2-isoprostane

↑ NO synthesis ↑ Nitrite, nitrate, nitrotyrosine

Xanthine oxidase activation ↑ Plasma xanthine oxidase

**Table 1.** Antioxidants neutralize the oxidative processes and modify levels in plasma. [150]

↓ Circulating antioxidants ↓ Uric acid, protein SH groups, Bilirubin

(unconjugated)

↓ Selenium, zinc

↓ GSH

lycopene

↓ Ascorbic acid, α-tocopherol, β-carotene,

↓ Antioxidant enzymes (GSHPx)

tion of excessive ROS production [150, 151].

370 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

### *7.1.1. Vitamin A precursors and derivatives*

Vitamin A precursors and derivatives are retinoids that consist of a beta-ionone ring attach‐ ed to an isoprenoid carbon chain. Foods high in vitamin A include liver, sweet potato, car‐ rot, pumpkin, and broccoli leaf. Initial interest in vitamin A-related compounds focused primarily on beta-carotene, given initial promising epidemiological data with respect to its cardioprotective effects and some correlation with higher plasma levels to lower blood pres‐ sure in men. However, concerns about beta-carotene's pro-oxidative potential came to light with a report suggesting adverse mitochondrial effects of beta-carotene cleavage products. Further, adverse mortality data with respect to beta-carotene has limited interest in this compound as an effective antihypertensive agent.

Recently, interest in vitamin A derivatives has turned to lycopene, itself a potent antioxidant [152], found concentrated in tomatoes. One small study has shown a reduction in blood pressure with a tomato-extract based intervention (containing a combination of potential an‐ ti-oxidant compounds including lycopene) in patients with stage I hypertension, [153] al‐ though second study showed no effect in pre-hypertensive patients [154].

### *7.1.2. Ascorbic acid (Vitamin C)*

L-ascorbic acid is a six-carbon lactone and, for humans, is an essential nutrient. In Western diets, commonly consumed foods that contain high levels of ascorbic acid include broccoli, lemons, limes, oranges, and strawberries. Toxicity potential of this compound is low, al‐ though an increased risk of oxalate renal calculi may exist at higher doses (exceeding 2 grams/day).

The initial purported mechanisms for the potential benefits of ascorbate supplementation were centered on quenching of single-electron free radicals. Subsequent research has dem‐ onstrated that the plasma concentrations of ascorbate required for this mechanism to be physiologically relevant are not attainable by oral supplementation [155]. However, vitamin C can concentrate in local tissues to levels an order of magnitude higher than that of plasma. At this ascorbate may to effectively compete for superoxide and reduce thiols [156]. Recent data also suggest potential suppressive effects of ascorbate on NADPH oxidase activity [157, 158]. Ascorbate appears to have limited pro-oxidant ability. [159].

Ascorbate's anti-hypertensive efficacy has been evaluated in multiple small studies [160-163] but not all, show modest reductions in blood pressure in both normotensive and hypertensive populations. These data also suggest that supplementation has limited effect on systemic antioxidant markers and little additional blood pressure benefits are seen be‐ yond the 500 mg daily dose. Large scale randomized trial data specific to ascorbate supple‐ mentation and its effects on hypertension are currently lacking. Data from Heart Protection Study (HPS) suggest no significant mortality from supplementation with 250mg/day of as‐ corbate supplementation. However, the relatively low dose of ascorbate, use of combination therapy, and high-risk patient population studied in HPS leave unanswered the key ques‐ tions of appropriate dosing and target. In the inflammatory processes follow next scheme in the therapy antioxidant [164].

trial, failed to show blood pressure or mortality benefit for patients at high risk for cardio‐

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 373

L-arginine is an amino acid and the main substrate for the production of NO from eNOS in a reaction that is dependent on tetrahydrobiopterin [173]. Potential dietary sources include milk products, beef, wheat germ, nuts, and soybeans. Reduced levels of tetrahydrobiopterin leads to uncoupling of reduced NADPH oxidation and NO synthesis, with oxygen as termi‐ nal electron acceptor instead of L-arginine, resulting in the generation of superoxide by eNOS [174-176]. Low cellular levels of L-arginine have been demonstrated in human hyper‐ tension. While L-arginine deficiency itself does not appear to lead to uncoupling of eNOS, [177] low levels of L-arginine may lead to reduced levels of bioavailable NO which could contribute to hypertension. Thus, L-arginine supplementation could theoretically reduce blood pressure by allowing for restoration of normal NO bioavailability, perhaps overcom‐ ing overall L-arginine deficiency as well as more successfully competing fo the eNOS active site with circulating asymmetric dimet hylarginine, a circulating competitor of L-arginine

This concept is supported by studies demonstrating the anti-hypertensive effect of L-argi‐ nine supplementation in salt-sensitive rats, healthy human subjects, hypertensive diabetics, patients with chronic kidney disease, and diabetic patients in combination with N-acetylcys‐ teine, a precursor of glutathione [178] L-arginine's anti-hypertensive response may be medi‐ ated in part by its suppressive effects on angiotensin II and endothelin-1, and its

However, recent concerns about potential deleterious increases in homocysteine in the set‐ ting of L-arginine supplementation have been raised. The majority of L-arginine is process‐ ed into creatine, which leads increased homocysteine levels. Homocysteine can increase oxidative stress. A recent study confirms that this mechanism is relevant to L-arginine me‐ tabolism in humans [179] suggesting a potential mechanism for neutralizing the eNOS-relat‐

vascular disease [172]. Vitamin E inhibits free radicals reactions.

**Figure 3.** Antioxidants function in the organism.

that may be increased in the setting of hypertension.

potentiating effects on insulin.

ed anti-oxidant effects of L-arginine.

*7.1.4. L-Arginine*

**Figure 2.** Restored normal endothelial function.

### *7.1.3. α-Tocopherol (Vitamin E)*

Vitamin E is a generic term for a group of compounds classified as tocopherols and tocotrie‐ nols [165]. While there are four isomers in each class of Vitamin E compounds, the over‐ whelming majority of the active form is α-tocopherol. [166, 167]. Dietary sources high in vitamin E include avocados, asparagus, vegetable oils, nuts, and leafy green vegetables.

Vitamin E is a potent antioxidant that inhibits LDL and membrane phospholipid oxidation. Interestingly, inflammatory cells and neurons have binding proteins for α-tocopherol, the actions of which may include inhibition of NADPH oxidase, lipoxygenase, and cyclo-oxy‐ genase, actions which may lower oxidative stress [168]. However, studies demonstrating vi‐ tamin E's pro-oxidant capacity under certain cellular conditions suggest that local condition may influence the vitamin E's redox activity [169]. Initial excitement for vitamin E supple‐ mentation was based on the reduction of cardiovascular events seen in the CHAOS study. However, follow-up studies have been largely disappointing [170-171]. While one small study that used vitamin E in combination with zinc, vitamin C, and beta-carotene showed a modest, significant reduction in blood pressure over 8 weeks of therapy, other small studies, show either no effect from vitamin E supplementation. Further, the more definitive HOPE trial, failed to show blood pressure or mortality benefit for patients at high risk for cardio‐ vascular disease [172]. Vitamin E inhibits free radicals reactions.

### *7.1.4. L-Arginine*

tions of appropriate dosing and target. In the inflammatory processes follow next scheme in

Vitamin E is a generic term for a group of compounds classified as tocopherols and tocotrie‐ nols [165]. While there are four isomers in each class of Vitamin E compounds, the over‐ whelming majority of the active form is α-tocopherol. [166, 167]. Dietary sources high in vitamin E include avocados, asparagus, vegetable oils, nuts, and leafy green vegetables.

Vitamin E is a potent antioxidant that inhibits LDL and membrane phospholipid oxidation. Interestingly, inflammatory cells and neurons have binding proteins for α-tocopherol, the actions of which may include inhibition of NADPH oxidase, lipoxygenase, and cyclo-oxy‐ genase, actions which may lower oxidative stress [168]. However, studies demonstrating vi‐ tamin E's pro-oxidant capacity under certain cellular conditions suggest that local condition may influence the vitamin E's redox activity [169]. Initial excitement for vitamin E supple‐ mentation was based on the reduction of cardiovascular events seen in the CHAOS study. However, follow-up studies have been largely disappointing [170-171]. While one small study that used vitamin E in combination with zinc, vitamin C, and beta-carotene showed a modest, significant reduction in blood pressure over 8 weeks of therapy, other small studies, show either no effect from vitamin E supplementation. Further, the more definitive HOPE

the therapy antioxidant [164].

372 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Figure 2.** Restored normal endothelial function.

*7.1.3. α-Tocopherol (Vitamin E)*

L-arginine is an amino acid and the main substrate for the production of NO from eNOS in a reaction that is dependent on tetrahydrobiopterin [173]. Potential dietary sources include milk products, beef, wheat germ, nuts, and soybeans. Reduced levels of tetrahydrobiopterin leads to uncoupling of reduced NADPH oxidation and NO synthesis, with oxygen as termi‐ nal electron acceptor instead of L-arginine, resulting in the generation of superoxide by eNOS [174-176]. Low cellular levels of L-arginine have been demonstrated in human hyper‐ tension. While L-arginine deficiency itself does not appear to lead to uncoupling of eNOS, [177] low levels of L-arginine may lead to reduced levels of bioavailable NO which could contribute to hypertension. Thus, L-arginine supplementation could theoretically reduce blood pressure by allowing for restoration of normal NO bioavailability, perhaps overcom‐ ing overall L-arginine deficiency as well as more successfully competing fo the eNOS active site with circulating asymmetric dimet hylarginine, a circulating competitor of L-arginine that may be increased in the setting of hypertension.

This concept is supported by studies demonstrating the anti-hypertensive effect of L-argi‐ nine supplementation in salt-sensitive rats, healthy human subjects, hypertensive diabetics, patients with chronic kidney disease, and diabetic patients in combination with N-acetylcys‐ teine, a precursor of glutathione [178] L-arginine's anti-hypertensive response may be medi‐ ated in part by its suppressive effects on angiotensin II and endothelin-1, and its potentiating effects on insulin.

However, recent concerns about potential deleterious increases in homocysteine in the set‐ ting of L-arginine supplementation have been raised. The majority of L-arginine is process‐ ed into creatine, which leads increased homocysteine levels. Homocysteine can increase oxidative stress. A recent study confirms that this mechanism is relevant to L-arginine me‐ tabolism in humans [179] suggesting a potential mechanism for neutralizing the eNOS-relat‐ ed anti-oxidant effects of L-arginine.

### *7.1.5. Flavonoids*

Flavonoids are polyphenolic compounds commonly found in concentrated amounts in multi‐ ple fruits, vegetables, and beverages, including apples, berries, grapes, onions, pomegranate, red wine, tea, cocoa, and dark chocolate. The exact structure and composition of the flavonoid compounds varies between food sources, and flavonoid content can be altered based on the manner of food preparation [180]. Interest in flavonoids as antioxidants therapy for cardiovas‐ cular disease originates from epidemiological data suggesting improved cardiovascular out‐ comes in individuals with high intake of food and beverages with high flavonoid content as well as cellular work suggesting a strong anti-oxidant effect of these compounds [181]. Howev‐ er, the limited oral bioavailability of flavonoids suggests cells signaling mechanism, rather than free radical quenching activity, is more likely to be root of sustained cardiovascular benefits from flavonoids [182, 183]. This concept is consistent with studies demondtrating that flavo‐ noids can inhibit NADPH oxidase through ACE inhibition, increase eNOS-specific NO pro‐ duction through the estrogen receptor, and alter COX-2 expression [184]. Studies investigating the anti-hypertensive effects of flavonoids are inconclusive. While multiple small studies of short duration of dark chocolate therapy have demonstrated blood pressure lowering effects in hypertensives [185], studies in normotensive and pre-hypertensive individuals have demon‐ strated no benefit [186], further tea intake may, at least temporarily, increase blood pressure certain populations [187, 188]. The specific flavonoids and combination of flavonoids that exert the largest beneficial effects remain unknown. The follow table indicates a function of antioxi‐ dants in therapy.

**Author details**

**References**

Marisela López Orozco

3(1), 53-63.

1364-1370.

cessed 2009 March 13.

trition j. , 14, 513-520.

Int. J. Biochem. Cell Biol. j. , 39, 44-84.

Redox Signal. , 8, 2039-2045.

Rev. 67 (Suppl. 1), SS144., 140.

Eva María Molina Trinidad, Sandra Luz de Ita Gutiérrez, Ana María Téllez López and

Universidad Autónoma del Estado de Hidalgo UAEH, Instituto de Ciencias de la Salud IC‐ Sa, Área Académica de Farmacia. ExHacienda la Concepción, Tilcuautla, Hidalgo, México

Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 375

[1] Huang, X. (2003). Iron overload and its association with cancer risk in humans: evi‐

[2] Markesbery WR, Lovell MA(2006). DNA oxidation in Alzheimer´s disease. Antioxid

[3] Halliwell, B. (2001). Role of free radicals in the neurodegenerative diseases: therapeu‐

[4] Vokurkova, M., Xu, S., & Touyz, R. M. (2007). Reactive oxygen species, cell growth, cell cycle progression and vascular remodeling in hypertension. Future Cardiol. Jan; ,

[5] Herrera, E., Jimenez, R., Aruoma, O. I., Hercberg, S., Sanchez-Garcia, I., & Fraga, C. (2009). Aspects of antioxidant foods and supplements in health and disease. Nutr.

[6] Dai, J., Jones, D. P., Goldberg, J., Ziegler, T. R., Bostick, R. M., Wilson, P. W., Mana‐ tunga, A. K., Shallenberger, L., Jones, L., & Vaccarino, V. (2008). Association between adherence to the Mediterranean diet and oxidative stress. Am. J. Clin. Nutr. , 88,

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**Table 2.** Antioxidants more indicated in treatments degeneratives chronics.

### **8. Conclusions**

The antioxidants present in food playing an important role in preventing chronic diseases. A balanced diet can prevent diseases associated with oxidative stress and help keep the body in top condition.

### **Author details**

*7.1.5. Flavonoids*

dants in therapy.

**8. Conclusions**

in top condition.

Selenium Septic ICU patients; major burns in

374 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Zinc Pneumonia in children: clinical course

Vitamin C (ascorbic acid) Burns, megadose during the first 24 h

**Table 2.** Antioxidants more indicated in treatments degeneratives chronics.

vitamin E

Cu-Se-Zn Burns: trials showing reduction of

patients

significantly shortened

Vitamin E (α-tocoferol) SIRS enteral supplementation Convincing animal data

Flavonoids are polyphenolic compounds commonly found in concentrated amounts in multi‐ ple fruits, vegetables, and beverages, including apples, berries, grapes, onions, pomegranate, red wine, tea, cocoa, and dark chocolate. The exact structure and composition of the flavonoid compounds varies between food sources, and flavonoid content can be altered based on the manner of food preparation [180]. Interest in flavonoids as antioxidants therapy for cardiovas‐ cular disease originates from epidemiological data suggesting improved cardiovascular out‐ comes in individuals with high intake of food and beverages with high flavonoid content as well as cellular work suggesting a strong anti-oxidant effect of these compounds [181]. Howev‐ er, the limited oral bioavailability of flavonoids suggests cells signaling mechanism, rather than free radical quenching activity, is more likely to be root of sustained cardiovascular benefits from flavonoids [182, 183]. This concept is consistent with studies demondtrating that flavo‐ noids can inhibit NADPH oxidase through ACE inhibition, increase eNOS-specific NO pro‐ duction through the estrogen receptor, and alter COX-2 expression [184]. Studies investigating the anti-hypertensive effects of flavonoids are inconclusive. While multiple small studies of short duration of dark chocolate therapy have demonstrated blood pressure lowering effects in hypertensives [185], studies in normotensive and pre-hypertensive individuals have demon‐ strated no benefit [186], further tea intake may, at least temporarily, increase blood pressure certain populations [187, 188]. The specific flavonoids and combination of flavonoids that exert the largest beneficial effects remain unknown. The follow table indicates a function of antioxi‐

combination with Cu and Zn; trauma

infectious complication (pneumonia) and improved wound healing

after injury; trauma, combined with

The antioxidants present in food playing an important role in preventing chronic diseases. A balanced diet can prevent diseases associated with oxidative stress and help keep the body

Ceiling "/>750 µg/day?

mg7day are provided

for the exudative losses

(189)

Immune depression if doses"/>50

Doses were calculated to compensate

Possible an endothelial mechanism

Eva María Molina Trinidad, Sandra Luz de Ita Gutiérrez, Ana María Téllez López and Marisela López Orozco

Universidad Autónoma del Estado de Hidalgo UAEH, Instituto de Ciencias de la Salud IC‐ Sa, Área Académica de Farmacia. ExHacienda la Concepción, Tilcuautla, Hidalgo, México

### **References**


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Disease and Therapy: A Role for Oxidants http://dx.doi.org/10.5772/51994 377

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388 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants


**Chapter 16**

**The Role of Natural Antioxidants in Cancer Disease**

Cell oxidation can lead to the onset and development of a wide range of diseases including Alzheimer and Parkinson, the pathologies caused by diabetes, rheumatoid arthritis, neuro‐ degeneration in motor neuron diseases, and cancer. Reactive species (RS) of various types are powerful oxidizing agents, capable of damaging DNA and other biomolecules. In‐ creased formation of RS can promote the development of malignancy, 'normal' rates of RS

Oxidants and free radicals are inevitably produced during the majority of physiological and metabolic processes and the human body has defensive antioxidant mechanisms; these mech‐ anisms vary according to cell and tissue type and may act antagonistically or synergistically. They include natural enzymes like Superoxide dismutase (SOD), Catalase (CAT), and Gluta‐ thione peroxidase (GPx), as well as antioxidants such as vitamins, carotenoids, polyphenols,

There has been a great deal of interest of late in the role of complementary and alternative drugs for the treatment of various acute and chronic diseases. Among the several classes of phytochemicals, interest has focused on the anti-inflammatory and antioxidant properties of the polyphenols that are found in various botanical agents. Plant vegetables and spices used in folk and traditional medicine have gained wide acceptance as one of the main sources of

> © 2013 Valadez-Vega et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and other natural antioxidants, which have attracted great interest in recent years.

Carmen Valadez-Vega, Luis Delgado-Olivares, José A. Morales González, Ernesto Alanís García,

Zuñiga Pérez Clara and Zuli Calderón Ramos

Additional information is available at the end of the chapter

Esther Ramírez Moreno , Manuel Sánchez Gutiérrez,

generation may account for the increased risk of cancer development.

prophylactic and chemopreventive drug discoveries and development.

José Roberto Villagomez Ibarra,

María Teresa Sumaya Martínez,

http://dx.doi.org/10.5772/51503

**1. Introduction**

### **The Role of Natural Antioxidants in Cancer Disease**

Carmen Valadez-Vega, Luis Delgado-Olivares, José A. Morales González, Ernesto Alanís García, José Roberto Villagomez Ibarra, Esther Ramírez Moreno , Manuel Sánchez Gutiérrez, María Teresa Sumaya Martínez, Zuñiga Pérez Clara and Zuli Calderón Ramos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51503

**1. Introduction**

Cell oxidation can lead to the onset and development of a wide range of diseases including Alzheimer and Parkinson, the pathologies caused by diabetes, rheumatoid arthritis, neuro‐ degeneration in motor neuron diseases, and cancer. Reactive species (RS) of various types are powerful oxidizing agents, capable of damaging DNA and other biomolecules. In‐ creased formation of RS can promote the development of malignancy, 'normal' rates of RS generation may account for the increased risk of cancer development.

Oxidants and free radicals are inevitably produced during the majority of physiological and metabolic processes and the human body has defensive antioxidant mechanisms; these mech‐ anisms vary according to cell and tissue type and may act antagonistically or synergistically. They include natural enzymes like Superoxide dismutase (SOD), Catalase (CAT), and Gluta‐ thione peroxidase (GPx), as well as antioxidants such as vitamins, carotenoids, polyphenols, and other natural antioxidants, which have attracted great interest in recent years.

There has been a great deal of interest of late in the role of complementary and alternative drugs for the treatment of various acute and chronic diseases. Among the several classes of phytochemicals, interest has focused on the anti-inflammatory and antioxidant properties of the polyphenols that are found in various botanical agents. Plant vegetables and spices used in folk and traditional medicine have gained wide acceptance as one of the main sources of prophylactic and chemopreventive drug discoveries and development.

© 2013 Valadez-Vega et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently, researches on medicinal plants has drawn global attention; large bodies of evidence have accumulated to demonstrate the promising potential of medicinal plants used in various traditional, complementary, and alternate treatment systems of human diseases. The plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, fla‐ vonoids, etc., which have been screened *in vivo* and *in vitro* and have indicated antioxidant and anticarcinogenic properties and which are used to developed drugs or dietary supplements.

reaction, can produce the hydroxyl radical (OH•‒

to produce the majority of oxidative damage [24]. On the other hand, exogenous antioxi‐ dants can be from animal and plant sources; however, those of plant origin are of great in‐ terest because they can contain major antioxidant activity [19]. Different reports show that persons with a high intake of a diet rich in fruit and vegetables have an important risk re‐ duction of developing cancer, mainly due to their antioxidant content [70]. Among the vege‐ table antioxidants are vitamins E and C, and ß-carotene, which are associated with diminished cardiovascular disease and a decreased risk of any cancer [48]. In particular, ßcarotene and vitamin E can reduce the risk of breast cancer, vitamin C, ß-carotene, and lu‐ tein/zeaxanthin possess a protector effect against ovarian cancer, and vitamin C, ß-carotene, and rivoflavin prevent colorectal cancer [70], while flavonoids such as plant phenolics and wine phenolics can inhibit lipid peroxidation and lipoxygenase enzymes. In addition, any

microelement, such as Se, Zn, Mn, and Cu, can exhibit antioxidant activity [48, 24].

due to that ROS can produce apoptosis in malignant cells [38, 101].

**3. Molecular Studies of Natural Antioxidants**

teractions are known as the "antioxidant network".

In recent years, interest has grown in the use of natural antioxidants for the prevention or treatment of different diseases related with OS; however despite the widespread informa‐ tion of the beneficial effects of antioxidants in the prevention of cancer, their use remains questionable, because different reports have shown that reducing the levels of ROS may have counterproductive effects because due to raising the risk of cancer; the latter may be

Different types of natural antioxidants are present in fruit and vegetables; they have syner‐ gistic interactions that are important due to their activity and regenerative potential. For ex‐ ample, ascorbate can regenerate into α-tocopherol [53], and the ascorbate radical is regenerated into other antioxidants via the thiol redox cycle. Taken together, all of these in‐

Vitamin E is an antioxidant that penetrates rapidly through the skin and is incorporated into the cellular membranes, inhibiting lipid peroxidation; specifically, α-tocotrienol, the vitamin E isoform, demonstrates greatest protection. Additionally, vitamin E possesses antiprolifera‐

Tumor necrosis factor-alpha (TNF-α) is a cytokine that, under normal conditions, induces inflammation, tumor inhibition, and apoptotic cell death. However, when the former under‐ goes deregulation, it acts as a breast tumor promoter, enhancing the proliferation of chemi‐ cally induced mammary tumors [113]. Phenolic antioxidants can block the increase of TNFα at the transcriptional level in the nucleus, which suggests the molecular mechanism of

tive properties that interfere in signal transduction and in inducing cell cycle arrest.

phenolic antioxidants through control of cytokine induction [81].

); wich is of more reactive the ROS, capable

The Role of Natural Antioxidants in Cancer Disease

http://dx.doi.org/10.5772/51503

393

Evidence suggests that the plant kingdom is considered a good candidate for chemopreven‐ tion and cancer therapy due to the high concentration and wide variety of antioxidants such as resveratrol, genestein, beicalein, vitamin A, vitamin C, polyphenols, (‒)‒Epigallocatechin 3-gallate, flavonoids, polyphenols, gallic acid, glycosides, verbascoside, calceorioside, epica‐ techin, quercetin, curcumin, lovastatin, and many other types of compounds with the capa‐ bility to inhibit the cell proliferation of different cancer cells *in vitro* and *in vitro*, such as colon cancer (HT-29, SW48, HCT116), breast (MCF7, MDA), cervix (HeLa, SiHa, Ca-Ski, C33-A), liver (Hep G2), skin (A 431), fibroblasts (3T3 SV40), and many other malignant cells; studies have indicated that antioxidants can be employed efficiently as chemopreventives and as effective inhibitors of cell proliferation, promoting cell apoptosis, and increasing de‐ toxification enzymes, and inhibiting gene expression and scavenger Reactive oxygen species (ROS). Thus, many researchers are working with different types of natural antioxidants with the aim of finding those with the greatest capacity to inhibit the development of cancer both *in vitro* as well as *in vivo*, because these compounds have exhibited high potential for use not only in the treatment of this disease, but they also act as good chemoprotective agents.

### **2. Antioxidants**

The production of ROS during metabolism is an inevitable phenomenon associated with the process of aerobic metabolism; on the other hand, we are exposed at all times to several exogenous sources of oxidant molecules, for example, environmental and pollutant factors and many dietary compounds, which increase their levels. ROS participate in different cellu‐ lar processes; their intracellular levels are relatively low. However, because ROS are highly toxic when their concentration increases, the phenomenon denominated Oxidative stress (OS) is produced [123], which can injure various cellular biomolecules, causing serious dam‐ age to tissues and organs and resulting in chronic diseases [24]. Oxidative damage can be prevented by antioxidants, which are present within the cell at low concentrations com‐ pared with oxidant molecules [141, 50].

Antioxidants are capable of donating electrons to stabilize ROS and to inhibit their detri‐ mental effects, including both endogenous (synthesized by the body itself) and exogenous molecules (those from external sources to the body) [141]. Endogenous antioxidants include Superoxide dismutase (SOD), which catalyzes the dismutation reaction of superoxide (O2•‒ ) into hydrogen peroxide (H2O2), which is in turn transformed into oxygen and water for the Catalase (CT), and in addition Glutathione peroxidase (GPx) can catalyze its reduction; however, if in the presence of transition metals such as iron, H2O2, by means of the Fenton reaction, can produce the hydroxyl radical (OH•‒ ); wich is of more reactive the ROS, capable to produce the majority of oxidative damage [24]. On the other hand, exogenous antioxi‐ dants can be from animal and plant sources; however, those of plant origin are of great in‐ terest because they can contain major antioxidant activity [19]. Different reports show that persons with a high intake of a diet rich in fruit and vegetables have an important risk re‐ duction of developing cancer, mainly due to their antioxidant content [70]. Among the vege‐ table antioxidants are vitamins E and C, and ß-carotene, which are associated with diminished cardiovascular disease and a decreased risk of any cancer [48]. In particular, ßcarotene and vitamin E can reduce the risk of breast cancer, vitamin C, ß-carotene, and lu‐ tein/zeaxanthin possess a protector effect against ovarian cancer, and vitamin C, ß-carotene, and rivoflavin prevent colorectal cancer [70], while flavonoids such as plant phenolics and wine phenolics can inhibit lipid peroxidation and lipoxygenase enzymes. In addition, any microelement, such as Se, Zn, Mn, and Cu, can exhibit antioxidant activity [48, 24].

In recent years, interest has grown in the use of natural antioxidants for the prevention or treatment of different diseases related with OS; however despite the widespread informa‐ tion of the beneficial effects of antioxidants in the prevention of cancer, their use remains questionable, because different reports have shown that reducing the levels of ROS may have counterproductive effects because due to raising the risk of cancer; the latter may be due to that ROS can produce apoptosis in malignant cells [38, 101].

### **3. Molecular Studies of Natural Antioxidants**

Recently, researches on medicinal plants has drawn global attention; large bodies of evidence have accumulated to demonstrate the promising potential of medicinal plants used in various traditional, complementary, and alternate treatment systems of human diseases. The plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, fla‐ vonoids, etc., which have been screened *in vivo* and *in vitro* and have indicated antioxidant and anticarcinogenic properties and which are used to developed drugs or dietary supplements.

392 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Evidence suggests that the plant kingdom is considered a good candidate for chemopreven‐ tion and cancer therapy due to the high concentration and wide variety of antioxidants such as resveratrol, genestein, beicalein, vitamin A, vitamin C, polyphenols, (‒)‒Epigallocatechin 3-gallate, flavonoids, polyphenols, gallic acid, glycosides, verbascoside, calceorioside, epica‐ techin, quercetin, curcumin, lovastatin, and many other types of compounds with the capa‐ bility to inhibit the cell proliferation of different cancer cells *in vitro* and *in vitro*, such as colon cancer (HT-29, SW48, HCT116), breast (MCF7, MDA), cervix (HeLa, SiHa, Ca-Ski, C33-A), liver (Hep G2), skin (A 431), fibroblasts (3T3 SV40), and many other malignant cells; studies have indicated that antioxidants can be employed efficiently as chemopreventives and as effective inhibitors of cell proliferation, promoting cell apoptosis, and increasing de‐ toxification enzymes, and inhibiting gene expression and scavenger Reactive oxygen species (ROS). Thus, many researchers are working with different types of natural antioxidants with the aim of finding those with the greatest capacity to inhibit the development of cancer both *in vitro* as well as *in vivo*, because these compounds have exhibited high potential for use not only in the treatment of this disease, but they also act as good chemoprotective agents.

The production of ROS during metabolism is an inevitable phenomenon associated with the process of aerobic metabolism; on the other hand, we are exposed at all times to several exogenous sources of oxidant molecules, for example, environmental and pollutant factors and many dietary compounds, which increase their levels. ROS participate in different cellu‐ lar processes; their intracellular levels are relatively low. However, because ROS are highly toxic when their concentration increases, the phenomenon denominated Oxidative stress (OS) is produced [123], which can injure various cellular biomolecules, causing serious dam‐ age to tissues and organs and resulting in chronic diseases [24]. Oxidative damage can be prevented by antioxidants, which are present within the cell at low concentrations com‐

Antioxidants are capable of donating electrons to stabilize ROS and to inhibit their detri‐ mental effects, including both endogenous (synthesized by the body itself) and exogenous molecules (those from external sources to the body) [141]. Endogenous antioxidants include Superoxide dismutase (SOD), which catalyzes the dismutation reaction of superoxide (O2•‒

into hydrogen peroxide (H2O2), which is in turn transformed into oxygen and water for the Catalase (CT), and in addition Glutathione peroxidase (GPx) can catalyze its reduction; however, if in the presence of transition metals such as iron, H2O2, by means of the Fenton

)

**2. Antioxidants**

pared with oxidant molecules [141, 50].

Different types of natural antioxidants are present in fruit and vegetables; they have syner‐ gistic interactions that are important due to their activity and regenerative potential. For ex‐ ample, ascorbate can regenerate into α-tocopherol [53], and the ascorbate radical is regenerated into other antioxidants via the thiol redox cycle. Taken together, all of these in‐ teractions are known as the "antioxidant network".

Vitamin E is an antioxidant that penetrates rapidly through the skin and is incorporated into the cellular membranes, inhibiting lipid peroxidation; specifically, α-tocotrienol, the vitamin E isoform, demonstrates greatest protection. Additionally, vitamin E possesses antiprolifera‐ tive properties that interfere in signal transduction and in inducing cell cycle arrest.

Tumor necrosis factor-alpha (TNF-α) is a cytokine that, under normal conditions, induces inflammation, tumor inhibition, and apoptotic cell death. However, when the former under‐ goes deregulation, it acts as a breast tumor promoter, enhancing the proliferation of chemi‐ cally induced mammary tumors [113]. Phenolic antioxidants can block the increase of TNFα at the transcriptional level in the nucleus, which suggests the molecular mechanism of phenolic antioxidants through control of cytokine induction [81].

### **4. Oxidative Stress and Diseases**

The ROS, as the superoxide anion (O2•‒ ), hydrogen peroxide (H2O2), and the hydroxyl radi‐ cal (OH•), are produced during cell metabolism in the lysosomes, peroxisomes, endoplas‐ mic reticulum in the process carried out to obtain energy such as Adenosine triphosphate (ATP) [108]. There are other sources of oxidant molecules, such as pollution, the environ‐ ment, and certain foods. During recent years, it has been discovered that during aging, the mitochondria increase the levels of ROS production and antioxidant endogens are diminish‐ ed [98, 13]. ROS play an important role in the physiological process; however, due to their toxicity, their levels must be controlled by the endogenous antioxidant system. But when ROS formation is increased, an imbalance is promoted between these and the antioxidant molecules; phenomenon known as Oxidative stress (OS) [123]. OS can cause oxidative dam‐ age of proteins, lipids, and nucleic acids, macromolecules involved in the cell function, membrane integrity, or in maintaining genetic information (nucleic acids) [44, 45, 65].

both RNA [97]. RNA oxidation can diminish the capacity of replacement oxidation of pro‐ teins [65, 44] and the inhibition of protein synthesis, cell cycle arrest, and cell death. Oxida‐ tion of RNA is involved in the development of cancer, viral infections, AIDS, hepatitis (VIH-1; HCV; 107, 148], and neurological diseases. It has been reported that each neurologi‐ cal disease, present a damage oxidative of RNA in a specific region on the brain, for example in Alzheimer disease, there are increased RNA oxidation in the hippocampus and cerebral neocortex, while in Parkinson disease, RNA oxidation is localized in the *sustancia nigra* [97]. On the other hand, high-fat diets induce obesity and insulin resistance, resulting in in‐ creased ROS production, which modifies sympathetic brain activity, which in turn contrib‐ utes to the rise in blood pressure, increase in insulin resistance, and obesity [6]. Obesity is the principal factor in the development of the metabolic syndrome, due to that persons with obesity have deficient antioxidant defense and increased production of ROS [126, 30, 75], which leads to spoilage and subsequently cell death, resulting in tissue and organ damage, to tissues causing serious health problems such as insulin resistance [7], diabetes mellitus, and hypertension [82]. Moreover, in the metabolic syndrome, NAD(P)H oxidase, the major source of ROS in several tissues, is up-regulated, resulting in an increase of ROS production and the down-regulation of several antioxidant enzymes (SOD isoforms, GPx, and heme oxygenase) [114]. This enzyme, specifically in the type 4 isoform (NOX4), is implicated in

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The scientific literature has shown that oxidative stress is involved in the development of a wide range of disease, such as heart diseases, Hutchinson-Gilford syndrome or progeria, hy‐ pertensive brain injury, muscular dystrophy, multiple sclerosis, congenital cataract, retinal degeneration, retinopathy of the premature, autoimmune diseases, cardiovascular abnor‐ malities, nephrological disorders, emphysema, stroke, rheumatoid arthritis, anemia, hepati‐ tis, pancreatitis, aging, premature wrinkles and dry skin, endothelial dysfunction, and

However the most important damage caused by OS are the DNA modifications, which can result in permanent mutations, due to that oxidative damage also affects the proteins in‐ volved in repairing the harm or reducing the OS (the endogenous antioxidant); thus, oxida‐ tive damage to DNA can be the cause of the development of various diseases, such as cancer

Cancer is unnatural cell growth, in which cells can lose their natural function and spread throughout the blood in the entire body. Breast cancer is the most commonly diagnosed can‐ cer in industrialized countries and has the highest death toll [88]. OS is involved in the proc‐ ess of the development of cancer and tumors, due to that ROS can damage the macromolecules as lipids, which react with metals (such as free iron and copper) and pro‐ duce aldehydes and synthesize malondialdehyde-inducing mutations [96] or cause breaks in the double chain, produce modifications in guanine and thymine bases, and sister chroma‐

the damage due to OS during cerebral ischemia [67].

dermatitis, among others [83, 7, 137, 91, 23, ¸102].

[13, 51].

**5. Cancer**

Proteins are responsible for different cell processes (enzymatic, hormonal, structural sup‐ port). The oxidation of proteins produces disulfide crosslinks, nitration, or tyrosine residues, and carbonylation, resulting in the loss of the structure and function of proteins and frag‐ mentation [11, 97]. But because the chaperones are susceptible to oxidative damage, allow‐ ing the accumulation of misfolding proteins and increasing their susceptibility to protease degradation [115], however, the proteasome also undergoes oxidation and its activity is di‐ minished, which makes the aggregates accumulate in the cell wich have been associated with aging and various pathologies, such as cancer and neurodegenerative disorders, such as Parkinson, Huntington, and Alzheimer disease [98].

The brain is the organ with the highest oxygen consumption; it has high levels of fatty acids, iron, and low antioxidant defenses. This is an organ with major susceptibility to oxidative damage [141], producing neurodegeneration that results in different diseases such as Par‐ kinson disease, Alzheimer disease, Down syndrome, autism, bipolar disorder, and epilepsy [23, 24], and the cognitive alteration known as Mild cognitive impairment (MCI), which is produced preferentially in regions of the brain involved in regulating cognition, contribu‐ ting to the development of dementia [65]. Similar processes occur during aging, resulting in the genetic response of increasing levels of antioxidant enzymes and chaperone proteins [73]. Reduction of OS causes improvement of the long-term memory [102].

Polyunsaturated fatty acids (mainly compounds of the membranes) are susceptible to peroxi‐ dation, which affects the integrity of the membranes of organelles of the cell membrane and the respiratory chain, in turn affecting cell viability. Lipid peroxidation produces aldehydes such as 4-hydroxy-2 *E*-nonenal, which is toxic and is involved in alterations in Alzheimer disease and DNA damage, causing mutations associated with the development of cancer [38, 20].

Ribosomal RNA and transfer RNA constitute the majority of stable species of cellular RNA, which possess a greater oxidation rate than DNA. The major modification for oxidation into RNA comprises 8-hydroxyGuanine (8-oxoG), which under normal conditions is present three times more in non-ribosomal that in ribosomal RNA; however, when the cell is ex‐ posed to H2O2, the concentration of 8-oxoG in ribosomal RNA increases at the same levels in both RNA [97]. RNA oxidation can diminish the capacity of replacement oxidation of pro‐ teins [65, 44] and the inhibition of protein synthesis, cell cycle arrest, and cell death. Oxida‐ tion of RNA is involved in the development of cancer, viral infections, AIDS, hepatitis (VIH-1; HCV; 107, 148], and neurological diseases. It has been reported that each neurologi‐ cal disease, present a damage oxidative of RNA in a specific region on the brain, for example in Alzheimer disease, there are increased RNA oxidation in the hippocampus and cerebral neocortex, while in Parkinson disease, RNA oxidation is localized in the *sustancia nigra* [97].

On the other hand, high-fat diets induce obesity and insulin resistance, resulting in in‐ creased ROS production, which modifies sympathetic brain activity, which in turn contrib‐ utes to the rise in blood pressure, increase in insulin resistance, and obesity [6]. Obesity is the principal factor in the development of the metabolic syndrome, due to that persons with obesity have deficient antioxidant defense and increased production of ROS [126, 30, 75], which leads to spoilage and subsequently cell death, resulting in tissue and organ damage, to tissues causing serious health problems such as insulin resistance [7], diabetes mellitus, and hypertension [82]. Moreover, in the metabolic syndrome, NAD(P)H oxidase, the major source of ROS in several tissues, is up-regulated, resulting in an increase of ROS production and the down-regulation of several antioxidant enzymes (SOD isoforms, GPx, and heme oxygenase) [114]. This enzyme, specifically in the type 4 isoform (NOX4), is implicated in the damage due to OS during cerebral ischemia [67].

The scientific literature has shown that oxidative stress is involved in the development of a wide range of disease, such as heart diseases, Hutchinson-Gilford syndrome or progeria, hy‐ pertensive brain injury, muscular dystrophy, multiple sclerosis, congenital cataract, retinal degeneration, retinopathy of the premature, autoimmune diseases, cardiovascular abnor‐ malities, nephrological disorders, emphysema, stroke, rheumatoid arthritis, anemia, hepati‐ tis, pancreatitis, aging, premature wrinkles and dry skin, endothelial dysfunction, and dermatitis, among others [83, 7, 137, 91, 23, ¸102].

However the most important damage caused by OS are the DNA modifications, which can result in permanent mutations, due to that oxidative damage also affects the proteins in‐ volved in repairing the harm or reducing the OS (the endogenous antioxidant); thus, oxida‐ tive damage to DNA can be the cause of the development of various diseases, such as cancer [13, 51].

### **5. Cancer**

**4. Oxidative Stress and Diseases**

394 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

The ROS, as the superoxide anion (O2•‒

as Parkinson, Huntington, and Alzheimer disease [98].

), hydrogen peroxide (H2O2), and the hydroxyl radi‐

cal (OH•), are produced during cell metabolism in the lysosomes, peroxisomes, endoplas‐ mic reticulum in the process carried out to obtain energy such as Adenosine triphosphate (ATP) [108]. There are other sources of oxidant molecules, such as pollution, the environ‐ ment, and certain foods. During recent years, it has been discovered that during aging, the mitochondria increase the levels of ROS production and antioxidant endogens are diminish‐ ed [98, 13]. ROS play an important role in the physiological process; however, due to their toxicity, their levels must be controlled by the endogenous antioxidant system. But when ROS formation is increased, an imbalance is promoted between these and the antioxidant molecules; phenomenon known as Oxidative stress (OS) [123]. OS can cause oxidative dam‐ age of proteins, lipids, and nucleic acids, macromolecules involved in the cell function,

membrane integrity, or in maintaining genetic information (nucleic acids) [44, 45, 65].

Proteins are responsible for different cell processes (enzymatic, hormonal, structural sup‐ port). The oxidation of proteins produces disulfide crosslinks, nitration, or tyrosine residues, and carbonylation, resulting in the loss of the structure and function of proteins and frag‐ mentation [11, 97]. But because the chaperones are susceptible to oxidative damage, allow‐ ing the accumulation of misfolding proteins and increasing their susceptibility to protease degradation [115], however, the proteasome also undergoes oxidation and its activity is di‐ minished, which makes the aggregates accumulate in the cell wich have been associated with aging and various pathologies, such as cancer and neurodegenerative disorders, such

The brain is the organ with the highest oxygen consumption; it has high levels of fatty acids, iron, and low antioxidant defenses. This is an organ with major susceptibility to oxidative damage [141], producing neurodegeneration that results in different diseases such as Par‐ kinson disease, Alzheimer disease, Down syndrome, autism, bipolar disorder, and epilepsy [23, 24], and the cognitive alteration known as Mild cognitive impairment (MCI), which is produced preferentially in regions of the brain involved in regulating cognition, contribu‐ ting to the development of dementia [65]. Similar processes occur during aging, resulting in the genetic response of increasing levels of antioxidant enzymes and chaperone proteins

Polyunsaturated fatty acids (mainly compounds of the membranes) are susceptible to peroxi‐ dation, which affects the integrity of the membranes of organelles of the cell membrane and the respiratory chain, in turn affecting cell viability. Lipid peroxidation produces aldehydes such as 4-hydroxy-2 *E*-nonenal, which is toxic and is involved in alterations in Alzheimer disease and DNA damage, causing mutations associated with the development of cancer [38, 20].

Ribosomal RNA and transfer RNA constitute the majority of stable species of cellular RNA, which possess a greater oxidation rate than DNA. The major modification for oxidation into RNA comprises 8-hydroxyGuanine (8-oxoG), which under normal conditions is present three times more in non-ribosomal that in ribosomal RNA; however, when the cell is ex‐ posed to H2O2, the concentration of 8-oxoG in ribosomal RNA increases at the same levels in

[73]. Reduction of OS causes improvement of the long-term memory [102].

Cancer is unnatural cell growth, in which cells can lose their natural function and spread throughout the blood in the entire body. Breast cancer is the most commonly diagnosed can‐ cer in industrialized countries and has the highest death toll [88]. OS is involved in the proc‐ ess of the development of cancer and tumors, due to that ROS can damage the macromolecules as lipids, which react with metals (such as free iron and copper) and pro‐ duce aldehydes and synthesize malondialdehyde-inducing mutations [96] or cause breaks in the double chain, produce modifications in guanine and thymine bases, and sister chroma‐ tid exchanges [16], which can affect the activities of signal transduction, transcription fac‐ tors, and gene tumor suppressors such as *p53*, which is a gene important in apoptosis and in cell cycle control. This inactivation can increase the expression of proto-oncogenes [96] which can produce major damage. Oxidative damage or genetic defects that result in some defective enzymes are incapable of repairing the mutations increase the incidence of age-de‐ pendent cancer [51].

macromolecular machinery of a cell, particularly lipids, proteins, and DNA. It is well known and in great detail the manner in which ROS react with lipids, leading to the peroxidation of biological membranes and resulting in necrotic lesions [43] and the way ROS react with the

The Role of Natural Antioxidants in Cancer Disease

http://dx.doi.org/10.5772/51503

397

When produced in excess, ROS (some of which are free radicals) can seriously alter the structure of biological substrates such as proteins, lipids, lipoproteins, and Deoxyribonucleic acid (DNA). They possess a huge range of potential actions on cells, and one could easily envisage them as anti-cancer (e.g., by promoting cell-cycle stasis, senescence, apoptosis, ne‐ crosis or other types of cell death, and inhibiting angiogenesis), or as pro-cancer (promoting

Active oxygen may be involved in carcinogenesis through two possible mechanisms: induc‐ tion of gene mutations that result from cell injury [34], and the effects on signal transduction and transcription factors. Which mechanism it follows depends on factors such as the type of active oxygen species involved and the intensity of stress [86]. Cellular targets affected by oxidative stress include DNA, phospholipids, proteins, and carbohydrates on the cell mem‐ brane. Oxidized and injured DNA has the potential to induce genetic mutation. That some telomere genes are highly susceptible to mutation in the presence of free radicals is now ap‐ parent, and it is known that tumor suppressor genes such as *p53* and cell cycle-related genes may undergo DNA damage. In addition, oxidized lipids react with metals to produce active substances (e.g., epoxides and aldehydes) or synthesize malondialdehyde, which has the po‐ tential to induce mutation. Active oxygen species act directly or indirectly via DNA damage on gene expression (DNA binding of transcription factors) and signaling at the cellular level.

Targets of free radicals include all types of molecules in the body. Among these, lipids, nu‐ cleic acids, and proteins are the major targets. Because free radicals are usually generated near membranes (cytoplasmic membrane, mitochondria, or endoplasmic reticulum), lipid peroxidation is the first reaction to occur. Lipid peroxidation products can be detected as classical Thiobarbituric acid (TBA)-reactive substances. Recently, the detection of 4-Hy‐ droxy-2-nonenal (HNE) or Malondialdehyde (MDA) is favored due to their high specificity [32], aldehydes are end-products of lipid peroxidation but continue to be reactive with cell

Exposure to free radicals from a variety of sources has led organisms to develop a series of

proliferation, invasiveness, angiogenesis, metastasis, and suppressing apoptosis).

nucleotides of DNA, leading to potential mutations [17, 43, 139].

Markers for OS can be divided into three categories:

**3.** activation or inhibition of transcription factors.

defense mechanisms that involve the following:

**1.** preventative mechanisms;

**2.** repair mechanisms; **3.** physical defenses, and

proteins [136].

**1.** formation of modified molecules by free radical reactions;

**2.** consumption or induction of antioxidant molecules or enzymes, and

On the other hand, treatments with anticancer drugs and radiation increase ROS and de‐ crease antioxidants content, producing a state of severe oxidative stress and causing apopto‐ sis, resulting in side effects [96], while persistent oxidative stress at sublethal levels can result in resistance to apoptosis [16].

Some microorganisms, as bacteria and viruses, are involved, via OS, in the process of the production of certain cancers such as, for example *Helicobacter pylori*, inducing gastric cancer and colon cancer through the production of SO•‒ [96]. It has been proposed that lower anti‐ oxidant activity increases the risk of developing cancer; thus, ingestion of antioxidants can prevent cancerogenesis. However is not clear the decrease of antioxidants levels is not clear, in as much as in freshly cancerous tissue, MnSOD levels are elevated; therefore, some inves‐ tigators have proposed that this antioxidant enzyme is involved in tumor invasion; thus, it is possible that antioxidants have a role as pro-oxidants. Another point to consider is that when the 8-oxodG level in DNA increases, cancer rates do not increase [96, 51]. However, OS is a factor for cancer and other diseases, but not the sole factor for diseases, because oth‐ ers, such as genetic factors (genetic predisposition) are involved.

### **6. Antioxidants and Cancer**

Humans are constantly bombarded by exogenous factors such as Ultraviolet (UV) rays, to‐ bacco smoke, and many others agents that cause OS. Such stress can also arise from the drugs that are employed in medical practice. On the other hand, under physiological condi‐ tions, normal aerobic metabolism gives rise to active and potentially dangerous oxidants in cells and tissues; these endogenous sources of OS include those derived from the activities of mitochondria or microsomes and peroxisomes in the electron transfer system and from the activities of the NADPH enzyme present in macrophages and neutrophils as a mecha‐ nism of protection against infection. Various reducing substances in the human body control the status of oxidation-reduction (redox), and a continuing imbalance in favor of oxidation causes several problems when it exceeds the capacity of such a control [96].

Otto Warburg was the first scientist to implicate oxygen in cancer [147] as far back as the 1920s. However, the underlying mechanism by which oxygen might contribute to the carci‐ nogenic process was undetermined for many years. The discovery of superoxide dismutase in 1968 by [90] led to an explosion of research on the role of reactive oxygen in the patholo‐ gies of biological organisms. Reactive oxygen has been specifically connected with not only cancer, but also many other human diseases [5, 57]. For many years, research on OS focused primarily on determining how ROS damage cells through indiscriminate reactions with the macromolecular machinery of a cell, particularly lipids, proteins, and DNA. It is well known and in great detail the manner in which ROS react with lipids, leading to the peroxidation of biological membranes and resulting in necrotic lesions [43] and the way ROS react with the nucleotides of DNA, leading to potential mutations [17, 43, 139].

When produced in excess, ROS (some of which are free radicals) can seriously alter the structure of biological substrates such as proteins, lipids, lipoproteins, and Deoxyribonucleic acid (DNA). They possess a huge range of potential actions on cells, and one could easily envisage them as anti-cancer (e.g., by promoting cell-cycle stasis, senescence, apoptosis, ne‐ crosis or other types of cell death, and inhibiting angiogenesis), or as pro-cancer (promoting proliferation, invasiveness, angiogenesis, metastasis, and suppressing apoptosis).

Active oxygen may be involved in carcinogenesis through two possible mechanisms: induc‐ tion of gene mutations that result from cell injury [34], and the effects on signal transduction and transcription factors. Which mechanism it follows depends on factors such as the type of active oxygen species involved and the intensity of stress [86]. Cellular targets affected by oxidative stress include DNA, phospholipids, proteins, and carbohydrates on the cell mem‐ brane. Oxidized and injured DNA has the potential to induce genetic mutation. That some telomere genes are highly susceptible to mutation in the presence of free radicals is now ap‐ parent, and it is known that tumor suppressor genes such as *p53* and cell cycle-related genes may undergo DNA damage. In addition, oxidized lipids react with metals to produce active substances (e.g., epoxides and aldehydes) or synthesize malondialdehyde, which has the po‐ tential to induce mutation. Active oxygen species act directly or indirectly via DNA damage on gene expression (DNA binding of transcription factors) and signaling at the cellular level.

Markers for OS can be divided into three categories:


Targets of free radicals include all types of molecules in the body. Among these, lipids, nu‐ cleic acids, and proteins are the major targets. Because free radicals are usually generated near membranes (cytoplasmic membrane, mitochondria, or endoplasmic reticulum), lipid peroxidation is the first reaction to occur. Lipid peroxidation products can be detected as classical Thiobarbituric acid (TBA)-reactive substances. Recently, the detection of 4-Hy‐ droxy-2-nonenal (HNE) or Malondialdehyde (MDA) is favored due to their high specificity [32], aldehydes are end-products of lipid peroxidation but continue to be reactive with cell proteins [136].

Exposure to free radicals from a variety of sources has led organisms to develop a series of defense mechanisms that involve the following:


tid exchanges [16], which can affect the activities of signal transduction, transcription fac‐ tors, and gene tumor suppressors such as *p53*, which is a gene important in apoptosis and in cell cycle control. This inactivation can increase the expression of proto-oncogenes [96] which can produce major damage. Oxidative damage or genetic defects that result in some defective enzymes are incapable of repairing the mutations increase the incidence of age-de‐

On the other hand, treatments with anticancer drugs and radiation increase ROS and de‐ crease antioxidants content, producing a state of severe oxidative stress and causing apopto‐ sis, resulting in side effects [96], while persistent oxidative stress at sublethal levels can

Some microorganisms, as bacteria and viruses, are involved, via OS, in the process of the production of certain cancers such as, for example *Helicobacter pylori*, inducing gastric cancer and colon cancer through the production of SO•‒ [96]. It has been proposed that lower anti‐ oxidant activity increases the risk of developing cancer; thus, ingestion of antioxidants can prevent cancerogenesis. However is not clear the decrease of antioxidants levels is not clear, in as much as in freshly cancerous tissue, MnSOD levels are elevated; therefore, some inves‐ tigators have proposed that this antioxidant enzyme is involved in tumor invasion; thus, it is possible that antioxidants have a role as pro-oxidants. Another point to consider is that when the 8-oxodG level in DNA increases, cancer rates do not increase [96, 51]. However, OS is a factor for cancer and other diseases, but not the sole factor for diseases, because oth‐

Humans are constantly bombarded by exogenous factors such as Ultraviolet (UV) rays, to‐ bacco smoke, and many others agents that cause OS. Such stress can also arise from the drugs that are employed in medical practice. On the other hand, under physiological condi‐ tions, normal aerobic metabolism gives rise to active and potentially dangerous oxidants in cells and tissues; these endogenous sources of OS include those derived from the activities of mitochondria or microsomes and peroxisomes in the electron transfer system and from the activities of the NADPH enzyme present in macrophages and neutrophils as a mecha‐ nism of protection against infection. Various reducing substances in the human body control the status of oxidation-reduction (redox), and a continuing imbalance in favor of oxidation

Otto Warburg was the first scientist to implicate oxygen in cancer [147] as far back as the 1920s. However, the underlying mechanism by which oxygen might contribute to the carci‐ nogenic process was undetermined for many years. The discovery of superoxide dismutase in 1968 by [90] led to an explosion of research on the role of reactive oxygen in the patholo‐ gies of biological organisms. Reactive oxygen has been specifically connected with not only cancer, but also many other human diseases [5, 57]. For many years, research on OS focused primarily on determining how ROS damage cells through indiscriminate reactions with the

ers, such as genetic factors (genetic predisposition) are involved.

causes several problems when it exceeds the capacity of such a control [96].

pendent cancer [51].

result in resistance to apoptosis [16].

396 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**6. Antioxidants and Cancer**

**3.** physical defenses, and

#### **4.** antioxidant defenses.

Enzymatic antioxidant defenses include Superoxide dismutase (SOD), Glutathione peroxi‐ dase (GPx), and Catalase (CAT). Non-enzymatic antioxidants are represented by ascorbic acid (vitamin C), α-tocopherol (vitamin E), Glutathione (GSH), carotenoids, flavonoids, tannins, triterpepenoids, saponins, glycosides, steroids, and other antioxidants [46]. Under normal con‐ ditions, there is a balance between both the activities and the intracellular levels of these anti‐ oxidants: this equilibrium is essential for the survival of organisms and their health

Some reports indicate that the prevalence of use of complementary and alternative medicine by patients with cancer has been estimated at a range of 7‒64% [3, 4, 58]. At present, many patients with cancer combine some forms of complementary and alternative therapy with their conventional therapies [4, 58]. A recent survey of patients at a comprehensive cancer center placed the use of vitamin and minerals at 62.6%; of these patients, 76.6% combined

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These types of patients employ complementary and alternative therapies for a variety of rea‐ sons [31, 14]: to improve quality of life (77%); to improve immune function (71%); to prolong life (62%), or to relieve symptoms (44%) related with their disease [31]. Only 37.5% of the patients surveyed expected complementary and alternative therapies to cure their disease. Whatever the reasons, alternative therapy use is on the rise and this includes the use of meg‐ avitamins, minerals, and cocktails of natural substances during chemotherapy administra‐ tion; these cocktails include antioxidants such as the commonly consumed antioxidants vitamin E (mixed tocopherols and tocotrienols), vitamin C, β-carotene (natural mixed caro‐ tenoids), polyphenols, tannins, terpenoids, alkaloids, flavonoids, vitamin A, and many oth‐ ers. Controversy exists concerning the use of antioxidants with chemotherapy, but increasing evidence suggests a benefit when antioxidants are added to chemotherapy [111,

It is widely accepted that diets rich in fruits and plants are rich sources of different types of antioxidants; phenolic compounds are the most studied of these and have been recognized to possess a wide range of properties including antioxidant, antibacterial, anti-inflammato‐ ry, hepatoprotective, and anticarcinogenic actions [3, 4, 63]. Many of the biological functions of flavonoid, phenolic, catechin, curcumin, resveratrol, and genistein compounds have been attributed to their free-radical scavenging, metal-ion chelating, and antioxidant activities [118, 152]. Antioxidant phenolic agents have been implicated in the mechanisms of chemo‐ prevention, which refers to the use of chemical substances of natural or of synthetic origin to

It has been shown that dietary phytochemicals can interfere with each stage of the devel‐ opment of carcinogenesis [130, 93]. As in the case of direct antioxidant effects, dietary pol‐ yphenols are most likely to exert their chemopreventive effects on the gastrointestinal tract, where they are present at highest concentrations [52, 49, 84, 75]. Indeed, studies have shown that various polyphenol-rich fruits and vegetables are particularly effective in protecting against several types of cancer development [84, 75, 59]. Dietary polyphe‐ nols may exert their anticancer effects through several possible mechanisms, such as remov‐ al of carcinogenic agents, modulation of cancer cell signaling and antioxidant enzymatic activities, and induction of apoptosis as well as of cell cycle arrest. Some of these ef‐ fects may be related, at least partly, with their antioxidant activities [59]. They may ex‐ ert protective effects against cancer development, particularly in the gastrointestinal tract, where they will be at their highest concentration. In fact, many studies have shown that various polyphenol-rich fruits and vegetables are particularly effective in protecting against

reverse, retard, or delay the multistage carcinogenic process [29].

the use of vitamins and minerals with conventional chemotherapy [58, 27].

112, 106, 151, 117, 105, 22, 27].

colon cancer development [84, 75].

### **7. Antioxidants in Cancer Assays**

Humans have evolved with antioxidant systems for protection against free radicals and ROS. These systems include some antioxidants produced in the body (endogenous) and oth‐ ers obtained from the diet (exogenous) [21]. The former include


The various defenses are complementary to each other because they act against different species in different cellular compartments. However, despite these defense antioxidants (able either to suppress free radical formation and chain initiation or to scavenge free radi‐ cals and chain propagation), some ROS escape to cause damage. Thus, the body's antioxi‐ dant system is also provided with repair antioxidants (able to repair damage) and based on proteases, lipases, transferases, and DNA repair enzymes [145, 103].

Owing to the incomplete efficiency of our endogenous defense systems and the existence of some physiopathological situations (cigarette smoke, air pollutants, UV radiation, a high, polyunsaturated fatty acid diet, inflammation, ischemia/reperfusion, etc.) in which ROS are produced in excess and at the wrong time and place, dietary antioxidants are required to diminish the cumulative effects of oxidative damage throughout the human lifespan [149, 47). Well known natural antioxidants derived from the diet, such as vitamins C, E, and A and the carotenoids, have been studied intensively [124]. In addition to these, antioxidants in plants might account for at least part of the health benefits associated with vegetable and fruit consumption [103].

The plants, vegetables, and spices used in folk and traditional medicine have gained wide acceptance as one of the main sources of prophylactic and chemopreventive drug discovery and development [85, 29].

Some reports indicate that the prevalence of use of complementary and alternative medicine by patients with cancer has been estimated at a range of 7‒64% [3, 4, 58]. At present, many patients with cancer combine some forms of complementary and alternative therapy with their conventional therapies [4, 58]. A recent survey of patients at a comprehensive cancer center placed the use of vitamin and minerals at 62.6%; of these patients, 76.6% combined the use of vitamins and minerals with conventional chemotherapy [58, 27].

**4.** antioxidant defenses.

**7. Antioxidants in Cancer Assays**

398 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ers obtained from the diet (exogenous) [21]. The former include

venting the majority of the formation of toxic HO∙, and

proteases, lipases, transferases, and DNA repair enzymes [145, 103].

to the radical-trapping capacity of plasma.

fruit consumption [103].

and development [85, 29].

Enzymatic antioxidant defenses include Superoxide dismutase (SOD), Glutathione peroxi‐ dase (GPx), and Catalase (CAT). Non-enzymatic antioxidants are represented by ascorbic acid (vitamin C), α-tocopherol (vitamin E), Glutathione (GSH), carotenoids, flavonoids, tannins, triterpepenoids, saponins, glycosides, steroids, and other antioxidants [46]. Under normal con‐ ditions, there is a balance between both the activities and the intracellular levels of these anti‐

Humans have evolved with antioxidant systems for protection against free radicals and ROS. These systems include some antioxidants produced in the body (endogenous) and oth‐

**1.** enzymatic defenses, such as *Se*-glutathione peroxidase, catalase, and superoxide dismu‐ tase, which metabolize superoxide, hydrogen peroxide, and lipid peroxides, thus pre‐

**2.** non-enzymatic defenses, such as glutathione, histidine peptides, the iron-binding trans‐ fer proteins and ferritin, and dihydrolipoic acid, reduced Coenzyme Q10, melatonin, ur‐ ate, and plasma protein thiols, with the latter two accounting for the major contribution

The various defenses are complementary to each other because they act against different species in different cellular compartments. However, despite these defense antioxidants (able either to suppress free radical formation and chain initiation or to scavenge free radi‐ cals and chain propagation), some ROS escape to cause damage. Thus, the body's antioxi‐ dant system is also provided with repair antioxidants (able to repair damage) and based on

Owing to the incomplete efficiency of our endogenous defense systems and the existence of some physiopathological situations (cigarette smoke, air pollutants, UV radiation, a high, polyunsaturated fatty acid diet, inflammation, ischemia/reperfusion, etc.) in which ROS are produced in excess and at the wrong time and place, dietary antioxidants are required to diminish the cumulative effects of oxidative damage throughout the human lifespan [149, 47). Well known natural antioxidants derived from the diet, such as vitamins C, E, and A and the carotenoids, have been studied intensively [124]. In addition to these, antioxidants in plants might account for at least part of the health benefits associated with vegetable and

The plants, vegetables, and spices used in folk and traditional medicine have gained wide acceptance as one of the main sources of prophylactic and chemopreventive drug discovery

oxidants: this equilibrium is essential for the survival of organisms and their health

These types of patients employ complementary and alternative therapies for a variety of rea‐ sons [31, 14]: to improve quality of life (77%); to improve immune function (71%); to prolong life (62%), or to relieve symptoms (44%) related with their disease [31]. Only 37.5% of the patients surveyed expected complementary and alternative therapies to cure their disease. Whatever the reasons, alternative therapy use is on the rise and this includes the use of meg‐ avitamins, minerals, and cocktails of natural substances during chemotherapy administra‐ tion; these cocktails include antioxidants such as the commonly consumed antioxidants vitamin E (mixed tocopherols and tocotrienols), vitamin C, β-carotene (natural mixed caro‐ tenoids), polyphenols, tannins, terpenoids, alkaloids, flavonoids, vitamin A, and many oth‐ ers. Controversy exists concerning the use of antioxidants with chemotherapy, but increasing evidence suggests a benefit when antioxidants are added to chemotherapy [111, 112, 106, 151, 117, 105, 22, 27].

It is widely accepted that diets rich in fruits and plants are rich sources of different types of antioxidants; phenolic compounds are the most studied of these and have been recognized to possess a wide range of properties including antioxidant, antibacterial, anti-inflammato‐ ry, hepatoprotective, and anticarcinogenic actions [3, 4, 63]. Many of the biological functions of flavonoid, phenolic, catechin, curcumin, resveratrol, and genistein compounds have been attributed to their free-radical scavenging, metal-ion chelating, and antioxidant activities [118, 152]. Antioxidant phenolic agents have been implicated in the mechanisms of chemo‐ prevention, which refers to the use of chemical substances of natural or of synthetic origin to reverse, retard, or delay the multistage carcinogenic process [29].

It has been shown that dietary phytochemicals can interfere with each stage of the devel‐ opment of carcinogenesis [130, 93]. As in the case of direct antioxidant effects, dietary pol‐ yphenols are most likely to exert their chemopreventive effects on the gastrointestinal tract, where they are present at highest concentrations [52, 49, 84, 75]. Indeed, studies have shown that various polyphenol-rich fruits and vegetables are particularly effective in protecting against several types of cancer development [84, 75, 59]. Dietary polyphe‐ nols may exert their anticancer effects through several possible mechanisms, such as remov‐ al of carcinogenic agents, modulation of cancer cell signaling and antioxidant enzymatic activities, and induction of apoptosis as well as of cell cycle arrest. Some of these ef‐ fects may be related, at least partly, with their antioxidant activities [59]. They may ex‐ ert protective effects against cancer development, particularly in the gastrointestinal tract, where they will be at their highest concentration. In fact, many studies have shown that various polyphenol-rich fruits and vegetables are particularly effective in protecting against colon cancer development [84, 75].

At the cellular level, there is good evidence that polyphenols present in tea, red wine, cocoa, fruit juices, and olive oil; at some level, they are able to stimulate carcinogenesis and tumor development [93]. For example, they may interact with reactive intermediates [28] and acti‐ vated carcinogens and mutagens [18], they may modulate the activity of the key proteins in‐ volved in controlling cell cycle progression [104], and they may influence the expression of many cancer-associated genes [142]. Perhaps most notably, the anticancer properties of green tea flavanols have been reported in animal models and in human cell lines *(* Takada *et al.*, 2002], as well as in human intervention studies [60]. On the other hand, green tea con‐ sumption has been proposed as significantly reducing the risk of cancer of the biliary tract [133], bladder [110], breast [74], and colon [72]. Many of the anti-cancer properties associated with green tea are thought to be mediated by the flavanol Epigallocatechin gallate (EGCG), which has been shown to induce apoptosis and inhibit cancer cell growth by altering the ex‐ pression of cell cycle regulatory proteins and the activity of signaling proteins involved in cell proliferation, transformation, and metastasis [66]. In addition to flavonoids, phenolic al‐ cohols, lignans, and secoiridoids (all found at high concentrations in olive oil) are also thought to induce anti-carcinogenic effects [99] and have been reported in large intestinal cancer cell models [79], in animals [10, 128], and in humans [99]. These effects may be medi‐ ated by the ability of olive oil phenolics to inhibit initiation, promotion, and metastasis in human colon adenocarcinoma cells [42, 55] and to down-regulate the expression of COX-2 and Bcl-2 proteins, which play a crucial role in colorectal carcinogenesis [79, 146].

and colon carcinogenesis in specific bioassay models. The properties of the tea's polyphe‐ nols make them effective chemopreventive agents against the initiation, promotion, and pro‐ gression stages of multistage carcinogenesis [64]. Rosmanic acid had demonstrated to possess potent anticancer and apoptotic effect in mouse-induced skin cancer [121], curcu‐ min, (‒)‒epigallocatechin-3-gallate, and lovastatin in combination were able to suppress esophageal cancer in mouse [154], and melatonin demonstrated diminishing the develop‐ ment and mortality of mouse implanted with murine hepatoma cells MN22a [39]. It was demonstrated that beta-ionone, a precursor of carotenoids, ameliorated lung carcinogenesis; the latter is attributed to the antiproliferative and antioxidant potential of beta-ionone through free radical scavenging properties [9]. Α-tocopherol showed down-regulation of the expression of the stress-activated genes *PKC-α*, *c-Myc*, and *Lactate dehydrogenase A (LDHA)* in cancerous mice, decreasing cancer cell proliferation [120]. It has been suggested that ros‐ manic acid suppresses oral carcinogenesis by stimulating the activities of detoxification en‐ zymes, improving the status of lipid peroxidation and antioxidants, and down-regulating the expression of *p53* and *bcl-2* during 7,12 dimethylbenz(a)anthracene-induced oral carcino‐ genesis in hamster [8]. In the same manner, the methanolic extract of fennel seed exhibited an antitumoral affect by modulating lipid peroxidation and augmenting the antioxidant de‐ fense system in Ehrlich ascites carcinoma- bearing mice with or without exposure to radia‐ tion [94]. Silymarin, a natural flavonoid from the milk thistle seed, displayed chemopreventive action against 1,2-dimethylhydrazine plus dextran sodium sulfate-in‐ duced inflammation associated with colon carcinogenesis [135]. Quercetin, a flavonoid found in many natural foods, demonstrated to exert a direct oro-apoptotic affect on tumor cells and can indeed block the growth of several human cancer-cell lines in different cell-cy‐ cle phases, which have been demonstrated in several animal models [41]. The methanolic extract of *Indigofera cassioides* was evaluated in terms of their antitumor activity on Ehrlich ascites carcinoma- bearing mice; the extract showed a potent antitumoral effect against tu‐ mor cells due its preventing lipid peroxidation and promoting the enzymatic antioxidant defense system in animals [69]. Brucine, a natural plant alkaloid, was reported to possess cy‐ totoxic and antiproliferative activities and also had showed to be a potential anti-metastatic

The Role of Natural Antioxidants in Cancer Disease

http://dx.doi.org/10.5772/51503

401

An *in vitro* assay demonstrated that the mechanism's antioxidant action, according to Halli‐

**1.** suppressing ROS formation either by inhibiting the enzymes or chelating the trace ele‐

Flavonoids have been identified as fulfilling the majority of the criteria previously descri‐

**1.** Flavonoids inhibit the enzymes responsible for superoxide anion production, such as

and -angiogenic agent [2].

**2.** scavenging ROS, and

well [52], can include the following:

ments involved in free radical production;

**3.** up-regulating or protecting antioxidant defenses.

xanthine oxidase [54] and Protein kinase C (PKC) [140], and

bed. Thus, their effects are two-fold as follows:

*In vivo* studies have demonstrated that many natural compounds found in plants and fruits have the capability to inhibit many types of human and animal cancer. Vitamins such as C, E, and A have shown that they can diminish cervical, bladder, prostate, intestinal, skin, and other gastrointestinal cancer types and that they have the capability to inhibit ROS produc‐ tion in patients [36, 37, 89, 134, 131, 62, 127]. In addition, it was demonstrated that these vita‐ mins can inhibit progression and pathogenesis in colorectal cancer [12]. In animal models, vitamins showed promise for chemopreventive agents against several types of gastrointesti‐ nal cancer [62].

With the use of a combination of vitamins, selenium, β-carotene, essential fatty acids, and coenzyme Q10 in patients with breast cancer, it was observed that during the study no pa‐ tient died, no patient showed signs of further distant metastasis, quality of life improved, and six patients showed apparent partial remission [80]. Human studies demonstrated that consumption of total antioxidants in the diet (fruits and vegetables) is inversely associated with the risk of distal gastric cancer [87]. Antioxidants, especially polyphenols, have been found to be promising agents against cervical cancer, including induction of apoptosis, growth arrest, inhibition of DNA synthesis, and modulation of signal transduction pathway; additionally, polyphenols can interfere with each stage of carcinogenesis initiation, promo‐ tion, and progression for the prevention of cancer development [26].

*Camelia sinensis* tea, which contains a great quantity of polyphenols (epichatechin, (‒)‒epi‐ gallocatechin-3-gallate) is the most widely consumed beverage worldwide, and it was dem‐ onstrated that consumption of this beverage has shown to afford protection against chemical carcinogen-induced stomach, lung, esophagus, duodenum, pancreas, liver, breast, and colon carcinogenesis in specific bioassay models. The properties of the tea's polyphe‐ nols make them effective chemopreventive agents against the initiation, promotion, and pro‐ gression stages of multistage carcinogenesis [64]. Rosmanic acid had demonstrated to possess potent anticancer and apoptotic effect in mouse-induced skin cancer [121], curcu‐ min, (‒)‒epigallocatechin-3-gallate, and lovastatin in combination were able to suppress esophageal cancer in mouse [154], and melatonin demonstrated diminishing the develop‐ ment and mortality of mouse implanted with murine hepatoma cells MN22a [39]. It was demonstrated that beta-ionone, a precursor of carotenoids, ameliorated lung carcinogenesis; the latter is attributed to the antiproliferative and antioxidant potential of beta-ionone through free radical scavenging properties [9]. Α-tocopherol showed down-regulation of the expression of the stress-activated genes *PKC-α*, *c-Myc*, and *Lactate dehydrogenase A (LDHA)* in cancerous mice, decreasing cancer cell proliferation [120]. It has been suggested that ros‐ manic acid suppresses oral carcinogenesis by stimulating the activities of detoxification en‐ zymes, improving the status of lipid peroxidation and antioxidants, and down-regulating the expression of *p53* and *bcl-2* during 7,12 dimethylbenz(a)anthracene-induced oral carcino‐ genesis in hamster [8]. In the same manner, the methanolic extract of fennel seed exhibited an antitumoral affect by modulating lipid peroxidation and augmenting the antioxidant de‐ fense system in Ehrlich ascites carcinoma- bearing mice with or without exposure to radia‐ tion [94]. Silymarin, a natural flavonoid from the milk thistle seed, displayed chemopreventive action against 1,2-dimethylhydrazine plus dextran sodium sulfate-in‐ duced inflammation associated with colon carcinogenesis [135]. Quercetin, a flavonoid found in many natural foods, demonstrated to exert a direct oro-apoptotic affect on tumor cells and can indeed block the growth of several human cancer-cell lines in different cell-cy‐ cle phases, which have been demonstrated in several animal models [41]. The methanolic extract of *Indigofera cassioides* was evaluated in terms of their antitumor activity on Ehrlich ascites carcinoma- bearing mice; the extract showed a potent antitumoral effect against tu‐ mor cells due its preventing lipid peroxidation and promoting the enzymatic antioxidant defense system in animals [69]. Brucine, a natural plant alkaloid, was reported to possess cy‐ totoxic and antiproliferative activities and also had showed to be a potential anti-metastatic and -angiogenic agent [2].

An *in vitro* assay demonstrated that the mechanism's antioxidant action, according to Halli‐ well [52], can include the following:


At the cellular level, there is good evidence that polyphenols present in tea, red wine, cocoa, fruit juices, and olive oil; at some level, they are able to stimulate carcinogenesis and tumor development [93]. For example, they may interact with reactive intermediates [28] and acti‐ vated carcinogens and mutagens [18], they may modulate the activity of the key proteins in‐ volved in controlling cell cycle progression [104], and they may influence the expression of many cancer-associated genes [142]. Perhaps most notably, the anticancer properties of green tea flavanols have been reported in animal models and in human cell lines *(* Takada *et al.*, 2002], as well as in human intervention studies [60]. On the other hand, green tea con‐ sumption has been proposed as significantly reducing the risk of cancer of the biliary tract [133], bladder [110], breast [74], and colon [72]. Many of the anti-cancer properties associated with green tea are thought to be mediated by the flavanol Epigallocatechin gallate (EGCG), which has been shown to induce apoptosis and inhibit cancer cell growth by altering the ex‐ pression of cell cycle regulatory proteins and the activity of signaling proteins involved in cell proliferation, transformation, and metastasis [66]. In addition to flavonoids, phenolic al‐ cohols, lignans, and secoiridoids (all found at high concentrations in olive oil) are also thought to induce anti-carcinogenic effects [99] and have been reported in large intestinal cancer cell models [79], in animals [10, 128], and in humans [99]. These effects may be medi‐ ated by the ability of olive oil phenolics to inhibit initiation, promotion, and metastasis in human colon adenocarcinoma cells [42, 55] and to down-regulate the expression of COX-2

400 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

and Bcl-2 proteins, which play a crucial role in colorectal carcinogenesis [79, 146].

nal cancer [62].

*In vivo* studies have demonstrated that many natural compounds found in plants and fruits have the capability to inhibit many types of human and animal cancer. Vitamins such as C, E, and A have shown that they can diminish cervical, bladder, prostate, intestinal, skin, and other gastrointestinal cancer types and that they have the capability to inhibit ROS produc‐ tion in patients [36, 37, 89, 134, 131, 62, 127]. In addition, it was demonstrated that these vita‐ mins can inhibit progression and pathogenesis in colorectal cancer [12]. In animal models, vitamins showed promise for chemopreventive agents against several types of gastrointesti‐

With the use of a combination of vitamins, selenium, β-carotene, essential fatty acids, and coenzyme Q10 in patients with breast cancer, it was observed that during the study no pa‐ tient died, no patient showed signs of further distant metastasis, quality of life improved, and six patients showed apparent partial remission [80]. Human studies demonstrated that consumption of total antioxidants in the diet (fruits and vegetables) is inversely associated with the risk of distal gastric cancer [87]. Antioxidants, especially polyphenols, have been found to be promising agents against cervical cancer, including induction of apoptosis, growth arrest, inhibition of DNA synthesis, and modulation of signal transduction pathway; additionally, polyphenols can interfere with each stage of carcinogenesis initiation, promo‐

*Camelia sinensis* tea, which contains a great quantity of polyphenols (epichatechin, (‒)‒epi‐ gallocatechin-3-gallate) is the most widely consumed beverage worldwide, and it was dem‐ onstrated that consumption of this beverage has shown to afford protection against chemical carcinogen-induced stomach, lung, esophagus, duodenum, pancreas, liver, breast,

tion, and progression for the prevention of cancer development [26].

**3.** up-regulating or protecting antioxidant defenses.

Flavonoids have been identified as fulfilling the majority of the criteria previously descri‐ bed. Thus, their effects are two-fold as follows:

**1.** Flavonoids inhibit the enzymes responsible for superoxide anion production, such as xanthine oxidase [54] and Protein kinase C (PKC) [140], and

**2.** Flavonoids have also shown to inhibit cyclo-oxygenase, lipoxygenase, microsomal mono-oxygenase, glutathione *S*-transferase, mitochondrial succinoxidase, and (Nicotinamide adenine denucleotide (NADH) oxidase, all of which are involved in ROS gener‐ ation [68, 15].

tent as antioxidants and antioxidant agents, suggesting its possible use as an adjuvant agent

The Role of Natural Antioxidants in Cancer Disease

http://dx.doi.org/10.5772/51503

403

The extract of *Calluna vulgaris* exhibited a photoprotective effect on human keratinocytes (HaCaT) exposed to Ultraviolet B (UVB) radiation [100]. *Cachrys pungens Jan* was analyzed in a human tumor- cell line, amelanotic melanoma, and it was found that its extract contains antioxidants, such as coumarins, which are responsible for their cytotoxicity in A375 cells [92]. *Inonotus obliquus* and *Peperomia pellucida*, plants employed as folk remedies for cancer treatment, were evaluated in several tumor cell-line types and it was found that these plants contains several antioxidants, such as lanosterol, inotodiols, ergosterol, phytol, 2-naphthale‐ nol, decahydro hexadecanoic acid, methyl ester, and 9,12 octadecadienoic acid, indicating that these antioxidant compounds are responsible for the anticarcinogenic activity of the plant extract [129, 150]. The extract of *Indigofera cassioides* indicated the presence antioxidant activity, preventing lipid peroxidation and promoting the enzymatic antioxidant defense system, and also showed potent antitumoral and cytotoxic affect against EAC, DLA, HeLa,

Hesperetin, hesperetin analog, carnocine, and resveratrol were evaluated for their antioxi‐ dant and anticarcinogenic activity on HT-29, HCT116, and mouse skin carcinogenesis; their studies demonstrated that these compounds can inhibit cell proliferation, induce apoptosis, affect glycolysis, and decrease tumoration [125, 161, 40]. Honey, a natural product common‐ ly used throughout the world, contains antioxidant properties and exerts a preventive effect against disease. Chrysin is a natural flavone commonly found in honey, and it was demon‐ strated that this compound induced apoptosis in PC-3 cells [116], fennel seeds (*Foeniculum vulgare*) are present in antioxidants that have an anticancer potential against HepG2 and MCF-7 cell lines [94). It was indicated that compounds such as quercetin, flavonoids, and brucine have chemopreventive action against the osteosarcoma cell line (MG63), C6 glioma cells, and Ehrlich ascites cells, and that they can be used as anticancer, antigenotoxic agents

Oxidative stress causes injury to cells, induces gene mutation, and is involved in carcino‐ genesis and other degenerative diseases by directly or indirectly influencing intracellular signal transduction and transcription factors. The state of OS under carcinogenesis and tu‐ mor-bearing conditions is an intricate one in which various substances are involved in com‐

The data discussed in this paper show that the biological effects of antioxidants on humans and animals can be controversial. Due to that the action of antioxidants depends on the oxi‐ dative status of cells, antioxidants can be protective against cancer; because ROS induce oxi‐ dative carcinogenic damage in DNA, antioxidants can prevent cancer in healthy persons

in the treatment of cancer [56, 1].

Hep-2, HepG-2, MCF-7, Ht-29, and NIH 3T3 cells [69].

and can induce apoptosis [135, 119, 2].

**8. Conclusion**

plex interactions.

harboring increased ROS levels.

A number of flavonoids efficiently chelate trace metals, which play an important role in oxy‐ gen metabolism. Free iron and copper are potential enhancers of ROS formation, as exempli‐ fied by the reduction of hydrogen peroxide with the generation of the highly aggressive hydroxyl radical [103].

On the other hand*, in* vitro studies showed that the compounds present in fruits and vegeta‐ bles, such as resveratrol, genestein, baicalein, and many others are attractive candidates for improved chemotherapeutic agents [35]. Resveratrol in combination with platinum drugs and oxaliplatin demonstrated that resveratrol administered 2 h prior to platinum drugs may sensitize ovarian cancer cells to platinum, inducing apoptosis and providing a means of overcoming resistance [95].

Ren [109] demonstrated that (‒)‒epigallocatechin-3-gallate induces reduction in IM9 myelo‐ ma cells and that its activity was dose- and time-dependent on the induction of apoptotic cell death; additionally, this natural metabolite combined with curcumin and lovastatin pos‐ sessed the ability to suppress esophageal cancer-cell growth [154]. In multilla berries, it was found that their high levels of polyphenols, flavonoids, and flavonols and their antioxidants have a strong ability to reduce the viability of colon-cancer HT-29 and SW480 cell lines [33]. The anticancer activity of baicalein, a flavonoid found in several plants, was evaluated in a cutaneous squamous carcinoma-cell line, A431; it was found that this compound reduced the migration and invasiveness of the cells through inhibition of ezrin expression, which leads to the suppression of tumor metastasis [153].

In beans, it was found that these contain several compounds with cytotoxic activity on ani‐ mals and human cell lines ( C33-A, SW480, and 3T3), which can be attributed to the antioxi‐ dants and damage to DNA caused by tannins, saponins, lectins, and others compounds found in the seed [143, 144].

*Melastoma malabathricum* showed to have the ability to inhibit the proliferation of Caov-3, HL-60, CEM-SS, MCF-7, HeLa, and MDA-MB-231 cell lines, indicating that the leaves of this plant possess potential antiproliferative and antioxidant activities that could be attributed to its high content of phenolic compounds [122]. Melatonin, a naturally occurring compound, showed cytotoxic activity toward transformed 3T3-SV40 fibroblasts [143] and murine hepa‐ toma cells MN22a, and it was shown that the sensitivities of both cell types to lysis by killer cells fell sharply [139].T he potent antioxidant activity of *Kalanchoe gracili*s (L.) DC stems due to that the polyphenolic compound found in this medicinal plant showed to have the ability to inhibit HepG2 cell proliferation [171], and the flavonoids found in *Rosa canina* L. are re‐ sponsible for the antiproliferative activity in HeLa, MCF7, and HT-29 cancer-cell lines [138]. Analysis of the fruit of *Phelaria macrocarpa* (Boerl.) Scheff and of *Olea europaea* L. indicated that all parts of the fruit possess cytotoxic activity against HT-29, MCF-7, HeLa, BPH-1, and Chang cells, indicating that these fruits are a sources of bioactive compounds that are as po‐ tent as antioxidants and antioxidant agents, suggesting its possible use as an adjuvant agent in the treatment of cancer [56, 1].

The extract of *Calluna vulgaris* exhibited a photoprotective effect on human keratinocytes (HaCaT) exposed to Ultraviolet B (UVB) radiation [100]. *Cachrys pungens Jan* was analyzed in a human tumor- cell line, amelanotic melanoma, and it was found that its extract contains antioxidants, such as coumarins, which are responsible for their cytotoxicity in A375 cells [92]. *Inonotus obliquus* and *Peperomia pellucida*, plants employed as folk remedies for cancer treatment, were evaluated in several tumor cell-line types and it was found that these plants contains several antioxidants, such as lanosterol, inotodiols, ergosterol, phytol, 2-naphthale‐ nol, decahydro hexadecanoic acid, methyl ester, and 9,12 octadecadienoic acid, indicating that these antioxidant compounds are responsible for the anticarcinogenic activity of the plant extract [129, 150]. The extract of *Indigofera cassioides* indicated the presence antioxidant activity, preventing lipid peroxidation and promoting the enzymatic antioxidant defense system, and also showed potent antitumoral and cytotoxic affect against EAC, DLA, HeLa, Hep-2, HepG-2, MCF-7, Ht-29, and NIH 3T3 cells [69].

Hesperetin, hesperetin analog, carnocine, and resveratrol were evaluated for their antioxi‐ dant and anticarcinogenic activity on HT-29, HCT116, and mouse skin carcinogenesis; their studies demonstrated that these compounds can inhibit cell proliferation, induce apoptosis, affect glycolysis, and decrease tumoration [125, 161, 40]. Honey, a natural product common‐ ly used throughout the world, contains antioxidant properties and exerts a preventive effect against disease. Chrysin is a natural flavone commonly found in honey, and it was demon‐ strated that this compound induced apoptosis in PC-3 cells [116], fennel seeds (*Foeniculum vulgare*) are present in antioxidants that have an anticancer potential against HepG2 and MCF-7 cell lines [94). It was indicated that compounds such as quercetin, flavonoids, and brucine have chemopreventive action against the osteosarcoma cell line (MG63), C6 glioma cells, and Ehrlich ascites cells, and that they can be used as anticancer, antigenotoxic agents and can induce apoptosis [135, 119, 2].

### **8. Conclusion**

**2.** Flavonoids have also shown to inhibit cyclo-oxygenase, lipoxygenase, microsomal mono-oxygenase, glutathione *S*-transferase, mitochondrial succinoxidase, and (Nicotinamide adenine denucleotide (NADH) oxidase, all of which are involved in ROS gener‐

A number of flavonoids efficiently chelate trace metals, which play an important role in oxy‐ gen metabolism. Free iron and copper are potential enhancers of ROS formation, as exempli‐ fied by the reduction of hydrogen peroxide with the generation of the highly aggressive

On the other hand*, in* vitro studies showed that the compounds present in fruits and vegeta‐ bles, such as resveratrol, genestein, baicalein, and many others are attractive candidates for improved chemotherapeutic agents [35]. Resveratrol in combination with platinum drugs and oxaliplatin demonstrated that resveratrol administered 2 h prior to platinum drugs may sensitize ovarian cancer cells to platinum, inducing apoptosis and providing a means of

Ren [109] demonstrated that (‒)‒epigallocatechin-3-gallate induces reduction in IM9 myelo‐ ma cells and that its activity was dose- and time-dependent on the induction of apoptotic cell death; additionally, this natural metabolite combined with curcumin and lovastatin pos‐ sessed the ability to suppress esophageal cancer-cell growth [154]. In multilla berries, it was found that their high levels of polyphenols, flavonoids, and flavonols and their antioxidants have a strong ability to reduce the viability of colon-cancer HT-29 and SW480 cell lines [33]. The anticancer activity of baicalein, a flavonoid found in several plants, was evaluated in a cutaneous squamous carcinoma-cell line, A431; it was found that this compound reduced the migration and invasiveness of the cells through inhibition of ezrin expression, which

In beans, it was found that these contain several compounds with cytotoxic activity on ani‐ mals and human cell lines ( C33-A, SW480, and 3T3), which can be attributed to the antioxi‐ dants and damage to DNA caused by tannins, saponins, lectins, and others compounds

*Melastoma malabathricum* showed to have the ability to inhibit the proliferation of Caov-3, HL-60, CEM-SS, MCF-7, HeLa, and MDA-MB-231 cell lines, indicating that the leaves of this plant possess potential antiproliferative and antioxidant activities that could be attributed to its high content of phenolic compounds [122]. Melatonin, a naturally occurring compound, showed cytotoxic activity toward transformed 3T3-SV40 fibroblasts [143] and murine hepa‐ toma cells MN22a, and it was shown that the sensitivities of both cell types to lysis by killer cells fell sharply [139].T he potent antioxidant activity of *Kalanchoe gracili*s (L.) DC stems due to that the polyphenolic compound found in this medicinal plant showed to have the ability to inhibit HepG2 cell proliferation [171], and the flavonoids found in *Rosa canina* L. are re‐ sponsible for the antiproliferative activity in HeLa, MCF7, and HT-29 cancer-cell lines [138]. Analysis of the fruit of *Phelaria macrocarpa* (Boerl.) Scheff and of *Olea europaea* L. indicated that all parts of the fruit possess cytotoxic activity against HT-29, MCF-7, HeLa, BPH-1, and Chang cells, indicating that these fruits are a sources of bioactive compounds that are as po‐

ation [68, 15].

hydroxyl radical [103].

overcoming resistance [95].

found in the seed [143, 144].

leads to the suppression of tumor metastasis [153].

402 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Oxidative stress causes injury to cells, induces gene mutation, and is involved in carcino‐ genesis and other degenerative diseases by directly or indirectly influencing intracellular signal transduction and transcription factors. The state of OS under carcinogenesis and tu‐ mor-bearing conditions is an intricate one in which various substances are involved in com‐ plex interactions.

The data discussed in this paper show that the biological effects of antioxidants on humans and animals can be controversial. Due to that the action of antioxidants depends on the oxi‐ dative status of cells, antioxidants can be protective against cancer; because ROS induce oxi‐ dative carcinogenic damage in DNA, antioxidants can prevent cancer in healthy persons harboring increased ROS levels.

Oxidative stress as cause and effect is not the sole factor in the development of cancer. It is important to take into account that there are other factors involved in its development, such as genetic predisposition, eating habits, environment, etc. Because ROS at moderate concen‐ trations act as indispensable mediators of cancer-protective apoptosis and phagocytosis, an excess of antioxidants in persons with low ROS levels can block these cancer-preventive mechanisms. High doses of antioxidants can reduce the ROS level in persons who overpro‐ duce ROS and protect them against cancer and other ROS-dependent morbid conditions.

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For individuals with low ROS levels, high doses of antioxidants can be deleterious, sup‐ pressing the already low rate of ROS generation and ROS-dependent cancer-preventive apoptosis. Screening and monitoring the human population regarding their ROS level can transform antioxidants into safe and powerful disease-preventive tools that could signifi‐ cantly contribute to the nation's health.

Many *in vivo* and *in vitro* studies performed to evaluate the capability of antioxidants against cancer, such as chemopreventive or therapeutic agents, were conduced employing natural antioxidants from fruits and vegetables; these are mainly supplied through food, which of‐ ten do not provide sufficient input for these to function as chemoprotectors. Thus, humans are forced to consume antioxidants in a more direct manner, either in the form of a tablet, a pill, or any other form in order to supply the levels that the body requires of these com‐ pounds to protect it against cell damage caused by oxidation reactions, thus reducing the risk of certain cancer types, especially those of the epithelial surface and in the upper part of the body, such as breast, lung, kidney, liver, intestine, and many others that have been well documented. However, further investigations are expected before our better understanding of the function of many antioxidants and their utilization in the prevention and treatment of cancer and other degenerative diseases.

### **Author details**

Carmen Valadez-Vega1 , Luis Delgado-Olivares1 , José A. Morales González1 , Ernesto Alanís García1 , José Roberto Villagomez Ibarra2 , Esther Ramírez Moreno 1 , Manuel Sánchez Gutiérrez1 , María Teresa Sumaya Martínez3 , Zuñiga Pérez Clara1 and Zuli Calderón Ramos1

1 Institute of Health Sciences, Autonomous University of Hidalgo State, Ex-Hacienda de la Concepción, Tilcuautla, Hgo, Mexico. C.P.42080., Mexico

2 Institute of Basic Sciences, Autonomous University of Hidalgo State, Km 4.5 Carretera Pa‐ chuca-Tulancingo, Ciudad del Conocimiento, Mineral de la Reforma Hidalgo, C.P. 42076, Mexico

3 Secretary of Research and Graduate Studies, Autonomous University of Nayarit, Ciudad de la Cultura "Amado Nervo", Boulevard Tepic-Xalisco S/N. Tepic, Nayarit, Mexico

### **References**

Oxidative stress as cause and effect is not the sole factor in the development of cancer. It is important to take into account that there are other factors involved in its development, such as genetic predisposition, eating habits, environment, etc. Because ROS at moderate concen‐ trations act as indispensable mediators of cancer-protective apoptosis and phagocytosis, an excess of antioxidants in persons with low ROS levels can block these cancer-preventive mechanisms. High doses of antioxidants can reduce the ROS level in persons who overpro‐ duce ROS and protect them against cancer and other ROS-dependent morbid conditions.

For individuals with low ROS levels, high doses of antioxidants can be deleterious, sup‐ pressing the already low rate of ROS generation and ROS-dependent cancer-preventive apoptosis. Screening and monitoring the human population regarding their ROS level can transform antioxidants into safe and powerful disease-preventive tools that could signifi‐

Many *in vivo* and *in vitro* studies performed to evaluate the capability of antioxidants against cancer, such as chemopreventive or therapeutic agents, were conduced employing natural antioxidants from fruits and vegetables; these are mainly supplied through food, which of‐ ten do not provide sufficient input for these to function as chemoprotectors. Thus, humans are forced to consume antioxidants in a more direct manner, either in the form of a tablet, a pill, or any other form in order to supply the levels that the body requires of these com‐ pounds to protect it against cell damage caused by oxidation reactions, thus reducing the risk of certain cancer types, especially those of the epithelial surface and in the upper part of the body, such as breast, lung, kidney, liver, intestine, and many others that have been well documented. However, further investigations are expected before our better understanding of the function of many antioxidants and their utilization in the prevention and treatment of

, José A. Morales González1

,

,

and

, Esther Ramírez Moreno 1

, Zuñiga Pérez Clara1

cantly contribute to the nation's health.

404 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cancer and other degenerative diseases.

, Luis Delgado-Olivares1

Concepción, Tilcuautla, Hgo, Mexico. C.P.42080., Mexico

, José Roberto Villagomez Ibarra2

, María Teresa Sumaya Martínez3

1 Institute of Health Sciences, Autonomous University of Hidalgo State, Ex-Hacienda de la

2 Institute of Basic Sciences, Autonomous University of Hidalgo State, Km 4.5 Carretera Pa‐ chuca-Tulancingo, Ciudad del Conocimiento, Mineral de la Reforma Hidalgo, C.P. 42076,

3 Secretary of Research and Graduate Studies, Autonomous University of Nayarit, Ciudad

de la Cultura "Amado Nervo", Boulevard Tepic-Xalisco S/N. Tepic, Nayarit, Mexico

**Author details**

Carmen Valadez-Vega1

Ernesto Alanís García1

Zuli Calderón Ramos1

Mexico

Manuel Sánchez Gutiérrez1


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[135] Toyoda-Hokaiwado, N, Yasui, Y, Muramatsu, M, Masumura, K, Takamune, M, Ya‐ mada, M, Ohta, T, Tanaka, T, & Nohmi, T. (2011). Chemopreventive effects of sily‐ marin against 1,2-dimethylhydrazine plus dextran sodium sulfate-induced inflammation-associated carcinogenicity and genotoxicity in the colon of gpt delta

[136] Toyokuni, MD. (1998). Oxidative Stress and Cancer: The Role of Redox Regulation

[137] Tsaluchidu, S., Cocchi, M., Tonello, L., & Puri, B. K. (2008). Fatty acids and oxidative stress in psychiatric disorders. *BMC Psychiatry*, 8(1), S1-S5, 0147-1244 X, Online.

[138] Tumbas, V. T., Canadanović-Brunet, J. M., Cetojević-Simin, D. D., Cetković, G. S., Ethilas, S. M., & Gille, L. (2012). Effect of rosehip (Rosa canina L.) phytochemicals on stable free radicals and human cancer cells. *Journal of the Science of Food and Agricul‐*

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*ture*, 92, 1273-1281, 0022-5142, Print, 1097-0010, Online.

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416 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants


[152] Winslow, LC, & Krol, DJ. (1998). Herbs as medicines. *Archives Internal Medicine*, 0003-9926, Print, 1538-3679, Online, 1258, 2192-219.

**Chapter 17**

**Emerging Role of Natural Antioxidants in Chronic**

**Selenium**

Manuel Soriano García

http://dx.doi.org/10.5772/51610

**1. Introduction**

antioxidant nutrients.

Additional information is available at the end of the chapter

**Disease Prevention with an Emphasis on Vitamin E and**

The possibility has arisen within the last three decades that major diseases that directly af‐ fect humankind worldwide may be preventable by the simple improving the dietary intake

There is no doubt that successful prevention is the key to controlling morbidity and mortali‐ ty from chronic diseases affecting humankind. Prevention provides: the methods to avoid occurrence of disease and most population-based health promotion efforts are of this type; methods to diagnose and treat extant disease in early stages before it causes significant mor‐ bidity; methods to reduce negative impact of extant disease by restoring function and reduc‐ ing disease-related complications; and finally, the methods to mitigate or avoid results of

The quality and quantity of diet with respect to the intake of fresh food (fruits, seeds and vegetables) may improve our health and consequently decrease the risk of any disease. Cur‐ rently, the antioxidant nutrients are the vitamins C and E and β-carotene. However, it is worthy to mention that these compounds are involved in other functions a part from being

Selenium (Se), a trace mineral. Is the 34th element and is located between sulfur and telluri‐ um in Group 16 in the periodic table. It is a nonmetallic element and its properties are inter‐ mediate between adjacent sulfur and tellurium. It was originally discovered by a German chemist Martin Heinric Klaproth, but misidentified as tellurium. Later, in 1818 a Swedish chemist Jons Jacob Berzelius discovered selenium and was named after the Greek goddess of the moon, Selene [1] and its name was associated with tellurium, a name for earth. He

> © 2013 Soriano García; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of those nutrient substances that have become called "antioxidant nutrients".

unnecessary or excessive interventions in the health system.


### **Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium**

Manuel Soriano García

[152] Winslow, LC, & Krol, DJ. (1998). Herbs as medicines. *Archives Internal Medicine*,

[153] Wu, B, Li, J, Huang, D, Wang, W, Chen, Y, Liao, Y, Tang, X, Xie, H, & Tang, F. (2011). Baicalein mediates inhibition of migration and invasiveness of skin carcinoma

[154] Ye, F, Zhang, GH, Guan, BX, & Xu, XC. (2012). Suppression of esophageal cancer cell growth using curcumin, (-)-epigallocatechin-3-gallate and lovastatin. *World Journal of*

*Gastroenterology*, 1007-9327, Print, 2219-2840, Online, 18, 126-135.

0003-9926, Print, 1538-3679, Online, 1258, 2192-219.

418 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

through Ezrin in A431 cells. 1471-2407, 11, 527-536.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51610

### **1. Introduction**

The possibility has arisen within the last three decades that major diseases that directly af‐ fect humankind worldwide may be preventable by the simple improving the dietary intake of those nutrient substances that have become called "antioxidant nutrients".

There is no doubt that successful prevention is the key to controlling morbidity and mortali‐ ty from chronic diseases affecting humankind. Prevention provides: the methods to avoid occurrence of disease and most population-based health promotion efforts are of this type; methods to diagnose and treat extant disease in early stages before it causes significant mor‐ bidity; methods to reduce negative impact of extant disease by restoring function and reduc‐ ing disease-related complications; and finally, the methods to mitigate or avoid results of unnecessary or excessive interventions in the health system.

The quality and quantity of diet with respect to the intake of fresh food (fruits, seeds and vegetables) may improve our health and consequently decrease the risk of any disease. Cur‐ rently, the antioxidant nutrients are the vitamins C and E and β-carotene. However, it is worthy to mention that these compounds are involved in other functions a part from being antioxidant nutrients.

Selenium (Se), a trace mineral. Is the 34th element and is located between sulfur and telluri‐ um in Group 16 in the periodic table. It is a nonmetallic element and its properties are inter‐ mediate between adjacent sulfur and tellurium. It was originally discovered by a German chemist Martin Heinric Klaproth, but misidentified as tellurium. Later, in 1818 a Swedish chemist Jons Jacob Berzelius discovered selenium and was named after the Greek goddess of the moon, Selene [1] and its name was associated with tellurium, a name for earth. He

© 2013 Soriano García; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. observed the element as a deposit following the oxidation of sulfur dioxide from cooper pyr‐ ites. It ranks seventieth in abundance among the elements and is distributed in the Earth's crust at concentrations averaging 0.09 mg/kg [2]. Selenium has six major stable isotopes have been reported and the most abundant in nature are 80Se (49.6%) and 78Se (23.8%) [3]. In general, selenium is present in the environment in elemental form or in the form of sele‐ nide (Se2-), selenate (SeO4 2-), or selenite (SeO3 2-). The identity and amounts of the various oxi‐ dation-state species in soils depends enormously on the redox-potential conditions. The lower oxidation states predominate in anaerobic conditions, acidic soils, and the higher oxi‐ dation states are favored in alkaline and aerobic conditions. Both selenites and selenates are taken up by plants and converted to protein-bound selenocysteine and selenomethionine, soluble inorganic forms, several free amino acids, and volatile organoselenium compounds. The elemental form of selenium, selenium dioxide, and volatile organoselenium compounds produced by industries and plants are incorporated in the environment. Selenium occurs naturally in water in trace amounts as a result of geochemical processes, such as weathering of rocks and erosion of soils, and is usually present in water as selenate or selenite; however the elemental form may be carried in suspension [4].

tremendous effort has been directed toward the synthesis of stable organoselenium com‐ pounds that could be used as antioxidants, enzyme modulators, antitumor, antimicrobials, antihypertensive agents, antivirals and cytokine inducers. Several excellent books and re‐ views appeared in literature describing the biological function of organoselenium com‐

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

421

The role of organoselenium compounds as antioxidants, as enzyme modulators, photo-chy‐ motherapeutic agents, cytokine inducers and immunomodulators, and antihypertensive and

The essentiality of selenium results as a necessary component of the active center of a num‐ ber of selenoenzymes. Selenium functions as a redox center. The term selenoprotein is any protein that includes in its primary sequence of amino acids, the selenocysteine (Se-Cys) res‐ idue [24]. There are at least 30 selenoproteins that have been identified in mammals, and it has been estimated that humans have about 25 selenoproteins, including glutathione peroxi‐ dase, thioredoxin reductase, iodothyronine, deiodinase, and selenoproteins P, W, and R [25-27]. GPx accounts for 10–30% of plasma selenium, and selenoprotein P accounts for an‐ other 50% [28]. These enzymes protect cells from free radical damage and regulate DNA transcription and cell proliferation. The glutathione and thioredoxin systems in particular have long been considered the major pathways through which selenium exerts its potential chemopreventive effect [24], while some investigations have also suggested growth inhibito‐ ry, proapoptotic activity for selenometabolites in premalignant cells [29]. Selenium is also in‐ volved in thyroid function, T cell immunity, and spermatogenesis [28], and is a competitive antagonist of potentially carcinogenic heavy metals such as arsenic and cadmium [30].

The organism has several biological defense mechanisms against intracellular oxidative stress such as superoxide dismutase, catalase, glutathione peroxidase and nonenzymatic an‐ tioxidants such as glutathione, vitamins A, C and E, riboflavin, a B vitamin and selenium

Vitamin E is a fat-soluble vitamin known for its antioxidant capacity that is why it is well known as a lipophilic antioxidant that protects membranes from being oxidatively damaged as an electron donor to free radicals [32]. Vitamin E belongs to a group of the eight naturally occurring vitamer forms, four tocopherols (α, β, γ, δ) and four tocotrienols (α, β, γ, δ) based on the hydroxyl and methyl substitution in their phenolic rings, all of which have saturated and three double bonds in their phytyl tails. α-tocopherol (from the Greek *tokos* = child, *phero* = to bear and *ol* indicating that the substance is an alcohol, is the most abundant form in nature; it's the most active and corrects human E deficiency symptoms [33]. The most abundant sources of vitamin E are vegetable oils, which typically contain all four tocopherol (α, β, γ, δ) in varying proportions, Other important source are nuts and seeds such as sun‐

It is well known that all forms of vitamin E are lipid soluble they easily absorbed from the intestinal lumen after dietary intake via micelles created by biliary and pancreatic secretions [34-35]. Vitamin E is then incorporated into chylomicrons and secreted into the circulation where, transported by various lipoproteins, it travels to the liver [36]. Plasma α-tocopherol

cardiotonic agents have been recently described in literature [23].

can also contribute to overcome oxidative stress [31].

flower and amaranth seeds.

pounds [20-22].

Interest in selenium and health was focused primarily on the potentially toxic effects of high intakes in humans, stimulated by reports of alkali disease in livestock raised in seleniferous areas, in the last century [5]. Selenium is a trace mineral that is essential to good health but required only small amounts [6,7]. Selenium is considered as essential human micronutrient and is incorporated into proteins to make selenoproteins. Selenium is present in the seleno‐ proteins, as the aminoacid selenocysteine (Se-Cys) [8-12].

Dietary levels of the desired amount of Se are in a very narrow range: consumption of foods containing less than 0.1 mg kg-1 of this element will result in Se deficiency, whereas dietary levels above 1 mg kg-1 will lead to toxic manifestations [13]. Se status varies significantly across different populations and different ethnic groups [14-15].

Selenium enters the food chain through plants, and the amount and bioavailability of seleni‐ um in the soil typically reflects the plant level. Selenium is provided by the diet in humans, but may also be provided from drinking water, environmental pollution, and in recent years through supplementation [16,17]. Plants convert Se mainly into selenomethionine (Se-Met) and incorporated it into protein place of methionine. More than 50% of the total Se content of the plant exist as Se-Met, the rest exist as selenocysteine (Se-Cys), methyl-Se-Cys and cglutamyl-Se-methyl-Cys. The later compounds are not significantly incorporated into plant protein. Higher animals are unable to synthesize Se-Met and only Se-Cys was detected in rats supplemented with Se as selenite [18]. Animals that eat grains (Brazil nuts, sunflower seeds, walnuts and grains) that were grown in selenium rich soil have higher levels of sele‐ nium in their muscles, liver, kidney, heart, spleen and fingernails. Other natural selenium sources are butter, eggs, brewer's yeast, wheat germ, garlic, raspberry leaf, radish, horserad‐ ish, onions, shellfish, broccoli, fennel seed and ginseng, among other sources.

Most ingested forms of selenium ultimately are metabolized to low molecular weight inor‐ ganic and organic compounds that play a central role in human health either via incorpora‐ tion into selenoproteins or binding to selenium binding proteins [19]. Therefore, a tremendous effort has been directed toward the synthesis of stable organoselenium com‐ pounds that could be used as antioxidants, enzyme modulators, antitumor, antimicrobials, antihypertensive agents, antivirals and cytokine inducers. Several excellent books and re‐ views appeared in literature describing the biological function of organoselenium com‐ pounds [20-22].

observed the element as a deposit following the oxidation of sulfur dioxide from cooper pyr‐ ites. It ranks seventieth in abundance among the elements and is distributed in the Earth's crust at concentrations averaging 0.09 mg/kg [2]. Selenium has six major stable isotopes have been reported and the most abundant in nature are 80Se (49.6%) and 78Se (23.8%) [3]. In general, selenium is present in the environment in elemental form or in the form of sele‐

dation-state species in soils depends enormously on the redox-potential conditions. The lower oxidation states predominate in anaerobic conditions, acidic soils, and the higher oxi‐ dation states are favored in alkaline and aerobic conditions. Both selenites and selenates are taken up by plants and converted to protein-bound selenocysteine and selenomethionine, soluble inorganic forms, several free amino acids, and volatile organoselenium compounds. The elemental form of selenium, selenium dioxide, and volatile organoselenium compounds produced by industries and plants are incorporated in the environment. Selenium occurs naturally in water in trace amounts as a result of geochemical processes, such as weathering of rocks and erosion of soils, and is usually present in water as selenate or selenite; however

Interest in selenium and health was focused primarily on the potentially toxic effects of high intakes in humans, stimulated by reports of alkali disease in livestock raised in seleniferous areas, in the last century [5]. Selenium is a trace mineral that is essential to good health but required only small amounts [6,7]. Selenium is considered as essential human micronutrient and is incorporated into proteins to make selenoproteins. Selenium is present in the seleno‐

Dietary levels of the desired amount of Se are in a very narrow range: consumption of foods containing less than 0.1 mg kg-1 of this element will result in Se deficiency, whereas dietary levels above 1 mg kg-1 will lead to toxic manifestations [13]. Se status varies significantly

Selenium enters the food chain through plants, and the amount and bioavailability of seleni‐ um in the soil typically reflects the plant level. Selenium is provided by the diet in humans, but may also be provided from drinking water, environmental pollution, and in recent years through supplementation [16,17]. Plants convert Se mainly into selenomethionine (Se-Met) and incorporated it into protein place of methionine. More than 50% of the total Se content of the plant exist as Se-Met, the rest exist as selenocysteine (Se-Cys), methyl-Se-Cys and cglutamyl-Se-methyl-Cys. The later compounds are not significantly incorporated into plant protein. Higher animals are unable to synthesize Se-Met and only Se-Cys was detected in rats supplemented with Se as selenite [18]. Animals that eat grains (Brazil nuts, sunflower seeds, walnuts and grains) that were grown in selenium rich soil have higher levels of sele‐ nium in their muscles, liver, kidney, heart, spleen and fingernails. Other natural selenium sources are butter, eggs, brewer's yeast, wheat germ, garlic, raspberry leaf, radish, horserad‐

Most ingested forms of selenium ultimately are metabolized to low molecular weight inor‐ ganic and organic compounds that play a central role in human health either via incorpora‐ tion into selenoproteins or binding to selenium binding proteins [19]. Therefore, a

2-). The identity and amounts of the various oxi‐

2-), or selenite (SeO3

the elemental form may be carried in suspension [4].

420 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

proteins, as the aminoacid selenocysteine (Se-Cys) [8-12].

across different populations and different ethnic groups [14-15].

ish, onions, shellfish, broccoli, fennel seed and ginseng, among other sources.

nide (Se2-), selenate (SeO4

The role of organoselenium compounds as antioxidants, as enzyme modulators, photo-chy‐ motherapeutic agents, cytokine inducers and immunomodulators, and antihypertensive and cardiotonic agents have been recently described in literature [23].

The essentiality of selenium results as a necessary component of the active center of a num‐ ber of selenoenzymes. Selenium functions as a redox center. The term selenoprotein is any protein that includes in its primary sequence of amino acids, the selenocysteine (Se-Cys) res‐ idue [24]. There are at least 30 selenoproteins that have been identified in mammals, and it has been estimated that humans have about 25 selenoproteins, including glutathione peroxi‐ dase, thioredoxin reductase, iodothyronine, deiodinase, and selenoproteins P, W, and R [25-27]. GPx accounts for 10–30% of plasma selenium, and selenoprotein P accounts for an‐ other 50% [28]. These enzymes protect cells from free radical damage and regulate DNA transcription and cell proliferation. The glutathione and thioredoxin systems in particular have long been considered the major pathways through which selenium exerts its potential chemopreventive effect [24], while some investigations have also suggested growth inhibito‐ ry, proapoptotic activity for selenometabolites in premalignant cells [29]. Selenium is also in‐ volved in thyroid function, T cell immunity, and spermatogenesis [28], and is a competitive antagonist of potentially carcinogenic heavy metals such as arsenic and cadmium [30].

The organism has several biological defense mechanisms against intracellular oxidative stress such as superoxide dismutase, catalase, glutathione peroxidase and nonenzymatic an‐ tioxidants such as glutathione, vitamins A, C and E, riboflavin, a B vitamin and selenium can also contribute to overcome oxidative stress [31].

Vitamin E is a fat-soluble vitamin known for its antioxidant capacity that is why it is well known as a lipophilic antioxidant that protects membranes from being oxidatively damaged as an electron donor to free radicals [32]. Vitamin E belongs to a group of the eight naturally occurring vitamer forms, four tocopherols (α, β, γ, δ) and four tocotrienols (α, β, γ, δ) based on the hydroxyl and methyl substitution in their phenolic rings, all of which have saturated and three double bonds in their phytyl tails. α-tocopherol (from the Greek *tokos* = child, *phero* = to bear and *ol* indicating that the substance is an alcohol, is the most abundant form in nature; it's the most active and corrects human E deficiency symptoms [33]. The most abundant sources of vitamin E are vegetable oils, which typically contain all four tocopherol (α, β, γ, δ) in varying proportions, Other important source are nuts and seeds such as sun‐ flower and amaranth seeds.

It is well known that all forms of vitamin E are lipid soluble they easily absorbed from the intestinal lumen after dietary intake via micelles created by biliary and pancreatic secretions [34-35]. Vitamin E is then incorporated into chylomicrons and secreted into the circulation where, transported by various lipoproteins, it travels to the liver [36]. Plasma α-tocopherol concentrations in humans range from 11 to 37 µmol/L, whereas γ-tocopherol are between 2 and 5 µmol/L. The liver plays a central role in regulating α-tocopherol levels by directly act‐ ing on the distribution, metabolism, and excretion of this vitamin [37]. The major hepatic regulatory mechanism is the α-tocopherol transfer protein, α-TTP, which has been identified in a variety of mammals, including humans [38]. This protein facilitates secretion of α-toco‐ pherol from the liver into the bloodstream, by acquiring it from endosomes and then deliv‐ ering it to the plasma membrane where it is released and promptly associates with the different nascent lipoproteins [39]. Plasma concentration of vitamin E depends completely on the absorption, tissue delivery, and excretion rate. The estimated α-tocopherol half-life in plasma of healthy individuals is ~ 48 to 60 H, which is much longer than the half-life of γtocopherol approximately 15 H. These kinetic data underscore an interesting concept that while α-tocopherol levels are maintained, the other forms of vitamin E are removed much more rapidly [40].

selenoenzymes with strong antioxidant activity are GPx, GPx1, GPx3, GPx4, GPx5 and GPx6. In Humans GPx1 through GPx4 and GPx6 are selenocysteine containing enzymes. These GPx play a significant role in protecting cells against oxidative damage from reactive oxygen species (ROS) and reactive nitrogen species (RNS), which include superoxide, hy‐ drogen peroxide, hydroxyl radicals, nitric oxide and peroxynitrite [43-44]. The other essen‐ tial antioxidant selenoenzymes are the thioredoxin reductase (TrxR) where they use thioredoxin (Trx) as a substrate to maintain a Trx/TrxR system in a reduced state for remov‐ al of harmful hydrogen peroxide and there are three types of TrxR. Iodothyronine deiodi‐

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

423

In [46] selenium's function as an antioxidant, and as a constituent of selenoproteins that are important in redox homeostasis, warrants further investigation as a risk factor for depres‐ sion, and suggest a potentially novel modifiable factor in the primary prevention and man‐ agement of depression. Depression is becoming recognized as an inflammatory disorder, accompanied by an accumulation of highly reactive oxygen species that overwhelm usual defensive physiological processes [47-51]. Several indicators support a role for selenium in normal brain function. During times of selenium deficiency, there is preferential storage of selenium in the brain [52]. Selenium has significant modulatory effects on dopamine [53] and dopamine plays a role in the pathophysiology of depression and other psychiatric ill‐ nesses [54]. Diminished levels of selenium in the brain are associated with cognitive decline [55] and Alzheimer's disease [56]. Selenium supplementation has been linked with improve‐ ments in mood [57] and protection against postpartum depression [58]. What is unclear is if low dietary selenium is a risk factor for the development of depression. In recognition of se‐ lenium's biological activity, it has been hypothesized that low levels of dietary selenium would be associated with an increased risk of major depressive disorder (MDD) in a repre‐

Alterations in redox biology are established in depression; however, there are no prospec‐ tive epidemiological data on redox-active selenium in depression. It is known that seleni‐ um's function as an antioxidant, and as a constituent of selenoproteins that are important in redox homeostasis, warrants further investigation as a risk factor for depression, and sug‐ gest a potentially novel modifiable factor in the primary prevention and management of de‐

The International Study of Asthma and Allergies in Childhood (ISAAC) found that one in four New Zealand children aged 6–7 years had experienced asthma symptoms, which placed New Zealand in the top four countries for asthma prevalence [59]. The reasons for the high prevalence and severity of this condition or the increased prevalence of asthma over the last 20 years are not well understood. One of a number of environmental factors that have been proposed as a reason for the escalation in asthma prevalence is a decreasing intake of dietary antioxidants [60]. It is well known that selenium is essential for the optimal

nase (DIO) have three subtypes, DIO 1, 2, and 3 [45].

sentative population-based sample of women.

**2.2. Se and depression**

pression.

**2.3. Selenium and allergies**

### **2. Selenium and Health**

Selenium deficiency is associated with the pathogenesis of wide variety of processes that af‐ fects our health and disease including the antioxidant activity, depression, allergies, pre‐ venting oxidative stress, HIV infection, in the brain, thyroid metabolism, cancer, diabetes mellitus, male fertility, asthma, cardiovascular disorders, rheumatoid arthritis, pre-eclamp‐ sia, in immune function, in alleviate bone impairments, aging, gastrointestinal problems, se‐ lenium interactions and toxicity, anti-inflammatory effects, and hypertension. The list of clinical disorders expected to be influenced by Se deficiency is rapidly growing with time. Some selected issues regarding the role of Se in health and disease have been briefly out‐ lined as follows:

#### **2.1. Se and antioxidant activity**

Selenocysteine is recognized as the 21st amino acid, and it forms a predominant residue of selenoproteins and selenoenzymes in biological tissues. The molecular structure of seleno‐ cystiene is an analogue of cysteine where a sulphur atom is replaced by Se. Even though Se and sulphur share some similar chemical properties, there are also some differences. The R-SeH with a pKa 5.2 is more is more acidic than R-SH with a pKa 8.5, and readily dissociated at physiological pH, which may contribute to its biological reactivity. In the body, both or‐ ganic [selenocysteine(SeCys) and selenomethionine (SeMet)] and inorganic (selenite, selen‐ ate) Se compounds are readily metabolized to various forms of Se metabolites [41]. Of particular importance during this metabolic process is the formation of hydrogen selenide (H2Se) from selenite after the action of glutathione-coupled reactions *via* selenodiglutathione (GS-Se-SG) and glutathione selenopersulfide (GS-SeH). H2Se is further metabolized and in‐ volved in the formation of methylselenol and dimethylselenide, which are exhaled or secret‐ ed *via* the skin. Selenium is also excreted in urine as trimethylselenonium ion and selenosugar compounds [42]. The selenoproteins are classified on the basis of their biologi‐ cal function [25]. The first identified selenoprotein was glutathione peroxidase 1 (GPx1). The selenoenzymes with strong antioxidant activity are GPx, GPx1, GPx3, GPx4, GPx5 and GPx6. In Humans GPx1 through GPx4 and GPx6 are selenocysteine containing enzymes. These GPx play a significant role in protecting cells against oxidative damage from reactive oxygen species (ROS) and reactive nitrogen species (RNS), which include superoxide, hy‐ drogen peroxide, hydroxyl radicals, nitric oxide and peroxynitrite [43-44]. The other essen‐ tial antioxidant selenoenzymes are the thioredoxin reductase (TrxR) where they use thioredoxin (Trx) as a substrate to maintain a Trx/TrxR system in a reduced state for remov‐ al of harmful hydrogen peroxide and there are three types of TrxR. Iodothyronine deiodi‐ nase (DIO) have three subtypes, DIO 1, 2, and 3 [45].

### **2.2. Se and depression**

concentrations in humans range from 11 to 37 µmol/L, whereas γ-tocopherol are between 2 and 5 µmol/L. The liver plays a central role in regulating α-tocopherol levels by directly act‐ ing on the distribution, metabolism, and excretion of this vitamin [37]. The major hepatic regulatory mechanism is the α-tocopherol transfer protein, α-TTP, which has been identified in a variety of mammals, including humans [38]. This protein facilitates secretion of α-toco‐ pherol from the liver into the bloodstream, by acquiring it from endosomes and then deliv‐ ering it to the plasma membrane where it is released and promptly associates with the different nascent lipoproteins [39]. Plasma concentration of vitamin E depends completely on the absorption, tissue delivery, and excretion rate. The estimated α-tocopherol half-life in plasma of healthy individuals is ~ 48 to 60 H, which is much longer than the half-life of γtocopherol approximately 15 H. These kinetic data underscore an interesting concept that while α-tocopherol levels are maintained, the other forms of vitamin E are removed much

422 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Selenium deficiency is associated with the pathogenesis of wide variety of processes that af‐ fects our health and disease including the antioxidant activity, depression, allergies, pre‐ venting oxidative stress, HIV infection, in the brain, thyroid metabolism, cancer, diabetes mellitus, male fertility, asthma, cardiovascular disorders, rheumatoid arthritis, pre-eclamp‐ sia, in immune function, in alleviate bone impairments, aging, gastrointestinal problems, se‐ lenium interactions and toxicity, anti-inflammatory effects, and hypertension. The list of clinical disorders expected to be influenced by Se deficiency is rapidly growing with time. Some selected issues regarding the role of Se in health and disease have been briefly out‐

Selenocysteine is recognized as the 21st amino acid, and it forms a predominant residue of selenoproteins and selenoenzymes in biological tissues. The molecular structure of seleno‐ cystiene is an analogue of cysteine where a sulphur atom is replaced by Se. Even though Se and sulphur share some similar chemical properties, there are also some differences. The R-SeH with a pKa 5.2 is more is more acidic than R-SH with a pKa 8.5, and readily dissociated at physiological pH, which may contribute to its biological reactivity. In the body, both or‐ ganic [selenocysteine(SeCys) and selenomethionine (SeMet)] and inorganic (selenite, selen‐ ate) Se compounds are readily metabolized to various forms of Se metabolites [41]. Of particular importance during this metabolic process is the formation of hydrogen selenide (H2Se) from selenite after the action of glutathione-coupled reactions *via* selenodiglutathione (GS-Se-SG) and glutathione selenopersulfide (GS-SeH). H2Se is further metabolized and in‐ volved in the formation of methylselenol and dimethylselenide, which are exhaled or secret‐ ed *via* the skin. Selenium is also excreted in urine as trimethylselenonium ion and selenosugar compounds [42]. The selenoproteins are classified on the basis of their biologi‐ cal function [25]. The first identified selenoprotein was glutathione peroxidase 1 (GPx1). The

more rapidly [40].

lined as follows:

**2. Selenium and Health**

**2.1. Se and antioxidant activity**

In [46] selenium's function as an antioxidant, and as a constituent of selenoproteins that are important in redox homeostasis, warrants further investigation as a risk factor for depres‐ sion, and suggest a potentially novel modifiable factor in the primary prevention and man‐ agement of depression. Depression is becoming recognized as an inflammatory disorder, accompanied by an accumulation of highly reactive oxygen species that overwhelm usual defensive physiological processes [47-51]. Several indicators support a role for selenium in normal brain function. During times of selenium deficiency, there is preferential storage of selenium in the brain [52]. Selenium has significant modulatory effects on dopamine [53] and dopamine plays a role in the pathophysiology of depression and other psychiatric ill‐ nesses [54]. Diminished levels of selenium in the brain are associated with cognitive decline [55] and Alzheimer's disease [56]. Selenium supplementation has been linked with improve‐ ments in mood [57] and protection against postpartum depression [58]. What is unclear is if low dietary selenium is a risk factor for the development of depression. In recognition of se‐ lenium's biological activity, it has been hypothesized that low levels of dietary selenium would be associated with an increased risk of major depressive disorder (MDD) in a repre‐ sentative population-based sample of women.

Alterations in redox biology are established in depression; however, there are no prospec‐ tive epidemiological data on redox-active selenium in depression. It is known that seleni‐ um's function as an antioxidant, and as a constituent of selenoproteins that are important in redox homeostasis, warrants further investigation as a risk factor for depression, and sug‐ gest a potentially novel modifiable factor in the primary prevention and management of de‐ pression.

### **2.3. Selenium and allergies**

The International Study of Asthma and Allergies in Childhood (ISAAC) found that one in four New Zealand children aged 6–7 years had experienced asthma symptoms, which placed New Zealand in the top four countries for asthma prevalence [59]. The reasons for the high prevalence and severity of this condition or the increased prevalence of asthma over the last 20 years are not well understood. One of a number of environmental factors that have been proposed as a reason for the escalation in asthma prevalence is a decreasing intake of dietary antioxidants [60]. It is well known that selenium is essential for the optimal functioning of the selenoenzymes glutathione peroxidases (GPx) and thioredoxin reductas‐ es, powerful antioxidants, and is found abundantly in lung tissue and the extracellular fluid of the respiratory system [61]. Selenium has been implicated in inflammation by reducing the severity of the inflammatory response through modulation of the pro-inflammatory leu‐ kotrienes, important mediators of acute asthmatic reactions as well as sustaining the inflam‐ matory process causing a late allergic reaction metabolism [62]. Evidence from randomized controlled trials [63] and basic mechanistic work investigating the effect of selenium on markers of inflammation and oxidative stress [62]. Evidences have supported a protective role for selenium in asthma, although other studies have not [64-66]. The ISAAC study does not support a strong association between selenium status and the high incidence of asthma in New Zealand. However, there was a modest association between lower plasma selenium and whole blood glutathione peroxidase activity and higher incidence of persistent wheeze [67].

the marginalized and socially disadvantaged. It is currently a leading cause of death in many parts of the world, and a disease that disproportionately affects the marginalized and socially disadvantaged. Many of those affected also suffer from chronic food insecurity and malnutrition, so therapies that could potentially target both HIV disease and malnutrition, such as multivitamins, have been extensively researched for potential benefits [76]. Among such therapies, the antioxidant micronutrients theorized to have potential benefits in HIV

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

425

Selenium has an inhibitory effect on HIV in vitro through antioxidant effects of glutathione peroxidase and other selenoproteins. Numerous studies have reported low selenium status in HIV-infected individuals, and serum selenium concentration declines with disease pro‐ gression. Some cohort studies have shown an association between selenium deficiency and progression to AIDS or mortality. In several randomized controlled trials, selenium supple‐ mentation has reduced hospitalizations and diarrheal morbidity, and improved CD4+ cell counts, but the evidence remains mixed. Additional trials are recommended to study the ef‐ fect of selenium supplementation on opportunistic infections, and other HIV disease-related comorbidities in the context of highly active antiretroviral therapy in both developing and

There is a historical record showing that organoselenium compounds can be used as antivi‐

In addition to the well-documented functions of Se as an antioxidant and in the regulation of the thyroid and immune function [80]. Recent advances have indicated a role of Se in the maintenance of brain function [81]. Selenium is widely distributed throughout the body, but is particularly well maintained in the brain, even upon prolonged dietary Se deficiency [82]. In the brain, the highest concentration of Se is found in the gray matter, an area responsible for chemical synaptic communication [83]. It has been shown that rats on a Se-deficient diet for thirteen weeks retained Se in their brain, while their plasma Se concentrations were de‐ pleted [84]. After intraperitoneal injection of 75SeO32- into Se-deficient rats, the brain rapidly sequesters a large portion of the available Se [85]. In the brain, it was found that the cerebel‐ lum accumulated the highest concentration of Se, followed by the cortex, medulla oblonga‐ ta, cerebral hemisphere, and the spinal cord. Interestingly, Se retention in the brain depends on Selenoprotein P expression [86]. Because the body preferentially allocates available Se to the brain during Se deficiency, Se may play an essential role in the brain. More evidence for the brain being at the apex of Se retention is provided by a study showing that a six genera‐ tion Se deficiency in rats caused a more than 99% reduction of Se concentration in the liver, blood, skeletal tissue, and muscle, while the brain retained a 60% of the Se [87]. Se concen‐ tration in Alzheimer's brains was found to be 60% of the age-matched control individuals [88]. Accumulated lines of evidence indicate important roles of selenoproteins in the mainte‐ nance of optimal brain functions via redox regulation. Decreased expression of several sele‐ noproteins is associated with the pathologies of a few age-associated neurodisorders,

ral and antibacterial agents. This topic has been reviewed by [22,23].

including Parkinson's disease, Alzheimer's disease and epilepsy [81].

disease, apart from correcting deficiencies, have been examined frequently [77,78].

developed countries [79].

**2.6. Selenium in the brain**

### **2.4. Selenium in preventing oxidative stress**

The reactivity of organoselenium compounds [22,68] characterized by high nucleophilicity and antioxidant potential, and provides the basis for their pharmacological activities in mammalian models. Organochalcogens have been widely studied given their antioxidant activity, which confers neuroprotection, antiulcer, and antidiabetic properties. Given the complexity of mammalian models, understanding the cellular and molecular effects of orga‐ nochalcogens has been hampered. In reference [69] the nematode worm *Caenorhabditis ele‐ gans* is an alternative experimental model that affords easy genetic manipulations, green fluorescent protein tagging, and in vivo live analysis of toxicity. Manganese (Mn)-exposed worms exhibit oxidative-stress-induced neurodegeneration and life-span reduction. Dieth‐ yl-2-phenyl-2-tellurophenyl vinyl phosphonate (DPTVP) and 2-Phenyl-1,2-benzoisoselena‐ zol-3-(2H)-one (Ebselen) were tested for reversing the Mn-induced reduction in survival and lifespan in this nematode. DPTVP was the most efficacious compound as compared to Ebse‐ len in reversing the Mn-induced toxicity and increasing in survival and life span. DPTVP and ebselen act as antiaging agents in a model of Mn-induced toxicity and aging by regulat‐ ing DAF-16/FOXO signaling and attenuating oxidative stress.

Bone is a specialized connective tissue, which forms the framework of the body. Various physiological conditions can adversely affect femoral bone metabolism. These physiological conditions could be food deprivation [70], and iodine and/or selenium (Se) deficiency [71,72] and antithyroid drugs [73] affects bone maturation. Selenium is an important protective ele‐ ment that may be used as a dietary supplement protecting against oxidative stress, cellular damage and bone impairments [74].

### **2.5. Selenium in HIV infection**

The HIV pandemic has placed a great demand upon the scientific community to develop ef‐ fective prevention and treatment methods. Since the beginning of the pandemic in 1981, over 25 million people are estimated to have died from the disease [75]. It is currently a lead‐ ing cause of death in many parts of the world, and a disease that disproportionately affects the marginalized and socially disadvantaged. It is currently a leading cause of death in many parts of the world, and a disease that disproportionately affects the marginalized and socially disadvantaged. Many of those affected also suffer from chronic food insecurity and malnutrition, so therapies that could potentially target both HIV disease and malnutrition, such as multivitamins, have been extensively researched for potential benefits [76]. Among such therapies, the antioxidant micronutrients theorized to have potential benefits in HIV disease, apart from correcting deficiencies, have been examined frequently [77,78].

Selenium has an inhibitory effect on HIV in vitro through antioxidant effects of glutathione peroxidase and other selenoproteins. Numerous studies have reported low selenium status in HIV-infected individuals, and serum selenium concentration declines with disease pro‐ gression. Some cohort studies have shown an association between selenium deficiency and progression to AIDS or mortality. In several randomized controlled trials, selenium supple‐ mentation has reduced hospitalizations and diarrheal morbidity, and improved CD4+ cell counts, but the evidence remains mixed. Additional trials are recommended to study the ef‐ fect of selenium supplementation on opportunistic infections, and other HIV disease-related comorbidities in the context of highly active antiretroviral therapy in both developing and developed countries [79].

There is a historical record showing that organoselenium compounds can be used as antivi‐ ral and antibacterial agents. This topic has been reviewed by [22,23].

### **2.6. Selenium in the brain**

functioning of the selenoenzymes glutathione peroxidases (GPx) and thioredoxin reductas‐ es, powerful antioxidants, and is found abundantly in lung tissue and the extracellular fluid of the respiratory system [61]. Selenium has been implicated in inflammation by reducing the severity of the inflammatory response through modulation of the pro-inflammatory leu‐ kotrienes, important mediators of acute asthmatic reactions as well as sustaining the inflam‐ matory process causing a late allergic reaction metabolism [62]. Evidence from randomized controlled trials [63] and basic mechanistic work investigating the effect of selenium on markers of inflammation and oxidative stress [62]. Evidences have supported a protective role for selenium in asthma, although other studies have not [64-66]. The ISAAC study does not support a strong association between selenium status and the high incidence of asthma in New Zealand. However, there was a modest association between lower plasma selenium and whole blood glutathione peroxidase activity and higher incidence of persistent wheeze

The reactivity of organoselenium compounds [22,68] characterized by high nucleophilicity and antioxidant potential, and provides the basis for their pharmacological activities in mammalian models. Organochalcogens have been widely studied given their antioxidant activity, which confers neuroprotection, antiulcer, and antidiabetic properties. Given the complexity of mammalian models, understanding the cellular and molecular effects of orga‐ nochalcogens has been hampered. In reference [69] the nematode worm *Caenorhabditis ele‐ gans* is an alternative experimental model that affords easy genetic manipulations, green fluorescent protein tagging, and in vivo live analysis of toxicity. Manganese (Mn)-exposed worms exhibit oxidative-stress-induced neurodegeneration and life-span reduction. Dieth‐ yl-2-phenyl-2-tellurophenyl vinyl phosphonate (DPTVP) and 2-Phenyl-1,2-benzoisoselena‐ zol-3-(2H)-one (Ebselen) were tested for reversing the Mn-induced reduction in survival and lifespan in this nematode. DPTVP was the most efficacious compound as compared to Ebse‐ len in reversing the Mn-induced toxicity and increasing in survival and life span. DPTVP and ebselen act as antiaging agents in a model of Mn-induced toxicity and aging by regulat‐

Bone is a specialized connective tissue, which forms the framework of the body. Various physiological conditions can adversely affect femoral bone metabolism. These physiological conditions could be food deprivation [70], and iodine and/or selenium (Se) deficiency [71,72] and antithyroid drugs [73] affects bone maturation. Selenium is an important protective ele‐ ment that may be used as a dietary supplement protecting against oxidative stress, cellular

The HIV pandemic has placed a great demand upon the scientific community to develop ef‐ fective prevention and treatment methods. Since the beginning of the pandemic in 1981, over 25 million people are estimated to have died from the disease [75]. It is currently a lead‐ ing cause of death in many parts of the world, and a disease that disproportionately affects

[67].

**2.4. Selenium in preventing oxidative stress**

424 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ing DAF-16/FOXO signaling and attenuating oxidative stress.

damage and bone impairments [74].

**2.5. Selenium in HIV infection**

In addition to the well-documented functions of Se as an antioxidant and in the regulation of the thyroid and immune function [80]. Recent advances have indicated a role of Se in the maintenance of brain function [81]. Selenium is widely distributed throughout the body, but is particularly well maintained in the brain, even upon prolonged dietary Se deficiency [82]. In the brain, the highest concentration of Se is found in the gray matter, an area responsible for chemical synaptic communication [83]. It has been shown that rats on a Se-deficient diet for thirteen weeks retained Se in their brain, while their plasma Se concentrations were de‐ pleted [84]. After intraperitoneal injection of 75SeO32- into Se-deficient rats, the brain rapidly sequesters a large portion of the available Se [85]. In the brain, it was found that the cerebel‐ lum accumulated the highest concentration of Se, followed by the cortex, medulla oblonga‐ ta, cerebral hemisphere, and the spinal cord. Interestingly, Se retention in the brain depends on Selenoprotein P expression [86]. Because the body preferentially allocates available Se to the brain during Se deficiency, Se may play an essential role in the brain. More evidence for the brain being at the apex of Se retention is provided by a study showing that a six genera‐ tion Se deficiency in rats caused a more than 99% reduction of Se concentration in the liver, blood, skeletal tissue, and muscle, while the brain retained a 60% of the Se [87]. Se concen‐ tration in Alzheimer's brains was found to be 60% of the age-matched control individuals [88]. Accumulated lines of evidence indicate important roles of selenoproteins in the mainte‐ nance of optimal brain functions via redox regulation. Decreased expression of several sele‐ noproteins is associated with the pathologies of a few age-associated neurodisorders, including Parkinson's disease, Alzheimer's disease and epilepsy [81].

Oxidative stress and generation of reactive oxygen species are strongly implicated in a num‐ ber of neuronal and neuromuscular disorders, including epilepsy. The functions of selenium as an antioxidant trace element are believed to be carried out by selenoproteins that possess antioxidant activities and the ability to promote neuronal cell survival [89]. It is known the role of selenium in a detoxifying enzyme, glutathione peroxidase, this element has been demonstrated to have a positive biological function in various aspects of human health [90]. Oxidative stress and generation of reactive oxygen species are strongly implicated in a num‐ ber of neurologic disorders including seizure disorders. Oxidative phosphorylation occur‐ ring in the mitochondria produces oxygen radicals routinely in all tissues as well as the nervous system. One important defense may be to remove the oxygen radicals. Seleniumrequiring processes are involved in normal maintenance of cell function. However, when the system is overused or chronically activated beyond its normal state, such as recurrent or intractable seizures, abnormal increases in by-products can produce neuronal cell damage. Selenium provides protection from reactive oxygen species–induced cell damage. The pro‐ posed mechanisms are mainly through the functions of seleno-dependent enzymes and sele‐ noproteins [82,91]. It seems that selenium plays an important role in stopping the vicious cycle of oxidative stress and neuronal damage in patients with intractable seizures by restor‐ ing the defense mechanism.

pact of the basal antithyroid peroxidase level, and the effect of disease duration on the treat‐ ment outcome. Clearly, further in-depth studies and evaluation are required concerning the mechanism of action of selenium as well as the choice of supplements or dietary intake.

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

427

The reactive oxygen species (ROS) are derived from cellular oxygen metabolism and from exogenous sources. An excess of ROS results in oxidative stress and may eventually cause cell death. ROS levels within cells and in extracellular body fluids are controlled by concert‐ ed action of enzymatic and non-enzymatic antioxidants. The essential trace element seleni‐ um exerts its antioxidant function mainly in the form of selenocysteine residues as an integral constituent of ROS-detoxifying selenoenzymes such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and possibly selenoprotein P (SeP). In particular, the dual role of selenoprotein P as selenium transporter and antioxidant enzyme is highlighted herein. A cytoprotective effect of selenium supplementation has been demonstrated for vari‐ ous cell types including neurons and astrocytes as well as endothelial cells. Maintenance of full GPx and TrxR activity by adequate dietary selenium supply has been proposed to be useful for the prevention of several cardiovascular and neurological disorders. On the other hand, selenium supplementation at supranutritional levels has been utilized for cancer pre‐ vention: antioxidant selenoenzymes as well as prooxidant effects of selenocompounds on tu‐ mor cells are thought to be involved in the anti-carcinogenic action of selenium [95,96].

Among various antioxidant minerals, selenium it may prove to be of major significance as a prophylactic agent against cancer. Low blood selenium concentration and incidence of carci‐ nogenesis have been well observed in both animals [97] as well as in human studies [98]. In addition, it has been demonstrated in a double blind randomized cancer prevention trial in humans that increased selenium intake has a significant role in the treatment of cancer [99]. A similar prospective study could also be designed for other cancers to determine the che‐ mopreventive effect of Se. Selenium has also been reported to have a beneficial effect on the

Although selenium is reported to play a significant role in cancer development, its exact an‐ ticancer mechanism of action at molecular levels is not fully understood. However, it has been hypothesized that the most possible mechanistic action of Se as chemoprevention is its role in the antioxidant defense systems to reduce oxidative stress and limit DNA damage [24,102]. Experiments carried out within the framework of a canine model using male beagle dogs to mimic prostate cancer in humans showed that the damage to DNA was significantly reduced when the animals were exposed to increased Se dietary supplements [103]. The ef‐ fectiveness of Se in the prevention of DNA damage, however, depends on its chemical forms. In an *in vitro* study [104] found that selenocysteine inhibited DNA damage more strongly than the selenomethionine. Other possible anticancer mechanisms of Se include the induction of apoptosis, cell-cycle arrest and DNA-repair genes, inhibition of protein kinase C activity and cell growth and effect on estrogen- and androgen-receptor expression [102]. In [105] knowledge of the plasma selenium levels are associated with optimized concentra‐ tion or activity of specific selenoproteins can provide considerable insights from epidemio‐

incidence of gastrointestinal and bladder cancers [100,101].

**2.8. Selenium in cancer**

#### **2.7. Selenium and the thyroid**

Some selenoproteins of the human selenoproteome display multiple genes performing simi‐ lar functions. The main selenoprotein families are the glutathione peroxidases (GPxs; seven genes), the thioredoxin reductases (TRxs; three genes) and the iodothyronine deiodinases (DIs; three genes) [92,93]. The GPxs, which possess oxidoreductase functions, protect the cell from oxidative stress. The TRxs form a cellular redox system, existing in many organisms, which is essential for cell development and proliferation. The DIs that catalyzes the conver‐ sion of T4 to T3 provides the sources of T3 production. It may thus be hypothesized that the essential micronutrient selenium, in the form of Se-Cys, modulates redox-sensitive signaling pathways and thereby potentially modifies selenoprotein gene expression. These findings have aroused growing interest of the scientific community in this multifaceted element. In this context, whereas selenium administration for cancer chemoprevention produced ques‐ tionable results, those of selenium supplementation in patients with autoimmune thyroid disease have been more encouraging. In [94] comprises an in-depth discussion of the link between selenium and thyroid function; it provides a critical analysis of the data contained in recent studies, an update and evaluation of current knowledge with regard to the mecha‐ nisms of action of selenium, and reflections on the prospects for selenium supplementation in thyroid pathology.

Evidence in support of selenium supplementation in thyroid autoimmune disease is evalu‐ ated; the results herein presented demonstrating the potential effectiveness of selenium in reducing the antithyroid peroxidase titer and improving the echostructure in the ultrasound examination. However, considerable discord remains as to who should comprise target groups for selenium treatment, who will most benefit from such treatment, the precise im‐ pact of the basal antithyroid peroxidase level, and the effect of disease duration on the treat‐ ment outcome. Clearly, further in-depth studies and evaluation are required concerning the mechanism of action of selenium as well as the choice of supplements or dietary intake.

### **2.8. Selenium in cancer**

Oxidative stress and generation of reactive oxygen species are strongly implicated in a num‐ ber of neuronal and neuromuscular disorders, including epilepsy. The functions of selenium as an antioxidant trace element are believed to be carried out by selenoproteins that possess antioxidant activities and the ability to promote neuronal cell survival [89]. It is known the role of selenium in a detoxifying enzyme, glutathione peroxidase, this element has been demonstrated to have a positive biological function in various aspects of human health [90]. Oxidative stress and generation of reactive oxygen species are strongly implicated in a num‐ ber of neurologic disorders including seizure disorders. Oxidative phosphorylation occur‐ ring in the mitochondria produces oxygen radicals routinely in all tissues as well as the nervous system. One important defense may be to remove the oxygen radicals. Seleniumrequiring processes are involved in normal maintenance of cell function. However, when the system is overused or chronically activated beyond its normal state, such as recurrent or intractable seizures, abnormal increases in by-products can produce neuronal cell damage. Selenium provides protection from reactive oxygen species–induced cell damage. The pro‐ posed mechanisms are mainly through the functions of seleno-dependent enzymes and sele‐ noproteins [82,91]. It seems that selenium plays an important role in stopping the vicious cycle of oxidative stress and neuronal damage in patients with intractable seizures by restor‐

426 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Some selenoproteins of the human selenoproteome display multiple genes performing simi‐ lar functions. The main selenoprotein families are the glutathione peroxidases (GPxs; seven genes), the thioredoxin reductases (TRxs; three genes) and the iodothyronine deiodinases (DIs; three genes) [92,93]. The GPxs, which possess oxidoreductase functions, protect the cell from oxidative stress. The TRxs form a cellular redox system, existing in many organisms, which is essential for cell development and proliferation. The DIs that catalyzes the conver‐ sion of T4 to T3 provides the sources of T3 production. It may thus be hypothesized that the essential micronutrient selenium, in the form of Se-Cys, modulates redox-sensitive signaling pathways and thereby potentially modifies selenoprotein gene expression. These findings have aroused growing interest of the scientific community in this multifaceted element. In this context, whereas selenium administration for cancer chemoprevention produced ques‐ tionable results, those of selenium supplementation in patients with autoimmune thyroid disease have been more encouraging. In [94] comprises an in-depth discussion of the link between selenium and thyroid function; it provides a critical analysis of the data contained in recent studies, an update and evaluation of current knowledge with regard to the mecha‐ nisms of action of selenium, and reflections on the prospects for selenium supplementation

Evidence in support of selenium supplementation in thyroid autoimmune disease is evalu‐ ated; the results herein presented demonstrating the potential effectiveness of selenium in reducing the antithyroid peroxidase titer and improving the echostructure in the ultrasound examination. However, considerable discord remains as to who should comprise target groups for selenium treatment, who will most benefit from such treatment, the precise im‐

ing the defense mechanism.

in thyroid pathology.

**2.7. Selenium and the thyroid**

The reactive oxygen species (ROS) are derived from cellular oxygen metabolism and from exogenous sources. An excess of ROS results in oxidative stress and may eventually cause cell death. ROS levels within cells and in extracellular body fluids are controlled by concert‐ ed action of enzymatic and non-enzymatic antioxidants. The essential trace element seleni‐ um exerts its antioxidant function mainly in the form of selenocysteine residues as an integral constituent of ROS-detoxifying selenoenzymes such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and possibly selenoprotein P (SeP). In particular, the dual role of selenoprotein P as selenium transporter and antioxidant enzyme is highlighted herein. A cytoprotective effect of selenium supplementation has been demonstrated for vari‐ ous cell types including neurons and astrocytes as well as endothelial cells. Maintenance of full GPx and TrxR activity by adequate dietary selenium supply has been proposed to be useful for the prevention of several cardiovascular and neurological disorders. On the other hand, selenium supplementation at supranutritional levels has been utilized for cancer pre‐ vention: antioxidant selenoenzymes as well as prooxidant effects of selenocompounds on tu‐ mor cells are thought to be involved in the anti-carcinogenic action of selenium [95,96].

Among various antioxidant minerals, selenium it may prove to be of major significance as a prophylactic agent against cancer. Low blood selenium concentration and incidence of carci‐ nogenesis have been well observed in both animals [97] as well as in human studies [98]. In addition, it has been demonstrated in a double blind randomized cancer prevention trial in humans that increased selenium intake has a significant role in the treatment of cancer [99]. A similar prospective study could also be designed for other cancers to determine the che‐ mopreventive effect of Se. Selenium has also been reported to have a beneficial effect on the incidence of gastrointestinal and bladder cancers [100,101].

Although selenium is reported to play a significant role in cancer development, its exact an‐ ticancer mechanism of action at molecular levels is not fully understood. However, it has been hypothesized that the most possible mechanistic action of Se as chemoprevention is its role in the antioxidant defense systems to reduce oxidative stress and limit DNA damage [24,102]. Experiments carried out within the framework of a canine model using male beagle dogs to mimic prostate cancer in humans showed that the damage to DNA was significantly reduced when the animals were exposed to increased Se dietary supplements [103]. The ef‐ fectiveness of Se in the prevention of DNA damage, however, depends on its chemical forms. In an *in vitro* study [104] found that selenocysteine inhibited DNA damage more strongly than the selenomethionine. Other possible anticancer mechanisms of Se include the induction of apoptosis, cell-cycle arrest and DNA-repair genes, inhibition of protein kinase C activity and cell growth and effect on estrogen- and androgen-receptor expression [102].

In [105] knowledge of the plasma selenium levels are associated with optimized concentra‐ tion or activity of specific selenoproteins can provide considerable insights from epidemio‐ logical data on the possible involvement of those selenoproteins in health, most notably with respect to cancer. For cohort studies, if selenoproteins such as glutathione peroxidase and selenoprotein P are relevant to cancer, one might only expect to see an effect on risk when the concentrations in the cohort range from below, to above, the level needed to opti‐ mize the activity or concentration of these enzymes. Similarly, trials would only show a ben‐ eficial effect of supplementation if selenium status were raised from below, to above, the optimal concentration for the selenoproteins likely to be implicated in cancer risk, as occur‐ red in the Nutritional Prevention of Cancer (NPC) trial but not in Selenium and Vitamin E Cancer Prevention Trial (SELECT). The most powerful evidence for the involvement of sele‐ noproteins in human health comes from epidemiological studies that have related single nu‐ cleotide polymorphisms in selenoproteins to disease risk. The totality of the evidence currently implicates GPx1, GPx4, SEPS1, Sep15, SEPP1 and TXNRD1 in conditions such as cardiovascular disease, pre-eclampsia and cancer. Future studies therefore need to deter‐ mine not only selenium status, but genotype, both in selenoproteins and related pathways, when investigating the relationship of selenium with disease risk.

present in spermatids and forms the structural part in the mid piece of mature spermatozoa. Some well known effects of selenium deficiency include instability of the middle piece lead‐ ing to defective sperm motility [112], low reproductive ability and abnormal development of spermatozoa [113]. Selenium is also required for testosterone synthesis and sequential de‐ velopment of flagella [114]. It can restore the physiological constitution of polyunsaturated fatty acid in the cell membrane [115]. Testes are extremely resistant to Se depletion and have high Se content. Recent studies have shown that sperm and testicular Se was unaffected by the supplementation, suggesting that testes are protected from Se excess as well as from Se

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

429

Se status is decreased in patients with asthma, as is activity of glutathione peroxidase in pla‐ telets and erythrocytes. There is an associated marked oxidant/antioxidant imbalance in the blood of asthmatics, which reflects poor antioxidant status and enhanced inflammatory mediated oxidative stress [117]. According to the University of Maryland Medical Center, a 2004 study of 24 asthmatics that were given selenium supplements for 14 weeks had signifi‐ cant improvement in their symptoms when compared to a control group given a placebo. Although this is a small study done over a short amount of time, it's encouraging [118].

Free radicals are toxic to the myocardium and can cause tissue damage that leads to exten‐ sive necrosis, myocytolysis and cellular edema [119]. Atherosclerotic plaque formation may be a reflection of sub-optimal GPx4 activity in the prevention of LDL oxidation, with subse‐ quent uptake by endothelial cells and macrophages in arterial blood vessels [120]. Selenium via GPx reduces phospholipids, hydro peroxides and cholestryl esters associated with lipo‐ proteins and may therefore, not only reduce the accumulation of oxidized LDL in arterial wall but also reduce platelet aggregation and activation of monocyte and macrophages [121]. Selenium owing to its antithrombotic effect on the interaction between platelets and endothelial cells via GPx, also provides concrete evidence in the prevention of atherosclero‐

The study on acute myocardial infarction (AMI) patients, it was observed that selenium de‐ pendent GPx level decreases significantly in AMI patients and explained it as an imperative consequent of GPx activity in annihilating oxygen toxicity by metabolizing H2O2 and inhibit‐ ing further free oxygen radical production in early phase of myocardial infarction [123].

Scientific research shows that people with rheumatoid arthritis have low levels of selenium. A study suggests, it is part of the body's defense mechanism [124]. In reference [125] the au‐ thors found lower selenium levels in patients with rheumatoid arthritis who were treated with arthritis medication compared with people without the condition. In people without

deficiency [116].

sis [122].

**2.11. Selenium in asthma**

**2.12. Selenium in cardiovascular disorders**

**2.13. Selenium in rheumatoid arthritis**

### **2.9. Selenium in diabetes**

The evidence supporting an effect of selenium on the risk of diabetes is variable, occasional‐ ly conflicting, and limited to very few human studies. Following a trial investigating the ef‐ fect of selenium supplementation (200 µg/day) on skin cancer, subsequent analysis showed that there was an increased risk of developing type 2 diabetes in the supplemented group. Evidence from analysis of NHANES III [106] supports these findings; the adjusted mean se‐ rum selenium concentrations were slightly, but significantly, higher in diabetics compared with those without the disease. This study, conducted in an elderly French population, found a sex-specific protective effect of higher selenium status at baseline on later occur‐ rence of dysglycemia; that is, risk of dysglycemia was significantly lower in men with plas‐ ma selenium, but no significant relationship was observed in women [107].

The role of selenium as an antioxidant, particularly within the GPxs, selenium is likely to be important in reducing oxidative stress, an important risk factor for developing diabetes. There are also plausible suggestions that selenium can influence glucose metabolism. How‐ ever, at high intakes it is also conceivable that reactive oxygen species could be generated or selenium may accumulate in the organs associated with glucose metabolism [108]. In pa‐ tients with diabetes, selenium supplementation (960 µg/day) reduced NF-κB levels to those comparable with nondiabetic controls [109]. In addition, further analysis of the Nutritional Prevention of Cancer trial data has shown an increased risk of self-reported Type-2 diabetes in those supplemented with Se, though the effect was significant only in those in the top ter‐ tile of plasma Se at baseline [110].

#### **2.10. Selenium and male fertility**

Selenoprotein P transports selenium particularly to testis and brain [111]. Among the five enzymes of GPx, GPx1 prevents apoptosis induced by oxidative stress and GPx4 acts direct‐ ly on membrane phospholipid hydroperoxides and detoxifies them. Selenium as GPx, is present in spermatids and forms the structural part in the mid piece of mature spermatozoa. Some well known effects of selenium deficiency include instability of the middle piece lead‐ ing to defective sperm motility [112], low reproductive ability and abnormal development of spermatozoa [113]. Selenium is also required for testosterone synthesis and sequential de‐ velopment of flagella [114]. It can restore the physiological constitution of polyunsaturated fatty acid in the cell membrane [115]. Testes are extremely resistant to Se depletion and have high Se content. Recent studies have shown that sperm and testicular Se was unaffected by the supplementation, suggesting that testes are protected from Se excess as well as from Se deficiency [116].

### **2.11. Selenium in asthma**

logical data on the possible involvement of those selenoproteins in health, most notably with respect to cancer. For cohort studies, if selenoproteins such as glutathione peroxidase and selenoprotein P are relevant to cancer, one might only expect to see an effect on risk when the concentrations in the cohort range from below, to above, the level needed to opti‐ mize the activity or concentration of these enzymes. Similarly, trials would only show a ben‐ eficial effect of supplementation if selenium status were raised from below, to above, the optimal concentration for the selenoproteins likely to be implicated in cancer risk, as occur‐ red in the Nutritional Prevention of Cancer (NPC) trial but not in Selenium and Vitamin E Cancer Prevention Trial (SELECT). The most powerful evidence for the involvement of sele‐ noproteins in human health comes from epidemiological studies that have related single nu‐ cleotide polymorphisms in selenoproteins to disease risk. The totality of the evidence currently implicates GPx1, GPx4, SEPS1, Sep15, SEPP1 and TXNRD1 in conditions such as cardiovascular disease, pre-eclampsia and cancer. Future studies therefore need to deter‐ mine not only selenium status, but genotype, both in selenoproteins and related pathways,

The evidence supporting an effect of selenium on the risk of diabetes is variable, occasional‐ ly conflicting, and limited to very few human studies. Following a trial investigating the ef‐ fect of selenium supplementation (200 µg/day) on skin cancer, subsequent analysis showed that there was an increased risk of developing type 2 diabetes in the supplemented group. Evidence from analysis of NHANES III [106] supports these findings; the adjusted mean se‐ rum selenium concentrations were slightly, but significantly, higher in diabetics compared with those without the disease. This study, conducted in an elderly French population, found a sex-specific protective effect of higher selenium status at baseline on later occur‐ rence of dysglycemia; that is, risk of dysglycemia was significantly lower in men with plas‐

The role of selenium as an antioxidant, particularly within the GPxs, selenium is likely to be important in reducing oxidative stress, an important risk factor for developing diabetes. There are also plausible suggestions that selenium can influence glucose metabolism. How‐ ever, at high intakes it is also conceivable that reactive oxygen species could be generated or selenium may accumulate in the organs associated with glucose metabolism [108]. In pa‐ tients with diabetes, selenium supplementation (960 µg/day) reduced NF-κB levels to those comparable with nondiabetic controls [109]. In addition, further analysis of the Nutritional Prevention of Cancer trial data has shown an increased risk of self-reported Type-2 diabetes in those supplemented with Se, though the effect was significant only in those in the top ter‐

Selenoprotein P transports selenium particularly to testis and brain [111]. Among the five enzymes of GPx, GPx1 prevents apoptosis induced by oxidative stress and GPx4 acts direct‐ ly on membrane phospholipid hydroperoxides and detoxifies them. Selenium as GPx, is

when investigating the relationship of selenium with disease risk.

428 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

ma selenium, but no significant relationship was observed in women [107].

**2.9. Selenium in diabetes**

tile of plasma Se at baseline [110].

**2.10. Selenium and male fertility**

Se status is decreased in patients with asthma, as is activity of glutathione peroxidase in pla‐ telets and erythrocytes. There is an associated marked oxidant/antioxidant imbalance in the blood of asthmatics, which reflects poor antioxidant status and enhanced inflammatory mediated oxidative stress [117]. According to the University of Maryland Medical Center, a 2004 study of 24 asthmatics that were given selenium supplements for 14 weeks had signifi‐ cant improvement in their symptoms when compared to a control group given a placebo. Although this is a small study done over a short amount of time, it's encouraging [118].

### **2.12. Selenium in cardiovascular disorders**

Free radicals are toxic to the myocardium and can cause tissue damage that leads to exten‐ sive necrosis, myocytolysis and cellular edema [119]. Atherosclerotic plaque formation may be a reflection of sub-optimal GPx4 activity in the prevention of LDL oxidation, with subse‐ quent uptake by endothelial cells and macrophages in arterial blood vessels [120]. Selenium via GPx reduces phospholipids, hydro peroxides and cholestryl esters associated with lipo‐ proteins and may therefore, not only reduce the accumulation of oxidized LDL in arterial wall but also reduce platelet aggregation and activation of monocyte and macrophages [121]. Selenium owing to its antithrombotic effect on the interaction between platelets and endothelial cells via GPx, also provides concrete evidence in the prevention of atherosclero‐ sis [122].

The study on acute myocardial infarction (AMI) patients, it was observed that selenium de‐ pendent GPx level decreases significantly in AMI patients and explained it as an imperative consequent of GPx activity in annihilating oxygen toxicity by metabolizing H2O2 and inhibit‐ ing further free oxygen radical production in early phase of myocardial infarction [123].

### **2.13. Selenium in rheumatoid arthritis**

Scientific research shows that people with rheumatoid arthritis have low levels of selenium. A study suggests, it is part of the body's defense mechanism [124]. In reference [125] the au‐ thors found lower selenium levels in patients with rheumatoid arthritis who were treated with arthritis medication compared with people without the condition. In people without rheumatoid arthritis or a family history of the condition, low levels of the mineral may in‐ crease the risk of developing rheumatoid arthritis [126].

well as antioxidant enzymes play a significant role in fracture healing. However, to the best of our knowledge, there are no reports about the use of vitamins A, C, E, and selenium as antioxidant therapy to explore their effects in the levels of bone-healing markers and oxida‐ tive stress parameters of osteoporotic patients. In [139] suggests that selenium is an impor‐ tant protective element that may be used as a dietary supplement protecting against bone

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

431

All forms of vitamin E meet the chemical definition of an antioxidant moiety: ''chain-break‐ ing free radical scavenger.'' Indeed consistent data have shown that all isoforms act as po‐ tent antioxidants in conventional in vitro paradigms. The free hydroxyl group on the aromatic ring is thought to be responsible for this property, and a relatively stable form of the original vitamin E is formed when hydrogen from this group is donated to a free radical. Yet, definitive proof that vitamin E possesses antioxidant properties has been hampered for a long time because of a lack of sensitive and specific analytical techniques to measure this biologic event *in vivo* [36]. Apart from antioxidant properties, more recent studies have clearly demonstrated that vitamin E also possesses important non-antioxidant cellular and molecular functions. One of the first roles of α-tocopherol in cell signaling was the report that it inhibits smooth muscle cell proliferation, decrease protein kinase C activity, and con‐

One of the major vitamin E-deficiency symptoms are neurological disorders. Furthermore, vitamin E deficiency is related to female infertility. The frame to pinpoint the physiological action of vitamin E is set by its chemical nature: (i) It is a redox-active compound prone to undergo 1- and 2-electron transitions and (ii) it is highly lipophilic, although this property may be modulated by phosphorylation [141]. In [142] oxidative stress is a developing re‐ search field and is being examined in female infertility. Prooxidants, also called free radicals or reactive oxygen species (ROS), and their neutralizing agents the antioxidants are the main chemicals of the oxidation mechanism. The term oxidative stress refers to the dysequilibri‐ um between the free radicals and the antioxidants in favor of the free radicals. In actuality, free radicals are not so frightening, since they are necessary for the adequate reproductive functions within the ovary and the endometrium. Vit E administration may improve the en‐ dometrial response in unexplained infertile women *via* the likely antioxidant and the antico‐ agulant effects. It may also modulate the antiestrogenic effect of clomiphene citrate and the

Some non-antioxidant properties of vitamin E could play a key role in neuroprotection. It has been recently shown that α-tocotrienol, at nanomolar concentrations, protects mouse hippocampal and cortical neurons from cell death by modulating neurodegenerative signal‐ ing cascades. Furthermore, it has been shown that α-tocotrienol modulates 12-lipoxygenase and phospholipase A2 activities, which are implicated in glutamate-induced neuronal cell

impairments.

**3.1. Vitamin E**

**3. Vitamin E and Health**

trols expression of the α-tropomyosin gene [140].

problem of a thin endometrium in these cycles may be adjusted.

### **2.14. Selenium in pre-eclampsia**

In reference [124], pre-eclampsia (pregnancy induced hypertension; PIH), is an important cause of maternal morbidity and mortality with essentially unknown etiology. However, the precise factors involved in the pathogenesis of PIH are still unknown [127]. It has been con‐ ceived that free radical mediated oxidative stress may contribute to the development of preeclampsia. Selenium and its related enzymes especially GPx play a crucial role in annihilating oxygen toxicity and there by controlling the progression of disease [128]. In ad‐ dition, selenium deficiency in women may result in infertility, miscarriages and retention of the placenta [129].

### **2.15. Selenium in immunity**

In reference [130], the generation of ROS in a limited dose is one of the processes induced by the immune system to destroy microbial pathogens and viruses. However, the over-produc‐ tion of ROS can also cause damage to the host cells that need to be protected by Se at various stages in the immune system. Keshan disease, an endemic cardiomyopathy in China that de‐ velops as a result of Se deficiency, may also be complicated with viral infection, and this has led to the investigation of the effects of viruses, such as coxsackievirus, on Se-deficient ani‐ mals [131,132]. Results from animal studies have demonstrated that Se deficiency can lead to an impairment of immune functions that result in the inability of phagocytic neutrophils and macrophages to destroy antigens. A low Se status in humans has been reported to cause a decreased immune response to poliovirus vaccination [133]. This study also demonstrated that the subjects supplemented with Se showed fewer mutations in poliovirus than those who received a placebo. The involvement of Se in the immune system may be associated with a number of mechanisms, including the increased activity of natural killer (NK) cells, the proliferation of T-lymphocytes, increased production of interferon c, increased high-af‐ finity interleukin-2 receptors, stimulation of vaccine-induced immunity and increased anti‐ body-producing B-cell numbers [134,135].

#### **2.16. Selenium in bone impairments**

Osteoblasts (bone-forming cells) and osteoclasts (bone-resorption cells) are involved in bone remodeling. Therefore, any loss of osteoblastic activity or an increase in osteoclastic activity could lead to a decrease in bone-mineral densities (BMD), bone mass, and make the bones more likely to osteoporosis, and ultimately to fractures [136]. In addition, high levels of reac‐ tive oxygen species (ROS) and many other factors such as genetic race, hormonal, mechani‐ cal, and nutritional statues are involved in bone weakness and fractures. ROS shift cells into a state of oxidative stress [137] which contributes to the etiology of various degenerative dis‐ eases that cause tissue injury [136,137]. Studies have demonstrated that the ischemia-reper‐ fusion processes that occur after a fracture are associated with oxidative stress development [136,138]. It is believed that bone markers such as osteocalcin and alkaline phosphatase as well as antioxidant enzymes play a significant role in fracture healing. However, to the best of our knowledge, there are no reports about the use of vitamins A, C, E, and selenium as antioxidant therapy to explore their effects in the levels of bone-healing markers and oxida‐ tive stress parameters of osteoporotic patients. In [139] suggests that selenium is an impor‐ tant protective element that may be used as a dietary supplement protecting against bone impairments.

### **3. Vitamin E and Health**

### **3.1. Vitamin E**

rheumatoid arthritis or a family history of the condition, low levels of the mineral may in‐

In reference [124], pre-eclampsia (pregnancy induced hypertension; PIH), is an important cause of maternal morbidity and mortality with essentially unknown etiology. However, the precise factors involved in the pathogenesis of PIH are still unknown [127]. It has been con‐ ceived that free radical mediated oxidative stress may contribute to the development of preeclampsia. Selenium and its related enzymes especially GPx play a crucial role in annihilating oxygen toxicity and there by controlling the progression of disease [128]. In ad‐ dition, selenium deficiency in women may result in infertility, miscarriages and retention of

In reference [130], the generation of ROS in a limited dose is one of the processes induced by the immune system to destroy microbial pathogens and viruses. However, the over-produc‐ tion of ROS can also cause damage to the host cells that need to be protected by Se at various stages in the immune system. Keshan disease, an endemic cardiomyopathy in China that de‐ velops as a result of Se deficiency, may also be complicated with viral infection, and this has led to the investigation of the effects of viruses, such as coxsackievirus, on Se-deficient ani‐ mals [131,132]. Results from animal studies have demonstrated that Se deficiency can lead to an impairment of immune functions that result in the inability of phagocytic neutrophils and macrophages to destroy antigens. A low Se status in humans has been reported to cause a decreased immune response to poliovirus vaccination [133]. This study also demonstrated that the subjects supplemented with Se showed fewer mutations in poliovirus than those who received a placebo. The involvement of Se in the immune system may be associated with a number of mechanisms, including the increased activity of natural killer (NK) cells, the proliferation of T-lymphocytes, increased production of interferon c, increased high-af‐ finity interleukin-2 receptors, stimulation of vaccine-induced immunity and increased anti‐

Osteoblasts (bone-forming cells) and osteoclasts (bone-resorption cells) are involved in bone remodeling. Therefore, any loss of osteoblastic activity or an increase in osteoclastic activity could lead to a decrease in bone-mineral densities (BMD), bone mass, and make the bones more likely to osteoporosis, and ultimately to fractures [136]. In addition, high levels of reac‐ tive oxygen species (ROS) and many other factors such as genetic race, hormonal, mechani‐ cal, and nutritional statues are involved in bone weakness and fractures. ROS shift cells into a state of oxidative stress [137] which contributes to the etiology of various degenerative dis‐ eases that cause tissue injury [136,137]. Studies have demonstrated that the ischemia-reper‐ fusion processes that occur after a fracture are associated with oxidative stress development [136,138]. It is believed that bone markers such as osteocalcin and alkaline phosphatase as

crease the risk of developing rheumatoid arthritis [126].

430 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**2.14. Selenium in pre-eclampsia**

the placenta [129].

**2.15. Selenium in immunity**

body-producing B-cell numbers [134,135].

**2.16. Selenium in bone impairments**

All forms of vitamin E meet the chemical definition of an antioxidant moiety: ''chain-break‐ ing free radical scavenger.'' Indeed consistent data have shown that all isoforms act as po‐ tent antioxidants in conventional in vitro paradigms. The free hydroxyl group on the aromatic ring is thought to be responsible for this property, and a relatively stable form of the original vitamin E is formed when hydrogen from this group is donated to a free radical. Yet, definitive proof that vitamin E possesses antioxidant properties has been hampered for a long time because of a lack of sensitive and specific analytical techniques to measure this biologic event *in vivo* [36]. Apart from antioxidant properties, more recent studies have clearly demonstrated that vitamin E also possesses important non-antioxidant cellular and molecular functions. One of the first roles of α-tocopherol in cell signaling was the report that it inhibits smooth muscle cell proliferation, decrease protein kinase C activity, and con‐ trols expression of the α-tropomyosin gene [140].

One of the major vitamin E-deficiency symptoms are neurological disorders. Furthermore, vitamin E deficiency is related to female infertility. The frame to pinpoint the physiological action of vitamin E is set by its chemical nature: (i) It is a redox-active compound prone to undergo 1- and 2-electron transitions and (ii) it is highly lipophilic, although this property may be modulated by phosphorylation [141]. In [142] oxidative stress is a developing re‐ search field and is being examined in female infertility. Prooxidants, also called free radicals or reactive oxygen species (ROS), and their neutralizing agents the antioxidants are the main chemicals of the oxidation mechanism. The term oxidative stress refers to the dysequilibri‐ um between the free radicals and the antioxidants in favor of the free radicals. In actuality, free radicals are not so frightening, since they are necessary for the adequate reproductive functions within the ovary and the endometrium. Vit E administration may improve the en‐ dometrial response in unexplained infertile women *via* the likely antioxidant and the antico‐ agulant effects. It may also modulate the antiestrogenic effect of clomiphene citrate and the problem of a thin endometrium in these cycles may be adjusted.

Some non-antioxidant properties of vitamin E could play a key role in neuroprotection. It has been recently shown that α-tocotrienol, at nanomolar concentrations, protects mouse hippocampal and cortical neurons from cell death by modulating neurodegenerative signal‐ ing cascades. Furthermore, it has been shown that α-tocotrienol modulates 12-lipoxygenase and phospholipase A2 activities, which are implicated in glutamate-induced neuronal cell death [143]. Some vitamin E forms (α- and γ-tocopherol, tocotrienols) also exhibit potent an‐ ti-inflammatory properties [144,145]. The introduction of the free radical theory of brain ag‐ ing has propelled a renewed interest in this vitamin. As result, by preventing and/or minimizing the oxidative stress dependent brain damage, this vitamin plays important role in brain aging, cognition, and Alzheimer's dementia.

In [166] blader cancer represents an important cause of morbidity and mortality. In 2010 it was again the second most common genitourinary cancer in the United States with an estab‐ lished 70,530 new cases and 14,680 deaths [167]. Currently it is estimated that more than 500,000 men and women in the United States have a history of bladder cancer. The etiology of most bladder urothelial carcinoma is associated with tobacco exposure, occupational ex‐ posure to aromatic amines, and exposure to the chemical and rubber industries [168]. Blad‐ der cancer is the most expensive cancer in the United States, accounting for almost \$3.7 billion (2001 value) in direct costs [169]. There is substantial epidemiological and biological evidence that selenium and vitamin E may prevent bladder cancer. A recent meta-analysis of 7 published epidemiological studies, including 3 case-control, 3 nested case-control and 1 case cohort series, examined the association between selenium levels and bladder cancer [170]. In the analysis stratified by gender only women showed a significantly decreased risk associated with selenium. An opposite gender pattern, with protective effects in men but not in women, was reported in a meta-analysis of selenium supplementation, primary cancer in‐ cidence and mortality [171]. Epidemiological and biological evidence suggests a preventive effect of selenium and vitamin E on bladder cancer. These researches assessed the effect of

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

433

Selenium and vitamin E are essential components of the human diet and have been studied as antioxidants and/or potential agents for a variety of human diseases. Various formula‐ tions of both selenium and vitamin E have been shown to possess a therapeutic and preven‐ tive effect against prostate cancer. The Selenium and Vitamin E Cancer prevention Trial (SELECT) started in 2001 and was a phase III, randomized placebo/controlled human trial to investigate the prostate cancer chemopreventive effects of selenium and vitamin E or their

Sselenium an essential trace element, and vitamin E, a lipid soluble antioxidant, are impor‐ tant mediators for protection against oxidative stress. Deficiencies in either Se or vitamin E result in increased viral pathogenicity and altered immune responses. Furthermore, defi‐ ciencies in either Se or vitamin E results in specific viral mutations, changing relatively be‐ nign viruses into virulent ones. Thus, host nutritional status should be considered a driving force for the emergence of new viral strains or newly pathogenic strains of known viruses

Several studies have evaluated the possible association between antioxidants vitamins or se‐ lenium supplement and the risk of prostate cancer, but the evidence is still inconsistent. We systematically searched PubMed, EMBASE, the Cochrane Library, Science Citation Index Expanded, Chinese biomedicine literature database, and bibliographies of retrieved articles up to January 2009. We included 9 randomized controlled trials with 165,056 participants; methodological quality of included trials was generally high. Meta-analysis showed that no significant effects of supplementation with β-carotene (3 trials), vitamin C (2 trials), vitamin E (5 trials), and selenium (2 trials)versus placebo on prostate cancer incidence. The mortality of prostate cancer did not differ significantly by supplement of β-carotene (1 trial), vitamin

selenium and/or vitamin E on bladder cancer development.

**3.2. Selenium and vitamin E**

combination [172,173].

[174].

Vitamin E is a potent peroxyl radical scavenger that prevents lipid peroxidation [146] and is found in high concentrations in immune cells [147]. Deficiency in vitamin E is associated with increased oxidative stress [148] and impaired immune function, including both humor‐ al and cell-mediated immunity, phagocyte function, and lymphocyte proliferation [149]. Age-related declines in immune function can be restored by vitamin E supplementation [150]. This vitamin is an exogenous, lipidsoluble antioxidant molecule. It is thought to be a direct free radical scavenger by activating the intracellular antioxidant enzymes and saving the cell membranes from lipid peroxidation, which was demonstrated on sperm membrane components [151]. Its antioxidant effect was concluded in cancer therapy, high-risk pregnan‐ cy and male infertility [152-154].

Vitamin E (α−tocopherol acetate) is found within the phospholipid bilayer of cell mem‐ branes where it functions as an electron donor to free radicals. It has been recognized as one of the body's major natural antioxidants. Another antioxidant, Se appears to function as an antimutagenic agent, preventing the malignant transformation of normal cells. Its protective effects seem to be primarily associated with its presence in the seleno-enzymes which are known to protect DNA and other cellular components from oxidative damage [44].

Selenium, vitamin A (retinol) and vitamin E (α-tocopherol) are essential micronutrients for human health. Both selenium and vitamin E are important in host antioxidant defense and immune function. It has been reported that deficiency of selenium and vitamins may pro‐ mote peroxidation events leading to the release of free radicals. All have free-radical-scav‐ enging properties that allow them to function as physiologic antioxidants in protecting a number of chronic diseases, such as cancer and cardiovascular disease. In addition to its an‐ tioxidant capacity, α-tocopherol regulates expression of genes involved in a wide range of cell functions, including cell cycle regulation, inflammation and cell adhesion, cell signaling, and lipid uptake [155].

Selenium also has an important role in antioxidant defense and immune function. Due to its incorporation as selenocysteine into glutathione peroxidase (GPX) [156] and thioredoxin re‐ ductase [157], selenium is important for the control of oxidative stress and, therefore, the re‐ dox tone of the cell. In total, there are 25 identified selenoproteins (24 in rodents), many with unknown function [25]. Selenium is important for cytotoxic T-lymphocyte and natural killer cell activity [158], respiratory burst [159], and protection against endotoxin-induced oxida‐ tive stress [160]. Multiple studies have shown that NF-kB activation can be affected by sele‐ nium status [161,162], and selenium deficiency can alter chemokine and cytokine expression during viral infections [163]. Various investigators have reported the role of selenium as an inhibitor of carcinogenesis in various organs including liver, skin, stomach, mammary gland, gastrointestinal and oral cavity [164,165].

In [166] blader cancer represents an important cause of morbidity and mortality. In 2010 it was again the second most common genitourinary cancer in the United States with an estab‐ lished 70,530 new cases and 14,680 deaths [167]. Currently it is estimated that more than 500,000 men and women in the United States have a history of bladder cancer. The etiology of most bladder urothelial carcinoma is associated with tobacco exposure, occupational ex‐ posure to aromatic amines, and exposure to the chemical and rubber industries [168]. Blad‐ der cancer is the most expensive cancer in the United States, accounting for almost \$3.7 billion (2001 value) in direct costs [169]. There is substantial epidemiological and biological evidence that selenium and vitamin E may prevent bladder cancer. A recent meta-analysis of 7 published epidemiological studies, including 3 case-control, 3 nested case-control and 1 case cohort series, examined the association between selenium levels and bladder cancer [170]. In the analysis stratified by gender only women showed a significantly decreased risk associated with selenium. An opposite gender pattern, with protective effects in men but not in women, was reported in a meta-analysis of selenium supplementation, primary cancer in‐ cidence and mortality [171]. Epidemiological and biological evidence suggests a preventive effect of selenium and vitamin E on bladder cancer. These researches assessed the effect of selenium and/or vitamin E on bladder cancer development.

### **3.2. Selenium and vitamin E**

death [143]. Some vitamin E forms (α- and γ-tocopherol, tocotrienols) also exhibit potent an‐ ti-inflammatory properties [144,145]. The introduction of the free radical theory of brain ag‐ ing has propelled a renewed interest in this vitamin. As result, by preventing and/or minimizing the oxidative stress dependent brain damage, this vitamin plays important role

Vitamin E is a potent peroxyl radical scavenger that prevents lipid peroxidation [146] and is found in high concentrations in immune cells [147]. Deficiency in vitamin E is associated with increased oxidative stress [148] and impaired immune function, including both humor‐ al and cell-mediated immunity, phagocyte function, and lymphocyte proliferation [149]. Age-related declines in immune function can be restored by vitamin E supplementation [150]. This vitamin is an exogenous, lipidsoluble antioxidant molecule. It is thought to be a direct free radical scavenger by activating the intracellular antioxidant enzymes and saving the cell membranes from lipid peroxidation, which was demonstrated on sperm membrane components [151]. Its antioxidant effect was concluded in cancer therapy, high-risk pregnan‐

Vitamin E (α−tocopherol acetate) is found within the phospholipid bilayer of cell mem‐ branes where it functions as an electron donor to free radicals. It has been recognized as one of the body's major natural antioxidants. Another antioxidant, Se appears to function as an antimutagenic agent, preventing the malignant transformation of normal cells. Its protective effects seem to be primarily associated with its presence in the seleno-enzymes which are

Selenium, vitamin A (retinol) and vitamin E (α-tocopherol) are essential micronutrients for human health. Both selenium and vitamin E are important in host antioxidant defense and immune function. It has been reported that deficiency of selenium and vitamins may pro‐ mote peroxidation events leading to the release of free radicals. All have free-radical-scav‐ enging properties that allow them to function as physiologic antioxidants in protecting a number of chronic diseases, such as cancer and cardiovascular disease. In addition to its an‐ tioxidant capacity, α-tocopherol regulates expression of genes involved in a wide range of cell functions, including cell cycle regulation, inflammation and cell adhesion, cell signaling,

Selenium also has an important role in antioxidant defense and immune function. Due to its incorporation as selenocysteine into glutathione peroxidase (GPX) [156] and thioredoxin re‐ ductase [157], selenium is important for the control of oxidative stress and, therefore, the re‐ dox tone of the cell. In total, there are 25 identified selenoproteins (24 in rodents), many with unknown function [25]. Selenium is important for cytotoxic T-lymphocyte and natural killer cell activity [158], respiratory burst [159], and protection against endotoxin-induced oxida‐ tive stress [160]. Multiple studies have shown that NF-kB activation can be affected by sele‐ nium status [161,162], and selenium deficiency can alter chemokine and cytokine expression during viral infections [163]. Various investigators have reported the role of selenium as an inhibitor of carcinogenesis in various organs including liver, skin, stomach, mammary

known to protect DNA and other cellular components from oxidative damage [44].

in brain aging, cognition, and Alzheimer's dementia.

432 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cy and male infertility [152-154].

and lipid uptake [155].

gland, gastrointestinal and oral cavity [164,165].

Selenium and vitamin E are essential components of the human diet and have been studied as antioxidants and/or potential agents for a variety of human diseases. Various formula‐ tions of both selenium and vitamin E have been shown to possess a therapeutic and preven‐ tive effect against prostate cancer. The Selenium and Vitamin E Cancer prevention Trial (SELECT) started in 2001 and was a phase III, randomized placebo/controlled human trial to investigate the prostate cancer chemopreventive effects of selenium and vitamin E or their combination [172,173].

Sselenium an essential trace element, and vitamin E, a lipid soluble antioxidant, are impor‐ tant mediators for protection against oxidative stress. Deficiencies in either Se or vitamin E result in increased viral pathogenicity and altered immune responses. Furthermore, defi‐ ciencies in either Se or vitamin E results in specific viral mutations, changing relatively be‐ nign viruses into virulent ones. Thus, host nutritional status should be considered a driving force for the emergence of new viral strains or newly pathogenic strains of known viruses [174].

Several studies have evaluated the possible association between antioxidants vitamins or se‐ lenium supplement and the risk of prostate cancer, but the evidence is still inconsistent. We systematically searched PubMed, EMBASE, the Cochrane Library, Science Citation Index Expanded, Chinese biomedicine literature database, and bibliographies of retrieved articles up to January 2009. We included 9 randomized controlled trials with 165,056 participants; methodological quality of included trials was generally high. Meta-analysis showed that no significant effects of supplementation with β-carotene (3 trials), vitamin C (2 trials), vitamin E (5 trials), and selenium (2 trials)versus placebo on prostate cancer incidence. The mortality of prostate cancer did not differ significantly by supplement of β-carotene (1 trial), vitamin C (1 trial), vitamin E (2 trials), and selenium (1 trial). This study indicates that antioxidant vitamins and selenium supplement did not reduce the incidence and mortality of prostate cancer; these data provide no support for the use of these supplements for the prevention of prostate cancer [175].

cellular level from damage due to oxidative stress; this is particularly relevant in heart dis‐ eases, which are still a major cause of death worldwide. Given the number of Se cancer pre‐ ventive trials that are currently being undertaken in many countries, the significant outcomes of these trials will not only provide us with more information on optimal Se in‐ take for the treatment and prevention of cancer, but they will also provide us with strategies in the management of other potential human diseases associated with low Se status. Until the specific biomarkers are identified that will directly link Se with disease prevention and

Emerging Role of Natural Antioxidants in Chronic Disease Prevention with an Emphasis on Vitamin E and Selenium

http://dx.doi.org/10.5772/51610

435

The Selenium and Vitamin E Cancer prevention Trial (SELECT) failed to show an effect in human population. However, pre- and pro-SELECT studies are still supporting the potential usefulness of selenium and/or vitamin E for prevention of prostate cancer and possibly oth‐ er conditions. Much remains to be understood about the absorption, metabolism and phys‐ iologic chemistry of these agents. Nonetheless, the existing evidence supporting selenium and vitamin E as potential prostate cancer chemopreventive agents is possibly enough to

My goal in putting this review together was to provide a wide range of subjects dealing with selenium and vitamin E supplementation, that are used in chronic disease prevention, due to their antiradical activities indicating that the combine effects of Se and vitamin E could provide an important dietary source of antioxidants and/or potential agents for a vari‐ ety of human diseases. It is my hope that readers will find this chapter to be useful in further

Chemistry of Biomacromolecules Department, Chemistry Institute, National Autonomus

[2] Shamberger, R. J. (1984). Selenium. *In Biochemistry of the Essential Ultratrace Elements*,

[3] Brasted, R. C. (1961). *In Comprehensive Inorganic Chemistry: Sulfur. Selenium, Tellurium, Polonium and Oxygen*, Robert C. Brasted, Ed.; D. Van Nostrand Co.; Princeton, NJ, 2. [4] Merian, E. (1984). Introduction on environmental chemistry and global cycles of chromium, nickel, cobalt beryllium, arsenic, cadmium and selenium, and their deriv‐

treatment, its use as supplements in health therapy should be taken with caution.

justify further efforts in this direction.

studies dealing with this subject.

University of México, University City, Mexico

[1] Berzelius, J. J. (1818). Afhandl. Fys. Kemi Minerag. 6, 42

Earl Frieden, Ed.; Plenum Press; New York, NY, 3, 201-237.

atives. *Toxicological and Environmental Chemistry*, 8, 9-38.

**Author details**

**References**

Manuel Soriano García\*

Epidemiological studies demonstrated that human exposure to methylmercury (MeHg) may contribute to the development and progression of metabolic and cardiovascular disorders. However, the mechanisms involved and the role of selenium (Se) and vitamin E (VE) sup‐ plementation in modulating MeHg cardiovascular toxicities remain unclear. The effects of Se and VE supplementation on MeHg-mediated systemic oxidative stress, antioxidant de‐ fense, inflammation, and endothelial dysfunction are carried out in an animal model. Male Sprague–Dawley rats were fed a starch-based casein diet or the same diet supplemented with 1 or 3 mg Se/kg diet and with or without 250 or 750 mg VE/kg diet. After 28 days of dietary treatment, rats were gavaged with 0 or 3 mg MeHg/kg BW for 14 consecutive days. Results suggested that exposure to MeHg may increase the risk of cardiovascular disease by decreasing circulating paraoxonase-1 activities, increasing serum oxidized low density lipo‐ protein levels, and associated systemic inflammation and endothelial dysfunction as reflect‐ ed by increased leukocyte counts and serum levels of intercellular adhesion molecule-1 and monocyte chemotactic protein-1. Se and VE supplementation may either alleviate or aug‐ ment the effects of MeHg, depending on their doses and combinations [176].

The analysis of the hepatotoxic effect of malathion in adult male rats and evaluate the possi‐ ble hepatoprotective effect of vitamin E and/or selenium. Oral administration of malathion for 45 days significantly induced marked hepatic injury as revealed by increased activity of the plasma enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), lac‐ tate dehydrogenase (LDH) and gamma-glutamyl transferase GGT). Oral administration of vitamin E and selenium in combination with malathion exhibited a significant protective ef‐ fect by lowering the elevated plasma levels of the previous enzymes. Light microscopic in‐ vestigation revealed that malathion exposure was associated with necrosis of hepatocytes, marked changes of liver tissues in the form of dilated veins, hemorrhagic spots and some degenerative signs of hepatocytes [177].

### **4. Conclusion**

Research on Se during the last few years has produced a great deal of evidence demonstrat‐ ing the important role that Se and its metabolites play in human diseases. In particular, our knowledge of the functional roles of the GPx and TrxR groups as essential antioxidant sele‐ noenzymes in protecting cells from oxidative stress has greatly increased, as has the link be‐ tween these enzymes and various diseases. However, there are still areas of research that require in-depth study, including the mechanistic modes of action of Se in cancer etiology, how Se delivers its anticancer activity at the molecular and genetic levels, and what bio‐ markers can be used to accurately measure the efficacy of Se for use in chemoprevention. It is not well understood the specific mechanism by which Se protects cells and tissue at the cellular level from damage due to oxidative stress; this is particularly relevant in heart dis‐ eases, which are still a major cause of death worldwide. Given the number of Se cancer pre‐ ventive trials that are currently being undertaken in many countries, the significant outcomes of these trials will not only provide us with more information on optimal Se in‐ take for the treatment and prevention of cancer, but they will also provide us with strategies in the management of other potential human diseases associated with low Se status. Until the specific biomarkers are identified that will directly link Se with disease prevention and treatment, its use as supplements in health therapy should be taken with caution.

The Selenium and Vitamin E Cancer prevention Trial (SELECT) failed to show an effect in human population. However, pre- and pro-SELECT studies are still supporting the potential usefulness of selenium and/or vitamin E for prevention of prostate cancer and possibly oth‐ er conditions. Much remains to be understood about the absorption, metabolism and phys‐ iologic chemistry of these agents. Nonetheless, the existing evidence supporting selenium and vitamin E as potential prostate cancer chemopreventive agents is possibly enough to justify further efforts in this direction.

My goal in putting this review together was to provide a wide range of subjects dealing with selenium and vitamin E supplementation, that are used in chronic disease prevention, due to their antiradical activities indicating that the combine effects of Se and vitamin E could provide an important dietary source of antioxidants and/or potential agents for a vari‐ ety of human diseases. It is my hope that readers will find this chapter to be useful in further studies dealing with this subject.

### **Author details**

C (1 trial), vitamin E (2 trials), and selenium (1 trial). This study indicates that antioxidant vitamins and selenium supplement did not reduce the incidence and mortality of prostate cancer; these data provide no support for the use of these supplements for the prevention of

Epidemiological studies demonstrated that human exposure to methylmercury (MeHg) may contribute to the development and progression of metabolic and cardiovascular disorders. However, the mechanisms involved and the role of selenium (Se) and vitamin E (VE) sup‐ plementation in modulating MeHg cardiovascular toxicities remain unclear. The effects of Se and VE supplementation on MeHg-mediated systemic oxidative stress, antioxidant de‐ fense, inflammation, and endothelial dysfunction are carried out in an animal model. Male Sprague–Dawley rats were fed a starch-based casein diet or the same diet supplemented with 1 or 3 mg Se/kg diet and with or without 250 or 750 mg VE/kg diet. After 28 days of dietary treatment, rats were gavaged with 0 or 3 mg MeHg/kg BW for 14 consecutive days. Results suggested that exposure to MeHg may increase the risk of cardiovascular disease by decreasing circulating paraoxonase-1 activities, increasing serum oxidized low density lipo‐ protein levels, and associated systemic inflammation and endothelial dysfunction as reflect‐ ed by increased leukocyte counts and serum levels of intercellular adhesion molecule-1 and monocyte chemotactic protein-1. Se and VE supplementation may either alleviate or aug‐

ment the effects of MeHg, depending on their doses and combinations [176].

degenerative signs of hepatocytes [177].

**4. Conclusion**

The analysis of the hepatotoxic effect of malathion in adult male rats and evaluate the possi‐ ble hepatoprotective effect of vitamin E and/or selenium. Oral administration of malathion for 45 days significantly induced marked hepatic injury as revealed by increased activity of the plasma enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), lac‐ tate dehydrogenase (LDH) and gamma-glutamyl transferase GGT). Oral administration of vitamin E and selenium in combination with malathion exhibited a significant protective ef‐ fect by lowering the elevated plasma levels of the previous enzymes. Light microscopic in‐ vestigation revealed that malathion exposure was associated with necrosis of hepatocytes, marked changes of liver tissues in the form of dilated veins, hemorrhagic spots and some

Research on Se during the last few years has produced a great deal of evidence demonstrat‐ ing the important role that Se and its metabolites play in human diseases. In particular, our knowledge of the functional roles of the GPx and TrxR groups as essential antioxidant sele‐ noenzymes in protecting cells from oxidative stress has greatly increased, as has the link be‐ tween these enzymes and various diseases. However, there are still areas of research that require in-depth study, including the mechanistic modes of action of Se in cancer etiology, how Se delivers its anticancer activity at the molecular and genetic levels, and what bio‐ markers can be used to accurately measure the efficacy of Se for use in chemoprevention. It is not well understood the specific mechanism by which Se protects cells and tissue at the

prostate cancer [175].

434 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Manuel Soriano García\*

Chemistry of Biomacromolecules Department, Chemistry Institute, National Autonomus University of México, University City, Mexico

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**Chapter 18**

**Antioxidant Role of Ascorbic Acid and**

José Luis Silencio Barrita and

http://dx.doi.org/10.5772/52181

for reducing inflammatory symptoms.

**1. Introduction**

of hydroxyproline.

María del Socorro Santiago Sánchez

Additional information is available at the end of the chapter

**His Protective Effects on Chronic Diseases**

Ascorbic acid (AA), commonly known as vitamin C, plays an important role in the human body, although its function at the cellular level is not yet clear. It is necessary for the synthe‐ sis of collagen, a protein that has many connective functions in the body. Among the sub‐ stances and structures that contain collagen are bone, cartilage and the surrounding material, as well as carrier substances and materials of union muscle, skin and other tissues. It also requires (AA) for the synthesis of hormones, neurotransmitters and in the metabolism of certain amino acids and vitamins. Participate in the liver for detoxification of toxic sub‐ stances and blood level for immunity. As an antioxidant reacts with histamine and peroxide

Its antioxidant capacity is associated with reduced incidence of cancer. The requirement for vitamin C for adults is well defined but they have not been uniform across different cul‐ tures, so their need has been defined as culture-specific. They have also defined other roles in cellular processes and reactions. Some epidemiological data mentioned its usefulness in reducing cold with increasing consumption of foods rich in vitamin, so people sometimes ingest an overdose of it. In most reports mention that discrete increases in blood levels of this vitamin reduces the risk of death in all conditions. Although there are many functions of vitamin C, his role in health is discussed mostly in relation to its role as an antioxidant and its effects on cancer, blood pressure, immunity, drug metabolism and urinary excretion

Antioxidants play important roles in cellular function and have been implicated in processes associated with aging, including vascular, inflammatory damage and cancer. In the case of

> © 2013 Barrita and Sánchez; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


### **Chapter 18**

### **Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases**

José Luis Silencio Barrita and María del Socorro Santiago Sánchez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52181

### **1. Introduction**

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min E Cancer Prevention Trial) status. *Molecular Medicine*, 17, 134-143.

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448 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

*Journal of American Medical Association*, 301, 39-51.

Controlled Trials. *Nutrition and Cancer*, 62, 719-727.

*Journal of Nutrition*, 137, 1338-1340.

10-24.

*cology*, 6, 806-813.

*ics*, 21, 1315-1330.

Ascorbic acid (AA), commonly known as vitamin C, plays an important role in the human body, although its function at the cellular level is not yet clear. It is necessary for the synthe‐ sis of collagen, a protein that has many connective functions in the body. Among the sub‐ stances and structures that contain collagen are bone, cartilage and the surrounding material, as well as carrier substances and materials of union muscle, skin and other tissues. It also requires (AA) for the synthesis of hormones, neurotransmitters and in the metabolism of certain amino acids and vitamins. Participate in the liver for detoxification of toxic sub‐ stances and blood level for immunity. As an antioxidant reacts with histamine and peroxide for reducing inflammatory symptoms.

Its antioxidant capacity is associated with reduced incidence of cancer. The requirement for vitamin C for adults is well defined but they have not been uniform across different cul‐ tures, so their need has been defined as culture-specific. They have also defined other roles in cellular processes and reactions. Some epidemiological data mentioned its usefulness in reducing cold with increasing consumption of foods rich in vitamin, so people sometimes ingest an overdose of it. In most reports mention that discrete increases in blood levels of this vitamin reduces the risk of death in all conditions. Although there are many functions of vitamin C, his role in health is discussed mostly in relation to its role as an antioxidant and its effects on cancer, blood pressure, immunity, drug metabolism and urinary excretion of hydroxyproline.

Antioxidants play important roles in cellular function and have been implicated in processes associated with aging, including vascular, inflammatory damage and cancer. In the case of

© 2013 Barrita and Sánchez; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

AA its antioxidant role is useful since it contributes to the maintenance of the vascular sys‐ tem and the reduction of atherogenesis through regulation in collagen synthesis, production of prostacyclin and nitric oxide. In addition to this antioxidant role, the AA has actions at the molecular level because it acts as a cofactor of enzymes such as dopamine hydroxylase (EC 1.14.17.1), influencing neurotransmitter concentration, improves lysosomal protein deg‐ radation and mediates consumer monosodium glutamate.

### **1.1. History**

Since the nineteenth and early twentieth century research on these compounds led to the discovery of vitamins.4 Since 1901, a publication of Wildiers first described the stimulating effect of small amounts of organic material in the growth of yeast; this effect was the subject of many publications and only after several years was universally accepted. Wilders gave the name "bios" to the substance or substances causing increased growth of yeast. In the years since it was shown that bios were multiple in nature, and changes fractionated as bios I, bios IIA, IIB and others.5

Name (IUPAC) systematic: (R) -3, 4-dihydroxy-5-((S) -1, 2-dihydroxyethyl) furan-2 (5H)-one;

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

http://dx.doi.org/10.5772/52181

451

Ascorbic acid contains several structural elements that contribute to their chemical behavior: the structure of the lactones and two enolic hydroxyl groups and a primary and secondary alcohol group. Enediol structure motivates their antioxidant properties, as can be oxidized easily enediols to diketones. Therefore, the carbonyl groups endioles neighbors are also

Ascorbic acid forms two bonds intermolecular hydrogen bonds (shown in red in the figure) that contribute substantially to the stability and with it the chemical qualities of the structure

Ascorbic acid is rapidly interconvert in two unstable diketone tautomers by proton transfer, though it is most stable in the enol form. The proton of the enol is lost, and again acquired by the electrons from the double bond to produce a diketone. This reaction is an enol. There

**Figure 1.** Structure of ascorbic acid and dehydroascorbic acid.

**2.2. Physical and chemical properties**

**Figure 2.** Hydrogen bridges formed by ascorbic acid

are two possible ways: 1, 2-diketone and 1, 3-diketone (figure 3).

called reductive.

endiol.

CAS Number 50-81-7: Formula: C6H8O6; mol wt. 176.13 g / mol

1747. - Lind cured scurvy in British sailors with oranges and lemons.

1907. - It is reproduced experimental scurvy in guinea pigs.

1928. - Eascott identified bios I as meso-inositol. Szent-Gyorgy and Glen published the isola‐ tion of vitamin C or hexuronic acid.

1933. - Allison, Hoover and Burk describe a compound that promotes the respiration and growth of Rhizobium, which designated "Coenzyme R". Then, it is defined molecular struc‐ ture and synthesis of vitamin C.

1937. - the Nobel Prize in Chemistry was awarded to Walter Haworth for his work in deter‐ mining the structure of ascorbic acid (shared with Paul Karrer for his work on vitamins) and the Nobel Prize for medicine was awarded to Albert von Szent-Györgyi Nagyrápolt for his studies on the biological functions of ascorbic acid.

### **2. Definition**

Vitamin C is defined as hexuronic acid, cevitáminic acid or xiloascórbic acid. The term vita‐ min C is generally used to describe all these compounds, although the representative of which is ascorbic acid.

#### **2.1. Structure, formula and chemical characteristics**

Ascorbic acid is the enolic form of one α-ketolactone. Ascorbic acid solution is easily oxi‐ dized to the diketo form referred to as dehydroascorbic acid, which can easily be converted into oxalic acid , diketogulonic acid or threonic acid.

**Figure 1.** Structure of ascorbic acid and dehydroascorbic acid.

Name (IUPAC) systematic: (R) -3, 4-dihydroxy-5-((S) -1, 2-dihydroxyethyl) furan-2 (5H)-one; CAS Number 50-81-7: Formula: C6H8O6; mol wt. 176.13 g / mol

### **2.2. Physical and chemical properties**

AA its antioxidant role is useful since it contributes to the maintenance of the vascular sys‐ tem and the reduction of atherogenesis through regulation in collagen synthesis, production of prostacyclin and nitric oxide. In addition to this antioxidant role, the AA has actions at the molecular level because it acts as a cofactor of enzymes such as dopamine hydroxylase (EC 1.14.17.1), influencing neurotransmitter concentration, improves lysosomal protein deg‐

Since the nineteenth and early twentieth century research on these compounds led to the

effect of small amounts of organic material in the growth of yeast; this effect was the subject of many publications and only after several years was universally accepted. Wilders gave the name "bios" to the substance or substances causing increased growth of yeast. In the years since it was shown that bios were multiple in nature, and changes fractionated as bios

1928. - Eascott identified bios I as meso-inositol. Szent-Gyorgy and Glen published the isola‐

1933. - Allison, Hoover and Burk describe a compound that promotes the respiration and growth of Rhizobium, which designated "Coenzyme R". Then, it is defined molecular struc‐

1937. - the Nobel Prize in Chemistry was awarded to Walter Haworth for his work in deter‐ mining the structure of ascorbic acid (shared with Paul Karrer for his work on vitamins) and the Nobel Prize for medicine was awarded to Albert von Szent-Györgyi Nagyrápolt for his

Vitamin C is defined as hexuronic acid, cevitáminic acid or xiloascórbic acid. The term vita‐ min C is generally used to describe all these compounds, although the representative of

Ascorbic acid is the enolic form of one α-ketolactone. Ascorbic acid solution is easily oxi‐ dized to the diketo form referred to as dehydroascorbic acid, which can easily be converted

Since 1901, a publication of Wildiers first described the stimulating

radation and mediates consumer monosodium glutamate.

450 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

1747. - Lind cured scurvy in British sailors with oranges and lemons.

1907. - It is reproduced experimental scurvy in guinea pigs.

studies on the biological functions of ascorbic acid.

**2.1. Structure, formula and chemical characteristics**

into oxalic acid , diketogulonic acid or threonic acid.

**1.1. History**

discovery of vitamins.4

I, bios IIA, IIB and others.5

tion of vitamin C or hexuronic acid.

ture and synthesis of vitamin C.

**2. Definition**

which is ascorbic acid.

Ascorbic acid contains several structural elements that contribute to their chemical behavior: the structure of the lactones and two enolic hydroxyl groups and a primary and secondary alcohol group. Enediol structure motivates their antioxidant properties, as can be oxidized easily enediols to diketones. Therefore, the carbonyl groups endioles neighbors are also called reductive.

Ascorbic acid forms two bonds intermolecular hydrogen bonds (shown in red in the figure) that contribute substantially to the stability and with it the chemical qualities of the structure endiol.

**Figure 2.** Hydrogen bridges formed by ascorbic acid

Ascorbic acid is rapidly interconvert in two unstable diketone tautomers by proton transfer, though it is most stable in the enol form. The proton of the enol is lost, and again acquired by the electrons from the double bond to produce a diketone. This reaction is an enol. There are two possible ways: 1, 2-diketone and 1, 3-diketone (figure 3).

Chemical data: IUPAC Name: D-isoascorbic acid, CAS Number: [89-65-6] Molecular formu‐

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

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453

A short-term supplementation with physiological doses of antioxidant vitamins, carote‐ noids and trace elements during alcohol rehabilitation clearly improves micronutrient status indicators. Heavy smokers in particular seem to respond to vitamin C supplemen‐

In the liver accumulate micronutrients such as Vitamin A, E and iron, and therefore, in pa‐ tients with hepatic impairment is a deficit of them due to reduced intake, as well as intesti‐ nal transport and liver stores. The alteration of fat soluble vitamins is especially important in

Moreover, in the alcoholic patient levels water-soluble vitamins are low due to the effect of ethanol on its metabolism, producing pyridoxine deficiency, retinol, cobalamin, folate and niacin. In fact, in chronic alcoholism may develop Wernicke encephalopathy. It has also shown a direct relationship between oxidative stress and disease severity liver, requiring the micronutrients with antioxidant activity, being increased the needs of vitamin E and C.

There are few data on vitamin needs and trace elements in burned patients. the large tissue loss, decreased gastrointestinal absorption, increased urinary losses, changes in distribution, and a high degree catabolism, that are made increased the needs of vitamins and trace ele‐ ments. Clinical guidelines recommend giving also established daily requirements and addi‐

Also, increase the intake of vitamin C (1,000 mg/day) as it promotes the healing process, and vitamin A (10,000 IU/day) for its effect immune and protective skin and mucous mem‐ branes. Also, is required additional vitamin D due to high risk of fractures in these group

Zinc supplement is suitable dose of 220 mg/day, as is involved in protein synthesis and tis‐ sue regeneration. Furthermore, Chan et al indicate that in the week post-injury, there are high losses exuding of copper, being necessary to increase their requirements (4.5 mg/day of

The increased production of oxygen species reactive in this clinical situation requires admin‐ istration of antioxidants (ascorbic acid, glutathione, carotenoids, vitamin A and E) been shown to reduce mortality, protecting the micro vascular circulation, reducing the peroxida‐ tion tissue lipid. According to some authors, surgical stress may necessitate supplementa‐ tion of ascorbic acid, alpha tocopherol and trace elements, associating too low preoperative levels of vitamin A (<0.77 mol/L) with an increase of postoperative infection and mortality. At present, it is unknown whether supplementation micronutrient for a short period of time

patients but have not yet been established Daily exact requirements.

la: C6H8O6, molar mass: 176.13 g/mol; Melting point: 169-172 ° C

**3. Relationship with other nutrients**

patients with steatorrhea or cholestasis.

tional doses of certain micronutrients.

copper sulfate).

**3.1. Vitamin. A and E**

tation (Gueguen, 2003)

**Figure 3.** Nucleophilic attack on the proton ascorbic enol to give 1,3 diketone.

#### **2.3. Vitamers or vitameric forms**

The vitamer of a particular vitamin is any chemical compound which generally has the same molecular structure and each shows a different vitamin activity in a biological system which is deficient of the vitamin.

The vitamin activity of multiple vitamers is due to the ability (sometimes limited) of the body to convert one or many vitamers in another vitamer for the same enzymatic cofactor which is active in the body as the most important form of the vitamin. As part of the defini‐ tion of the vitamin, the body can not completely synthesize an optimal amount of vitamin activity of foodstuffs simple, without a certain minimum amount of vitamer as base. Not all vitamers have the same vitamin power by mass or weight. This is due to differences in the absorption and the variable interconversion several vitamers in the vitamin.

For ascorbic acid per se, may be mentioned the following vitamers: dehydroascorbic acid, erythorbic acid (figure 4) and the following salts: sodium ascorbate, calcium ascorbate, and others. (Rogur, 2010)

**Figure 4.** Structure of erythorbic acid, isoascorbic or Arabian-ascorbic

Chemical data: IUPAC Name: D-isoascorbic acid, CAS Number: [89-65-6] Molecular formu‐ la: C6H8O6, molar mass: 176.13 g/mol; Melting point: 169-172 ° C

### **3. Relationship with other nutrients**

### **3.1. Vitamin. A and E**

**Figure 3.** Nucleophilic attack on the proton ascorbic enol to give 1,3 diketone.

452 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

The vitamer of a particular vitamin is any chemical compound which generally has the same molecular structure and each shows a different vitamin activity in a biological system which

The vitamin activity of multiple vitamers is due to the ability (sometimes limited) of the body to convert one or many vitamers in another vitamer for the same enzymatic cofactor which is active in the body as the most important form of the vitamin. As part of the defini‐ tion of the vitamin, the body can not completely synthesize an optimal amount of vitamin activity of foodstuffs simple, without a certain minimum amount of vitamer as base. Not all vitamers have the same vitamin power by mass or weight. This is due to differences in the

For ascorbic acid per se, may be mentioned the following vitamers: dehydroascorbic acid, erythorbic acid (figure 4) and the following salts: sodium ascorbate, calcium ascorbate, and

absorption and the variable interconversion several vitamers in the vitamin.

**Figure 4.** Structure of erythorbic acid, isoascorbic or Arabian-ascorbic

**2.3. Vitamers or vitameric forms**

is deficient of the vitamin.

others. (Rogur, 2010)

A short-term supplementation with physiological doses of antioxidant vitamins, carote‐ noids and trace elements during alcohol rehabilitation clearly improves micronutrient status indicators. Heavy smokers in particular seem to respond to vitamin C supplemen‐ tation (Gueguen, 2003)

In the liver accumulate micronutrients such as Vitamin A, E and iron, and therefore, in pa‐ tients with hepatic impairment is a deficit of them due to reduced intake, as well as intesti‐ nal transport and liver stores. The alteration of fat soluble vitamins is especially important in patients with steatorrhea or cholestasis.

Moreover, in the alcoholic patient levels water-soluble vitamins are low due to the effect of ethanol on its metabolism, producing pyridoxine deficiency, retinol, cobalamin, folate and niacin. In fact, in chronic alcoholism may develop Wernicke encephalopathy. It has also shown a direct relationship between oxidative stress and disease severity liver, requiring the micronutrients with antioxidant activity, being increased the needs of vitamin E and C.

There are few data on vitamin needs and trace elements in burned patients. the large tissue loss, decreased gastrointestinal absorption, increased urinary losses, changes in distribution, and a high degree catabolism, that are made increased the needs of vitamins and trace ele‐ ments. Clinical guidelines recommend giving also established daily requirements and addi‐ tional doses of certain micronutrients.

Also, increase the intake of vitamin C (1,000 mg/day) as it promotes the healing process, and vitamin A (10,000 IU/day) for its effect immune and protective skin and mucous mem‐ branes. Also, is required additional vitamin D due to high risk of fractures in these group patients but have not yet been established Daily exact requirements.

Zinc supplement is suitable dose of 220 mg/day, as is involved in protein synthesis and tis‐ sue regeneration. Furthermore, Chan et al indicate that in the week post-injury, there are high losses exuding of copper, being necessary to increase their requirements (4.5 mg/day of copper sulfate).

The increased production of oxygen species reactive in this clinical situation requires admin‐ istration of antioxidants (ascorbic acid, glutathione, carotenoids, vitamin A and E) been shown to reduce mortality, protecting the micro vascular circulation, reducing the peroxida‐ tion tissue lipid. According to some authors, surgical stress may necessitate supplementa‐ tion of ascorbic acid, alpha tocopherol and trace elements, associating too low preoperative levels of vitamin A (<0.77 mol/L) with an increase of postoperative infection and mortality. At present, it is unknown whether supplementation micronutrient for a short period of time could restore plasma antioxidant levels after surgery. Some authors suggest that antioxi‐ dants could lead to improved metabolism and ventricular function after cardiac surgery. Al‐ so state that patient's major surgery may benefit from selenium, even before surgery, to action at the level of oxidative stress. The ESPEN recommended in these surgical patients treated with parenteral nutrition, supplement micronutrient recommended daily doses, the vitamin supplementation is unnecessary if the patient is on concomitant enteral nutrition, oral or parenteral.

require thiamine, niacin, vitamin A, E and C, B complex, zinc (15-20 mg/day and 10 mg/L

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Consumption of 0.6 mg equivalents of alpha tocopherol/g linoleic acid is suitable for human adults. The minimum requirement of vitamin E related to the consumption of fatty acids with a high degree of unsaturation can be calculated with a specific formula that must take into account the peroxibility of polyunsaturated fatty acids is based on the results of animal experiments. But still no convincing evidence of how much vitamin E is required in relation

Studies so far show that the effects of supplementation with EPA and DHA lipid peroxidiza‐ cion increase even when the amounts of vitamin E present are adequate in relation to the oxida‐ tive potential of these fatty acids. On the other hand the calculation of the requirement for vitamin E using current data from recent consumption, show that a reduction in total fat intake with a concomitant increase in consumption of polyunsaturated fatty acids, including EPA and DHA results in an increased need intake of vitamin E. In fact the methods used to investigate the requirements of vitamin E and polyunsaturated fatty acid intake (erythrocyte hemolysis) and the techniques used to assess lipid peroxidation (malondialdehyde analysis, MDA) may

Therefore, further studies are needed to establish the requirement of vitamin E when intake of unsaturated fatty acids of longer chain increases. For this purpose it is necessary to use functional techniques based on the measurement of in vivo lipid peroxidation. Until then or until the available data suggest using the index of 0.6 mg of alpha tocopherol per gram of ingested PUFA. However it is likely that higher levels are necessary for vitamin fats are rich

The diet of our ancestors was less dense in calories, being higher in fiber, rich in fruits, vege‐ tables, lean meat and fish. As a result, the diet was significantly lower in total fat and satu‐ rated fat, but containing equal amounts of n-6 essential fatty acids and n-3. Linoleic acid (LA) is the major n-6 fatty acid and alpha-linolenic acid (ALA) is the major n-3 fatty acid.

In the body, LA is metabolized to arachidonic acid (ARA), and ALA is metabolized to eico‐ sapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The essential fatty acid ratio n-6: n-3 is 1 to 2:1, with higher levels of long chain polyunsaturated fatty acids (PUFA), such as EPA, DHA and ARA, today's diet. Today this ratio is about 10 to 1 or 20 and 25 to 1, indicat‐ ing that Western diets are deficient in n-3 fatty acids compared with the diet that humans

The n-3 and n-6 are not interconvertible in the human body and are important components of practically all cell membranes. The n-6 fatty acids and n-3 influence eicosanoid metabo‐ lism, gene expression, and intercellular communication cell to cell. The polyunsaturated fat‐ ty acid composition of cell membranes is largely dependent on food ingestion. Therefore, appropriate amounts of n-6 fatty acids and n-3 in the diet should be considered in making

to consumption of polyunsaturated fatty acids: EPA (20:5 n-3) and DHA (22:6 n-3).

be inappropriate for measuring a quantitative relationship between the two loads.

in fatty acids containing more than two double bonds (Valk, 2000).

evolved and established patterns genetic.

dietary recommendations.

intestinal leaks) and selenium (rise up to 120 ug/day) in patients with sepsis.

**3.3. Polyunsaturated fatty acids**

### **3.2. Minerals such as selenium, iron and zinc**

Recent studies have shown that in patients with inflammatory bowel disease there is a cor‐ relation between the level of some antioxidants such as selenium, vitamin C and E and clini‐ cal improvement and reduction in serum levels of TNF-α and decrease in steroid dose to 65%. Low serum zinc levels have been correlated with increased blood pressure, disease cor‐ onary type II diabetes mellitus and hyperlipidemia. Also, high intake of magnesium (> 500-1000 mg/day) can lower high blood pressure, and be effective in acute myocardial in‐ farction and atherosclerosis. Houston recommended to prevent the emergence and develop‐ ment of hypertension, administration of additional vitamins and trace elements. Finally, oxidative stress plays an important role in the initiation and maintenance of the pathogene‐ sis of cardiovascular disease and its complications. For some authors, antioxidants such as vitamins E and C, beta-carotene, selenium and zinc, may act by reducing the cardiovascular risk, although evidence is limited.

Malnutrition is a common feature of inflammatory intestinal diseases, being frequent the deficit of vitamins B12, A, D, E and K, steatorrhea, ileal resection or extensive lesions in the intestine. GI bleeding contributing to iron losses, and through diarrhea and fistulas is loss of electrolytes and trace elements (copper, magnesium, selenium and zinc).

The role of antioxidant micronutrients in the clinical and functional improvement has been described by different authors. Thus, a low intake of selenium, beta-carotene and vitamins E and C, can reduce the body's natural defenses and increase inflammation of the airways, and therefore the selenium (100-200 ug/d) been associated with improved lung function, es‐ pecially in smokers. Gazdik et al indicate that supplementation of 200 ug/d of selenium in asthmatic patients produced a statistically significant decrease in the use of corticosteroids. Loannidis and McClave et al indicate that antioxidants such as selenium, vitamin A, vitamin C and vitamin E reduce pancreatic inflammation and pain, and prevent the occurrence of exacerbations. Recently in a double blind study in patients with chronic pancreatitis were given daily supplements of 600 ug of selenium, 9,000 IU of beta-carotene, 540 mg of vitamin C, 270 IU of vitamin E and 2,000 mg methionine, the pain was reduced significantly, as well as stress markers oxidative normalized plasma concentrations of antioxidants.

For some authors, parenteral administration of ascorbic acid can lower the morbidity and mortality of these patients in a randomized, double-blind placebo-controlled; we observed that mortality at day 28 decreased in the group of patients who received ascorbate and vita‐ min E by intravenous infusion. Some authors recommend increasing the contribution of an‐ tioxidants such as vitamin C, retinol, vitamin E, beta-carotene and selenium. Also appear to require thiamine, niacin, vitamin A, E and C, B complex, zinc (15-20 mg/day and 10 mg/L intestinal leaks) and selenium (rise up to 120 ug/day) in patients with sepsis.

### **3.3. Polyunsaturated fatty acids**

could restore plasma antioxidant levels after surgery. Some authors suggest that antioxi‐ dants could lead to improved metabolism and ventricular function after cardiac surgery. Al‐ so state that patient's major surgery may benefit from selenium, even before surgery, to action at the level of oxidative stress. The ESPEN recommended in these surgical patients treated with parenteral nutrition, supplement micronutrient recommended daily doses, the vitamin supplementation is unnecessary if the patient is on concomitant enteral nutrition,

Recent studies have shown that in patients with inflammatory bowel disease there is a cor‐ relation between the level of some antioxidants such as selenium, vitamin C and E and clini‐ cal improvement and reduction in serum levels of TNF-α and decrease in steroid dose to 65%. Low serum zinc levels have been correlated with increased blood pressure, disease cor‐ onary type II diabetes mellitus and hyperlipidemia. Also, high intake of magnesium (> 500-1000 mg/day) can lower high blood pressure, and be effective in acute myocardial in‐ farction and atherosclerosis. Houston recommended to prevent the emergence and develop‐ ment of hypertension, administration of additional vitamins and trace elements. Finally, oxidative stress plays an important role in the initiation and maintenance of the pathogene‐ sis of cardiovascular disease and its complications. For some authors, antioxidants such as vitamins E and C, beta-carotene, selenium and zinc, may act by reducing the cardiovascular

Malnutrition is a common feature of inflammatory intestinal diseases, being frequent the deficit of vitamins B12, A, D, E and K, steatorrhea, ileal resection or extensive lesions in the intestine. GI bleeding contributing to iron losses, and through diarrhea and fistulas is loss of

The role of antioxidant micronutrients in the clinical and functional improvement has been described by different authors. Thus, a low intake of selenium, beta-carotene and vitamins E and C, can reduce the body's natural defenses and increase inflammation of the airways, and therefore the selenium (100-200 ug/d) been associated with improved lung function, es‐ pecially in smokers. Gazdik et al indicate that supplementation of 200 ug/d of selenium in asthmatic patients produced a statistically significant decrease in the use of corticosteroids. Loannidis and McClave et al indicate that antioxidants such as selenium, vitamin A, vitamin C and vitamin E reduce pancreatic inflammation and pain, and prevent the occurrence of exacerbations. Recently in a double blind study in patients with chronic pancreatitis were given daily supplements of 600 ug of selenium, 9,000 IU of beta-carotene, 540 mg of vitamin C, 270 IU of vitamin E and 2,000 mg methionine, the pain was reduced significantly, as well

For some authors, parenteral administration of ascorbic acid can lower the morbidity and mortality of these patients in a randomized, double-blind placebo-controlled; we observed that mortality at day 28 decreased in the group of patients who received ascorbate and vita‐ min E by intravenous infusion. Some authors recommend increasing the contribution of an‐ tioxidants such as vitamin C, retinol, vitamin E, beta-carotene and selenium. Also appear to

electrolytes and trace elements (copper, magnesium, selenium and zinc).

as stress markers oxidative normalized plasma concentrations of antioxidants.

oral or parenteral.

**3.2. Minerals such as selenium, iron and zinc**

454 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

risk, although evidence is limited.

Consumption of 0.6 mg equivalents of alpha tocopherol/g linoleic acid is suitable for human adults. The minimum requirement of vitamin E related to the consumption of fatty acids with a high degree of unsaturation can be calculated with a specific formula that must take into account the peroxibility of polyunsaturated fatty acids is based on the results of animal experiments. But still no convincing evidence of how much vitamin E is required in relation to consumption of polyunsaturated fatty acids: EPA (20:5 n-3) and DHA (22:6 n-3).

Studies so far show that the effects of supplementation with EPA and DHA lipid peroxidiza‐ cion increase even when the amounts of vitamin E present are adequate in relation to the oxida‐ tive potential of these fatty acids. On the other hand the calculation of the requirement for vitamin E using current data from recent consumption, show that a reduction in total fat intake with a concomitant increase in consumption of polyunsaturated fatty acids, including EPA and DHA results in an increased need intake of vitamin E. In fact the methods used to investigate the requirements of vitamin E and polyunsaturated fatty acid intake (erythrocyte hemolysis) and the techniques used to assess lipid peroxidation (malondialdehyde analysis, MDA) may be inappropriate for measuring a quantitative relationship between the two loads.

Therefore, further studies are needed to establish the requirement of vitamin E when intake of unsaturated fatty acids of longer chain increases. For this purpose it is necessary to use functional techniques based on the measurement of in vivo lipid peroxidation. Until then or until the available data suggest using the index of 0.6 mg of alpha tocopherol per gram of ingested PUFA. However it is likely that higher levels are necessary for vitamin fats are rich in fatty acids containing more than two double bonds (Valk, 2000).

The diet of our ancestors was less dense in calories, being higher in fiber, rich in fruits, vege‐ tables, lean meat and fish. As a result, the diet was significantly lower in total fat and satu‐ rated fat, but containing equal amounts of n-6 essential fatty acids and n-3. Linoleic acid (LA) is the major n-6 fatty acid and alpha-linolenic acid (ALA) is the major n-3 fatty acid.

In the body, LA is metabolized to arachidonic acid (ARA), and ALA is metabolized to eico‐ sapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The essential fatty acid ratio n-6: n-3 is 1 to 2:1, with higher levels of long chain polyunsaturated fatty acids (PUFA), such as EPA, DHA and ARA, today's diet. Today this ratio is about 10 to 1 or 20 and 25 to 1, indicat‐ ing that Western diets are deficient in n-3 fatty acids compared with the diet that humans evolved and established patterns genetic.

The n-3 and n-6 are not interconvertible in the human body and are important components of practically all cell membranes. The n-6 fatty acids and n-3 influence eicosanoid metabo‐ lism, gene expression, and intercellular communication cell to cell. The polyunsaturated fat‐ ty acid composition of cell membranes is largely dependent on food ingestion. Therefore, appropriate amounts of n-6 fatty acids and n-3 in the diet should be considered in making dietary recommendations.

These two classes of polyunsaturated fatty acids must be distinguished because they are metabolically and functionally distinct and have opposing physiological functions, balance is important for maintaining homeostasis and normal development. Studies with nonhuman primates and human newborns indicate that DHA is essential for normal functional devel‐ opment of the retina and the brain, especially in premature babies. A balanced n-6/n-3 ratio in the diet is essential for normal growth and development and should lead to reduced car‐ diovascular disease and other chronic diseases and improve mental health. Although a rec‐ ommended dietary allowance for essential fatty acids does not exist, an adequate intake (AI) has been estimated for essential fatty acids n-6 and n-3 by an international scientific working group. The final recommendations are for Western societies, reduce the consumption of n-6 fatty acids and increased intake of n-3 fatty acids. (Simopoulus, 2000)

**4.2. Fruits and vegetables**

Lemon juice Orange juice, canned Col common, dehydrated Peppers, sweet, ripe, red, raw Currants, black, European, raw

parsley, dry Orange juice green radish, raw Grape juice Orange peel, raw Pokeroot fruit Mustard seeds

leaves of kale, boiled, drained

Leaves and stems of cress, raw

Brussels sprouts, cooked in water

Broccoli cooked Brussels sprouts cooked

Cauliflower, raw

Cabbage, red, raw Strawberries Raw

Lamb crude

to quantify when using colorimetric methods.

Vitamin C is a major constituent of fruits and vegetables, which also contain citric acid, oxa‐ lates and substances such as anthocyanins, coloring agents and carotenoids that are difficult

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

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Currently there is great interest in relation to consumption of natural foods and mainly on the content of nutrients in fruits, vegetables and vitamin C. This interest is due in part to vitamin C is probably one of the most widely used nutrients in the food and pharmaceutical industry. Used as a supplement, additive, preservative, as an antioxidant in processed

> > 76 61 59

foods. Table 2 shows the main foods that are good sources of vitamin C.

Acerola 1743 Coriander leaves dry 567 red peppers, spicy, ripe, raw 369 Orange juice, dehydrated 359 Grape juice, dehydrated 350 common Guavas, whole, raw 242 Dried Tomato juice 239 red pepper, spicy, immature, raw 235

Papaya 56

**Table 2.** Acid content of ascorbic acid in different foods and different presentations

**FOOD CONCETRATION (mg/100g)**

### **4. Vitamin C sources**

#### **4.1. Food sources in Mexico**

The main sources of ascorbic acid are presented in Table 1. The results of vitamin C are shown as the mean and correspond to the official tables of composition of Mexican foods.


**Table 1.** Main sources of ascorbic acid in México

### **4.2. Fruits and vegetables**

These two classes of polyunsaturated fatty acids must be distinguished because they are metabolically and functionally distinct and have opposing physiological functions, balance is important for maintaining homeostasis and normal development. Studies with nonhuman primates and human newborns indicate that DHA is essential for normal functional devel‐ opment of the retina and the brain, especially in premature babies. A balanced n-6/n-3 ratio in the diet is essential for normal growth and development and should lead to reduced car‐ diovascular disease and other chronic diseases and improve mental health. Although a rec‐ ommended dietary allowance for essential fatty acids does not exist, an adequate intake (AI) has been estimated for essential fatty acids n-6 and n-3 by an international scientific working group. The final recommendations are for Western societies, reduce the consumption of n-6

The main sources of ascorbic acid are presented in Table 1. The results of vitamin C are shown as the mean and correspond to the official tables of composition of Mexican foods.

fatty acids and increased intake of n-3 fatty acids. (Simopoulus, 2000)

456 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**FOOD CONCENTRATION (mg/100g EP)**

poblan chili 364 "trompito" chili 320 yucca flower 273 guava 199 Marañon 167 cauliflower 127 red bell pepper 160 guajillo chili 100 garlic 99 Nanche fruit 86 orange 76 manila mango 76 serran chili 65 pumpkin 58 watercress 51 beet 20 cucumber 13

**4. Vitamin C sources**

**4.1. Food sources in Mexico**

EP = edible portion

**Table 1.** Main sources of ascorbic acid in México

Vitamin C is a major constituent of fruits and vegetables, which also contain citric acid, oxa‐ lates and substances such as anthocyanins, coloring agents and carotenoids that are difficult to quantify when using colorimetric methods.

Currently there is great interest in relation to consumption of natural foods and mainly on the content of nutrients in fruits, vegetables and vitamin C. This interest is due in part to vitamin C is probably one of the most widely used nutrients in the food and pharmaceutical industry. Used as a supplement, additive, preservative, as an antioxidant in processed foods. Table 2 shows the main foods that are good sources of vitamin C.


**Table 2.** Acid content of ascorbic acid in different foods and different presentations

In Table 3 shows the analysis of vitamin C in different parts of the food such as edi‐ ble portion thereof, the seed or plant center and the shell and stalks that are normally discarded.

**Food Concentration**

**Table 4.** Content of ascorbic acid in Mexican fruits and vegetables

rose apple and apple are the best examples.

The shell or skin of many fruits and vegetables is usually discarded, but these same wastes have significant amounts of ascorbic acid, which is shown in Table 5, where the lime peel,

**(mg / 100 g EP)**

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

squash 7.2±0.9 94.2 spinach 8.5±0.2 91.6 potatoes 74.6±6.7 79.7 cucumber 93.0±7.1 96.0 green tomato 222.8±10.7 90.9 poblano chile 191.0±7.8 91.9 green pepper 195.5±9.5 94.3 nopales 268.9±30.1 95.2 cambray onion 17.0±0.7 93.0 carrot 50.4±5.6 87.2 white cabbage 184.7±17.2 93.2 grapefruit 261.3±10.7 87.8 mango 319.6±5.3 84.6 watermelon 56.2±8.9 92.5 banana 333.7±6.3 76.5 orange 279.8±39.4 84.0 mamey 31.6±1.2 66.8 plum 331.1±17.9 88.1 grape 66.1±13.6 84.3 apple 458.1±17.8 84.5 beet 70.8±6.8 85.3 lemon 39.4±2.5 88.3 avocado 256.2±61.9 84.3 sweet lime 306.8±23.4 89.8

**Moisture (%)**

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**Table 3.** Distribution of ascorbic acid (mg/100g) in some fruits and vegetables produced in México

Below the edible portion the moisture (%) is indicated in parenthesis. The data shown in Tables 3 through 6 are original and have not been published yet. EP = Edible portion

Table 4 shows the concentration of ascorbic acid in the main fruits and vegetables con‐ sumed in Mexico. Data are presented as mean and standard deviation. It shows that even the concentration of vitamin C is lower in the edible portion in the shells report‐ ed in Table 5.


**Table 4.** Content of ascorbic acid in Mexican fruits and vegetables

In Table 3 shows the analysis of vitamin C in different parts of the food such as edi‐ ble portion thereof, the seed or plant center and the shell and stalks that are normally

> 848.9±11.2 (81.8)

> > 19.9±0.5 (94.2)

1916.5±186.7 (69.5)

623.0±35.1 (82.5)

> 82.1±4.9 (81.8)

115.8±1.2 (85.8)

> 4.2±1.0 (85.8)

Below the edible portion the moisture (%) is indicated in parenthesis. The data shown in

Table 4 shows the concentration of ascorbic acid in the main fruits and vegetables con‐ sumed in Mexico. Data are presented as mean and standard deviation. It shows that even the concentration of vitamin C is lower in the edible portion in the shells report‐

Tables 3 through 6 are original and have not been published yet. EP = Edible portion

**Table 3.** Distribution of ascorbic acid (mg/100g) in some fruits and vegetables produced in México




(93.0)


266.6±6.7 (90.5)


1044.5±50.9 (65.7)

**Food Edible portion Shell Seed Stem**

discarded.

Apple 458.1±17.8

chayote 7.2±0.9

onion 17.0±0.7

lime 256.2±17.2

pomarrosa 531.7±36.4

guanábana 140.7±3.6

beat 70.8±6.8

Potato 74.6±6.7

ed in Table 5.

(84.5)

458 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

(94.2)

(87.6)

(87.1)

(83.6)

(80.0)

(85.3)

(80.3)

The shell or skin of many fruits and vegetables is usually discarded, but these same wastes have significant amounts of ascorbic acid, which is shown in Table 5, where the lime peel, rose apple and apple are the best examples.


**Food-industrialized**

**Use as a preservative**

**5. Deficiencies**

gulated lymph (20-53 ug/108

ents on the label of the final product

acids (palmitate and ascorbyl stearate).

**5.1. Primary deficiency – scurvy. Signs and symptoms**

Vitamin C (ascorbic acid) is water soluble and sensitive to oxygen. For this reason, it may partially destroyed in foods during processing, if exposed to air during storage or if treated with water. Manufacturers can protect them from oxidation by adding vitamin C. The addi‐ tion of ascorbic acid as an antioxidant should be appropriately marked in the list of ingredi‐

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Ascorbic acid and its salts are practically insoluble in lipids (fats), for this reason that is often used in the food industry as an antioxidant and preservative greasy foods, in order to avoid rancid. Their salts are usually used with a solubilizing agent (usually a monoglyceride) to improve its implementation. Also this often used in the processing industry of cosmetics products. Sodium ascorbate is a sodium salt of ascorbic acid (vitamin C) and formula C6H7NaO6. This form is used in the food industry for their functions antiseptic, antioxidants, and preservatives. Ascorbyl palmitate is an ester formed by ascorbic acid (vitamin C) and palmitic acid creating a liposoluble form of vitamin C. It is used in the food industry as an

Is usually used as a food preservative and as antioxidant in the food industry, a typical case is found as a bread improver additive. In industry collecting fruit prevents the color oxida‐ tive change known browning. Is often added to foods treated with nitrite in order to reduce the generation of nitrosamines (a carcinogen), so commonly found in sausages and cold

Ascorbic acid and its sodium, potassium and calcium salts are used widely as antioxidants and additives. These compounds are soluble in water, so that fats do not protect against oxi‐ dation. For this purpose may be used ascorbic acid esters with fat soluble long chain fatty

A frank deficiency of vitamin C causes scurvy, a disease characterized by multiple hemorrhag‐ es. Scurvy in adults is manifested by lassitude, weakness, irritability, vague muscle pain, joint pain and weight loss. Early signs objectives are as bleeding gums, tooth loss and gingivitis.

The diagnosis of scurvy, is achieved by testing plasma ascorbic acid, low concentration indicates low levels in tissues. It is generally accepted that ascorbic acid concentration in the layer of coa‐

garding vitamin C and its concentration in tissues. In the most extreme cases scurvy shows: bleeding gums and skin, perifollicular bleeding and ocular petechiae, salivary and lachrymal glands "drier", functional neuropathy, lower limb edema, psychological disturbances, anemia

and poor healing of wounds. The consumption of snuff lowers blood levels of vitamin C.

leukocytes) is the most reliable indicator of nutritional status re‐

cuts. In the same way is generally used in the food industry as an acidity regulator.

antioxidant (code E 304). It is wrong to think that is a natural antioxidant

**Table 5.** Content of ascorbic acid in the shell of some fruits and vegetables

Table 6 shows the values of ascorbic acid in some plant species used as flavoring for Mexi‐ can dishes. In most cases the amount used for the preparation of food is very low and some‐ times do not amount to more than 2% by weight of the end plate. However their presence in cooked food gives organoleptic properties suitable for the acceptance of it and especially the potential of the flavors of food.


**Table 6.** Content of ascorbic acid in some food used as spices

### **Food-industrialized**

**Shell of concentration**

460 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**Table 5.** Content of ascorbic acid in the shell of some fruits and vegetables

**spices concentration**

**Table 6.** Content of ascorbic acid in some food used as spices

potential of the flavors of food.

**(mg/100g)**

Table 6 shows the values of ascorbic acid in some plant species used as flavoring for Mexi‐ can dishes. In most cases the amount used for the preparation of food is very low and some‐ times do not amount to more than 2% by weight of the end plate. However their presence in cooked food gives organoleptic properties suitable for the acceptance of it and especially the

**(mg /100g EP)**

coriander 56.0±7.3 85.3 yerbabuena 226.9±21.9 86.8 epazote leaves 18.3±1.1 86.3 chard leaves 8.5±0.9 92.7 chard stem 15.7±0.9 95.0 parsley leaves 243.9±17.2 84.5 parsley stem 182.1±11.0 90.2 celery leaves 5.6±0.2 88.4 celery stem 7.8±0.9 90.3 corn grain 35.3±6.0 70.7 corn "hair" 55.9±1.2 85.4

CARROT 129.1±10.1 88.5 APPLE 848.9±11.2 81.8 BITTER LEMON 39.4±2.5 71.7 AVOCADO 187.1±27.2 82.0 BEET 115.8±1.2 85.8 SMALL POTATO 4.2±1.0 85.5 BIG POTATO 2.1±0.1 80.3 POMARROSA 622.9±35.1 82.5 LIME 1916.4±186.7 69.4 CHAYOTE 19.9±0.5 94.2

**moisture (%)**

**moisture (%)**

Vitamin C (ascorbic acid) is water soluble and sensitive to oxygen. For this reason, it may partially destroyed in foods during processing, if exposed to air during storage or if treated with water. Manufacturers can protect them from oxidation by adding vitamin C. The addi‐ tion of ascorbic acid as an antioxidant should be appropriately marked in the list of ingredi‐ ents on the label of the final product

Ascorbic acid and its salts are practically insoluble in lipids (fats), for this reason that is often used in the food industry as an antioxidant and preservative greasy foods, in order to avoid rancid. Their salts are usually used with a solubilizing agent (usually a monoglyceride) to improve its implementation. Also this often used in the processing industry of cosmetics products. Sodium ascorbate is a sodium salt of ascorbic acid (vitamin C) and formula C6H7NaO6. This form is used in the food industry for their functions antiseptic, antioxidants, and preservatives. Ascorbyl palmitate is an ester formed by ascorbic acid (vitamin C) and palmitic acid creating a liposoluble form of vitamin C. It is used in the food industry as an antioxidant (code E 304). It is wrong to think that is a natural antioxidant

#### **Use as a preservative**

Is usually used as a food preservative and as antioxidant in the food industry, a typical case is found as a bread improver additive. In industry collecting fruit prevents the color oxida‐ tive change known browning. Is often added to foods treated with nitrite in order to reduce the generation of nitrosamines (a carcinogen), so commonly found in sausages and cold cuts. In the same way is generally used in the food industry as an acidity regulator.

Ascorbic acid and its sodium, potassium and calcium salts are used widely as antioxidants and additives. These compounds are soluble in water, so that fats do not protect against oxi‐ dation. For this purpose may be used ascorbic acid esters with fat soluble long chain fatty acids (palmitate and ascorbyl stearate).

### **5. Deficiencies**

### **5.1. Primary deficiency – scurvy. Signs and symptoms**

A frank deficiency of vitamin C causes scurvy, a disease characterized by multiple hemorrhag‐ es. Scurvy in adults is manifested by lassitude, weakness, irritability, vague muscle pain, joint pain and weight loss. Early signs objectives are as bleeding gums, tooth loss and gingivitis.

The diagnosis of scurvy, is achieved by testing plasma ascorbic acid, low concentration indicates low levels in tissues. It is generally accepted that ascorbic acid concentration in the layer of coa‐ gulated lymph (20-53 ug/108 leukocytes) is the most reliable indicator of nutritional status re‐ garding vitamin C and its concentration in tissues. In the most extreme cases scurvy shows: bleeding gums and skin, perifollicular bleeding and ocular petechiae, salivary and lachrymal glands "drier", functional neuropathy, lower limb edema, psychological disturbances, anemia and poor healing of wounds. The consumption of snuff lowers blood levels of vitamin C.

### **Clinical manifestations of Vitamin C deficiency**

Clinical manifestations of deficiency have been described at several levels: a) mesenchymal. - By the presence of petechiae, ecchymosis, curly hair, peri-follicular hemorrhages, bleeding gums, swollen, hyperkeratosis, Sjôgren syndrome, dyspnea, arthralgia, edema and poor heal‐ ing, b) systemics, characterized by fatigue, weakness and lassitude c) psychological and neuro‐ logical, by the presence of depression, hysteria, hypochondriasis and vasomotor instability.

wound healing. • Dry and separated strands in the hair. • Tendency to bruising. • Gingivi‐ tis (gum inflammation). • nosebleeds. • Possible weight gain because of slowed metabolism.

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**6. Laboratory methods for its measurement in foods and biological fluid**

In the analysis of vitamin C, for the methods commonly used consume many time and therefore overestimates the concentration, due to other oxydizable species different of vita‐ min C; the determination by liquid chromatography of high resolution (HPLC), with electro‐ chemical detection, for example, they require equipment not always available in smalls laboratories and also, is very expensive. However this method quantifies all the forms of the vitamin C present in the sample, and even it detects an epimer of ascorbic acid, the eritorbic acid or isoascorbic acid. The samples of vitamin C saturation are used to establish the defi‐ ciency of ascorbate in tissue and are useful to confirm the diagnostic of scurvy when the pa‐ tient has a normal absorption (Engelfried, 1944). It has been described 3 types of tests to determine the tissue saturation, the first 2 are easy to make but they don't cover the problem on totally, the third test is complicated and it's only useful in research work listed below:

**•** The vitamin C in the plasma is not found doing a metabolic function; it is rather in a

**•** from one tissue to another. Its lost or decrease does not indicates the intracellular sta‐ tus of this vitamin. A well-nourish adult with a free acid ascorbic diet decreases his serum levels of acid to cero in about 6 weeks; however only after many weeks of more deprivation the scurvy symptoms appears. So that for this reason the scurvy patients have low levels of ascorbic acid in the plasma. The vitamin C determination in plasma after a charge dose generally reflects the vitamin proportion, which has been stored by the tissues, however is tough to do a completely quantitative techni‐ que, because in high doses of this vitamin, the plasmatic concentration exceeds the kidney threshold which causes the lost of this vitamin in urine. For a specific meas‐ ure most be given multiple small doses, to avoid an excess in the blood levels above

**b.** Measurement of kidney excretion with and without sample dose: The most important problems in this measurement are those concerning to the collection of urine, more than the vitamin measurement. The excretion of the vitamin has been correlated with the cre‐ atinine excretion. This is because the creatinine is used as a real and simple indicator of

**c.** Tissue Measurement This is the one of the 3 techniques which gives a real representa‐ tion of desaturation of the vitamin. This measurement is difficult because of problems in the sampling of tissue. In the case of ascorbic acid is recommended two methods to measure tissue levels. In the first method is measured its concentration in the buffy coat

• Rough, dry and scaly. • Pain and swelling of the joints. • Weakened tooth enamel.

**a.** Measurement of blood levels with and without a test sample:

transit

the renal threshold.

glomerular filtration.

### **5.2. Deficiencies secondary and association with other diseases**

Severe deficiency of vitamin C leads to Scurvy. This rarely occurs but can be observed deficien‐ cies in those who consume a diet without vegetables and fruits, alcoholism, in older people with limited diets, severely ill patients with chronic stress and in infants fed cow's milk.

Symptoms of scurvy are follicular hyperkeratosis, gingival swelling and inflammation (in gums), bleeding gums, loose teeth, dry mouth and eyes, hair loss and dry skin, among other symptoms that can lead to death.

By deficiency of collagen, the wounds do not heal scars and wounds of previous rupture and may lead to secondary infections. Neurotic disorders are common, consisting of hyste‐ ria and depression, followed by decreased psychomotor activity. It is not safe indiscriminate administration of ascorbic acid, since as the body becomes saturated, decreased absorption, and giving large doses, abruptly deleted. So if you continue with diet low in vitamin, may appear "rebound scurvy". In addition to "rebound scurvy," gastric intolerance and kidney, its use decreases the cobalamin (vitamin B12), a substance synthesized by the body.

Eating a balanced and varied diet high in fruits and vegetables, the minimum dose of vita‐ min C, this completely covered. The daily requirement in an adult male is 90 mg/d and a woman of 75 mg/d (mg/day), although there are always situations where it is necessary to increase the dose of vitamin A through supplementation. Such circumstances or situations are:• Pregnancy and Lactation, • Alcoholics and smokers, • diabetics, • Allergic and asth‐ matic, • People who take daily medications or medications such as oral contraceptives, cor‐ tisone, antibiotics, etc.

#### **Anemia by Vitamin C Deficiency**

Anemia of vitamin C Deficiency is a rare type of anemia that is caused by a severe and very pro‐ longed lack of vitamin C. In this type of anemia, the bone marrow produces small red blood cells (microcytosis). This deficiency is diagnosed by measuring the values of vitamin C in white blood cells. One tablet of vitamin C per day corrects the deficiency and anemia cure.

#### **Associated symptoms**

Associated deficiency or lack of vitamin C (ascorbic acid) can produce or be reflected by: Swollen and bleeding gums, dry, rough skin, spontaneous bruising, Impaired wound heal‐ ing, bleeding nose, joint pain and swelling, anemia and dental enamel weakened. Very small amounts of vitamin C may be associated with signs and symptoms of deficiency, including: • Anemia, • Bleeding gums, • Decreased, ability to fight infection. • Decrease the rate of wound healing. • Dry and separated strands in the hair. • Tendency to bruising. • Gingivi‐ tis (gum inflammation). • nosebleeds. • Possible weight gain because of slowed metabolism. • Rough, dry and scaly. • Pain and swelling of the joints. • Weakened tooth enamel.

### **6. Laboratory methods for its measurement in foods and biological fluid**

In the analysis of vitamin C, for the methods commonly used consume many time and therefore overestimates the concentration, due to other oxydizable species different of vita‐ min C; the determination by liquid chromatography of high resolution (HPLC), with electro‐ chemical detection, for example, they require equipment not always available in smalls laboratories and also, is very expensive. However this method quantifies all the forms of the vitamin C present in the sample, and even it detects an epimer of ascorbic acid, the eritorbic acid or isoascorbic acid. The samples of vitamin C saturation are used to establish the defi‐ ciency of ascorbate in tissue and are useful to confirm the diagnostic of scurvy when the pa‐ tient has a normal absorption (Engelfried, 1944). It has been described 3 types of tests to determine the tissue saturation, the first 2 are easy to make but they don't cover the problem on totally, the third test is complicated and it's only useful in research work listed below:

**a.** Measurement of blood levels with and without a test sample:

**Clinical manifestations of Vitamin C deficiency**

462 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

symptoms that can lead to death.

tisone, antibiotics, etc.

**Associated symptoms**

**Anemia by Vitamin C Deficiency**

**5.2. Deficiencies secondary and association with other diseases**

Clinical manifestations of deficiency have been described at several levels: a) mesenchymal. - By the presence of petechiae, ecchymosis, curly hair, peri-follicular hemorrhages, bleeding gums, swollen, hyperkeratosis, Sjôgren syndrome, dyspnea, arthralgia, edema and poor heal‐ ing, b) systemics, characterized by fatigue, weakness and lassitude c) psychological and neuro‐ logical, by the presence of depression, hysteria, hypochondriasis and vasomotor instability.

Severe deficiency of vitamin C leads to Scurvy. This rarely occurs but can be observed deficien‐ cies in those who consume a diet without vegetables and fruits, alcoholism, in older people

Symptoms of scurvy are follicular hyperkeratosis, gingival swelling and inflammation (in gums), bleeding gums, loose teeth, dry mouth and eyes, hair loss and dry skin, among other

By deficiency of collagen, the wounds do not heal scars and wounds of previous rupture and may lead to secondary infections. Neurotic disorders are common, consisting of hyste‐ ria and depression, followed by decreased psychomotor activity. It is not safe indiscriminate administration of ascorbic acid, since as the body becomes saturated, decreased absorption, and giving large doses, abruptly deleted. So if you continue with diet low in vitamin, may appear "rebound scurvy". In addition to "rebound scurvy," gastric intolerance and kidney,

Eating a balanced and varied diet high in fruits and vegetables, the minimum dose of vita‐ min C, this completely covered. The daily requirement in an adult male is 90 mg/d and a woman of 75 mg/d (mg/day), although there are always situations where it is necessary to increase the dose of vitamin A through supplementation. Such circumstances or situations are:• Pregnancy and Lactation, • Alcoholics and smokers, • diabetics, • Allergic and asth‐ matic, • People who take daily medications or medications such as oral contraceptives, cor‐

Anemia of vitamin C Deficiency is a rare type of anemia that is caused by a severe and very pro‐ longed lack of vitamin C. In this type of anemia, the bone marrow produces small red blood cells (microcytosis). This deficiency is diagnosed by measuring the values of vitamin C in white

Associated deficiency or lack of vitamin C (ascorbic acid) can produce or be reflected by: Swollen and bleeding gums, dry, rough skin, spontaneous bruising, Impaired wound heal‐ ing, bleeding nose, joint pain and swelling, anemia and dental enamel weakened. Very small amounts of vitamin C may be associated with signs and symptoms of deficiency, including: • Anemia, • Bleeding gums, • Decreased, ability to fight infection. • Decrease the rate of

blood cells. One tablet of vitamin C per day corrects the deficiency and anemia cure.

with limited diets, severely ill patients with chronic stress and in infants fed cow's milk.

its use decreases the cobalamin (vitamin B12), a substance synthesized by the body.


and platelets which correlates good with the first signs of scurvy, making the most rec‐ ommended technique. The second method determines the tissue saturation grade for an intradermal test, using dichlorophenol indophenol, which depends of skin reductor substances, which made this nonspecific.

tain the greatest sensitivity and resolution in the chromatographic signal. These tests are

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465

The test is performed with the standard addition curve and calibration curve AA patterns between 1.0 to 25.0 mg/L phosphoric acid, 0.05 N, to determine the effect matrix in the meth‐ od of quantification. The proposal is a column Hypersil ODS C18, 5µm x 4.0 mm x 250 mm.

Ascorbic acid or Vitamin C (C6H8O6) can be determined by means of an iodometric titration. Vitamin C is a mild reducing agent that reacts rapidly with tri-iodide ion, this reaction is generated in a known excess of tri-iodide ion (I3-) by reacting iodate iodide, is allowed to react and then the excess is titrated by I3-back with a solution of thiosulphate. -The method

The recommended daily ingestion (intake) is of 60 to 100 mg to avoid the appearance of disease symptoms that are produced by deficiencies of this vitamin. The infants require a little more of 100mg/day, although there is controversy over the minimum amount of this vitamin. We must take into account that this vitamin is very labile at heat and oxy‐ gen presence. The ascorbic acid is specific in the treatment of scurvy; the required dose could be better measured by the urinary excretion after a saturation dose. Depending of the required saturation velocity is the daily dose recommended which varies between 0.2 and 2.0 g/d. In the vitamin C deficiency, the tissue saturation is obtained with 3 daily

performed with the following mobile phases:

Water-methanol-acetonitrile (74.4: 25.0: 0.6)

**6.3. Other methods reported**

is based on the following reactions:

<sup>8</sup>*<sup>I</sup>* <sup>−</sup> <sup>+</sup> *IO*<sup>3</sup> <sup>−</sup> <sup>+</sup> <sup>6</sup>*<sup>H</sup>* <sup>+</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>→</sup>3*H*2*<sup>O</sup>* <sup>+</sup> <sup>3</sup>*I*<sup>3</sup>

*<sup>I</sup>* <sup>3</sup><sup>−</sup> <sup>+</sup> 2(*S*2*O*3)<sup>−</sup><sup>2</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>→</sup><sup>3</sup> *<sup>I</sup>* <sup>−</sup> <sup>+</sup> (*S*4*O*6)<sup>−</sup><sup>2</sup> thiosulfate tetrathionate

Ascorbic acid dehydroascorbic acid

<sup>−</sup> <sup>+</sup> *<sup>H</sup>*2*<sup>O</sup>* <sup>+</sup> *<sup>I</sup>*<sup>3</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>→</sup>*C*6*H*8*O*<sup>6</sup> <sup>+</sup> <sup>2</sup>*<sup>H</sup>* <sup>+</sup> <sup>+</sup> <sup>3</sup>*<sup>I</sup>*

**7. Requirements and recommendations in Mexico**

Methanol-buffer solution: 0.03 M KH2PO4, pH = 2.7, (99:1)

2% KH2PO4, pH = 2.3

1% NaH2PO4, pH = 2.7

Iodometric titration

*C*6*H*8*O*<sup>6</sup>

Acetonitrile-Water (70:30)

### **6.1. Spectrophotometric method**

Another proposed method is highly sensitive colorimetric determination of ascorbic acid with 2,4-dinitrophenylhydrazine.

The method can be applied in virtually any laboratory is simple to perform and requires little complicated equipment compared to HPLC. Samples can be from serum to food. In foods the determination may be affected if the food contains natural dyes interfering reading the wavelength of detection. However, it is an easy to implement, since it is in‐ expensive and sensitive.

The use of UV-Visible spectroscopy (UV-Vis), for the determination of AA, is widely used in research with food, since this acid has strong electronic transitions in the UV region, facili‐ tating their identification and quantification by this technique.

Vitamin C is easily affected by such factors as moisture, light, air, heat, metal ions such as iron and copper, oxygen and the alkaline medium. After decomposition under these condi‐ tions is easily transformed into various compounds such as: oxalic acid, L-threonic acid, Lxylonic acid and L-dehydroascorbic acid, and in turn the latter are irreversibly transformed into acid-diketo 2.3 L gluconic acid, which is its main degradation product.

Within the official methods described for the analysis of vitamin C in tablet, one of the most widely used is the direct titration with 2,6-dichlorophenol indophenol by simple and rapid result. The method is valid if it is known that the composition of the sample no interfering substances and the concentration of dehydroascorbic acid is negligible, therefore, can be ap‐ plied to a freshly prepared sample, but not useful in stability studies of vitamin C.

### **6.2. Chromatographic method (HPLC)**

However, high performance liquid chromatography (HPLC) ensures detection and quantita‐ tion limits lower, which also facilitate the elimination of the effects caused by the matrix (in‐ terference to other methods of analysis); this technique used as an essential tool in detailed kinetic studies.

Quantification of Ascorbic Acid by HPLC.

Using a standard solution of 50 mg AA/L phosphoric acid, 0.05 N and taking into account the various reports, on the conditions for the quantification of AA in samples, it optimizes the mobile phase and the wavelength of the detector (scanning from 200 to 320 nm) to ob‐ tain the greatest sensitivity and resolution in the chromatographic signal. These tests are performed with the following mobile phases:

2% KH2PO4, pH = 2.3

and platelets which correlates good with the first signs of scurvy, making the most rec‐ ommended technique. The second method determines the tissue saturation grade for an intradermal test, using dichlorophenol indophenol, which depends of skin reductor

Another proposed method is highly sensitive colorimetric determination of ascorbic acid

The method can be applied in virtually any laboratory is simple to perform and requires little complicated equipment compared to HPLC. Samples can be from serum to food. In foods the determination may be affected if the food contains natural dyes interfering reading the wavelength of detection. However, it is an easy to implement, since it is in‐

The use of UV-Visible spectroscopy (UV-Vis), for the determination of AA, is widely used in research with food, since this acid has strong electronic transitions in the UV region, facili‐

Vitamin C is easily affected by such factors as moisture, light, air, heat, metal ions such as iron and copper, oxygen and the alkaline medium. After decomposition under these condi‐ tions is easily transformed into various compounds such as: oxalic acid, L-threonic acid, Lxylonic acid and L-dehydroascorbic acid, and in turn the latter are irreversibly transformed

Within the official methods described for the analysis of vitamin C in tablet, one of the most widely used is the direct titration with 2,6-dichlorophenol indophenol by simple and rapid result. The method is valid if it is known that the composition of the sample no interfering substances and the concentration of dehydroascorbic acid is negligible, therefore, can be ap‐

However, high performance liquid chromatography (HPLC) ensures detection and quantita‐ tion limits lower, which also facilitate the elimination of the effects caused by the matrix (in‐ terference to other methods of analysis); this technique used as an essential tool in detailed

Using a standard solution of 50 mg AA/L phosphoric acid, 0.05 N and taking into account the various reports, on the conditions for the quantification of AA in samples, it optimizes the mobile phase and the wavelength of the detector (scanning from 200 to 320 nm) to ob‐

substances, which made this nonspecific.

464 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

tating their identification and quantification by this technique.

**6.2. Chromatographic method (HPLC)**

Quantification of Ascorbic Acid by HPLC.

into acid-diketo 2.3 L gluconic acid, which is its main degradation product.

plied to a freshly prepared sample, but not useful in stability studies of vitamin C.

**6.1. Spectrophotometric method**

with 2,4-dinitrophenylhydrazine.

expensive and sensitive.

kinetic studies.

Acetonitrile-Water (70:30)

1% NaH2PO4, pH = 2.7

Methanol-buffer solution: 0.03 M KH2PO4, pH = 2.7, (99:1)

Water-methanol-acetonitrile (74.4: 25.0: 0.6)

The test is performed with the standard addition curve and calibration curve AA patterns between 1.0 to 25.0 mg/L phosphoric acid, 0.05 N, to determine the effect matrix in the meth‐ od of quantification. The proposal is a column Hypersil ODS C18, 5µm x 4.0 mm x 250 mm.

### **6.3. Other methods reported**

Iodometric titration

Ascorbic acid or Vitamin C (C6H8O6) can be determined by means of an iodometric titration. Vitamin C is a mild reducing agent that reacts rapidly with tri-iodide ion, this reaction is generated in a known excess of tri-iodide ion (I3-) by reacting iodate iodide, is allowed to react and then the excess is titrated by I3-back with a solution of thiosulphate. -The method is based on the following reactions:

<sup>8</sup>*<sup>I</sup>* <sup>−</sup> <sup>+</sup> *IO*<sup>3</sup> <sup>−</sup> <sup>+</sup> <sup>6</sup>*<sup>H</sup>* <sup>+</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>→</sup>3*H*2*<sup>O</sup>* <sup>+</sup> <sup>3</sup>*I*<sup>3</sup> *C*6*H*8*O*<sup>6</sup> <sup>−</sup> <sup>+</sup> *<sup>H</sup>*2*<sup>O</sup>* <sup>+</sup> *<sup>I</sup>*<sup>3</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>→</sup>*C*6*H*8*O*<sup>6</sup> <sup>+</sup> <sup>2</sup>*<sup>H</sup>* <sup>+</sup> <sup>+</sup> <sup>3</sup>*<sup>I</sup>* Ascorbic acid dehydroascorbic acid *<sup>I</sup>* <sup>3</sup><sup>−</sup> <sup>+</sup> 2(*S*2*O*3)<sup>−</sup><sup>2</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>→</sup><sup>3</sup> *<sup>I</sup>* <sup>−</sup> <sup>+</sup> (*S*4*O*6)<sup>−</sup><sup>2</sup> thiosulfate tetrathionate

### **7. Requirements and recommendations in Mexico**

The recommended daily ingestion (intake) is of 60 to 100 mg to avoid the appearance of disease symptoms that are produced by deficiencies of this vitamin. The infants require a little more of 100mg/day, although there is controversy over the minimum amount of this vitamin. We must take into account that this vitamin is very labile at heat and oxy‐ gen presence. The ascorbic acid is specific in the treatment of scurvy; the required dose could be better measured by the urinary excretion after a saturation dose. Depending of the required saturation velocity is the daily dose recommended which varies between 0.2 and 2.0 g/d. In the vitamin C deficiency, the tissue saturation is obtained with 3 daily doses of 700 mg each one for 3 days. Harris and cols. defined the tissue saturation as a sufficient storage of ascorbic acid where occurs a excretion of 50 mg or even more in a period of 4 to 5 hours after 1 dose of 700 mg/d.

**9.2. Interaction with ROS**

substrate of the oxidative damage16.

Vitamin C quickly debug the reactive species of oxygen and nitrogen just as superoxide, hi‐ droperoxile radicals, aqueous peroxyl radicals, singlet oxygen, ozone, peroxynitrite, nitro‐ gen dioxide, nitroxide radicals and hypochlorous acid thereby protecting in fact other

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

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Although the AA (ascorbic acid) reacts quickly with the hydroxyl radicals (constant speed > 109 Lmol-1s-1) is clumsy to debug this radical preferentially over other substrates. This is be‐ cause hydroxyl radicals are very reactive and they will combine immediately with nearest substratum in their environment at a limited speed because of its diffusion. Vitamin C can also act as a co-antioxidant when regenerate the α-tocopherol (vitamin E) from the α-toco‐ pheroxil radical produced when this is debugged from the lipid-soluble radicals just made. This is a function potentially important because in the *in vitro* experiments have shown that α-tocopherol can act as a pro-oxidant in absentia of co-oxidants just as vitamin C. However

AA can regenerate urates, glutathione and β-carotene *in vitro* from their respective oxidation products with one unpaired electron (urate radicals, glutathionil radicals and cations of β-caro‐ tene radicals). Two important properties of vitamin C make it an ideal antioxidant. The first one is its low potential reduction of ascorbate (282 mV) and its oxidation product with an elec‐ tron, the ascorbile radical (2174 mV), which is derivates from its functional group en-diol in the molecule. This low potential of reduction of the ascorbate and the ascorbile radical makes them potentially appropriate for oxidation-reduction reactions and that why the vitamin acts as a soluble antioxidant terminal molecule. The second property which makes the vitamin an ideal antioxidant is the stability and the low reactivity of the just made ascorbile radical when the as‐

The ascorbyl radical disproportionate rapidly to form ascorbate and dehydroascorbic acid (equations 1 and 2), or it is retro-reduced to ascorbate by an enzyme semi-dehydroascorbate reductase dependent of NADH. The oxidation product of two AA electrons, dehydroascor‐ bic acid, can be reduced by itself to ascorbate because of the glutathione, by enzymes de‐ pendents of glutathione: glutaredoxin (dehydroascorbate oxidoreductase [glutathione dehydrogenase (ascorbate)]) or by an enzyme dependent of selenium (seleno-enzyme): the tioredoxine reductase. Alternatively, the dehydroascorbic acid gets quickly and irreversibly

Where in equation 1 shows the reversible oxidation of 2 ascorbate electrons (AH2) to the as‐ corbile radical (A•2) and dehydroascorbic acid (A) respectively; the equation 2 shows the

« «• **AH A A 2 2** (1)

• • +®+ **A A AH A 22 2** (2)

**A ADCG** ® ® oxalate, threonate, etc. (3)

the relevance *in vivo* of the interaction of both vitamins it's not that clear yet.

corbate debug the reactive species of oxygen and nitrogen. (equation 1).

hydrolyzed to 2,3-dicetogulonic acid (ADCG) (equation 3):

The decreased levels in smokers are basically explained because they consume fewer sour‐ ces of the vitamin. In this kind of population will be required a 50% more of the recom‐ mended dose of the vitamin. Because of the daily recommendation (RDA) it is defined as the daily ingestion average of food which is enough to cover the nutriments required for healthy people in a group of the population, it's necessary to continually assess these recom‐ mendations for vitamin C. The totalities of the reviewed information suggest that a con‐ sumption of 90-100 mg of this vitamin is enough for the optimum reduction of chronic disease risk in non-smoking men and women. Although some reports are suggested amounts up to 120 mg/day.

### **8. Toxicity and hypersensitivity**

High doses of the vitamin (5-15 g/day), may cause osmotic diarrhea because it is ingested more vitamin of which can be absorbed. Also ascorbic acid can provoke intestinal cramps and acidification of the urine, leading to the formation of oxalate stones in the kidney of uri‐ nary tract. An exaggerated complementation during pregnancy may high the fetal require‐ ment and result in the presence of scurvy in the newborn. It is also credited with the destruction of vitamin B12 of food during the ingestion.

Since the oxalic acid is a metabolite of the catabolism of the ascorbic acid is likely to be the formation of oxalate crystals in kidney in patient's susceptible to nucleation and therefore the formation of crystals or "kidney stones" when it is consumed excess of the vitamin. This relation however does not extend to subjects which are not susceptible to the formation of these kidney stones.

### **9. Biochemical functions**

#### **9.1. Paper as an antioxidant**

Vitamin C is a soluble antioxidant important in biological fluids. An antioxidant is defined as "any substance which, when present in lower concentrations compared with the oxidable substrates (for example, proteins, lipids and carbohydrates and even nucleic acids) avoids or prevent significantly the oxidation of this substratum". The definition also given by the Food Nutrition Board is "A dietary antioxidant is a substance present in food which decreases sig‐ nificantly the adverse effects of the reactive species of oxygen (ROS), reactive species of ni‐ trogen (RNS), or both for the normal physiology function in humans.

### **9.2. Interaction with ROS**

doses of 700 mg each one for 3 days. Harris and cols. defined the tissue saturation as a sufficient storage of ascorbic acid where occurs a excretion of 50 mg or even more in a

The decreased levels in smokers are basically explained because they consume fewer sour‐ ces of the vitamin. In this kind of population will be required a 50% more of the recom‐ mended dose of the vitamin. Because of the daily recommendation (RDA) it is defined as the daily ingestion average of food which is enough to cover the nutriments required for healthy people in a group of the population, it's necessary to continually assess these recom‐ mendations for vitamin C. The totalities of the reviewed information suggest that a con‐ sumption of 90-100 mg of this vitamin is enough for the optimum reduction of chronic disease risk in non-smoking men and women. Although some reports are suggested

High doses of the vitamin (5-15 g/day), may cause osmotic diarrhea because it is ingested more vitamin of which can be absorbed. Also ascorbic acid can provoke intestinal cramps and acidification of the urine, leading to the formation of oxalate stones in the kidney of uri‐ nary tract. An exaggerated complementation during pregnancy may high the fetal require‐ ment and result in the presence of scurvy in the newborn. It is also credited with the

Since the oxalic acid is a metabolite of the catabolism of the ascorbic acid is likely to be the formation of oxalate crystals in kidney in patient's susceptible to nucleation and therefore the formation of crystals or "kidney stones" when it is consumed excess of the vitamin. This relation however does not extend to subjects which are not susceptible to the formation of

Vitamin C is a soluble antioxidant important in biological fluids. An antioxidant is defined as "any substance which, when present in lower concentrations compared with the oxidable substrates (for example, proteins, lipids and carbohydrates and even nucleic acids) avoids or prevent significantly the oxidation of this substratum". The definition also given by the Food Nutrition Board is "A dietary antioxidant is a substance present in food which decreases sig‐ nificantly the adverse effects of the reactive species of oxygen (ROS), reactive species of ni‐

trogen (RNS), or both for the normal physiology function in humans.

period of 4 to 5 hours after 1 dose of 700 mg/d.

466 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

amounts up to 120 mg/day.

these kidney stones.

**9. Biochemical functions**

**9.1. Paper as an antioxidant**

**8. Toxicity and hypersensitivity**

destruction of vitamin B12 of food during the ingestion.

Vitamin C quickly debug the reactive species of oxygen and nitrogen just as superoxide, hi‐ droperoxile radicals, aqueous peroxyl radicals, singlet oxygen, ozone, peroxynitrite, nitro‐ gen dioxide, nitroxide radicals and hypochlorous acid thereby protecting in fact other substrate of the oxidative damage16.

Although the AA (ascorbic acid) reacts quickly with the hydroxyl radicals (constant speed > 109 Lmol-1s-1) is clumsy to debug this radical preferentially over other substrates. This is be‐ cause hydroxyl radicals are very reactive and they will combine immediately with nearest substratum in their environment at a limited speed because of its diffusion. Vitamin C can also act as a co-antioxidant when regenerate the α-tocopherol (vitamin E) from the α-toco‐ pheroxil radical produced when this is debugged from the lipid-soluble radicals just made. This is a function potentially important because in the *in vitro* experiments have shown that α-tocopherol can act as a pro-oxidant in absentia of co-oxidants just as vitamin C. However the relevance *in vivo* of the interaction of both vitamins it's not that clear yet.

AA can regenerate urates, glutathione and β-carotene *in vitro* from their respective oxidation products with one unpaired electron (urate radicals, glutathionil radicals and cations of β-caro‐ tene radicals). Two important properties of vitamin C make it an ideal antioxidant. The first one is its low potential reduction of ascorbate (282 mV) and its oxidation product with an elec‐ tron, the ascorbile radical (2174 mV), which is derivates from its functional group en-diol in the molecule. This low potential of reduction of the ascorbate and the ascorbile radical makes them potentially appropriate for oxidation-reduction reactions and that why the vitamin acts as a soluble antioxidant terminal molecule. The second property which makes the vitamin an ideal antioxidant is the stability and the low reactivity of the just made ascorbile radical when the as‐ corbate debug the reactive species of oxygen and nitrogen. (equation 1).

The ascorbyl radical disproportionate rapidly to form ascorbate and dehydroascorbic acid (equations 1 and 2), or it is retro-reduced to ascorbate by an enzyme semi-dehydroascorbate reductase dependent of NADH. The oxidation product of two AA electrons, dehydroascor‐ bic acid, can be reduced by itself to ascorbate because of the glutathione, by enzymes de‐ pendents of glutathione: glutaredoxin (dehydroascorbate oxidoreductase [glutathione dehydrogenase (ascorbate)]) or by an enzyme dependent of selenium (seleno-enzyme): the tioredoxine reductase. Alternatively, the dehydroascorbic acid gets quickly and irreversibly hydrolyzed to 2,3-dicetogulonic acid (ADCG) (equation 3):

$$\mathbf{^\star A}\mathbf{H}\_2 \leftrightarrow \mathbf{A} \bullet \mathbf{^\star}\_2 \leftrightarrow \mathbf{A} \tag{1}$$

$$\mathbf{A}\bullet\_{2} + \mathbf{A}\bullet\_{2} \to \mathbf{A}\mathbf{H}\_{2} + \mathbf{A} \tag{2}$$

$$\mathbf{A} \rightarrow \mathbf{A} \mathbf{DCG} \rightarrow \text{oxalate}, \text{ threeone}, \text{etc.} \tag{3}$$

Where in equation 1 shows the reversible oxidation of 2 ascorbate electrons (AH2) to the as‐ corbile radical (A•2) and dehydroascorbic acid (A) respectively; the equation 2 shows the dismutation of the ascorbile radical to transform ascorbate and dehydroascorbic acid; and equation 3 show the hydrolysis of the dehydroascorbic acid to ADCG, which decomposes to oxalate, treonate y many other products. Vitamin C has been recognized and accepted by the FDA as one of 4 dietary antioxidants, the other 3 are vitamin E, β-carotene precursor of vitamin A and selenium as an essential component of antioxidant enzymes glutathione per‐ oxidase (GPx) and thioredoxin reductase.

Although there is substantial scientific evidence of the role of antioxidant vitamin C and its effects on human health are needed more research that guarantee the role of vitamin both *in vivo* and *in vitro*, particularly because the AA is a redox active compound, which may act not only as an antioxidant but also as pro-oxidant in the presence of ion redox active transition metal.

The reduction of metallic ions like iron and copper for the vitamin C *in vitro* (equation 4) results in the formation of hydroxyl radicals highly reactive way to the reaction of this ions with hydrogen peroxide, a process known as the Fenton chemistry (equation 5), The lipid hydroperoxides can also "break" because of reduced metallic ions, forming lipid alkoxy radi‐ cals (equation 6) which can begin and spread chain reactions of the lipidic peroxidation. However the shown mechanism in the equation 5 requires the availability of free ions, redox active metallic ions and a low index vitamin C/metallic ion, conditions unlikely under nor‐ mal conditions *in vivo*. Although has been shown that in biological fluids like plasma, the vitamin C acts like an antioxidant towards the lipids even in presence of free active ions.

$$\mathbf{A}\mathbf{H}\_2 + \mathbf{M}(n+1) \to \mathbf{A}\bullet\_2 + \mathbf{M}n + \mathbf{H} + \tag{4}$$

**9.3. Molecular mechanisms intracellular**

quired for the synthesis of catecholamine.

**10. Ascorbic acid in cancer**

The molecular mechanisms of the anti-scurvy effect of vitamin C are very broad and so low studied. Also Vitamin C is a cofactor of many involved enzymes in the collagen biosynthe‐ sis, carnitine and neurotransmitters. The pro-collagen dioxygenase (proline hydroxylase) and the procolagene-lisine-5-dioxigenese are two enzymes involved in the synthesis of the collagen which needs ascorbic acids for maximum activity. The posttranslational hydroxyla‐ tion of the lysine and proline residues of these enzymes are indispensable for the synthesis and formation of the stable helix which forms the collagen. So that the difference of the vita‐ min leads to the formation of weak structures causing lost of teeth, pain in joints, disorders

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

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469

Two dioxigenases involved in the carnitine synthesis also require vitamin C for its activity. Carnitine is essential for the transport of long chain fatty acids to the mitochondria so one deficiency of vitamin C will bring consequences just as fatigue and lethargy which are late symptoms of scurvy. Besides that the vitamin C is a cofactor for the synthesis of catechola‐ mines, in particular for the conversion of dopamine to norepinephrine catalyzed by the en‐ zyme dopamine-β-monooxygenase. Depression, hypochondriasis, and behavioral changes

Other kind of enzymes where vitamin C acts as a cofactor are the ones involved in the pepti‐ des amidations and in the tyrosine metabolism (this are also of the mono and dioxygenases kind). It is also implicated in the cholesterol metabolism to bile acids, way of 7-α-monooxy‐ genase and in the adrenal steroids metabolism. The hydroxylation of aromatic drugs and

The role of vitamin C, due to its redox potential is to reduce metal ions present in the active sites of enzymes mono and dioxygenases. Ascorbate for instance acts as a co substrate in these reactions, not as a coenzyme. The reduction of iron, involved by the presence of vita‐ min improves the intestinal absorption of dietary non heme iron. Other proposals include the maintenance of the thiol groups of proteins, keeping in its reduced form of glutathione addition, a cellular antioxidant and enzyme cofactor, and tetrahydrofolate as a cofactor re‐

The recommended daily allowance (RDA) for ascorbic acid varies from 100 to 120 mg/day for adults. Have been attributed many benefits just like its antioxidant power, antiathero‐ genic, anticarcinogenic, immunomodulatory and anti-cold. However these benefits have been subject of debate and controversies because of the danger in the use of mega doses of‐ ten used and its prooxidant effects and antioxidants. Discussed even if ascorbic acid cause cancer or promote or interfere with cancer therapy, the experts panels of dietary antioxi‐

carcinogens by cythocrome P-450, gets better also by reducing agents like vitamin C.

of the connective tissue, and poor healing, characteristic signs of scurvy.

are common in scurvy as a result of deficient dopamine hydroxylation.

*9.3.1. As an enzyme cofactor*

$$\bullet \text{ H}\_2\text{O}\_2 + \text{Mn} \rightarrow \bullet \text{OH}^\cdot + \text{OH}\_2 + \text{M} \,(n+1) \tag{5}$$

$$\bullet \text{ LOOH} + \text{Mn} \rightarrow \text{LO} \\ \bullet \text{ } + \text{OH}\_2 + \text{M} \\ \text{(}n+\text{1)} \tag{6}$$

Where in equation 4 shows the reduction of metallic active redox ions M(*n*+1)] because of the ascorbate to form de ascorbile radical and the reduced metal (M*n),* the equation 5 shows the productions of hydroxyl radicals highly reactive (•OH) of the reaction of the hydrogen peroxide (H2O2) with the reduced metallic ions and the equation 6 shows the reaction of the lipid hydroperoxides (LOOH) with the reduced metallic ions to form alkoxy radicals (LO•). Although there no convincing evidence of a prooxidant effect of vitamin C on humans, exist a substantial evidence of its antioxidant activity. Interestingly its antioxidant activity does not correlate directly in its anti-curvy effect.

Because of this, the experts considerate that if the antioxidant activity of Vitamin C is accept‐ ed *in vivo* and that if this is relevant for human health, then scurvy should not be considerate as the only criterion for the nutritional fitness or for determine the ideal quantity or required of the vitamin.

### **9.3. Molecular mechanisms intracellular**

#### *9.3.1. As an enzyme cofactor*

dismutation of the ascorbile radical to transform ascorbate and dehydroascorbic acid; and equation 3 show the hydrolysis of the dehydroascorbic acid to ADCG, which decomposes to oxalate, treonate y many other products. Vitamin C has been recognized and accepted by the FDA as one of 4 dietary antioxidants, the other 3 are vitamin E, β-carotene precursor of vitamin A and selenium as an essential component of antioxidant enzymes glutathione per‐

Although there is substantial scientific evidence of the role of antioxidant vitamin C and its effects on human health are needed more research that guarantee the role of vitamin both *in vivo* and *in vitro*, particularly because the AA is a redox active compound, which may act not only as an antioxidant but also as pro-oxidant in the presence of ion redox

The reduction of metallic ions like iron and copper for the vitamin C *in vitro* (equation 4) results in the formation of hydroxyl radicals highly reactive way to the reaction of this ions with hydrogen peroxide, a process known as the Fenton chemistry (equation 5), The lipid hydroperoxides can also "break" because of reduced metallic ions, forming lipid alkoxy radi‐ cals (equation 6) which can begin and spread chain reactions of the lipidic peroxidation. However the shown mechanism in the equation 5 requires the availability of free ions, redox active metallic ions and a low index vitamin C/metallic ion, conditions unlikely under nor‐ mal conditions *in vivo*. Although has been shown that in biological fluids like plasma, the vitamin C acts like an antioxidant towards the lipids even in presence of free active ions.

Where in equation 4 shows the reduction of metallic active redox ions M(*n*+1)] because of the ascorbate to form de ascorbile radical and the reduced metal (M*n),* the equation 5 shows the productions of hydroxyl radicals highly reactive (•OH) of the reaction of the hydrogen peroxide (H2O2) with the reduced metallic ions and the equation 6 shows the reaction of the lipid hydroperoxides (LOOH) with the reduced metallic ions to form alkoxy radicals (LO•). Although there no convincing evidence of a prooxidant effect of vitamin C on humans, exist a substantial evidence of its antioxidant activity. Interestingly its antioxidant activity does

Because of this, the experts considerate that if the antioxidant activity of Vitamin C is accept‐ ed *in vivo* and that if this is relevant for human health, then scurvy should not be considerate as the only criterion for the nutritional fitness or for determine the ideal quantity or required

+ +® + + + • ( ) **AH M 1 A M H 2 2** *n n* (4)

+® + + + • ( ) **H O M OH OH M 1 2 2 <sup>2</sup>** *n n* (5)

• +® + + + ( ) **<sup>2</sup> LOOH M LO OH M 1** *n n* (6)

oxidase (GPx) and thioredoxin reductase.

468 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

not correlate directly in its anti-curvy effect.

of the vitamin.

active transition metal.

The molecular mechanisms of the anti-scurvy effect of vitamin C are very broad and so low studied. Also Vitamin C is a cofactor of many involved enzymes in the collagen biosynthe‐ sis, carnitine and neurotransmitters. The pro-collagen dioxygenase (proline hydroxylase) and the procolagene-lisine-5-dioxigenese are two enzymes involved in the synthesis of the collagen which needs ascorbic acids for maximum activity. The posttranslational hydroxyla‐ tion of the lysine and proline residues of these enzymes are indispensable for the synthesis and formation of the stable helix which forms the collagen. So that the difference of the vita‐ min leads to the formation of weak structures causing lost of teeth, pain in joints, disorders of the connective tissue, and poor healing, characteristic signs of scurvy.

Two dioxigenases involved in the carnitine synthesis also require vitamin C for its activity. Carnitine is essential for the transport of long chain fatty acids to the mitochondria so one deficiency of vitamin C will bring consequences just as fatigue and lethargy which are late symptoms of scurvy. Besides that the vitamin C is a cofactor for the synthesis of catechola‐ mines, in particular for the conversion of dopamine to norepinephrine catalyzed by the en‐ zyme dopamine-β-monooxygenase. Depression, hypochondriasis, and behavioral changes are common in scurvy as a result of deficient dopamine hydroxylation.

Other kind of enzymes where vitamin C acts as a cofactor are the ones involved in the pepti‐ des amidations and in the tyrosine metabolism (this are also of the mono and dioxygenases kind). It is also implicated in the cholesterol metabolism to bile acids, way of 7-α-monooxy‐ genase and in the adrenal steroids metabolism. The hydroxylation of aromatic drugs and carcinogens by cythocrome P-450, gets better also by reducing agents like vitamin C.

The role of vitamin C, due to its redox potential is to reduce metal ions present in the active sites of enzymes mono and dioxygenases. Ascorbate for instance acts as a co substrate in these reactions, not as a coenzyme. The reduction of iron, involved by the presence of vita‐ min improves the intestinal absorption of dietary non heme iron. Other proposals include the maintenance of the thiol groups of proteins, keeping in its reduced form of glutathione addition, a cellular antioxidant and enzyme cofactor, and tetrahydrofolate as a cofactor re‐ quired for the synthesis of catecholamine.

### **10. Ascorbic acid in cancer**

The recommended daily allowance (RDA) for ascorbic acid varies from 100 to 120 mg/day for adults. Have been attributed many benefits just like its antioxidant power, antiathero‐ genic, anticarcinogenic, immunomodulatory and anti-cold. However these benefits have been subject of debate and controversies because of the danger in the use of mega doses of‐ ten used and its prooxidant effects and antioxidants. Discussed even if ascorbic acid cause cancer or promote or interfere with cancer therapy, the experts panels of dietary antioxi‐ dants and related compounds have been concluded that the data *in vivo* does not shows clearly a direct relation between the excess ingestion and the formation of kidney stones, the prooxidant effects and the excess absorption of iron.

that the cytotoxicity induced by vitamin C is mediated primarily by the formation of hy‐ drogen peroxide in the cell surface. The cytotoxic activity may also be mediated by the presence of cupric ions (Cu2+) in malignant melanoma cells that react with vitamin C to form free radicals in solution. Vitamin C also invests into cells, transforming them chem‐

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

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471

Studies of cell surface and ultrastructure suggest that cancer cells after administration of vi‐ tamin C had cytolysis, cell membrane damage, mitochondrial changes, nuclear and nucleo‐ lar reduction and an increase in the formation of phagolysosomes. Changes in cell surface as cytolysis showed predominantly increased synthesis of collagen and disruption of the cell

The quantitative estimation of cellular organelles shown that vitamin C affects the intracel‐ lular distribution of the organelles, event that plays an important role in the citodifferentia‐ tion of the carcinogenic cell and this is the shared effect that not only vitamin C has, but also vitamin A and E. Changes in the Golgi complex and apoptotic activity and autophagic addi‐ tion to changes in cell surface and in some cases even the reversal of transformed cells to their normal cell types are needed in the possible reduction in incidence of various cancers.

Have also been associated changes in the protein synthesis, DNA and ARA with the dif‐ ferentiation and proliferation cell. But these mechanisms are not clear yet. It have been mentioned that many of this metabolic effects are mediated by the transcription and translation at genomic level. This vitamins modulate the DNA synthesis and the genetic expression in a similarly to hormones and steroids.Their effects can affect the chemical mutagenicity and the cell status. The vitamins can control the cell replication affecting the DNA, RNA and proteins in specific places which are target of electrophyles, promot‐ ing the rearrangement of codons in the altered cells and the translocation of specific genes or carcinogens. In this way, vitamins A, E and C affects directly the DNA, RNA

The vitamin C administration decrease the DNA synthesis in the core, the RNA synthesis in the nucleolus and the protein in the cytoplasm of these cells. This inhibition is accompanied

Have been proposed many mechanisms of the vitamin C activity in the prevention and

**3.** Inhibition of the hyaluronidase, keeping the substances around the tumor intact avoid‐

**1.** Would improve the immune system by increasing the lymphocytes production.

**5.** Correction of a likely ascorbate deficiency, seen in patients with cancer

by ultrastructural changes mentioned which decreases the cancer progression.

ically to a normal phenotype fine.

membrane with increased phagocytic activity and apoptotic.

and the protein synthesis in the carcinogen cells.

**2.** Stimulation in the collagen synthesis.

**Mechanism of action:**

treatment of cancer:

ing metastasis.

**4.** Inhibition of carcinogen virus

The epidemiological and clinic study does not shows conclusive benefic effects in many kinds of cancer, with the exception of stomach cancer. Recently it has tested several deriva‐ tives of ascorbic acid on cancer cells as ascorbic acid spheres. The ascorbyl stearate is a com‐ pound which inhibits the human carcinogenic cell proliferation, by interfering with the progression of the cellular cycle and inducing apoptosis by modulation of signal transduc‐ tion pathways. The cancer is a global public health problem with increasing levels of mortal‐ ity. Although exists a great variety and types of cancer, we can remark the role of vitamin C and its effects in this suffering. AA is effective protecting against the oxidative damage in tissue and also suppressing the carcinogens formation like nitrosamines.

Although vitamin C is a cytotoxic agent for tumor cells and non toxic for normal cells, in modern medicine and conventional favors more the use of powerful toxic chemotherapeutic agents. A great amount of studies have shown that the consumption of vitamin C is inverse‐ ly related with cancer with protective effects in cancer of lung, pancreas, stomach, cervix, rectum and oral cavity.

The guanine oxidation, a DNA purine, gets reduced significantly after the vitamin C supple‐ mentation, but the adenine oxidation, another purine, it's up high which suggest the antioxi‐ dant role of the vitamin. Other extensive studies both *in vivo* and *in vitro* have shown its ability to prevent, reduce or increase the adverse effects of chemotherapy. The combination of vitamin C and vitamin K already given in the chemotherapy increases the survival and the effects of various chemotherapeutic agents in a tumor-ascitic-murine model. The vitamin C has shown be safe even with the radiotherapy. The co administration of vitamin A, β-caro‐ tene, E and C can reduce the incidence and delay the progression of several cancers, such as skin, colon, stomach, esophagus, mammary gland and matrix.

Epidemiologic studies have revealed an inverse relation between the consumption of vi‐ tamin A, β-carotene, E and C and the incidence of several human cancers. There are a decrease in the risk and incidence of cancer in populations with high content of vitamins in plasma. The carcinogenesis is related with the cell differentiation, progression, metabo‐ lism and synthesis of collagen. The basic mechanism for the carcinogenesis is the cell dif‐ ferentiation because the cancer develops when a lost in this differentiation exists. And here is where the mentioned vitamins have a wide influence over de cell growth and its differentiation. Vitamin C is a strong antioxidant that acts synergistically with vitamin E in the purification of free radicals which are carcinogenic. The AA as sodium ascorbate exerts marked cytotoxic effects over many human cell lines when they are cultured. These effects are dose dependent.

Lupulescu reported that vitamin C (up to 200 ug/mL) did not cause any morphological change in mouse melanoma, neuroblastoma, and mouse and rat gliomas but is lethal for neuroblastoma cells. Cytotoxic effects are dependent cell also because they are stronger in human melanoma cells compared to mouse melanoma. It has even been suggested that the cytotoxicity induced by vitamin C is mediated primarily by the formation of hy‐ drogen peroxide in the cell surface. The cytotoxic activity may also be mediated by the presence of cupric ions (Cu2+) in malignant melanoma cells that react with vitamin C to form free radicals in solution. Vitamin C also invests into cells, transforming them chem‐ ically to a normal phenotype fine.

Studies of cell surface and ultrastructure suggest that cancer cells after administration of vi‐ tamin C had cytolysis, cell membrane damage, mitochondrial changes, nuclear and nucleo‐ lar reduction and an increase in the formation of phagolysosomes. Changes in cell surface as cytolysis showed predominantly increased synthesis of collagen and disruption of the cell membrane with increased phagocytic activity and apoptotic.

The quantitative estimation of cellular organelles shown that vitamin C affects the intracel‐ lular distribution of the organelles, event that plays an important role in the citodifferentia‐ tion of the carcinogenic cell and this is the shared effect that not only vitamin C has, but also vitamin A and E. Changes in the Golgi complex and apoptotic activity and autophagic addi‐ tion to changes in cell surface and in some cases even the reversal of transformed cells to their normal cell types are needed in the possible reduction in incidence of various cancers.

Have also been associated changes in the protein synthesis, DNA and ARA with the dif‐ ferentiation and proliferation cell. But these mechanisms are not clear yet. It have been mentioned that many of this metabolic effects are mediated by the transcription and translation at genomic level. This vitamins modulate the DNA synthesis and the genetic expression in a similarly to hormones and steroids.Their effects can affect the chemical mutagenicity and the cell status. The vitamins can control the cell replication affecting the DNA, RNA and proteins in specific places which are target of electrophyles, promot‐ ing the rearrangement of codons in the altered cells and the translocation of specific genes or carcinogens. In this way, vitamins A, E and C affects directly the DNA, RNA and the protein synthesis in the carcinogen cells.

The vitamin C administration decrease the DNA synthesis in the core, the RNA synthesis in the nucleolus and the protein in the cytoplasm of these cells. This inhibition is accompanied by ultrastructural changes mentioned which decreases the cancer progression.

#### **Mechanism of action:**

dants and related compounds have been concluded that the data *in vivo* does not shows clearly a direct relation between the excess ingestion and the formation of kidney stones, the

The epidemiological and clinic study does not shows conclusive benefic effects in many kinds of cancer, with the exception of stomach cancer. Recently it has tested several deriva‐ tives of ascorbic acid on cancer cells as ascorbic acid spheres. The ascorbyl stearate is a com‐ pound which inhibits the human carcinogenic cell proliferation, by interfering with the progression of the cellular cycle and inducing apoptosis by modulation of signal transduc‐ tion pathways. The cancer is a global public health problem with increasing levels of mortal‐ ity. Although exists a great variety and types of cancer, we can remark the role of vitamin C and its effects in this suffering. AA is effective protecting against the oxidative damage in

Although vitamin C is a cytotoxic agent for tumor cells and non toxic for normal cells, in modern medicine and conventional favors more the use of powerful toxic chemotherapeutic agents. A great amount of studies have shown that the consumption of vitamin C is inverse‐ ly related with cancer with protective effects in cancer of lung, pancreas, stomach, cervix,

The guanine oxidation, a DNA purine, gets reduced significantly after the vitamin C supple‐ mentation, but the adenine oxidation, another purine, it's up high which suggest the antioxi‐ dant role of the vitamin. Other extensive studies both *in vivo* and *in vitro* have shown its ability to prevent, reduce or increase the adverse effects of chemotherapy. The combination of vitamin C and vitamin K already given in the chemotherapy increases the survival and the effects of various chemotherapeutic agents in a tumor-ascitic-murine model. The vitamin C has shown be safe even with the radiotherapy. The co administration of vitamin A, β-caro‐ tene, E and C can reduce the incidence and delay the progression of several cancers, such as

Epidemiologic studies have revealed an inverse relation between the consumption of vi‐ tamin A, β-carotene, E and C and the incidence of several human cancers. There are a decrease in the risk and incidence of cancer in populations with high content of vitamins in plasma. The carcinogenesis is related with the cell differentiation, progression, metabo‐ lism and synthesis of collagen. The basic mechanism for the carcinogenesis is the cell dif‐ ferentiation because the cancer develops when a lost in this differentiation exists. And here is where the mentioned vitamins have a wide influence over de cell growth and its differentiation. Vitamin C is a strong antioxidant that acts synergistically with vitamin E in the purification of free radicals which are carcinogenic. The AA as sodium ascorbate exerts marked cytotoxic effects over many human cell lines when they are cultured.

Lupulescu reported that vitamin C (up to 200 ug/mL) did not cause any morphological change in mouse melanoma, neuroblastoma, and mouse and rat gliomas but is lethal for neuroblastoma cells. Cytotoxic effects are dependent cell also because they are stronger in human melanoma cells compared to mouse melanoma. It has even been suggested

tissue and also suppressing the carcinogens formation like nitrosamines.

skin, colon, stomach, esophagus, mammary gland and matrix.

prooxidant effects and the excess absorption of iron.

470 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

rectum and oral cavity.

These effects are dose dependent.

Have been proposed many mechanisms of the vitamin C activity in the prevention and treatment of cancer:


quired to maintain the normal properties of collagen. The decrease in AA concentration in plasma and Prolasa activity in diabetes can be normalized by the inhibitor of aldose reduc‐ tase. We conclude that in diabetic animals there is a deficiency of AA, which may be respon‐

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

http://dx.doi.org/10.5772/52181

473

The decrease in plasma ascorbic acid in diabetes plays an important role in the abnormali‐ ties of collagen and proteoglycans. These are the 2 major constituents of the extracellular matrix and its abnormalities are associated with the pathogenesis and complications of dia‐ betes. The structural similarity between glucose metabolism and AA and can interact at the

Ascorbic acid enhances the collagen and proteoglycans in fibroblast culture media. This stimulatory action is inhibited by high concentrations of glucose (25 mM). This effect how‐ ever, is not mediated AA consumption of fibroblasts. Insulin removes the inhibitory effect of glucose on the production of collagen, but the mechanism is not yet known. Thus high con‐ centrations of glucose in diabetes damage the action of ascorbic acid at the cellular level.

High blood pressure is a powerful indicator of heart disease and stroke. And in many cases is "asymptomatic" or people who have it doing not give importance. However there have been great efforts to use its measurement in the detection of primary or secondary essential

Virtually the observed declines in blood pressure and its control in recent years due to better control among individuals diagnosed as hypertensive. In this regard dietary factor has been the best for control. Obesity, dietary sodium and alcohol consumption are strongly associat‐

A high intake of polyunsaturated fatty acids and magnesium are associated with for in‐ stance with low pressure. It has also shown an inverse association between plasma vita‐ min C and blood pressure. Subjects with serum levels of vitamin C equal to 0.5 mg/dL have a systolic pressure 122 mm Hg average compared to subjects with average pressure is 113 mm Hg and whose values of serum vitamin C are 0.9 mg/dL, showing a relative

These subjects have a similar difference in diastolic pressure ranging from 78 to 73 mm Hg, a difference of 6%. The prevalence of hypertension was 7.5% in the group of subjects with low serum vitamin C and only 1% in subjects with high values of the vitamin in the serum. These results were consistent in several studies regardless of quintiles being compared.

Such relationships have also been identified in Chinese-American population; both men and women aged 60-96 years without antihypertensive treatment. This study revealed a statisti‐ cally significant difference between the values of systolic and diastolic pressure in upper and lower quintiles of 14% (21 mm Hg) and 9% (8mmHg) respectively. It appears that vita‐

sible for the observed changes in collagen in diabetes.

**12. Ascorbic acid in essential hypertension (HAS)**

level of the membrane and transporters.

hypertension for decades.

difference of 7%.

ed with low or high blood pressure values.


Patients with cancer tend to immune-undertake, showing low levels of ascorbate in their lymphocytes. The survival of immune system is important both for inhibit the carcino‐ gen cell growth phase and to prevent its proliferation. The supplementation with ascor‐ bate increases the number and the effectiveness of the lymphocytes and upgrades the phagocythosis

The characteristics of the neoplastic cell and its behavior (invasiveness, selective nutrition and possibly accelerated growth) are caused by microenvironmental depolymerization. This destabilization of the matrix is favored by constant exposure to lysosomal glycosidases con‐ tinually released by the neoplastic cell. The AA is then involved in the control and restric‐ tion of this degradative enzyme activity.

The synthesis of collagen is a major factor for the encapsulation of tumors or metastases de‐ creased via the development of a nearly impermeable barrier. AA is necessary for synthesis of collagen and its stabilization. A loss of ascorbate significantly reduces the hydroxylation of proline and hydroxyproline and hydroxylysine to lysine respectively, affecting the cross linking of collagen. This disrupts the structure of collagen triple helix, which increases its catabolism s. *In vitro*, vitamin C also increases the synthesis of collagen in fibroblasts.

### **11. Ascorbic acid in diabetes mellitus**

It has been demonstrated *in vitro* competition between glucose and ascorbic acid by the cell membrane transporter, granulocytes and fibroblasts, and under conditions of substantial and significant changes in chemotaxis of PMN leukocytes and mononuclear cells. There are significant changes to various chemoattractants, and a significant correlation with the de‐ crease in AA.

These results are consistent with the hypothesis that the chronic hyperglycemia associated with leukocyte AA deficiency, an acute inflammatory response damaged and altered sus‐ ceptibility to infections, and failure to repair bleeding in these patients, further changes are observed sustained hyperglycemia.

The concentrations of ascorbic acid (AA) are decreased in tissues and plasma in diabetes. These values can be normalized with extra supplements of 20-40 mg/d or corresponding to its maximum synthetic rate. Treating diabetic rats with this scheme prevents the decreased activity of granulation tissue of proline hydroxylase (Prolasa) an AA-dependent enzyme, re‐ quired to maintain the normal properties of collagen. The decrease in AA concentration in plasma and Prolasa activity in diabetes can be normalized by the inhibitor of aldose reduc‐ tase. We conclude that in diabetic animals there is a deficiency of AA, which may be respon‐ sible for the observed changes in collagen in diabetes.

The decrease in plasma ascorbic acid in diabetes plays an important role in the abnormali‐ ties of collagen and proteoglycans. These are the 2 major constituents of the extracellular matrix and its abnormalities are associated with the pathogenesis and complications of dia‐ betes. The structural similarity between glucose metabolism and AA and can interact at the level of the membrane and transporters.

Ascorbic acid enhances the collagen and proteoglycans in fibroblast culture media. This stimulatory action is inhibited by high concentrations of glucose (25 mM). This effect how‐ ever, is not mediated AA consumption of fibroblasts. Insulin removes the inhibitory effect of glucose on the production of collagen, but the mechanism is not yet known. Thus high con‐ centrations of glucose in diabetes damage the action of ascorbic acid at the cellular level.

### **12. Ascorbic acid in essential hypertension (HAS)**

**6.** Adequate healing after the surgery.

**9.** Prevention of the cell damage by free radicals.

472 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**10.** Neutralization of carcinogenic substances.

tion of this degradative enzyme activity.

**11. Ascorbic acid in diabetes mellitus**

observed sustained hyperglycemia.

tina, DTIC and others.

phagocythosis

crease in AA.

**7.** Improvement in the effect of some chemotherapeutic agents, just like tamoxifen, cispla‐

Patients with cancer tend to immune-undertake, showing low levels of ascorbate in their lymphocytes. The survival of immune system is important both for inhibit the carcino‐ gen cell growth phase and to prevent its proliferation. The supplementation with ascor‐ bate increases the number and the effectiveness of the lymphocytes and upgrades the

The characteristics of the neoplastic cell and its behavior (invasiveness, selective nutrition and possibly accelerated growth) are caused by microenvironmental depolymerization. This destabilization of the matrix is favored by constant exposure to lysosomal glycosidases con‐ tinually released by the neoplastic cell. The AA is then involved in the control and restric‐

The synthesis of collagen is a major factor for the encapsulation of tumors or metastases de‐ creased via the development of a nearly impermeable barrier. AA is necessary for synthesis of collagen and its stabilization. A loss of ascorbate significantly reduces the hydroxylation of proline and hydroxyproline and hydroxylysine to lysine respectively, affecting the cross linking of collagen. This disrupts the structure of collagen triple helix, which increases its

It has been demonstrated *in vitro* competition between glucose and ascorbic acid by the cell membrane transporter, granulocytes and fibroblasts, and under conditions of substantial and significant changes in chemotaxis of PMN leukocytes and mononuclear cells. There are significant changes to various chemoattractants, and a significant correlation with the de‐

These results are consistent with the hypothesis that the chronic hyperglycemia associated with leukocyte AA deficiency, an acute inflammatory response damaged and altered sus‐ ceptibility to infections, and failure to repair bleeding in these patients, further changes are

The concentrations of ascorbic acid (AA) are decreased in tissues and plasma in diabetes. These values can be normalized with extra supplements of 20-40 mg/d or corresponding to its maximum synthetic rate. Treating diabetic rats with this scheme prevents the decreased activity of granulation tissue of proline hydroxylase (Prolasa) an AA-dependent enzyme, re‐

catabolism s. *In vitro*, vitamin C also increases the synthesis of collagen in fibroblasts.

**8.** Reduction of the toxicity of other chemotherapeutic agents like adriamicine.

High blood pressure is a powerful indicator of heart disease and stroke. And in many cases is "asymptomatic" or people who have it doing not give importance. However there have been great efforts to use its measurement in the detection of primary or secondary essential hypertension for decades.

Virtually the observed declines in blood pressure and its control in recent years due to better control among individuals diagnosed as hypertensive. In this regard dietary factor has been the best for control. Obesity, dietary sodium and alcohol consumption are strongly associat‐ ed with low or high blood pressure values.

A high intake of polyunsaturated fatty acids and magnesium are associated with for in‐ stance with low pressure. It has also shown an inverse association between plasma vita‐ min C and blood pressure. Subjects with serum levels of vitamin C equal to 0.5 mg/dL have a systolic pressure 122 mm Hg average compared to subjects with average pressure is 113 mm Hg and whose values of serum vitamin C are 0.9 mg/dL, showing a relative difference of 7%.

These subjects have a similar difference in diastolic pressure ranging from 78 to 73 mm Hg, a difference of 6%. The prevalence of hypertension was 7.5% in the group of subjects with low serum vitamin C and only 1% in subjects with high values of the vitamin in the serum. These results were consistent in several studies regardless of quintiles being compared.

Such relationships have also been identified in Chinese-American population; both men and women aged 60-96 years without antihypertensive treatment. This study revealed a statisti‐ cally significant difference between the values of systolic and diastolic pressure in upper and lower quintiles of 14% (21 mm Hg) and 9% (8mmHg) respectively. It appears that vita‐ min C has a lowering effect on systolic rather than diastolic pressure. Supplementation with vitamin C (1g/day) does not influence the diastolic pressure. Subjects with low vitamin C levels in serum have a high risk of developing stroke compared with those with high values in plasma of the vitamin. Hypertensive subjects, usually overweight, and low levels of se‐ rum vitamin C have the same risk.

but when cigarette smokers were excluded from the analysis, we obtain an association where smoking becomes a confounding variable. The relationship between fibrinogen, Fac‐ tor VIIc and ascorbate concentrations were consistent in subjects taking supplements of vita‐ min C during 1 year, which indicates that the homeostatic factors relate the variability in the

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

http://dx.doi.org/10.5772/52181

475

The inverse association between homeostatic factors and serum concentrations of ascorbate is strong and consistent, however only some markers of infection (e.g. C-reactive protein and α1-antichymotrypsin) are related inversely and significantly with serum ascorbate. It is possible that this low concentration of ascorbate may be the result rather than the cause, of a biological response to infection. The strong relationship between serum ascorbate and diet‐ ary intake suggest however that their serum concentrations reflect the nutritional status of

The various studies reported in the literature indicate that vitamin does not prevent respira‐ tory infection but may modulate the biological response, leading to less severe disease, so it

It has been suggested a protective effect of antioxidants such as vitamin C, A (β-carotene) and E plus selenium in cardiovascular disease. Prospective studies so far have documented an inverse relationship between vitamin C intake and cardiovascular disease, and a strong

Finnish and Swiss studies showed that blood levels of ascorbate and therefore a diminished nutritional status of vitamin predicts myocardial infarction. Low levels of vitamin C in‐ creased to 2.7 times the risk of myocardial infarction and this is independent of other risk factors. Mediterranean studies showed a 70% reduction in mortality and risk of myocardial infarction independent of the effect on blood pressure and lipids. Any protective effect on heart disease of these antioxidants is mediated by the oxidation of LDL cholesterol, but there

Fibrinogen and factor VII are recognized risk factors of myocardial infarction and stroke, in the same way that acute and chronic infection and increased white blood cell count are risk factors for cardiovascular disease. The infection may contribute to the inflammatory process

C-reactive protein and alpha-1 antichymotrypsin are acute phase proteins are synthesized in hepatocytes in large numbers in inflammatory processes. This synthesis is mediated primar‐ ily by IL-6 produced by monocytes and macrophages. Elevated fibrinogen favors these mechanisms and therefore an increased cardiovascular risk. In this way a reduction in diet‐ ary intake in winter for instance, would lead to lower serum ascorbate levels, an increase in susceptibility to infection and the factors haemostatic factors and therefore to an increase in

status of vitamin C within a range usual dietary.

has a protective function in lung function.

**13.1. Effect of antioxidants in cardiovascular disease**

protective effect of vitamin E supplementation on coronary patients.

may be other mechanisms of homeostasis and inflammation.

**13.2. Infection, homeostasis, and cardiovascular disease**

observed in atherosclerosis.

cardiovascular mortality.

the vitamin.

The increase in the consumption of vitamin C during periods of fat restriction occurs on the one hand a reduction in blood pressure. Low levels of AA in plasma are also associated with low concentrations of 6-keto-prostaglandin F, a prostacyclin. Thus dietary antioxidants en‐ hance the production of prostacyclin for the purification of free radicals and peroxides that inhibit prostacyclin synthase. Vitamin C and blood pressure then are related, because it has a lowering effect on blood pressure especially when fat intake is low.

### **13. Ascorbic acid and cardiovascular disease**

Vitamin C acts as a regulator of the catabolism of cholesterol into bile acids in the guinea pig and is an important factor in the regulation of lipid in several animal species (rabbit, horse, and rat).

Correlation studies in humans have shown an inverse relationship between vitamin C in‐ take and mortality from cardiovascular disease.

Experimental and observational studies in humans have been inconsistent but indicate that individuals with high cholesterol consumption, greater than or equal to 5.20mM/L (200mg/dL) and lower in tissue saturation, increase concentration of vitamin C, which may have a beneficial effect on total cholesterol. This effect is explained by the promotion or in‐ hibition of degradation of prostacyclin and its implications for thrombosis and atherogene‐ sis, in addition to its protective effect on lipid peroxidation. In patients with high cardiovascular risk, supplementation with antioxidant vitamins shows no reduction in over‐ all mortality or incidence of any vascular disease, cancer or other adverse events.

Recent findings indicate a relationship between the nutritional status of vitamin C (as meas‐ ured by the concentration of ascorbate in serum), biological markers of infection and haemo‐ static factors and support the hypothesis that vitamin C may protect against cardiovascular events through effects on the haemostatic factors in response to infection.

This relationship is surprising given the uncertainty and potential error in the estimation of consumption of vitamin and vitamin C status assessment (determined mostly by food intake records of 24 h blood samples isolated). Add to this the wide variation between subjects is greater than within the same subject.

Lower socioeconomic status and smoking are associated with low concentrations of ascor‐ bate and high concentrations of homeostatic factors that may be confounding factors in cross-sectional studies.

As expected smokers have lower concentrations of AA in serum than non-smokers. The re‐ lationship between concentrations of ascorbate and homeostatic factors is very consistent, but when cigarette smokers were excluded from the analysis, we obtain an association where smoking becomes a confounding variable. The relationship between fibrinogen, Fac‐ tor VIIc and ascorbate concentrations were consistent in subjects taking supplements of vita‐ min C during 1 year, which indicates that the homeostatic factors relate the variability in the status of vitamin C within a range usual dietary.

The inverse association between homeostatic factors and serum concentrations of ascorbate is strong and consistent, however only some markers of infection (e.g. C-reactive protein and α1-antichymotrypsin) are related inversely and significantly with serum ascorbate. It is possible that this low concentration of ascorbate may be the result rather than the cause, of a biological response to infection. The strong relationship between serum ascorbate and diet‐ ary intake suggest however that their serum concentrations reflect the nutritional status of the vitamin.

The various studies reported in the literature indicate that vitamin does not prevent respira‐ tory infection but may modulate the biological response, leading to less severe disease, so it has a protective function in lung function.

### **13.1. Effect of antioxidants in cardiovascular disease**

min C has a lowering effect on systolic rather than diastolic pressure. Supplementation with vitamin C (1g/day) does not influence the diastolic pressure. Subjects with low vitamin C levels in serum have a high risk of developing stroke compared with those with high values in plasma of the vitamin. Hypertensive subjects, usually overweight, and low levels of se‐

The increase in the consumption of vitamin C during periods of fat restriction occurs on the one hand a reduction in blood pressure. Low levels of AA in plasma are also associated with low concentrations of 6-keto-prostaglandin F, a prostacyclin. Thus dietary antioxidants en‐ hance the production of prostacyclin for the purification of free radicals and peroxides that inhibit prostacyclin synthase. Vitamin C and blood pressure then are related, because it has

Vitamin C acts as a regulator of the catabolism of cholesterol into bile acids in the guinea pig and is an important factor in the regulation of lipid in several animal species (rabbit, horse,

Correlation studies in humans have shown an inverse relationship between vitamin C in‐

Experimental and observational studies in humans have been inconsistent but indicate that individuals with high cholesterol consumption, greater than or equal to 5.20mM/L (200mg/dL) and lower in tissue saturation, increase concentration of vitamin C, which may have a beneficial effect on total cholesterol. This effect is explained by the promotion or in‐ hibition of degradation of prostacyclin and its implications for thrombosis and atherogene‐ sis, in addition to its protective effect on lipid peroxidation. In patients with high cardiovascular risk, supplementation with antioxidant vitamins shows no reduction in over‐

Recent findings indicate a relationship between the nutritional status of vitamin C (as meas‐ ured by the concentration of ascorbate in serum), biological markers of infection and haemo‐ static factors and support the hypothesis that vitamin C may protect against cardiovascular

This relationship is surprising given the uncertainty and potential error in the estimation of consumption of vitamin and vitamin C status assessment (determined mostly by food intake records of 24 h blood samples isolated). Add to this the wide variation between subjects is

Lower socioeconomic status and smoking are associated with low concentrations of ascor‐ bate and high concentrations of homeostatic factors that may be confounding factors in

As expected smokers have lower concentrations of AA in serum than non-smokers. The re‐ lationship between concentrations of ascorbate and homeostatic factors is very consistent,

all mortality or incidence of any vascular disease, cancer or other adverse events.

events through effects on the haemostatic factors in response to infection.

a lowering effect on blood pressure especially when fat intake is low.

**13. Ascorbic acid and cardiovascular disease**

474 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

take and mortality from cardiovascular disease.

greater than within the same subject.

cross-sectional studies.

rum vitamin C have the same risk.

and rat).

It has been suggested a protective effect of antioxidants such as vitamin C, A (β-carotene) and E plus selenium in cardiovascular disease. Prospective studies so far have documented an inverse relationship between vitamin C intake and cardiovascular disease, and a strong protective effect of vitamin E supplementation on coronary patients.

Finnish and Swiss studies showed that blood levels of ascorbate and therefore a diminished nutritional status of vitamin predicts myocardial infarction. Low levels of vitamin C in‐ creased to 2.7 times the risk of myocardial infarction and this is independent of other risk factors. Mediterranean studies showed a 70% reduction in mortality and risk of myocardial infarction independent of the effect on blood pressure and lipids. Any protective effect on heart disease of these antioxidants is mediated by the oxidation of LDL cholesterol, but there may be other mechanisms of homeostasis and inflammation.

#### **13.2. Infection, homeostasis, and cardiovascular disease**

Fibrinogen and factor VII are recognized risk factors of myocardial infarction and stroke, in the same way that acute and chronic infection and increased white blood cell count are risk factors for cardiovascular disease. The infection may contribute to the inflammatory process observed in atherosclerosis.

C-reactive protein and alpha-1 antichymotrypsin are acute phase proteins are synthesized in hepatocytes in large numbers in inflammatory processes. This synthesis is mediated primar‐ ily by IL-6 produced by monocytes and macrophages. Elevated fibrinogen favors these mechanisms and therefore an increased cardiovascular risk. In this way a reduction in diet‐ ary intake in winter for instance, would lead to lower serum ascorbate levels, an increase in susceptibility to infection and the factors haemostatic factors and therefore to an increase in cardiovascular mortality.

According to the seasonal variations of vitamin C intake may be relatively low (<80 mg/day on average), corresponding to serum ascorbate concentrations of 50 umol/L. within this range may be variations in infection and homeostatic markers. Increased intake of vitamin C to 90-100 mg/day can increase in these subjects more than 60 umol/L, which has a significant effect on all risk factors.

**15. Ascorbic acid and gallbladder**

common development of cholesterol gallstones.

ings from different countries agree to respect.

**16. Ascorbic acid in other conditions**

*16.1.1. Vitamin C and Sjögren syndrome*

**16.1. Sjögren's syndrome**

The gallbladder disease is highly prevalent in the U.S. and in Mexico, remains a serious pub‐ lic health problem. It has been estimated that only about in U.S. 20 million Americans have gallstones partially or entirely composed of cholesterol. Gallstones form when bile supersa‐ turated with cholesterol is destabilized. AA affects a limiting step in the catabolism of cho‐ lesterol into bile acids in experimental animals, as described AA-deficient guinea pig

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

http://dx.doi.org/10.5772/52181

477

Because of this it has been hypothesized that the deficiency in humans may be a risk factor for this disease in humans. The main findings in humans have shown an inverse relation‐ ship between serum levels of AA and biliary disease prevalence among women. It was also observed a low prevalence of clinical biliary disease between women taking ascorbic acid supplements. However, this prevalence has not been observed in males. Almost all the find‐

In another study, Simon showed that the use of ascorbic acid supplementation correlates with biliary disease among postmenopausal women with coronary disease. Among women who consumed alcohol, the use of ascorbic acid supplementation was associated independ‐ ently with a 50% reduction in the prevalence of gallstones and 62% for cholecystectomies.

Within the NHANES III study was not observed association, linear or nonlinear, between serum ascorbic acid and prevalence of biliary disease in men. Reflecting the low prevalence

It has been hypothesized that the inverse relationship between AA and biliary disease, dem‐ onstrated in animals, affects the activity of the enzyme cholesterol-7-α-hydroxylase, which is the limiting step that regulates the metabolism of cholesterol into bile acids. Supplementa‐ tion with ascorbic acid increases the activity of the enzyme up to 15 times compared with the vitamin-deficient animals that develop the formation of cholesterol gallstones. Addition‐ ally there is a hypersecretion of mucin, a glycoprotein that is secreted by the epithelium of the gallbladder, which precedes cholesterol destabilization and gallstone formation. Because oxygen and hydroxyl radicals stimulate mucin hypersecretion, inhibition of the oxidative

Primary Sjögren's syndrome (SSP) is a chronic disorder of unknown cause, characterized by dry eyes and mouth. It usually occurs in middle-aged women with a prevalence of 1:5000. Patients may have swelling of joints, muscles, nerves, thyroid, kidneys or other

of the disease in men and reduced statistical power to detect such an association.

changes in the vesicle due to AA can decrease the production of this glycoprotein.

### **14. Ascorbic acid and immunity**

In stress situations the adrenal glands react liberating a large number of active and ready hormones. It has been suggested that 200 mg of vitamin C per day can reduce stress levels caused by these hormones. The stress suppresses the immune response. Megadoses of vita‐ min C increases the body levels of antibodies in animal models (rats stressed and un‐ stressed) having the highest values stressed rats. Stressed animals may need more vitamin C for proper immune system function.

Healing is characterized by synthesis of connective tissue, whose main component is colla‐ gen. This molecule AA required for cross linking of the fibers in hydroxylated residues of prolyne and lysine. Ascorbic acid supplementation is necessary for healing since this is oxi‐ dized during the synthesis of collagen.

There is an undeniable evidence of the interaction of vitamin C and phagocytes. The collect‐ ed cells from the blood, peritoneal or alveolar fluid usually contain high concentrations of vitamin C (1-2 ug/mg protein). Guinea pig neutrophils produced H2O2 and destroy staphy‐ lococci in the same way they do control cells. Both ascorbate as dehydroascorbic acid are used for phagocytic process. Neutrophils can avoid self-poisoning absorb extra amounts of ascorbic acid, which can neutralize the antioxidants. However glucolytic activity does not increase much in the neutrophils of guinea pigs supplemented and the stimulation of NADPH oxidase activity is depressed. The addition of ascorbate to the culture media of nor‐ mal macrophages increases the concentration of cyclic GMP (cGMP) in addition to the route of pentose or hexose monophosphate.

Although the addition of large amounts of ascorbate can inhibit myeloperoxidase activity is not altered its bactericidal capacity. It has been an increase in the bactericidal activity in mouse peritoneal macrophages by the addition of ascorbate to the medium. Besides ascor‐ bate increase the motility and chemotactic activity of these cells. The motor functions of cells as the random motion and chemotactic migration of neutrophils and macrophages is dam‐ aged in the absence of vitamin C. Ascorbic acid can also influence the ability of certain cell lines to produce interferon. The addition of AA to cultures of skin embryonic or fibroblasts leads to the production of interferon.

Vitamin C is also necessary for thymic function and operation of certain cells involved in the production of thymic humoral factor. Thymic content of dehydroascorbate diminishes in di‐ rect proportion to vitamin C intake. The hormonal activity of thymic extracts correlates with thymic ascorbate and inversely with dehydroascorbate.

### **15. Ascorbic acid and gallbladder**

According to the seasonal variations of vitamin C intake may be relatively low (<80 mg/day on average), corresponding to serum ascorbate concentrations of 50 umol/L. within this range may be variations in infection and homeostatic markers. Increased intake of vitamin C to 90-100 mg/day can increase in these subjects more than 60 umol/L, which has a significant

In stress situations the adrenal glands react liberating a large number of active and ready hormones. It has been suggested that 200 mg of vitamin C per day can reduce stress levels caused by these hormones. The stress suppresses the immune response. Megadoses of vita‐ min C increases the body levels of antibodies in animal models (rats stressed and un‐ stressed) having the highest values stressed rats. Stressed animals may need more vitamin C

Healing is characterized by synthesis of connective tissue, whose main component is colla‐ gen. This molecule AA required for cross linking of the fibers in hydroxylated residues of prolyne and lysine. Ascorbic acid supplementation is necessary for healing since this is oxi‐

There is an undeniable evidence of the interaction of vitamin C and phagocytes. The collect‐ ed cells from the blood, peritoneal or alveolar fluid usually contain high concentrations of vitamin C (1-2 ug/mg protein). Guinea pig neutrophils produced H2O2 and destroy staphy‐ lococci in the same way they do control cells. Both ascorbate as dehydroascorbic acid are used for phagocytic process. Neutrophils can avoid self-poisoning absorb extra amounts of ascorbic acid, which can neutralize the antioxidants. However glucolytic activity does not increase much in the neutrophils of guinea pigs supplemented and the stimulation of NADPH oxidase activity is depressed. The addition of ascorbate to the culture media of nor‐ mal macrophages increases the concentration of cyclic GMP (cGMP) in addition to the route

Although the addition of large amounts of ascorbate can inhibit myeloperoxidase activity is not altered its bactericidal capacity. It has been an increase in the bactericidal activity in mouse peritoneal macrophages by the addition of ascorbate to the medium. Besides ascor‐ bate increase the motility and chemotactic activity of these cells. The motor functions of cells as the random motion and chemotactic migration of neutrophils and macrophages is dam‐ aged in the absence of vitamin C. Ascorbic acid can also influence the ability of certain cell lines to produce interferon. The addition of AA to cultures of skin embryonic or fibroblasts

Vitamin C is also necessary for thymic function and operation of certain cells involved in the production of thymic humoral factor. Thymic content of dehydroascorbate diminishes in di‐ rect proportion to vitamin C intake. The hormonal activity of thymic extracts correlates with

effect on all risk factors.

**14. Ascorbic acid and immunity**

476 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

for proper immune system function.

dized during the synthesis of collagen.

of pentose or hexose monophosphate.

leads to the production of interferon.

thymic ascorbate and inversely with dehydroascorbate.

The gallbladder disease is highly prevalent in the U.S. and in Mexico, remains a serious pub‐ lic health problem. It has been estimated that only about in U.S. 20 million Americans have gallstones partially or entirely composed of cholesterol. Gallstones form when bile supersa‐ turated with cholesterol is destabilized. AA affects a limiting step in the catabolism of cho‐ lesterol into bile acids in experimental animals, as described AA-deficient guinea pig common development of cholesterol gallstones.

Because of this it has been hypothesized that the deficiency in humans may be a risk factor for this disease in humans. The main findings in humans have shown an inverse relation‐ ship between serum levels of AA and biliary disease prevalence among women. It was also observed a low prevalence of clinical biliary disease between women taking ascorbic acid supplements. However, this prevalence has not been observed in males. Almost all the find‐ ings from different countries agree to respect.

In another study, Simon showed that the use of ascorbic acid supplementation correlates with biliary disease among postmenopausal women with coronary disease. Among women who consumed alcohol, the use of ascorbic acid supplementation was associated independ‐ ently with a 50% reduction in the prevalence of gallstones and 62% for cholecystectomies.

Within the NHANES III study was not observed association, linear or nonlinear, between serum ascorbic acid and prevalence of biliary disease in men. Reflecting the low prevalence of the disease in men and reduced statistical power to detect such an association.

It has been hypothesized that the inverse relationship between AA and biliary disease, dem‐ onstrated in animals, affects the activity of the enzyme cholesterol-7-α-hydroxylase, which is the limiting step that regulates the metabolism of cholesterol into bile acids. Supplementa‐ tion with ascorbic acid increases the activity of the enzyme up to 15 times compared with the vitamin-deficient animals that develop the formation of cholesterol gallstones. Addition‐ ally there is a hypersecretion of mucin, a glycoprotein that is secreted by the epithelium of the gallbladder, which precedes cholesterol destabilization and gallstone formation. Because oxygen and hydroxyl radicals stimulate mucin hypersecretion, inhibition of the oxidative changes in the vesicle due to AA can decrease the production of this glycoprotein.

### **16. Ascorbic acid in other conditions**

### **16.1. Sjögren's syndrome**

### *16.1.1. Vitamin C and Sjögren syndrome*

Primary Sjögren's syndrome (SSP) is a chronic disorder of unknown cause, characterized by dry eyes and mouth. It usually occurs in middle-aged women with a prevalence of 1:5000. Patients may have swelling of joints, muscles, nerves, thyroid, kidneys or other body areas. These symptoms result from lymphocytic infiltration and destruction of these tissues.

dins, particularly PGE1, is important in the immune defects associated with the

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

http://dx.doi.org/10.5772/52181

479

In 1992, Gomez et al, from the National Institute of Medical Sciences and Nutrition "Salva‐ dor Zubiran", observed values less than 0.2 mg AA/dl in plasma of patients with SSP (refer‐ ence values were from 0.4 to 2.0 mg/dL), representing a frank deficiency of vitamin in 100%

It is reported that the diabetic individual has low levels of vitamin C in plasma and leuko‐ cytes, which is our immune defense. However, more clinical studies, in a large scale, are needed to determine whether the supplementation with large doses of the vitamin are bene‐ ficial or not. Some studies have shown that supplementation with 2 g/d, decreased glucose

Megadoses of vitamin C can still be toxic in diabetics with kidney disorders. It was men‐ tioned that vitamin C also helps to reduce body glycosylation, which shows abnormali‐ ties in the binding of sugars and proteins. In addition vitamin C reduces the accumulation of sugar sorbitol which damages eyes and kidneys. Vitamin C lowers blood pressure and plasma cholesterol helping to keep the blood flowing and protected from oxidation in a synergistic action with vitamin E. In doses of 1g/day protects the

Atherosclerosis is the best contributor to heart disease. Vitamin C prevents the formation of atheromatous plaque by inhibiting the oxidative modification of LDL's, which contrib‐ utes to atherosclerotic process for their cytotoxic effects, inhibition of receptor radical

Vitamin C also helps to prevent atherosclerosis through the synthesis of collagen in the arterial wall and prevent undesirable adhesion of leukocytes to the damaged artery.

Supplementation with 2 g/day reduces the adhesion of monocytes to blood vessels, effec‐ tively reverses the vasomotor dysfunction observed in patients with atherosclerosis. In addi‐ tion these doses increase HDL, being highly protective against heart attacks and stroke. Risk is reduced by up to 62% in subjects consuming 700 mg/day compared with those consuming 60mg/day or less. Only Joel study has shown that low levels of ascorbic acid in serum (AAs) are marginally associated with an increased risk and fatal cardiovascular disease was signif‐ icantly associated with an increased risk of mortality for all causes. Low levels of AAs are also a risk factor for cancer death in men, but unexpectedly it was associated with a de‐

Vitamin C has an effect antihistaminic. Subjects with low plasma vitamin C levels have ele‐ vated blood histamine and vitamin supplementation, reduces these levels. Table 7 shows the

scavengers and their influence on the motility of monocytes and macrophages.

syndrome.

of cases with SSP.

**16.4. Role of vitamin C in other body disorders**

levels in diabetics and reduce capillary fragility.

body against LDL lipoproteins.

creased risk of cancer death in women.

relationship of vitamin C reported in different conditions.

The diagnosis is based on clinical examination of the eyes and mouth, blood tests specif‐ ic (auto antibodies) and biopsy of minor salivary gland (taken from inside the inner lip). Sjögren's syndrome is not fatal, but must be addressed quickly to prevent complications due to dry mouth (caries, abscesses, gingivitis) and eyes (corneal erosion, infection). However, there is no therapy available that "removes" these symptoms because all thera‐ pies are directed at eliminating the symptoms and prevent complications.

On the other hand there are several studies that reported a direct relationship between the clinical manifestations of Sjögren's syndrome and those caused by deficiency of ascorbic acid (vitamin C): scurvy. A frank deficiency of vitamin C causes scurvy, a disease character‐ ized by multiple hemorrhages. Scurvy in adults is manifested by latitude, weakness, irrita‐ bility, vague muscle pain, joint pain and weight loss. Early signs are objective as bleeding gums, tooth loss and gingivitis.

The diagnosis of scurvy, is achieved by testing plasma ascorbic acid, low concentration indi‐ cates low levels in tissues. It is generally accepted that ascorbic acid concentration in the lay‐ er of coagulated lymph (20-53 ug/108 leukocytes) is the most reliable indicator of nutritional status regarding vitamin C and its concentration in tissues and serum.

### **16.2. Pharmacological data**

Ascorbic acid is specific in the treatment of scurvy; the dose required can best be meas‐ ured by determining urinary excretion after a dose of saturation, depending on the speed at which the saturation is required is the recommended daily dose ranging from 0.2 and 2.0 g/day. In the vitamin deficiency C tissue saturation is achieved with three daily doses of 700 mg c/u, for three days. Harris defined as saturation of tissues, a suffi‐ cient store where an ascorbic acid excretion 50 mg or more occurs in a period of 4 to 5 hours after a dose of 700 mg/day.

#### **16.3. Previous experience in animals**

Kessler in his study reported that rats which have been induced vitamin C deficiency, devel‐ op various manifestations of primary Sjögren's syndrome (SSP), such as infiltration of mono‐ nuclear cells in salivary and lachrymal glands and that these were more severe is female rats than in male rats, concluding that these pathological changes are similar to those that char‐ acterize the syndrome in humans.

In previous studies Hood reported a direct relationship between the manifestations of primary Sjögren's syndrome (xerostomia and xerophthalmia) and clinical signs of scurvy such as gingivitis, periodontal bleeding and loss of teeth. In their study, Hood study 5 subjects men whose diets did not contain ascorbic acid, for 84 to 97 days. During the de‐ ficiency, in the demonstrations that make Sjögren syndrome, observed that prostaglan‐ dins, particularly PGE1, is important in the immune defects associated with the syndrome.

In 1992, Gomez et al, from the National Institute of Medical Sciences and Nutrition "Salva‐ dor Zubiran", observed values less than 0.2 mg AA/dl in plasma of patients with SSP (refer‐ ence values were from 0.4 to 2.0 mg/dL), representing a frank deficiency of vitamin in 100% of cases with SSP.

### **16.4. Role of vitamin C in other body disorders**

body areas. These symptoms result from lymphocytic infiltration and destruction of

The diagnosis is based on clinical examination of the eyes and mouth, blood tests specif‐ ic (auto antibodies) and biopsy of minor salivary gland (taken from inside the inner lip). Sjögren's syndrome is not fatal, but must be addressed quickly to prevent complications due to dry mouth (caries, abscesses, gingivitis) and eyes (corneal erosion, infection). However, there is no therapy available that "removes" these symptoms because all thera‐

On the other hand there are several studies that reported a direct relationship between the clinical manifestations of Sjögren's syndrome and those caused by deficiency of ascorbic acid (vitamin C): scurvy. A frank deficiency of vitamin C causes scurvy, a disease character‐ ized by multiple hemorrhages. Scurvy in adults is manifested by latitude, weakness, irrita‐ bility, vague muscle pain, joint pain and weight loss. Early signs are objective as bleeding

The diagnosis of scurvy, is achieved by testing plasma ascorbic acid, low concentration indi‐ cates low levels in tissues. It is generally accepted that ascorbic acid concentration in the lay‐

Ascorbic acid is specific in the treatment of scurvy; the dose required can best be meas‐ ured by determining urinary excretion after a dose of saturation, depending on the speed at which the saturation is required is the recommended daily dose ranging from 0.2 and 2.0 g/day. In the vitamin deficiency C tissue saturation is achieved with three daily doses of 700 mg c/u, for three days. Harris defined as saturation of tissues, a suffi‐ cient store where an ascorbic acid excretion 50 mg or more occurs in a period of 4 to 5

Kessler in his study reported that rats which have been induced vitamin C deficiency, devel‐ op various manifestations of primary Sjögren's syndrome (SSP), such as infiltration of mono‐ nuclear cells in salivary and lachrymal glands and that these were more severe is female rats than in male rats, concluding that these pathological changes are similar to those that char‐

In previous studies Hood reported a direct relationship between the manifestations of primary Sjögren's syndrome (xerostomia and xerophthalmia) and clinical signs of scurvy such as gingivitis, periodontal bleeding and loss of teeth. In their study, Hood study 5 subjects men whose diets did not contain ascorbic acid, for 84 to 97 days. During the de‐ ficiency, in the demonstrations that make Sjögren syndrome, observed that prostaglan‐

leukocytes) is the most reliable indicator of nutritional

pies are directed at eliminating the symptoms and prevent complications.

478 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

status regarding vitamin C and its concentration in tissues and serum.

these tissues.

gums, tooth loss and gingivitis.

**16.2. Pharmacological data**

er of coagulated lymph (20-53 ug/108

hours after a dose of 700 mg/day.

**16.3. Previous experience in animals**

acterize the syndrome in humans.

It is reported that the diabetic individual has low levels of vitamin C in plasma and leuko‐ cytes, which is our immune defense. However, more clinical studies, in a large scale, are needed to determine whether the supplementation with large doses of the vitamin are bene‐ ficial or not. Some studies have shown that supplementation with 2 g/d, decreased glucose levels in diabetics and reduce capillary fragility.

Megadoses of vitamin C can still be toxic in diabetics with kidney disorders. It was men‐ tioned that vitamin C also helps to reduce body glycosylation, which shows abnormali‐ ties in the binding of sugars and proteins. In addition vitamin C reduces the accumulation of sugar sorbitol which damages eyes and kidneys. Vitamin C lowers blood pressure and plasma cholesterol helping to keep the blood flowing and protected from oxidation in a synergistic action with vitamin E. In doses of 1g/day protects the body against LDL lipoproteins.

Atherosclerosis is the best contributor to heart disease. Vitamin C prevents the formation of atheromatous plaque by inhibiting the oxidative modification of LDL's, which contrib‐ utes to atherosclerotic process for their cytotoxic effects, inhibition of receptor radical scavengers and their influence on the motility of monocytes and macrophages.

Vitamin C also helps to prevent atherosclerosis through the synthesis of collagen in the arterial wall and prevent undesirable adhesion of leukocytes to the damaged artery.

Supplementation with 2 g/day reduces the adhesion of monocytes to blood vessels, effec‐ tively reverses the vasomotor dysfunction observed in patients with atherosclerosis. In addi‐ tion these doses increase HDL, being highly protective against heart attacks and stroke. Risk is reduced by up to 62% in subjects consuming 700 mg/day compared with those consuming 60mg/day or less. Only Joel study has shown that low levels of ascorbic acid in serum (AAs) are marginally associated with an increased risk and fatal cardiovascular disease was signif‐ icantly associated with an increased risk of mortality for all causes. Low levels of AAs are also a risk factor for cancer death in men, but unexpectedly it was associated with a de‐ creased risk of cancer death in women.

Vitamin C has an effect antihistaminic. Subjects with low plasma vitamin C levels have ele‐ vated blood histamine and vitamin supplementation, reduces these levels. Table 7 shows the relationship of vitamin C reported in different conditions.


[7] Bourges H. Madrigal H. Chavéz A., Tablas del valor nutritivo de los alimentosmexi‐

Antioxidant Role of Ascorbic Acid and His Protective Effects on Chronic Diseases

http://dx.doi.org/10.5772/52181

481

[9] Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: Prolongation of survival times in terminal human cancer. Proc Natl Acad Sci 1976;

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[11] Crary, E.J. and M.F. McCarty, 1984. Potential clinical applications for high-dose nu‐

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[15] Fisher E, McLennan SV, Tada H, Heffernan S, Yue DK, Turtle JR. (Interaction of as‐ corbic acid and glucose on production of collagen and proteoglycan by fibroblasts.

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[17] Gomez E, Silencio JL, Bourges H. Vitamin B2, B6 and C status in patients with pri‐ mary sjögren's syndrome. 7th International congress of mucosal immunology, august

[18] Harris J.L. Vitamin C saturation test. Standarization meassurements at graded levels

[19] Head KA, Ascorbic Acid in the Prevention and Treatment of Cancer. Altern Med Rev

[20] Herrera, V. Más evidencia en contra del uso de vitaminas y antioxidantes en la pre‐ vención de enfermedades crónicas Evidencia Actualización en la Práctica Ambulato‐

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tritional antioxidants. Med. Hypoth., 13: 77-98.

Med Clin (Barc) 1993; 101: 653-656

bilitation. J Am Coll Nutr, 2003, 22:303-310

16-20, 1992, Prague Csechoslovaquia.

of intake. Lancet 1943, 1:515,

1998; 3(3):174-186)

ria- 2002, 5(6): Nov-Dic

Diabetes, 1991, 40(3):371-6)

73:3685-3689.

13604-13609

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Figures in parentheses indicate the reference.

**Table 7.** Association of serum vitamin C (ascorbic acid) in serum or plasma in different symptoms and diseases.

### **Author details**

José Luis Silencio Barrita1 and María del Socorro Santiago Sánchez2

1 Association of Chemists, National Institute for Medical Sciences and Nutrition "Salvador Zubiran", Mexico

2 Department of Nutrition and Dietetics, General Hospital No, 30 "Iztacalco", Mexico

### **References**


**Low concentrations of AA High concentration of AA**

480 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

overview. Nutr J 2003, 2:1475-2891-2-7

Cancer in women (Joel) Uric Acid Excretion (Stein) Back pain and spinal discs. Antioxidant (Kahn) Allergic process (Ruskin) Blood pressure (Ringsdorff) Constipation (Sindair)

**Table 7.** Association of serum vitamin C (ascorbic acid) in serum or plasma in different symptoms and diseases.

and María del Socorro Santiago Sánchez2

1 Association of Chemists, National Institute for Medical Sciences and Nutrition "Salvador

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[4] Bergman F, Curstedt T, Eriksson H, van der Linden W, Sjo°vall J. Gallstoneformation in guinea pigs under different dietary conditions: effect of vitamin C on bile acid pat‐

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of Mucosal Immunology. August 16-20, 1992, Prague, Czechoslovaquia.

2 Department of Nutrition and Dietetics, General Hospital No, 30 "Iztacalco", Mexico

Probable association with menopause. (Smith)

Rheumatoid arthritis (Lunec) Cancer in Men (Joel) Asthma (Ruskin) Bronchospasm Cataract (Jackes) Aging (Jackes) Retinopathy (Crary) (Macular Degeneration)

**Author details**

Zubiran", Mexico

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**Chapter 19**

**Protective Effect of Silymarin on Liver Damage by**

Cesar Esquivel-Chirino, Manuel García-Luna y González-Rubio and

The liver is the vertebrates' largest internal organ. It weighs nearly 1.5 kg, is dark red in col‐ or, and is situated in the upper right quadrant of the abdominal cavity. Among the functions that it performs are the following: the metabolism of lipids and carbohydrates, and the syn‐ thesis of proteins, coagulation factors, and biliary salts. Eighty percent of the hepatic paren‐ chyma is made up of hepatocytes, which are the cells mainly responsible for maintaining every function that the liver in its entirety requires to sustain the body's normal physiologi‐ cal functions in general. In addition to hepatocytes, the liver possesses other cells, such as the so-called Kupffer cells (hepatic macrophages), Ito cells, endothelial cells. The hepato‐ cytes are disposed in the liver in groups denominated lobules, which have a central orifice comprised of the bile duct and by means of which the biliary salts are excreted. The anatom‐ ical loss of the structure of the hepatic lobule is considered a symptom of severe damage to the liver; it can be accompanied by partial or total loss of some physiological function, as in

Liver regeneration is a fundamental response of the liver on encountering tissue damage. The complex interaction of factors that determine this response involves a stimulus (experi‐

> © 2013 Morales-González et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

José A. Morales-González, Evila Gayosso-Islas, Cecilia Sánchez-Moreno, Carmen Valadez-Vega, Ángel Morales-González, Jaime Esquivel-Soto,

Additional information is available at the end of the chapter

the case of alcohol-related hepatic cirrhosis. [23].

**2. Hepatic regeneration**

**Xenobiotics**

Eduardo Madrigal-Santillán

http://dx.doi.org/10.5772/51502

**1. Introduction**


### **Protective Effect of Silymarin on Liver Damage by Xenobiotics**

José A. Morales-González, Evila Gayosso-Islas, Cecilia Sánchez-Moreno, Carmen Valadez-Vega, Ángel Morales-González, Jaime Esquivel-Soto, Cesar Esquivel-Chirino, Manuel García-Luna y González-Rubio and Eduardo Madrigal-Santillán

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51502

**1. Introduction**

[55] Simon J.A Vitamin C and cardiovascular disease: a review J Ame Collage of Nutr,

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2000 Jul;79(7):961-70.

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484 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

therapy. Ann. Intern.Med., 84: 385-388.

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Departament of Agriculture, USA, 1975.

Gerontology 1992, 38:266-274

Chicago med, 1984.

The liver is the vertebrates' largest internal organ. It weighs nearly 1.5 kg, is dark red in col‐ or, and is situated in the upper right quadrant of the abdominal cavity. Among the functions that it performs are the following: the metabolism of lipids and carbohydrates, and the syn‐ thesis of proteins, coagulation factors, and biliary salts. Eighty percent of the hepatic paren‐ chyma is made up of hepatocytes, which are the cells mainly responsible for maintaining every function that the liver in its entirety requires to sustain the body's normal physiologi‐ cal functions in general. In addition to hepatocytes, the liver possesses other cells, such as the so-called Kupffer cells (hepatic macrophages), Ito cells, endothelial cells. The hepato‐ cytes are disposed in the liver in groups denominated lobules, which have a central orifice comprised of the bile duct and by means of which the biliary salts are excreted. The anatom‐ ical loss of the structure of the hepatic lobule is considered a symptom of severe damage to the liver; it can be accompanied by partial or total loss of some physiological function, as in the case of alcohol-related hepatic cirrhosis. [23].

### **2. Hepatic regeneration**

Liver regeneration is a fundamental response of the liver on encountering tissue damage. The complex interaction of factors that determine this response involves a stimulus (experi‐

© 2013 Morales-González et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mentally, a hepatectomy), gene expression, and the interaction of other factors that modu‐ late the response. This proliferation depends on the hepatocytes, epithelial bile cells, Kupffer cells, and Ito cells. [24].

Once ethanol is absorbed, it is distributed to all of the tissues, being concentrated in greatest proportion in brain, blood, eye, and cerebrospinal fluid, crossing the feto-placentary and hematocephalic barrier [44]. Gender difference is a factor that modifies the distributed etha‐ nol volume; this is due to its hydrosolubility and to that it is not distributed in body fats, which explains why in females this parameter is found diminished compared with males.

Protective Effect of Silymarin on Liver Damage by Xenobiotics

http://dx.doi.org/10.5772/51502

487

Ethanol is eliminated mainly (> 90%) by the liver through the enzymatic oxidation path‐ way; 5-10% is excreted without changes by the kidneys, lungs, and in sweat [14, 30]. The liver is the primary site of ethanol metabolism through the following three differ‐ ent enzymatic systems: Alcohol dehydrogenases (ADH); Microsomal ethanol oxidation

Ethanol is a well known hepatotoxic xenobiotic because hepatotoxicity has been well docu‐ mented in humans as well as in animals. Although aspects concerning the pathogenesis of liver damage have been widely studied, it is known that liver regeneration restores the func‐ tional hepatic mass after hepatic damage caused by toxins. Suppression of the regenerating capacity of the liver by ethanol is the major factor of liver damage. [45]. Although the effects of acute or chronic administration of ethanol on the proliferative capacity of the liver to re‐ generate itself has been studied, the precise mechanism by which ethanol affects hepatocel‐

Liver regeneration induced by partial hepatectomy in rats represents an ideal model of con‐ trolled hepatocellular growth. This surgical procedure has been sufficiently employed to study the factors than can be implicated in the growth of the liver. Endogenous signals have been described to control hepatic regeneration. The first marker of DNA synthesis in partial‐ ly hepatectomized rats (70%) occurring normally 24-28 hours postsurgery comprises an enormous action of growth factors and cytokines affecting expression of the gene of the hep‐ atocytes, associated with initiation of the cell cycle. [2]. It has indicated that the hepatocytes enter into a state denominated "priming" to thus begin replication and response to growth factors, that is, which range from the quiescent to the G 1 phase of the cell cycle. The pro‐ gression of hepatic cells requires the activation of cyclin-dependent kinases that are regulat‐ ed by cyclins and cyclin-dependent kinase inhibitors. [39]. It has also been demonstrated that a dose of ethanol importantly diminishes the specific activity of two enzymes related with the metabolism of DNA synthesis, which are thymidine kinase and thymidylate syn‐

Free radicals are the result of the organism's own physiological processes, such as the me‐ tabolism of food, respiration, exercise, or even those generated by environmental factors,

lular function and the regenerative process are poorly explained. [31, 29, 38].

system (MEOS); Catalase system.

thetase. [47, 11, 32].

**6. Free radicals**

**5. Liver regeneration and ethanol**

The mechanisms of hepatic growth have been studied in detail in experimental models. In the latter, regeneration is induced whether by tissue resection (partial hepatectomy) or by death of the hepatocytes (toxic damage). The principles that govern the growth of this organ in these systems also apply to clinical situations, such as, for example, fulminating liver failure, acute and chronic hepatitis, partial hepatectomy for treating liver cancer, or even in liver transplant donors. Evidence that there is a humoral growth factor of the hepatocyte has been observed in animal models and in patients with liver disease from the 1980s. [1, 13, 34, 10].

### **3. Ethanol**

On being ingested, alcohol (also called ethanol) produces a series of biochemical reactions that lead to the affectation of numerous organs involving economy, having as the endpoint the development of hepatic diseases such as alcoholic hepatitis and cirrhosis. Despite that much is known about the physiopathological mechanisms that trigger ethanol within the or‐ ganism, it has been observed that a sole mechanism of damage cannot fully explain all of the adverse effects that ethanol produces in the organism or in one organ in particular. [37, 30].

A factor that is referred as playing a central role in the many adverse effects that ethanol exerts on the organism and that has been the focus of attention of many researchers is the excessive generation of molecules called free radicals, which can produce a condition known as oxidative stress, which triggers diverse alterations in the cell's biochemical processes that can finally activate the mechanism of programmed cell death, also known as apoptosis. [28, 26, 17, 19, 25].

Of particular importance for the objective of this chapter is the focus on a particular class of free radicals that are oxygen derivatives, because these are the main chemical entities that are produced within the organism and that affect it in general.

### **4. Ethanol metabolism**

Ethanol is absorbed rapidly in the gastrointestinal tract; the surface of greatest adsorption is the first portion of the small intestine with 70%; 20% is absorbed in the stomach, and the re‐ mainder, in the colon. Diverse factors can cause the increase in absorption speed, such as gastric emptying, ingestion without food, ethanol dilution (maximum absorption occurs at a 20% concentration), and carbonation. Under optimal conditions, 80-90% of the ingested dose is completely absorbed within 60 minutes. Similarly, there are factors that can delay ethanol absorption (from 2-6 hours), including high concentrations of the latter, the presence of food, the co-existence of gastrointestinal diseases, the administration of drugs, and individu‐ al variations [14, 37].

Once ethanol is absorbed, it is distributed to all of the tissues, being concentrated in greatest proportion in brain, blood, eye, and cerebrospinal fluid, crossing the feto-placentary and hematocephalic barrier [44]. Gender difference is a factor that modifies the distributed etha‐ nol volume; this is due to its hydrosolubility and to that it is not distributed in body fats, which explains why in females this parameter is found diminished compared with males.

Ethanol is eliminated mainly (> 90%) by the liver through the enzymatic oxidation path‐ way; 5-10% is excreted without changes by the kidneys, lungs, and in sweat [14, 30]. The liver is the primary site of ethanol metabolism through the following three differ‐ ent enzymatic systems: Alcohol dehydrogenases (ADH); Microsomal ethanol oxidation system (MEOS); Catalase system.

### **5. Liver regeneration and ethanol**

mentally, a hepatectomy), gene expression, and the interaction of other factors that modu‐ late the response. This proliferation depends on the hepatocytes, epithelial bile cells, Kupffer

The mechanisms of hepatic growth have been studied in detail in experimental models. In the latter, regeneration is induced whether by tissue resection (partial hepatectomy) or by death of the hepatocytes (toxic damage). The principles that govern the growth of this organ in these systems also apply to clinical situations, such as, for example, fulminating liver failure, acute and chronic hepatitis, partial hepatectomy for treating liver cancer, or even in liver transplant donors. Evidence that there is a humoral growth factor of the hepatocyte has been observed in

On being ingested, alcohol (also called ethanol) produces a series of biochemical reactions that lead to the affectation of numerous organs involving economy, having as the endpoint the development of hepatic diseases such as alcoholic hepatitis and cirrhosis. Despite that much is known about the physiopathological mechanisms that trigger ethanol within the or‐ ganism, it has been observed that a sole mechanism of damage cannot fully explain all of the adverse effects that ethanol produces in the organism or in one organ in particular. [37, 30]. A factor that is referred as playing a central role in the many adverse effects that ethanol exerts on the organism and that has been the focus of attention of many researchers is the excessive generation of molecules called free radicals, which can produce a condition known as oxidative stress, which triggers diverse alterations in the cell's biochemical processes that can finally activate the mechanism of programmed cell death, also known as apoptosis. [28,

Of particular importance for the objective of this chapter is the focus on a particular class of free radicals that are oxygen derivatives, because these are the main chemical entities that

Ethanol is absorbed rapidly in the gastrointestinal tract; the surface of greatest adsorption is the first portion of the small intestine with 70%; 20% is absorbed in the stomach, and the re‐ mainder, in the colon. Diverse factors can cause the increase in absorption speed, such as gastric emptying, ingestion without food, ethanol dilution (maximum absorption occurs at a 20% concentration), and carbonation. Under optimal conditions, 80-90% of the ingested dose is completely absorbed within 60 minutes. Similarly, there are factors that can delay ethanol absorption (from 2-6 hours), including high concentrations of the latter, the presence of food, the co-existence of gastrointestinal diseases, the administration of drugs, and individu‐

are produced within the organism and that affect it in general.

animal models and in patients with liver disease from the 1980s. [1, 13, 34, 10].

486 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

cells, and Ito cells. [24].

**3. Ethanol**

26, 17, 19, 25].

**4. Ethanol metabolism**

al variations [14, 37].

Ethanol is a well known hepatotoxic xenobiotic because hepatotoxicity has been well docu‐ mented in humans as well as in animals. Although aspects concerning the pathogenesis of liver damage have been widely studied, it is known that liver regeneration restores the func‐ tional hepatic mass after hepatic damage caused by toxins. Suppression of the regenerating capacity of the liver by ethanol is the major factor of liver damage. [45]. Although the effects of acute or chronic administration of ethanol on the proliferative capacity of the liver to re‐ generate itself has been studied, the precise mechanism by which ethanol affects hepatocel‐ lular function and the regenerative process are poorly explained. [31, 29, 38].

Liver regeneration induced by partial hepatectomy in rats represents an ideal model of con‐ trolled hepatocellular growth. This surgical procedure has been sufficiently employed to study the factors than can be implicated in the growth of the liver. Endogenous signals have been described to control hepatic regeneration. The first marker of DNA synthesis in partial‐ ly hepatectomized rats (70%) occurring normally 24-28 hours postsurgery comprises an enormous action of growth factors and cytokines affecting expression of the gene of the hep‐ atocytes, associated with initiation of the cell cycle. [2]. It has indicated that the hepatocytes enter into a state denominated "priming" to thus begin replication and response to growth factors, that is, which range from the quiescent to the G 1 phase of the cell cycle. The pro‐ gression of hepatic cells requires the activation of cyclin-dependent kinases that are regulat‐ ed by cyclins and cyclin-dependent kinase inhibitors. [39]. It has also been demonstrated that a dose of ethanol importantly diminishes the specific activity of two enzymes related with the metabolism of DNA synthesis, which are thymidine kinase and thymidylate syn‐ thetase. [47, 11, 32].

### **6. Free radicals**

Free radicals are the result of the organism's own physiological processes, such as the me‐ tabolism of food, respiration, exercise, or even those generated by environmental factors, such as contamination, tobacco, or by drugs, chemical additives, etc. Free radicals (FR) are atoms or groups of atoms that in their atomic structure present one or more unpaired elec‐ trons (odd in number) in the outer orbit. This spatial configuration generates in the molecule distinct physical and chemical properties such as heightened reactivity and diminished life‐ time, respectively. [5, 27].

chain reaction in which the fatty acid, on being oxygenated, becomes a fatty acid radical with the capacity of oxidizing another, neighboring molecule. This process is known as lipid peroxidation and it generates numerous subproducts, many of these, such as Malondialde‐ hyde (MDA), whose determination in tissues, plasma, or urine is one of the methods for evaluating oxidative stress. In the case of proteins, these preferentially oxidize the amino acids (phenylalanine, tyrosine, triptophan, histidine, and methionine), and consequently form peptide chain overlapping, protein fragmentation, and the formation of carbonyl groups, and these impede the normal development of their functions (ionic membrane transporters, receptors, and cellular messengers), enzymes that regulate the cell's metabo‐

Protective Effect of Silymarin on Liver Damage by Xenobiotics

http://dx.doi.org/10.5772/51502

489

While ROS and FR are generated during ethanol metabolism, causing oxidative stress and lipoperoxidation in the liver, they can also form a significant pathway of damage to the re‐ generative process of the hepatocyte. In this process, ethanol-induced FR and the generation of ROS involve the mitochondria, the microsomal cytochrome P450 2E1, the iron (FE) ion, and less frequently, peroxisomes, cytosolic xanthines, and aldehyde oxidases, to regulate

In general, ROS-derived FR intervene in the persistent bombardment of molecules by reac‐ tive oxygen radicals, thus maintaining redox homeostasis, in such a manner that during liv‐ er regeneration, these can modify the metabolic response necessary for carrying out cellular mitosis in the hepatocyte. While FR are generated and utilized by the cells as neutrophils, monocytes, macrophages, eosinophils, and fibroblasts for eliminating foreign organisms or toxic substances, the increase of FR due to exposure to ethanol leads to cellular deterioration that in turn produces hepatic alterations, with an unfavorable influence on cell proliferative

Halliwell defines an antioxidant as all substances that on being found present at low concen‐ trations with respect to those of an oxidizable substrate (biomolecule), delays or prevents the oxidation of this substrate. The antioxidant, on colliding with FR, is ceded to an electron, in turn oxidizing itself and transforming itself into a non-toxic, weak FR and, in some cases such as with vitamin E, it can regenerate itself into its primitive state due to the action of other antioxidants. Not all antioxidants act in this way: the so-called enzymatic antioxidants catalyze or accelerate chemical reactions that utilize substrates that in turn react with FR. Of the numerous classifications of antioxidants, it is recommended to adopt that which divides these into the following: exogenes or antioxidants that enter through the alimentary chain, and endogenes that are synthesized by the cell. Each antioxidant possesses an affinity for a

lism, etc.). [7, 33].

action. [25].

**9. Antioxidants**

**8. Liver regeneration, ethanol, and free radicals**

cellular proliferation, acting as direct or indirect factors. [37, 2].

This instability confers on these physical avidity for the uptake of an electron of any other molecule in its ambit (stable molecules), causing the affected structure to remain unstable with the purpose of reaching its electrochemical stability. Once the free radical has achieved trapping the electron that it requires for pairing with its free electron, the stable molecule that cedes the latter to it in turn becomes a free radical, due to its remaining with an un‐ paired electron, this initiating a true chain reaction that destroys our cells. [4, 7].

In aerobic cells, there are diverse pathways that lead to the production of Oxygen-derived free radicals (OFR). The main sources are enzymes associated with the metabolism of arachi‐ donic acid, such as cycloxygenase, lipoxygenase, and cytochrome P-450. The presence and ubiquity of enzymes (superoxide dismutase, catalase, and peroxidase) that eliminate secon‐ dary products in a univalent pathway in aerobic cells suggest that the superoxide anions and hydrogen peroxide are important secondary products of oxidative metabolism. [40, 7]. Reactive oxygen species (ROS) can damage macromolecules such as DNA, carbohydrates, and proteins. These cytotoxic oxygen species can be classified as two types:


The instable radicals attack cell components, causing damage to the lipids, proteins, and the DNA, which can trigger a chain of events that result in cellular damage. [7, 21]. These reduc‐ tive processes are accelerated by the presence of trace metals such as iron (Fe) and copper (Cu) and of specific enzymes such as monoxygenases and certain oxidases. [7, 21]

### **7. Oxidative stress**

In 1954, an Argentine researcher, Rebeca Gerschman, suggested for the first time that FR were toxic agents and generators of disease. [12].

Due to the atomic instability of FR, the latter collide with a biomolecule and subtract an elec‐ tron, oxidating it, losing in this manner its specific function in the cell. If lipids are involved (polyunsaturated fatty acids), the structures rich in these are damaged, such as the cell membranes and the lipoproteins. In the former, the permeability is altered, leading to edema and cell death, and in the latter, to oxygenation of the Low-density lipoproteins (LDL) and genesis of the atheromatous plaque. The characteristics of lipid oxygenation by FR involve a chain reaction in which the fatty acid, on being oxygenated, becomes a fatty acid radical with the capacity of oxidizing another, neighboring molecule. This process is known as lipid peroxidation and it generates numerous subproducts, many of these, such as Malondialde‐ hyde (MDA), whose determination in tissues, plasma, or urine is one of the methods for evaluating oxidative stress. In the case of proteins, these preferentially oxidize the amino acids (phenylalanine, tyrosine, triptophan, histidine, and methionine), and consequently form peptide chain overlapping, protein fragmentation, and the formation of carbonyl groups, and these impede the normal development of their functions (ionic membrane transporters, receptors, and cellular messengers), enzymes that regulate the cell's metabo‐ lism, etc.). [7, 33].

### **8. Liver regeneration, ethanol, and free radicals**

While ROS and FR are generated during ethanol metabolism, causing oxidative stress and lipoperoxidation in the liver, they can also form a significant pathway of damage to the re‐ generative process of the hepatocyte. In this process, ethanol-induced FR and the generation of ROS involve the mitochondria, the microsomal cytochrome P450 2E1, the iron (FE) ion, and less frequently, peroxisomes, cytosolic xanthines, and aldehyde oxidases, to regulate cellular proliferation, acting as direct or indirect factors. [37, 2].

In general, ROS-derived FR intervene in the persistent bombardment of molecules by reac‐ tive oxygen radicals, thus maintaining redox homeostasis, in such a manner that during liv‐ er regeneration, these can modify the metabolic response necessary for carrying out cellular mitosis in the hepatocyte. While FR are generated and utilized by the cells as neutrophils, monocytes, macrophages, eosinophils, and fibroblasts for eliminating foreign organisms or toxic substances, the increase of FR due to exposure to ethanol leads to cellular deterioration that in turn produces hepatic alterations, with an unfavorable influence on cell proliferative action. [25].

### **9. Antioxidants**

such as contamination, tobacco, or by drugs, chemical additives, etc. Free radicals (FR) are atoms or groups of atoms that in their atomic structure present one or more unpaired elec‐ trons (odd in number) in the outer orbit. This spatial configuration generates in the molecule distinct physical and chemical properties such as heightened reactivity and diminished life‐

This instability confers on these physical avidity for the uptake of an electron of any other molecule in its ambit (stable molecules), causing the affected structure to remain unstable with the purpose of reaching its electrochemical stability. Once the free radical has achieved trapping the electron that it requires for pairing with its free electron, the stable molecule that cedes the latter to it in turn becomes a free radical, due to its remaining with an un‐

In aerobic cells, there are diverse pathways that lead to the production of Oxygen-derived free radicals (OFR). The main sources are enzymes associated with the metabolism of arachi‐ donic acid, such as cycloxygenase, lipoxygenase, and cytochrome P-450. The presence and ubiquity of enzymes (superoxide dismutase, catalase, and peroxidase) that eliminate secon‐ dary products in a univalent pathway in aerobic cells suggest that the superoxide anions and hydrogen peroxide are important secondary products of oxidative metabolism. [40, 7]. Reactive oxygen species (ROS) can damage macromolecules such as DNA, carbohydrates,

**1.** the free radicals, such as the superoxide radical (O2) and the hydroxyl radical (˙OH),

**2.** non-radical oxygen species, such as hydrogen peroxide (H2O2), the oxygen singlette (O1), which is a very toxic species, peroxynitrite (ONOO), and Hypochlorous acid

The instable radicals attack cell components, causing damage to the lipids, proteins, and the DNA, which can trigger a chain of events that result in cellular damage. [7, 21]. These reduc‐ tive processes are accelerated by the presence of trace metals such as iron (Fe) and copper

In 1954, an Argentine researcher, Rebeca Gerschman, suggested for the first time that FR

Due to the atomic instability of FR, the latter collide with a biomolecule and subtract an elec‐ tron, oxidating it, losing in this manner its specific function in the cell. If lipids are involved (polyunsaturated fatty acids), the structures rich in these are damaged, such as the cell membranes and the lipoproteins. In the former, the permeability is altered, leading to edema and cell death, and in the latter, to oxygenation of the Low-density lipoproteins (LDL) and genesis of the atheromatous plaque. The characteristics of lipid oxygenation by FR involve a

(Cu) and of specific enzymes such as monoxygenases and certain oxidases. [7, 21]

paired electron, this initiating a true chain reaction that destroys our cells. [4, 7].

and proteins. These cytotoxic oxygen species can be classified as two types:

time, respectively. [5, 27].

488 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

and

(HOCL).

**7. Oxidative stress**

were toxic agents and generators of disease. [12].

Halliwell defines an antioxidant as all substances that on being found present at low concen‐ trations with respect to those of an oxidizable substrate (biomolecule), delays or prevents the oxidation of this substrate. The antioxidant, on colliding with FR, is ceded to an electron, in turn oxidizing itself and transforming itself into a non-toxic, weak FR and, in some cases such as with vitamin E, it can regenerate itself into its primitive state due to the action of other antioxidants. Not all antioxidants act in this way: the so-called enzymatic antioxidants catalyze or accelerate chemical reactions that utilize substrates that in turn react with FR. Of the numerous classifications of antioxidants, it is recommended to adopt that which divides these into the following: exogenes or antioxidants that enter through the alimentary chain, and endogenes that are synthesized by the cell. Each antioxidant possesses an affinity for a determined FR or for several. Vitamin E, beta-carotene, and lycopene act within the liposol‐ uble medium of the cell and their absorption and transport are found to be very much linked with that of the lipids. Vitamin E is considered the most important protector of lipid molecules. [27].

**9.5. Fifth level**

tablishment of genetic information.

ic compounds [46, 36].

gen donation, avoiding chain reactions. [7, 21, 33].

ganisms. [36].

**10. Antioxidants and their role in hepatoprotection**

Once the molecular damage is produced, there is a fifth level of defense that consists of re‐ pair. It has been demonstrated that FR were capable of causing breaks in the DNA chain and even of inducing mutagenesis, but there are enzymatic repair mechanisms that permit rees‐

Protective Effect of Silymarin on Liver Damage by Xenobiotics

http://dx.doi.org/10.5772/51502

491

The term antioxidant was originally utilized to refer specifically to a chemical product that prevented the consumption of oxygen [6]; thus, antioxidants are defined as molecules whose function is to delay or prevent the oxygenation of other molecules. The importance of anti‐ oxidants lies in their mission to end oxidation reactions that are found in the process and to impede their generating new oxidation reactions on acting in a type of sacrifice on oxidating themselves. There are endogenous and exogenous antioxidants in nature. Some of the bestknown exogenous antioxidant substances are the following: β‐carotene (pro‐vitamin A); reti‐ nol (vitamin (A); ascorbic acid (vitamin C); α‐tocopherol (vitamin E); oligoelements such as selenium; amino acids such as glycine, and flavonoids such as *silymarin*, among other organ‐

Historically, it is known that the first investigations on the role that antioxidants play in Bi‐ ology were centered on their intervention in preventing the oxidation of unsaturated fats, which is the main cause of rancidity in food. However, it was the identification of vitamins A, C, and E as antioxidant substances that revolutionized the study area of antioxidants and that led to elucidating the importance of these substances in the defense system of live or‐

Due to their solubilizing nature, antioxidant compounds have been divided into hydrophil‐ ics (phenolic compounds and vitamin C) and lipophilics (carotenoids and vitamin E). The antioxidant capacity of phenolic compounds is due principally to their redox properties, which allow them to act as reducing agents, hydrogen and electron donors, and individual oxygen inhibitors, while vitamin C's antioxidant action is due to its possessing two free elec‐ trons that can be taken up by Free radicals (FR), as well as by other Reactive oxygen species (ROS), which lack an electron in their molecular structure. Carotenoids are deactivators of electronically excited sensitizing molecules, which are involved in the generation of radicals and individual oxygen, and the antioxidant activity of vitamin A is characterized by hydro‐

The antioxidant defense system is composed of a group of substances that, on being present at low concentrations with respect to the oxidizable substrate, delay or significantly prevent oxygenation of the latter. Given that FR such as ROS are inevitably produced constantly during metabolic processes, in general it may be considered as an oxidizable substrate to nearly all organic or inorganic molecules that are found in living cells, such as proteins, lip‐ ids, carbohydrates, and DNA molecules. Antioxidants impede other molecules from binding

Life in the presence of molecular oxygen requires the possession of a multiple battery of de‐ fenses against the diverse oxygen FR, which on the one hand tend to impede their formation and on the other, neutralize them once they are formed. These defenses exert an effect at five levels [7, 21, 33]:

### **9.1. First level**

This consists of editing univalent oxygen reduction through enzymatic systems capable of effecting consecutive tetravalent reduction without releasing the partially reduced interme‐ diaries; this is achieved with great effectiveness by the cytochrome-oxidase system of the mitochondrial respiratory chain, which is responsible for more than 90% of oxygen reduc‐ tion in the human organism.

### **9.2. Second level**

This is constituted of enzymes specialized in the uptake of the superoxide anion radical (O 2 ‒). These are Superoxide dismutase (SOD), the methaloenzyme that catalyzes the dismuta‐ tion of the superoxide anion radical to provide molecular oxygen and hydrogen peroxide, with such great effectiveness that it approaches the theoretical limit of diffusion. In the cells of the eukaryotic organisms, there are two of these: one is cytoplasmatic, and the other is mitochondrial. SOD was described by Fridovich in 1975.

### **9.3. Third level**

This is conferred by a group of specialized enzymes on neutralizing hydrogen peroxide. Among these is catalase, which is found in the peroxisomes and which catalyzes the dismu‐ tation reaction.

Also in mammals, glutathione peroxidase (a cytoplasmic enzyme that contains selenium) is the most important.

#### **9.4. Fourth level**

Here the hydroxyl radical produced in the Haber-Weiss cycle can neutralized by vitamin E or alpha-tocopherol, which is an effective antioxidant and that due to its hydrophobicity is found in biological membranes in which its protection is particularly important. In addition, vitamin C or ascorbic acid is a reducer agent or electron donor and reacts rapidly with the OH‒ radical and with the superoxide anion.

### **9.5. Fifth level**

determined FR or for several. Vitamin E, beta-carotene, and lycopene act within the liposol‐ uble medium of the cell and their absorption and transport are found to be very much linked with that of the lipids. Vitamin E is considered the most important protector of lipid

Life in the presence of molecular oxygen requires the possession of a multiple battery of de‐ fenses against the diverse oxygen FR, which on the one hand tend to impede their formation and on the other, neutralize them once they are formed. These defenses exert an effect at five

This consists of editing univalent oxygen reduction through enzymatic systems capable of effecting consecutive tetravalent reduction without releasing the partially reduced interme‐ diaries; this is achieved with great effectiveness by the cytochrome-oxidase system of the mitochondrial respiratory chain, which is responsible for more than 90% of oxygen reduc‐

This is constituted of enzymes specialized in the uptake of the superoxide anion radical (O 2 ‒). These are Superoxide dismutase (SOD), the methaloenzyme that catalyzes the dismuta‐ tion of the superoxide anion radical to provide molecular oxygen and hydrogen peroxide, with such great effectiveness that it approaches the theoretical limit of diffusion. In the cells of the eukaryotic organisms, there are two of these: one is cytoplasmatic, and the other is

This is conferred by a group of specialized enzymes on neutralizing hydrogen peroxide. Among these is catalase, which is found in the peroxisomes and which catalyzes the dismu‐

Also in mammals, glutathione peroxidase (a cytoplasmic enzyme that contains selenium) is

Here the hydroxyl radical produced in the Haber-Weiss cycle can neutralized by vitamin E or alpha-tocopherol, which is an effective antioxidant and that due to its hydrophobicity is found in biological membranes in which its protection is particularly important. In addition, vitamin C or ascorbic acid is a reducer agent or electron donor and reacts rapidly with the

molecules. [27].

levels [7, 21, 33]:

**9.1. First level**

**9.2. Second level**

**9.3. Third level**

tation reaction.

the most important.

**9.4. Fourth level**

tion in the human organism.

mitochondrial. SOD was described by Fridovich in 1975.

490 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

OH‒ radical and with the superoxide anion.

Once the molecular damage is produced, there is a fifth level of defense that consists of re‐ pair. It has been demonstrated that FR were capable of causing breaks in the DNA chain and even of inducing mutagenesis, but there are enzymatic repair mechanisms that permit rees‐ tablishment of genetic information.

### **10. Antioxidants and their role in hepatoprotection**

The term antioxidant was originally utilized to refer specifically to a chemical product that prevented the consumption of oxygen [6]; thus, antioxidants are defined as molecules whose function is to delay or prevent the oxygenation of other molecules. The importance of anti‐ oxidants lies in their mission to end oxidation reactions that are found in the process and to impede their generating new oxidation reactions on acting in a type of sacrifice on oxidating themselves. There are endogenous and exogenous antioxidants in nature. Some of the bestknown exogenous antioxidant substances are the following: β‐carotene (pro‐vitamin A); reti‐ nol (vitamin (A); ascorbic acid (vitamin C); α‐tocopherol (vitamin E); oligoelements such as selenium; amino acids such as glycine, and flavonoids such as *silymarin*, among other organ‐ ic compounds [46, 36].

Historically, it is known that the first investigations on the role that antioxidants play in Bi‐ ology were centered on their intervention in preventing the oxidation of unsaturated fats, which is the main cause of rancidity in food. However, it was the identification of vitamins A, C, and E as antioxidant substances that revolutionized the study area of antioxidants and that led to elucidating the importance of these substances in the defense system of live or‐ ganisms. [36].

Due to their solubilizing nature, antioxidant compounds have been divided into hydrophil‐ ics (phenolic compounds and vitamin C) and lipophilics (carotenoids and vitamin E). The antioxidant capacity of phenolic compounds is due principally to their redox properties, which allow them to act as reducing agents, hydrogen and electron donors, and individual oxygen inhibitors, while vitamin C's antioxidant action is due to its possessing two free elec‐ trons that can be taken up by Free radicals (FR), as well as by other Reactive oxygen species (ROS), which lack an electron in their molecular structure. Carotenoids are deactivators of electronically excited sensitizing molecules, which are involved in the generation of radicals and individual oxygen, and the antioxidant activity of vitamin A is characterized by hydro‐ gen donation, avoiding chain reactions. [7, 21, 33].

The antioxidant defense system is composed of a group of substances that, on being present at low concentrations with respect to the oxidizable substrate, delay or significantly prevent oxygenation of the latter. Given that FR such as ROS are inevitably produced constantly during metabolic processes, in general it may be considered as an oxidizable substrate to nearly all organic or inorganic molecules that are found in living cells, such as proteins, lip‐ ids, carbohydrates, and DNA molecules. Antioxidants impede other molecules from binding to oxygen on reacting or interacting more rapidly with FR and ROS than with the remainder of molecules that are present in the microenvironment in which they are found (plasma membrane, cytosol, the nucleus, or Extracellular fluid [ECF]). Antioxidant action is one of the sacrifices of its own molecular integrity in order to avoid alterations in the remainder of vitally functioning or more important molecules. In the case of the exogenic antioxidants, re‐ placement through consumption in the diet is of highest importance, because these act as suicide molecules on encountering FR, as previously mentioned. [7, 21, 33].

More research is needed because great majority of the former has been conducted in ani‐ mals, to determine effects and dosage. Flavonoids in the menopause result in controversial effects due to the population type studied, that is, Asiatic, absorption, metabolism, the bind‐ ing of isoflavones to estrogen receptors, etc.; however, they appear to possess a beneficial

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493

The flavonoids absorb Ultraviolet light (UV) from the sun and possess direct and indirect antioxidant effects (through the induction of cytoprotector proteins). Topical application (on the human skin) of the polyphenolic fraction of green tea protects against immunosuppres‐ sion and inhibits the erythema and the formation of pyrimidine dimers in DNA caused by UV. On representing one of the most important lifestyle factors, alimentation can important‐ ly affect the incidence and initiation of cardiovascular or neurodegenerative diseases. The cardioprotector effect of flavonoids is based on reducing oxidation and blood concentrations of the binding of cholesterol to Low-density lipoproteins (LDL); flavonoids reduce endothe‐ lial dysfunction and blood pressure and increase the HDL-bound cholesterol concentration. Flavonoids possess a neuroprotector effect because they protect the neurons from oxidative stress by means of induction of antioxidant defenses, modulation of signaling cascades, mi‐ tochondrial interactions, apoptotic processes, or by synthesis/degradation of the β-amyloid peptide. The potential effect of flavonoids as neuroprotectors is due to three main factors: they prevent neurodegeneration; inhibit neuroinflammation, and reduce the diminution of

In cancer, the flavonoids have been classified as chemopreventive, as blockers as well as in‐ hibitors, given their functions in carcinogenesis, in which they modulate transduction sig‐ naling in cellular proliferation and angiogenesis, modulate enzymes for the metabolic activation of procarcinogens and the detoxification of carcinogens, and modulate enzymes in the biosynthesis of anti-oxidant-pro-oxidant estrogen activity estrogen (promoting oxida‐ tive homeostasis, rendering its antioxidative capacity as a contribution to antineoplastic as well as preventive as well as therapeutic activity due to inhibiting the activation of mitogen‐ ic kinases and transduction factors, while pro-oxidative activity increases the cell damage that promotes detention of the cell cycle and apoptosis). In obesity, the flavonoids have been identified as reducer factors of fat mass and as inhibitors of fat mass deposition and catabo‐

The procyanidins and proanthocyanidins have demonstrated, in human population, to di‐ minish visceral fatty mass (depending on the dose) with an associated increase of adiponec‐ tin. This diminution is linked with the malabsorption of carbohydrates and lipids due to enzyme inhibition. It has been observed that the procyanidins increase β-oxidation and in‐ hibit the expression of genes that promote the synthesis of fatty acids. Epigallocatechin gal‐ late can increase energy expenditure and lipid oxidation in humans; it is thought that this is possible because of the increase of thermogenesis and the inhibition of the activity of the li‐ pase, as well as, according to studies *in vitro,* the inhibition of lipogenesis and apoptosis of the adipocytes. Catechins that alter the deposition of adipose tissue related with diminution of the respiratory co-efficient and greater oxygen consumption, and thermogenesis induced by the sympathetic nervous system. Phytoestrogens can improve obesity and its alterations

effect in terms of the prevention of certain types of cancer and osteoporosis. [16].

age-related cognitive functions. [16].

lic activity. [16].

This is the reason that, for several years, diverse researchers have been carrying out experi‐ mental studies that demonstrate the importance of the role of antioxidants in protection and/or hepatic regeneration in animals. Thus, in this chapter, the principal antioxidants will be described that play an important role in the regeneration of hepatic cells and in the pre‐ vention of damage deriving from alcohol.

### **11. Flavonoids**

Flavonoids are compounds that make up part of the polyphenols and are also considered essentials nutrients. Their basic chemical structure consists of two benzene rings bound by means of a three-atom heterocyclic carbon chain. Oxidation of the structure gives rise to sev‐ eral families of flavonoids (flavons, flavonols, flavanons, anthocyanins, flavanols, and isofla‐ vons), and the chemical modifications that each family can undergo give rise to >5,000 compounds identified by their particular properties. [16].

Flavonoid digestion, absorption, and metabolism have common pathways with small differ‐ ences, such as, for example, unconjugated/non-conjugated flavonoids can be absorbed at the stomach level, while conjugated flavonoids are digested and absorbed at the intestinal level by extracellular enzymes on the enterocyte brush border. After absorption, flavonoids are conjugated by methylation, sukfonation, ands glucoronidation reactions due to their biologi‐ cal activity, such as facilitating their excretion by biliary or urinary route. The conjugation type the site where this occurs determine that metabolite's biological action, together with the protein binding for its circulation and interaction with cellular membranes and lipopro‐ teins. Flavonoid metabolites (conjugated or not) penetrate the tissues in which they possess some function (mainly antioxidant), or are metabolized. [27].

On the other hand, the flavonoids possess implications in health; in recent years, the proper‐ ties of these compounds have been studied in relation to diverse pathologies. In diabetes, these compounds present regulation of glycemia through diverse mechanisms that include the inhibition of some enzymes such as α-glucosidase, glucose 6 phosphatase, and phos‐ phorylated glycogen. The flavonoids possess other characteristics such as the trapping of molecules of glioxal and methyl-glioxal molecules, which propitiate the formation of ad‐ vanced final products of glycosylation that are found to be directly related with micro- and macrovascular complications. They also regulate the rise or fall of transporter proteins; the structure of some flavonoids appears to have important participation with regard to the studied benefits. [16].

More research is needed because great majority of the former has been conducted in ani‐ mals, to determine effects and dosage. Flavonoids in the menopause result in controversial effects due to the population type studied, that is, Asiatic, absorption, metabolism, the bind‐ ing of isoflavones to estrogen receptors, etc.; however, they appear to possess a beneficial effect in terms of the prevention of certain types of cancer and osteoporosis. [16].

to oxygen on reacting or interacting more rapidly with FR and ROS than with the remainder of molecules that are present in the microenvironment in which they are found (plasma membrane, cytosol, the nucleus, or Extracellular fluid [ECF]). Antioxidant action is one of the sacrifices of its own molecular integrity in order to avoid alterations in the remainder of vitally functioning or more important molecules. In the case of the exogenic antioxidants, re‐ placement through consumption in the diet is of highest importance, because these act as

This is the reason that, for several years, diverse researchers have been carrying out experi‐ mental studies that demonstrate the importance of the role of antioxidants in protection and/or hepatic regeneration in animals. Thus, in this chapter, the principal antioxidants will be described that play an important role in the regeneration of hepatic cells and in the pre‐

Flavonoids are compounds that make up part of the polyphenols and are also considered essentials nutrients. Their basic chemical structure consists of two benzene rings bound by means of a three-atom heterocyclic carbon chain. Oxidation of the structure gives rise to sev‐ eral families of flavonoids (flavons, flavonols, flavanons, anthocyanins, flavanols, and isofla‐ vons), and the chemical modifications that each family can undergo give rise to >5,000

Flavonoid digestion, absorption, and metabolism have common pathways with small differ‐ ences, such as, for example, unconjugated/non-conjugated flavonoids can be absorbed at the stomach level, while conjugated flavonoids are digested and absorbed at the intestinal level by extracellular enzymes on the enterocyte brush border. After absorption, flavonoids are conjugated by methylation, sukfonation, ands glucoronidation reactions due to their biologi‐ cal activity, such as facilitating their excretion by biliary or urinary route. The conjugation type the site where this occurs determine that metabolite's biological action, together with the protein binding for its circulation and interaction with cellular membranes and lipopro‐ teins. Flavonoid metabolites (conjugated or not) penetrate the tissues in which they possess

On the other hand, the flavonoids possess implications in health; in recent years, the proper‐ ties of these compounds have been studied in relation to diverse pathologies. In diabetes, these compounds present regulation of glycemia through diverse mechanisms that include the inhibition of some enzymes such as α-glucosidase, glucose 6 phosphatase, and phos‐ phorylated glycogen. The flavonoids possess other characteristics such as the trapping of molecules of glioxal and methyl-glioxal molecules, which propitiate the formation of ad‐ vanced final products of glycosylation that are found to be directly related with micro- and macrovascular complications. They also regulate the rise or fall of transporter proteins; the structure of some flavonoids appears to have important participation with regard to the

suicide molecules on encountering FR, as previously mentioned. [7, 21, 33].

vention of damage deriving from alcohol.

492 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

compounds identified by their particular properties. [16].

some function (mainly antioxidant), or are metabolized. [27].

**11. Flavonoids**

studied benefits. [16].

The flavonoids absorb Ultraviolet light (UV) from the sun and possess direct and indirect antioxidant effects (through the induction of cytoprotector proteins). Topical application (on the human skin) of the polyphenolic fraction of green tea protects against immunosuppres‐ sion and inhibits the erythema and the formation of pyrimidine dimers in DNA caused by UV. On representing one of the most important lifestyle factors, alimentation can important‐ ly affect the incidence and initiation of cardiovascular or neurodegenerative diseases. The cardioprotector effect of flavonoids is based on reducing oxidation and blood concentrations of the binding of cholesterol to Low-density lipoproteins (LDL); flavonoids reduce endothe‐ lial dysfunction and blood pressure and increase the HDL-bound cholesterol concentration. Flavonoids possess a neuroprotector effect because they protect the neurons from oxidative stress by means of induction of antioxidant defenses, modulation of signaling cascades, mi‐ tochondrial interactions, apoptotic processes, or by synthesis/degradation of the β-amyloid peptide. The potential effect of flavonoids as neuroprotectors is due to three main factors: they prevent neurodegeneration; inhibit neuroinflammation, and reduce the diminution of age-related cognitive functions. [16].

In cancer, the flavonoids have been classified as chemopreventive, as blockers as well as in‐ hibitors, given their functions in carcinogenesis, in which they modulate transduction sig‐ naling in cellular proliferation and angiogenesis, modulate enzymes for the metabolic activation of procarcinogens and the detoxification of carcinogens, and modulate enzymes in the biosynthesis of anti-oxidant-pro-oxidant estrogen activity estrogen (promoting oxida‐ tive homeostasis, rendering its antioxidative capacity as a contribution to antineoplastic as well as preventive as well as therapeutic activity due to inhibiting the activation of mitogen‐ ic kinases and transduction factors, while pro-oxidative activity increases the cell damage that promotes detention of the cell cycle and apoptosis). In obesity, the flavonoids have been identified as reducer factors of fat mass and as inhibitors of fat mass deposition and catabo‐ lic activity. [16].

The procyanidins and proanthocyanidins have demonstrated, in human population, to di‐ minish visceral fatty mass (depending on the dose) with an associated increase of adiponec‐ tin. This diminution is linked with the malabsorption of carbohydrates and lipids due to enzyme inhibition. It has been observed that the procyanidins increase β-oxidation and in‐ hibit the expression of genes that promote the synthesis of fatty acids. Epigallocatechin gal‐ late can increase energy expenditure and lipid oxidation in humans; it is thought that this is possible because of the increase of thermogenesis and the inhibition of the activity of the li‐ pase, as well as, according to studies *in vitro,* the inhibition of lipogenesis and apoptosis of the adipocytes. Catechins that alter the deposition of adipose tissue related with diminution of the respiratory co-efficient and greater oxygen consumption, and thermogenesis induced by the sympathetic nervous system. Phytoestrogens can improve obesity and its alterations on diminishing insulin resistance, thus lipogenesis, as well as inhibition of the mechanisms for cell differentiation and proliferation. The study of flavonoids and their effects on the pre‐ vention and treatment of obesity is a widespread, yet incomplete research field. [16].

The main active agent of the milk thistle is *silymarin*, a mixture of flavonolignans, silydia‐ nine, silycristin, and silybinin, the latter the most biologically active extract; the flavonoids appear to be activated as trappers of free radicals and as plasmatic membrane stabilizers. Concentrations of *silymarin* are localized in the fruit of the plant, as well as in the seeds and leaves, from which *silymarin* is extracted with 95%-proof ethanol, achieving a brilliant yel‐

Protective Effect of Silymarin on Liver Damage by Xenobiotics

http://dx.doi.org/10.5772/51502

495

The standardized extract of *silymarin* contains 70% *silymarin*. Pharmacokinetic studies have shown that there is rapid absorption of silybinin into the bloodstream after an oral dose. Steady-state plasma concentrations are reached after 2 hours and the elimination half-life is 6 hours. [Lorenz et al., 1984, 3]. From 3-8% of an oral dose is excreted in the urine and from

Silybinin works as an antioxidant, reacting rapidly with oxygen free radicals as demonstrat‐ ed *in vitro* with hydroxyl anions and hypochlorous acid. Reported activities include the in‐ hibition of hepatocyte lipoperoxidation, the microsomal membrane in rats, and protection against genomic damage through the suppression of hydrogen peroxide, superoxide anions, and lipoxygenase. It is thought that silybinin also increases the synthesis of the proteins of the hepatocyte through stimulation of the activity of the ribosomal RNA (rRNA) polymer‐ ase. In addition, silybinin diminishes hepatic and mitochondrial oxidation induced by an

*Silymarin* is an active principle that possesses hepatoprotector and regenerative action; its mechanism of action derives from its capacity to counterarrest the action of FR, which are formed due to the action of toxins that damage the cell membranes (lipid peroxidation), competitive inhibition through external cell membrane modification of hepatocytes; it forms a complex that impedes the entrance of toxins into the interior of liver cells and, on the other hand, metabolically stimulates hepatic cells, in addition to activating RNA biosythesis of the ribosomes, stimulating protein formation. In a study published by [41], the authors ob‐ served that *silymarin*'s protector effect on hepatic cells in rats when they employed this as a comparison factor on measuring liver weight/animal weight % (hepatomegaly), their values always being less that those of other groups administered with other possibly antioxidant substances; no significant difference was observed between the *silymarin* group and the *sily‐ marin*-alcohol group, thus demonstrating the protection of *silymarin*. On the other hand, *sily‐ marin* diminishes Kupffer cell activity and the production of glutathione, also inhibiting its oxidation. Participation has also been shown in the increase of protein synthesis in the hepa‐ tocyte on stimulating polymerase I RNA activity. *Silymarin* reduces collagen accumulation by 30% in biliary fibrosis induced in rat. An assay in humans reported a slight increase in the survival of persons with cirrhotic alcoholism compared with untreated controls [2].

*Silymarin* is a flavonoid derived from the *Silybum marianum* plant that has been employed for some 2,000 years for the treatment of liver diseases. At present, its use as an alternative

low liquid. The term flavonoid is derived from *flavus*, which denotes yellow. [20, 16].

20-40% is recovered in the bile as glucuronide and sulfate. [42].

iron overcharge and acts as an iron chelate. [16].

**13. Antioxidant and hepatoprotector action**

The metabolism of phytoestrogens and their maximum concentration in serum presents great variability, depending on genetic differences and estrogen exposure in early life stages. [16].

### **12. Silimarina (***silybum marianum***)**

*Silymarin* is a compound of natural origin extracted from the *Silybum marianum* plant, popu‐ larly known as St. Mary's thistle, whose active ingredients are flavonoids such as silybin, si‐ lydianin, and silycristin. This compound has attracted attention because of its possessing antifibrogenic properties, which have permitted it to be studied for its very promising ac‐ tions in experimental hepatic damage. In general, it possesses functions such as its antioxi‐ dant one, and it can diminish hepatic damage because of its cytoprotection as well as due to its inhibition of Kupffer cell function. [41].

*Silymarin*, derived from the milk thistle plant named *Silybum marianum*, has been used since time past as a natural remedy for combating liver diseases. *Silymarin* and its active constitu‐ ents (silybinin, silycristin, and silydianine, among others), have been classified as uptakers of free radicals and inhibitors of lipoperoxidation; some studies also suggest that that they increase the synthesis of hepatocytes, diminish the activity of tumor promoters, stabilize mastocyte cells, and act as iron chelates. [8].

*Silybum marianum* belongs to the Aster family (Asteraceae or Compositae), which includes daisies and thistles. The milk thistle is distributed widely throughout Europe, was the first plant that appeared in North America to the European colonizers, and is at present estab‐ lished in the South of the U.S., California, and South America. [22].

The name milk thistle is derived from the characteristics of its thorny leaves with white veins, which, according to the legend, were carried by the Virgin Mary. Its name *Cardo lecho‐ so* derives from the same tradition. The mature plant has large flowers, of a brilliant purple color, and abundant thorns of significant appearance. The milk thistle grows in places where exposure to the sun is abundant. [15].

Extracts of the milk thistle have been used as medical remedies from ancestral Greece, when Dioscorides, a Greek herbalist, wrote that the seeds of the milk thistle could cure the bite of a poisonous snake. Pliny noted that the mixture of the juice of the plant and its honey were excellent for bile tract disorders. [9]. In 1596, Gerard mentioned *Silybum marianum* as a major remedy against melancholy or black bile. The milk thistle was sold for treating liver diseas‐ es. In the 1960s, observed that milk thistle was an excellent remedy for cleaning obstructions of the liver and spleen, notwithstanding that infusions of the fresh roots and seeds were ef‐ fective for counteracting jaundice.

The main active agent of the milk thistle is *silymarin*, a mixture of flavonolignans, silydia‐ nine, silycristin, and silybinin, the latter the most biologically active extract; the flavonoids appear to be activated as trappers of free radicals and as plasmatic membrane stabilizers. Concentrations of *silymarin* are localized in the fruit of the plant, as well as in the seeds and leaves, from which *silymarin* is extracted with 95%-proof ethanol, achieving a brilliant yel‐ low liquid. The term flavonoid is derived from *flavus*, which denotes yellow. [20, 16].

The standardized extract of *silymarin* contains 70% *silymarin*. Pharmacokinetic studies have shown that there is rapid absorption of silybinin into the bloodstream after an oral dose. Steady-state plasma concentrations are reached after 2 hours and the elimination half-life is 6 hours. [Lorenz et al., 1984, 3]. From 3-8% of an oral dose is excreted in the urine and from 20-40% is recovered in the bile as glucuronide and sulfate. [42].

Silybinin works as an antioxidant, reacting rapidly with oxygen free radicals as demonstrat‐ ed *in vitro* with hydroxyl anions and hypochlorous acid. Reported activities include the in‐ hibition of hepatocyte lipoperoxidation, the microsomal membrane in rats, and protection against genomic damage through the suppression of hydrogen peroxide, superoxide anions, and lipoxygenase. It is thought that silybinin also increases the synthesis of the proteins of the hepatocyte through stimulation of the activity of the ribosomal RNA (rRNA) polymer‐ ase. In addition, silybinin diminishes hepatic and mitochondrial oxidation induced by an iron overcharge and acts as an iron chelate. [16].

### **13. Antioxidant and hepatoprotector action**

on diminishing insulin resistance, thus lipogenesis, as well as inhibition of the mechanisms for cell differentiation and proliferation. The study of flavonoids and their effects on the pre‐

The metabolism of phytoestrogens and their maximum concentration in serum presents great variability, depending on genetic differences and estrogen exposure in early life

*Silymarin* is a compound of natural origin extracted from the *Silybum marianum* plant, popu‐ larly known as St. Mary's thistle, whose active ingredients are flavonoids such as silybin, si‐ lydianin, and silycristin. This compound has attracted attention because of its possessing antifibrogenic properties, which have permitted it to be studied for its very promising ac‐ tions in experimental hepatic damage. In general, it possesses functions such as its antioxi‐ dant one, and it can diminish hepatic damage because of its cytoprotection as well as due to

*Silymarin*, derived from the milk thistle plant named *Silybum marianum*, has been used since time past as a natural remedy for combating liver diseases. *Silymarin* and its active constitu‐ ents (silybinin, silycristin, and silydianine, among others), have been classified as uptakers of free radicals and inhibitors of lipoperoxidation; some studies also suggest that that they increase the synthesis of hepatocytes, diminish the activity of tumor promoters, stabilize

*Silybum marianum* belongs to the Aster family (Asteraceae or Compositae), which includes daisies and thistles. The milk thistle is distributed widely throughout Europe, was the first plant that appeared in North America to the European colonizers, and is at present estab‐

The name milk thistle is derived from the characteristics of its thorny leaves with white veins, which, according to the legend, were carried by the Virgin Mary. Its name *Cardo lecho‐ so* derives from the same tradition. The mature plant has large flowers, of a brilliant purple color, and abundant thorns of significant appearance. The milk thistle grows in places where

Extracts of the milk thistle have been used as medical remedies from ancestral Greece, when Dioscorides, a Greek herbalist, wrote that the seeds of the milk thistle could cure the bite of a poisonous snake. Pliny noted that the mixture of the juice of the plant and its honey were excellent for bile tract disorders. [9]. In 1596, Gerard mentioned *Silybum marianum* as a major remedy against melancholy or black bile. The milk thistle was sold for treating liver diseas‐ es. In the 1960s, observed that milk thistle was an excellent remedy for cleaning obstructions of the liver and spleen, notwithstanding that infusions of the fresh roots and seeds were ef‐

vention and treatment of obesity is a widespread, yet incomplete research field. [16].

stages. [16].

**12. Silimarina (***silybum marianum***)**

494 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

its inhibition of Kupffer cell function. [41].

mastocyte cells, and act as iron chelates. [8].

exposure to the sun is abundant. [15].

fective for counteracting jaundice.

lished in the South of the U.S., California, and South America. [22].

*Silymarin* is an active principle that possesses hepatoprotector and regenerative action; its mechanism of action derives from its capacity to counterarrest the action of FR, which are formed due to the action of toxins that damage the cell membranes (lipid peroxidation), competitive inhibition through external cell membrane modification of hepatocytes; it forms a complex that impedes the entrance of toxins into the interior of liver cells and, on the other hand, metabolically stimulates hepatic cells, in addition to activating RNA biosythesis of the ribosomes, stimulating protein formation. In a study published by [41], the authors ob‐ served that *silymarin*'s protector effect on hepatic cells in rats when they employed this as a comparison factor on measuring liver weight/animal weight % (hepatomegaly), their values always being less that those of other groups administered with other possibly antioxidant substances; no significant difference was observed between the *silymarin* group and the *sily‐ marin*-alcohol group, thus demonstrating the protection of *silymarin*. On the other hand, *sily‐ marin* diminishes Kupffer cell activity and the production of glutathione, also inhibiting its oxidation. Participation has also been shown in the increase of protein synthesis in the hepa‐ tocyte on stimulating polymerase I RNA activity. *Silymarin* reduces collagen accumulation by 30% in biliary fibrosis induced in rat. An assay in humans reported a slight increase in the survival of persons with cirrhotic alcoholism compared with untreated controls [2].

*Silymarin* is a flavonoid derived from the *Silybum marianum* plant that has been employed for some 2,000 years for the treatment of liver diseases. At present, its use as an alternative drug has extended throughout Europe and the U.S. *Silymarin* acts as a hepatoprotector due to its antioxidant effect, which has been observed to inhibit liver damage due to the releas‐ ing of the substances of free radicals, such as ethanol, acetaminophen, and Carbon tetra‐ chloride (CCL4), in addition to increasing the activity of SOD and glutathione. As a uptaker of free radicals, *silymarin* can inhibit the lipid peroxidation cascade in the cell membranes. The hepatoprotector effect of this flavonoid also can be explained by an anti-inflammatory effect, in which it has been observed that *silymarin* acts on the functions of the Kupffer cells. Inhibition also has been reported in the activation of the Nuclear kappa-Beta [NK-B) tran‐ scription factor. [2, 16, 7, 21, 33].

velopment of oxidative stress depends in part on whether acute or chronic intoxication is in‐ volved. The factors that contribute to the development of oxidative stress imply disequilibrium among pro- and antioxidant factors. It can occur that oxidative stress devel‐ ops if the xenobiotic increases the pro-oxidant factors (the generation of Oxygen-generated free radicals [OFR]) or decreases intracellular antioxidant factors. In whichever of the two cases, the general result is important damage to the hepatocyte that can lead to general dam‐ age to the DNA that, in turn, can comprise a determining factor in the induction of the apop‐ totic system (programmed cell death) of the cell, thus accelerating its death and destruction.

Protective Effect of Silymarin on Liver Damage by Xenobiotics

http://dx.doi.org/10.5772/51502

497

The study of the factors that determine the increase in the generation of OFR in the liver, originating due to acute or chronic intoxication with a xenobiotic, is of great importance be‐ cause it will allow diminishing the damage that these reactive species produce within the hepatocyte. On the other hand, despite that at present much is known concerning the physi‐ opathological mechanisms of ethanol ingestion-related liver damage and the role that the production of oxygen-generated free radicals plays in these processes, the exact extent of this damage, as well as how to prevent it, remains unknown with precision. There is evi‐ dence obtained from laboratory models that the ingestion of natural antioxidants, such as vitamins A, C, and E, oligoelements (selenium), amino acids (glycine), and principally flavo‐ noids, such as *silymarin*, can in the future be a potential treatment for all persons who present hepatic alterations. However, beyond the remedy, the cooperation of the patient is required to regulate his/her ethanol consumption; as long as this does not take place, taking antioxidant vitamins can be considered within the regular therapy of a patient with alcohol‐ ism, taking care above all that this supplement does not reach toxic concentrations, in partic‐

ular in the case of the vitamin that possess the tendency to accumulate in the liver.

, Evila Gayosso-Islas1

, Ángel Morales-González2

, Manuel García-Luna y González-Rubio3

of the latter.

**Author details**

José A. Morales-González1

Carmen Valadez-Vega1

Cesar Esquivel-Chirino3

Eduardo Madrigal-Santillán1

1 Instituto de Ciencias de la Salud, UAEH, México

2 Escuela Superior de Computo, IPN, México

3 Facultad de Odontología, UNAM, México

The use of novel experimental procedures that determine the degree of damage caused by xenobiotics, and in particular by free radicals, is of great importance in the management of diseases caused by this type of substance, especially if they damage the liver, because this organ comprises a vital part of our organism on having in its charge the metabolic support

, Cecilia Sánchez-Moreno1

, Jaime Esquivel-Soto3

,

,

and

### **14.** *Silymarin* **and Exercise**

During physical activity, oxygen consumption increases, which produces oxidative stress that leads to the generation of free radicals, which are highly toxic for the cell, because these interact with organic molecules susceptible to being oxidized, such as unsaturated fatty acids, which causes lipoperoxidation. To avoid this damage by FR, there are the following antioxidant systems: Superoxide-dismutase (SOD), and Catalase (CAT), in addition to other protector substances such as vitamins A, C, and E and the flavonoids, which trap free radi‐ cals. (unpublished data)

In experiments conducted by our research group on groups of rats that were submitted to daily aerobic exercise in a physical-activity cage for 20 minutes during 4 weeks (5 days/ week) and on another group of rats submitted to physical activity plus administration of *silymarin* (200 mg/kg of weight) prior to exercise, with daily quantification of physical per‐ formance and at the at the end of the experiment, quantification of DNA in serum and of SOD and CAT activity in liver. We found that in the group with physical activity, MDA in‐ creased 134% (in serum) and 123% (in liver) vs. control rats. In the group with exercise plus *silymarin*, MDA returned to normality (in serum and in liver). Catalase activity increases during exercise (118%) and with exercise plus *silymarin* (137%). SOD activity exhibited no modifications in any treatment. Finally, we found an increase of physical activity in the group administered *silymarin* (27%) in comparison with the group in which no *silymarin* was administered. (unpublished data)

A protector effect was found of *silymarin* during exercise, because it diminishes MDA levels in serum as well as in liver, which translates into diminution of the production of free radi‐ cals, causing as a consequence less cellular damage, which in turn leads to an increase in physical performance.

### **15. Conclusions**

The process of the induction of oxidative stress generated in the liver due to the presence of ethanol implies the conjugation of various factors. The role that these factors play in the de‐ velopment of oxidative stress depends in part on whether acute or chronic intoxication is in‐ volved. The factors that contribute to the development of oxidative stress imply disequilibrium among pro- and antioxidant factors. It can occur that oxidative stress devel‐ ops if the xenobiotic increases the pro-oxidant factors (the generation of Oxygen-generated free radicals [OFR]) or decreases intracellular antioxidant factors. In whichever of the two cases, the general result is important damage to the hepatocyte that can lead to general dam‐ age to the DNA that, in turn, can comprise a determining factor in the induction of the apop‐ totic system (programmed cell death) of the cell, thus accelerating its death and destruction.

The study of the factors that determine the increase in the generation of OFR in the liver, originating due to acute or chronic intoxication with a xenobiotic, is of great importance be‐ cause it will allow diminishing the damage that these reactive species produce within the hepatocyte. On the other hand, despite that at present much is known concerning the physi‐ opathological mechanisms of ethanol ingestion-related liver damage and the role that the production of oxygen-generated free radicals plays in these processes, the exact extent of this damage, as well as how to prevent it, remains unknown with precision. There is evi‐ dence obtained from laboratory models that the ingestion of natural antioxidants, such as vitamins A, C, and E, oligoelements (selenium), amino acids (glycine), and principally flavo‐ noids, such as *silymarin*, can in the future be a potential treatment for all persons who present hepatic alterations. However, beyond the remedy, the cooperation of the patient is required to regulate his/her ethanol consumption; as long as this does not take place, taking antioxidant vitamins can be considered within the regular therapy of a patient with alcohol‐ ism, taking care above all that this supplement does not reach toxic concentrations, in partic‐ ular in the case of the vitamin that possess the tendency to accumulate in the liver.

The use of novel experimental procedures that determine the degree of damage caused by xenobiotics, and in particular by free radicals, is of great importance in the management of diseases caused by this type of substance, especially if they damage the liver, because this organ comprises a vital part of our organism on having in its charge the metabolic support of the latter.

### **Author details**

drug has extended throughout Europe and the U.S. *Silymarin* acts as a hepatoprotector due to its antioxidant effect, which has been observed to inhibit liver damage due to the releas‐ ing of the substances of free radicals, such as ethanol, acetaminophen, and Carbon tetra‐ chloride (CCL4), in addition to increasing the activity of SOD and glutathione. As a uptaker of free radicals, *silymarin* can inhibit the lipid peroxidation cascade in the cell membranes. The hepatoprotector effect of this flavonoid also can be explained by an anti-inflammatory effect, in which it has been observed that *silymarin* acts on the functions of the Kupffer cells. Inhibition also has been reported in the activation of the Nuclear kappa-Beta [NK-B) tran‐

During physical activity, oxygen consumption increases, which produces oxidative stress that leads to the generation of free radicals, which are highly toxic for the cell, because these interact with organic molecules susceptible to being oxidized, such as unsaturated fatty acids, which causes lipoperoxidation. To avoid this damage by FR, there are the following antioxidant systems: Superoxide-dismutase (SOD), and Catalase (CAT), in addition to other protector substances such as vitamins A, C, and E and the flavonoids, which trap free radi‐

In experiments conducted by our research group on groups of rats that were submitted to daily aerobic exercise in a physical-activity cage for 20 minutes during 4 weeks (5 days/ week) and on another group of rats submitted to physical activity plus administration of *silymarin* (200 mg/kg of weight) prior to exercise, with daily quantification of physical per‐ formance and at the at the end of the experiment, quantification of DNA in serum and of SOD and CAT activity in liver. We found that in the group with physical activity, MDA in‐ creased 134% (in serum) and 123% (in liver) vs. control rats. In the group with exercise plus *silymarin*, MDA returned to normality (in serum and in liver). Catalase activity increases during exercise (118%) and with exercise plus *silymarin* (137%). SOD activity exhibited no modifications in any treatment. Finally, we found an increase of physical activity in the group administered *silymarin* (27%) in comparison with the group in which no *silymarin* was

A protector effect was found of *silymarin* during exercise, because it diminishes MDA levels in serum as well as in liver, which translates into diminution of the production of free radi‐ cals, causing as a consequence less cellular damage, which in turn leads to an increase in

The process of the induction of oxidative stress generated in the liver due to the presence of ethanol implies the conjugation of various factors. The role that these factors play in the de‐

scription factor. [2, 16, 7, 21, 33].

496 Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

**14.** *Silymarin* **and Exercise**

cals. (unpublished data)

administered. (unpublished data)

physical performance.

**15. Conclusions**

José A. Morales-González1 , Evila Gayosso-Islas1 , Cecilia Sánchez-Moreno1 , Carmen Valadez-Vega1 , Ángel Morales-González2 , Jaime Esquivel-Soto3 , Cesar Esquivel-Chirino3 , Manuel García-Luna y González-Rubio3 and Eduardo Madrigal-Santillán1

1 Instituto de Ciencias de la Salud, UAEH, México

2 Escuela Superior de Computo, IPN, México

3 Facultad de Odontología, UNAM, México

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### *Edited by José A. Morales-González*

This work responds to the need to find, in a sole document, the affect of oxidative stress at different levels, as well as treatment with antioxidants to revert and diminish the damage. Oxidative Stress and Chronic Degenerative Diseases - a Role for Antioxidants is written for health professionals by researchers at diverse educative institutions (Mexico, Brazil, USA, Spain, Australia, and Slovenia). I would like to underscore that of the 19 chapters, 14 are by Mexican researchers, which demonstrates the commitment of Mexican institutions to academic life and to the prevention and treatment of chronic degenerative diseases.

Oxidative Stress and Chronic Degenerative Diseases - A Role for Antioxidants

Oxidative Stress and Chronic

Degenerative Diseases

A Role for Antioxidants

*Edited by José A. Morales-González*

Photo by M-image / iStock