Preface

Chapter 8 **Antimicrobial and Antioxidant Properties of Essential Oil**

Chapter 9 **Vascular Calcification and Oxidative DNA Damage as**

**Stress Conditions 107**

**Renal Disease 121**

Guillermina Miranda-Díaz

Kolluru

**VI** Contents

**Isolated from Coleus zeylanicus under Normal and Salinity**

Divya Kotagiri, Khasim Beebi Shaik and Viswanatha Chaitanya

Edith Viridiana Alatorre-Moreno, José Ignacio Cerrillos-Gutiérrez, Jorge Andrade-Sierra, Enrique Rojas-Campos, Sandra Carrillo-Ibarra, Sonia Sifuentes-Franco, Andrés García-Sánchez and Alejandra

**Nontraditional Cardiovascular Risk Factors in Chronic**

The idea of this book project comes after the much-acclaimed volume in this series, *Free Rad‐ icals and Diseases* published by InTech in 2016 (downloaded more than 11,000 times). Free radicals have been extensively studied in recent years, but with very few exceptions, their roles in the etiologies of specific disorders remain largely undefined. As our understanding of molecular regulation improves, the roles that free radicals have in both preserving and adversely affecting functions will become more clearly defined.

Antioxidants may play a role in the management or prevention of some medical conditions, such as some cancers, macular degeneration, Alzheimer's diseases, and autoimmune disor‐ ders. This volume entitled *Free Radicals, Antioxidants, and Diseases* gives an idea of detecting free radicals in vivo by newer techniques and provides insights into the roles played by var‐ ious antioxidants in combating diseases caused by oxidative stress.

The book covers topics beginning from the basics of free radicals and the role of antioxi‐ dants in human health, including mechanisms of their actions in disease prevention such as the role of dimethylformamide in cerebral ischemia, essential oil from *Coleus zeylanicus*, car‐ dioprotective effects of S-nitrosothiols, and physical exercise as prooxidant and antioxidant. Analysis of various antioxidants by EPR is also described. Some of the chapters present re‐ views on subjects of oxidative DNA damage in chronic renal diseases and aging process. The role of microRNAs as possible regulators of gene expression in aging is also envisaged.

Since this field is rapidly expanding with new discoveries of implications of free radicals in many diseases and roles of various antioxidants being reported every year, we hope that these two books will also provide a reference to free radical research having easy-to-follow ap‐ proach for the academic use or laboratory manual for students in basic and medical sciences.

I am very much delighted to be involved in this book project. It really gives me the chance to go through the diversified aspects of this astonishing field. I am indebted to Dr. Abdullah Al-Rubaish, President of IAU; Dr. Fahad Al-Muhanna, Vice President; Prof. Ali Al-Sultan, Dean of the College of Medicine; and Prof. Abdulaziz Al-Sebiani, Vice Dean of Academic Affairs for providing me the time and necessary facilities for this accomplishment. Editorial assistance by Ms. Marijana Francetic is gratefully acknowledged.

> **Rizwan Ahmad, MPhil, PhD** College of Medicine Imam Abdulrahman Bin Faisal University Dammam, Saudi Arabia

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Basics of Free Radicals and**

**Introductory Chapter: Basics of Free Radicals and** 

DOI: 10.5772/intechopen.76689

Do you know the scientist who had discovered the free radical? It was a stunning Professor of Chemistry named Moses Gomberg in 1900 at Michigan State University. In June 25, 2000, after a century the discovery was commemorated by the American Chemical Society as a National Historic Chemical Landmark in a ceremony at the University of Michigan, Michigan [1]. Earlier evidence by a British Chemist Fenton also suggests that hydrogen peroxide which reacts with ferrous sulfate results in violet color which is nothing but oxidation of tartaric acid upon addition of alkali. This is the basis of production of hydroxyl radical, and the reaction is

The most common definition of free radicals is "molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals" [3]. Free radicals are uncharged, very reactive, and short-lived molecules. Human beings contain 10,000–20,000 free radicals which attack each and individual cell of our body. Many of these radicals are beneficial in that they work for immune cells responsible for killing bacterial cells and toning of smooth muscles, which in turn regulate the normal working of blood vessels and internal organs. Uncontrolled generation of free radicals in our body may lead to various ill effects

such as autoimmune diseases, heart and neurodegenerative diseases, cancers, etc. [4].

Now, coming to their very existence, one should know the basics. The free radicals are produced during ATP generation through mitochondria. They are generally divided into two

> © 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76689

**Antioxidants**

**Antioxidants**

Rizwan Ahmad

Rizwan Ahmad

**1. Historical Aspect**

called as Fenton reaction [2].

**2. Free Radicals**

#### **Introductory Chapter: Basics of Free Radicals and Antioxidants Introductory Chapter: Basics of Free Radicals and Antioxidants**

DOI: 10.5772/intechopen.76689

#### Rizwan Ahmad Rizwan Ahmad

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76689

### **1. Historical Aspect**

Do you know the scientist who had discovered the free radical? It was a stunning Professor of Chemistry named Moses Gomberg in 1900 at Michigan State University. In June 25, 2000, after a century the discovery was commemorated by the American Chemical Society as a National Historic Chemical Landmark in a ceremony at the University of Michigan, Michigan [1]. Earlier evidence by a British Chemist Fenton also suggests that hydrogen peroxide which reacts with ferrous sulfate results in violet color which is nothing but oxidation of tartaric acid upon addition of alkali. This is the basis of production of hydroxyl radical, and the reaction is called as Fenton reaction [2].

### **2. Free Radicals**

The most common definition of free radicals is "molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals" [3]. Free radicals are uncharged, very reactive, and short-lived molecules. Human beings contain 10,000–20,000 free radicals which attack each and individual cell of our body. Many of these radicals are beneficial in that they work for immune cells responsible for killing bacterial cells and toning of smooth muscles, which in turn regulate the normal working of blood vessels and internal organs. Uncontrolled generation of free radicals in our body may lead to various ill effects such as autoimmune diseases, heart and neurodegenerative diseases, cancers, etc. [4].

Now, coming to their very existence, one should know the basics. The free radicals are produced during ATP generation through mitochondria. They are generally divided into two

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

well-known entities: reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS collectively form all the radical and non-radical (oxidants) entities. Radicals are more reactive and less stable than their non-radical counterparts. Non-radical derivatives or oxidants can be easily converted into free radicals by various reactions in living organisms [5].

and chronic inflammation. Aging is also a form of the deleterious effect of free radical damage (protein oxidation, lipid peroxidation). Mutation of DNA is a crucial step in carcinogenesis, and elevated levels of DNA lesions are found in many tumors, strongly correlating the oxidative damage in the etiology of cancer. ROS-induced changes are found in various diseases of the heart such as cardiomyopathies, ischemic heart disease, hypertension, atherosclerosis, and heart failure [9]. Free radicals induced cell damage that is found in many autoimmune diseases. Oxidative injury and inflammatory status were proven by raised levels of isoprostanes and prostaglandins in serum and synovial fluid compared to controls [10]. Increased oxidative stress has been proposed as one of the major factors causing hyperglycemia which in turn trigger various diabetic conditions [11]. Oxidative damage advances with age, and this is the reason that it is considered as one of the causative factors in various neurological disorders such as

Introductory Chapter: Basics of Free Radicals and Antioxidants

http://dx.doi.org/10.5772/intechopen.76689

3

In this book, we have tried to incorporate themes and concepts which eventually give the insights into the role of antioxidants in combating various disorders caused by free radical damage. In one of the chapter the author discusses the unique property of essential oil extracted from *Coleus zeylanicus* using the bioautography technique. It reveals the antimicrobial and antioxidant properties of oil from *Coleus zeylanicus* which further increases upon exposure

A novel concept of immune-spin trapping combined with molecular magnetic resonance imaging is also explored in this book. This would give the readers an insight to detect free radicals *in vivo* formed in different tissues. Various therapeutic agents can also be traced to

The very motive of including a chapter on ethnic aspects of oxidative stress-induced TIDM is that the racial variability was not studied and there is no conclusive evidence about lipid peroxidation and antioxidant defense system in patients of a region. Their comparative study

Scavenging effect of dimethylformamide (DMF) in cerebral ischemia (CI) is also analyzed. The results and discussion will lead to future research and more clinical trials to establish

The role of physical exercise on oxidative stress and vascular diseases is reviewed extensively. Generation of oxidants and simultaneously antioxidants during physical exercise which is in

The recent findings in free radical-induced damage in aging and how antioxidants are employed as an anti-aging agent in the preclinical model are discussed in the book. This would give an idea of their therapeutic potential. Micro-RNAs (miRNAs) are the novel regulators of gene expressions which induce aging process. In the chapter of miRNAs in the regulation of hyperglycemia-induced oxidative stress and cellular senescence, authors explain the critical role of miRNAs in age-related disorders which could open the front of new findings in geriatrics.

Alzheimer's and Parkinson's diseases [4].

their role in free radical generation and quenching.

turn responsible for its prevention is summarized.

recommends a highly significant approach to this aspect.

**5. Book Overview**

to salinity stress condition.

DMF as an antioxidant in CI.

Oxidative phosphorylation occurring in mitochondria is a nonenzymatic process which results in the production of ROS and RNS. For example, excess generation of hydroxyl radical and peroxynitrite causes damage to cell membranes and lipoproteins, and the process referred to as lipid peroxidation. This results in the formation of mutagenic and cytotoxic compounds such as malondialdehyde (MDA) and other diene derivatives. ROS/RNS affects proteins which may lead to the loss of enzyme activity and structural deformity. These species attack DNA, thus affecting the functional growth and formation of oxidative lesions which can lead to mutagenesis. The body has various enzymes and antioxidants to combat the damage incurred by oxidative stress [6, 7].

### **3. Antioxidants**

Antioxidants are chemicals that bind with free radicals and nullify their effect from causing damage to biological molecules. Endogenous antioxidants are produced by our body which is used to combat various free radicals. However, most of them are obtained from external sources, primarily through diet called as exogenous antioxidants or dietary antioxidants. Major sources of this class of antioxidants are brightly colored vegetables, fruits, and grains. Other very effective sources are berries, green tea, and dark chocolate. Nowadays, many oral supplements are available in the market labeled as dietary antioxidants.

Antioxidants bind with free radical by giving up their own electrons. These results in the termination of oxidative chain reactions, and the free radicals are no longer able to attack the cell. Antioxidant attains free radical state after donating its electron. It can accommodate the change in electrons without becoming reactive, and that's why they are not harmful. There are two lines of antioxidant defense inside the cell. Vitamin E, beta-carotene, and coenzyme Q constitute the first line which is found in the fat-soluble cell membrane. Vitamin E is considered as the most potent chain-breaking antioxidant within the cell membrane. The water-soluble antioxidant is present inside the cell. These are vitamin C, superoxide dismutase (SOD), glutathione peroxidase, and catalase [8].

### **4. Role in Diseases**

Despite the antagonistic role played by various antioxidants, the free radical damage has been implicated in various degenerative conditions such as cardiovascular diseases, neurological disorders, diabetes, ischemia-reperfusion injury, and aging. These diseases may be divided into two groups: (i) the first group includes cancer and diabetes which occurs due to impairing glucose tolerance in which the condition is better known as "mitochondrial oxidative stress" and (ii) the second group is characterized by inflammatory oxidative conditions leading to atherosclerosis and chronic inflammation. Aging is also a form of the deleterious effect of free radical damage (protein oxidation, lipid peroxidation). Mutation of DNA is a crucial step in carcinogenesis, and elevated levels of DNA lesions are found in many tumors, strongly correlating the oxidative damage in the etiology of cancer. ROS-induced changes are found in various diseases of the heart such as cardiomyopathies, ischemic heart disease, hypertension, atherosclerosis, and heart failure [9].

Free radicals induced cell damage that is found in many autoimmune diseases. Oxidative injury and inflammatory status were proven by raised levels of isoprostanes and prostaglandins in serum and synovial fluid compared to controls [10]. Increased oxidative stress has been proposed as one of the major factors causing hyperglycemia which in turn trigger various diabetic conditions [11]. Oxidative damage advances with age, and this is the reason that it is considered as one of the causative factors in various neurological disorders such as Alzheimer's and Parkinson's diseases [4].

### **5. Book Overview**

well-known entities: reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS collectively form all the radical and non-radical (oxidants) entities. Radicals are more reactive and less stable than their non-radical counterparts. Non-radical derivatives or oxidants can be easily converted into free radicals by various reactions in living organisms [5]. Oxidative phosphorylation occurring in mitochondria is a nonenzymatic process which results in the production of ROS and RNS. For example, excess generation of hydroxyl radical and peroxynitrite causes damage to cell membranes and lipoproteins, and the process referred to as lipid peroxidation. This results in the formation of mutagenic and cytotoxic compounds such as malondialdehyde (MDA) and other diene derivatives. ROS/RNS affects proteins which may lead to the loss of enzyme activity and structural deformity. These species attack DNA, thus affecting the functional growth and formation of oxidative lesions which can lead to mutagenesis. The body has various enzymes and antioxidants to combat

Antioxidants are chemicals that bind with free radicals and nullify their effect from causing damage to biological molecules. Endogenous antioxidants are produced by our body which is used to combat various free radicals. However, most of them are obtained from external sources, primarily through diet called as exogenous antioxidants or dietary antioxidants. Major sources of this class of antioxidants are brightly colored vegetables, fruits, and grains. Other very effective sources are berries, green tea, and dark chocolate. Nowadays, many oral

Antioxidants bind with free radical by giving up their own electrons. These results in the termination of oxidative chain reactions, and the free radicals are no longer able to attack the cell. Antioxidant attains free radical state after donating its electron. It can accommodate the change in electrons without becoming reactive, and that's why they are not harmful. There are two lines of antioxidant defense inside the cell. Vitamin E, beta-carotene, and coenzyme Q constitute the first line which is found in the fat-soluble cell membrane. Vitamin E is considered as the most potent chain-breaking antioxidant within the cell membrane. The water-soluble antioxidant is present inside the cell. These are vitamin C, superoxide dismutase (SOD),

Despite the antagonistic role played by various antioxidants, the free radical damage has been implicated in various degenerative conditions such as cardiovascular diseases, neurological disorders, diabetes, ischemia-reperfusion injury, and aging. These diseases may be divided into two groups: (i) the first group includes cancer and diabetes which occurs due to impairing glucose tolerance in which the condition is better known as "mitochondrial oxidative stress" and (ii) the second group is characterized by inflammatory oxidative conditions leading to atherosclerosis

supplements are available in the market labeled as dietary antioxidants.

the damage incurred by oxidative stress [6, 7].

glutathione peroxidase, and catalase [8].

**4. Role in Diseases**

**3. Antioxidants**

2 Free Radicals, Antioxidants and Diseases

In this book, we have tried to incorporate themes and concepts which eventually give the insights into the role of antioxidants in combating various disorders caused by free radical damage. In one of the chapter the author discusses the unique property of essential oil extracted from *Coleus zeylanicus* using the bioautography technique. It reveals the antimicrobial and antioxidant properties of oil from *Coleus zeylanicus* which further increases upon exposure to salinity stress condition.

A novel concept of immune-spin trapping combined with molecular magnetic resonance imaging is also explored in this book. This would give the readers an insight to detect free radicals *in vivo* formed in different tissues. Various therapeutic agents can also be traced to their role in free radical generation and quenching.

The very motive of including a chapter on ethnic aspects of oxidative stress-induced TIDM is that the racial variability was not studied and there is no conclusive evidence about lipid peroxidation and antioxidant defense system in patients of a region. Their comparative study recommends a highly significant approach to this aspect.

Scavenging effect of dimethylformamide (DMF) in cerebral ischemia (CI) is also analyzed. The results and discussion will lead to future research and more clinical trials to establish DMF as an antioxidant in CI.

The role of physical exercise on oxidative stress and vascular diseases is reviewed extensively. Generation of oxidants and simultaneously antioxidants during physical exercise which is in turn responsible for its prevention is summarized.

The recent findings in free radical-induced damage in aging and how antioxidants are employed as an anti-aging agent in the preclinical model are discussed in the book. This would give an idea of their therapeutic potential. Micro-RNAs (miRNAs) are the novel regulators of gene expressions which induce aging process. In the chapter of miRNAs in the regulation of hyperglycemia-induced oxidative stress and cellular senescence, authors explain the critical role of miRNAs in age-related disorders which could open the front of new findings in geriatrics.

### **Author details**

#### Rizwan Ahmad

Address all correspondence to: ahmadriz.biochem@gmail.com

College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia

**Chapter 2**

**Provisional chapter**

**Detecting** *In Vivo* **Free Radicals in Various Disease**

**Detecting** *In Vivo* **Free Radicals in Various Disease** 

DOI: 10.5772/intechopen.74106

*In vivo* free radical imaging in pre-clinical models of disease is now possible. Free radicals have traditionally been characterized by ESR or EPR spin trapping spectroscopy. The disadvantage of the ESR/EPR approach is that spin adducts are short-lived due to biological reductive and/or oxidative processes. Immuno-spin trapping (IST) involves the use of an antibody that recognizes macromolecular DMPO-spin adducts (anti-DMPO antibody), regardless of the oxidative/reductive state of trapped radical adducts. The IST approach has been extended to an *in vivo* application that combines IST with molecular magnetic resonance imaging (mMRI). This combined IST-mMRI approach involves the use of a spin trapping agent, DMPO, to trap free radicals in disease models, and administration of a mMRI probe, an anti-DMPO probe, that combines an antibody against DMPO-radical adducts and a MRI contrast agent, resulting in targeted free radical adduct detection. The combined IST-mMRI approach has been used in several rodent disease models, including diabetes, ALS, gliomas, and septic encephalopathy. The advantage of this approach is that heterogeneous levels of trapped free radicals can be detected directly *in vivo* and *in situ* to pin-point where free radicals are formed in different tissues. The approach can also be used to assess therapeutic agents that are either free radical scavengers or generate free radicals. The focus of this review will be on the different applications that have

been studied, advantages and limitations, and future directions.

(ALS), glioma, septic encephalopathy, mice, rats

**Keywords:** immuno-spin trapping (IST), molecular magnetic resonance imaging (mMRI), targeted free radical imaging, *in vivo*, diabetes, amyotrophic lateral sclerosis

> © 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

**Models**

**Models**

Rheal A. Towner and Nataliya Smith

Rheal A. Towner and Nataliya Smith

http://dx.doi.org/10.5772/intechopen.74106

**Abstract**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

### **References**


#### **Detecting** *In Vivo* **Free Radicals in Various Disease Models Detecting** *In Vivo* **Free Radicals in Various Disease Models**

DOI: 10.5772/intechopen.74106

Rheal A. Towner and Nataliya Smith Rheal A. Towner and Nataliya Smith

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74106

#### **Abstract**

**Author details**

4 Free Radicals, Antioxidants and Diseases

Rizwan Ahmad

**References**

day.com

Research. 1992;**26**:641-655

Biology. 2007;**39**(1):44-84

Society. 1894;**65**:899-910

Molecular Biology. 2008;**1**:1-9

Address all correspondence to: ahmadriz.biochem@gmail.com

education/whatischemistry/landmarks/freeradicals

College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia

[1] American Chemical Society National Historic Chemical Landmarks. Moses Gomberg and Organic Free Radicals. 2000. Available from: http://www.acs.org/content/acs/en/

[2] Fenton HJH. Oxidation of tartaric acid in presence of iron. Journal of the Chemical

[3] Halliwell B, Gutteridge JMC. Free radicals, other reactive species and disease. In: Free Radicals in Biology and Medicine. 3rd ed. Oxford: Oxford University Press; 1999. pp. 617-783

[4] Pala FS, Gurkan H. The role of free radicals in etiopathogenesis of diseases. Advances in

[5] Diplock AT, Charleux JL, Crozier-Willi G, et al. Functional food science and defence against reactive oxygen species. British Journal of Nutrition. 1998;**80**(1):S77-S112

[6] Bouayed J, Bohn T. Exogenous antioxidants—double-edged swords in cellular redoc state: health beneficial effects at physiologic doses versus deleterious effects at high

[7] Ahmad R, Ahsan H. Role of peroxynitrite, a nitrogen free radical in the etiopathogenesis of systemic *Lupus Erythematosus*. Clinical and Experimental Medicine. 2014;**14**(1):1-11 [8] Villines Z. Antioxidants and free radicals. 2017. Available from: www.medicalnewsto-

[9] Kukreja RC, Hess ML. The oxygen free-radical system-From equations through membrane-protein interactions to cardiovascular injury and protection. Cardiovascular

[10] Firestein GS, Echeverri F, Yeo M, et al. Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. Proceedings of the National Academy of

[11] Valko M, Leibfritz D, Moncola J, et al. Free radicals and antioxidants in normal physiological functions and human disease. International Journal of Biochemistry and Cell

doses. Oxidative Medicine and Cellular Longevity. 2010;**3**(4):228-237

Sciences of the United States of America. 1997;**94**:10895-10900

*In vivo* free radical imaging in pre-clinical models of disease is now possible. Free radicals have traditionally been characterized by ESR or EPR spin trapping spectroscopy. The disadvantage of the ESR/EPR approach is that spin adducts are short-lived due to biological reductive and/or oxidative processes. Immuno-spin trapping (IST) involves the use of an antibody that recognizes macromolecular DMPO-spin adducts (anti-DMPO antibody), regardless of the oxidative/reductive state of trapped radical adducts. The IST approach has been extended to an *in vivo* application that combines IST with molecular magnetic resonance imaging (mMRI). This combined IST-mMRI approach involves the use of a spin trapping agent, DMPO, to trap free radicals in disease models, and administration of a mMRI probe, an anti-DMPO probe, that combines an antibody against DMPO-radical adducts and a MRI contrast agent, resulting in targeted free radical adduct detection. The combined IST-mMRI approach has been used in several rodent disease models, including diabetes, ALS, gliomas, and septic encephalopathy. The advantage of this approach is that heterogeneous levels of trapped free radicals can be detected directly *in vivo* and *in situ* to pin-point where free radicals are formed in different tissues. The approach can also be used to assess therapeutic agents that are either free radical scavengers or generate free radicals. The focus of this review will be on the different applications that have been studied, advantages and limitations, and future directions.

**Keywords:** immuno-spin trapping (IST), molecular magnetic resonance imaging (mMRI), targeted free radical imaging, *in vivo*, diabetes, amyotrophic lateral sclerosis (ALS), glioma, septic encephalopathy, mice, rats

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

### **1. Introduction**

#### **1.1. Free radicals in various diseases**

Reactive oxygen and nitrogen species (RONS) lead to structural and functional modifications of cellular proteins and lipids, resulting in cellular dysfunction, such as impaired energy metabolism, altered cell signaling and cell cycle control, impaired cell transport processes and dysfunctional biological activities, immune activation, and inflammation [1]. RONS can be involved in several disease processes as causative agents or result as an effect of the pathogenesis. It is well known that free radicals play a role in the pathogenesis associated with various diseases such as diabetes, septic encephalopathy, neurodegenerative diseases, and cancers, to mention a few.

The formation of excessive superoxide radicals also depletes ambient nitric oxide in the cerebrovascular bed, forming peroxynitrite, which irreversibly inhibits the mitochondrial electron transport chain, resulting in an increase in mitochondrial release of free radicals, and leads to mitochondrial dysfunction and neuronal bioenergetics failure [4]. Additionally, free radicals trigger apoptosis via altering intracellular calcium homeostasis in brain regions such as the cerebral cortex and hippocampus, further exacerbating local inflammatory responses further [4].

Detecting *In Vivo* Free Radicals in Various Disease Models

http://dx.doi.org/10.5772/intechopen.74106

7

Oxidative stress has been proposed as a contributory factor in the pathogenesis of several neurodegenerative diseases [5]. For instance, in familial ALS (amyotrophic lateral sclerosis) (accounting for 5–10% of ALS cases) there is a mutation in superoxide dismutase 1 (SOD1) which results in dysfunctional superoxide radical clearance, leading to increased oxidative stress [5]. NADPH oxidases have emerged as possible drug targets for the treatment of neurodegeneration, due to

In cancer cells, RONS accumulation can result in damaging DNA, directly through an increase in cellular mutations and/or increase in oncogenic phenotypes, or indirectly by acting as secondary messengers intracellular signaling cascades [7]. It is thought that impaired cellular repair mechanisms induced by RONS oxidative stress on DNA can lead to cell injury and subsequently to genomic instability, mutagenesis and tumorigenesis [7]. It is also known that ROS can promote cell proliferation activating growth-related signaling pathways [7]. ROS may be involved in the multistep oncogenesis process at various different phases related to tumor initiation and progression, ROS-related mechanisms during tumor promotion, maintenance of the transformed state through extracellular superoxide radical formation by NADPH oxidase 1, and resistance to oxidative stress signals through membrane-associated

For over half a century, free radicals were characterized by electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy coupled with spin trapping. Nitrone spin traps (*N*-oxides of imines), such as PBN (α-phenyl-*tert*-butyl nitrone), DMPO (5,5-dimethylpyrroline-*N*-oxide) or 4-POBN (α(4-pyridyl-1-oxide)-N-tert-butyl nitrone), are the most commonly used for biological systems [8–18], and have been administered *in vivo* in various

The disadvantage of the ESR/EPR approach is that the spin adducts (spin trapping agent free radical adducts or aminoxyls) are short-lived due to reductive and/or oxidative processes

Mason *et al*. developed an antibody that recognizes macromolecular DMPO spin adducts, regardless of the oxidative/reductive state of the trapped radical adducts, and called the methodology immuno-spin trapping (IST) [23–28] (see **Figure 2** for an illustrative description), that has been applied in over 80 publications. The anti-DMPO antibody is attached to a fluorescent dye, allowing the *in vitro* or *ex vivo* detection of trapped DMPO-radical adducts,

their role in generating oxidants and also regulating microglia activation [6].

catalase expression [7].

**1.2. Spin trapping and ESR/EPR spectroscopy**

pre-clinical disease models [19–22] for several decades.

in biological systems [8, 9] (see **Figure 1**).

**1.3. Immuno-spin trapping (IST)**

Nutritional stress, that for instance may result from excessive high-fat and/or carbohydrates, can promote oxidative stress, subsequently forming lipid peroxidation products, protein carbonylation, as well as decreased antioxidant levels [1]. Chronic oxidative stress and inflammation, both associated with obesity, can lead to insulin resistance, dysregulated metabolic pathways, diabetes and cardiovascular diseases, via impaired signaling and metabolism that result in insulin secretion dysfunction, insulin action, and immune responses [1]. In type 1 diabetes mellitus, RONS released from phagocytes may damage adjacent cells, which can lead to excessive inflammation and an autoimmune attack against pancreatic islet β-cells, and contribute to a rapid progression of pathogenesis [1]. Immune system-associated enzymes (such as NADPH oxidase) can trigger the formation of reactive oxygen species (ROS) [1]. Excessive glucose and lipid levels, endocrine factors and numerous pro-inflammatory cytokines are known to activate NADPH oxidase [1]. Pro-inflammatory cytokines can also upregulate nitric oxide synthase 2 (NOS2), producing excessive nitric oxide, which can subsequently lead to the formation of peroxynitrite, and lead to further oxidative stress [1]. In Type 2 diabetes mellitus, excessive RONS production from chronic hyperglycemia increases oxidative stress in tissues that exacerbate the disease, such as pancreatic islets, muscle, adipose and hepatic, as well as influences secondary diabetic complications, including nephropathy, vascular disease and retinopathy, leading to oxidized lipids and proteins [1, 2].

Sepsis-associated encephalopathy pathophysiology is still poorly understood, but a number of mechanisms-of-action (MOA) have been proposed, including mitochondrial and vascular dysfunction, oxidative damage, neurotransmission disturbances, inflammation, and cellular death [3, 4]. Oxidative stress is a central MOA of acute brain damage [3]. Systemic inflammation induces mitochondrial dysfunction, which is involved in both apoptotic and necrotic cell death pathways, and increased glucose uptake by brain tissues, which results in the diversion of glucose to the pentose phosphate pathway that may contribute to oxidative stress by producing excessive superoxide radicals via NADPH oxidase [3, 4]. In addition, microglia activation results in the secretion of nitric oxide, ROS (reactive oxygen species) and matrix metalloproteinases (MMPs) that can all contribute to blood-brain barrier (BBB) and neuronal damage [3]. Regarding brain dysfunction in sepsis, it is thought that RONS, generated during a systemic inflammatory response, triggers lipid peroxidation due to a decreased antioxidant activity [4]. Free radical-induced structural membrane damage also induces neuro-inflammation [4]. The formation of excessive superoxide radicals also depletes ambient nitric oxide in the cerebrovascular bed, forming peroxynitrite, which irreversibly inhibits the mitochondrial electron transport chain, resulting in an increase in mitochondrial release of free radicals, and leads to mitochondrial dysfunction and neuronal bioenergetics failure [4]. Additionally, free radicals trigger apoptosis via altering intracellular calcium homeostasis in brain regions such as the cerebral cortex and hippocampus, further exacerbating local inflammatory responses further [4].

Oxidative stress has been proposed as a contributory factor in the pathogenesis of several neurodegenerative diseases [5]. For instance, in familial ALS (amyotrophic lateral sclerosis) (accounting for 5–10% of ALS cases) there is a mutation in superoxide dismutase 1 (SOD1) which results in dysfunctional superoxide radical clearance, leading to increased oxidative stress [5]. NADPH oxidases have emerged as possible drug targets for the treatment of neurodegeneration, due to their role in generating oxidants and also regulating microglia activation [6].

In cancer cells, RONS accumulation can result in damaging DNA, directly through an increase in cellular mutations and/or increase in oncogenic phenotypes, or indirectly by acting as secondary messengers intracellular signaling cascades [7]. It is thought that impaired cellular repair mechanisms induced by RONS oxidative stress on DNA can lead to cell injury and subsequently to genomic instability, mutagenesis and tumorigenesis [7]. It is also known that ROS can promote cell proliferation activating growth-related signaling pathways [7]. ROS may be involved in the multistep oncogenesis process at various different phases related to tumor initiation and progression, ROS-related mechanisms during tumor promotion, maintenance of the transformed state through extracellular superoxide radical formation by NADPH oxidase 1, and resistance to oxidative stress signals through membrane-associated catalase expression [7].

#### **1.2. Spin trapping and ESR/EPR spectroscopy**

**1. Introduction**

6 Free Radicals, Antioxidants and Diseases

mention a few.

**1.1. Free radicals in various diseases**

Reactive oxygen and nitrogen species (RONS) lead to structural and functional modifications of cellular proteins and lipids, resulting in cellular dysfunction, such as impaired energy metabolism, altered cell signaling and cell cycle control, impaired cell transport processes and dysfunctional biological activities, immune activation, and inflammation [1]. RONS can be involved in several disease processes as causative agents or result as an effect of the pathogenesis. It is well known that free radicals play a role in the pathogenesis associated with various diseases such as diabetes, septic encephalopathy, neurodegenerative diseases, and cancers, to

Nutritional stress, that for instance may result from excessive high-fat and/or carbohydrates, can promote oxidative stress, subsequently forming lipid peroxidation products, protein carbonylation, as well as decreased antioxidant levels [1]. Chronic oxidative stress and inflammation, both associated with obesity, can lead to insulin resistance, dysregulated metabolic pathways, diabetes and cardiovascular diseases, via impaired signaling and metabolism that result in insulin secretion dysfunction, insulin action, and immune responses [1]. In type 1 diabetes mellitus, RONS released from phagocytes may damage adjacent cells, which can lead to excessive inflammation and an autoimmune attack against pancreatic islet β-cells, and contribute to a rapid progression of pathogenesis [1]. Immune system-associated enzymes (such as NADPH oxidase) can trigger the formation of reactive oxygen species (ROS) [1]. Excessive glucose and lipid levels, endocrine factors and numerous pro-inflammatory cytokines are known to activate NADPH oxidase [1]. Pro-inflammatory cytokines can also upregulate nitric oxide synthase 2 (NOS2), producing excessive nitric oxide, which can subsequently lead to the formation of peroxynitrite, and lead to further oxidative stress [1]. In Type 2 diabetes mellitus, excessive RONS production from chronic hyperglycemia increases oxidative stress in tissues that exacerbate the disease, such as pancreatic islets, muscle, adipose and hepatic, as well as influences secondary diabetic complications, including nephropathy, vascular disease

Sepsis-associated encephalopathy pathophysiology is still poorly understood, but a number of mechanisms-of-action (MOA) have been proposed, including mitochondrial and vascular dysfunction, oxidative damage, neurotransmission disturbances, inflammation, and cellular death [3, 4]. Oxidative stress is a central MOA of acute brain damage [3]. Systemic inflammation induces mitochondrial dysfunction, which is involved in both apoptotic and necrotic cell death pathways, and increased glucose uptake by brain tissues, which results in the diversion of glucose to the pentose phosphate pathway that may contribute to oxidative stress by producing excessive superoxide radicals via NADPH oxidase [3, 4]. In addition, microglia activation results in the secretion of nitric oxide, ROS (reactive oxygen species) and matrix metalloproteinases (MMPs) that can all contribute to blood-brain barrier (BBB) and neuronal damage [3]. Regarding brain dysfunction in sepsis, it is thought that RONS, generated during a systemic inflammatory response, triggers lipid peroxidation due to a decreased antioxidant activity [4]. Free radical-induced structural membrane damage also induces neuro-inflammation [4].

and retinopathy, leading to oxidized lipids and proteins [1, 2].

For over half a century, free radicals were characterized by electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy coupled with spin trapping. Nitrone spin traps (*N*-oxides of imines), such as PBN (α-phenyl-*tert*-butyl nitrone), DMPO (5,5-dimethylpyrroline-*N*-oxide) or 4-POBN (α(4-pyridyl-1-oxide)-N-tert-butyl nitrone), are the most commonly used for biological systems [8–18], and have been administered *in vivo* in various pre-clinical disease models [19–22] for several decades.

The disadvantage of the ESR/EPR approach is that the spin adducts (spin trapping agent free radical adducts or aminoxyls) are short-lived due to reductive and/or oxidative processes in biological systems [8, 9] (see **Figure 1**).

### **1.3. Immuno-spin trapping (IST)**

Mason *et al*. developed an antibody that recognizes macromolecular DMPO spin adducts, regardless of the oxidative/reductive state of the trapped radical adducts, and called the methodology immuno-spin trapping (IST) [23–28] (see **Figure 2** for an illustrative description), that has been applied in over 80 publications. The anti-DMPO antibody is attached to a fluorescent dye, allowing the *in vitro* or *ex vivo* detection of trapped DMPO-radical adducts,

involves the use of a spin trapping agent, DMPO, which is used to trap free radicals in an oxidative stress-related disease model, and administration of a mMRI probe, called an anti-DMPO probe (see **Figure 3**), that combines an antibody against DMPO-radical adducts and a MRI contrast agent, resulting in targeted free radical adduct mMRI (see **Figure 4** for methodology scheme). The contrast agent used in the Towner approach includes an albumin-Gd-DTPA (gadolinium diethylene tri-amine penta-acetic acid)-biotin construct, where the anti-DMPO antibody is covalently linked to the cysteine residues of albumin, forming an anti-DMPOadduct antibody-albumin-Gd-DTPA-biotin entity. The Gd-DTPA moiety acts as the MRI

probe. The biotin moiety can be used for *ex vivo* validation of the presence of the anti-DMPO probe in tissues, by using a streptavidin-fluorescent dye (e.g. Cy3) or streptavidin-HRP (horse

It is well known that intensity-based fluorescent methods (particularly 2′,7′-dicholorofluorescin [DCFH]) for ROS (includes the non-radical hydrogen peroxide) detection/quantification are sensitive and readily used, however, these agents lack the specificity for ROS or reactive nitrogen species (RNS), and often produce artifacts resulting in false-positive signals [35, 36]. An interesting recent study by Liu et al. used a new fluorescent probe, MPT-Cy2, which can be used to detect endogenous *in vivo* hydroxyl radicals in cells and zebrafish [37]. MPT-Cy2 becomes a fluorescent product, OMPT-Cy2, when it binds hydroxyl radicals [37]. In a similar fashion, Hu et al. reported on a fluorescent probe, HKSOX-1, for the imaging and

**Figure 3.** Illustration of the anti-DMPO probe, consisting of an albumin link that binds a MRI contrast agent, Gd-DTPA (for detection by MRI), the anti-DMPO antibody (Ab) (that binds to DMPO-free radical adducts), and biotin (that can be

used for ex vivo fluorescence microscopic imaging). Modified from Gomez-Mejiba et al. [28].

relaxation in a T1

relaxation) can be used to assess the presence of the anti-DMPO


9

map image. Both of these

Detecting *In Vivo* Free Radicals in Various Disease Models

http://dx.doi.org/10.5772/intechopen.74106

signaling component, which will increase MRI signal intensity (SI) in a T1

**1.5. Other approaches used to detect** *in vivo* **and** *in situ* **free radicals in animal** 

logical MR imaging sequence, and decrease T1

radish peroxidase) to tag the biotin in the anti-DMPO probe.

parameters, MRI SI or T1

**models and cells**

**Figure 1.** Spin trapping agents (nitrones) can be used to trap free radical compounds (R•) to form a spin adduct (nitroxide or aminoxyl), which are detected by EPR spectroscopy. However, in biological systems spin adducts can be either reduced (R'H) or oxidized ([O]). Reduced or oxidized spin adducts are EPR silent.

**Figure 2.** Immuno-spin trapping (IST) involves tagging a fluorescent-labeled-anti-DMPO antibody to DMPO-spin adducts (either as the free radical (aminoxyl), reduced or oxidized products, i.e. regardless of whether they are EPR detectable or not). The "F" designates a fluorescent dye.

either as the free radical (aminoxyl), reduced or oxidized products, with fluorescence microscopy, regardless of whether they are EPR detectable or not.

#### **1.4. Combined IST and molecular MRI (mMRI) detection of** *in vivo* **and** *in situ* **free radicals**

Towner *et al*. extended the fluorescence *in vitro*/*ex vivo* approach to an *in vivo* approach that involves the use of immuno-spin trapping (IST) in conjunction with targeted molecular magnetic resonance imaging (mMRI), currently published in six publications [29–34]. This involves the use of a spin trapping agent, DMPO, which is used to trap free radicals in an oxidative stress-related disease model, and administration of a mMRI probe, called an anti-DMPO probe (see **Figure 3**), that combines an antibody against DMPO-radical adducts and a MRI contrast agent, resulting in targeted free radical adduct mMRI (see **Figure 4** for methodology scheme). The contrast agent used in the Towner approach includes an albumin-Gd-DTPA (gadolinium diethylene tri-amine penta-acetic acid)-biotin construct, where the anti-DMPO antibody is covalently linked to the cysteine residues of albumin, forming an anti-DMPOadduct antibody-albumin-Gd-DTPA-biotin entity. The Gd-DTPA moiety acts as the MRI signaling component, which will increase MRI signal intensity (SI) in a T1 -weighted morphological MR imaging sequence, and decrease T1 relaxation in a T1 map image. Both of these parameters, MRI SI or T1 relaxation) can be used to assess the presence of the anti-DMPO probe. The biotin moiety can be used for *ex vivo* validation of the presence of the anti-DMPO probe in tissues, by using a streptavidin-fluorescent dye (e.g. Cy3) or streptavidin-HRP (horse radish peroxidase) to tag the biotin in the anti-DMPO probe.

#### **1.5. Other approaches used to detect** *in vivo* **and** *in situ* **free radicals in animal models and cells**

It is well known that intensity-based fluorescent methods (particularly 2′,7′-dicholorofluorescin [DCFH]) for ROS (includes the non-radical hydrogen peroxide) detection/quantification are sensitive and readily used, however, these agents lack the specificity for ROS or reactive nitrogen species (RNS), and often produce artifacts resulting in false-positive signals [35, 36]. An interesting recent study by Liu et al. used a new fluorescent probe, MPT-Cy2, which can be used to detect endogenous *in vivo* hydroxyl radicals in cells and zebrafish [37]. MPT-Cy2 becomes a fluorescent product, OMPT-Cy2, when it binds hydroxyl radicals [37]. In a similar fashion, Hu et al. reported on a fluorescent probe, HKSOX-1, for the imaging and

either as the free radical (aminoxyl), reduced or oxidized products, with fluorescence micros-

**Figure 2.** Immuno-spin trapping (IST) involves tagging a fluorescent-labeled-anti-DMPO antibody to DMPO-spin adducts (either as the free radical (aminoxyl), reduced or oxidized products, i.e. regardless of whether they are EPR

**Figure 1.** Spin trapping agents (nitrones) can be used to trap free radical compounds (R•) to form a spin adduct (nitroxide or aminoxyl), which are detected by EPR spectroscopy. However, in biological systems spin adducts can be

either reduced (R'H) or oxidized ([O]). Reduced or oxidized spin adducts are EPR silent.

Towner *et al*. extended the fluorescence *in vitro*/*ex vivo* approach to an *in vivo* approach that involves the use of immuno-spin trapping (IST) in conjunction with targeted molecular magnetic resonance imaging (mMRI), currently published in six publications [29–34]. This

**1.4. Combined IST and molecular MRI (mMRI) detection of** *in vivo* **and** *in situ* **free** 

copy, regardless of whether they are EPR detectable or not.

detectable or not). The "F" designates a fluorescent dye.

8 Free Radicals, Antioxidants and Diseases

**radicals**

**Figure 3.** Illustration of the anti-DMPO probe, consisting of an albumin link that binds a MRI contrast agent, Gd-DTPA (for detection by MRI), the anti-DMPO antibody (Ab) (that binds to DMPO-free radical adducts), and biotin (that can be used for ex vivo fluorescence microscopic imaging). Modified from Gomez-Mejiba et al. [28].

Another interesting approach using nitrone functionalized gold nanoparticles (Au@EMPO, EMPO = 2-(ethoxycarbonyl)-2-methyl-3,4-dihydr-2H-pyrrole 1-oxide)) to trap hydroxyl radicals was demonstrated by Du et al. [48] and may be potentially important for pre-clinical *in* 

oxidative stress changes in rodent models [49, 50]. Quest MRI detected pathologic free radical production in MnSOD (manganese superoxide dismutase) knockout mouse retinas with laminar resolution *in vivo*, where in particular dark-adapted RPE-specific MnSOD knockout

MRI technique was also used to report on high levels of free radicals in the hippocampus region in mouse models for neurological diseases such as Alzheimer's disease and Angelman syndrome [50]. However, it should be noted that paramagnetic oxygen [49–51] and hydrogen

interpretation of the presence of free radicals. In addition, temperature and pH also can influ-

Quest MRI is an interesting approach that provides information on total free radical burden, somewhat similar to the combined IST and mMRI approach, but without the use of a MRI

Endogenous reactive oxygen species (ROS) contrast MRI was also recently used by Tain et al.

study by Eto et al. used *in vivo* nuclear polarization MRI (DNP-MRI) with nitroxyl radicals (carbamoyl-PROXYL (cell permeable) and carboxy-PROXYL (cell impermeable)) to assess the redox status (measured as an increase in image intensity) in the skeletal muscle of mice that had an acute local inflammation (induced with i.m. injection of bupivacaine) [54]. The signal decay of carbamoyl-PROXYL in bupivacaine-exposed mice was confirmed by *in vivo* L-band EPR spectroscopy [54]. The nitroxyl radical probes, are paramagnetic which broaden the MRI signal, bind free radicals, and thus results in an increase in MRI signal intensity [55]. Another group used DNP-MRI to visualize endogenous free radical intermediates of FMNH (flavin mononucleotide-hydrogen) and FADH (flavin adenine dinucleotide-hydrogen) *in* vitro [56], which could potentially be detected *in vivo* in the future. DNP-MRI, also used for PEDRI (proton electron double-resonance imaging) or OMRI (Overhauser enhanced magnetic resonance imaging), is a relatively new imaging approach for detecting free radical species *in vivo* [57, 58]. Yamamoto et al. recently developed a combined PET (positron emission tomography)/OMRI system to detect radionucleotide and nitroxyl radical probes for small

The focus of this review is on *in vivo* and *in situ* IST-mMRI applications in different experimental oxidative stress-associated animal disease models that have been studied, advantages and limitations of the technique, and future directions in further applications, improvements on the methodology that can be made, and subsequent free radical identifi-

values in the outer retina, compared to relevant controls [49] The Quest

contrast effect, which could complicate the

Detecting *In Vivo* Free Radicals in Various Disease Models

http://dx.doi.org/10.5772/intechopen.74106

) in rotenone-treated mouse brains [51]. Another

contrast [52, 53]. Nonetheless,

MRI to measure

11

*vivo* applications in combination with micro-computed tomography (CT).

mice had elevated 1/T1

contrast agent [49, 50].

animal imaging [59].

**1.6. Overall scope**

cation approaches.

peroxide [50, 51] can also provide a dominant 1/T1

to detect ROS (measured as a reduction in T1

ence the rates of proton exchange which will also affect 1/T<sup>1</sup>

Recent studies by Berkowitz et al. have used quench-assisted (Quest) 1/T<sup>1</sup>

**Figure 4.** Combined IST and free radical-targeted molecular MRI (mMRI) approach. Initially mice are administered DMPO (i.p.) to trap free radicals resulting from an oxidative stress-associated disease or process. Any cell membranebound radicals (e.g. oxidized proteins or lipids) can then be detected with the anti-DMPO probe (administered via a tail-vein catheter). Modified from Towner et al. [29].

detection of endogenous *in vivo* superoxide in cells and zebrafish embryos [38]. A fluorescence probe, o-phenylene diamine-Phe-Phe-OH, has also been developed for the detection of nitric oxide (NO), and used in living cells [39]. Another fluorescence probe, LyNP-NO, was used to detect NO in C6 glioma cells [40]. It was also found that single-walled carbon nanotubes have fluorescent properties, and have been used to detect *in vivo* levels of NO, which quenches the fluorescence signal, in mice [41]. However, for most of the fluorescence probes *in vivo* applications will be limited to a depth-of-penetration detection of the fluorescence signal and may only be applicable to small animal models.

Another group, Li et al. used a near infrared (NIR)-light excited luminescence resonance energy transfer based nanoprobe for *in vivo* detection of hydroxyl radicals [42]. NIR fluorescence probes (Hcy-Mito and Hcy-Biot) were also recently used for the *in situ* detection of superoxide anion and hydrogen polysulfides in living cells and in mouse tumor models [43]. Also, a phosphinate-based NIR fluorescence probe, CyR, was recently also used to detect superoxide radical anion *in vivo* within zebrafish [44]. NIR-fluorescent single-walled carbon nanotubes have also been used to detect *in vivo* NO levels in mice [45]. It should be pointed out, however, that there is ESR spectroscopy evidence for *in vivo* formation of free radicals in the tissues (lungs, heart and liver) of mice exposed to single-walled carbon nanotubes with no oxidative stress [46].

Rayner et al. used a reversible pro-fluorescent probe containing a redox sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN) for the *in vivo* detection of retinal oxidative status within rat retina following acute ischemia-reperfusion injury [47]. Another interesting approach using nitrone functionalized gold nanoparticles (Au@EMPO, EMPO = 2-(ethoxycarbonyl)-2-methyl-3,4-dihydr-2H-pyrrole 1-oxide)) to trap hydroxyl radicals was demonstrated by Du et al. [48] and may be potentially important for pre-clinical *in vivo* applications in combination with micro-computed tomography (CT).

Recent studies by Berkowitz et al. have used quench-assisted (Quest) 1/T<sup>1</sup> MRI to measure oxidative stress changes in rodent models [49, 50]. Quest MRI detected pathologic free radical production in MnSOD (manganese superoxide dismutase) knockout mouse retinas with laminar resolution *in vivo*, where in particular dark-adapted RPE-specific MnSOD knockout mice had elevated 1/T1 values in the outer retina, compared to relevant controls [49] The Quest MRI technique was also used to report on high levels of free radicals in the hippocampus region in mouse models for neurological diseases such as Alzheimer's disease and Angelman syndrome [50]. However, it should be noted that paramagnetic oxygen [49–51] and hydrogen peroxide [50, 51] can also provide a dominant 1/T1 contrast effect, which could complicate the interpretation of the presence of free radicals. In addition, temperature and pH also can influence the rates of proton exchange which will also affect 1/T<sup>1</sup> contrast [52, 53]. Nonetheless, Quest MRI is an interesting approach that provides information on total free radical burden, somewhat similar to the combined IST and mMRI approach, but without the use of a MRI contrast agent [49, 50].

Endogenous reactive oxygen species (ROS) contrast MRI was also recently used by Tain et al. to detect ROS (measured as a reduction in T1 ) in rotenone-treated mouse brains [51]. Another study by Eto et al. used *in vivo* nuclear polarization MRI (DNP-MRI) with nitroxyl radicals (carbamoyl-PROXYL (cell permeable) and carboxy-PROXYL (cell impermeable)) to assess the redox status (measured as an increase in image intensity) in the skeletal muscle of mice that had an acute local inflammation (induced with i.m. injection of bupivacaine) [54]. The signal decay of carbamoyl-PROXYL in bupivacaine-exposed mice was confirmed by *in vivo* L-band EPR spectroscopy [54]. The nitroxyl radical probes, are paramagnetic which broaden the MRI signal, bind free radicals, and thus results in an increase in MRI signal intensity [55]. Another group used DNP-MRI to visualize endogenous free radical intermediates of FMNH (flavin mononucleotide-hydrogen) and FADH (flavin adenine dinucleotide-hydrogen) *in* vitro [56], which could potentially be detected *in vivo* in the future. DNP-MRI, also used for PEDRI (proton electron double-resonance imaging) or OMRI (Overhauser enhanced magnetic resonance imaging), is a relatively new imaging approach for detecting free radical species *in vivo* [57, 58]. Yamamoto et al. recently developed a combined PET (positron emission tomography)/OMRI system to detect radionucleotide and nitroxyl radical probes for small animal imaging [59].

#### **1.6. Overall scope**

detection of endogenous *in vivo* superoxide in cells and zebrafish embryos [38]. A fluorescence probe, o-phenylene diamine-Phe-Phe-OH, has also been developed for the detection of nitric oxide (NO), and used in living cells [39]. Another fluorescence probe, LyNP-NO, was used to detect NO in C6 glioma cells [40]. It was also found that single-walled carbon nanotubes have fluorescent properties, and have been used to detect *in vivo* levels of NO, which quenches the fluorescence signal, in mice [41]. However, for most of the fluorescence probes *in vivo* applications will be limited to a depth-of-penetration detection of the fluorescence

**Figure 4.** Combined IST and free radical-targeted molecular MRI (mMRI) approach. Initially mice are administered DMPO (i.p.) to trap free radicals resulting from an oxidative stress-associated disease or process. Any cell membranebound radicals (e.g. oxidized proteins or lipids) can then be detected with the anti-DMPO probe (administered via a

Another group, Li et al. used a near infrared (NIR)-light excited luminescence resonance energy transfer based nanoprobe for *in vivo* detection of hydroxyl radicals [42]. NIR fluorescence probes (Hcy-Mito and Hcy-Biot) were also recently used for the *in situ* detection of superoxide anion and hydrogen polysulfides in living cells and in mouse tumor models [43]. Also, a phosphinate-based NIR fluorescence probe, CyR, was recently also used to detect superoxide radical anion *in vivo* within zebrafish [44]. NIR-fluorescent single-walled carbon nanotubes have also been used to detect *in vivo* NO levels in mice [45]. It should be pointed out, however, that there is ESR spectroscopy evidence for *in vivo* formation of free radicals in the tissues (lungs, heart and liver) of mice exposed to single-walled carbon nanotubes with

Rayner et al. used a reversible pro-fluorescent probe containing a redox sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN) for the *in vivo* detection of retinal oxidative status within rat retina following acute ischemia-reperfusion injury [47].

signal and may only be applicable to small animal models.

tail-vein catheter). Modified from Towner et al. [29].

10 Free Radicals, Antioxidants and Diseases

no oxidative stress [46].

The focus of this review is on *in vivo* and *in situ* IST-mMRI applications in different experimental oxidative stress-associated animal disease models that have been studied, advantages and limitations of the technique, and future directions in further applications, improvements on the methodology that can be made, and subsequent free radical identification approaches.

### **2.** *In vivo* **and** *In situ* **targeted free radical detection: various models**

The combined IST-mMRI approach has been used in several *in vivo* disease models, including multi-tissue assessment in diabetic mice [29] (see **Figure 5**) with further assessment of cardiomyopathy [30] (see **Figure 6**), and in neurological applications, such as rodent models septic encephalopathy [31] (see **Figure 8**), for amyotrophic lateral sclerosis (ALS) [32] (see **Figure 7**), and gliomas [33], to target *in vivo* and *in situ* free radical detection.

#### **2.1. Diabetes**

The initial proof-of-concept combined IST and mMRI approach to detect *in vivo* free radicals was assessed in a STZ (streptozotocin)-induced diabetes model in mice. From this study, all major organs excluding the heart, such as the lungs, liver and kidneys, were assessed regarding levels of trapped *in vivo* and *in situ* DMPO-radical adducts [29]. **Figure 5** depicts the data obtained in the liver of diabetic (STZ-induced) and non-diabetic mice (wild-type

**Figure 5.** Combined IST and free radical-targeted mMRI in a STZ-induced mouse diabetic model. (A) *In vivo* anti-DMPO probe distribution map (based on MRI signal intensity (SI) change). Anatomical assignments: ln = lung, h = heart, s = stomach, lv = liver, k = kidney, i = intestine, and b = bladder. (B) Stretavidin-Cy3 biotin-tagged *ex vivo* liver image in Diab mice. (C) iNOS immunohistochemistry (IHC) from the liver of a diabetic mouse. (D) Fluorescence intensity of iNOS IHC in non-diabetic (control) and diabetic mouse livers. N = 5 for each. (E) Percent (%) change in MRI SI in diabetic mice administered DMPO + anti-DMPO probe (Diab), DMPO + a non-specific IgG contrast agent (Cont), saline + anti-DMPO probe (Sal), and wild-type non-diabetic mice administered DMPO + anti-DMPO probe. N = 5 for each group. (F) Fluorescent-IST image of the liver of a diabetic mouse. Modified from Towner et al. [29].

mice not administered STZ), and appropriate controls (e.g. diabetic mice given saline rather than DMPO plus the anti-DMPO probe — spin trap control; or a diabetic mouse administered DMPO, but given a non-specific IgG contrast agent [IgG-albumin-Gd-DTPA-biotin] instead

of diabetic and non-diabetic mice. N = 5 for each. Modified from Towner et al. [30].

**Figure 6.** Combined IST and free radical-targeted mMRI in a STZ-induced mouse diabetic heart. (A) MR image of a mouse heart with an *in vivo* anti-DMPO probe cardiac map overlay (based on MRI SI change). Anatomical assignments: 1 = lung, 2 = left ventricle chamber, 3 = cardiac muscle, 4 = liver, and 5 = thoracic muscle. (B) Percent (%) change in MRI SI in diabetic mouse hearts after administered DMPO + anti-DMPO probe (Diab+D + P), DMPO + a non-specific IgG contrast agent (Diab+D + IgG), and non-diabetic mice administered DMPO + anti-DMPO probe (nDiab+D + P). N = 5 for each group. (C) Stretavidin-Cy3 biotin-tagged *ex vivo* cardiac image in Diab mice. (D) Fluorescent-IST image of the heart of a diabetic mouse. (E) 3-Nitrotyrosine (3-NT) adducts and (F) malondialdehyde (MDA) ELISAs from the hearts

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13

**2.** *In vivo* **and** *In situ* **targeted free radical detection: various models**

and gliomas [33], to target *in vivo* and *in situ* free radical detection.

**2.1. Diabetes**

12 Free Radicals, Antioxidants and Diseases

The combined IST-mMRI approach has been used in several *in vivo* disease models, including multi-tissue assessment in diabetic mice [29] (see **Figure 5**) with further assessment of cardiomyopathy [30] (see **Figure 6**), and in neurological applications, such as rodent models septic encephalopathy [31] (see **Figure 8**), for amyotrophic lateral sclerosis (ALS) [32] (see **Figure 7**),

The initial proof-of-concept combined IST and mMRI approach to detect *in vivo* free radicals was assessed in a STZ (streptozotocin)-induced diabetes model in mice. From this study, all major organs excluding the heart, such as the lungs, liver and kidneys, were assessed regarding levels of trapped *in vivo* and *in situ* DMPO-radical adducts [29]. **Figure 5** depicts the data obtained in the liver of diabetic (STZ-induced) and non-diabetic mice (wild-type

**Figure 5.** Combined IST and free radical-targeted mMRI in a STZ-induced mouse diabetic model. (A) *In vivo* anti-DMPO probe distribution map (based on MRI signal intensity (SI) change). Anatomical assignments: ln = lung, h = heart, s = stomach, lv = liver, k = kidney, i = intestine, and b = bladder. (B) Stretavidin-Cy3 biotin-tagged *ex vivo* liver image in Diab mice. (C) iNOS immunohistochemistry (IHC) from the liver of a diabetic mouse. (D) Fluorescence intensity of iNOS IHC in non-diabetic (control) and diabetic mouse livers. N = 5 for each. (E) Percent (%) change in MRI SI in diabetic mice administered DMPO + anti-DMPO probe (Diab), DMPO + a non-specific IgG contrast agent (Cont), saline + anti-DMPO probe (Sal), and wild-type non-diabetic mice administered DMPO + anti-DMPO probe. N = 5 for each group. (F)

Fluorescent-IST image of the liver of a diabetic mouse. Modified from Towner et al. [29].

**Figure 6.** Combined IST and free radical-targeted mMRI in a STZ-induced mouse diabetic heart. (A) MR image of a mouse heart with an *in vivo* anti-DMPO probe cardiac map overlay (based on MRI SI change). Anatomical assignments: 1 = lung, 2 = left ventricle chamber, 3 = cardiac muscle, 4 = liver, and 5 = thoracic muscle. (B) Percent (%) change in MRI SI in diabetic mouse hearts after administered DMPO + anti-DMPO probe (Diab+D + P), DMPO + a non-specific IgG contrast agent (Diab+D + IgG), and non-diabetic mice administered DMPO + anti-DMPO probe (nDiab+D + P). N = 5 for each group. (C) Stretavidin-Cy3 biotin-tagged *ex vivo* cardiac image in Diab mice. (D) Fluorescent-IST image of the heart of a diabetic mouse. (E) 3-Nitrotyrosine (3-NT) adducts and (F) malondialdehyde (MDA) ELISAs from the hearts of diabetic and non-diabetic mice. N = 5 for each. Modified from Towner et al. [30].

mice not administered STZ), and appropriate controls (e.g. diabetic mice given saline rather than DMPO plus the anti-DMPO probe — spin trap control; or a diabetic mouse administered DMPO, but given a non-specific IgG contrast agent [IgG-albumin-Gd-DTPA-biotin] instead

**Figure 7.** Combined IST and free radical-targeted mMRI in a septic encephalopathy CLP-induced mouse model. (A) *In vivo* anti-DMPO probe brain map of a septic mouse (based on MRI SI change). (B) Stretavidin-Cy3 biotin-tagged *ex vivo* septic brain image. (C) Fluorescent-IST image of the septic brain of a mouse. (D) Percent (%) T1 relaxation change in septic and sham mice administered DMPO + anti-DMPO probe in different brain regions (hippocampus, striatum, occipital lobe, and medial cortex). N = 5 for each group. (E) 4-Hydroxynonenal (4HNE) levels from western blots of brains of septic and non-septic mice. (F) 3-Nitro-tyrosine (3-NT) levels from western blots of brains of septic and nonseptic mice. N = 5 for each. Modified from Towner et al. [31].

of the anti-DMPO probe). (**Figure 5**) Diabetic mice that were administered DMPO and the anti-DMPO probe, had significantly higher levels of the anti-DMPO probe (detected by an increase in percent (%) change in MRI SI), than non-diabetic mice or diabetic mouse controls, in their lungs, kidneys and livers (see **Figure 5A** for overall distribution of the anti-DMPO probe in a horizontal image; and **Figure 5E** for quantitative liver data). A post-contrast image minus a pre-contrast image was obtained, and the gray-scale image was false-colored red, and overlaid on top of the morphological image. Non-specific biodistribution of the anti-DMPO was also found in the stomach, intestines, and bladder (**Figure 5A**). From kinetics assessment, it was found that the anti-DMPO probe persisted in certain tissues (e.g. lungs, liver and kidneys) for over 3 hours. Verification of the presence of the anti-DMPO probe in ex vivo tissues was done by using streptavidin-Cy3 which bound to the biotin moiety of the anti-DMPO probe (see **Figure 5B** for example in liver tissue). It was also confirmed that DMPO-radical adducts were formed by using a fluorescent-anti-DMPO antibody (IST approach) in diabetic (see **Figure 5C**) and non-diabetic mouse livers. In addition, inducible nitric oxide synthase (iNOS) levels were assessed in the livers of diabetic and non-diabetic mice (see **Figure 5D** for immunohistochemistry (IHC) detection of iNOS in a diabetic mouse liver; and **Figure 5F** for quantitative levels of iNOS in diabetic and non-diabetic mouse livers) as an additional marker of oxidative stress. This was the first *in vivo* study to demonstrate that diabetic mice had elevated *in situ* free radical levels in organs/tissues such as the lungs, liver and kidneys.

At a later stage it was found that the cardiac muscle in diabetic mice also retained the anti-DMPO probe [30] (see **Figure 6**). A morphological MR image of a mouse heart is shown in **Figure 6A**. The post-contrast minus pre-contrast image in a diabetic mouse with false coloration is shown in **Figure 6A**, overlaid on top of a horizontal morphological image of the heart. Significantly higher quantitative levels of the anti-DMPO probe in diabetic (Diab) mice administered DMPO (D) and the anti-DMPO probe (P) were found when compared to diabetic (administered the isotype IgG contrast agent instead of the anti-DMPO probe) and nondiabetic (non-STZ exposed WT mice administered DMPO and the anti-DMPO probe) controls (see **Figure 6B**). Confirmation of the presence of the anti-DMPO probe in cardiac muscle of a diabetic mouse is shown in **Figure 6C**. Verification of the presence of DMPO-radical adducts is shown in **Figure 6D**, where a fluorescent-labeled anti-DMPO antibody was used. In the diabetic cardiomyopathy study, it was also established that diabetic mice had significantly higher levels of 3-nitrotyrosine (3-NT) (oxidized protein marker) (**Figure 6E**) and malondi aldehyde (MDA) adducts (oxidized lipid marker) (**Figure 6F**) in cardiac muscle, when compared

spinal cord. (F) Fluorescent-IST image of the spinal cord of an ALS mouse. Modified from Towner et al. [32].

**Figure 8.** Combined IST and free radical-targeted mMRI in an ALS mouse model. (A) *In vivo* anti-DMPO probe map (based on MRI signal intensity (SI) change). (B) DMPO probe concentration in ALS mice administered DMPO + anti-DMPO probe (ALS-D), DMPO + non-specific IgG contrast agent (ALS-C), and wild-type non-ALS mice administered DMPO + anti-DMPO probe (non-ALS-D). N = 5 for each group. (C) Diffusion-weighted image of an ALS mouse with increased apparent diffusion coefficient (ADC) in lumbar region (outlined). (D) Stretavidin-Cy3 biotin-tagged *ex vivo* spinal cord in ALS-D. (E) Co-localized DMPO probe (red) and neuronal marker (green) fluorescence image in mouse

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**Figure 8.** Combined IST and free radical-targeted mMRI in an ALS mouse model. (A) *In vivo* anti-DMPO probe map (based on MRI signal intensity (SI) change). (B) DMPO probe concentration in ALS mice administered DMPO + anti-DMPO probe (ALS-D), DMPO + non-specific IgG contrast agent (ALS-C), and wild-type non-ALS mice administered DMPO + anti-DMPO probe (non-ALS-D). N = 5 for each group. (C) Diffusion-weighted image of an ALS mouse with increased apparent diffusion coefficient (ADC) in lumbar region (outlined). (D) Stretavidin-Cy3 biotin-tagged *ex vivo* spinal cord in ALS-D. (E) Co-localized DMPO probe (red) and neuronal marker (green) fluorescence image in mouse spinal cord. (F) Fluorescent-IST image of the spinal cord of an ALS mouse. Modified from Towner et al. [32].

of the anti-DMPO probe). (**Figure 5**) Diabetic mice that were administered DMPO and the anti-DMPO probe, had significantly higher levels of the anti-DMPO probe (detected by an increase in percent (%) change in MRI SI), than non-diabetic mice or diabetic mouse controls, in their lungs, kidneys and livers (see **Figure 5A** for overall distribution of the anti-DMPO probe in a horizontal image; and **Figure 5E** for quantitative liver data). A post-contrast image minus a pre-contrast image was obtained, and the gray-scale image was false-colored red, and overlaid on top of the morphological image. Non-specific biodistribution of the anti-DMPO was also found in the stomach, intestines, and bladder (**Figure 5A**). From kinetics assessment, it was found that the anti-DMPO probe persisted in certain tissues (e.g. lungs, liver and kidneys) for over 3 hours. Verification of the presence of the anti-DMPO probe in ex vivo tissues was done by using streptavidin-Cy3 which bound to the biotin moiety of the anti-DMPO probe (see **Figure 5B** for example in liver tissue). It was also confirmed that DMPO-radical adducts were formed by using a fluorescent-anti-DMPO antibody (IST approach) in diabetic (see **Figure 5C**) and non-diabetic mouse livers. In addition, inducible nitric oxide synthase (iNOS) levels were assessed in the livers of diabetic and non-diabetic mice (see **Figure 5D** for immunohistochemistry (IHC) detection of iNOS in a diabetic mouse liver; and **Figure 5F** for quantitative levels of iNOS in diabetic and non-diabetic mouse livers) as an additional marker of oxidative stress. This was the first *in vivo* study to demonstrate that diabetic mice had elevated *in situ* free radical levels in organs/tissues such as the lungs, liver and kidneys.

septic mice. N = 5 for each. Modified from Towner et al. [31].

14 Free Radicals, Antioxidants and Diseases

**Figure 7.** Combined IST and free radical-targeted mMRI in a septic encephalopathy CLP-induced mouse model. (A) *In vivo* anti-DMPO probe brain map of a septic mouse (based on MRI SI change). (B) Stretavidin-Cy3 biotin-tagged *ex vivo* septic brain image. (C) Fluorescent-IST image of the septic brain of a mouse. (D) Percent (%) T1 relaxation change in septic and sham mice administered DMPO + anti-DMPO probe in different brain regions (hippocampus, striatum, occipital lobe, and medial cortex). N = 5 for each group. (E) 4-Hydroxynonenal (4HNE) levels from western blots of brains of septic and non-septic mice. (F) 3-Nitro-tyrosine (3-NT) levels from western blots of brains of septic and non-

> At a later stage it was found that the cardiac muscle in diabetic mice also retained the anti-DMPO probe [30] (see **Figure 6**). A morphological MR image of a mouse heart is shown in **Figure 6A**. The post-contrast minus pre-contrast image in a diabetic mouse with false coloration is shown in **Figure 6A**, overlaid on top of a horizontal morphological image of the heart.

> Significantly higher quantitative levels of the anti-DMPO probe in diabetic (Diab) mice administered DMPO (D) and the anti-DMPO probe (P) were found when compared to diabetic (administered the isotype IgG contrast agent instead of the anti-DMPO probe) and nondiabetic (non-STZ exposed WT mice administered DMPO and the anti-DMPO probe) controls (see **Figure 6B**). Confirmation of the presence of the anti-DMPO probe in cardiac muscle of a diabetic mouse is shown in **Figure 6C**. Verification of the presence of DMPO-radical adducts is shown in **Figure 6D**, where a fluorescent-labeled anti-DMPO antibody was used. In the diabetic cardiomyopathy study, it was also established that diabetic mice had significantly higher levels of 3-nitrotyrosine (3-NT) (oxidized protein marker) (**Figure 6E**) and malondi aldehyde (MDA) adducts (oxidized lipid marker) (**Figure 6F**) in cardiac muscle, when compared

to non-diabetic mice. This was the first *in vivo* study to demonstrate increased *in situ* free radical levels in diabetic cardiomyopathy. The correlation with oxidized lipids and proteins was done, as it is suspected that the combined IST and mMRI approach primarily reports on macromolecular free radicals that are cell-membrane bound. Mason et al. have previously reported on the application of IST to trap oxidized proteins [23–25]. As both an increase in oxidized lipids and proteins were detected in diabetic cardiac muscle via ELISA, it is possible that the combined IST and mMRI method detects both oxidized lipids as well as proteins. Further verification would require a mass spectrometry approach to confirm this assumption.

(NrCAM) (green) co-localizes with the location of the anti-DMPO probe (red) in some regions (yellow). Confirmation of DMPO-radical adducts is depicted in **Figure 8F**. This is the first direct *in vivo* detection of elevated *in situ* free radicals in the lumbar region of ALS-like mice.

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Lastly, the combined IST and mMRI *in vivo* free radical detection method was applied to assess glioma models, both for untreated and anti-cancer agent treated tumors. It was initially demonstrated that free radicals can be detected in a GL261 mouse glioma model [33]. The DMPO-trapped radicals were found to be heterogeneously distributed primarily in the tumor core region, possibly associated with increased cell proliferation [33]. In addition to confirming the presence of the anti-DMPO probe in glioma tumor tissue and detection of anti-DMPO trapped radicals, it was also possible to demonstrate that GL261 gliomas had elevated levels

**Figure 9.** Combined IST and free radical-targeted mMRI in a F98 rat glioma model. *In vivo* anti-DMPO probe brain maps of (A) untreated (UT) and (B) OKN-007-treated rat F98 tumors (based on MRI SI change). (C) Trapped free radical levels (% change in MRI SI) in UT and OKN-007-treated F98 gliomas administered DMPO + anti-DMPO probe. N = 5 for each group. Stretavidin-HRP biotin-tagged ex vivo F98 gliomas that were (D) UT or (E) OKN-007-treated. (F) Malondialdehyde (MDA) levels from IHC of F98 tumors of OKN-007-treated or UT rats. (G) 3-Nitrotyrosine (3-NT) levels from IHC of F98

tumors of OKN-007-treated or UT rats. N = 5 for each. Modified from Coutinho de Souza et al. [34].

of MDA protein adducts and 3-NT, compared to normal mouse brain tissue [33].

**2.4. Gliomas**

#### **2.2. Septic encephalopathy**

It was then decided to assess the combined IST and mMRI free radical-targeted approach in other disease models, such as septic encephalopathy. Mice with septic encephalopathy (induced by cecal ligation and puncture (CLP)) were also found to have higher levels of trapped DMPOradical adducts compared to sham animals (abdominal incision without CLP and sutured) [31] (**Figure 7**). **Figure 7A** depicts a MRI SI difference image (false-colored red) overlaid on top of a morphological image of the brain region of a septic mouse. Confirmation of the presence of the anti-DMPO in the cortical brain tissue of a septic mouse is shown in **Figure 7B**, and verification of DMPO-radical adducts in a septic mouse brain is depicted in **Figure 7C**. The distribution of the anti-DMPO probe is dispersed throughout the brain, and was found to be significantly higher in septic mice vs. sham animals in the hippocampus, striatum, occipital lobe and medial cortex regions of the brain (**Figure 7D**), as measured by a % change (overall decrease in T1 relaxation). Oxidized lipid levels (measured from Western blots for 4-hydroxynonenal (4-HNE) (**Figure 7E**) and oxidized protein levels (measured from Western blots for 3-NT) (**Figure 7F**) were found to be significantly higher in septic mice (CLP) after 6 hours, compared to sham controls. This study also indicates that both oxidized lipids and proteins may play a role in the free radical-associated pathology of ALS. This is the first reported *in vivo* detection of elevated *in situ* free radicals in a mouse model for septic encephalopathy.

#### **2.3. Amyotrophic lateral sclerosis (ALS)**

The combined IST and mMRI approach for detecting *in vivo* free radicals was extended to other neurological disorders, such as ALS. High levels of trapped DMPO-radical adducts were also found in a transgenic mouse model for ALS (superoxide dismutase (SOD) mutation) [32] (see **Figure 8**). As SOD1 plays an important role in O2 −• clearance, the loss of SOD1 can lead to increased levels of free radicals [5]. A pre-contrast minus post-contrast difference sagittal image is shown in **Figure 8A** for an ALS mouse, with false coloration depicted in the lumbar region of the spinal cord. From T1 relaxation values the estimated anti-DMPO probe concentration can be obtained, and it was found that ALS mice administered DMPO and the anti-DMPO probe (ALS-D) had significantly higher levels of the anti-DMPO probe, compared to ALS (administered the IgG contrast agent instead of the anti-DMPO probe) and non-ALS (administered both DMPO and the anti-DMPO probe) controls (see **Figure 8B**). A diffusionweighted image is shown in **Figure 8C** depicting a high signal intensity in the lumbar region of the spinal cord. *Ex vivo* detection of the anti-DMPO probe in the lumbar region of the spinal cord of an ALS mouse is shown in **Figure 8D**. **Figure 8E** illustrates that a neuronal marker (NrCAM) (green) co-localizes with the location of the anti-DMPO probe (red) in some regions (yellow). Confirmation of DMPO-radical adducts is depicted in **Figure 8F**. This is the first direct *in vivo* detection of elevated *in situ* free radicals in the lumbar region of ALS-like mice.

#### **2.4. Gliomas**

to non-diabetic mice. This was the first *in vivo* study to demonstrate increased *in situ* free radical levels in diabetic cardiomyopathy. The correlation with oxidized lipids and proteins was done, as it is suspected that the combined IST and mMRI approach primarily reports on macromolecular free radicals that are cell-membrane bound. Mason et al. have previously reported on the application of IST to trap oxidized proteins [23–25]. As both an increase in oxidized lipids and proteins were detected in diabetic cardiac muscle via ELISA, it is possible that the combined IST and mMRI method detects both oxidized lipids as well as proteins. Further verification would require a mass spectrometry approach to confirm this assumption.

It was then decided to assess the combined IST and mMRI free radical-targeted approach in other disease models, such as septic encephalopathy. Mice with septic encephalopathy (induced by cecal ligation and puncture (CLP)) were also found to have higher levels of trapped DMPOradical adducts compared to sham animals (abdominal incision without CLP and sutured) [31] (**Figure 7**). **Figure 7A** depicts a MRI SI difference image (false-colored red) overlaid on top of a morphological image of the brain region of a septic mouse. Confirmation of the presence of the anti-DMPO in the cortical brain tissue of a septic mouse is shown in **Figure 7B**, and verification of DMPO-radical adducts in a septic mouse brain is depicted in **Figure 7C**. The distribution of the anti-DMPO probe is dispersed throughout the brain, and was found to be significantly higher in septic mice vs. sham animals in the hippocampus, striatum, occipital lobe and medial cortex regions of the brain (**Figure 7D**), as measured by a % change (overall decrease in T1 relaxation). Oxidized lipid levels (measured from Western blots for 4-hydroxynonenal (4-HNE) (**Figure 7E**) and oxidized protein levels (measured from Western blots for 3-NT) (**Figure 7F**) were found to be significantly higher in septic mice (CLP) after 6 hours, compared to sham controls. This study also indicates that both oxidized lipids and proteins may play a role in the free radical-associated pathology of ALS. This is the first reported *in vivo* detection of elevated

The combined IST and mMRI approach for detecting *in vivo* free radicals was extended to other neurological disorders, such as ALS. High levels of trapped DMPO-radical adducts were also found in a transgenic mouse model for ALS (superoxide dismutase (SOD) muta-

can lead to increased levels of free radicals [5]. A pre-contrast minus post-contrast difference sagittal image is shown in **Figure 8A** for an ALS mouse, with false coloration depicted in the

concentration can be obtained, and it was found that ALS mice administered DMPO and the anti-DMPO probe (ALS-D) had significantly higher levels of the anti-DMPO probe, compared to ALS (administered the IgG contrast agent instead of the anti-DMPO probe) and non-ALS (administered both DMPO and the anti-DMPO probe) controls (see **Figure 8B**). A diffusionweighted image is shown in **Figure 8C** depicting a high signal intensity in the lumbar region of the spinal cord. *Ex vivo* detection of the anti-DMPO probe in the lumbar region of the spinal cord of an ALS mouse is shown in **Figure 8D**. **Figure 8E** illustrates that a neuronal marker

−• clearance, the loss of SOD1

relaxation values the estimated anti-DMPO probe

*in situ* free radicals in a mouse model for septic encephalopathy.

tion) [32] (see **Figure 8**). As SOD1 plays an important role in O2

**2.3. Amyotrophic lateral sclerosis (ALS)**

lumbar region of the spinal cord. From T1

**2.2. Septic encephalopathy**

16 Free Radicals, Antioxidants and Diseases

Lastly, the combined IST and mMRI *in vivo* free radical detection method was applied to assess glioma models, both for untreated and anti-cancer agent treated tumors. It was initially demonstrated that free radicals can be detected in a GL261 mouse glioma model [33]. The DMPO-trapped radicals were found to be heterogeneously distributed primarily in the tumor core region, possibly associated with increased cell proliferation [33]. In addition to confirming the presence of the anti-DMPO probe in glioma tumor tissue and detection of anti-DMPO trapped radicals, it was also possible to demonstrate that GL261 gliomas had elevated levels of MDA protein adducts and 3-NT, compared to normal mouse brain tissue [33].

**Figure 9.** Combined IST and free radical-targeted mMRI in a F98 rat glioma model. *In vivo* anti-DMPO probe brain maps of (A) untreated (UT) and (B) OKN-007-treated rat F98 tumors (based on MRI SI change). (C) Trapped free radical levels (% change in MRI SI) in UT and OKN-007-treated F98 gliomas administered DMPO + anti-DMPO probe. N = 5 for each group. Stretavidin-HRP biotin-tagged ex vivo F98 gliomas that were (D) UT or (E) OKN-007-treated. (F) Malondialdehyde (MDA) levels from IHC of F98 tumors of OKN-007-treated or UT rats. (G) 3-Nitrotyrosine (3-NT) levels from IHC of F98 tumors of OKN-007-treated or UT rats. N = 5 for each. Modified from Coutinho de Souza et al. [34].

pin-point where high levels of free radicals are formed in different heterogeneous regions of specific tissues. It should be noted that MRI has no depth-of-penetration limitation. The current anti-DMPO-albumin-Gd-DTPA-biotin construct allows rapid vascular delivery, and binding to macromolecular DMPO-trapped free radicals in the plasma membrane, as well as

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This review has discussed all of the current studies that have utilized combined IST and mMRI to detect targeted trapped macromolecular DMPO-radical adducts *in vivo* and *in situ* within various animal models where oxidative stress plays a major role. It was established for all oxidative stress-associated disease models studied thus far, that levels of free radicals were found to be significantly increased in all cases for animals treated with DMPO and the anti-DMPO probe, when compared to controls, including disease controls (e.g. wild-type rodents or shams), non-DMPO controls (i.e. administered saline instead of DMPO), and/or mMRI probe controls (i.e. a non-specific IgG was covalently bound to the albumin of the MRI contrast agent construct instead of the anti-DMPO antibody). An example of assessing an anti-cancer agent with free radical scavenging activity was also presented. The biotin moiety of the anti-DMPO probe also allowed *ex vivo* validation of the presence of trapped DMPO macromolecular adducts in various tissues. IST was also used in all cases to confirm the presence of trapped free radicals with fluorescence or optical (e.g. HRP) microscopy. Finally, IEM was used to confirm the presence of the anti-DMPO probe in plasma and nuclear membranes.

Some of the disadvantages with the methodology include limited access to pre-clinical MRI systems, availability of the anti-DMPO antibody, and further identifying the radical source that is being trapped. For non-neurological studies, this approach can be easily utilized in numerous pathological/toxicological models. However, for neurological studies, the approach will be limited to whether there is BBB permeability, in order to allow the anti-DMPO probe,

The IST-mMRI approach can certainly be further applied to study free radicals associated longitudinally in oxidative stress-related disease processes, as well as assess the effect of therapeutic agents that alter free radical levels. Mass spectrometry may need to be used to not only further assess whether the anti-DMPO probe detected in heterogeneous tissue regions are essentially oxidized proteins or oxidized lipids, or a combination of both, before the type of protein or lipid is identified. The current size of the probe may prohibit use in neurological studies with an intact blood-brain barrier (BBB). The development of a smaller nanoparticle-based anti-DMPO probe, which may allow access through an intact BBB, is currently being considered.

*ex vivo* confirmation with microscopy.

and possibly DMPO, to access the target tissue.

**Author details**

Oklahoma City, OK, USA

Rheal A. Towner\* and Nataliya Smith

\*Address all correspondence to: rheal-towner@omrf.org

Advanced Magnetic Resonance Center, Oklahoma Medical Research Foundation,

**Figure 10.** Immuno-electron microscopy detection of the anti-DMPO probe in the plasma membrane/ cytoplasm and cell nuclei in F98 rat gliomas. The biotin moiety of the anti-DMPO probe was targeted with gold-anti-biotin. Gold-antibiotin colloids were detected within the plasma membrane/cytoplasm (black arrows) and cell nuclei membranes of F98 tumor cells administered the anti-DMPO probe. Scale bar = 1 μm. Magnification = 20,000×. n = nucleus; c = cytoplasm; pm = plasma membrane. Modified from Coutinho de Souza et al. [34].

The combined IST and mMRI approach in glioma models can also be used to assess possible therapeutic agents that are either free radical scavengers or generate free radicals. For example, this approach was used to assess the free radical scavenging ability of an anti-cancer agent, OKN-007, in a rat glioma model [34] (see **Figure 9**). Representative difference images (falsecolored red) of an untreated F98 glioma and an OKN-007-treated F98 glioma, overlaid over appropriate morphological images, are shown in **Figure 9A** and **B**, respectively. Quantitative levels of trapped free radical levels (measured from % changes in MRI signal intensities) for untreated and OKN-007-treated F98 gliomas is shown in **Figure 9C**. Significantly lower levels of MDA (**Figure 9F**) and 3-NT (**Figure 9G**) were found for F98 gliomas treated with OKN-007 compared to untreated (UT) tumors. IHC levels for MDA and 3-NT were quantitated in several OKN-007-treated and UT F98 tumor-bearing rats. These results indicate that OKN-007 acts as a free radical scavenger when used as an anti-cancer agent. This is the first time *in vivo* detection of *in situ* free radicals had been reported for an anti-cancer agent with free radical scavenging capability. It was previously demonstrated that OKN-007 can significantly increase animal survival and significantly decrease tumor volumes, when compared to UT animals. The combined IST and mMRI approach can be taken to assess any therapeutic agents that would either increase or decrease free radical levels in different disease models.

Immuno-electron microscopy (IEM) with gold-anti-biotin, targeting the biotin moiety of the anti-DMPO probe, was also used to confirm the *ex vivo* presence of the anti-DMPO probe in the plasma membrane of rat glioma cells following *in vivo* administration [34] (see **Figure 10**). The IEM data is confirmation that the combined IST and mMRI approach is detecting macromolecular membrane-bound (both plasma membrane and possibly nuclear membrane) free radicals.

### **3. Concluding statements**

The novelty of the IST-mMRI approach is that heterogeneous levels of trapped free radicals can be detected directly *in vivo* and *in situ* with high image resolution, and can be used to pin-point where high levels of free radicals are formed in different heterogeneous regions of specific tissues. It should be noted that MRI has no depth-of-penetration limitation. The current anti-DMPO-albumin-Gd-DTPA-biotin construct allows rapid vascular delivery, and binding to macromolecular DMPO-trapped free radicals in the plasma membrane, as well as *ex vivo* confirmation with microscopy.

This review has discussed all of the current studies that have utilized combined IST and mMRI to detect targeted trapped macromolecular DMPO-radical adducts *in vivo* and *in situ* within various animal models where oxidative stress plays a major role. It was established for all oxidative stress-associated disease models studied thus far, that levels of free radicals were found to be significantly increased in all cases for animals treated with DMPO and the anti-DMPO probe, when compared to controls, including disease controls (e.g. wild-type rodents or shams), non-DMPO controls (i.e. administered saline instead of DMPO), and/or mMRI probe controls (i.e. a non-specific IgG was covalently bound to the albumin of the MRI contrast agent construct instead of the anti-DMPO antibody). An example of assessing an anti-cancer agent with free radical scavenging activity was also presented. The biotin moiety of the anti-DMPO probe also allowed *ex vivo* validation of the presence of trapped DMPO macromolecular adducts in various tissues. IST was also used in all cases to confirm the presence of trapped free radicals with fluorescence or optical (e.g. HRP) microscopy. Finally, IEM was used to confirm the presence of the anti-DMPO probe in plasma and nuclear membranes.

Some of the disadvantages with the methodology include limited access to pre-clinical MRI systems, availability of the anti-DMPO antibody, and further identifying the radical source that is being trapped. For non-neurological studies, this approach can be easily utilized in numerous pathological/toxicological models. However, for neurological studies, the approach will be limited to whether there is BBB permeability, in order to allow the anti-DMPO probe, and possibly DMPO, to access the target tissue.

The IST-mMRI approach can certainly be further applied to study free radicals associated longitudinally in oxidative stress-related disease processes, as well as assess the effect of therapeutic agents that alter free radical levels. Mass spectrometry may need to be used to not only further assess whether the anti-DMPO probe detected in heterogeneous tissue regions are essentially oxidized proteins or oxidized lipids, or a combination of both, before the type of protein or lipid is identified. The current size of the probe may prohibit use in neurological studies with an intact blood-brain barrier (BBB). The development of a smaller nanoparticle-based anti-DMPO probe, which may allow access through an intact BBB, is currently being considered.

### **Author details**

The combined IST and mMRI approach in glioma models can also be used to assess possible therapeutic agents that are either free radical scavengers or generate free radicals. For example, this approach was used to assess the free radical scavenging ability of an anti-cancer agent, OKN-007, in a rat glioma model [34] (see **Figure 9**). Representative difference images (falsecolored red) of an untreated F98 glioma and an OKN-007-treated F98 glioma, overlaid over appropriate morphological images, are shown in **Figure 9A** and **B**, respectively. Quantitative levels of trapped free radical levels (measured from % changes in MRI signal intensities) for untreated and OKN-007-treated F98 gliomas is shown in **Figure 9C**. Significantly lower levels of MDA (**Figure 9F**) and 3-NT (**Figure 9G**) were found for F98 gliomas treated with OKN-007 compared to untreated (UT) tumors. IHC levels for MDA and 3-NT were quantitated in several OKN-007-treated and UT F98 tumor-bearing rats. These results indicate that OKN-007 acts as a free radical scavenger when used as an anti-cancer agent. This is the first time *in vivo* detection of *in situ* free radicals had been reported for an anti-cancer agent with free radical scavenging capability. It was previously demonstrated that OKN-007 can significantly increase animal survival and significantly decrease tumor volumes, when compared to UT animals. The combined IST and mMRI approach can be taken to assess any therapeutic agents

pm = plasma membrane. Modified from Coutinho de Souza et al. [34].

18 Free Radicals, Antioxidants and Diseases

**Figure 10.** Immuno-electron microscopy detection of the anti-DMPO probe in the plasma membrane/ cytoplasm and cell nuclei in F98 rat gliomas. The biotin moiety of the anti-DMPO probe was targeted with gold-anti-biotin. Gold-antibiotin colloids were detected within the plasma membrane/cytoplasm (black arrows) and cell nuclei membranes of F98 tumor cells administered the anti-DMPO probe. Scale bar = 1 μm. Magnification = 20,000×. n = nucleus; c = cytoplasm;

that would either increase or decrease free radical levels in different disease models.

**3. Concluding statements**

Immuno-electron microscopy (IEM) with gold-anti-biotin, targeting the biotin moiety of the anti-DMPO probe, was also used to confirm the *ex vivo* presence of the anti-DMPO probe in the plasma membrane of rat glioma cells following *in vivo* administration [34] (see **Figure 10**). The IEM data is confirmation that the combined IST and mMRI approach is detecting macromolecular membrane-bound (both plasma membrane and possibly nuclear membrane) free radicals.

The novelty of the IST-mMRI approach is that heterogeneous levels of trapped free radicals can be detected directly *in vivo* and *in situ* with high image resolution, and can be used to Rheal A. Towner\* and Nataliya Smith

\*Address all correspondence to: rheal-towner@omrf.org

Advanced Magnetic Resonance Center, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

### **References**

[1] Newsholme P, Cruzat VF, Keane KN, Carlessi R, Homen de Bittencourt PI Jr. Molecular mechanisms of ROS production and oxidative stress in diabetes. The Biochemical Journal. 2016;**473**:4527-4550

[14] Davies MJ, Slater TF. The use of electron-spin-resonance techniques to detect free-radical formation and tissue damage. The Proceedings of the Nutrition Society. 1988;**47**:397-405

Detecting *In Vivo* Free Radicals in Various Disease Models

http://dx.doi.org/10.5772/intechopen.74106

21

[15] Villamena FA, Zweier JL. Detection of reactive oxygen and nitrogen species by EPR spin

[16] Hawkins CL, Davies MJ. Detection and characterisation of radicals in biological materials

[17] Barriga-González G, Olea-Azar C, Zuñiga-López MC, Folch-Cano C, Aguilera-Venegas B, Porcal W, González M, Cerecetto H. Spin trapping: An essential tool for the study of diseases caused by oxidative stress. Current Topics in Medicinal Chemistry. 2015;

[18] Davies MJ. Detection and characterisation of radicals using electron paramagnetic reso-

[19] Mason RP. Assay of *in situ* radicals by electron spin resonance. Methods in Enzymology.

[20] Mason RP. In: Rhodes C, editor. *In vivo* Spin Trapping – From Chemistry to Toxicology, in Toxicology of the Human Environment: The Critical Role of Free Radicals. London:

[21] Berliner LJ, Khramtsov V, Fujii H, Clanton TL. Unique in vivo applications of spin traps.

[22] Berliner LJ. From spin-labeled proteins to *in vivo* EPR applications. European Biophysics

[23] Mason RP. Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping. Free Radical Biology & Medicine.

[24] Ramirez DC, Mason RP. Immuno-spin trapping: Detection of protein-centered radicals.

[25] Gomez-Mejiba SE, Zhai Z, Akram H, Deterding LJ, Hensley K, Smith N, Towner RA, Tomer KB, Mason RP, Ramirez DC. Immuno-spin trapping of protein and DNA radicals: "Tagging" free radicals to locate and understand the redox process. Free Radical

[26] Khoo NK, Cantu-Medellin N, St Croix C, Kelley EE. *In vivo* immuno-spin trapping: Imaging the footprints of oxidative stress. Current Protocols in Cytometry. 2015;**74**:

[27] Mason RP. Imaging free radicals in organelles, cells, tissue, and *in vivo* with immuno-

[28] Gomez-Mejiba SE, Zhai Z, Della-Vedova MC, Muñoz MD, Chatterjee S, Towner RA, Hensley K, Floyd RA, Mason RP, Ramirez DC. Immuno-spin trapping from biochemistry

using EPR methodology. Biochimica et Biophysica Acta. 2014;**1840**:708-721

nance (EPR) spin trapping and related methods. Methods. 2016;**109**:21-31

trapping. Antioxidants & Redox Signaling. 2004;**6**:619-629

**15**:484-495

1984;**105**:416-422

Taylor and Francis; 2000. pp. 49-70

Journal. 2010;**39**:579-588

2004;**36**:1214-1223

12.42.1-12.4211

Free Radical Biology & Medicine. 2011;**30**:489-499

Current Protocols in Toxicology. 2005;**17**:17

spin trapping. Redox Biology. 2016;**8**:422-429

Biology & Medicine. 2009;**46**:853-865


[14] Davies MJ, Slater TF. The use of electron-spin-resonance techniques to detect free-radical formation and tissue damage. The Proceedings of the Nutrition Society. 1988;**47**:397-405

**References**

20 Free Radicals, Antioxidants and Diseases

Journal. 2016;**473**:4527-4550

halopathy. Shock. 2013;**39**:10-16

Cerebral Blood Flow and Metabolism. 2011;**31**:1532-1544

Sciences. 2016;**17**(6). Pii: E984. DOI: 10.3390/ijms17060984

Environmental Health Perspectives. 1985;**64**:151-170

[9] Janzen EG. Spin trapping. Methods in Enzymology. 1984;**104**:188-198

**2017**:8379327

2000. pp. 7-24

**60**:133-137

Medicine. 1987;**3**:259-303

Biophysical Methods. 1995;**30**:239-247

[1] Newsholme P, Cruzat VF, Keane KN, Carlessi R, Homen de Bittencourt PI Jr. Molecular mechanisms of ROS production and oxidative stress in diabetes. The Biochemical

[2] Fakhruddin S, Alanzi W, Jackson KE. Diabetes-induced reactive oxygen species: Mechanism of their generation and role in renal injury. Journal of Diabetes Research. 2017;

[3] Bozza FA, D'Avila JC, Ritter C, Sonneville R, Sharshar T, Dal-Pizzol F. Bioenergetics, mitochondrial dysfunction, and oxidative stress in the pathophysiology of septic encep-

[4] Berg RMG, Moller K, Bailey DM. Neuro-oxidative-nitrosative stress in sepsis. Journal of

[5] Niedzielska E, Smaga I, Gawlik M, Moniczewski A, Stankowicz P, Pera J, Filip M.Oxidative stress in neurodegenerative diseases. Molecular Neurobiology. 2016;**53**:4094-4125

[6] Sorce S, Stocker R, Seredenina T, Holmdahl R, Aguzzi A, Chio A, Depaulis A, Heitz F, Olosson P, Olsson T, Duveau V, Sanoudou D, Skosgater S, Vlahou A, Wasquel D, Krause KH, Jaquet V. NADPH oxidases as drug targets and biomarkers in neurodegenerative diseases: What is the evidence? Free Radical Biology & Medicine. 2017;**112**:387-396

[7] Rinaldi M, Caffo M, Minutoli L, Marini H, Abbritti RV, Squadrito F, Trichilo V, Valenti A, Barresi V, Altavilla D, Passalacqua M, Caruso G. ROS and brain gliomas: An overview of potential and innovative therapeutic strategies. International Journal of Molecular

[8] Towner RA. In: Rhodes C, editor. Chemistry of Spin Trapping, in Toxicology of the Human Environment: The Critical Role of Free Radicals. London: Taylor and Francis;

[10] Janzen EG, Stronks HJ, Dubose CM, Poyer JL, McCay PB. Chemistry and biology of spin-trapping radicals associated with halocarbon metabolism in vitro and in vivo.

[11] Buettner GR. Spin trapping: ESR parameters of spin adducts. Free Radical Biology &

[12] McCay PB. Application of ESR spectroscopy in toxicology. Archives of Toxicology. 1987;

[13] Janzen EG, Poyer JL, Schaefer CF, Downs PE, DuBose CM. Biological spin trapping. II. Toxicity of nitrone spin traps: Dose-ranging in the rat. Journal of Biochemical and


to medicine: Advances, challenges, and pitfalls. Focus on protein-centered radicals. Biochimica et Biophysica Acta. 2014;**1840**(2):722-729

[40] Gupta N, Reja SI, Bhalla V, Gupta M, Kaur G, Kumar M. An approach for the selective detection of nitric oxide in biological systems: An *in vitro* and *in vivo* perspective.

Detecting *In Vivo* Free Radicals in Various Disease Models

http://dx.doi.org/10.5772/intechopen.74106

23

[41] Iverson NM, Strano MS, Wogan GN. *In vivo* delivery of nitric oxide-sensing, singlewalled carbon nanotubes. Current Protocols in Chemical Biology. 2015;**7**:93-102

[42] Li Z, Liang T, Lv S, Zhuang Q, Liu Z. A rationally designed upconversion nanoprobe for *in vivo* detection of hydroxyl radical. Journal of the American Chemical Society.

[43] Huang Y, Yu F, Wang J, Chen L. Near-infrared fluorescence probe for *in situ* detection of superoxide anion and hydrogen polysulfides in mitochondrial oxidative stress. Ana-

[44] Zhang J, Li C, Zhang R, Zhang F, Liu W, Liu X, Le SM-Y, Zhang H. A phosphinate-based near-infrared fluorescence probe for imaging the superoxide radical anion *in vitro* and *in* 

[45] Iverson NM, Barone PW, Shandell M, Trudel LJ, Sen S, Sen F, Ivanov V, Atolia E, Farias E, McNicholas TP, Reuel N, Parry NM, Wogan GN, Strano MS. *In vivo* biosensing via tissue-localizable near-infrared-fluorecent single-walled carbon nanotubes. Nature Nano-

[46] Shvedova AA, Kisin ER, Murray AR, Mouithys-Mickalad A, Stadler K, Mason RP, Kadiiska M. ESR evidence for *in vivo* formation of free radicals in tissue of mice exposed to single-walled carbon nanotubes. Free Radical Biology & Medicine. 2014;**73**:154-165

[47] Rayner CL, Gole GA, Bottle SE, Barnett NL. Dynamic, *in vivo*, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe. Experimental Eye Research. 2014;**129**:48-56

[48] Du L, Huang S, Zhuang Q, Jia H, Rockenbauer A, Liu Y, Liu KJ, Liu Y. Highly sensitive free radical detection by nitrone-functionalized gold nanoparticles. Nanoscale. 2014;

[49] Berkowitz BA, Lewin AS, Biswal MR, Bredell BX, Davis C, Roberts R. MRI of retinal free radical production with laminar resolution in vivo. Investigative Ophthalmology &

[50] Berkowitz BA, Lenning J, Khetarpal N, Tran C, Wu JY, Berri AM, Dernay K, Haacke EM, Shafie-Khorassani F, Podolsky RH, Gant JC, Maimaiti S, Thibault O, Murphy GG, Bennett BM, Roberts R. In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome. The FASEB Journal.

[51] Tain RW, Scotti AM, Li W, Zhou XJ, Cai K. Imaging short-lived reactive oxygen species (ROS) with endogenous contrast MRI. Journal of Magnetic Resonance Imaging. 2017.

Chemistry, an Asian Journal. 2016;**11**:1020-1027

2015;**137**:11179-11185

lytical Chemistry. 2016;**88**:4122-4129

technology. 2013;**8**:873-880

Visual Science. 2016;**57**:577-585

2017. Epub ahead of print. DOI: 10.1096/fj.201700229R

Epub ahead of print. DOI: 10.1002/jmri.25763

**6**:1646-1652

*vivo*. Chemical Communications. 2016;**52**:2679-2682


[40] Gupta N, Reja SI, Bhalla V, Gupta M, Kaur G, Kumar M. An approach for the selective detection of nitric oxide in biological systems: An *in vitro* and *in vivo* perspective. Chemistry, an Asian Journal. 2016;**11**:1020-1027

to medicine: Advances, challenges, and pitfalls. Focus on protein-centered radicals. Bio-

[29] Towner RA, Smith N, Saunders D, Henderson M, Downum K, Lupu F, Silasi-Mansat R, Ramirez DC, Gomez-Mejiba SE, Bonini MG, Ehrenshaft M, Mason RP. *In vivo* imaging of immuno-spin trapped radicals with molecular MRI in a mouse diabetes model.

[30] Towner RA, Smith N, Saunders D, Carrizales J, Lupu F, Silasi-Mansat R, Ehrenshaft M, Mason RP. *In vivo* targeted molecular magnetic resonance imaging of free radicals in

[32] Towner RA, Smith N, Saunders D, Lupu F, Silasi-Mansat R, West M, Ramirez DC, Gomez-Mejiba SE, Bonini MG, Mason RP, Ehrenshaft M, Hensley K. *In vivo* detection of free radicals using molecular MRI and immuno-spin-trapping in a mouse model for amyotrophic lateral sclerosis (ALS). Free Radical Biology and Medicine. 2013;**63**:351-360

[33] Towner RA, Smith N, Saunders D, De Souza PC, Henry L, Lupu F, Silasi-Mansat R, Ehrenshaft M, Mason RP, Gomez-Mejiba SE, Ramirez DC. Combined molecular MRI and immuno-spin-trapping for *in vivo* detection of free radicals in orthotopic mouse

[34] Coutinho de Souza P, Smith N, Atolagbe O, Ziegler J, Nijoku C, Lerner M, Ehrenshaft M, Mason RP, Meek B, Plafker SM, Saunders D, Mamedova N, Towner RA. OKN-007 decreases free radicals levels in a preclinical F98 rat glioma model. Free Radical Biology

[35] Ribou AC. Synthetic sensors for reactive oxygen species detection and quantification: A critical review of current methods. Antioxidants & Redox Signaling. 2016;**25**:520-533

[36] Bonini MG, Rota C, Tomasi A, Mason RP. The oxidation of 2′,7′-dichorofluorescin to reactive oxygen species: A self-fulfilling prophesy? Free Radical Biology & Medicine.

[37] Liu F, Du J, Song D, Xu M, Sun G. A sensitive fluorescent sensor for the detection of endogenous hydroxyl radicals in living cells and bacteria and direct imaging with respect to its ecotoxicity in living zebra fish. Chemical Communications. 2016;**52**:4636-4639 [38] Hu JJ, Wong NK, Ye S, Chen X, Lu MY, Zhao AQ, Guo Y, Ma AC, Leung AY, Shen J, Yang D. Fluorescent probe HKSOX-1 for imaging and detection of endogenous superoxide in live cells and *in vivo*. Journal of the American Chemical Society. 2015;**137**:6837-6843 [39] Liu X, Liu S, Liang G. Fluorescence turn-on for the highly selective detection of nitric

GL261 gliomas. Biochimica et Biophysica Acta. 2013;**1832**:2153-2161

oxide *in vitro* and in living cells. Analyst. 2016;**141**:2600-2605

diabetic cardiomyopathy within mice. Free Radical Research. 2015;**49**:1140-1146 [31] Towner RA, Garteiser P, Bozza F, Smith N, Saunders D, d'Avila JCP, Magno F, Oliveira MF, Ehrenshaft M, Lupu F, Silasi-Mansat R, Ramirez DC, Gomez-Mejiba SE, Mason RP, Faria-Neto HCC. *In vivo* detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin-trapping. Free Radical Biology & Medicine.

chimica et Biophysica Acta. 2014;**1840**(2):722-729

Diabetes. 2012;**61**:2405-2413

22 Free Radicals, Antioxidants and Diseases

& Medicine. 2015;**87**:157-168

2006;**40**:968-975

2013;**65**:828-837


[52] Jerome NP, Papoutsaki MV, Orton MR, Parkes HG, Winfield JM, Boss MA, Leach MO, deSouza NM, Collins DJ. Development of a temperature-controlled phantom for magnetic resonance quality assurance of diffusion, dynamic, and relaxometry measurements. Medical Physics. 2016;**43**:2998

**Chapter 3**

**Provisional chapter**

**Oxidative Stress and Vascular Diseases: Effect of**

**Oxidative Stress and Vascular Diseases: Effect of** 

DOI: 10.5772/intechopen.76576

Investigations have shown that worldwide the vascular diseases are considered independent risk factors for an increased mortality. Despite these diseases being related to factors such as sedentary lifestyle, poor diet and stress, the oxidative stress been the which most strongly explained the genesis of these vascular diseases. In this sense, the body of evidence involving an analysis and understanding of the factors and preventive variables of these diseases available in the literature is necessary. Considering this, we aimed to available information about the role of the oxidants and antioxidants enzymes in the prevention or causes of vascular diseases, and how the physical exercise can prevent the development of these vascular diseases. It was observed that endothelin-1 is an important oxidant involved in the atherosclerotic process, while the hydrogen sulfide and glutathione peroxidase have an antioxidant role. About the physical exercise, there is greater production of oxidants, however, as a mechanism of compensation, there is also greater production and release of antioxidants such as nitric oxide and glutathiones after some sessions when compared with the baseline. We conclude that the stress oxidative is involved in the vascular disease and the physical exercise could be used like prevention.

**Keywords:** vascular disease, oxidant enzyme, antioxidant enzyme, oxidative stress,

© 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

**Physical Exercise**

**Physical Exercise**

Aline de Freitas Brito,

Aline de Freitas Brito,

Glêbia Alexa Cardoso,

**Abstract**

physical exercise

Glêbia Alexa Cardoso,

Caio Victor Coutinho de Oliveira,

Caio Victor Coutinho de Oliveira,

Joana Marcela Sales de Lucena,

Joana Marcela Sales de Lucena,

http://dx.doi.org/10.5772/intechopen.76576

Alesandra Araújo de Souza

Alesandra Araújo de Souza

Jordanna di Paula dos Santos Sousa and

Jordanna di Paula dos Santos Sousa and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **Oxidative Stress and Vascular Diseases: Effect of Physical Exercise Oxidative Stress and Vascular Diseases: Effect of Physical Exercise**

DOI: 10.5772/intechopen.76576

Aline de Freitas Brito, Caio Victor Coutinho de Oliveira, Glêbia Alexa Cardoso, Joana Marcela Sales de Lucena, Jordanna di Paula dos Santos Sousa and Alesandra Araújo de Souza Aline de Freitas Brito, Caio Victor Coutinho de Oliveira, Glêbia Alexa Cardoso, Joana Marcela Sales de Lucena, Jordanna di Paula dos Santos Sousa and Alesandra Araújo de Souza

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76576

#### **Abstract**

[52] Jerome NP, Papoutsaki MV, Orton MR, Parkes HG, Winfield JM, Boss MA, Leach MO, deSouza NM, Collins DJ. Development of a temperature-controlled phantom for magnetic resonance quality assurance of diffusion, dynamic, and relaxometry measure-

[53] Spear JT, Gore JC. New insights into rotating frame relaxation at high field. NMR in

[54] Eto H, Hyodo F, Kosem N, Kobayashi R, Yasukawa K, Nakao M, Kiniwa M, Utsumi H. Redox imaging of skeletal muscle using in vivo DNP-MRI and its application to an animal model of local inflammation. Free Radical Biology & Medicine. 2015;**89**:1097-1104

[55] Janzen EG, Towner RA. Aminoxyl radicals as MRI contrast agents. In: Zhdanov R, editor.

[56] Hyodo F, Ito S, Eto H, Nakaji T, Yasukawa K, Kobayashi R, Utsumi H. Development of redox metabolic imaging using endogenous molecules. Yakugaku Zasshi. 2016;

[57] Utsumi H, Hyodo F. Free radical imaging using in vivo dynamic nuclear polarization-

[58] Ichikawa K, Yasukawa K. Imaging in vivo redox status in high spatial resolution with

[59] Yamamoto S, Watabe T, Ikeda H, Ichikawa K, Nakao M, Kato K, Hatazawa J.Development of a PET/OMRI combined system for simultaneous imaging of positron and free radical

Bioactive Spin Labels. Berlin: Springer-Verlag; 1992. pp. 573-583

MRI. Methods in Enzymology. 2015;**564**:553-571

OMRI. Free Radical Research. 2012;**46**:1004-1010

probes for small animals. Medical Physics. 2016;**43**:5676

ments. Medical Physics. 2016;**43**:2998

Biomedicine. 2016;**29**:1258-1273

24 Free Radicals, Antioxidants and Diseases

**136**:1107-1114

Investigations have shown that worldwide the vascular diseases are considered independent risk factors for an increased mortality. Despite these diseases being related to factors such as sedentary lifestyle, poor diet and stress, the oxidative stress been the which most strongly explained the genesis of these vascular diseases. In this sense, the body of evidence involving an analysis and understanding of the factors and preventive variables of these diseases available in the literature is necessary. Considering this, we aimed to available information about the role of the oxidants and antioxidants enzymes in the prevention or causes of vascular diseases, and how the physical exercise can prevent the development of these vascular diseases. It was observed that endothelin-1 is an important oxidant involved in the atherosclerotic process, while the hydrogen sulfide and glutathione peroxidase have an antioxidant role. About the physical exercise, there is greater production of oxidants, however, as a mechanism of compensation, there is also greater production and release of antioxidants such as nitric oxide and glutathiones after some sessions when compared with the baseline. We conclude that the stress oxidative is involved in the vascular disease and the physical exercise could be used like prevention.

**Keywords:** vascular disease, oxidant enzyme, antioxidant enzyme, oxidative stress, physical exercise

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

### **1. Introduction**

Vascular diseases have been reported worldwide as an independent risk factor for premature mortality [1]. It is now understood that the point in common of all vascular, cardiovascular or chronic non-cardiovascular degenerative diseases is the imbalance between oxidation and reduction profile caused by free radicals culminating in a situation denominated oxidative stress [1, 2]. This reactive oxygen species are generated from NADPH oxidases (NOx), responsible for the bioavailability of nitric oxide (NO) in vascular pathologies, through of a direct inactivation of NO, together with a reduction in NO synthesis and in oxidation of your receptor, denominated guanylyl cyclase soluble [3]. In this sense, the development of studies about the antioxidant action in vascular function are important, considering that alterations in this functions, characterized for one increase in vasoconstrictor responses, decrease in vasodilatory capacity and one increase in reactive oxygen species production, and the reduction of the activity of antioxidants enzymes are associated with the cardiovascular risk factor, as arterial hypertension and atherosclerosis [4–9].

**2. What is the role of oxidative stress in vascular diseases?**

general term that refers not only to radicals derived from superoxide (O2

Singlet oxygen <sup>1</sup>

Superoxide anion O2

Hydrogen peroxide H2

Hydroxyl radical HO• Perhydroxyl radical HO2

Alcoxila radical RO• Peroxyl radical ROO• Hydroperoxyl radical ROOH• Hypochlorous acid HCLO Ozone O3

Nitric oxide NO Nitric oxide NO2 Peroxynitrite ONO2

age and increased proinflammatory responses [14].

includes non-radical O2

**Oxigen-reactive species**

**Nitrogen reactive species**

**Table 1.** Reactive oxygen and nitrogen species.

At first, it is necessary to understand that free radicals are molecules that contain one or more unpaired electrons, are generated independently, and are considered highly reactive due to their ability to accept electrons from other molecules until a terminal reaction occurs [2]. Excessive production of these radicals can trigger cumulative cellular damage in proteins, lipids, deoxyribonucleic acid (DNA) and other components, resulting in several pathological processes [1]. Thus, in vitro studies have demonstrated that these molecules are important intracellular signaling factors that contribute to vascular remodeling, modulating vascular contraction/ dilatation, migration, apoptosis and protein turnover of the extracellular matrix [14]. Thus, increased reactive oxygen species (ROS) formation is identified in vascular diseases such as hypertension, atherosclerosis and stroke, and is associated with a reduction in levels of nitric oxide (NO) and other vasodilators, endothelial tissue damage, protein oxidation, DNA dam-

There are two types of reactive species, one of which is called the reactive oxygen species, a

class is known as nitrogen reactive species (RNS), it refers to nitrogen radicals reactive with other molecules in which the reactive center is nitrogen [15]. The most common ROS and RNS are shown in **Table 1** in an order of the ones that are the most to the least reactive in the cell [15]. A variety of enzymatic and non-enzymatic processes can generate the reactive species in mammalian cells [1]. The primary sources are: mitochondrial respiratory chain, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, dissociated nitric


) metabolism, but also

) [15]. Similarly, the other

O2

−•

O2

•

−

O2

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

27

Therefore, researches have analyzed effective therapeutic strategies in the treatment of pathologies that affect the vascular musculature. Among them, drug therapy is still the most indicated, due to its antithrombotic, vasodilatory and hypocholesterolemic pharmacological efficacy, such as statins, capable of improving the endothelial functions, due to yours antioxidants, anti-inflammatories and anti-atherosclerotic properties [10, 11]. Although the drugs administered are effective, their clinical utility is limited due to the development of tolerance and resistance. For that reason, other therapeutic strategies, with fewer collateral effects, have been indicated together with the drug treatment, since they are also able to reduce the deleterious vascular effects, such as nutraceutical biology with a diet rich in fruits, vegetables or red wine, for example [12, 13]. In the present study, the use of polyphenols in the treatment of high levels of polyphenols was studied. Then, it seems plausible to affirm that a better understanding of the relationship between oxidants and antioxidants functions in the prevention or treatment of vascular diseases should have a body of evidence amply constructed to collaborate in the prevention and treatment of diseases.

Physical exercise, in this context, has been presented as one of the elements of a healthy lifestyle capable of modulating oxidative stress, by promoting the increase of endothelial nitric oxide synthase (eNOS) activity, of the early oncogenesis protein tyrosine kinase (c-Src) [73] and the bioavailability of NO and antioxidant enzymes, leading to significant vascular protection [12, 13] .

Although to date the investigations have demonstrated the endogenous antioxidant effect on the prevention of vascular disease, obtained with or without the contribution of short- or long-term physical exercise, other mechanisms are need to be better understood, especially with regard to exercise. It is not yet possible to assert the understanding that in addition to modulation in the antioxidant action, there is also an action on inflammatory factors, reducing the inflammation so present in vascular diseases, if there is modification of the membrane proteins of some vascular cells, or if this regulation also occurs at the downstream level. The collection of the literature regarding these gaps will be best seen in Chapters 2 and 3 of this book.

Considering this, we aimed to avail information about the role of the oxidants and antioxidants enzymes in the prevention or causes of vascular diseases, and how the physical exercise can prevent the development of these same diseases.

## **2. What is the role of oxidative stress in vascular diseases?**

**1. Introduction**

26 Free Radicals, Antioxidants and Diseases

sclerosis [4–9].

laborate in the prevention and treatment of diseases.

can prevent the development of these same diseases.

Vascular diseases have been reported worldwide as an independent risk factor for premature mortality [1]. It is now understood that the point in common of all vascular, cardiovascular or chronic non-cardiovascular degenerative diseases is the imbalance between oxidation and reduction profile caused by free radicals culminating in a situation denominated oxidative stress [1, 2]. This reactive oxygen species are generated from NADPH oxidases (NOx), responsible for the bioavailability of nitric oxide (NO) in vascular pathologies, through of a direct inactivation of NO, together with a reduction in NO synthesis and in oxidation of your receptor, denominated guanylyl cyclase soluble [3]. In this sense, the development of studies about the antioxidant action in vascular function are important, considering that alterations in this functions, characterized for one increase in vasoconstrictor responses, decrease in vasodilatory capacity and one increase in reactive oxygen species production, and the reduction of the activity of antioxidants enzymes are associated with the cardiovascular risk factor, as arterial hypertension and athero-

Therefore, researches have analyzed effective therapeutic strategies in the treatment of pathologies that affect the vascular musculature. Among them, drug therapy is still the most indicated, due to its antithrombotic, vasodilatory and hypocholesterolemic pharmacological efficacy, such as statins, capable of improving the endothelial functions, due to yours antioxidants, anti-inflammatories and anti-atherosclerotic properties [10, 11]. Although the drugs administered are effective, their clinical utility is limited due to the development of tolerance and resistance. For that reason, other therapeutic strategies, with fewer collateral effects, have been indicated together with the drug treatment, since they are also able to reduce the deleterious vascular effects, such as nutraceutical biology with a diet rich in fruits, vegetables or red wine, for example [12, 13]. In the present study, the use of polyphenols in the treatment of high levels of polyphenols was studied. Then, it seems plausible to affirm that a better understanding of the relationship between oxidants and antioxidants functions in the prevention or treatment of vascular diseases should have a body of evidence amply constructed to col-

Physical exercise, in this context, has been presented as one of the elements of a healthy lifestyle capable of modulating oxidative stress, by promoting the increase of endothelial nitric oxide synthase (eNOS) activity, of the early oncogenesis protein tyrosine kinase (c-Src) [73] and the bioavailability of NO and antioxidant enzymes, leading to significant vascular protection [12, 13] . Although to date the investigations have demonstrated the endogenous antioxidant effect on the prevention of vascular disease, obtained with or without the contribution of short- or long-term physical exercise, other mechanisms are need to be better understood, especially with regard to exercise. It is not yet possible to assert the understanding that in addition to modulation in the antioxidant action, there is also an action on inflammatory factors, reducing the inflammation so present in vascular diseases, if there is modification of the membrane proteins of some vascular cells, or if this regulation also occurs at the downstream level. The collection of the literature regarding these gaps will be best seen in Chapters 2 and 3 of this book. Considering this, we aimed to avail information about the role of the oxidants and antioxidants enzymes in the prevention or causes of vascular diseases, and how the physical exercise At first, it is necessary to understand that free radicals are molecules that contain one or more unpaired electrons, are generated independently, and are considered highly reactive due to their ability to accept electrons from other molecules until a terminal reaction occurs [2]. Excessive production of these radicals can trigger cumulative cellular damage in proteins, lipids, deoxyribonucleic acid (DNA) and other components, resulting in several pathological processes [1].

Thus, in vitro studies have demonstrated that these molecules are important intracellular signaling factors that contribute to vascular remodeling, modulating vascular contraction/ dilatation, migration, apoptosis and protein turnover of the extracellular matrix [14]. Thus, increased reactive oxygen species (ROS) formation is identified in vascular diseases such as hypertension, atherosclerosis and stroke, and is associated with a reduction in levels of nitric oxide (NO) and other vasodilators, endothelial tissue damage, protein oxidation, DNA damage and increased proinflammatory responses [14].

There are two types of reactive species, one of which is called the reactive oxygen species, a general term that refers not only to radicals derived from superoxide (O2 ) metabolism, but also includes non-radical O2 -reactive derivative (e.g., hydrogen—H2 O2 ) [15]. Similarly, the other class is known as nitrogen reactive species (RNS), it refers to nitrogen radicals reactive with other molecules in which the reactive center is nitrogen [15]. The most common ROS and RNS are shown in **Table 1** in an order of the ones that are the most to the least reactive in the cell [15].

A variety of enzymatic and non-enzymatic processes can generate the reactive species in mammalian cells [1]. The primary sources are: mitochondrial respiratory chain, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, dissociated nitric


**Table 1.** Reactive oxygen and nitrogen species.

oxide synthase (NOS), lipoxygenase and myeloperoxidase (MPO), the first four of which are responsible for aggravating the vascular diseases [1].

l-arginine to l-citrulline and NO. The production of NO via eNOS involves the transfer of electrons from the NADPH cofactor to adenine and flavin dinucleotide and the mononucleo-

cofactor (tetrahydrobiopterin—BH4) conditions. The BH4 enzyme is highly susceptible to oxidative degradation, and the initial oxidative loss of BH4 in response to increased EROS production by NADPH oxidases amplifies oxidative stress through the resulting loss in NO

The coupling of the electron flow through the eNOS to l-arginine is dependent on adequate levels of cofactors and under specific circumstances, eNOS can become "decoupled" and reduces the oxygen molecule rather than transfer electrons to l-arginine, generating O2

Thus, the impact of eNOS on vasculature may depend on adequate levels of cofactors to support endothelial function. In fact, studies have shown the decoupling of eNOS from the arter-

Xanthine and xanthine dehydrogenase are forms of the same enzyme, known as xanthine oxidoreductase. This enzyme is widely expressed in the capillary endothelium and catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid, however, only the oxidase

under conditions of stress or, for example, in hypoxia induced by a process of atherosclerosis, the oxidase isoform predominates. Therefore, xanthine oxidase has been implicated as a source of EROS after reperfusion of ischemic tissue in various organs [2], and its expression is

The development of vascular disease originates from an initial injury to the vessel wall by biological or mechanical factors. Both produced in response to injury can stimulate ROS production in macrophages, endothelial cells, smooth muscle cells and adventitial layer. These then impair vessel tone through endothelial dysfunction, which is characterized by inflammatory response, pro-constrictive response, increased migration of smooth muscle cells, proliferation and apoptosis (**Figure 1**), contributing to diseases such as stroke, atherosclerosis and hypertension [5–8, 24].

. The enzyme typically exists in the form of dehydrogenase, but

•− under substrate (arginine) or

http://dx.doi.org/10.5772/intechopen.76576

•−.

29

•− dependent of us. Most of the evidence

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

tide of adenine and flavin to heme [20]. All can generate O2

linking NOS to EROS production belongs to the eNOS isoform [20].

production and an increase in the generation of O2

ies of individuals with diabetes [21] or atherosclerosis [22].

O2

**Figure 1.** Role of reactive oxygen species in response to an injury.

•− and H2

upregulated by NADPH oxidase [23].

form generates O2

The mitochondrial respiratory chain is the main pathway for radical generation in biological systems involving the transport of mitochondrial electrons, where oxygen is used for the production of ATP. Under physiological conditions, most of the oxygen consumed by cells is reduced to water in the mitochondria by serial oxy-reduction reactions through the action of the cytochrome oxidase complex. The reduction of oxygen to water takes place inside it in a way that leaves no intermediates. In fact, it is necessary to receive the oxygen atom to form two molecules of H2 O and this is done when it receives four electrons (H+ ) and, upon receiving them, the oxygen goes through intermediate stages: superoxide, hydrogen peroxide, radical hydroxyl and finally water. All this happens inside the cytochrome oxidase complex and it does not let these intermediates leak. However, if there is an accumulation of electron flow in the chain, this increases the probability that some electron will leak out of the chain. From 1 to 5% of the passage of oxygen along the respiratory chain may give rise to O2 •−, which results in other non-radical species (H2 O2 ) and radicals (HO•). This may result from the reduction of an ubiquinone (coenzyme Q) electron, generating ubisemiquinone, which then binds its unpaired electron to O2 to form O2 •−. However, there may be other free radical generation sites in the electron transport chain [2]. Mitochondria also generate NO, which can react with O2 •− to form peroxynitrite (ONOO<sup>−</sup> ), a very potent oxidant [16].

Nicotinamides adenine dinucleotide phosphate oxidases (NADPH) are a family of enzymes with multiple complex subunits that generate O2 •− by reducing one of electron oxygen using NADPH as the source of electrons [17]. They comprise a cytochrome b558 that crosses the plasma membrane, is composed of a large catalytic subunit, gp91*phox* (nox2), and a small subunit, p22*phox* (the term "phox" is derived from "phagocytic oxidase") together with cytosolic regulatory subunits p47*phox*, p67*phox*, p40*phox* and the small GTPase Rac [14, 18]. Activation of NADPH oxidase is initiated by phosphorylation (in serine) of the p47*phox* cytoplasmic subunit, triggering its migration to the membrane, where, along with Rac, it associates with cytochrome b558, initiating the catalytic activity of the enzyme. The identification of subunits homologous to gp91*phox* resulted in the formation of the Nox family (of "Nonphagocytic NADPH Oxidase") (Nox1, Nox2 [formally known as gp91*phox*], Nox3, Nox4, Nox5, Duox1 and Duox2 [Dual oxidase]). The main components of the complex enzymatic are nox1, nox2 and nox4 being the major catalytic subunits in vascular endothelial cells, smooth muscle cells, fibroblasts and cardiomyocytes. In the large arteries, p22*phox*, p47*phox* and Rac subunits are found. While in cells of small arteries of resistance, gp91*phox* (nox2), p22*phox*, p47*phox* and e p67*phox*, were identified as the main responsible for the formation of intracellular ROS [19]. In cell stimulation, p47*phox* becomes phosphorylated and the cytosolic subunits form a complex, which then migrates to the membrane, where it associates with cytochrome b558 to leave the active oxidase, which transfers electrons from the O2 substrate, leading to formation of de O2 •− [18]. In vascular cells, nox4 is abundantly expressed and plays an important role in the production of de O2 •− and has been associated with vascular pathophysiology [17].

There are three isoforms of nitric oxide synthase (NOS) decoupling enzymes that are termed: neuronal nitric oxide synthase (nNOS) expressed in most neural tissues, endothelial nitric oxide synthase (eNOS) expressed in cardiovascular tissues and inducible iNOS), induced by pro-inflammatory mediators [20]. NO synthesizing enzymes catalyze the conversion of l-arginine to l-citrulline and NO. The production of NO via eNOS involves the transfer of electrons from the NADPH cofactor to adenine and flavin dinucleotide and the mononucleotide of adenine and flavin to heme [20]. All can generate O2 •− under substrate (arginine) or cofactor (tetrahydrobiopterin—BH4) conditions. The BH4 enzyme is highly susceptible to oxidative degradation, and the initial oxidative loss of BH4 in response to increased EROS production by NADPH oxidases amplifies oxidative stress through the resulting loss in NO production and an increase in the generation of O2 •− dependent of us. Most of the evidence linking NOS to EROS production belongs to the eNOS isoform [20].

oxide synthase (NOS), lipoxygenase and myeloperoxidase (MPO), the first four of which are

The mitochondrial respiratory chain is the main pathway for radical generation in biological systems involving the transport of mitochondrial electrons, where oxygen is used for the production of ATP. Under physiological conditions, most of the oxygen consumed by cells is reduced to water in the mitochondria by serial oxy-reduction reactions through the action of the cytochrome oxidase complex. The reduction of oxygen to water takes place inside it in a way that leaves no intermediates. In fact, it is necessary to receive the oxygen atom to form

O and this is done when it receives four electrons (H+

) and radicals (HO•). This may result from the reduction

•−. However, there may be other free radical generation

•− by reducing one of electron oxygen using NADPH

ing them, the oxygen goes through intermediate stages: superoxide, hydrogen peroxide, radical hydroxyl and finally water. All this happens inside the cytochrome oxidase complex and it does not let these intermediates leak. However, if there is an accumulation of electron flow in the chain, this increases the probability that some electron will leak out of the chain. From 1

of an ubiquinone (coenzyme Q) electron, generating ubisemiquinone, which then binds its

sites in the electron transport chain [2]. Mitochondria also generate NO, which can react with

Nicotinamides adenine dinucleotide phosphate oxidases (NADPH) are a family of enzymes with

as the source of electrons [17]. They comprise a cytochrome b558 that crosses the plasma membrane, is composed of a large catalytic subunit, gp91*phox* (nox2), and a small subunit, p22*phox* (the term "phox" is derived from "phagocytic oxidase") together with cytosolic regulatory subunits p47*phox*, p67*phox*, p40*phox* and the small GTPase Rac [14, 18]. Activation of NADPH oxidase is initiated by phosphorylation (in serine) of the p47*phox* cytoplasmic subunit, triggering its migration to the membrane, where, along with Rac, it associates with cytochrome b558, initiating the catalytic activity of the enzyme. The identification of subunits homologous to gp91*phox* resulted in the formation of the Nox family (of "Nonphagocytic NADPH Oxidase") (Nox1, Nox2 [formally known as gp91*phox*], Nox3, Nox4, Nox5, Duox1 and Duox2 [Dual oxidase]). The main components of the complex enzymatic are nox1, nox2 and nox4 being the major catalytic subunits in vascular endothelial cells, smooth muscle cells, fibroblasts and cardiomyocytes. In the large arteries, p22*phox*, p47*phox* and Rac subunits are found. While in cells of small arteries of resistance, gp91*phox* (nox2), p22*phox*, p47*phox* and e p67*phox*, were identified as the main responsible for the formation of intracellular ROS [19]. In cell stimulation, p47*phox* becomes phosphorylated and the cytosolic subunits form a complex, which then migrates to the membrane, where it associates with cytochrome

), a very potent oxidant [16].

•− [18]. In vascular cells, nox4 is abundantly expressed and plays an important

There are three isoforms of nitric oxide synthase (NOS) decoupling enzymes that are termed: neuronal nitric oxide synthase (nNOS) expressed in most neural tissues, endothelial nitric oxide synthase (eNOS) expressed in cardiovascular tissues and inducible iNOS), induced by pro-inflammatory mediators [20]. NO synthesizing enzymes catalyze the conversion of

•− and has been associated with vascular pathophysiology [17].

to 5% of the passage of oxygen along the respiratory chain may give rise to O2

O2

b558 to leave the active oxidase, which transfers electrons from the O2

to form O2

) and, upon receiv-

•−, which results

substrate, leading to for-

responsible for aggravating the vascular diseases [1].

two molecules of H2

28 Free Radicals, Antioxidants and Diseases

in other non-radical species (H2

•− to form peroxynitrite (ONOO<sup>−</sup>

multiple complex subunits that generate O2

unpaired electron to O2

mation of de O2

role in the production of de O2

O2

The coupling of the electron flow through the eNOS to l-arginine is dependent on adequate levels of cofactors and under specific circumstances, eNOS can become "decoupled" and reduces the oxygen molecule rather than transfer electrons to l-arginine, generating O2 •−. Thus, the impact of eNOS on vasculature may depend on adequate levels of cofactors to support endothelial function. In fact, studies have shown the decoupling of eNOS from the arteries of individuals with diabetes [21] or atherosclerosis [22].

Xanthine and xanthine dehydrogenase are forms of the same enzyme, known as xanthine oxidoreductase. This enzyme is widely expressed in the capillary endothelium and catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid, however, only the oxidase form generates O2 •− and H2 O2 . The enzyme typically exists in the form of dehydrogenase, but under conditions of stress or, for example, in hypoxia induced by a process of atherosclerosis, the oxidase isoform predominates. Therefore, xanthine oxidase has been implicated as a source of EROS after reperfusion of ischemic tissue in various organs [2], and its expression is upregulated by NADPH oxidase [23].

The development of vascular disease originates from an initial injury to the vessel wall by biological or mechanical factors. Both produced in response to injury can stimulate ROS production in macrophages, endothelial cells, smooth muscle cells and adventitial layer. These then impair vessel tone through endothelial dysfunction, which is characterized by inflammatory response, pro-constrictive response, increased migration of smooth muscle cells, proliferation and apoptosis (**Figure 1**), contributing to diseases such as stroke, atherosclerosis and hypertension [5–8, 24].

**Figure 1.** Role of reactive oxygen species in response to an injury.

During vascular injury, when oxidative stress is greatest, there is vessel remodeling, where agonists such as angiotensin II, platelet growth factor, cytokines/chemokines, IL-6 and IL-8, thrombin and endothelin stimulate increased activity of NADPH oxidase and its p22*phox* domain, increased proliferation, migration and adhesion activity of vascular smooth muscle cells and reduced NO bioavailability [4, 14, 20].

DNA bases [18]. Antioxidants obtained from the diet are extremely important in interception. Another mechanism is the repair of the lesions by removing damages of the DNA molecule

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

31

Antioxidant defense mechanisms are grouped into enzymatic systems (are the first to act) and non-enzymatic [17]. Important antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione-dependent enzymes, such as glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), glutathione reductase (GSH) and glutathione synthetase [17]. There are three types of SOD: SOD-1, found in the cytosol and in the nucleus, dependent on zinc and copper; SOD-2, present in the inner mitochondria, dependent on manganese and SOD-3, found in the extracellular medium. All require a transitional redox active metal in the

Physiologically, our body adapts daily so that the concentration of antioxidant/antioxidant substances is balanced. However, in pathological conditions, such as in the development and maintenance of vascular diseases, antioxidant defenses are unable to maintain the oxidoreduction balance, causing higher levels of active reactive oxygen species which have the need

In a vicious cycle and difficult to break, oxidative stress can generate an inflammatory process and vice versa, so that there is the progression to an inflamed environment being it internal or external to the cell, resulting in an increase in the concentration of adhesion molecules vascular wall, endothelial dysfunction and onset of atherosclerosis, progressing to stroke, peripheral obstructive arterial disease, diabetic foot, coronary artery disease or abdominal aneurysm [34–37]. One of the oxidizing mechanisms that may explain the onset of vascular diseases such as atherosclerosis and peripheral arterial obstructive disease [38, 39] refers to the exacerbated concentrations of endothelin-1 (ET-1). ET-1 is a peptide with pro-inflammatory and pro-oxidant properties commonly secreted when there is damage to the endothelium as a signaling medium for tissue repair mechanisms. ET-1 directly causes increased NADPH activity and consequent increase in the concentration of reactive oxygen species. This is only the beginning of a reaction cascade that leads to an increase in the activity of adhesion molecules in the vascular cell (VCAM-1), with a consequent increase in macrophage and monocyte infiltration, calcium influx and vasoconstriction. Already indirectly, ET-1 participates in the generational process of atherosclerosis since it decreases the vasodilatory property of the arteries considering that there is redistribution of the eNOS to the mitochondria, thus decreasing the NO

bioavailability [38]. In **Figure 2**, we observe the above-mentioned effects of ET-1.

rosis and obstructive arterial disease peripheral [39–43].

role in the prevention of vascular diseases.

H2

For the antioxidant processes of prevention or deceleration of vascular diseases, proteins, enzymes or transmitting gases are involved [39–43]. In view of this, hydrogen sulfide (H<sup>2</sup>

paraoxonase and glutathione peroxidase (GSH-PX) are reported in the literature as the main antioxidant sources capable of preventing or treating vascular diseases, especially atheroscle-

S is currently considered the third gas transmitter after NO and carbon monoxide (CO) [40, 41]. It is known that due to its interaction with ion channels [44], second messengers [45–47], post-translational modification [44, 48, 49] and antioxidant defense [50], this compound plays an important

S),

active site to perform the catalytic decomposition of the superoxide anion.

of interaction with other cells, substances or membranes [34–37].

and the reconstitution of damaged cell membranes [2].

In this sense, it has been observed in diseases, such as hypertension, that in the aorta and mesenteric arteries there is an increase in vascular activation of NADPH oxidase, xanthine oxidase and decoupling of eNOS resulting in an increase in O2 •− generation, whereas levels of glutathione antioxidant and activity of the endotoxin superoxide dismutase (SOD) are reduced [25]. An increase in the local and systemic vascular inflammatory process (C-reactive protein) is also observed [26]. Activation of the renin-angiotensin system stimulates NADPH oxidase activation and production of O2 •− [27]. Vecchione et al. [28] in 2009 have shown that the reduction in endothelium-dependent vasodilation in rat arteries is associated with vascular increase in superoxide production and increased NADPH oxidase activity. However, transgenic mice with overexpression of thioredoxin 2, peroxidase that helps conversion of hydrogen peroxide into water, are resistant to hypertension induced by angiotensin II, oxidative stress and endothelial dysfunction [29]. In this sense, the present chapter aims to present the main vascular diseases, the role of the redox balance and physical exercise, in its prevention.

### **3. Prevention of vascular diseases**

### **3.1. What is the role of antioxidants?**

A vascular inflammation, risk of vascular disease development and oxidative stress have been widely discussed in the literature [30]. In general, oxidative stress and the inflammatory process are closely related to vascular diseases such as atherosclerosis, peripheral obstructive arterial disease, stroke, coronary artery disease and abdominal aneurysm [31].

Vascular diseases are chronic, progressive and multifactorial inflammation in which, at present, the immunological disorder, more precisely inflammatory, is perceived as a factor that plays an important role in the onset and maintenance of diseases [32, 33]. Already, oxidative stress is defined as the state of unbalance without qualifying favoring of oxidants at the expense of antioxidants that culminate in damaging effects on cells and membranes [34]. Oxidants may be referred to as reactive oxygen species, and free radicals such as superoxide (O2 •−), peroxynitrite (ONOO•−) and hydroxyl (OH•), in addition to non-radicals such as hydrogen peroxide (H2 O2 ).

Antioxidant defenses consistently protect tissues and body fluids from injury caused by free radicals produced by normal metabolism, disease response or from external sources [15]. For this, they are strategically arranged throughout the cytoplasm, within several organelles, extracellular space and vascular [15].

The first defense mechanism against free radicals is to prevent their formation, mainly by inhibiting chain reactions with iron and copper. A second mechanism is through the interception of free radicals, preventing the attack on the lipids of the cell membrane causing lipid peroxidation, protein amino acids, the double bond of polyunsaturated fatty acids and DNA bases [18]. Antioxidants obtained from the diet are extremely important in interception. Another mechanism is the repair of the lesions by removing damages of the DNA molecule and the reconstitution of damaged cell membranes [2].

During vascular injury, when oxidative stress is greatest, there is vessel remodeling, where agonists such as angiotensin II, platelet growth factor, cytokines/chemokines, IL-6 and IL-8, thrombin and endothelin stimulate increased activity of NADPH oxidase and its p22*phox* domain, increased proliferation, migration and adhesion activity of vascular smooth muscle

In this sense, it has been observed in diseases, such as hypertension, that in the aorta and mesenteric arteries there is an increase in vascular activation of NADPH oxidase, xanthine

els of glutathione antioxidant and activity of the endotoxin superoxide dismutase (SOD) are reduced [25]. An increase in the local and systemic vascular inflammatory process (C-reactive protein) is also observed [26]. Activation of the renin-angiotensin system stimulates NADPH

reduction in endothelium-dependent vasodilation in rat arteries is associated with vascular increase in superoxide production and increased NADPH oxidase activity. However, transgenic mice with overexpression of thioredoxin 2, peroxidase that helps conversion of hydrogen peroxide into water, are resistant to hypertension induced by angiotensin II, oxidative stress and endothelial dysfunction [29]. In this sense, the present chapter aims to present the main vascular diseases, the role of the redox balance and physical exercise, in its prevention.

A vascular inflammation, risk of vascular disease development and oxidative stress have been widely discussed in the literature [30]. In general, oxidative stress and the inflammatory process are closely related to vascular diseases such as atherosclerosis, peripheral obstructive

Vascular diseases are chronic, progressive and multifactorial inflammation in which, at present, the immunological disorder, more precisely inflammatory, is perceived as a factor that plays an important role in the onset and maintenance of diseases [32, 33]. Already, oxidative stress is defined as the state of unbalance without qualifying favoring of oxidants at the expense of antioxidants that culminate in damaging effects on cells and membranes [34]. Oxidants may be

(ONOO•−) and hydroxyl (OH•), in addition to non-radicals such as hydrogen peroxide (H2

Antioxidant defenses consistently protect tissues and body fluids from injury caused by free radicals produced by normal metabolism, disease response or from external sources [15]. For this, they are strategically arranged throughout the cytoplasm, within several organelles,

The first defense mechanism against free radicals is to prevent their formation, mainly by inhibiting chain reactions with iron and copper. A second mechanism is through the interception of free radicals, preventing the attack on the lipids of the cell membrane causing lipid peroxidation, protein amino acids, the double bond of polyunsaturated fatty acids and

arterial disease, stroke, coronary artery disease and abdominal aneurysm [31].

referred to as reactive oxygen species, and free radicals such as superoxide (O2

•− generation, whereas lev-

•−), peroxynitrite

O2 ).

•− [27]. Vecchione et al. [28] in 2009 have shown that the

cells and reduced NO bioavailability [4, 14, 20].

30 Free Radicals, Antioxidants and Diseases

oxidase activation and production of O2

**3. Prevention of vascular diseases**

**3.1. What is the role of antioxidants?**

extracellular space and vascular [15].

oxidase and decoupling of eNOS resulting in an increase in O2

Antioxidant defense mechanisms are grouped into enzymatic systems (are the first to act) and non-enzymatic [17]. Important antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione-dependent enzymes, such as glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), glutathione reductase (GSH) and glutathione synthetase [17].

There are three types of SOD: SOD-1, found in the cytosol and in the nucleus, dependent on zinc and copper; SOD-2, present in the inner mitochondria, dependent on manganese and SOD-3, found in the extracellular medium. All require a transitional redox active metal in the active site to perform the catalytic decomposition of the superoxide anion.

Physiologically, our body adapts daily so that the concentration of antioxidant/antioxidant substances is balanced. However, in pathological conditions, such as in the development and maintenance of vascular diseases, antioxidant defenses are unable to maintain the oxidoreduction balance, causing higher levels of active reactive oxygen species which have the need of interaction with other cells, substances or membranes [34–37].

In a vicious cycle and difficult to break, oxidative stress can generate an inflammatory process and vice versa, so that there is the progression to an inflamed environment being it internal or external to the cell, resulting in an increase in the concentration of adhesion molecules vascular wall, endothelial dysfunction and onset of atherosclerosis, progressing to stroke, peripheral obstructive arterial disease, diabetic foot, coronary artery disease or abdominal aneurysm [34–37].

One of the oxidizing mechanisms that may explain the onset of vascular diseases such as atherosclerosis and peripheral arterial obstructive disease [38, 39] refers to the exacerbated concentrations of endothelin-1 (ET-1). ET-1 is a peptide with pro-inflammatory and pro-oxidant properties commonly secreted when there is damage to the endothelium as a signaling medium for tissue repair mechanisms. ET-1 directly causes increased NADPH activity and consequent increase in the concentration of reactive oxygen species. This is only the beginning of a reaction cascade that leads to an increase in the activity of adhesion molecules in the vascular cell (VCAM-1), with a consequent increase in macrophage and monocyte infiltration, calcium influx and vasoconstriction. Already indirectly, ET-1 participates in the generational process of atherosclerosis since it decreases the vasodilatory property of the arteries considering that there is redistribution of the eNOS to the mitochondria, thus decreasing the NO bioavailability [38]. In **Figure 2**, we observe the above-mentioned effects of ET-1.

For the antioxidant processes of prevention or deceleration of vascular diseases, proteins, enzymes or transmitting gases are involved [39–43]. In view of this, hydrogen sulfide (H<sup>2</sup> S), paraoxonase and glutathione peroxidase (GSH-PX) are reported in the literature as the main antioxidant sources capable of preventing or treating vascular diseases, especially atherosclerosis and obstructive arterial disease peripheral [39–43].

H2 S is currently considered the third gas transmitter after NO and carbon monoxide (CO) [40, 41]. It is known that due to its interaction with ion channels [44], second messengers [45–47], post-translational modification [44, 48, 49] and antioxidant defense [50], this compound plays an important role in the prevention of vascular diseases.

**Figure 2.** Principal effects of ET-1 on the vascular system and surrounding tissues. VCAM-1, vascular cell adhesion molecule 1.

Vasoprotection promoted by H2 S involves a cascade of effects that culminate in the prevention or deceleration of the atherosclerotic process once it has been initiated. Thus, the effects cascade is composed of: (1) inhibition of atherogenesis by modifying low density lipoprotein (LDL) molecules [51]; (2) inhibition of monocyte aggregation in endothelium [1, 52]; (3) inhibition of proliferation and migration of vascular smooth muscle cells in the atherosclerotic process once it is established in the vascular wall [53, 54]; (4) inhibition of the formation of spongy cells [55]; (5) angiogenesis [56]; (6) improvement of vasorelaxative mechanisms [57]; (7) reduction of vascular wall stiffening or calcification [58] and (8) prevention of platelet aggregation and thrombogenesis [59, 60]. Some of these effects can be visualized in **Figure 3**.

H2 S demonstrates its antioxidant and, consequently, vasoprotective action when it reduces pexonitrite (ONOO<sup>−</sup> ) molecules to nitrous acid (HSNO2 ) and hydroxyl radical (OH<sup>−</sup> ) in a chemical representation, proposed by Filipovic et al. [61], as described below:

$$\mathrm{H\_2S} + \mathrm{ONOC}^- \rightarrow \mathrm{H\_2NO\_2} + \mathrm{OH^-} \tag{1}$$

Another proposed antioxidant and vasoprotective mechanism involves the action of paraoxonases, universally accepted proteins as capable of protecting cells from oxidative stress [4]. The paraoxonase family includes paraoxonase-1, 2 and 3 (PON-1, PON-2 and PON-3), with PON-1 and PON-3 being found in plasma and directly associated with high density lipoprotein fractions (HDL) promoting action against the formation of reactive oxygen species, oxidation of low density lipoprotein (LDL) and macrophages leading to blockage or reduction of atherosclerotic lesions [4]. As for PON-2, it is a cell-associated complex, it is not found free in plasma, but in some tissues, especially in the kidneys, in which its antioxidant and anti-inflammatory

purposes. LDL, low density lipoprotein; LDL-ox, oxidized low density lipoprotein.

**Figure 3.** Main effects of hydrogen sulfide (H2S) in vascular system. 1—inhibition oxidation of low density lipoprotein; 2—diminution of monocyte aggregation in tunica intima; 3—inhibition of formation of spongy cells; 4—inhibition of proliferation and migration of vascular smooth muscular cells. The order of these effects was adopted for didactic

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

In recent years, it has been observed that glutathione peroxidase (GSH-PX), an endogenous antioxidant enzyme, attenuates the development of atherosclerosis in a similar action to H<sup>2</sup>

that is, reducing hydroperoxides [64]. In fact, when blocked, GSH-Px elevates oxidative stress in macrophages and increases ox-LDL activity. In addition, some elements when in non-physiological concentrations decrease GSH-PX activity, such as homocysteine [64]. Porter et al. [65] and Blann et al. [66] demonstrated that volunteers affected by atherosclerotic disease showed a reduction of approximately 29% of the peroxidase activity compared to healthy volunteers, demonstrating that the performance of this enzyme may be more related to pre-

**3.2. Effects of physical activity and physical exercise on oxidative stress: molecular** 

The antioxidant defense system has the function of inhibiting and/or reducing the damage caused by the action of free radicals. For this, the mechanisms of action may be the impediment in formation of free radicals or non-radical species (prevention systems), preventing the action of these molecules (sweep systems) or favoring the repair and reconstitution of damaged bio-

In response to the increased oxygen consumption that occurs in intense physical exercise, the reactive oxygen species (ROS) are generated by activating at least three main mechanisms: mitochondrial, cytoplasmic and favored production by iron and copper ions [68]. At the same time,

S,

33

effects are more evident [42].

logical structures [67].

vention than to the repair process after vascular disease.

**mechanisms and antioxidant effect of physical exercise**

Carballal et al. [62] in 2011 proposed that the antioxidant action of H2 S is minimal, or of no physiological significance. However, Filipovic et al. [61] demonstrated that, unlike the initial hypothesis, H2 S has a potent antioxidant and vasoprotective effect and is similar to glutathione.

It is also emphasized that all cells are susceptible to the action of reactive oxygen species, however, the lipid matrix of cell membranes is one of the most affected sites of these active species causing lipid peroxidation [63]. In this sense, although H2 S is able to reduce peroxynitrite molecules, its main and vasoprotective antioxidant action is to reduce lipid hydroperoxides by limiting the pathobiological potential for the development of vascular diseases through lipid peroxidation. The chemical representation can be seen below:

$$\text{LCOOH} + \text{H}\_2\text{S} + \text{O}\_2 \xrightarrow{} \text{LOH} + \text{SO}\_2 + \text{H}\_2\text{O} \tag{2}$$

**Figure 3.** Main effects of hydrogen sulfide (H2S) in vascular system. 1—inhibition oxidation of low density lipoprotein; 2—diminution of monocyte aggregation in tunica intima; 3—inhibition of formation of spongy cells; 4—inhibition of proliferation and migration of vascular smooth muscular cells. The order of these effects was adopted for didactic purposes. LDL, low density lipoprotein; LDL-ox, oxidized low density lipoprotein.

Another proposed antioxidant and vasoprotective mechanism involves the action of paraoxonases, universally accepted proteins as capable of protecting cells from oxidative stress [4]. The paraoxonase family includes paraoxonase-1, 2 and 3 (PON-1, PON-2 and PON-3), with PON-1 and PON-3 being found in plasma and directly associated with high density lipoprotein fractions (HDL) promoting action against the formation of reactive oxygen species, oxidation of low density lipoprotein (LDL) and macrophages leading to blockage or reduction of atherosclerotic lesions [4]. As for PON-2, it is a cell-associated complex, it is not found free in plasma, but in some tissues, especially in the kidneys, in which its antioxidant and anti-inflammatory effects are more evident [42].

Vasoprotection promoted by H2

32 Free Radicals, Antioxidants and Diseases

H2

molecule 1.

pexonitrite (ONOO<sup>−</sup>

hypothesis, H2

S involves a cascade of effects that culminate in the prevention

) and hydroxyl radical (OH<sup>−</sup>

) in a

S is minimal, or of no

S is able to reduce peroxynitrite

or deceleration of the atherosclerotic process once it has been initiated. Thus, the effects cascade is composed of: (1) inhibition of atherogenesis by modifying low density lipoprotein (LDL) molecules [51]; (2) inhibition of monocyte aggregation in endothelium [1, 52]; (3) inhibition of proliferation and migration of vascular smooth muscle cells in the atherosclerotic process once it is established in the vascular wall [53, 54]; (4) inhibition of the formation of spongy cells [55]; (5) angiogenesis [56]; (6) improvement of vasorelaxative mechanisms [57]; (7) reduction of vascular wall stiffening or calcification [58] and (8) prevention of platelet aggregation

**Figure 2.** Principal effects of ET-1 on the vascular system and surrounding tissues. VCAM-1, vascular cell adhesion

S demonstrates its antioxidant and, consequently, vasoprotective action when it reduces

H2 S + ONOO<sup>−</sup> HSNO2 + OH<sup>−</sup> (1)

physiological significance. However, Filipovic et al. [61] demonstrated that, unlike the initial

It is also emphasized that all cells are susceptible to the action of reactive oxygen species, however, the lipid matrix of cell membranes is one of the most affected sites of these active species

molecules, its main and vasoprotective antioxidant action is to reduce lipid hydroperoxides by limiting the pathobiological potential for the development of vascular diseases through

LOOH + H2 S + O2  LOH + SO2 + H2 O (2)

S has a potent antioxidant and vasoprotective effect and is similar to glutathione.

and thrombogenesis [59, 60]. Some of these effects can be visualized in **Figure 3**.

) molecules to nitrous acid (HSNO2

chemical representation, proposed by Filipovic et al. [61], as described below:

Carballal et al. [62] in 2011 proposed that the antioxidant action of H2

causing lipid peroxidation [63]. In this sense, although H2

lipid peroxidation. The chemical representation can be seen below:

In recent years, it has been observed that glutathione peroxidase (GSH-PX), an endogenous antioxidant enzyme, attenuates the development of atherosclerosis in a similar action to H<sup>2</sup> S, that is, reducing hydroperoxides [64]. In fact, when blocked, GSH-Px elevates oxidative stress in macrophages and increases ox-LDL activity. In addition, some elements when in non-physiological concentrations decrease GSH-PX activity, such as homocysteine [64]. Porter et al. [65] and Blann et al. [66] demonstrated that volunteers affected by atherosclerotic disease showed a reduction of approximately 29% of the peroxidase activity compared to healthy volunteers, demonstrating that the performance of this enzyme may be more related to prevention than to the repair process after vascular disease.

### **3.2. Effects of physical activity and physical exercise on oxidative stress: molecular mechanisms and antioxidant effect of physical exercise**

The antioxidant defense system has the function of inhibiting and/or reducing the damage caused by the action of free radicals. For this, the mechanisms of action may be the impediment in formation of free radicals or non-radical species (prevention systems), preventing the action of these molecules (sweep systems) or favoring the repair and reconstitution of damaged biological structures [67].

In response to the increased oxygen consumption that occurs in intense physical exercise, the reactive oxygen species (ROS) are generated by activating at least three main mechanisms: mitochondrial, cytoplasmic and favored production by iron and copper ions [68]. At the same time, physical exercise may also promote adaptation able to reduces the oxidative damage caused by the action of such agents. One such mechanism is the increased expression of the enzyme nitric oxide synthase (eNOS), through phosphorylation of residues by the proto-oncogenesis protein tyrosine kinase (c-SRC) and the activation of eNOS induced by other ROS in response by shear stress; while other mechanisms are triggered by oxidative stress, such as concomitant production of the enzyme superoxide dismutase (SOD), increased glutathione dismutase (GPx) activity, increased NO production induced by adenosine and the NO signaling pathway dependent on cyclic guanosine monophosphate (cGMP) (**Figure 4**). In addition, there is reduced mRNA expression and activity of pro-oxidant enzymes such as NADPH oxidase, angiotensin II receptor type I and increased expression of angiotensin II receptor type II in mammalian arteries [69].

hence the profile of the shear waveform, may activate highly different signaling pathways than do increases in average shear stress. More research is warranted to isolate the influence of shear patterns from other exercise-related signals to fully evaluate the hypothesis that exercise-induced acute changes in shear waveforms modulate endothelial health systemically with training [75]. Several considerations should be taken into account when viewing the hypothesis that shear stress is an exercise-induced signal for endothelial adaptations in nonworking tissues. Shear stress is directly related to blood flow and viscosity but inversely related to arterial diameter [76]. Given vascular tone (and hence diameter) is constantly regulated by central and local factors (e.g., shear stress), changes in blood flow through a given vessel do not always correspond with alterations in shear stress. In this regard, the extent to which enhanced blood flow or viscosity results in increased shear stress may be dependent on the caliber of the vessel and/or its ability to dilate in response to shear. Contrary to conduit arteries, given the remarkable capacity of arterioles to dilate and constrict, it is unclear to what degree changes in blood flow in the micro-

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

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35

Moreover, shear stress may not be the only hemodynamic exercise-induced signal for systemic endothelial adaptations. Endothelial cells are also exposed to stress from distention of arteries caused by relaxation of smooth muscle in the wall or by increased transmural pressure across the arterial wall. Since endothelial cells are exposed to cyclic distention within each cardiac cycle and during exercise the frequency and magnitude of this distention is augmented, cyclic strain should be considered as a potential exercise-induced signal. In this regard, Awolesi et al. [77] have shown that cyclic strain increases transcription of eNOS in cultured endothelial cells. Similarly, it is demonstrated that distention of isolated arteries is a

However, it is important to note that cyclic strain has also been associated with increased production of ROS and expression of adhesion molecules including vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin and monocyte chemoattractant protein-1 (MCP-1) [79]. Although chronic exposure of endothelial cells increasing cyclic strain (as occurs with hypertension) may produce negative adaptations, based on the classical physiological concept of hormesis, it is plausible that recurring periods of exerciseinduced cyclic strain and consequent oxidative stress may increase the tolerance of endothelial cells to withstand subsequent doses and hence stimulate a long-term protective effect [75].

In addition to evidence in the literature that exercise-induced adaptations of the endothelium result from increases in shear stress and/or cyclic strain [80], there is also growing evidence suggesting that changes in chemical signaling (i.e., hormones, cytokines and adipokines) may contribute to systemic benefits of chronic exercise on endothelial cells. Hemodynamic forces may interact with anti-atherogenic mediators such as insulin, adiponectin and IL-6 and with inflammatory cytokines (pro-atherogenic mediators) in the determination of endothelial cell phenotype/function. It appears that substances such as signal remodeling and altered phenotype of endothelial and smooth muscle cells are also released in response to increased shear stress [71].

Regarding the positive adaptations on coronary smooth muscle (CSM) by physical training, some other points need discussion. The beneficial adaptations of physical training can occur both at the sarcoplasmic level (rianodine-sensitive Ca2+ channels—RyR-) and sarcolemma

vasculature translate into alterations in shear [75].

stimulus for increased expression of the eNOS gene [78].

(voltage-dependent Ca2+ channels and K+ channels) [70].

Exercise-induced cardioprotection is probably a multifaceted phenomenon, with potential effector tissues including the myocardium, endothelial cells, inflammatory cells and coronary smooth muscle (CSM) [70]. Several mechanisms explain the positive effects of physical training regarding vascular adaptations [71].

The improvement of endothelial function by physical exercise is dependent on factors such as frequency and magnitude of physical exercise, which can cause shear stress and autoregulation of eNOS expression in endothelial cells [72]. Physical exercise results in increased heart rate and, consequently, increased blood flow and shear stress, which increases the activity of the early oncogenesis protein tyrosine kinase (c-Src) and increased eNOS production.

In the same sense, apparently the adaptations to physical exercise also occur in those vessels where there is no change in perfusion/blood flow during exercise [73]. It is important to highlight that, during exercise, the signal triggering endothelial adaptations in blood vessels perfusing tissues outside actively contracting muscle may not only be increased mean shear stress but also the alteration in shear profiles [74] that result from hemodynamic changes (e.g., heart rate and pressure) during exercise. It is suggested that alterations in the frequency of cyclic shear, and

**Figure 4.** Possible pathways of antioxidant effect occurring as a result of physical exercise.

hence the profile of the shear waveform, may activate highly different signaling pathways than do increases in average shear stress. More research is warranted to isolate the influence of shear patterns from other exercise-related signals to fully evaluate the hypothesis that exercise-induced acute changes in shear waveforms modulate endothelial health systemically with training [75].

physical exercise may also promote adaptation able to reduces the oxidative damage caused by the action of such agents. One such mechanism is the increased expression of the enzyme nitric oxide synthase (eNOS), through phosphorylation of residues by the proto-oncogenesis protein tyrosine kinase (c-SRC) and the activation of eNOS induced by other ROS in response by shear stress; while other mechanisms are triggered by oxidative stress, such as concomitant production of the enzyme superoxide dismutase (SOD), increased glutathione dismutase (GPx) activity, increased NO production induced by adenosine and the NO signaling pathway dependent on cyclic guanosine monophosphate (cGMP) (**Figure 4**). In addition, there is reduced mRNA expression and activity of pro-oxidant enzymes such as NADPH oxidase, angiotensin II receptor type I and increased expression of angiotensin II receptor type II in mammalian arteries [69]. Exercise-induced cardioprotection is probably a multifaceted phenomenon, with potential effector tissues including the myocardium, endothelial cells, inflammatory cells and coronary smooth muscle (CSM) [70]. Several mechanisms explain the positive effects of physical train-

The improvement of endothelial function by physical exercise is dependent on factors such as frequency and magnitude of physical exercise, which can cause shear stress and autoregulation of eNOS expression in endothelial cells [72]. Physical exercise results in increased heart rate and, consequently, increased blood flow and shear stress, which increases the activity of

In the same sense, apparently the adaptations to physical exercise also occur in those vessels where there is no change in perfusion/blood flow during exercise [73]. It is important to highlight that, during exercise, the signal triggering endothelial adaptations in blood vessels perfusing tissues outside actively contracting muscle may not only be increased mean shear stress but also the alteration in shear profiles [74] that result from hemodynamic changes (e.g., heart rate and pressure) during exercise. It is suggested that alterations in the frequency of cyclic shear, and

the early oncogenesis protein tyrosine kinase (c-Src) and increased eNOS production.

**Figure 4.** Possible pathways of antioxidant effect occurring as a result of physical exercise.

ing regarding vascular adaptations [71].

34 Free Radicals, Antioxidants and Diseases

Several considerations should be taken into account when viewing the hypothesis that shear stress is an exercise-induced signal for endothelial adaptations in nonworking tissues. Shear stress is directly related to blood flow and viscosity but inversely related to arterial diameter [76]. Given vascular tone (and hence diameter) is constantly regulated by central and local factors (e.g., shear stress), changes in blood flow through a given vessel do not always correspond with alterations in shear stress. In this regard, the extent to which enhanced blood flow or viscosity results in increased shear stress may be dependent on the caliber of the vessel and/or its ability to dilate in response to shear. Contrary to conduit arteries, given the remarkable capacity of arterioles to dilate and constrict, it is unclear to what degree changes in blood flow in the microvasculature translate into alterations in shear [75].

Moreover, shear stress may not be the only hemodynamic exercise-induced signal for systemic endothelial adaptations. Endothelial cells are also exposed to stress from distention of arteries caused by relaxation of smooth muscle in the wall or by increased transmural pressure across the arterial wall. Since endothelial cells are exposed to cyclic distention within each cardiac cycle and during exercise the frequency and magnitude of this distention is augmented, cyclic strain should be considered as a potential exercise-induced signal. In this regard, Awolesi et al. [77] have shown that cyclic strain increases transcription of eNOS in cultured endothelial cells. Similarly, it is demonstrated that distention of isolated arteries is a stimulus for increased expression of the eNOS gene [78].

However, it is important to note that cyclic strain has also been associated with increased production of ROS and expression of adhesion molecules including vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin and monocyte chemoattractant protein-1 (MCP-1) [79]. Although chronic exposure of endothelial cells increasing cyclic strain (as occurs with hypertension) may produce negative adaptations, based on the classical physiological concept of hormesis, it is plausible that recurring periods of exerciseinduced cyclic strain and consequent oxidative stress may increase the tolerance of endothelial cells to withstand subsequent doses and hence stimulate a long-term protective effect [75].

In addition to evidence in the literature that exercise-induced adaptations of the endothelium result from increases in shear stress and/or cyclic strain [80], there is also growing evidence suggesting that changes in chemical signaling (i.e., hormones, cytokines and adipokines) may contribute to systemic benefits of chronic exercise on endothelial cells. Hemodynamic forces may interact with anti-atherogenic mediators such as insulin, adiponectin and IL-6 and with inflammatory cytokines (pro-atherogenic mediators) in the determination of endothelial cell phenotype/function. It appears that substances such as signal remodeling and altered phenotype of endothelial and smooth muscle cells are also released in response to increased shear stress [71].

Regarding the positive adaptations on coronary smooth muscle (CSM) by physical training, some other points need discussion. The beneficial adaptations of physical training can occur both at the sarcoplasmic level (rianodine-sensitive Ca2+ channels—RyR-) and sarcolemma (voltage-dependent Ca2+ channels and K+ channels) [70].

A study by Newcomer et al. [78] demonstrated a postdepolarization, time-dependent decline in the caffeine-releasable SR Ca2+ store in cells from exercise-trained animals, but not sedentary control subjects. This phenomenon (termed SR Ca2+ unloading) was further determined to result from a slow release of SR Ca2+ via RyR. Of noteworth is that the Ca2+ released through SR Ca2+ unloading seems to be extruded from the cell, not resequestered by the SR or other organelles, and was demonstrated to occur with no increase in bulk Cam (myoplasmic free Ca2+ concentration) [81]. It was concluded that the net effect of this training-induced SR Ca2+ unloading would be a lower SR Ca2+ content and an increased subsarcolemmal Ca2+ gradient, with no effect on bulk Cam. In this way, it was proposed that a lower SR Ca2+ content caused by SR Ca2+ unloading may contribute to attenuated contractile responses to vasoactive agonists in the exercise trained state because of both diminished SR Ca2+ release and increased SR buffering of influx Ca2+.

guanosine monophosphate (cGMP), refers to endothelial cells that present mechanoreceptors. These receptors directly activate G-proteins, enzymes and other protein kinases that generate a second messenger, such as cGMP, which leads to vasodilation. Another mechanism increased NO production stimulated by adenosine. Studies have shown that erythrocyte membranes tend to release ATP in response to shear stress and that during the strenuous physical exercise in the

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

37

Despite the exhaustive studies, there are controversies regarding the antioxidant effect of physical exercise related to issues such as eNOS measurement after a training period, eNOS status in the animal and human baseline, and the existence of polymorphisms in the gene promoter of eNOS [83]. It has been shown that eNOS activation induced by shear stress does not depend on the increase of intracellular calcium, but on enzyme phosphorylation [85]. This post-transcription modification occurs at serine 1177 and is mediated by the serine/threonine protein kinase Akt (protein kinase B) [86]. This alters the sensitivity of the enzyme to Ca2+, making its activity maximal at subphysiological concentrations of Ca2+. In the presence of Ca2+/calmodulin, the serine 1177-mediated eNOS phosphorylation occurs in the skeletal and cardiac muscle of rats by the activation of AMPK (activated protein kinase), an enzyme activated by vigorous exercise and ischemic stress [87]. Boo et al. [88] suggested that a coordinated interaction between Akt and PKA may be an important mechanism by regulating eNOS activity in response to shear stress. These results are confirmed in humans with coronary artery disease who underwent 4 weeks of supervised physical exercise training lasting 60 min/day. The increase in the levels of phosphorylation of eNOS-mediated enzyme ser-1177 increased fourfold in the left mammary artery region. This was associated with a two-fold increase in eNOS and a significant increase in endothelium-dependent vasodilation in this artery [89]. Together, the current evidence suggests that phosphorylation induced by shear stress caused by physical exercise contributes to the improvement of endothelium-dependent vasodilation. The acute effects of aerobic and anaerobic physical exercise are related to the increase of vascular oxidative stress and damages to lipid cells, nucleic acids and the glutathione system (GSH). Very intense physical exercise for 4 weeks may induce increased plasmatic activity of the glutathione peroxidase enzyme (GPx) and decrease of antioxidant substances in the resting plasma, in the pre-exercise period, and mainly, in the post-exercise period and accompanied by a reduced glutathione (GSH) and oxidized glutathione (GSSG, GSH/GSSG) and an increase in plasma thiobarbituric acid reactive substances (TBARS) [90]. Thus, acute periods of exhaustive training may decrease the antioxidative capacity of tissues, such as skeletal muscle and vascular cells [83]. On the other hand, adaptations to moderate exercise appear to occur after a few weeks of training; in fact, endurance training has been shown to be able to reduce oxidative stress, such as lipid peroxidation in membrane erythrocytes, when compared to exhaustive exercise in young men trained [91]. The increase in eNOS expression by physical exercise is followed by increased expression of SOD3 [84]. Self-regulation of SOD by physical exercise not only provides efficient detoxification of superoxide but also reduces the generation of peroxynitrite, a strong oxidant with important pathophysiological effects [92]. While manganese protein levels, superoxide dismutase (SOD2) was not altered, levels of the p67phox protein, a subunit of the pro-oxidant enzyme NADPH oxidase, were reduced by physical training [93]. These observations demonstrate that the antioxidant effects of physical exercise can not only be mediated by increased expression of antioxidant enzymes, but also

cincunflex artery, there is NO production stimulated by adenosine [84].

by reduced expression of pro-oxidant enzymes [83].

L-type Ca2+ channels are associated with endothelin response, so they may be associated with changes due to physical exercise. It is known that endurance training increases L-type Ca2+ channel current density approximately twofold in all three arterial sizes, with no effect on voltage-dependent activation or inactivation characteristics [81]. Additionally, a significant correlation between treadmill endurance time and peak L type Ca2+ current density was demonstrated in all three arterial sizes, supporting a direct association between endurance capacity and coronary smooth muscle L-type Ca2+ current density. The increase in L-type Ca2+ current density could result from an increase in the number of L-type Ca2+ channels in the sarcolemmal membrane and/or increased activity of existing channels. Future studies will be necessary to determine the basis for this training-induced adaptation.

Another channels that plays major role in control of smooth muscle tone are K+ channels, by determining Cam, through regulation of membrane potential (Vm) and voltage-gated Ca2+ channel activity. Activation of K+ channels produces membrane hyperpolarization, acting as a negative feedback on voltage-gated Ca2+ channel activation to limit contraction or produce vasodilation. Various K+ channels are expressed in vascular smooth muscle, including largeconductance, Ca2+-activated K+ channels (KCa), voltage-dependent K+ channels (Kv ), inward rectifier K<sup>+</sup> channels (Kir) and ATP sensitive K+ channels (KATP).

Preliminary research [82] indicates that exercise training increases the relative contribution of both KCa and Kv channels to regulate basal tone of coronary arteries. Thus, K+ channels play a greater role in regulating basal coronary tone in the exercise-trained state. Interestingly, it is concluded that stretch is a requisite factor for expression of this training-induced adaptation because KCa and Kv , currents, determined by voltage-clamp in enzymatically isolated smooth muscle cells, exhibited no difference in current density in cells from exercise trained and sedentary groups.

An intense physical exercise session can generate large amounts of reactive oxygen species, which increases oxidative stress and superoxide production (O2 •−) [83]. The superoxide radical is highly reactive, however, it crosses with difficulty the plasma membrane, being converted quickly to hydrogen peroxide (H2 O2 ) by the enzyme superoxide dismutase (SOD), whose concentration also increases with physical exercise [84]. Hydrogen peroxide (H2 O2 ) is catalyzed by the enzyme glutathione oxidase (GPX), which reduces it to two molecules of water, reducing the oxidant damage of the superoxide radical.

The antioxidant mechanisms induced by shear stress are not yet completely clarified by the literature, so other mechanisms are studied [83]. The mechanism of NO signaling dependent on cyclic guanosine monophosphate (cGMP), refers to endothelial cells that present mechanoreceptors. These receptors directly activate G-proteins, enzymes and other protein kinases that generate a second messenger, such as cGMP, which leads to vasodilation. Another mechanism increased NO production stimulated by adenosine. Studies have shown that erythrocyte membranes tend to release ATP in response to shear stress and that during the strenuous physical exercise in the cincunflex artery, there is NO production stimulated by adenosine [84].

A study by Newcomer et al. [78] demonstrated a postdepolarization, time-dependent decline in the caffeine-releasable SR Ca2+ store in cells from exercise-trained animals, but not sedentary control subjects. This phenomenon (termed SR Ca2+ unloading) was further determined to result from a slow release of SR Ca2+ via RyR. Of noteworth is that the Ca2+ released through SR Ca2+ unloading seems to be extruded from the cell, not resequestered by the SR or other organelles, and was demonstrated to occur with no increase in bulk Cam (myoplasmic free Ca2+ concentration) [81]. It was concluded that the net effect of this training-induced SR Ca2+ unloading would be a lower SR Ca2+ content and an increased subsarcolemmal Ca2+ gradient, with no effect on bulk Cam. In this way, it was proposed that a lower SR Ca2+ content caused by SR Ca2+ unloading may contribute to attenuated contractile responses to vasoactive agonists in the exercise trained state because of both diminished SR Ca2+ release and increased SR buffering of influx Ca2+.

L-type Ca2+ channels are associated with endothelin response, so they may be associated with changes due to physical exercise. It is known that endurance training increases L-type Ca2+ channel current density approximately twofold in all three arterial sizes, with no effect on voltage-dependent activation or inactivation characteristics [81]. Additionally, a significant correlation between treadmill endurance time and peak L type Ca2+ current density was demonstrated in all three arterial sizes, supporting a direct association between endurance capacity and coronary smooth muscle L-type Ca2+ current density. The increase in L-type Ca2+ current density could result from an increase in the number of L-type Ca2+ channels in the sarcolemmal membrane and/or increased activity of existing channels. Future studies will be

necessary to determine the basis for this training-induced adaptation.

channels (Kir) and ATP sensitive K+

which increases oxidative stress and superoxide production (O2

water, reducing the oxidant damage of the superoxide radical.

verted quickly to hydrogen peroxide (H2

channel activity. Activation of K+

conductance, Ca2+-activated K+

vasodilation. Various K+

36 Free Radicals, Antioxidants and Diseases

rectifier K<sup>+</sup>

KCa and Kv

and Kv

Another channels that plays major role in control of smooth muscle tone are K+

channels to regulate basal tone of coronary arteries. Thus, K+

by determining Cam, through regulation of membrane potential (Vm) and voltage-gated Ca2+

a negative feedback on voltage-gated Ca2+ channel activation to limit contraction or produce

Preliminary research [82] indicates that exercise training increases the relative contribution of both

role in regulating basal coronary tone in the exercise-trained state. Interestingly, it is concluded that stretch is a requisite factor for expression of this training-induced adaptation because KCa

exhibited no difference in current density in cells from exercise trained and sedentary groups.

An intense physical exercise session can generate large amounts of reactive oxygen species,

cal is highly reactive, however, it crosses with difficulty the plasma membrane, being con-

is catalyzed by the enzyme glutathione oxidase (GPX), which reduces it to two molecules of

The antioxidant mechanisms induced by shear stress are not yet completely clarified by the literature, so other mechanisms are studied [83]. The mechanism of NO signaling dependent on cyclic

O2

whose concentration also increases with physical exercise [84]. Hydrogen peroxide (H2

channels (KCa), voltage-dependent K+

, currents, determined by voltage-clamp in enzymatically isolated smooth muscle cells,

channels (KATP).

channels produces membrane hyperpolarization, acting as

channels are expressed in vascular smooth muscle, including large-

channels,

), inward

O2 )

channels (Kv

channels play a greater

•−) [83]. The superoxide radi-

) by the enzyme superoxide dismutase (SOD),

Despite the exhaustive studies, there are controversies regarding the antioxidant effect of physical exercise related to issues such as eNOS measurement after a training period, eNOS status in the animal and human baseline, and the existence of polymorphisms in the gene promoter of eNOS [83]. It has been shown that eNOS activation induced by shear stress does not depend on the increase of intracellular calcium, but on enzyme phosphorylation [85]. This post-transcription modification occurs at serine 1177 and is mediated by the serine/threonine protein kinase Akt (protein kinase B) [86]. This alters the sensitivity of the enzyme to Ca2+, making its activity maximal at subphysiological concentrations of Ca2+. In the presence of Ca2+/calmodulin, the serine 1177-mediated eNOS phosphorylation occurs in the skeletal and cardiac muscle of rats by the activation of AMPK (activated protein kinase), an enzyme activated by vigorous exercise and ischemic stress [87]. Boo et al. [88] suggested that a coordinated interaction between Akt and PKA may be an important mechanism by regulating eNOS activity in response to shear stress. These results are confirmed in humans with coronary artery disease who underwent 4 weeks of supervised physical exercise training lasting 60 min/day. The increase in the levels of phosphorylation of eNOS-mediated enzyme ser-1177 increased fourfold in the left mammary artery region. This was associated with a two-fold increase in eNOS and a significant increase in endothelium-dependent vasodilation in this artery [89]. Together, the current evidence suggests that phosphorylation induced by shear stress caused by physical exercise contributes to the improvement of endothelium-dependent vasodilation.

The acute effects of aerobic and anaerobic physical exercise are related to the increase of vascular oxidative stress and damages to lipid cells, nucleic acids and the glutathione system (GSH). Very intense physical exercise for 4 weeks may induce increased plasmatic activity of the glutathione peroxidase enzyme (GPx) and decrease of antioxidant substances in the resting plasma, in the pre-exercise period, and mainly, in the post-exercise period and accompanied by a reduced glutathione (GSH) and oxidized glutathione (GSSG, GSH/GSSG) and an increase in plasma thiobarbituric acid reactive substances (TBARS) [90]. Thus, acute periods of exhaustive training may decrease the antioxidative capacity of tissues, such as skeletal muscle and vascular cells [83]. On the other hand, adaptations to moderate exercise appear to occur after a few weeks of training; in fact, endurance training has been shown to be able to reduce oxidative stress, such as lipid peroxidation in membrane erythrocytes, when compared to exhaustive exercise in young men trained [91]. The increase in eNOS expression by physical exercise is followed by increased expression of SOD3 [84]. Self-regulation of SOD by physical exercise not only provides efficient detoxification of superoxide but also reduces the generation of peroxynitrite, a strong oxidant with important pathophysiological effects [92]. While manganese protein levels, superoxide dismutase (SOD2) was not altered, levels of the p67phox protein, a subunit of the pro-oxidant enzyme NADPH oxidase, were reduced by physical training [93]. These observations demonstrate that the antioxidant effects of physical exercise can not only be mediated by increased expression of antioxidant enzymes, but also by reduced expression of pro-oxidant enzymes [83].


A review with studies that verified the influence of physical exercise on oxidative stress is presented in **Table 2**. Overall, human studies with interventions ranging from 4 weeks to 12 months, either with strength training [91, 94, 95] or aerobic training of moderate to vigorous intensity [94, 96–99] have demonstrated an improvement in antioxidant capacity by increasing SOD, CAT, GPx, total antioxidant capacity (TAC) and/or decrease of malondialdehyde (MDA). Only one study that demonstrated the acute effect of horse racing did not show significant changes in GPx, SOD, GSSG and glutathione redox ratio (GRR) [100]. In diabetic rats that performed moderate intensity exercise also did not significantly alter SOD, GPx and

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

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39

Considering that the acute effects of vigorous physical exercise are related to the increase in oxidative stress, while the chronic effects of training with moderate exercise can favor changes in gene expression and increase of the antioxidant effect, it is possible to speculate that the antioxidant effect of physical exercise is dependent of the occurrence of oxidative stress in an intermittent way [83]. Briefly, physical exercise may, in the medium term (about 3 weeks, for example) increase vascular hydrogen peroxide and, consequently, eNOS expression [83, 87]. It is possible that physical exercise training in the medium term reduces oxidative stress by the measurement of lipid peroxidation in the erythrocyte membrane in response to strenuous exercise in young, untrained males [91]. Furthermore, eNOS activity was shown to be a crucial factor for vascular expression of the antioxidant enzyme SOD3, and 4 weeks of physical training reduced the expression of potentially pro-oxidant proteins, such as NADPH oxidase and type 1 angiotensin II receptor, while the expression of vascular antioxidant proteins such as angiotensin II receptor type 2 is reduced [104]. Additionally, the potentially beneficial effects of exercise and/or regular physical activity as increased eNOS expression is reversible

Thus, regular physical exercise becomes beneficial for healthy people and patients with cardiovascular disease. While exercise training may hinder the development of pro-oxidative vascular gene expression associated with endothelial dysfunction in individuals, it corrects and/or improves already established endothelial dysfunction and increased vascular oxidative stress in cardiovascular diseases such as hypertension, diabetes, encephalic stroke, coro-

The present chapter demonstrates that the oxidative stress is heavily involved in most of vascular diseases considering your effect for elevated the aggregation of monocytes in endothelium, low density lipoprotein oxidation, proliferation of vascular smooth muscular cell, among others. On the other hand, endogenous enzymes or compounds like glutathione peroxidase and sulfide hydrogen have the antagonistic effects like inhibition of the pathophysiological processes involved in vascular diseases. Therefore, although the physical exercise be able to elevated the concentration of reactive oxygen species after your practice, is be able to promoted the elevation in the production and secretion of antioxidants like superoxide dismutase, resulting in scavenging effects, responsible for the defenses against the development of the atherosclerotic process, present in the most of the vascular diseases can be observed.

oxygen radical absorbance capacity (ORAC) [101] or increased MDA [102].

by a sedentary lifestyle as induced by forced physical inactivity [105].

nary artery disease and heart failure [83, 103, 106].

**4. Conclusion**

**Table 2.** Review of articles about effects of physical exercise and oxidative stress in humans and animals.

A review with studies that verified the influence of physical exercise on oxidative stress is presented in **Table 2**. Overall, human studies with interventions ranging from 4 weeks to 12 months, either with strength training [91, 94, 95] or aerobic training of moderate to vigorous intensity [94, 96–99] have demonstrated an improvement in antioxidant capacity by increasing SOD, CAT, GPx, total antioxidant capacity (TAC) and/or decrease of malondialdehyde (MDA). Only one study that demonstrated the acute effect of horse racing did not show significant changes in GPx, SOD, GSSG and glutathione redox ratio (GRR) [100]. In diabetic rats that performed moderate intensity exercise also did not significantly alter SOD, GPx and oxygen radical absorbance capacity (ORAC) [101] or increased MDA [102].

Considering that the acute effects of vigorous physical exercise are related to the increase in oxidative stress, while the chronic effects of training with moderate exercise can favor changes in gene expression and increase of the antioxidant effect, it is possible to speculate that the antioxidant effect of physical exercise is dependent of the occurrence of oxidative stress in an intermittent way [83]. Briefly, physical exercise may, in the medium term (about 3 weeks, for example) increase vascular hydrogen peroxide and, consequently, eNOS expression [83, 87]. It is possible that physical exercise training in the medium term reduces oxidative stress by the measurement of lipid peroxidation in the erythrocyte membrane in response to strenuous exercise in young, untrained males [91]. Furthermore, eNOS activity was shown to be a crucial factor for vascular expression of the antioxidant enzyme SOD3, and 4 weeks of physical training reduced the expression of potentially pro-oxidant proteins, such as NADPH oxidase and type 1 angiotensin II receptor, while the expression of vascular antioxidant proteins such as angiotensin II receptor type 2 is reduced [104]. Additionally, the potentially beneficial effects of exercise and/or regular physical activity as increased eNOS expression is reversible by a sedentary lifestyle as induced by forced physical inactivity [105].

Thus, regular physical exercise becomes beneficial for healthy people and patients with cardiovascular disease. While exercise training may hinder the development of pro-oxidative vascular gene expression associated with endothelial dysfunction in individuals, it corrects and/or improves already established endothelial dysfunction and increased vascular oxidative stress in cardiovascular diseases such as hypertension, diabetes, encephalic stroke, coronary artery disease and heart failure [83, 103, 106].

### **4. Conclusion**

**Table 2.** Review of articles about effects of physical exercise and oxidative stress in humans and animals.

38 Free Radicals, Antioxidants and Diseases

The present chapter demonstrates that the oxidative stress is heavily involved in most of vascular diseases considering your effect for elevated the aggregation of monocytes in endothelium, low density lipoprotein oxidation, proliferation of vascular smooth muscular cell, among others. On the other hand, endogenous enzymes or compounds like glutathione peroxidase and sulfide hydrogen have the antagonistic effects like inhibition of the pathophysiological processes involved in vascular diseases. Therefore, although the physical exercise be able to elevated the concentration of reactive oxygen species after your practice, is be able to promoted the elevation in the production and secretion of antioxidants like superoxide dismutase, resulting in scavenging effects, responsible for the defenses against the development of the atherosclerotic process, present in the most of the vascular diseases can be observed.

### **Conflict of interest**

There is no conflict of interest.

### **Author details**

Aline de Freitas Brito1,2\*, Caio Victor Coutinho de Oliveira3 , Glêbia Alexa Cardoso2 , Joana Marcela Sales de Lucena3 , Jordanna di Paula dos Santos Sousa5 and Alesandra Araújo de Souza4

[8] Virdis A, Neves MF, Duranti E, Bernini G, Taddei S. Microvascular endothelial dysfunction in obesity and hypertension. Current Pharmaceutical Design. 2013;**19**(13):2382-2389

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

41

[9] Durgin BG, Straub AC. Redox control of vascular smooth muscle cell function and plas-

[10] Davignon J. Beneficial cardiovascular pleiotropic effects of statins. Circulation. 2004;

[11] Liao JK, Laufs U. Pleiotropic effects of statins. Annual Review of Pharmacology and

[12] Korsager Larsen M, Matchkov VV. Hypertension and physical exercise: The role of oxidative stress. Medicina (Kaunas, Lithuania). 2016;**52**(1):19-27. DOI: 10.1016/j.medici.

[13] Boccatonda A, Tripaldi R, Davì G, Santilli F. Oxidative stress modulation through habitual physical activity. Current Pharmaceutical Design. 2016;**22**(24):3648-3680

[14] Xu S, Touyz RM. Reactive oxygen species and vascular remodelling in hypertension:

[15] Powers SK, Jackson MJ. Exercise-induced oxidative stress: Cellular mechanisms and impact on muscle force production. Physiological Reviews. 2008;**88**(4):1243-1276 [16] Giulivi C. Functional implications of nitric oxide produced by mitochondria in mito-

[17] Bedard K, Krause H. The NOX family of ROS-generating NADPH oxidases: Physiology

[18] Schramm A, Matusik P, Osmenda G, Guzik TJ. Targeting NADPH oxidases in vascular

[19] Lassègue B, Clempus RE. Vascular NAD(P)H oxidases: Specific features, expression, and regulation. American Journal of Physiology. Regulatory, Integrative and Comparative

[20] Papaharalambus CA, Griendling KK. Basic mechanisms of oxidative stress and reactive oxygen species in cardiovascular injury. Trends in Cardiovascular Medicine. 2007;

[21] Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM. Mechanisms of increased vascular superoxide production in human diabetes mellitus: Role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002;

[22] Antoniades C, Shirodaria C, Leeson P, Antonopoulos A, Warrick N, Van-Assche T, Cunnington C, Tousoulis D, Pillai R, Ratnatunga C, Stefanadis C, Channon KM.Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: Implications for endothelial function in human atherosclerosis. European Heart Journal. 2009;**30**:1142-1150.

Still alive. Canadian Journal of Cardiology. 2006;**22**(11):947-951

chondrial metabolism. Biochemical Journal. 1998;**332**(3):673-679

and pathophysiology, Physiological Reviews. 2007;**87**:245-313

pharmacology. Vascular Pharmacology. 2012;**56**(5-6):216-231

Physiology. 2003;**285**(2):R277-R297

DOI: 10.1093/eurheartj/ehp061

ticity. Laboratory Investigation. 2018. DOI: 10.1038/s41374-018-0032-9

**109**(23):III39-III43

2016.01.005

**17**(2):48-54

**105**:1656-1662

Toxicology. 2005;**45**(1):89-118

\*Address all correspondence to: alineebritoo@gmail.com

1 School of Physical Education, University of Pernambuco, Recife, Brazil

2 Post-Graduation Program in Physical Education UPE/UFPB, João Pessoa, Brazil

3 Faculties of Medical Sciences, University Center Unifacisa, Campina Grande, Paraíba, Brazil

4 Licentiate in Physical Education, Federal University of Tocantins, Tocantinopolis, Tocantins, Brazil

5 Pharmacy School, Santo Angostinho University Center, Teresina, Piauí, Brazil

### **References**


[8] Virdis A, Neves MF, Duranti E, Bernini G, Taddei S. Microvascular endothelial dysfunction in obesity and hypertension. Current Pharmaceutical Design. 2013;**19**(13):2382-2389

**Conflict of interest**

40 Free Radicals, Antioxidants and Diseases

**Author details**

Brazil

Tocantins, Brazil

**References**

There is no conflict of interest.

Joana Marcela Sales de Lucena3

Alesandra Araújo de Souza4

Aline de Freitas Brito1,2\*, Caio Victor Coutinho de Oliveira3

\*Address all correspondence to: alineebritoo@gmail.com

Radical Biology & Medicine. 2011;**51**(5):978-992

2017;**116**:57-69. DOI: 10.1016/j.phrs.2016.12.017

Hypertension Research. 2012;**35**(11):1039-1047

Chemistry. 2008;**46**:1-50

s11906-010-0150-2

1 School of Physical Education, University of Pernambuco, Recife, Brazil

2 Post-Graduation Program in Physical Education UPE/UFPB, João Pessoa, Brazil

4 Licentiate in Physical Education, Federal University of Tocantins, Tocantinopolis,

5 Pharmacy School, Santo Angostinho University Center, Teresina, Piauí, Brazil

3 Faculties of Medical Sciences, University Center Unifacisa, Campina Grande, Paraíba,

[1] Sugamura K, Keaney JF Jr. Reactive oxygen species in cardiovascular disease. Free

[2] Bloomer RJ. Effect of exercise on oxidative stress biomarkers. Advances in Clinical

[3] Ritchie RH, Drummond GR, Sobey CG, De Silva TM, Kemp-Harper BK. The opposing roles of NO and oxidative stress in cardiovascular disease. Pharmacological Research.

[4] Dharmashankar K, Widlansky ME. Vascular endothelial function and hypertension: Insights and directions. Current Hypertension Reports. 2010;**12**(6):448-455. DOI: 10.1007/

[5] Weseler AR, Bast A. Oxidative stress and vascular function: Implications for pharmaco-

[6] Virdis A, Taddei S. How to evaluate microvascular organ damage in hypertension: Assessment of endothelial function. High Blood Pressure & Cardiovascular Prevention.

[7] Higashi Y, Kihara Y, Noma K. Endothelial dysfunction and hypertension in aging.

logic treatments. Current Hypertension Reports. 2010;**12**(3):154-161

2011;**18**(4):163-167. DOI: 10.2165/11593630-000000000-00000

, Glêbia Alexa Cardoso2

and

, Jordanna di Paula dos Santos Sousa5

,


[23] Mcnally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalo S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. American Journal of Physiology. Heart and Circulatory Physiology. 2003;**285**:2290-2297

[37] Majzunova M, Dovinova I, Barancik M, Chan JYH. Redox signaling in pathophysiology

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

43

[38] Lemkens P, Nelissen J, Meens MJPMT, Schiffers PMH, De Mey JGR. Dual neural peptidase/endothelin-converting enzyme inhibition improves endothelial function in mesenteric resistance arteries of young spontaneously hypertensive rats. Journal of

[39] Poole KM, Nelson CE, Joshi RV, Martin JR, Gupta MK, Haws SC, Kavanaugh TE, Skala1 MC, Duvall CL. ROS-responsive microspheres for on demand antioxidant therapy in a

[40] Szabo C. Hydrogen sulphide and its therapeutic potential. Nature Reviews. Drug

[41] Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, Snyder SH, Wang R. H2S as a physiologic vasorelaxant: Hypertension inmice with dele-

[42] Borovkova EI, Antipova NV, Komeenko TV, Shakhparonov MI, Borovkov IM. Paraoxonase: The universal factor of antioxidant Defense in human body. Vestnik Rossiĭskoĭ Akademii

[43] Maddipati KR, Marnett LJ. Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione

[44] Mustafa AK, Sikka G, Gazi SK, Steppan J, Jung SM, Bhunia AK, Barodka VM, Gazi FK, Barrow RK, Wang R, Amzel LM, Berkowitz DE, Snyder SH. Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels. Circulation

[45] Moccia F, Bertoni G, Pla AF, Dragoni S, Pupo E, Merlino A, Mancardi D, Munaron L, Tanzi F. Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells

from excised rat aorta. Current Pharmaceutical Biotechnology. 2011;**12**:1416-1426 [46] Bauer CC, Boyle JP, Porter KE, Peers C. Modulation of Ca(2+) signalling in human vascu-

[47] Pupo E, Pla AF, Avanzato D, Moccia F, Cruz JE, Tanzi F, Merlino A, Mancardi D, Munaron L. Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothe-

[48] Zhang D, Macinkovic I, Devarie-Baez NO, Pan J, Park CM, Carroll KS, Filipovic MR, Xian M. Detection of protein S-sulfhydration by a tag-switch technique. Angewandte Chemie (International Ed. in English). 2014;**53**:575-581. DOI: 10.1002/anie.201305876 [49] Park CM, Macinkovic I, Filipovic MR, Xian M. Use of the "tag-switch" method for the detection of protein S-sulfhydration. Methods in Enzymology. 2015;**555**:39-56

[50] Yu Q, Lu Z, Tao L, Yang L, Guo Y, Yang Y, Sun X, Ding Q. ROS-dependent neuroprotective effects of NaHS in ischemia brain injury involves the PARP/AIF pathway. Cellular

lar endothelial cells by hydrogen sulfide. Atherosclerosis. 2010;**209**:374-380

lial cells. Free Radical Biology & Medicine. 2011;**51**:1765-1773

Physiology and Biochemistry. 2015;**36**:1539-1551

model of diabetic peripheral arterial disease. Biomaterials. 2015;**41**:166-175

tion of cystathionine gamma-lyase. Science. 2008;**322**(5901):587-590

peroxidase. The Journal of Biological Chemistry. 1987;**262**(17):398-403

Meditsinskikh Nauk. 2017;**72**(1):5-10. DOI: 10.15690/vramn764

of hypertension. Journal of Biomedical Science. 2013;**20**(1):69

Hypertension. 2012;**30**(9):1799-1808

Discovery. 2007;**6**:917-935

Research. 2011;**109**:1259-1268


[37] Majzunova M, Dovinova I, Barancik M, Chan JYH. Redox signaling in pathophysiology of hypertension. Journal of Biomedical Science. 2013;**20**(1):69

[23] Mcnally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalo S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. American Journal of Physiology. Heart and

[24] Rodrigo R, González J, Paoletto F. The role of oxidative stress in the pathophysiology of

[25] Kedziora-Kornatowska K, Czuczejko J, Pawluk H, Kornatowski T, Motyl J, Szadujkis-Szadurski L, Szewczyk-Golec K, Kedziora J. The markers of oxidative stress and activity of the antioxidant system in the blood of elderly patients with essential arterial hyper-

[26] Hage FG, Oparil S, Xing D, Chen YF, McCrory MA, Szalai AJ. C-reactive protein-mediated vascular injury requires complement. Arteriosclerosis, Thrombosis, and Vascular Biology.

[27] Touyz RM, Schiffrin EL. Reactive oxygen species in vascular biology: Implications in

[28] Vecchione C, Carnevale D, Di Pardo A, Gentile MT, Damato A, Cocozza G, Antenucci G, Mascio G, Bettarini U, Landolfi A, Iorio L, Maffei A, Lembo G. Pressure-induced vascular oxidative stress is mediated through activation of integrin-linked kinase 1/betaPIX/

[29] Widder JD, Fraccarollo D, Galuppo P, Hansen JM, Jones DP, Ertl G, Bauersachs J. Attenuation of angiotensin II-induced vascular dysfunction and hypertension by overex-

[30] Hawa NS, Yusof K, Kamsiah J. The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review). Vascular Pharmacology. 2015;**40-56**

[31] Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury part 1: Basic mechanisms and in vivo monitoring of ROS. Circulation. 2003;**108**:1912-1916

[32] Libby P. Mechanisms of acute coronary syndromes and their implications for therapy. The New England Journal of Medicine. 2013;**368**:2004-2013. DOI: 10.1056/NEJMra1216063

[33] Xu S, Bai P, Little PJ, Liu P. Poly(ADP-ribose) Polymerase-1 (PARP1) in atherosclerosis: From molecular mechanisms to therapeutic implications. Medicinal Research Reviews.

[34] Sies H. Oxidative stress: Oxidants and antioxidants. Experimental Physiology. 1997;

[35] Vaziri ND. Causal link between oxidative stress, inflammation, and hypertension.

[36] Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: The role of

Circulatory Physiology. 2003;**285**:2290-2297

42 Free Radicals, Antioxidants and Diseases

hypertension. Hypertension Research. 2010;**34**(4):431-440

2010;**30**:1189-1195. DOI: 10.1161/ATVBAHA.110.205377

Rac-1 pathway. Hypertension. 2009;**54**:1028-1034

Iranian Journal of Kidney Diseases. 2008;**2**(1):1-10

oxidant stress. Circulation Research. 2000;**87**:840-844

(2015):71

2013;**34**(3):644-675

**82**(2):291-295

pression of thioredoxin 2. Hypertension. 2009;**54**:338-344

tension. Cellular & Molecular Biology Letters. 2004;**9**(4A):635-641

hypertension. Histochemistry and Cell Biology. 2004;**122**:339-352


[51] Laggner H, Muellner MK, Schreier S, Sturm B, Hermann M, Exner M, Gmeiner BM, Kapiotis S. Hydrogen sulphide: A novel physiological inhibitor of LDL atherogenic modification by HOCl. Free Radical Research. 2007;**41**(7):741

[63] Mello Filho AC, Hoffman ME, Meneghini R. Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron. Biochemical Journal. 1983;**218**(1):273-275 [64] Chen N, Liu Y, Greiner CD, Holtzman JL. Physiologic concentrations of homocysteine inhibit the human plasma GSH peroxidase that reduces organic hydroperoxides. The

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

45

[65] Porter M, Pearson DJ, Suarez-Mendez VJ, Blann AD. Plasma, platelet and erythrocyte glutathione peroxidases as risk factors in ischaemic heart disease in man. Clinical

[66] Blann AD, Maxwell SR, Burrows G, Miller JP. Antioxidants, von Willebrand factor and endothelial cell injury in hypercholesterolaemia and vascular disease. Atherosclerosis.

[67] Barbosa KBF, Costa NMB, Alfenas RDCG, De Paula SO, Minim VPR, Bressan J. Oxidative stress; concept, implications and modulating factors. Revista de Nutrição. 2010;**23**(4):629-643

[68] Koury JC, Donangelo CM. Zinco, estresse oxidativo e atividade física. Revista de Nutrição.

[69] Bruning RS, Sturek M. Benefits of exercise training on coronary blood flow in coronary artery disease patients. Progress in Cardiovascular Diseases. 2015;**57**(5):443-453

[70] Bowles DK, Wamhoff BR. Coronary smooth muscle adaptation to exercise: Does it play a role in cardioprotection? Acta Physiologica Scandinavica. 2003;**178**(2):117-121

[71] Muller-Delp JM, Hotta K, Chen B, Behnke BJ, Maraj JJ, Delp MD, Lucero TR, Bramy JA, Alarcon DB, Morgan HE, Cowan MR, Haynes AD. Effects of age and exercise training on coronary microvascular smooth muscle phenotype and function. Journal of Applied

[72] Di Francescomarino S, Sciartilli A, Di Valerio V, Di Baldassarre A, Gallina S. The effect of physical exercise on endothelial function. Sports Medicine. 2009;**39**(10):797-812

[73] Dora KA. Endothelial-smooth muscle cell interactions in the regulation of vascular tone

[74] Simmons GH, Padilla J, Young CN, Wong BJ, Lang JA, Davis MJ, Laughlin MH, Fadel PJ. Increased brachial artery retrograde shear rate at exercise onset is abolished during prolonged cycling: Role of thermoregulatory vasodilation. Journal of Applied Physiology

[75] Padilla J, Simmons GH, Bender SB, Arce-Esquivel AA, Whyte JJ, Laughlin MH. Vascular

[76] M1 T, Yamamoto K, Shimizu N, Obi S, Kumagaya S, Igarashi T, Kamiya A, Ando J. Differential gene responses in endothelial cells exposed to a combination of shear stress

[77] Awolesi MA, Sessa WC, Sumpio BE. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. The Journal of Clinical Investigation. 1995;**96**(3):1449-1454

Physiology (1985). 2018;**124**(1):140-149. DOI: 10.1152/japplphysiol.00459.2017

in skeletal muscle. Microcirculation. 2016;**23**(8):626-630

(1985). 2011;**110**(2):389-397. DOI: 10.1152/japplphysiol.00936.2010

and cyclic stretch. Journal of Biotechnology. 2008;**133**(2):239-244

adaptations beyond active vascular beds. Physiology. 2011;**26**(3):132-145

Journal of Laboratory and Clinical Medicine. 2000;**136**(1):58-65

Science. 1992;**83**(3):343-345

1995;**116**(2):191-198

2003;**16**(4):433-441


[63] Mello Filho AC, Hoffman ME, Meneghini R. Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron. Biochemical Journal. 1983;**218**(1):273-275

[51] Laggner H, Muellner MK, Schreier S, Sturm B, Hermann M, Exner M, Gmeiner BM, Kapiotis S. Hydrogen sulphide: A novel physiological inhibitor of LDL atherogenic

[52] Wang Y, Zhao X, Jin H, et al. Role of hydrogen sulfide in the development of atherosclerotic lesions in apolipoprotein E knockout mice. Arteriosclerosis, Thrombosis, and

[53] Meng QH, Yang G, Yang W, Jiang B, Wu L, Wang R. Protective effect of hydrogen sulphide on balloon injury-induced neointima hyperplasia in rat carotid arteries. The

[54] G1 Y, Li H, Tang G, Wu L, Zhao K, Cao Q, Xu C, Wang R. Increased neointimal formation in cystathionine gamma-lyase deficient mice: Role of hydrogen sulfide in alpha5beta1 integrin and matrix metalloproteinase-2 expression in smooth muscle cells. Journal of

[55] Zhao ZZ, Wang Z, Li GH, Wang R, Tan JM, Cao X, Suo R, Jiang ZS. Hydrogen sulfide inhibits macrophage-derived foam cell formation. Experimental Biology and Medicine

[56] Lynn EG, Austin RC. Hydrogen sulfide in the pathogenesis of atherosclerosis and its therapeutic potential. Expert Review of Clinical Pharmacology. 2011;**4**(1):97-108

[57] Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, Zhao Y, Baskar R, Tan CH, Moore PK. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): New insights into the biology of hydrogen sulfide. Circulation. 2008;

[58] Wu SY, Pan CS, Geng B, Zhao J, Yu F, Pang YZ, Tang CS, Qi YF. Hydrogen sulfide ameliorates vascular calcification induced by vitamin D3 plus nicotine in rats. Acta

[59] Zagli G, Patacchini R, Trevisani M, Abbate R, Cinotti S, Gensini FG, Masotti G, Geppetti P. Hydrogen sulfide inhibits human platelet aggregation. European Journal of

[60] Grambow E, Mueller-Graf F, Delyagina E, Frank M, Kuhla A, Vollmar B. Effect of the hydrogen sulfide donor GYY4137 on platelet activation and microvascular thrombus

[61] Filipovic MR, Miljkovic J, Allgauer A, Chaurio R, Shubina T, Herrmann M, Ivanovic-Burmazovic I. Biochemical insight into physiological effects of H2S: Reaction with peroxynitrite and formation of a new nitric oxide donor, sulfinyl nitrite. Biochemical

[62] Carballal S, Trujillo M, Cuevasanta E, Bartesaghi S, Moller MN, Folkes LK, Garcia-Bereguiain MA, Gutierrez-Merino C, Wardman P, Denicola A, Radi R, Alvarez B. Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological inter-

modification by HOCl. Free Radical Research. 2007;**41**(7):741

American Journal of Pathology. 2007;**170**(4):1406-1414

Molecular and Cellular Cardiology. 2012;**52**(3):677-688

Vascular Biology. 2009;**29**:173-179

44 Free Radicals, Antioxidants and Diseases

(Maywood, N.J.). 2011;**236**(2):169-176

Pharmacolica Sinica. 2006;**27**(3):299-306

formation in mice. Platelets. 2014;**25**(3):166-174

est. Free Radical Biology & Medicine. 2011;**50**(1):196-205

Pharmacology. 2007;**559**(559):65-68

Journal. 2012;**441**(2):609-621

**117**(18):2351-2360


[78] Newcomer SC, Thijssen DHJ, Green DJ. Mechanism of beneficial effects of physical activity on atherosclerosis and coronary heart disease effects of exercise on endothelium and endothelium/smooth muscle cross talk : Role of exercise-induced hemodynamics. Journal of Applied Physiology (1985). 2011;**111**:311-320

[91] Miyazaki H, Oh-ishi S, Ookawara T, Kizaki T, Toshinai K, Ha S, Haga S, Ji LL, Ohno H. Strenuous endurance training in humans reduces oxidative stress following exhaust-

Oxidative Stress and Vascular Diseases: Effect of Physical Exercise

http://dx.doi.org/10.5772/intechopen.76576

47

[92] Gewaltig MT, Kojda G. Vasoprotection by nitric oxide: Mechanisms and therapeutic

[93] Rush JW, Turk JR, Laughlin MH. Exercise training regulates SOD-1 and oxidative stress in porcine aortic endothelium. American Journal of Physiology-Heart and Circulatory

[94] Venojärvi M, Korkmaz A, Wasenius N, Manderoos S, Heinonen OJ, Lindholm H, Aunola S, Eriksson JG, Atalay M. 12 weeks' aerobic and resistance training without dietary intervention did not influence oxidative stress but aerobic training decreased atherogenic index in middle-aged men with impaired glucose regulation. Food and

[95] Dantas FF, Brasileiro-Santos Mdo S, Batista RM, do Nascimento LS, Castellano LR, Ritti-Dias RM, Lima KC, Santos Ada C. Effect of strength training on oxidative stress and the correlation of the same with forearm vasodilatation and blood pressure of hypertensive

[96] Wycherley YP, Brinkworth GD, Noakes M, Buckley JD, Clifton PM. Effect of caloric restriction with and without exercise training on oxidative stress and endothelial function in obese subjects with type 2 diabetes. Diabetes, Obesity & Metabolism. 2008;**10**(11):1062-1073

[97] Duggan Duggan C, Tapsoba JD, Wang CY, Campbell KL, Foster-Schubert K, Gross MD, McTiernan A. Dietary weight loss, exercise and oxidative stress in postmenopausal women: A randomized controlled trial. Cancer Prevention Research (Philadelphia, PA).

[98] Li C, Feng F, Xiong X, Li R, Chen N. Exercise coupled with dietary restriction reduces oxidative stress in male adolescents with obesity. Journal of Sports Sciences. 2017;**35**(7):

[99] Alghadir AH, Gabr SA, Al-Eisa ES. Effects of moderate aerobic exercise on cognitive abilities and redox state biomarkers in older adults. Oxidative Medicine and Cellular

[100] De Moffarts B, Kirschvink N, Art T, Pincemail J, Lekeux P. Effect of exercise on blood oxidant/antioxidant markers in standardbred horses: Comparison between treadmill

[101] Malardé L, Gratas-Delamarche A, Le Douairon-Lahaye S, Zguira MS, Vincent S, Lemoine-Morel S, Groussard C. Endurance training and insulin therapy need to be associated to fully exert their respective beneficial effects on oxidant stress and glycemic

[102] Alaca N, Uslu S, Gulec Suyen G, Ince U, Serteser M, Kurtel H. Effects of different aerobic exercise frequencies on streptozotocin-nicotinamide-induced type 2 diabetic rats:

and race track tests. Equine Veterinary Journal. Supplement. 2006;**36**:254-257

regulation in diabetic rats. Free Radical Research. 2014;**48**(4):412-419

elderly women: A randomized clinical trial. PLoS One. 2016;**11**(8):e0161178

ing exercise. European Journal of Applied Physiology. 2001;**84**(1-2):1-6

potential. Cardiovascular Research. 2002;**55**(2):250-260

Physiology. 2003;**284**(4):H1378-H1387

Chemical Toxicology. 2013;**61**:127-135

2016;**9**(11):835-843

Longevity. 2016;**2016**:2545168

663-668


[91] Miyazaki H, Oh-ishi S, Ookawara T, Kizaki T, Toshinai K, Ha S, Haga S, Ji LL, Ohno H. Strenuous endurance training in humans reduces oxidative stress following exhausting exercise. European Journal of Applied Physiology. 2001;**84**(1-2):1-6

[78] Newcomer SC, Thijssen DHJ, Green DJ. Mechanism of beneficial effects of physical activity on atherosclerosis and coronary heart disease effects of exercise on endothelium and endothelium/smooth muscle cross talk : Role of exercise-induced hemodynamics.

[79] Yun JK, Anderson JM, Ziats NP. Cyclic-strain-induced endothelial cell expression of adhesion molecules and their roles in monocyte-endothelial interaction. Journal of

[80] Laughlin M, Newcomer S, Bender S. Importance of hemodynamic forces as signals for exercise-induced changes in endothelial cell phenotype. Journal of Applied Physiology.

[81] Bowles DK, Woodman CR, Laughlin MH. Coronary smooth muscle and endothelial adaptations to exercise training. Exercise and Sport Sciences Reviews. 2000;28(2):57-62

[82] Bowles DK, Hu Q, Laughlin MH, Sturek M. Exercise training increases L-type calcium current density in coronary smooth muscle. The American Journal of Physiology.

[83] Kojda G, Hambrecht R. Molecular mechanisms of vascular adaptations to exercise. Physical activity as an effective antioxidant therapy? Cardiovascular Research.

[84] Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, Harrison DG. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training.

[85] Corson MA, James NL, Latta SE, Nerem RM, Berk BC, Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circulation Research.

[86] Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Aktdependent phosphorylation. Nature.

[87] Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Kemp BE. AMP activated protein kinase phosphorylation of endothelial NO synthase. FEBS

[88] Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, Jo H. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms role of protein kinase A. Journal of Biological Chemistry. 2002;277(5):3388-3396 [89] Hambrecht R, Adams V, Erbs S, Linke A, Kränkel N, Shu Y, Baither Y, Gielen S, Thiele H, Gummert JF, Mohr FW, Schuler G. Regular physical activity improves endothelial function in patients with coronary artery disease by increasing phosphorylation of endo-

[90] Gómez-Cabrera MC, Pallardó FV, Sastre J, Viña J, García-del-Moral L. Allopurinol and markers of muscle damage among participants in the Tour de France. Journal of the

The Journal of Clinical Investigation. 2000;105(11):1631-1639

thelial nitric oxide synthase. Circulation. 2003;107(25):3152-3158

American Medical Association. 2003;**289**(19):2503-2504

Journal of Applied Physiology (1985). 2011;**111**:311-320

Biomedical Materials Research. 1999;**44**(1):87-97

2008;**104**(3):588-600

46 Free Radicals, Antioxidants and Diseases

1998;275(6 Pt 2):2159-2169

2005;67(2):187-197

1996;79(5):984-991

1999;399:601-605

Letters. 1999;443(3):285-289


Continuous versus short bouts and weekend warrior exercises. Journal of Diabetes. 2018;**10**(1):73-84

**Chapter 4**

Provisional chapter

**EPR Analysis of Antioxidant Compounds**

A free radical is a molecular species having an unpaired electron and it is a highly reactive entity and unstable. A free radical is a molecule with one or more unpaired electrons in its outer shell. Free radicals can be formed by chemical bond breakage from molecules or by redox reactions. When cells use oxygen, the oxidative stress occurs. The oxidative stress causes free radical formation. Free radicals can also be generated from ionizing radiations, ozone, heavy metal poisoning, cigarette smoking, and chronic alcohol intake. These free radicals are highly reactive and oxidize biomolecules leading to tissue injury and cell death. They also cause toxic effects and diseases. Antioxidants neutralize free radicals resulting from oxidative stress. Antioxidants play an important role in the treatment of diseases. The most suitable method for the analysis of free radicals is the electron paramagnetic resonance (EPR) spectroscopy method. The EPR method detects a paramagnetic center with a single electron. It gives information about the interactions with other nuclei around one electron. It provides information on the structure and environment of radicals.

DOI: 10.5772/intechopen.74294

Keywords: free radical, radiation damage center, antioxidant, electron paramagnetic

Free radicals are an atom or a molecule that bears an unpaired electron and is extremely reactive, capable of engaging in a rapid change reaction that destabilizes other molecules and generates many more free radicals. In plants and animals, these free radicals are deactivated by antioxidants. These antioxidants act as an inhibitor of the process of oxidation, even at

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

EPR Analysis of Antioxidant Compounds

Betül Çalişkan and Ali Cengiz Çalişkan

Betül Çalişkan and Ali Cengiz Çalişkan

http://dx.doi.org/10.5772/intechopen.74294

Abstract

resonance

1. Introduction

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **EPR Analysis of Antioxidant Compounds** EPR Analysis of Antioxidant Compounds

Betül Çalişkan and Ali Cengiz Çalişkan Betül Çalişkan and Ali Cengiz Çalişkan

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74294

#### Abstract

Continuous versus short bouts and weekend warrior exercises. Journal of Diabetes.

[103] Seawright JW, Luttrell M, Trache A, Woodman CR. Short-term increases in pressure and shear stress attenuate age-related declines in endothelial function in skeletal mus-

[105] Suvorava T, Lauer N, Kojda G. Physical inactivity causes endothelial dysfunction in healthy young mice. Journal of the American College of Cardiology. 2004;**44**(6):1320-1327

[106] Kojda G, Laursen JB, Ramasamy S, Kent JD, Kurz S, Burchfield J, et al. Protein expression, vascular reactivity and soluble guanylate cyclase activity in mice lacking the endothelial cell nitric oxide synthase: Contributions of NOS isoforms to blood pressure and

cle feed arteries. European Journal of Applied Physiology. 2016;**116**:1305-1311 [104] Adams V, Linke A, Kränkel N, Erbs S, Gielen S, Möbius-Winkler S, et al. Impact of regular physical activity on the NAD (P) H oxidase and angiotensin receptor system in

patients with coronary artery disease. Circulation. 2005;**111**(5):555-562

heart rate control. Cardiovascular Research. 1999;**42**(1):206-213

2018;**10**(1):73-84

48 Free Radicals, Antioxidants and Diseases

A free radical is a molecular species having an unpaired electron and it is a highly reactive entity and unstable. A free radical is a molecule with one or more unpaired electrons in its outer shell. Free radicals can be formed by chemical bond breakage from molecules or by redox reactions. When cells use oxygen, the oxidative stress occurs. The oxidative stress causes free radical formation. Free radicals can also be generated from ionizing radiations, ozone, heavy metal poisoning, cigarette smoking, and chronic alcohol intake. These free radicals are highly reactive and oxidize biomolecules leading to tissue injury and cell death. They also cause toxic effects and diseases. Antioxidants neutralize free radicals resulting from oxidative stress. Antioxidants play an important role in the treatment of diseases. The most suitable method for the analysis of free radicals is the electron paramagnetic resonance (EPR) spectroscopy method. The EPR method detects a paramagnetic center with a single electron. It gives information about the interactions with other nuclei around one electron. It provides information on the structure and environment of radicals.

DOI: 10.5772/intechopen.74294

Keywords: free radical, radiation damage center, antioxidant, electron paramagnetic resonance

#### 1. Introduction

Free radicals are an atom or a molecule that bears an unpaired electron and is extremely reactive, capable of engaging in a rapid change reaction that destabilizes other molecules and generates many more free radicals. In plants and animals, these free radicals are deactivated by antioxidants. These antioxidants act as an inhibitor of the process of oxidation, even at

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

relatively small concentrations, and thus have diverse physiological roles in the body. The body is constantly exposed to the negative and sometimes lethal effects of oxidants during normal physiological processes. The harmful free radicals such as hydroxyl, peroxyl and the superoxide anion are constantly being produced as a result of metabolic reactions in living systems. On a daily basis, up to 5% of inhaled oxygen may be converted to reactive oxygen species (ROS). These ROS have the ability to bind to cellular structures and have been implicated in a number of pathological processes such as aging, inflammation, reoxygenation of ischemic tissues, atherosclerosis, cancer and even Parkinson's disease in men [1]. Two processes, which produce free radicals in vivo, have been identified and named the Fenton reaction and the Haber-Weiss reaction [2]. Antioxidants play an important role in animal health. Conventional antioxidants have been shown to improve animal performance during conditions characterized by increased tissue oxidant levels such as stress, injury and infections [3].

2. EPR studies of antioxidant compounds

tions can be drawn concerning modes of bond rupture.

are shown in Figure 1 [6].

in PHM are included in Tables 2 and 3.

obtained from the oxidation of PHM single crystal.

radicals.

2.01477 and aCHð Þ<sup>β</sup>

0.568 mT, respectively.

2.1. Gamma-irradiated potassium hydroquinone monosulfonate single crystal

Free radicals or other odd electrons have often been postulated as intermediates in the decomposition of organic compounds by ionizing radiation. It is still important for formulating mechanisms in radiation chemistry to identify the radicals that are formed. If several organic compounds are irradiated and resulting radicals are identified, one may hope that generaliza-

The quinone structure is widespread in nature: for example, quinones play an integral role in many biological electron-transfer processes, particularly respiration and photosynthesis [9, 10]. Quinones have long been considered for their fungicide, antibacterial and anticancer properties [11]. The quinone derivatives are especially interesting because they have significant properties due to the ready reversibility of the quinone-semiquinone redox system [12].

The single crystal of PHM was irradiated with gamma rays at room temperature and studied by the EPR technique at 125 K. The spectra were dependent on the orientation of H in the planes, which are perpendicular to each other. The paramagnetic centers formed from PHM

The experimental spectrum in Figure 2 has 1:1:1:2:1 intensity ratios and exhibits the superimposition due to the two PHM anion radicals. Because of the radical A, the spectra exhibit 2-lines with intensity ratios 1:1. Owing to the C6 β-proton, the spectra exhibit a doublet (1:1). Because of the radical B, the spectra exhibit 3-lines with intensity ratios 1:2:1. Owing to the C3 and C4 β-protons, the spectra exhibit a triplet (1:2:1). The spectrum in Figure 3 has 1:2:2:1 intensity ratios and exhibits the superimposition due to the two PHM anion

The simulation values of the hyperfine coupling constants of the spectra in Figures 2 and 3 are given in Table 1. The EPR parameters belonging to two semiquinone anion radicals observed

The EPR measurements have shown the existence of two semiquinone anion radicals. The semiquinone anion radicals were stable. The observed semiquinone anion radicals were

The angular dependences of EPR spectra were obtained for different orientations of the static magnetic field with respect to the crystalline axes. For the radical A and radical B, the spectroscopic splitting factor and the hyperfine coupling constants are anisotropic. For the radical A, the average values of the g-factor and the hyperfine coupling constant were obtained as gA =

factor and the hyperfine coupling constant were obtained as gB = 2.01054 and aC2H2ð Þ<sup>β</sup>

<sup>A</sup> = 0.326 mT, respectively. For the radical B, the average values of the g-

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294 51

B =

Free radicals can be classified as reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS). Even though free radicals are mainly produced through regular metabolic routes, there are also some external factors that promote their production, including smoking, environmental pollutants, radiation and drugs, among others [4]. In healthy organisms, there is a delicate balance between the production and the removal of free radicals, which guarantees that they remain in adequate concentrations. However, when this balance is broken, these reactive species start producing chemical damages to proteins, lipids, DNA, RNA and sugars generating the so-called oxidative stress of body cells [4]. These processes have been associated with several diseases including cardiovascular, liver, neurological and renal disorders, as well as cancer, auto-immune deficiency and degenerative disorders associated with aging, diabetes, obesity, autism, Alzheimer's, Parkinson's and Huntington's diseases [4]. Although humans have developed different endogenous defense mechanisms to protect cells from the excess of free radicals and to avoid the oxidative stress, often these mechanisms are not enough. Therefore, to increase protection from oxidative damage, dietary supplements with antioxidants are recommended as a way to maintain the concentration of free radicals as low as possible [5].

Antioxidants destroy free radicals. Antioxidants neutralize the radical, and thus the radicalantioxidant association maintains itself in a stationary state. For all these situations, antioxidants must be in a structure that will quench the radical. Therefore, unpaired electrons in the structure of antioxidants must be either radical ions or free radicals. So, they must form a paramagnetic center. Paramagnetic structures are analyzed by electron paramagnetic resonance (EPR) spectroscopy. Thus, the EPR analysis of antioxidants is as valuable and important as the investigation of the paramagnetic centers of radicals. There are many studies done for this purpose. Among these studies, especially the works we have done recently are noteworthy.

Potassium hydroquinone monosulfonate (PHM), succinic anhydride and 3-nitroacetophenone compounds are antioxidants. Paramagnetic centers formed by gamma irradiation effect on single crystals of these materials were analyzed by the EPR method [6–8].

## 2. EPR studies of antioxidant compounds

relatively small concentrations, and thus have diverse physiological roles in the body. The body is constantly exposed to the negative and sometimes lethal effects of oxidants during normal physiological processes. The harmful free radicals such as hydroxyl, peroxyl and the superoxide anion are constantly being produced as a result of metabolic reactions in living systems. On a daily basis, up to 5% of inhaled oxygen may be converted to reactive oxygen species (ROS). These ROS have the ability to bind to cellular structures and have been implicated in a number of pathological processes such as aging, inflammation, reoxygenation of ischemic tissues, atherosclerosis, cancer and even Parkinson's disease in men [1]. Two processes, which produce free radicals in vivo, have been identified and named the Fenton reaction and the Haber-Weiss reaction [2]. Antioxidants play an important role in animal health. Conventional antioxidants have been shown to improve animal performance during conditions characterized by increased tissue oxidant levels such as stress, injury and

Free radicals can be classified as reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS). Even though free radicals are mainly produced through regular metabolic routes, there are also some external factors that promote their production, including smoking, environmental pollutants, radiation and drugs, among others [4]. In healthy organisms, there is a delicate balance between the production and the removal of free radicals, which guarantees that they remain in adequate concentrations. However, when this balance is broken, these reactive species start producing chemical damages to proteins, lipids, DNA, RNA and sugars generating the so-called oxidative stress of body cells [4]. These processes have been associated with several diseases including cardiovascular, liver, neurological and renal disorders, as well as cancer, auto-immune deficiency and degenerative disorders associated with aging, diabetes, obesity, autism, Alzheimer's, Parkinson's and Huntington's diseases [4]. Although humans have developed different endogenous defense mechanisms to protect cells from the excess of free radicals and to avoid the oxidative stress, often these mechanisms are not enough. Therefore, to increase protection from oxidative damage, dietary supplements with antioxidants are recommended as a way to maintain the concentration of

Antioxidants destroy free radicals. Antioxidants neutralize the radical, and thus the radicalantioxidant association maintains itself in a stationary state. For all these situations, antioxidants must be in a structure that will quench the radical. Therefore, unpaired electrons in the structure of antioxidants must be either radical ions or free radicals. So, they must form a paramagnetic center. Paramagnetic structures are analyzed by electron paramagnetic resonance (EPR) spectroscopy. Thus, the EPR analysis of antioxidants is as valuable and important as the investigation of the paramagnetic centers of radicals. There are many studies done for this purpose. Among these studies, especially the works we have done recently are

Potassium hydroquinone monosulfonate (PHM), succinic anhydride and 3-nitroacetophenone compounds are antioxidants. Paramagnetic centers formed by gamma irradiation effect on

single crystals of these materials were analyzed by the EPR method [6–8].

infections [3].

50 Free Radicals, Antioxidants and Diseases

noteworthy.

free radicals as low as possible [5].

### 2.1. Gamma-irradiated potassium hydroquinone monosulfonate single crystal

Free radicals or other odd electrons have often been postulated as intermediates in the decomposition of organic compounds by ionizing radiation. It is still important for formulating mechanisms in radiation chemistry to identify the radicals that are formed. If several organic compounds are irradiated and resulting radicals are identified, one may hope that generalizations can be drawn concerning modes of bond rupture.

The quinone structure is widespread in nature: for example, quinones play an integral role in many biological electron-transfer processes, particularly respiration and photosynthesis [9, 10]. Quinones have long been considered for their fungicide, antibacterial and anticancer properties [11]. The quinone derivatives are especially interesting because they have significant properties due to the ready reversibility of the quinone-semiquinone redox system [12].

The single crystal of PHM was irradiated with gamma rays at room temperature and studied by the EPR technique at 125 K. The spectra were dependent on the orientation of H in the planes, which are perpendicular to each other. The paramagnetic centers formed from PHM are shown in Figure 1 [6].

The experimental spectrum in Figure 2 has 1:1:1:2:1 intensity ratios and exhibits the superimposition due to the two PHM anion radicals. Because of the radical A, the spectra exhibit 2-lines with intensity ratios 1:1. Owing to the C6 β-proton, the spectra exhibit a doublet (1:1). Because of the radical B, the spectra exhibit 3-lines with intensity ratios 1:2:1. Owing to the C3 and C4 β-protons, the spectra exhibit a triplet (1:2:1). The spectrum in Figure 3 has 1:2:2:1 intensity ratios and exhibits the superimposition due to the two PHM anion radicals.

The simulation values of the hyperfine coupling constants of the spectra in Figures 2 and 3 are given in Table 1. The EPR parameters belonging to two semiquinone anion radicals observed in PHM are included in Tables 2 and 3.

The EPR measurements have shown the existence of two semiquinone anion radicals. The semiquinone anion radicals were stable. The observed semiquinone anion radicals were obtained from the oxidation of PHM single crystal.

The angular dependences of EPR spectra were obtained for different orientations of the static magnetic field with respect to the crystalline axes. For the radical A and radical B, the spectroscopic splitting factor and the hyperfine coupling constants are anisotropic. For the radical A, the average values of the g-factor and the hyperfine coupling constant were obtained as gA = 2.01477 and aCHð Þ<sup>β</sup> <sup>A</sup> = 0.326 mT, respectively. For the radical B, the average values of the gfactor and the hyperfine coupling constant were obtained as gB = 2.01054 and aC2H2ð Þ<sup>β</sup> B = 0.568 mT, respectively.

Figure 1. Structure of two semiquinone anion radicals observed in PHM single crystal.

#### 2.2. Gamma-irradiated succinic anhydride single crystal

Succinate:quinone reductase (SQR) of complex II occupies a unique central point in the mitochondrial respiratory system as a major source of electrons driving reactive oxygen species (ROS) production. It is an ideal pharmaceutical target for modulating ROS levels in normal cells to prevent oxidative stress-induced damage or alternatively, increase ROS in cancer cells, inducing cell death [13].

exhibits the superimposition due to the two CH2CH2 atom groups. Because of the radical A, the spectra exhibit 9-lines with intensity ratios 1:2:1:2:4:2:1:2:1. Owing to the β-protons, the spectra exhibit a triplet (1:2:1). Each of the lines (1:2:1) splits into triplets (1:2:1) due to the two equivalent γ-protons. Because of the radical B, the spectra exhibit 9-lines with intensity ratios 1:2:1:2:4:2:1:2:1. Owing to the β-protons, the spectra exhibit a triplet (1:2:1). Each of the lines (1:2:1) splits into triplets (1:2:1) due to the two equivalent γ-protons. Total 18-lines are observed due to the two anion radicals. The spectrum in Figure 6 has 1:1:1:1:1:1:1:1:2:2:2:2:2:2:2:2:1:1:1:1:1:1:1:1 intensity ratios and exhibits the superimposition due to the two (CH2)βCHγ1Hγ<sup>2</sup> atom groups. Because of the radical A, the spectra exhibit 12-lines with intensity ratios 1:1:1:1:2:2:2:2:1:1:1:1. Owing to the β-protons, the spectra exhibit a triplet (1:2:1). Each of the lines (1:2:1) splits into doublets (1:1) due to the γ1-proton. Each of the lines (1:1:2:2:1:1) splits into doublets (1:1) due to the γ2-proton. The hyperfine structure splittings in the radical A are the same as that of the radical

Figure 2. EPR spectrum of gamma-irradiated PHM single crystal at 125 K when the magnetic field is in the ab plane at an

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294 53

We have obtained the computer simulations of the spectra that give the best agreement with experimental values. The simulations of the EPR spectra have been carried out using the

B. The total 24-lines are observed due to the two anion radicals.

Bruker's WINEPR software.

angle 0 toward the axis.

The single crystals of succinic anhydride were irradiated gamma-rays at room temperature and studied by the EPR technique at 125 K [7]. The spectra were dependent on the orientation of H in the planes which are perpendicular to each other The paramagnetic centers formed from succinic anhydride are shown in Figure 4.

For only a few angles, the spectra have 1:2:1:1:2:1:2:4:2:2:4:2:1:2:1:1:2:1 intensity ratios. For many angles, the spectra have 1:1:1:1:1:1:1:1:2:2:2:2:2:2:2:2:1:1:1:1:1:1:1:1 intensity ratios. The experimental spectrum in Figure 5 has 1:2:1:1:2:1:2:4:2:2:4:2:1:2:1:1:2:1 intensity ratios and

Figure 2. EPR spectrum of gamma-irradiated PHM single crystal at 125 K when the magnetic field is in the ab plane at an angle 0 toward the axis.

exhibits the superimposition due to the two CH2CH2 atom groups. Because of the radical A, the spectra exhibit 9-lines with intensity ratios 1:2:1:2:4:2:1:2:1. Owing to the β-protons, the spectra exhibit a triplet (1:2:1). Each of the lines (1:2:1) splits into triplets (1:2:1) due to the two equivalent γ-protons. Because of the radical B, the spectra exhibit 9-lines with intensity ratios 1:2:1:2:4:2:1:2:1. Owing to the β-protons, the spectra exhibit a triplet (1:2:1). Each of the lines (1:2:1) splits into triplets (1:2:1) due to the two equivalent γ-protons. Total 18-lines are observed due to the two anion radicals. The spectrum in Figure 6 has 1:1:1:1:1:1:1:1:2:2:2:2:2:2:2:2:1:1:1:1:1:1:1:1 intensity ratios and exhibits the superimposition due to the two (CH2)βCHγ1Hγ<sup>2</sup> atom groups. Because of the radical A, the spectra exhibit 12-lines with intensity ratios 1:1:1:1:2:2:2:2:1:1:1:1. Owing to the β-protons, the spectra exhibit a triplet (1:2:1). Each of the lines (1:2:1) splits into doublets (1:1) due to the γ1-proton. Each of the lines (1:1:2:2:1:1) splits into doublets (1:1) due to the γ2-proton. The hyperfine structure splittings in the radical A are the same as that of the radical B. The total 24-lines are observed due to the two anion radicals.

2.2. Gamma-irradiated succinic anhydride single crystal

Figure 1. Structure of two semiquinone anion radicals observed in PHM single crystal.

from succinic anhydride are shown in Figure 4.

inducing cell death [13].

52 Free Radicals, Antioxidants and Diseases

Succinate:quinone reductase (SQR) of complex II occupies a unique central point in the mitochondrial respiratory system as a major source of electrons driving reactive oxygen species (ROS) production. It is an ideal pharmaceutical target for modulating ROS levels in normal cells to prevent oxidative stress-induced damage or alternatively, increase ROS in cancer cells,

The single crystals of succinic anhydride were irradiated gamma-rays at room temperature and studied by the EPR technique at 125 K [7]. The spectra were dependent on the orientation of H in the planes which are perpendicular to each other The paramagnetic centers formed

For only a few angles, the spectra have 1:2:1:1:2:1:2:4:2:2:4:2:1:2:1:1:2:1 intensity ratios. For many angles, the spectra have 1:1:1:1:1:1:1:1:2:2:2:2:2:2:2:2:1:1:1:1:1:1:1:1 intensity ratios. The experimental spectrum in Figure 5 has 1:2:1:1:2:1:2:4:2:2:4:2:1:2:1:1:2:1 intensity ratios and We have obtained the computer simulations of the spectra that give the best agreement with experimental values. The simulations of the EPR spectra have been carried out using the Bruker's WINEPR software.

Figure 3. EPR spectrum of gamma-irradiated PHM single crystal at 125 K when the magnetic field is in the ac plane at an angle 70� toward the axis.

The simulation values of the hyperfine coupling constants of the spectra in Figures 5 and 6 are given in Table 4. The EPR parameters belonging to two succinic anhydride anion radicals

Principal values Direction cosines

�0.198271 0.556143

0.353981 0.565109

Note: The errors are estimated to be �0.00005 and �0.005 mT for all the calculated g- and A-values, respectively.

�0.736026 0.274800

0.787949 �0.349608

Note: The errors are estimated to be �0.00005 and �0.005 mT for all the calculated g- and A-values, respectively.

Figure 4. Structure of two succinic anhydride anion radicals observed in succinic anhydride single crystal.

0.370051 Ayy = 0.56

0.474185 gyy = 2.01056

Principal values Direction cosines

Axx = 0.662 0.618669

0.745467 Ayy = 0.324

0.235582 gyy = 2.01479

0.452748 0.812426 �0.367402

0.366680 0.490298 �0.790667

0.457813 0.053485 �0.887439

�0.262535 0.527402 0.808037

�0.378980 0.548318

55

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294

> �0.556952 0.796643

0.638480 0.674838

0.821075 �0.317780

<sup>A</sup> (mT) Axx = 0.347 0.807090

Azz = 0.307 aav = 0.326 gA gxx = 2.01690 0.745218

> gzz = 2.01263 gav = 2.01477

Table 2. The EPR parameters of radical A observed in PHM at 125 K.

Azz = 0.483 aav = 0.568 gB gxx = 2.01119 0.506864

> gzz = 2.00986 gav = 2.01054

Table 3. The EPR parameters of radical B observed in PHM at 125 K.

ACHð Þ<sup>β</sup> 

AC2H2 ð Þ<sup>β</sup> 

B

observed in succinic anhydride are included in Tables 5 and 6.


Table 1. EPR parameters of simulated spectra.


Note: The errors are estimated to be �0.00005 and �0.005 mT for all the calculated g- and A-values, respectively.

Table 2. The EPR parameters of radical A observed in PHM at 125 K.


Table 3. The EPR parameters of radical B observed in PHM at 125 K.

Figure 3. EPR spectrum of gamma-irradiated PHM single crystal at 125 K when the magnetic field is in the ac plane at an

Radical A Radical B

<sup>A</sup> = 0.328 mT aCH<sup>2</sup>ð Þ<sup>β</sup>

ν = 9.38 GHz ν = 9.408 GHz

<sup>A</sup> = 0.327 mT aCH<sup>2</sup>ð Þ<sup>β</sup>

ν = 9.38 GHz ν = 9.401 GHz Line width = 0.22 mT Line width = 0.22 mT

Center field = 335.5 mT Center field = 335.5 mT

Line width = 0.2 mT Line width = 0.215 mT

Center field = 335.5 mT Center field = 335.5 mT

<sup>B</sup> <sup>=</sup> aH<sup>β</sup> 

<sup>B</sup> <sup>=</sup> aH<sup>β</sup> 

<sup>B</sup> = 0.572 mT

<sup>B</sup> = 0.55 mT

angle 70� toward the axis.

54 Free Radicals, Antioxidants and Diseases

Figure 2 aCHð Þ<sup>β</sup>

Figure 3 aCHð Þ<sup>β</sup>

Table 1. EPR parameters of simulated spectra.

The simulation values of the hyperfine coupling constants of the spectra in Figures 5 and 6 are given in Table 4. The EPR parameters belonging to two succinic anhydride anion radicals observed in succinic anhydride are included in Tables 5 and 6.

Figure 4. Structure of two succinic anhydride anion radicals observed in succinic anhydride single crystal.

Radical A Radical B

<sup>A</sup> = 3.26 mT aCH<sup>2</sup>ð Þ<sup>β</sup>

<sup>A</sup> = 0.47 mT <sup>a</sup>

<sup>A</sup> = 0.43 mT <sup>a</sup>

Center field = 335.576 mT Center field = 335.576 mT

<sup>A</sup> = 3.245 mT aCH<sup>2</sup>ð Þ<sup>β</sup>

<sup>A</sup> = 0.39 mT <sup>a</sup>

<sup>A</sup> = 0.52 mT <sup>a</sup>

Center field = 335.576 mT Center field = 335.576 mT

�0.723568 0.662390

0.016324 0.574669

0.775337 �0.541185

�0.054529 �0.022518

0.675662 Ayy = 3.285

0.426473 Ayy = 0.44

0.583838 Ayy = 0.494

0.692883 gyy = 2.01513

ν = 9.368 GHz ν = 9.423 GHz Line width = 0.3 mT Line width = 0.42 mT

ν = 9.368 GHz ν = 9.423 GHz Line width = 0.4 mT Line width = 0.4 mT

Hγ<sup>1</sup> CHγ1Hγ<sup>2</sup> 

Hγ<sup>2</sup> CHγ1Hγ<sup>2</sup> 

Hγ<sup>1</sup> CHγ1Hγ<sup>2</sup> 

Hγ<sup>2</sup> CHγ1Hγ<sup>2</sup> 

> 0.945231 �0.042637 �0.323606

> 0.479980 0.530794 �0.698482

> �0.489384 0.627894 0.605188

> 0.021593 0.692912 �0.720698

<sup>B</sup> <sup>=</sup> aH<sup>β</sup> 

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294

> B = aHγ<sup>1</sup>

> B = aHγ<sup>2</sup>

<sup>B</sup> <sup>=</sup> aH<sup>β</sup> 

> B = aHγ<sup>1</sup>

> B = aHγ<sup>2</sup>

<sup>B</sup> = 3.281 mT

<sup>B</sup> = 0.505 mT

57

<sup>B</sup> = 0.47 mT

<sup>B</sup> = 3.253 mT

<sup>B</sup> = 0.44 mT

<sup>B</sup> = 0.5 mT

0.262393 0.688935

�0.316433 0.847344

0.809031 0.067839

0.054902 0.718957

Figure 5 aCH<sup>2</sup>ð Þ<sup>β</sup>

Figure 6 aCH<sup>2</sup>ð Þ<sup>β</sup>

Table 4. EPR parameters of simulated spectra.

ACH<sup>2</sup>ð Þ<sup>β</sup> 

AHγ<sup>1</sup> CHγ1Hγ<sup>2</sup> 

AHγ<sup>2</sup> CHγ1Hγ<sup>2</sup> 

<sup>A</sup> <sup>=</sup> AH<sup>β</sup> 

> A = AHγ<sup>1</sup>

> A = AHγ<sup>2</sup>

a Hγ<sup>1</sup> CHγ1Hγ<sup>2</sup> 

a Hγ<sup>2</sup> CHγ1Hγ<sup>2</sup> 

a Hγ<sup>1</sup> CHγ1Hγ<sup>2</sup> 

a Hγ<sup>2</sup> CHγ1Hγ<sup>2</sup> 

<sup>A</sup> <sup>=</sup> aH<sup>β</sup> 

> A = aHγ<sup>1</sup>

> A = aHγ<sup>2</sup>

<sup>A</sup> <sup>=</sup> aH<sup>β</sup> 

> A = aHγ<sup>1</sup>

> A = aHγ<sup>2</sup>

EPR parameters Principal values Direction cosines

<sup>A</sup> (mT) Axx = 3.381 0.194135

<sup>A</sup> (mT) Axx = 0.492 0.818223

<sup>A</sup> (mT) Axx = 0.62 0.325533

Azz = 3.227 aiso = 3.298

Azz = 0. 37 aiso = 0.434

Azz = 0.414 aiso = 0.509

gzz = 2.01428 giso = 2.01565

Table 5. The EPR parameters of radical A observed in succinic anhydride at 125 K.

gA gxx = 2.01753 0.998258

Figure 5. Experimental and simulated EPR spectra of gamma-irradiated succinic anhydride single crystal at 125 K when the magnetic field is in the bc plane at an angle 90� toward the axis.

Figure 6. Experimental and simulated EPR spectra of gamma-irradiated succinic anhydride single crystal at 125 K when the magnetic field is in the ac plane at an angle 110� toward the axis.


Table 4. EPR parameters of simulated spectra.

Figure 5. Experimental and simulated EPR spectra of gamma-irradiated succinic anhydride single crystal at 125 K when

Figure 6. Experimental and simulated EPR spectra of gamma-irradiated succinic anhydride single crystal at 125 K when

the magnetic field is in the bc plane at an angle 90� toward the axis.

56 Free Radicals, Antioxidants and Diseases

the magnetic field is in the ac plane at an angle 110� toward the axis.


Table 5. The EPR parameters of radical A observed in succinic anhydride at 125 K.


temperature range of 120–360 K, we decided that the radiation damage center in the sample was due to the break of the pi bond in the carbon-oxygen double bond. It was seen that the

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294 59

In the EPR spectra, especially the impact of the methyl protons was felt. The hyperfine coupling constants of the methyl protons were measured at 300 K. The angular variations of EPR parameters of the 3NAP only at 300 K could be investigated. The spectra were clear only in a few angles, at 120 K. Therefore, only the simulations of these spectra were made, at 120 K.

The EPR spectra of irradiated 3-nitroacetophenone single crystals consist of four hyperfine structure lines with a 1:3:3:1 intensity ratio, which is caused by the interaction of the unpaired electron with the three equivalent protons of the methyl group, at most orientations of the crystal in the magnetic field, at 300 K. The H(2) and the H(6) protons are also considered

In Figures 8–11, the experimental spectra of the gamma-irradiated 3NAP single crystals and

The simulation values of the hyperfine coupling constants of the spectra in Figures 8–11 are given in Table 7. The EPR parameters of the 3-nitroacetophenone anion radical are given in

In the EPR spectra, especially the impact of the methyl protons was felt. Only the hyperfine coupling constants of the methyl protons could be experimentally measured at 300 K. The EPR spectra of irradiated 3-nitroacetophenone single crystals consist of four hyperfine structure lines with a 1:3:3:1 intensity ratio, which is caused by interaction of the unpaired electron with the three equivalent protons of the methyl group, at most orientations of the crystal in the magnetic field, at 300 K. The H(2) and the H(6) protons are also considered approximately equivalent. The hyperfine coupling for the CH3 group is anisotropic and their average values

unpaired electron interacted with all the protons in the molecular structure [8].

In addition, the simulation was also made for a single angle, at 300 K.

Figure 7. Structure of the 3NAP anion radical observed in 3NAP.

approximately equivalent.

being aCH<sup>3</sup> = 1.402 mT.

Table 8.

their simulation spectra were compared.

Table 6. The EPR parameters of the radical B observed in succinic anhydride at 125 K.

The radical formation mechanism in the present work is the same as that of potassium hydroquinone monosulfonate [6]. The angular dependences of EPR spectra were obtained for different orientations of the static magnetic field with respect to the crystalline axes. For the radical A and radical B, the spectroscopic splitting factor and the hyperfine coupling constants of the Hβ, Hγ<sup>1</sup> and Hγ<sup>2</sup> protons are anisotropic. For the radical A, the average values of the gfactor and the hyperfine coupling constants were obtained as gA = 2.01565, aH<sup>β</sup> A = 3.298 mT, aHγ<sup>1</sup> A = 0.434 mT, aHγ<sup>2</sup> <sup>A</sup> = 0.509 mT, respectively. For the radical B, the average values of the g-factor and the hyperfine coupling constants were obtained as gB = 2.00457 and aH<sup>β</sup> B = 3.298 mT, aHγ<sup>1</sup> B = 0.483 mT, aHγ<sup>2</sup> <sup>B</sup> = 0.455 mT, respectively.

#### 2.3. Gamma-irradiated 3-nitroacetophenone single crystal

Nitroaromatic compounds have widespread actual or potential use in medicine and cancer therapy [14]. Nitroaromatic compounds have been extensively studied and reviewed mainly because of their interesting biological activities as well as their extended use in the chemical industry [15].

The radical in 3NAP identified as 3-nitroacetophenone anion radical is shown in Figure 7. The spectra were found to be temperature dependent. When we examined the EPR spectra at a

Figure 7. Structure of the 3NAP anion radical observed in 3NAP.

The radical formation mechanism in the present work is the same as that of potassium hydroquinone monosulfonate [6]. The angular dependences of EPR spectra were obtained for different orientations of the static magnetic field with respect to the crystalline axes. For the radical A and radical B, the spectroscopic splitting factor and the hyperfine coupling constants of the Hβ, Hγ<sup>1</sup> and Hγ<sup>2</sup> protons are anisotropic. For the radical A, the average values of the g-

<sup>A</sup> = 0.509 mT, respectively. For the radical B, the average values of

 A

= 3.298 mT,

 B =

factor and the hyperfine coupling constants were obtained as gA = 2.01565, aH<sup>β</sup>

the g-factor and the hyperfine coupling constants were obtained as gB = 2.00457 and aH<sup>β</sup>

<sup>B</sup> = 0.455 mT, respectively.

Nitroaromatic compounds have widespread actual or potential use in medicine and cancer therapy [14]. Nitroaromatic compounds have been extensively studied and reviewed mainly because of their interesting biological activities as well as their extended use in the chemical

The radical in 3NAP identified as 3-nitroacetophenone anion radical is shown in Figure 7. The spectra were found to be temperature dependent. When we examined the EPR spectra at a

aHγ<sup>1</sup> 

ACH<sup>2</sup>ð Þ<sup>β</sup> 

AHγ<sup>1</sup> CHγ1Hγ<sup>2</sup> 

AHγ<sup>2</sup> CHγ1Hγ<sup>2</sup> 

<sup>B</sup> <sup>=</sup> AH<sup>β</sup> 

58 Free Radicals, Antioxidants and Diseases

<sup>B</sup> <sup>=</sup> AHγ<sup>1</sup> 

<sup>B</sup> <sup>=</sup> AHγ<sup>2</sup> 

A

3.298 mT, aHγ<sup>1</sup>

industry [15].

= 0.434 mT, aHγ<sup>2</sup>

B

= 0.483 mT, aHγ<sup>2</sup>

2.3. Gamma-irradiated 3-nitroacetophenone single crystal

EPR parameters Principal values Direction cosines

<sup>B</sup> (mT) Axx = 3.36 0.021180

<sup>B</sup> (mT) Axx = 0.586 0.066077

<sup>B</sup> (mT) Axx = 0.612 0.759274

Azz = 3.251 aiso = 3.298

Azz = 0.379 aiso = 0.483

Azz = 0.288 aiso = 0.455

gzz = 2.00300 giso = 2.00457

Table 6. The EPR parameters of the radical B observed in succinic anhydride at 125 K.

gB gxx = 2.00660 0.977193

0.829840 �0.557599

�0.896800 0.437475

�0.632874 �0.151571

�0.096452 0.189184

0.820500 Ayy = 3.283

0.839778 Ayy = 0.485

0.021600 Ayy = 0.464

0.553111 gyy = 2.00410

�0.990260 �0.059355 �0.125949

0.942855 �0.087383 �0.321541

�0.102410 0.113810 �0.988210

0.210889 0.545205 �0.811343 �0.137614 0.554835

0.326586 0.433722

0.642663 0.765844

�0.024888 0.832736

temperature range of 120–360 K, we decided that the radiation damage center in the sample was due to the break of the pi bond in the carbon-oxygen double bond. It was seen that the unpaired electron interacted with all the protons in the molecular structure [8].

In the EPR spectra, especially the impact of the methyl protons was felt. The hyperfine coupling constants of the methyl protons were measured at 300 K. The angular variations of EPR parameters of the 3NAP only at 300 K could be investigated. The spectra were clear only in a few angles, at 120 K. Therefore, only the simulations of these spectra were made, at 120 K. In addition, the simulation was also made for a single angle, at 300 K.

The EPR spectra of irradiated 3-nitroacetophenone single crystals consist of four hyperfine structure lines with a 1:3:3:1 intensity ratio, which is caused by the interaction of the unpaired electron with the three equivalent protons of the methyl group, at most orientations of the crystal in the magnetic field, at 300 K. The H(2) and the H(6) protons are also considered approximately equivalent.

In Figures 8–11, the experimental spectra of the gamma-irradiated 3NAP single crystals and their simulation spectra were compared.

The simulation values of the hyperfine coupling constants of the spectra in Figures 8–11 are given in Table 7. The EPR parameters of the 3-nitroacetophenone anion radical are given in Table 8.

In the EPR spectra, especially the impact of the methyl protons was felt. Only the hyperfine coupling constants of the methyl protons could be experimentally measured at 300 K. The EPR spectra of irradiated 3-nitroacetophenone single crystals consist of four hyperfine structure lines with a 1:3:3:1 intensity ratio, which is caused by interaction of the unpaired electron with the three equivalent protons of the methyl group, at most orientations of the crystal in the magnetic field, at 300 K. The H(2) and the H(6) protons are also considered approximately equivalent. The hyperfine coupling for the CH3 group is anisotropic and their average values being aCH<sup>3</sup> = 1.402 mT.

Figure 8. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 300 K when (a) the magnetic field is in the a\*b-plane and is away 20� from the a\*-axis and (b) simulation of the spectrum; line width is 0.34 mT.

Figure 10. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 120 K when (a) the magnetic field is in the a\*c-plane

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294 61

Figure 11. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 120 K when (a) the magnetic field is in the a\*c-plane

and is away 165� from the a\*-axis and (b) simulation of the spectrum; line width is 0.34 mT.

and is away 150� from the a\*-axis and (b) simulation of the spectrum; line width is 0.32 mT.

Figure 9. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 120 K when (a) the magnetic field is in the a\*c-plane and is away 140� from the a\*-axis and (b) simulation of the spectrum; line width is 0.38 mT.

Figure 10. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 120 K when (a) the magnetic field is in the a\*c-plane and is away 150� from the a\*-axis and (b) simulation of the spectrum; line width is 0.32 mT.

Figure 8. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 300 K when (a) the magnetic field is in the a\*b-plane and

Figure 9. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 120 K when (a) the magnetic field is in the a\*c-plane and

is away 140� from the a\*-axis and (b) simulation of the spectrum; line width is 0.38 mT.

is away 20� from the a\*-axis and (b) simulation of the spectrum; line width is 0.34 mT.

60 Free Radicals, Antioxidants and Diseases

Figure 11. EPR spectra of 60Co-γ irradiated 3NAP single crystal at 120 K when (a) the magnetic field is in the a\*c-plane and is away 165� from the a\*-axis and (b) simulation of the spectrum; line width is 0.34 mT.


Author details

Betül Çalişkan<sup>1</sup>

References

2119

\* and Ali Cengiz Çalişkan<sup>2</sup>

1 Department of Physics, Faculty of Arts and Science, Pamukkale University, Denizli, Turkey

EPR Analysis of Antioxidant Compounds http://dx.doi.org/10.5772/intechopen.74294 63

[1] Setiadi DH, Chass GA, Torday LL, Varro A, Papp JG. Vitamin E models. Can the antioxidant and pro-oxidant dichotomy of α-tocopherol be related to ionic ring closing and radical ring opening redox reactions? Journal of Molecular Structure. 2003;620(2-3):93-106

[2] Halliwell B, Gutteridge JMC. Free radicals in Biology and Medicines. 4th ed. Oxford:

[3] Nickel W. The mystery of non-classical protein secretion: A current view on cargo proteins and potential export routes. European Journal of Biochemistry. 2003;270(10):2109-

[4] Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health.

[5] Pietta PG. Flavonoids as antioxidants. Journal of Natural Products. 2000;63(7):1035-1042 [6] Caliskan B, Caliskan AC, Er E. Electron paramagnetic resonance study of gammairradiated potassium hydroquinone monosulfonate single crystal. Radiation Effects and

[7] Caliskan B, Caliskan AC, Er E. Electron paramagnetic resonance study of radiationinduced paramagnetic centers in succinic anhydride single crystal. Journal of Molecular

[8] Caliskan B, Caliskan AC. EPR study of radiation damage in gamma irradiated 3-nitroacetophenone single crystal. Radiation Effects and Defects in Solids. 2017;172(5-6):398-

[9] Thomson RH. Naturally Occurring Quinones. 2nd ed. New York: Academic Press; 1971

[11] O'Brien PJ. Molecular mechanisms of quinone cytotoxicity. Chemico-Biological Interac-

[12] Hashimoto K, Togo H, Morihashi K, Yokoyama Y, Kikuchi O. Conformational change of benzoquinone crown ether radical anions induced by complexation with alkali metal

Defects in Solids. 2016;171(5-6):440-450. DOI: 10.1080/10420150.2016.1203924

Structure. 2017;1144:421-431. DOI: 10.1016/j.molstruc.2017.05.039

[10] Morton RA. Biochemistry of Quinones. New York: Academic Press; 1965

2 Department of Chemistry, Faculty of Science, Gazi University, Ankara, Turkey

\*Address all correspondence to: bcaliska@gmail.com

Oxford University Press; 1985. pp. 120-123

410. DOI: 10.1080/10420150.2017.1320800

tions. 1991;80(1):1-41

International Journal of Biomedical Science. 2008;4(2):89-96

Table 7. Coupling constants values of simulated spectra.


Table 8. The EPR parameters of the 3-nitroacetophenone anion radical observed in 3-nitroacetophenone at 300 K.

The simulations of the EPR spectra were carried out using the Win-EPR software.

### 3. Conclusion

There are many factors that trigger the formation of free radicals. One of these is the radiation effect. The various compounds that undergo radiation effects have the radiation damage centers as a result of breaking bonds in their structures. The radiation damage center is a paramagnetic center, and the detection and motion of these centers are investigated in detail by EPR spectroscopy.

Antioxidants also show paramagnetic structure under radiation effect. EPR analysis of antioxidants is crucial to remove the effect of free radicals that cause many diseases. Potassium hydroquinone monosulfonate (PHM), succinic anhydride and 3-nitroacetophenone compounds show antioxidant properties. When EPR analyses of these materials were made, it was seen that their radical mechanisms were similar to each other. The anion radicals were observed in all three compounds. When the resonance structure and the formation mechanisms are carefully examined, it is seen that the oxygen atom takes an electron and forms a stable anion structure.

### Author details

Betül Çalişkan<sup>1</sup> \* and Ali Cengiz Çalişkan<sup>2</sup>


### References

The simulations of the EPR spectra were carried out using the Win-EPR software.

Principal values Direction cosines

There are many factors that trigger the formation of free radicals. One of these is the radiation effect. The various compounds that undergo radiation effects have the radiation damage centers as a result of breaking bonds in their structures. The radiation damage center is a paramagnetic center, and the detection and motion of these centers are investigated in detail

Figure 8 Figure 9 Figure 10 Figure 11

�0.717129 0.659816 0.224430

0.415100 0.515457 �0.749664 �0.180325 �0.486721

�0.540063 0.802728

aCH<sup>3</sup> ð Þ<sup>β</sup> mT 1.55 1.36 1.68 1.585 aHð Þ<sup>2</sup> ffi aHð Þ<sup>6</sup> ¼ aC2H<sup>2</sup> ð Þ <sup>γ</sup> mT 0.38 0.43 0.45 0.46 aHð Þ<sup>3</sup> ¼ aHð Þ<sup>δ</sup> mT 0.35 0.68 0.71 0.68 aHð Þ<sup>4</sup> ¼ aHð Þ <sup>σ</sup> mT 0.25 0.39 0.265 0.29

> 0.572490 0.468023

> 0.299886 0.611591

Table 8. The EPR parameters of the 3-nitroacetophenone anion radical observed in 3-nitroacetophenone at 300 K.

0.854743 Ayy = 1.447

0.252902 gyy = 2.006110

Table 7. Coupling constants values of simulated spectra.

62 Free Radicals, Antioxidants and Diseases

aCH<sup>3</sup> ð Þ<sup>β</sup> (mT) Axx = 1.743 0.673208

Azz = 1.015 aiso = 1.402 r = 0.52 g gxx = 2.006443 0.732137

> g zz = 2.004067 giso = 2.005540

Antioxidants also show paramagnetic structure under radiation effect. EPR analysis of antioxidants is crucial to remove the effect of free radicals that cause many diseases. Potassium hydroquinone monosulfonate (PHM), succinic anhydride and 3-nitroacetophenone compounds show antioxidant properties. When EPR analyses of these materials were made, it was seen that their radical mechanisms were similar to each other. The anion radicals were observed in all three compounds. When the resonance structure and the formation mechanisms are carefully examined, it is seen that the oxygen atom takes an electron and forms a stable anion structure.

3. Conclusion

by EPR spectroscopy.


cation: An ESR study. Bulletin of the Chemical Society of Japan. 1991;64(11):3245-3250. DOI: 10.1246/bcsj.64.3245

**Chapter 5**

**Provisional chapter**

**Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic**

Numerous researches show that data on an ethnic origin can give additional information for the personified approach in treatment of different diseases. The aim of this study was to evaluate the level of some lipid peroxidation components and antioxidant defense system in Mongoloid and Caucasian patients with Type 1 diabetes mellitus. Conjugated dienes, ketodienes and conjugated trienes, thiobarbituric acid reactants, total antioxidant activity, α-tocopherol, retinol, superoxide dismutase activity, reduced and oxidized glutathione, and oxidative stress coefficient levels were evaluated in 65 patients with type 1 diabetes (38 Mongoloids and 27 Caucasians) and in 82 healthy people (42 Mongoloids and 40 Caucasians). Spectrophotometric and fluorometric methods were used. The intensity of LPO in Mongoloid patients was lower than in Caucasians: the level of primary and intermediate products was by lower 1.53 and 1.83 times, while total antioxidant activity was elevated by 1.44 times, and decreased α-tocopherol level by 1.32 times, which was also supported by oxidative stress coefficient (1.35 in Mongoloids and 2.32 in Caucasians). Activity of the POL-AOD system in Mongoloids is low, which is probably due to the increase of antioxidant defense system work. These results are consistent with clinical characteristics of type 1 diabetes mellitus with infre-

quent development of complications in Mongoloids living in Eastern Siberia.

**Keywords:** type 1 diabetes mellitus, oxidative stress, antioxidant defense, ethnos

Type 1 diabetes mellitus (T1DM) is common in the world and is considered as one of the severe human diseases. This disease is the cause of heart disease, blindness, stroke, kidney

**Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic** 

© 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

DOI: 10.5772/intechopen.76512

Lyubov I. Kolesnikova, Marina A. Darenskaya, Lyudmila A. Grebenkina, Svetlana V. Gnusina and

Lyubov I. Kolesnikova, Marina A. Darenskaya, Lyudmila A. Grebenkina, Svetlana V. Gnusina and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76512

**Aspects**

**Aspects**

Sergey I. Kolesnikov

Sergey I. Kolesnikov

**Abstract**

**1. Introduction**


#### **Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic Aspects Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic Aspects**

DOI: 10.5772/intechopen.76512

Lyubov I. Kolesnikova, Marina A. Darenskaya, Lyudmila A. Grebenkina, Svetlana V. Gnusina and Sergey I. Kolesnikov Lyubov I. Kolesnikova, Marina A. Darenskaya, Lyudmila A. Grebenkina, Svetlana V. Gnusina and Sergey I. Kolesnikov

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76512

#### **Abstract**

cation: An ESR study. Bulletin of the Chemical Society of Japan. 1991;64(11):3245-3250.

[13] Ralph SJ, Moreno-Sánchez R, Neuzil J, Rodríguez-Enríquez S. Inhibitors of succinate: Quinone reductase/Complex II regulate production of mitochondrial reactive oxygen species and protect normal cells from ischemic damage but induce specific cancer cell death. Pharmaceutical Research. 2011;28(12):3274-3274. DOI: 10.1007/s11095-011-0566-7

[14] Wardman P. Some reactions and properties of nitro radical-anions important in biology

[15] Dincer S. The preferential reduction of 4,6 (5,7)-dinitro and 5,6-dinitrobenzimidazoles.

and medicine. Environ. Health Perspect. 1985;64:309-320

DOI: 10.1246/bcsj.64.3245

64 Free Radicals, Antioxidants and Diseases

Dyes Pigments. 2002;53:263-266

Numerous researches show that data on an ethnic origin can give additional information for the personified approach in treatment of different diseases. The aim of this study was to evaluate the level of some lipid peroxidation components and antioxidant defense system in Mongoloid and Caucasian patients with Type 1 diabetes mellitus. Conjugated dienes, ketodienes and conjugated trienes, thiobarbituric acid reactants, total antioxidant activity, α-tocopherol, retinol, superoxide dismutase activity, reduced and oxidized glutathione, and oxidative stress coefficient levels were evaluated in 65 patients with type 1 diabetes (38 Mongoloids and 27 Caucasians) and in 82 healthy people (42 Mongoloids and 40 Caucasians). Spectrophotometric and fluorometric methods were used. The intensity of LPO in Mongoloid patients was lower than in Caucasians: the level of primary and intermediate products was by lower 1.53 and 1.83 times, while total antioxidant activity was elevated by 1.44 times, and decreased α-tocopherol level by 1.32 times, which was also supported by oxidative stress coefficient (1.35 in Mongoloids and 2.32 in Caucasians). Activity of the POL-AOD system in Mongoloids is low, which is probably due to the increase of antioxidant defense system work. These results are consistent with clinical characteristics of type 1 diabetes mellitus with infrequent development of complications in Mongoloids living in Eastern Siberia.

**Keywords:** type 1 diabetes mellitus, oxidative stress, antioxidant defense, ethnos

#### **1. Introduction**

Type 1 diabetes mellitus (T1DM) is common in the world and is considered as one of the severe human diseases. This disease is the cause of heart disease, blindness, stroke, kidney

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

failure, foot ulcers, and so on. A number of studies have shown that complications rate of T1DM depends on different factors including geographic residence and human ethnicity [1–4]. The dominance of T1DM is there in the countries of Scandinavia—Finland and Sweden (63 cases per 100,000 population) [5], while the lowest prevalence is observed in the East and South-East Asia where representatives of the Mongoloid race live [6]. In Russian Federation, there are more than 250,000 patients with T1DM [7]. Some studies revealed that low morbidity of T1DM among the aboriginal people in Arctic and Siberian regions resulted from the presence of the protective allele's genes for this disorder [6]. The prevalence of this form of diabetes among indigenous people in the Buryatia Republic is 24.18 per 100,000, which is below the average level in Russia (224.5 per 100,000) [8, 9].

statistically significant differences in sex, age, duration of disease, Hb A1C, body mass index (BMI), arterial pressure in T1DM groups. There were no statistically significant differences in

Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic Aspects

http://dx.doi.org/10.5772/intechopen.76512

67

Blood samples were taken after 12 h of fasting during night, then were centrifuged for 5 min at 4°C, and erythrocytes were washed three times with NaCI 0.9% (wt/vol). Aliquots of EDTA plasma and washed erythrocytes were used immediately or kept frozen at −40°С but not more than 1 month. Blood plasma and hemolysate were used as the materials for analysis. The blood was taken from the cubital vein in accordance with accepted requirements. The intensity of LPO was evaluated by the level of diene conjugates (DC), ketodienes (KD), and conjugated trienes (CT). The concentration of DCs detected on absorbance of plasma heptanes extracts at 232 nm (μmol/liter), KD and CT at 278 nm (arb. units) [20]. Thiobarbituric acid reactants (TBARs) levels were identified by fluorometry methods and were considered in μmol/liter [21]. An evaluation of АОD activity was carried out on total radical-trapping antioxidant parameter (TRAР), which was measured by specific methods on 2,2′-azinobis- (3-ethylbensothazoline-6-sulphonate) radical action formation (absorbance at 734 nm) in con-

Also contents of other components of AOD system were determined: α-tocopherol and retinol [23], superoxide dismutase (SOD) [24], and reduced and oxidized glutathione (GSH and the GSSG) [25]. Measurements were carried out on the Shimadzu RF-1501 and Shimadzu RF-1650 spectrofluorometer. For more informative description of the LPO–AOD, coefficient of oxidative stress (COS) was calculated as the ratio of LPO–AOD system values in T1DM patient to the mean control group values. At COS > 1, oxidative stress was stated [26]. In our study,

**Mongoloids Caucasians**

) 23.7 ± 2.1 20.3 ± 1.3 24.6 ± 3.1 21.4 ± 3.4

**T1DM Control group T1DM Control group**

diets and physical activity between the patients of both ethnic groups with T1DM.

ditions of exogenous Н<sup>2</sup>

**Clinical Data**

Body mass index (kg/m<sup>2</sup>

О2

presence [22].

n 38 42 27 40 Sex (M / F) 15/23 22/20 15/12 20/20 Age (years) 34.4 ± 11.7 31.4 ± 8.0 32.7 ± 11.9 27.8 ± 7.7

Duration of disease (years) 12.1 ± 3.5 – 12.9 ± 4.0 – Hb A1C (%) 9.29 ± 3.06 – 8.74 ± 2.24 –

**Table 1.** General characteristics of patients with T1DM and control subjects.

Total cholesterol (mmol/l) 4.65 ± 1.2 4.28 ± 1.29 5.66 ± 1.22 4.24 ± 1.25 Triglycerides level (mmol/l) 1.01 ± 0.58 0.67 ± 0.21 1.53 ± 0.66 0.54 ± 0.19 Systolic pressure (mm Hg) 115 ± 11 116 ± 12 117 ± 10 113 ± 11 Diastolic pressure (mm Hg) 73 ± 12 71 ± 12 74 ± 9 76 ± 12

Oxidative stress is an imbalance between increase reactive oxygen species and an antioxidant ability to detoxify the reactive components. Oxidative stress is thought to be involved in the development of different diseases [10–12].

Many experimental [13] and clinical studies [14] suggest that free-radical processes are activated during different stages and in different types of diabetes mellitus, even in its subclinical forms [15, 16]. In patients with diabetes mellitus, oxidative stress (OS) caused by elevated production of reactive species of oxygen and decrease of antioxidant defense system (AOD) level, leads to activation of lipid peroxidation (LPO) and oxidative lipoprotein modification with increasing atherogenicity [17]. Hyperglycemia can induce damage of β-cells functions with development of OS and decrease of thioredoxine level [18]. Marra et al. suggest that T1DM patients with a short duration of disease and good metabolic control show an early imbalance in their antioxidant capacity and augmented levels of lipid hydroperoxides and conjugated dienes (CDs) [19]. Diabetic women show, independently from other factors, a decreased antioxidant capacity and an increased rate of lipoperoxidation compared with diabetic men [19].

At the same time, the link between certain metabolic characteristics in patients with T1DM and their race remains poorly studied. The **aim** of this study was to evaluate the level of some LPO components and AOD system in Mongoloid and Caucasian patients with T1DM.

## **2. An evaluation of the level of some lipid peroxidation components and antioxidant defense system in mongoloid and Caucasian patients with type 1 diabetes mellitus**

Biochemical parameters in 147 persons (healthy and with T1DM) both Mongoloids (ethnic group is Buryats) and Caucasians (ethnic group is Russians) living in the modern city Ulan-Ude (East-Siberia) were assessed. The diagnosis of T1DM was confirmed in all patients based on clinical and laboratory investigations, severe comorbidities and severe diabetic complications were excluded. Main group's characteristics are presented in **Table 1**. There were no statistically significant differences in sex, age, duration of disease, Hb A1C, body mass index (BMI), arterial pressure in T1DM groups. There were no statistically significant differences in diets and physical activity between the patients of both ethnic groups with T1DM.

failure, foot ulcers, and so on. A number of studies have shown that complications rate of T1DM depends on different factors including geographic residence and human ethnicity [1–4]. The dominance of T1DM is there in the countries of Scandinavia—Finland and Sweden (63 cases per 100,000 population) [5], while the lowest prevalence is observed in the East and South-East Asia where representatives of the Mongoloid race live [6]. In Russian Federation, there are more than 250,000 patients with T1DM [7]. Some studies revealed that low morbidity of T1DM among the aboriginal people in Arctic and Siberian regions resulted from the presence of the protective allele's genes for this disorder [6]. The prevalence of this form of diabetes among indigenous people in the Buryatia Republic is 24.18 per 100,000, which is

Oxidative stress is an imbalance between increase reactive oxygen species and an antioxidant ability to detoxify the reactive components. Oxidative stress is thought to be involved in the

Many experimental [13] and clinical studies [14] suggest that free-radical processes are activated during different stages and in different types of diabetes mellitus, even in its subclinical forms [15, 16]. In patients with diabetes mellitus, oxidative stress (OS) caused by elevated production of reactive species of oxygen and decrease of antioxidant defense system (AOD) level, leads to activation of lipid peroxidation (LPO) and oxidative lipoprotein modification with increasing atherogenicity [17]. Hyperglycemia can induce damage of β-cells functions with development of OS and decrease of thioredoxine level [18]. Marra et al. suggest that T1DM patients with a short duration of disease and good metabolic control show an early imbalance in their antioxidant capacity and augmented levels of lipid hydroperoxides and conjugated dienes (CDs) [19]. Diabetic women show, independently from other factors, a decreased antioxidant capacity and an increased rate of lipoperoxidation compared with

At the same time, the link between certain metabolic characteristics in patients with T1DM and their race remains poorly studied. The **aim** of this study was to evaluate the level of some LPO components and AOD system in Mongoloid and Caucasian patients with

**2. An evaluation of the level of some lipid peroxidation components** 

Biochemical parameters in 147 persons (healthy and with T1DM) both Mongoloids (ethnic group is Buryats) and Caucasians (ethnic group is Russians) living in the modern city Ulan-Ude (East-Siberia) were assessed. The diagnosis of T1DM was confirmed in all patients based on clinical and laboratory investigations, severe comorbidities and severe diabetic complications were excluded. Main group's characteristics are presented in **Table 1**. There were no

**and antioxidant defense system in mongoloid and Caucasian** 

**patients with type 1 diabetes mellitus**

below the average level in Russia (224.5 per 100,000) [8, 9].

development of different diseases [10–12].

66 Free Radicals, Antioxidants and Diseases

diabetic men [19].

T1DM.

Blood samples were taken after 12 h of fasting during night, then were centrifuged for 5 min at 4°C, and erythrocytes were washed three times with NaCI 0.9% (wt/vol). Aliquots of EDTA plasma and washed erythrocytes were used immediately or kept frozen at −40°С but not more than 1 month. Blood plasma and hemolysate were used as the materials for analysis. The blood was taken from the cubital vein in accordance with accepted requirements. The intensity of LPO was evaluated by the level of diene conjugates (DC), ketodienes (KD), and conjugated trienes (CT). The concentration of DCs detected on absorbance of plasma heptanes extracts at 232 nm (μmol/liter), KD and CT at 278 nm (arb. units) [20]. Thiobarbituric acid reactants (TBARs) levels were identified by fluorometry methods and were considered in μmol/liter [21]. An evaluation of АОD activity was carried out on total radical-trapping antioxidant parameter (TRAР), which was measured by specific methods on 2,2′-azinobis- (3-ethylbensothazoline-6-sulphonate) radical action formation (absorbance at 734 nm) in conditions of exogenous Н<sup>2</sup> О2 presence [22].

Also contents of other components of AOD system were determined: α-tocopherol and retinol [23], superoxide dismutase (SOD) [24], and reduced and oxidized glutathione (GSH and the GSSG) [25]. Measurements were carried out on the Shimadzu RF-1501 and Shimadzu RF-1650 spectrofluorometer. For more informative description of the LPO–AOD, coefficient of oxidative stress (COS) was calculated as the ratio of LPO–AOD system values in T1DM patient to the mean control group values. At COS > 1, oxidative stress was stated [26]. In our study,


**Table 1.** General characteristics of patients with T1DM and control subjects.

all patients and control groups signed informed consent according to the World Medical Association Declaration of Helsinki (1964, 2000). For statistic analysis of the data, Statistica 6.1 software (StatSoft Inc.) was used. To determine normal distribution of the quantitative data, graphic visual method and Kolmogorov-Smirnov Test with Lilliefors and Shapiro-Wilk corrections were used. The variances equality was verified by Fisher's Test. Descriptive statistics was applied for quantitative data description: mean ± error of the mean. The differences between parameters of groups by parametric Student's Test for independent samples and non-parametric Mann–Whitney Test were analyzed. The critical significance level was considered 5% (p < 0.05).

### **3. Results**

The oxidative stress is considered an imbalance in the redox-state with isolated or combined variations in the pro- or antioxidant components concentration. Our study has shown higher concentration of DC (by 1.35 times; *p* < 0.01) in Mongoloid patients as well as higher levels of DC (by 2.4 times; *p* < 0.001) and KD and CT (by 2.71 times; *p* < 0.05) in Caucasian patients in comparison to the corresponding control groups (**Table 2**).

p < 0.001) (**Table 2**). Also in this group in comparison with control, statistically significant differences in activity of SOD, α-tocopherol, and retinol levels were not noted. Initiation of processes of lipid peroxidation at primary and intermediate stages in the absence of natural AОD activity enhancement can lead to function impairment of different components of hemostasis and increased aggregation of blood cell, which will increase blооd viscosity, induce thickening of the vascular wall basal membrane, slower blооd flow in small and medium vessels, deterioration of micrоcirculation [24]. The given changes in parameters of lipid peroxidation can attest about the presence of risk factors for microangiopathy development in Caucasian patients with T1DM. Comparison of LPO values in Caucasian and Mongoloid patients showed decreased DC (by 1.56 times; p < 0.001), KD and CT (by 1.86 times; p < 0.05) values, increased TRAP (by 1.44 times; p < 0.001), and decreased α-tocopherol levels (1.22-times, p < 0.01) in Mongoloid patients with T1DM in compare with the same values in Caucasian patients with T1DM (**Tables 1** and **2**). Coefficient of oxidative stress (COS) in Mongoloid patients was 1.35, in Caucasian patients was 2.32 (p < 0.05). It is believed that CОS value>1 indicates activation of oxidative stress. The higher is CОS, the more insensitive are processes lipid peroxidation processes and less effective is the antioxidant defense system in the examined patients with different diseases. So, our results indicate increased LPO processes in grоups of patients with diabetes mellitus and intensity of LPO processes depends on ethnicity [29]. Noted changes in LPO-AOD system in Mongoloid patients with T1DM were less insensitive than in Caucasian patients, that allows to make a recommendation on highly individualized approaches to the

**Control group T1DM Control group T1DM**

, + 69

Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic Aspects

http://dx.doi.org/10.5772/intechopen.76512

**Parameters Mongoloids Caucasians**

**Table 3.** AOD values in T1DM in mongoloid and Caucasian patients (M ± m).

TRAP, arb. Units 14.35 ± 0.72 22.17 ± 1.08\* 17.68 ± 1.36 15.42 ± 0.96+ α-tocopherol, μmol/liter 6.85 ± 0.38 6.23 ± 0.32 6.72 ± 0.3 8.21 ± 0.78\*

retinol, μmol/liter 2.7 ± 0.18 2.29 ± 0.15 2.38 ± 0.13 2.33 ± 0.17 SOD, arb. Units 1.41 ± 0.04 1.26 ± 0.03 1.43 ± 0.44 1.57 ± 0.24 GSH, mmol/liter 2.79 ± 0.16 2.79 ± 0.11 2.90 ± 0.14 2.51 ± 0.13\* GSSG, mmol/liter 2.03 ± 0.15 2.15 ± 0.09 1.72 ± 0.09 2.16 ± 0.10\*

We suppose that ethnic factor plays one of the most important roles in the course of various diseases, including T1DM. It is quite possible that the low incidence in T1DM in Mongoloids is based on less LPO-AOD metabolic imbalance. Further studies of ethnically associated metabolic features can give more opportunities for developing specific approaches for diagnos-

tics, prophylactic and treatment of T1DM in patients of different ethnic groups.

cоmplex therapy.

**4. Conclusions**

No statistically significant differences between investigated groups in the TBA-reactive products level of lipid peroxidation processes were identified. In conditions of increased generation of LPO products are observed changes permeability of cell membranes for a lot of ions, nonelectrolytes, and macromolecules [27]. This processes in loss of barrier function of cell membranes that is the pathogenesis of different diseases, including development of vascular disorders in T1DM due to lipid peroxidation activation during prolonged hyperglycemia [9, 28]. The observed increase in LPO activity cannot provide sufficient information about the redox-status in patients with T1DM, because it indicates one aspect of the study, LPO excluding of AOD activity. The study of the AOD integral parameter level, TRAP in patients with T1DM, an increase (by 1.54 times; p < 0.001) in this indicator in Mongoloid patients in comparison with the control group was shown (**Table 3**).

In Caucasian patients with T1DM, statistically significant differences from the control group included reduced GSH values (by 1.16 times; p < 0.05) and increased GSSG level (1.26-times,


**Table 2.** Level of LPO products in DM1 mongoloid and Caucasian patients (M ± m).


**Table 3.** AOD values in T1DM in mongoloid and Caucasian patients (M ± m).

p < 0.001) (**Table 2**). Also in this group in comparison with control, statistically significant differences in activity of SOD, α-tocopherol, and retinol levels were not noted. Initiation of processes of lipid peroxidation at primary and intermediate stages in the absence of natural AОD activity enhancement can lead to function impairment of different components of hemostasis and increased aggregation of blood cell, which will increase blооd viscosity, induce thickening of the vascular wall basal membrane, slower blооd flow in small and medium vessels, deterioration of micrоcirculation [24]. The given changes in parameters of lipid peroxidation can attest about the presence of risk factors for microangiopathy development in Caucasian patients with T1DM. Comparison of LPO values in Caucasian and Mongoloid patients showed decreased DC (by 1.56 times; p < 0.001), KD and CT (by 1.86 times; p < 0.05) values, increased TRAP (by 1.44 times; p < 0.001), and decreased α-tocopherol levels (1.22-times, p < 0.01) in Mongoloid patients with T1DM in compare with the same values in Caucasian patients with T1DM (**Tables 1** and **2**). Coefficient of oxidative stress (COS) in Mongoloid patients was 1.35, in Caucasian patients was 2.32 (p < 0.05). It is believed that CОS value>1 indicates activation of oxidative stress. The higher is CОS, the more insensitive are processes lipid peroxidation processes and less effective is the antioxidant defense system in the examined patients with different diseases. So, our results indicate increased LPO processes in grоups of patients with diabetes mellitus and intensity of LPO processes depends on ethnicity [29]. Noted changes in LPO-AOD system in Mongoloid patients with T1DM were less insensitive than in Caucasian patients, that allows to make a recommendation on highly individualized approaches to the cоmplex therapy.

#### **4. Conclusions**

all patients and control groups signed informed consent according to the World Medical Association Declaration of Helsinki (1964, 2000). For statistic analysis of the data, Statistica 6.1 software (StatSoft Inc.) was used. To determine normal distribution of the quantitative data, graphic visual method and Kolmogorov-Smirnov Test with Lilliefors and Shapiro-Wilk corrections were used. The variances equality was verified by Fisher's Test. Descriptive statistics was applied for quantitative data description: mean ± error of the mean. The differences between parameters of groups by parametric Student's Test for independent samples and non-parametric Mann–Whitney Test were analyzed. The critical significance level was con-

The oxidative stress is considered an imbalance in the redox-state with isolated or combined variations in the pro- or antioxidant components concentration. Our study has shown higher concentration of DC (by 1.35 times; *p* < 0.01) in Mongoloid patients as well as higher levels of DC (by 2.4 times; *p* < 0.001) and KD and CT (by 2.71 times; *p* < 0.05) in Caucasian patients in

No statistically significant differences between investigated groups in the TBA-reactive products level of lipid peroxidation processes were identified. In conditions of increased generation of LPO products are observed changes permeability of cell membranes for a lot of ions, nonelectrolytes, and macromolecules [27]. This processes in loss of barrier function of cell membranes that is the pathogenesis of different diseases, including development of vascular disorders in T1DM due to lipid peroxidation activation during prolonged hyperglycemia [9, 28]. The observed increase in LPO activity cannot provide sufficient information about the redox-status in patients with T1DM, because it indicates one aspect of the study, LPO excluding of AOD activity. The study of the AOD integral parameter level, TRAP in patients with T1DM, an increase (by 1.54 times; p < 0.001) in this indicator in Mongoloid patients in

In Caucasian patients with T1DM, statistically significant differences from the control group included reduced GSH values (by 1.16 times; p < 0.05) and increased GSSG level (1.26-times,

**Control group T1DM Control group T1DM**

0.31 ± 0.05 0.35 ± 0.04 0.24 ± 0.06 0.65 ± 0.13\*

Caucasian T1DM patients.

, +

comparison to the corresponding control groups (**Table 2**).

comparison with the control group was shown (**Table 3**).

**Parameters Mongoloids Caucasians**

**Table 2.** Level of LPO products in DM1 mongoloid and Caucasian patients (M ± m).

Diene conjugates, μmol/liter 0.57 ± 0.03 0.77 ± 0.04\* 0.51 ± 0.07 1.2 ± 0.11\*,+

TBA-reactive products, μmol/liter 1.57 ± 0.10 1.81 ± 0.11 1.93 ± 0.10 2.05 ± 0.12

corresponding control, +

sidered 5% (p < 0.05).

68 Free Radicals, Antioxidants and Diseases

Ketodienes and conjugated trienes, arb. units

Here and in **Table 3**: *p* < 0.05 in comparison with \*

**3. Results**

We suppose that ethnic factor plays one of the most important roles in the course of various diseases, including T1DM. It is quite possible that the low incidence in T1DM in Mongoloids is based on less LPO-AOD metabolic imbalance. Further studies of ethnically associated metabolic features can give more opportunities for developing specific approaches for diagnostics, prophylactic and treatment of T1DM in patients of different ethnic groups.

## **Conflict of interest**

There is no conflict of interest.

### **Author details**

Lyubov I. Kolesnikova1 , Marina A. Darenskaya1 \*, Lyudmila A. Grebenkina1 , Svetlana V. Gnusina1 and Sergey I. Kolesnikov1,2

\*Address all correspondence to: marina\_darenskaya@inbox.ru

1 Scientific Centre for Family Health and Human Reproduction Problems, Irkutsk, Russian Federation

[8] Dedov II, Kolesnikova LI, Bardymova TP, Prokofiev SA, Ivanova ON. Clinical, genetic and metabolic features of diabetes mellitus in patients with Buryat population. Diabetes

Oxidative Stress in Type 1 Diabetes Mellitus: Ethnic Aspects

http://dx.doi.org/10.5772/intechopen.76512

71

[9] Kolesnikova LI, Vlasov BY, Kolesnikov SI, Darenskaya MA, Grebenkina LA, Semenova NV, Vanteeva OA. Intensity of oxidative stress in mongoloid and Caucasian patients with type 1 diabetes mellitus. Bulletin of Experimental Biology and Medicine. 2016;**161**(6):

[10] Kolesnikova LI, Vlasov B Ya, Kolesnikov SI, Darenskaya M.A., Grebenkina LA, Natyaganova LV, Semenova NV, Gnusina SV. The values of lactate, pyruvate and their ratio in patients with diabetes mellitus type I. Clinical Laboratory Diagnosis. 2016;

[11] Sies H. Oxidative stress: A concept in redox biology and medicine. Redox Biology.

[12] Kolesnikova LI, Kolesnikov SI, Darenskaya MA, Grebenkina LA, Nikitina OA, Lazareva LM, Suturina LV, Danusevich IN, Druzhinina EB, Semendyaev AA. Activity of LPO processes in women with polycystic ovarian syndrome and infertility. Bulletin of Experimental Biology and Medicine. 2017;**162**(3):320-322. DOI: 10.1007/s10517-017-3605-5

[13] Maritim AC, Sanders RA, Watkins JB. Diabetes, oxidative stress, and antioxidants: A review. Journal of Biochemical and Molecular Toxicology. 2003;**17**:24-38. DOI: 10.1002/

[14] Martin-Gallan P, Carrascosa A, Gussinye M, Dominguez C. Estimation of lipoperoxidative damage and antioxidant status in diabetic children: Relationship with individual

[15] Martin-Gallan P, Carrascosa A, Gussinye M, Dominguez C. Biomarkers of diabetesassociated oxidative stress and antioxidadant status in young diabetic patients with or without subclinical complications. Free Radical Biology & Medicine. 2003;**34**:1563-1574

[16] Takayanagi R, Inoguchi T, Ohnaka K. Clinical and experimental evidence for oxidative stress as an exacerbating factor of diabetes mellitus. Journal of Clinical Biochemistry and

[17] Taskinen MR. Diabetic deslipidaemia: From basic research to clinical practice.

[18] Miyzaki Y, Kawano H, Yoshido T, Miyamoto S, Hokamaki J, Nagayoshi Y, Yamabe H, Nakamura H, Yodoi J, Ogawa H. Pancreatic B-cell function is altered by oxidative stress

[19] Marra G, Cotroneo P, Pitocco D, Manto A, Di Leo MAS, Ruotolo V, Caputo S, Giardina B, Ghirlanda G, Santini SA. Early increase of oxidative stress and reduced antioxidant defenses in patients with uncomplicated type 1 diabetes. Diabetes Care. 2002;**25**:370-375.

Diabetologia. 2003;**146**:733-749. DOI: 10.1007/s00125-003-1111-y

unduced by acute hyperglicaemia. Diabetic Medicine. 2007;**24**:154-160

Mellitus. 2006;**9**(1):2-8. DOI: 10.14341/2072-0351-6166 (in Russian)

767-769. DOI: 10.1007/s10517-016-3505-0

**61**(7):405-407. DOI: 10.2337/dc14-2205 (in Russian)

2015;**4**:180-183. DOI: 10.1016/j.redox.2015.01.002

antioxidant. Free Radical Research. 2005;**39**:933-942

jbt.10058

Nutrition. 2010;**48**(1):72-77

DOI: 10.2337/diacare.25.2.370

2 Lomonosov Moscow State University, Moscow, Russian Federation

### **References**


[8] Dedov II, Kolesnikova LI, Bardymova TP, Prokofiev SA, Ivanova ON. Clinical, genetic and metabolic features of diabetes mellitus in patients with Buryat population. Diabetes Mellitus. 2006;**9**(1):2-8. DOI: 10.14341/2072-0351-6166 (in Russian)

**Conflict of interest**

70 Free Radicals, Antioxidants and Diseases

**Author details**

Lyubov I. Kolesnikova1

Svetlana V. Gnusina1

Federation

**References**

S0140-6736(11)60679-X

and Therapeutics. 2017;**19**(1):126

immunity Reviews. 2010;**9**:355-365

New York Academy of Sciences. 2006;**1079**:51-59

There is no conflict of interest.

, Marina A. Darenskaya1

1 Scientific Centre for Family Health and Human Reproduction Problems, Irkutsk, Russian

[1] Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions

[2] Danaei G, Finucane MM, Lu Y, Singh GM, Cowan MJ, Paciorek CJ, Lin JK, Farzadfar F, Khang YH, Stevens GA, Rao M, Ali MK, Riley LM, Robinson CA, Ezzati M. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: Systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2 metabolic features can. Lancet. 2011;**378**:31-40. DOI: 10.1016/

[3] Sivaprasad S, Gupta B, Crosby-Nwaobi R, Evans J. Prevalence of diabetic retinopathy in various ethnic groups: A worldwide perspective. Survey of Ophthalmology.

[4] Kolesnikova L, Kolesnikov S, Darenskaya M, Grebenkina L, Gnusina S. Lipid status in patients with diabetes mellitus type 1 from various ethnic groups. Diabetes Technology

[5] Borchers AT, Uibo R, Gershwin ME. The geoepidemiology of type 1 diabetes. Auto-

[6] Ikegami H, Fujisawa T, Kawabata Y, Noso S, Ogihara T. Genetics of type 1 diabetes: Similarities and diff erences between Asian and Caucasian populations. Annals of the

[7] Dedov II, Shestakova MV, Vikulova OK. Epidemiology of diabetes mellitus in the Russian Federation: Clinical and statistical analysis according to the Federal Register of Diabetes

Mellitus. Diabetes Mellitus. 2017;**20**(1):13-41. DOI: 10.14341/DM8664 (in Russian)

and Sergey I. Kolesnikov1,2

2 Lomonosov Moscow State University, Moscow, Russian Federation

\*Address all correspondence to: marina\_darenskaya@inbox.ru

in type 1 diabetes. Nature. 2010;**464**(7293):1293-1300

2012;**57**(4):347-370. DOI: 10.1016/j.survophthal.2012.01.004

\*, Lyudmila A. Grebenkina1

,


[20] Volchegorskii IA, Nalimov AG, Yarovinskii BG, Lifshits RI. A comparison of different approaches to measurements of LPO products in heptane-isopropanol extracts of the blood. Voprosy medicinskoj himii. 1989;**35**(1):127-131 (in Russian)

**Chapter 6**

**Provisional chapter**

**Dimethylformamide Reduces Cerebral Ischaemia in**

**in Diabetic Rats Hours after Its Occurrence; A New** 

The antioxidant properties of dimethylformamide (DMF) depend on its interference with the hydroxyl-radical-transduction pathway. Diabetes is a risk factor of cerebral ischaemia (CI), and both entities are associated with oxidative stress (OS). We evaluated DMF's effects on CI in non-diabetic rats (NDRs) and in diabetic rats (DRs). One hour after CI, the animals were divided into two treatment groups (300 μl subcutaneous): either DMF or isotonic saline solution. Treatment effects were analysed in NDRs or DRs without CI. Eight hours after CI, a neurophysiologic score (NS) was determined; CI and OS biomarkers were measured in the ischaemic cerebral hemisphere. Infarct/oedema volumes were measured on dyed brain slices. DMF reduced infarct volume in NDRs and DRs but only improved the NS in DRs. Basal concentrations of all the biomarkers were similar in the NDRs and DRs. Metalloproteinase 9 (MMP9) did not change with DMF. Malondialdehyde (MDA) increased with CI, and DMF only reduced it in DRs. RAGE, nitrite/nitrate and nitrotyrosine increased with CI only in DRs (all prevented by DMF). We conclude that DMF's benefits on CI were greater in the DRs due to a higher susceptibility of diabetic animals to the OS produced by CI. The results open a new hori-

**Dimethylformamide Reduces Cerebral Ischaemia** 

© 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

DOI: 10.5772/intechopen.73700

**Diabetic Rats Hours after Its Occurrence; A New**

**Horizon**

**Horizon**

Julio Amadeo Mendoza-Betancourt, Robert David Kross, Maria Angeles Moro,

Maria Angeles Moro, Ignacio Lizasoain,

http://dx.doi.org/10.5772/intechopen.73700

**Abstract**

antioxidant

Luis Humberto Pérez-Astudillo,

Ignacio Lizasoain, Luis Humberto Pérez-Astudillo,

Julio Amadeo Mendoza-Betancourt, Robert David Kross,

zon in CI treatment since DMF has not been investigated before.

**Keywords:** cerebral ischaemia, diabetes, oxidative stress, dimethylformamide,

Arturo Alva-Félix-Díaz and Cleva Villanueva

Arturo Alva-Félix-Díaz and Cleva Villanueva

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **Dimethylformamide Reduces Cerebral Ischaemia in Diabetic Rats Hours after Its Occurrence; A New Horizon Dimethylformamide Reduces Cerebral Ischaemia in Diabetic Rats Hours after Its Occurrence; A New Horizon**

DOI: 10.5772/intechopen.73700

Julio Amadeo Mendoza-Betancourt, Robert David Kross, Maria Angeles Moro, Ignacio Lizasoain, Luis Humberto Pérez-Astudillo, Arturo Alva-Félix-Díaz and Cleva Villanueva Julio Amadeo Mendoza-Betancourt, Robert David Kross, Maria Angeles Moro, Ignacio Lizasoain, Luis Humberto Pérez-Astudillo, Arturo Alva-Félix-Díaz and Cleva Villanueva

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73700

#### **Abstract**

[20] Volchegorskii IA, Nalimov AG, Yarovinskii BG, Lifshits RI. A comparison of different approaches to measurements of LPO products in heptane-isopropanol extracts of the

[21] Gavrilov VB, Gavrilova AR. Mazhulovandianalysis of methods for measurements of LPO products in the blood serum by the test with thiobarbituric acid. Voprosy medicin-

[22] Klebanov GI, Babenkova IV, Teselkin YO, Komarov OS, Vladimirov YA. Evaluation of antioxidant activity of blood plasma using yolk lipoprotein. Laboratornoe Delo.

[23] Cheryauskene RC, Varshkyavichene ZZ, Gribauskas PS. Parallel measurements of the concentrations of vitamins E and a in the serum. Laboratornoe Delo. 1984;**6**:362-365 (in

[24] Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry.

[25] Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Analytical Biochemistry. 1976;**74**(1):214-226. DOI: 10.1016/0003-

[26] Kolesnikova LI, Semyonova NV, Grebenkina LA, Darenskaya MA, Suturina LV, Gnusina SV. Integral indicator of oxidative stress in human blood. Bulletin of Experimental

[27] Kolesnikova LI, Kolesnikov SI, Darenskaya MA, Grebenkina LA, Semenova NV, Osipova EV, Gnusina SV, Bardymova TA. Lipid status and predisposing genes in patients with diabetes mellitus type 1 from various ethnic groups. Bulletin of Experimental Biology

and Medicine. 2015;**160**(2):278-280. DOI: 10.1007/s10517-015-3149-5 (in Russian)

[28] Kolesnikova LI, Darenskaya MA, Grebyonkina LA, Suturina LV, Labygina AV, Semenova NV, Tsyrenov TB, Darzhaev ZY, Kurashova NA, Tolpygina OA. State features of the antioxidant system at healthy people of the basic ethnic groups of Baikal Lake. Voprosy

[29] Stadler K. Oxidative stress in diabetes. In: Ahmad SI, editor. Diabetes. Advances in Experimental Medicine and Biology. New York: Springer; 2013. pp. 272-287. DOI: 10.1007/

Biology and Medicine. 2014;**157**(6):715-717. DOI: 10:1007/s10517-014-2649-z

blood. Voprosy medicinskoj himii. 1989;**35**(1):127-131 (in Russian)

skoj himii. 1987;**33**(1):118-121 (in Russian)

1988;**5**:59-62 (in Russian)

72 Free Radicals, Antioxidants and Diseases

1972;**247**(10):3170-3175

Pitaniya. 2012;**81**(3):46-51 (in Russian)

978-1-4614-5441-0\_21

2697(76)90326-2

Russian)

The antioxidant properties of dimethylformamide (DMF) depend on its interference with the hydroxyl-radical-transduction pathway. Diabetes is a risk factor of cerebral ischaemia (CI), and both entities are associated with oxidative stress (OS). We evaluated DMF's effects on CI in non-diabetic rats (NDRs) and in diabetic rats (DRs). One hour after CI, the animals were divided into two treatment groups (300 μl subcutaneous): either DMF or isotonic saline solution. Treatment effects were analysed in NDRs or DRs without CI. Eight hours after CI, a neurophysiologic score (NS) was determined; CI and OS biomarkers were measured in the ischaemic cerebral hemisphere. Infarct/oedema volumes were measured on dyed brain slices. DMF reduced infarct volume in NDRs and DRs but only improved the NS in DRs. Basal concentrations of all the biomarkers were similar in the NDRs and DRs. Metalloproteinase 9 (MMP9) did not change with DMF. Malondialdehyde (MDA) increased with CI, and DMF only reduced it in DRs. RAGE, nitrite/nitrate and nitrotyrosine increased with CI only in DRs (all prevented by DMF). We conclude that DMF's benefits on CI were greater in the DRs due to a higher susceptibility of diabetic animals to the OS produced by CI. The results open a new horizon in CI treatment since DMF has not been investigated before.

**Keywords:** cerebral ischaemia, diabetes, oxidative stress, dimethylformamide, antioxidant

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

### **1. Introduction**

Stroke is one of the leading causes of death and disability worldwide [1]. To date, thrombolysis is the only approved therapy for acute stroke, but the window of time for treatment is short (3–4 h after a stroke). There is an associated risk of haemorrhagic transformation with thrombolysis, which increases the morbidity and mortality of ischaemic stroke [2], and the efficacy of this treatment is small. There is an obvious need for pursuing new treatment strategies to address these deficiencies. Being diabetic is an important risk of suffering a stroke [3]. Oxidative stress (OS) has been associated with both diabetes [4] and stroke [5, 6]. Some antioxidants have shown beneficial effects in animal models of diabetes [7]. However, exogenous antioxidant effects on humans are difficult to evaluate. Indeed, the long-term ingestion of antioxidants included in one's diet seems to provide beneficial health effects [8, 9], whereas there is still some controversy with respect to the usefulness of long-term antioxidant supplements [10].

**2.1. Diabetes model**

ated haemoglobin at the end of the experiment.

**2.2. Cerebral ischaemia model**

animals per group).

**2.3. Experimental groups**

**2.4. Neurophysiological test**

score was 12 points.

**2.5. Infarct and oedema evaluation**

Type 1 diabetes mellitus was induced in 4-week old male Wistar rats (79.7 ± 1.8 g), through the intraperitoneal (ip) administration of streptozotocin (STZ, 75 mg/kg, Sigma St. Louis, USA). Diabetes was confirmed 3 days later by the onset of hyperglycaemia (>200 mg/dL). A control group (same characteristics, non-diabetic) was also maintained during the experiment. All the animals were fed Purina chow and water *ad libitum* and kept in light-darkness cycles of 12×12 h. The evolution of diabetes was evaluated by: (1) weighing the rats weekly and comparing the weights of the diabetic and the control animals and (2) measuring glycaemia and glucosyl-

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Six weeks after diabetes induction, cerebral ischaemia was produced in diabetic as well as the control animals by slightly modifying the model reported by De Cristóbal et al. [15]. In short, the animals were anaesthetised through intramuscular administration of an anaesthesia cocktail containing acepromazine (5 mg/kg), ketamine (5 mg/kg) and butorphanol (13 mg/kg). The right carotid and the middle cerebral arteries were permanently ligated. One hour later the animals were randomly divided into two treatment groups (eight control and eight diabetic

**1.** Isotonic saline solution (ISS, 300 μl) administered subcutaneously (sc)

**2.** DMF (300 μl, equivalent to 0.9 g/kg, Sigma, St. Louis, USA) also administered sc

Eight hours after cerebral ischaemia was induced, the neurological signs were scored as follows (sum of all): consciousness (0–3, where 3 was the response only after stimulation), decreased grip of the left forelimb when the tail was pulled (0–3, 3 was complete gripping), failure to grab a bar with the left forelimb (0–3, 0 was strongly grabbing with both forelimbs, 3 was no grabbing at all with the left forelimb), and walking in a circle to the left side (0–3, 3 was walking spontaneously in circles). If the animal did not respond to any stimulus, the

The animals were anaesthetised (pentobarbital, 75 mg/kg, ip). A blood sample (at least 5 ml) was taken from the heart and anticoagulated with ethylenediaminetetraacetic acid (EDTA, Sigma, St. Louis, USA). The blood glucose and glucosylated haemoglobin were measured. The animals were decapitated after taking the blood samples, and each brain was immediately extracted and sliced (2 mm slices). The slices were incubated in a 1% triphenyltetrazolium chloride (Sigma, St. Louis, USA)/ISS solution, at 37°C for 5 min). Both sides of each slice were photographed against

Most of the positive effects of antioxidants on stroke models are the result of prophylactic strategies (treatment administered before stroke) [11]. The applicability of such approaches in humans yields dubious results because, although there are risk factors that could identify stroke-susceptible patients, it is virtually impossible to predict who would actually suffer a stroke.

N-N-dimethylformamide (DMF) is an amphipathic solvent significantly miscible with water and most organic solvents. It has been shown that DMF significantly protected against neuronal damage produced by dopamine or hydrogen peroxide. Even though the results were attributed to an increase in antioxidant enzymes [12], it seems that DMF interferes with the signal transduction pathway originated by the hydroxyl radical, which includes changes in permeability and ion exchange in the different compartments of mitochondria [13]. Moreover, DMF seems to have anti-ageing effects associated with protein homeostasis, which are independent of its antioxidant properties [14].

Signal transduction mediated by free radicals (including the hydroxyl radical) acts as a double-edge sword. It could be harmful (*i.e.* oxidative damage to biomolecules—proteins, lipids, RNA/DNA-) or beneficial (leading to the synthesis of antioxidant enzymes through nuclear factor kappa B activation) [10]. Free radicals generated during cerebral ischaemia (a first wave within minutes followed by a second wave shortly thereafter) participate in the cascade of events generating neuronal damage, which includes changes in mitochondria function [6]. We postulate that signal transduction mediated by the hydroxyl radical (one of those free radicals liberated during cerebral ischaemia) is one of the harmful events in cerebral ischaemia, and DMF would reduce such ischaemic damage. Therefore, the goal of this study was to precisely investigate the effects of a single dose of DMF, administered 1 h after induced cerebral ischaemia in rats.

### **2. Material and methods**

The Institutional Ethical Committee approved the present work.

#### **2.1. Diabetes model**

**1. Introduction**

74 Free Radicals, Antioxidants and Diseases

Stroke is one of the leading causes of death and disability worldwide [1]. To date, thrombolysis is the only approved therapy for acute stroke, but the window of time for treatment is short (3–4 h after a stroke). There is an associated risk of haemorrhagic transformation with thrombolysis, which increases the morbidity and mortality of ischaemic stroke [2], and the efficacy of this treatment is small. There is an obvious need for pursuing new treatment strategies to address these deficiencies. Being diabetic is an important risk of suffering a stroke [3]. Oxidative stress (OS) has been associated with both diabetes [4] and stroke [5, 6]. Some antioxidants have shown beneficial effects in animal models of diabetes [7]. However, exogenous antioxidant effects on humans are difficult to evaluate. Indeed, the long-term ingestion of antioxidants included in one's diet seems to provide beneficial health effects [8, 9], whereas there is still some

controversy with respect to the usefulness of long-term antioxidant supplements [10].

tible patients, it is virtually impossible to predict who would actually suffer a stroke.

pendent of its antioxidant properties [14].

cerebral ischaemia in rats.

**2. Material and methods**

The Institutional Ethical Committee approved the present work.

Most of the positive effects of antioxidants on stroke models are the result of prophylactic strategies (treatment administered before stroke) [11]. The applicability of such approaches in humans yields dubious results because, although there are risk factors that could identify stroke-suscep-

N-N-dimethylformamide (DMF) is an amphipathic solvent significantly miscible with water and most organic solvents. It has been shown that DMF significantly protected against neuronal damage produced by dopamine or hydrogen peroxide. Even though the results were attributed to an increase in antioxidant enzymes [12], it seems that DMF interferes with the signal transduction pathway originated by the hydroxyl radical, which includes changes in permeability and ion exchange in the different compartments of mitochondria [13]. Moreover, DMF seems to have anti-ageing effects associated with protein homeostasis, which are inde-

Signal transduction mediated by free radicals (including the hydroxyl radical) acts as a double-edge sword. It could be harmful (*i.e.* oxidative damage to biomolecules—proteins, lipids, RNA/DNA-) or beneficial (leading to the synthesis of antioxidant enzymes through nuclear factor kappa B activation) [10]. Free radicals generated during cerebral ischaemia (a first wave within minutes followed by a second wave shortly thereafter) participate in the cascade of events generating neuronal damage, which includes changes in mitochondria function [6]. We postulate that signal transduction mediated by the hydroxyl radical (one of those free radicals liberated during cerebral ischaemia) is one of the harmful events in cerebral ischaemia, and DMF would reduce such ischaemic damage. Therefore, the goal of this study was to precisely investigate the effects of a single dose of DMF, administered 1 h after induced Type 1 diabetes mellitus was induced in 4-week old male Wistar rats (79.7 ± 1.8 g), through the intraperitoneal (ip) administration of streptozotocin (STZ, 75 mg/kg, Sigma St. Louis, USA). Diabetes was confirmed 3 days later by the onset of hyperglycaemia (>200 mg/dL). A control group (same characteristics, non-diabetic) was also maintained during the experiment. All the animals were fed Purina chow and water *ad libitum* and kept in light-darkness cycles of 12×12 h. The evolution of diabetes was evaluated by: (1) weighing the rats weekly and comparing the weights of the diabetic and the control animals and (2) measuring glycaemia and glucosylated haemoglobin at the end of the experiment.

### **2.2. Cerebral ischaemia model**

Six weeks after diabetes induction, cerebral ischaemia was produced in diabetic as well as the control animals by slightly modifying the model reported by De Cristóbal et al. [15]. In short, the animals were anaesthetised through intramuscular administration of an anaesthesia cocktail containing acepromazine (5 mg/kg), ketamine (5 mg/kg) and butorphanol (13 mg/kg). The right carotid and the middle cerebral arteries were permanently ligated. One hour later the animals were randomly divided into two treatment groups (eight control and eight diabetic animals per group).

#### **2.3. Experimental groups**


### **2.4. Neurophysiological test**

Eight hours after cerebral ischaemia was induced, the neurological signs were scored as follows (sum of all): consciousness (0–3, where 3 was the response only after stimulation), decreased grip of the left forelimb when the tail was pulled (0–3, 3 was complete gripping), failure to grab a bar with the left forelimb (0–3, 0 was strongly grabbing with both forelimbs, 3 was no grabbing at all with the left forelimb), and walking in a circle to the left side (0–3, 3 was walking spontaneously in circles). If the animal did not respond to any stimulus, the score was 12 points.

#### **2.5. Infarct and oedema evaluation**

The animals were anaesthetised (pentobarbital, 75 mg/kg, ip). A blood sample (at least 5 ml) was taken from the heart and anticoagulated with ethylenediaminetetraacetic acid (EDTA, Sigma, St. Louis, USA). The blood glucose and glucosylated haemoglobin were measured. The animals were decapitated after taking the blood samples, and each brain was immediately extracted and sliced (2 mm slices). The slices were incubated in a 1% triphenyltetrazolium chloride (Sigma, St. Louis, USA)/ISS solution, at 37°C for 5 min). Both sides of each slice were photographed against a millimetre paper background and measured using the ImageJ software (National Institutes of Health, USA). Areas were integrated as volume in mm**<sup>3</sup>** and infarct volume quantified as a percentage of the right hemisphere volume. Oedema was calculated (in percentage of the left brain hemisphere) by subtracting the left from the right hemisphere volumes.

ANOVA test (Tukey post-hoc) was used to compare biomarkers (diabetic, non-diabetic, with or without ischaemia). The neurophysiological score was analysed using the Kruskal-Wallis test (Dunn's multiple comparison post-hoc). Data are shown as the mean ± standard error of

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Non-diabetic and diabetic animals had the same weights at the beginning of the experiment (81.6 ± 0.8 g *vs.* 78.8 ± 2.7 g, respectively, p > 0.05). The time-course of their weights is shown in **Figure 1**. There was a significant difference beginning the third week after diabetes induction. Glycaemia at the beginning of the experiment (after diabetes induction) was higher in the diabetic animals compared with the non-diabetic animals (456.1 ± 2.3 mg/dL *vs.* 91.1 ± 2.3 mg/dL respectively, p < 0.0001). At the end of the experiment both glycaemia and glucosylated haemoglobin were also different: glycaemia 88.7 ± 3.0 mg/dL versus 467.5 ± 12.6 mg/dL (non-diabetic *vs.* diabetic, p < 0.0001); glucosylated haemoglobin 4.3 ± 0.1% versus 9.3 ± 0.1%

The neurophysiological score is shown in **Figure 2**. DMF significantly (p < 0.05) improves the

**Figure 1.** Time-course of the weight. The time-course of the weight was significantly different. Control (non-diabetic rats) gained more weight than diabetic animals during the experiment. The media + SEM are shown. Zero was at the

the mean (SEM). The p < 0.05 was considered significant.

**3. Results**

**3.1. Metabolic evolution**

(non-diabetic *vs.* diabetic, p < 0.0001).

**3.2. Cerebral ischaemia evaluation**

neurophysiological scores only in diabetic rats.

beginning of the experiment, when the rats were 4 weeks old.

*3.2.1. Neurophysiological score*

#### **2.6. Stroke and oxidative stress biomarkers' evaluation**

Two groups of animals (diabetic or non-diabetic, n = 12 per group) submitted to cerebral ischaemia (treatments—6 per group—and sacrifice as mentioned earlier) were used to measure biomarkers. Three hundred milligrams of the right hemispheres were homogenised in 1.5 ml of phosphate saline buffer (Sigma, St. Louis, USA; 0.1 M, pH 7.4). The homogenised tissue was centrifuged (13,000 ×*g*, 4°C, 10 min). The supernatant was isolated and kept at −80°C until measurement of the biomarkers was taken (within a month of taking the samples). Ten milligrams of the right hemisphere was homogenised in 0.5 ml of malondialdehyde (MDA) lysis buffer (Abcam, MA, USA) for the measurement of MDA.

Biomarkers evaluated were:


To establish reference values, and evaluate the effects of DMF, the biomarkers were measured in the brains of four non-diabetic and four diabetic rats that were not submitted to cerebral ischaemia.

#### **2.7. Statistical analysis**

The Kolmogorov-Smirnov test was used to analyse the normality of distribution. The unpaired "t" test was used to compare glycaemia and glucosylated haemoglobin between the nondiabetic and diabetic animals. The two-way ANOVA test (Bonferroni post-hoc) was used to analyse the time-course of weights (6 weeks, diabetic and non-diabetic animals). The one-way ANOVA test (Tukey post-hoc) was used to compare biomarkers (diabetic, non-diabetic, with or without ischaemia). The neurophysiological score was analysed using the Kruskal-Wallis test (Dunn's multiple comparison post-hoc). Data are shown as the mean ± standard error of the mean (SEM). The p < 0.05 was considered significant.

### **3. Results**

a millimetre paper background and measured using the ImageJ software (National Institutes

percentage of the right hemisphere volume. Oedema was calculated (in percentage of the left

Two groups of animals (diabetic or non-diabetic, n = 12 per group) submitted to cerebral ischaemia (treatments—6 per group—and sacrifice as mentioned earlier) were used to measure biomarkers. Three hundred milligrams of the right hemispheres were homogenised in 1.5 ml of phosphate saline buffer (Sigma, St. Louis, USA; 0.1 M, pH 7.4). The homogenised tissue was centrifuged (13,000 ×*g*, 4°C, 10 min). The supernatant was isolated and kept at −80°C until measurement of the biomarkers was taken (within a month of taking the samples). Ten milligrams of the right hemisphere was homogenised in 0.5 ml of malondialdehyde (MDA) lysis

**1.** Metalloproteinase 9 (MMP 9): It was evaluated through ELISA using a commercial kit (R&D Systems, MN, USA). The concentrations were expressed in ng/ml per mg of protein (proteins were measured using a commercial kit from Cayman Chemical Co, MI, USA). **2.** Advanced glycation end-product receptor (RAGE) was measured through ELISA using a commercial kit (Abcam MA, USA). The concentrations were expressed in ng/ml per mg of

by the modified Griess method, using a commercial kit (Cayman Chemical Co, MI, USA).

**4.** Nitrotyrosine (NT) was measured through ELISA using a commercial kit (Abcam MA,

**5.** MDA was measured using a commercial kit (Abcam MA, USA). The results were expressed

To establish reference values, and evaluate the effects of DMF, the biomarkers were measured in the brains of four non-diabetic and four diabetic rats that were not submitted to cerebral

The Kolmogorov-Smirnov test was used to analyse the normality of distribution. The unpaired "t" test was used to compare glycaemia and glucosylated haemoglobin between the nondiabetic and diabetic animals. The two-way ANOVA test (Bonferroni post-hoc) was used to analyse the time-course of weights (6 weeks, diabetic and non-diabetic animals). The one-way

USA). The concentrations were expressed in ng/ml per mg of protein.

ratios, an indirect measurement of nitric oxide) were measured

brain hemisphere) by subtracting the left from the right hemisphere volumes.

and infarct volume quantified as a

of Health, USA). Areas were integrated as volume in mm**<sup>3</sup>**

**2.6. Stroke and oxidative stress biomarkers' evaluation**

buffer (Abcam, MA, USA) for the measurement of MDA.

/NO**<sup>3</sup>**

Results were expressed as μM per mg of protein.

Biomarkers evaluated were:

76 Free Radicals, Antioxidants and Diseases

protein.

ischaemia.

**2.7. Statistical analysis**

**3.** Nitrite/nitrate (NO**<sup>2</sup>**

in nmol per mg of protein.

### **3.1. Metabolic evolution**

Non-diabetic and diabetic animals had the same weights at the beginning of the experiment (81.6 ± 0.8 g *vs.* 78.8 ± 2.7 g, respectively, p > 0.05). The time-course of their weights is shown in **Figure 1**. There was a significant difference beginning the third week after diabetes induction. Glycaemia at the beginning of the experiment (after diabetes induction) was higher in the diabetic animals compared with the non-diabetic animals (456.1 ± 2.3 mg/dL *vs.* 91.1 ± 2.3 mg/dL respectively, p < 0.0001). At the end of the experiment both glycaemia and glucosylated haemoglobin were also different: glycaemia 88.7 ± 3.0 mg/dL versus 467.5 ± 12.6 mg/dL (non-diabetic *vs.* diabetic, p < 0.0001); glucosylated haemoglobin 4.3 ± 0.1% versus 9.3 ± 0.1% (non-diabetic *vs.* diabetic, p < 0.0001).

#### **3.2. Cerebral ischaemia evaluation**

### *3.2.1. Neurophysiological score*

The neurophysiological score is shown in **Figure 2**. DMF significantly (p < 0.05) improves the neurophysiological scores only in diabetic rats.

**Figure 1.** Time-course of the weight. The time-course of the weight was significantly different. Control (non-diabetic rats) gained more weight than diabetic animals during the experiment. The media + SEM are shown. Zero was at the beginning of the experiment, when the rats were 4 weeks old.

**Figure 2.** Neurophysiological scores: the neurophysiological score was similar in the control (non-diabetic) rats without (C) or with dimethylformamide treatment (C + DMF), as well as in the diabetic animals without DMF treatment (D). DMF significantly decreased the score in diabetic rats treated with DMF (D + DMF). The media + SEM are shown.

#### *3.2.2. Infarct and oedema volume*

DMF significantly decreased the infarct volume in both non-diabetic (p = 0.02, **Figure 3**) and diabetic (p = 0.007) rats. However, oedema was the same in non-diabetic rats compared to diabetic rats and was not changed by DMF (not shown). **Figure 4** shows an example of the slices, dyed with triphenyltetrazolium.

#### *3.2.3. Cerebral ischaemia and stroke biomarkers*

Results are shown in **Figures 5**–**9**. The results in non-diabetic (Control, C) rats are shown in the left panels whereas results in the diabetic (D) animals are in the right panels. The figures

**Figure 3.** Infarct volume: this figure shows the infarct volume (expressed as percentage of the right hemisphere) in nondiabetic (C, panel A) and diabetic (D, panel B) animals. Dimethylformamide (DMF) significantly reduced infarct volume in non-diabetic as well as in diabetic rats.

show the basal data (without ischaemia) of animals without (C, D) or with treatment (C + DMF, D + DMF). Animals with ischaemia and without treatment are represented as C + I or D + I. The animals with ischaemia and treatment are shown as C + I + DMF or D + I + DM.

significantly higher concentrations of MMP9 than non-diabetic animals treated with DMF (C + DMF).

**Figure 5.** Concentration of metalloproteinase 9 (MMP9) in the brain: The concentration of MMP9 showed significant incremental increases in the brains of diabetic (D, panel B) animals with ischaemia with or without DMF treatment (D + I and D + I + DMF, respectively). DMF did not change MMP9 either in non-diabetic (C + DMF, panel A) or in diabetic (D + DMF, panel B) rats. Non-diabetic (C, panel A) animals with ischaemia and treated with DMF (C + I + DMF) had

**Figure 4.** Both sides of the slices of a representative animal per group are shown: C (non-diabetic without treatment), C + DMF (non-diabetic treated with DMF), D (diabetic without treatment), D + DMF (diabetic treated with DMF). The infarct shows no colour (in white), whereas the tissue that is not affected has a red colour. It is noticeable that animals

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treated with DMF (control and diabetic) have a smaller infarct.

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**Figure 4.** Both sides of the slices of a representative animal per group are shown: C (non-diabetic without treatment), C + DMF (non-diabetic treated with DMF), D (diabetic without treatment), D + DMF (diabetic treated with DMF). The infarct shows no colour (in white), whereas the tissue that is not affected has a red colour. It is noticeable that animals treated with DMF (control and diabetic) have a smaller infarct.

*3.2.2. Infarct and oedema volume*

78 Free Radicals, Antioxidants and Diseases

slices, dyed with triphenyltetrazolium.

in non-diabetic as well as in diabetic rats.

*3.2.3. Cerebral ischaemia and stroke biomarkers*

DMF significantly decreased the infarct volume in both non-diabetic (p = 0.02, **Figure 3**) and diabetic (p = 0.007) rats. However, oedema was the same in non-diabetic rats compared to diabetic rats and was not changed by DMF (not shown). **Figure 4** shows an example of the

**Figure 2.** Neurophysiological scores: the neurophysiological score was similar in the control (non-diabetic) rats without (C) or with dimethylformamide treatment (C + DMF), as well as in the diabetic animals without DMF treatment (D). DMF significantly decreased the score in diabetic rats treated with DMF (D + DMF). The media + SEM are shown.

Results are shown in **Figures 5**–**9**. The results in non-diabetic (Control, C) rats are shown in the left panels whereas results in the diabetic (D) animals are in the right panels. The figures

**Figure 3.** Infarct volume: this figure shows the infarct volume (expressed as percentage of the right hemisphere) in nondiabetic (C, panel A) and diabetic (D, panel B) animals. Dimethylformamide (DMF) significantly reduced infarct volume

**Figure 5.** Concentration of metalloproteinase 9 (MMP9) in the brain: The concentration of MMP9 showed significant incremental increases in the brains of diabetic (D, panel B) animals with ischaemia with or without DMF treatment (D + I and D + I + DMF, respectively). DMF did not change MMP9 either in non-diabetic (C + DMF, panel A) or in diabetic (D + DMF, panel B) rats. Non-diabetic (C, panel A) animals with ischaemia and treated with DMF (C + I + DMF) had significantly higher concentrations of MMP9 than non-diabetic animals treated with DMF (C + DMF).

show the basal data (without ischaemia) of animals without (C, D) or with treatment (C + DMF, D + DMF). Animals with ischaemia and without treatment are represented as C + I or D + I. The animals with ischaemia and treatment are shown as C + I + DMF or D + I + DM.

**Figure 6.** Concentration of the receptor for advanced glycation end products (RAGE) in the brain: RAGE did not change in the brains of non-diabetic (control, C) animals with or without ischaemia (I), either with or without dimethylformamide (C + DMF) treatment (panel A), whereas this biomarker significantly increased in the brains of diabetic animals submitted to ischaemia (D + I, panel B). DMF prevented the effect of ischaemia in diabetic animals (D + I + DMF). The mean + SEM are shown.

**Figure 7.** Nitrite and nitrate (NO**<sup>2</sup>** /NO**<sup>3</sup>** ) in the brain: NO**<sup>2</sup>** /NO**<sup>3</sup>** did not change in the brains of non-diabetic (control, C) animals with or without ischaemia (I), neither with nor without dimethylformamide (DMF) treatment (panel A) whereas this biomarker increased significantly in the brains of diabetic (D) rats submitted to ischaemia (panel B). DMF completely prevented the effect of ischaemia in diabetic animals. The mean + SEM are shown.

*3.2.3.2. RAGE*

*3.2.3.3. NO***<sup>2</sup>**

effect was prevented by DMF.

*/NO***<sup>3</sup>**

 *and NT*

ischaemia in diabetic animals. The mean + SEM are shown.

**Figure 6** shows that RAGE did not change in non-diabetic rats, with or without treatment or ischaemia, whereas it significantly increased in diabetic rats with ischaemia (p < 0.05), and the

**Figure 9.** Malondialdehyde (MDA) in the brain: dimethylformamide (DMF) non-significantly reduced MDA in the brains of non-diabetic (control, C, panel A) rats. Ischaemia (I) did not significantly increase MDA in control animals resulting in a significant difference between control rats without ischaemia with DMF treatment (C + DMF) and control rats with ischaemia (C + I). DMF significantly reduced DMF in diabetic (D, panel B) rats without ischaemia (D + DMF). Ischaemia significantly increased MDA in diabetic rats and the change was prevented by DMF. The mean + SEM are shown.

**Figure 8.** Nitrotyrosine (NT) in the brain: NT did not change in the brains of non-diabetic rats (control, C, panel A), with or without ischaemia (I), neither with nor without dimethylformamide (DMF) treatment, whereas this biomarker increased significantly in the brains of diabetic (D, panel B) rats submitted to ischaemia. DMF prevented the effect of

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Nitrite/nitrate ratios and nitrotyrosine results are shown in **Figures 7** and **8**. Results were similar to those of RAGE (**Figure 6**). Neither ischaemia nor DMF changes the basal concentrations

#### *3.2.3.1. MMP9*

**Figure 5** shows that MMP9 increased significantly (p < 0.05) in the right hemisphere of diabetic rats, and DMF did not prevent such increase. There was a slight (non-significant) increase in the right hemisphere of the non-diabetic rats with ischaemia. Likewise, DMF did not reduce MMP9 in non-diabetic animals with ischaemia. Interestingly, DMF slightly reduced MMP9 in non-diabetic animals without ischaemia; because of that change, nondiabetic animals treated with DMF had different concentrations (significantly higher with than without ischaemia).

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**Figure 8.** Nitrotyrosine (NT) in the brain: NT did not change in the brains of non-diabetic rats (control, C, panel A), with or without ischaemia (I), neither with nor without dimethylformamide (DMF) treatment, whereas this biomarker increased significantly in the brains of diabetic (D, panel B) rats submitted to ischaemia. DMF prevented the effect of ischaemia in diabetic animals. The mean + SEM are shown.

**Figure 9.** Malondialdehyde (MDA) in the brain: dimethylformamide (DMF) non-significantly reduced MDA in the brains of non-diabetic (control, C, panel A) rats. Ischaemia (I) did not significantly increase MDA in control animals resulting in a significant difference between control rats without ischaemia with DMF treatment (C + DMF) and control rats with ischaemia (C + I). DMF significantly reduced DMF in diabetic (D, panel B) rats without ischaemia (D + DMF). Ischaemia significantly increased MDA in diabetic rats and the change was prevented by DMF. The mean + SEM are shown.

#### *3.2.3.2. RAGE*

*3.2.3.1. MMP9*

are shown.

80 Free Radicals, Antioxidants and Diseases

than without ischaemia).

**Figure 7.** Nitrite and nitrate (NO**<sup>2</sup>**

/NO**<sup>3</sup>**

prevented the effect of ischaemia in diabetic animals. The mean + SEM are shown.

) in the brain: NO**<sup>2</sup>**

**Figure 5** shows that MMP9 increased significantly (p < 0.05) in the right hemisphere of diabetic rats, and DMF did not prevent such increase. There was a slight (non-significant) increase in the right hemisphere of the non-diabetic rats with ischaemia. Likewise, DMF did not reduce MMP9 in non-diabetic animals with ischaemia. Interestingly, DMF slightly reduced MMP9 in non-diabetic animals without ischaemia; because of that change, nondiabetic animals treated with DMF had different concentrations (significantly higher with

/NO**<sup>3</sup>**

animals with or without ischaemia (I), neither with nor without dimethylformamide (DMF) treatment (panel A) whereas this biomarker increased significantly in the brains of diabetic (D) rats submitted to ischaemia (panel B). DMF completely

did not change in the brains of non-diabetic (control, C)

**Figure 6.** Concentration of the receptor for advanced glycation end products (RAGE) in the brain: RAGE did not change in the brains of non-diabetic (control, C) animals with or without ischaemia (I), either with or without dimethylformamide (C + DMF) treatment (panel A), whereas this biomarker significantly increased in the brains of diabetic animals submitted to ischaemia (D + I, panel B). DMF prevented the effect of ischaemia in diabetic animals (D + I + DMF). The mean + SEM

> **Figure 6** shows that RAGE did not change in non-diabetic rats, with or without treatment or ischaemia, whereas it significantly increased in diabetic rats with ischaemia (p < 0.05), and the effect was prevented by DMF.

#### *3.2.3.3. NO***<sup>2</sup>** */NO***<sup>3</sup>**  *and NT*

Nitrite/nitrate ratios and nitrotyrosine results are shown in **Figures 7** and **8**. Results were similar to those of RAGE (**Figure 6**). Neither ischaemia nor DMF changes the basal concentrations of NO**<sup>2</sup>** /NO**<sup>3</sup>** or NT in the brains of non-diabetic rats, whereas in the diabetic animals nitrates/ nitrates as well as NT significantly increased with ischaemia, and the effect was prevented by DMF.

ated by inhibitors of the renin-angiotensin system, whose effects include the reduction in oxidative stress and probably inflammation [20–22]. In the present study, the diabetic animals had low weights, hyperglycaemia, and high glucosylated haemoglobin. Cerebral ischaemia was produced 5 weeks after the induction of diabetes. This means that diabetic animals were not supposed to have apparent cardiovascular complications of diabetes, although the pathophysiological events were certainly already triggered. Indeed, this could explain why even in basal conditions (brains of animals without ischaemia and without treatment) all the biomarkers had the same concentrations, and notably cerebral ischaemia significantly increased all the biomarkers only in diabetic animals, whereas MDA (biomarkers or OS effects on lipids)

Dimethylformamide Reduces Cerebral Ischaemia in Diabetic Rats Hours after Its Occurrence…

http://dx.doi.org/10.5772/intechopen.73700

83

The expression of MMP9 has been related to various steps of the pathophysiology of cerebral ischaemia, such as inflammation, excitotoxicity, neuronal damage, and blood brain barrier (BBB) disturbance [6, 23]. MMP9 increased clearly in diabetic but not in non-diabetic animals with ischaemia. DMF did not prevent MMP9 incremental increases. The lack of an increment in non-diabetic animals could be due to the short time that passed after ischaemia was

Nitrotyrosine (NT) is the fingerprint of peroxynitrite, a nitrogen active species produced by the interaction of superoxide and nitric oxide (NO) [24]. NT is a biomarker of inflammation and OS [24]. NT increases in the brain mitochondria of diabetic animals 21 days after STZ administration [25]. NT also increases in the brains 24 h after ischaemia induction [26]. In the present study, NT was evaluated in homogenised tissue, which could explain why the increment of this biomarker was not observed in the brains of diabetic animals without ischaemia. No increment of NT was observed in the brains of non-diabetic animals, whereas it significantly increased after cerebral ischaemia in diabetic animals. Nitrotyrosine changes in diabetic animals were completely prevented by DMF. Interestingly, nitrite/nitrate (indirect measurement of NO production) and RAGE (a biomarker of neuroinflammation in cerebral ischaemia [27]) changed similarly to NT; they increased after ischaemia only in diabetic ani-

The evaluation of cerebral ischaemia was performed early after arterial occlusion (8 h). It could be the reason for not observing changes in non-diabetic animals (only MDA increment). Diabetic animals were probably more susceptible to inflammation and OS after ischaemia due to the basal conditions produced by hyperglycaemia. Such basal conditions could explain the greater effects of DMF in diabetic animals. It is necessary to study the effects of DMF for longer periods after ischaemia and on other biomarkers. The results of this study open a new

We conclude that DMF has beneficial effects on cerebral ischaemia produced in rats. The protective effects of DMF in diabetic rats could be the result of interference with oxidative and inflammation-triggered pathways, which are exacerbated by diabetes. That could explain the

horizon in the design of therapeutic strategies for the treatment of stroke.

was the only biomarker increased in non-diabetic rats with ischaemia.

induced and the sacrifice (8 h).

**5. Conclusion**

mals and such increment was prevented by DMF.

#### *3.2.3.4. MDA*

**Figure 9** shows the concentrations of MDA in the brains. Basal (no ischaemia, no treatment) concentrations were similar in diabetic and non-diabetic animals. DMF slightly (non-significantly) reduced MDA in non-diabetic rats, whereas it significantly reduced MDA in diabetic animals (p < 0.05). Cerebral ischaemia increased MDA in both diabetic and non-diabetic animals but the change was significant only in diabetic rats (p < 0.05). DMF suppressed the effect of ischaemia in diabetic rats (p < 0.05). Due to the non-significant changes produced by DMF in non-diabetic animals without ischaemia, there was a significant difference (p < 0.05) between this group and non-diabetic animals with ischaemia.

### **4. Discussion**

This is the first time that DMF has been used and shown to be beneficial in treating cerebral ischaemia. In the present study DMF significantly reduced infarct volume in both diabetic and non-diabetic animals (similar reductions). It is important to note that infarct volume was evaluated just 8 h after cerebral ischaemia induction; thus, it could be interesting to observe if the similar effect occurs after a longer time (24 or 48 h). It is also important to note that treatment was administered 1 h after the onset of ischaemia, which is different from the experimental strategies that have been used for antioxidants (they usually are administered as a pre-treatment). One hour is a long period and results of the present study open a new horizon in the evaluation of therapeutic strategies that eventually could be used after the therapeutic window of thrombolysis, which is the current treatment (and not very efficient) in cerebral ischaemia.

Even though the reduction of infarct volume was similar in non-diabetic as in diabetic animals, DMF seems to have a better effect in diabetic rats because it significantly improved the neurophysiological score. One of the concerns of using DMF is the possibility of toxic effects. However, the toxic effect noted in workers in the synthetic leather industry is the result of chronic long-term exposure [16, 17]. Moreover, the single dose that we used in the present study (0.9 g/kg) is far from the reported LD**50** (7.2 g/kg) [18]. Since no apparent toxic effect was noted, it seems that DMF was safe and beneficial. Moreover, the treatment was administered 1 h after ischaemia in non-diabetic as well as diabetic animals, which places the study closer to clinical setting conditions where diabetes is a risk factor for stroke, and it is virtually impossible to predict who would suffer cerebral ischaemia.

The difference of DMF effects between groups seems to be related to special conditions of diabetes pathology. It is known that long-term inflammation and oxidative stress play an important role in the cardiovascular complications of diabetes [8, 19]. Indeed, it was reported that cardiovascular, renal, and neurological complications become apparent at least 2 months after diabetes induction in the rat streptozotocin model and that those complications were attenuated by inhibitors of the renin-angiotensin system, whose effects include the reduction in oxidative stress and probably inflammation [20–22]. In the present study, the diabetic animals had low weights, hyperglycaemia, and high glucosylated haemoglobin. Cerebral ischaemia was produced 5 weeks after the induction of diabetes. This means that diabetic animals were not supposed to have apparent cardiovascular complications of diabetes, although the pathophysiological events were certainly already triggered. Indeed, this could explain why even in basal conditions (brains of animals without ischaemia and without treatment) all the biomarkers had the same concentrations, and notably cerebral ischaemia significantly increased all the biomarkers only in diabetic animals, whereas MDA (biomarkers or OS effects on lipids) was the only biomarker increased in non-diabetic rats with ischaemia.

The expression of MMP9 has been related to various steps of the pathophysiology of cerebral ischaemia, such as inflammation, excitotoxicity, neuronal damage, and blood brain barrier (BBB) disturbance [6, 23]. MMP9 increased clearly in diabetic but not in non-diabetic animals with ischaemia. DMF did not prevent MMP9 incremental increases. The lack of an increment in non-diabetic animals could be due to the short time that passed after ischaemia was induced and the sacrifice (8 h).

Nitrotyrosine (NT) is the fingerprint of peroxynitrite, a nitrogen active species produced by the interaction of superoxide and nitric oxide (NO) [24]. NT is a biomarker of inflammation and OS [24]. NT increases in the brain mitochondria of diabetic animals 21 days after STZ administration [25]. NT also increases in the brains 24 h after ischaemia induction [26]. In the present study, NT was evaluated in homogenised tissue, which could explain why the increment of this biomarker was not observed in the brains of diabetic animals without ischaemia. No increment of NT was observed in the brains of non-diabetic animals, whereas it significantly increased after cerebral ischaemia in diabetic animals. Nitrotyrosine changes in diabetic animals were completely prevented by DMF. Interestingly, nitrite/nitrate (indirect measurement of NO production) and RAGE (a biomarker of neuroinflammation in cerebral ischaemia [27]) changed similarly to NT; they increased after ischaemia only in diabetic animals and such increment was prevented by DMF.

The evaluation of cerebral ischaemia was performed early after arterial occlusion (8 h). It could be the reason for not observing changes in non-diabetic animals (only MDA increment). Diabetic animals were probably more susceptible to inflammation and OS after ischaemia due to the basal conditions produced by hyperglycaemia. Such basal conditions could explain the greater effects of DMF in diabetic animals. It is necessary to study the effects of DMF for longer periods after ischaemia and on other biomarkers. The results of this study open a new horizon in the design of therapeutic strategies for the treatment of stroke.

### **5. Conclusion**

of NO**<sup>2</sup>**

DMF.

*3.2.3.4. MDA*

**4. Discussion**

/NO**<sup>3</sup>**

82 Free Radicals, Antioxidants and Diseases

or NT in the brains of non-diabetic rats, whereas in the diabetic animals nitrates/

nitrates as well as NT significantly increased with ischaemia, and the effect was prevented by

**Figure 9** shows the concentrations of MDA in the brains. Basal (no ischaemia, no treatment) concentrations were similar in diabetic and non-diabetic animals. DMF slightly (non-significantly) reduced MDA in non-diabetic rats, whereas it significantly reduced MDA in diabetic animals (p < 0.05). Cerebral ischaemia increased MDA in both diabetic and non-diabetic animals but the change was significant only in diabetic rats (p < 0.05). DMF suppressed the effect of ischaemia in diabetic rats (p < 0.05). Due to the non-significant changes produced by DMF in non-diabetic animals without ischaemia, there was a significant difference (p < 0.05)

This is the first time that DMF has been used and shown to be beneficial in treating cerebral ischaemia. In the present study DMF significantly reduced infarct volume in both diabetic and non-diabetic animals (similar reductions). It is important to note that infarct volume was evaluated just 8 h after cerebral ischaemia induction; thus, it could be interesting to observe if the similar effect occurs after a longer time (24 or 48 h). It is also important to note that treatment was administered 1 h after the onset of ischaemia, which is different from the experimental strategies that have been used for antioxidants (they usually are administered as a pre-treatment). One hour is a long period and results of the present study open a new horizon in the evaluation of therapeutic strategies that eventually could be used after the therapeutic window of thrombolysis, which is the current treatment (and not very efficient) in cerebral ischaemia. Even though the reduction of infarct volume was similar in non-diabetic as in diabetic animals, DMF seems to have a better effect in diabetic rats because it significantly improved the neurophysiological score. One of the concerns of using DMF is the possibility of toxic effects. However, the toxic effect noted in workers in the synthetic leather industry is the result of chronic long-term exposure [16, 17]. Moreover, the single dose that we used in the present study (0.9 g/kg) is far from the reported LD**50** (7.2 g/kg) [18]. Since no apparent toxic effect was noted, it seems that DMF was safe and beneficial. Moreover, the treatment was administered 1 h after ischaemia in non-diabetic as well as diabetic animals, which places the study closer to clinical setting conditions where diabetes is a risk factor for stroke, and it is virtually impos-

The difference of DMF effects between groups seems to be related to special conditions of diabetes pathology. It is known that long-term inflammation and oxidative stress play an important role in the cardiovascular complications of diabetes [8, 19]. Indeed, it was reported that cardiovascular, renal, and neurological complications become apparent at least 2 months after diabetes induction in the rat streptozotocin model and that those complications were attenu-

between this group and non-diabetic animals with ischaemia.

sible to predict who would suffer cerebral ischaemia.

We conclude that DMF has beneficial effects on cerebral ischaemia produced in rats. The protective effects of DMF in diabetic rats could be the result of interference with oxidative and inflammation-triggered pathways, which are exacerbated by diabetes. That could explain the reason for the better results in diabetic rather than in non-diabetic animals. The mechanisms of the protective effects in non-diabetic rats were not clear. It is necessary to explore other biomarkers and longer periods to elucidate those effects.

[4] Newsholme P, Cruzat VF, Keane KN, Carlessi R, de Bittencourt PI Jr. Molecular mechanisms of ROS production and oxidative stress in diabetes. The Biochemical Journal.

Dimethylformamide Reduces Cerebral Ischaemia in Diabetic Rats Hours after Its Occurrence…

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85

[5] Manzanero S, Santro T, Arumugam TV. Neuronal oxidative stress in acute ischemic stroke: Sources and contribution to cell injury. Neurochemistry International. 2013;**62**(5):712-718 [6] Villanueva C, Kross R, Perez-Astudillo L. Free radicals and neuronal recovery from an ischaemic penumbra: A review. In: Free Radicals and Diseases. Croatia: In Tech; 2016. pp. 331-346

[7] Kant V, Gopal A, Pathak NN, Kumar P, Tandan SK, Kumar D. Antioxidant and antiinflammatory potential of curcumin accelerated the cutaneous wound healing in streptozotocin-induced diabetic rats. International Immunopharmacology. 2014;**20**(2):322-330 [8] Ceriello A, Esposito K, La Sala L, Pujadas G, De Nigris V, Testa R, Bucciarelli L, Rondinelli M, Genovese S. The protective effect of the Mediterranean diet on endothelial resistance to GLP-1 in type 2 diabetes: A preliminary report. Cardiovascular Diabetology.

[9] Hjartaker A, Knudsen MD, Tretli S, Weiderpass E. Consumption of berries, fruits and vegetables and mortality among 10,000 Norwegian men followed for four decades.

[10] Villanueva C, Kross RD. Antioxidant-induced stress. International Journal of Molecular

[11] Saleh TM, Saleh MC, Connell BJ, Song YH. A co-drug conjugate of naringenin and lipoic acid mediates neuroprotection in a rat model of oxidative stress. Clinical and

[12] Duffy S, So A, Murphy TH. Activation of endogenous antioxidant defenses in neuronal cells prevents free radical-mediated damage. Journal of Neurochemistry. 1998;**71**(1):69-77

[13] Garlid AO, Jaburek M, Jacobs JP, Garlid KD. Mitochondrial reactive oxygen species: Which ROS signals cardioprotection? American Journal of Physiology. Heart and Cir-

[14] Frankowski H, Alavez S, Spilman P, Mark KA, Nelson JD, Mollahan P, Rao RV, Chen SF, Lithgow GJ, Ellerby HM. Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid. Mechanisms of Ageing and Development. 2013;**134**(3-4):69-78 [15] De Cristóbal J, Moro MA, Davalos A, Castillo J, Leza JC, Camarero J, Colado MI, Lorenzo P, Lizasoain I. Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats. Journal of Neurochemistry. 2001;**79**(2):456-459

[16] Chang HY, Shih TS, Guo YL, Tsai CY, Hsu PC. Sperm function in workers exposed to N,Ndimethylformamide in the synthetic leather industry. Fertility and Sterility. 2004;**81**(6):

[17] He J, Wang P, Zhu JQ, Wu G, Ji JM, Xue Y. Role of urinary biomarkers of N,Ndimethylformamide in the early detection of hepatic injury among occupational exposed workers. International Archives of Occupational and Environmental Health. 2010;**83**(4):

Experimental Pharmacology & Physiology. 2017;**44**(10):1008-1016

European Journal of Nutrition. 2015;**54**(4):599-608

culatory Physiology. 2013;**305**(7):H960-H968

Sciences. 2012;**13**(2):2091-2109

2016;**473**(24):4527-4550

2014;**13**:140

1589-1594

399-406

### **Acknowledgements**

This work was supported by Consejo Nacional de Ciencia y Tecnologia, Mexico (Grant CONACYT202483), Instituto Politecnico Nacional (Grant SIP20162031), Secretaria de la Defensa Nacional, Mexico, and Kross-Link Laboratories (NY, USA).

### **Author details**

Julio Amadeo Mendoza-Betancourt<sup>1</sup> , Robert David Kross2 , Maria Angeles Moro3 , Ignacio Lizasoain3 , Luis Humberto Pérez-Astudillo<sup>4</sup> , Arturo Alva-Félix-Díaz<sup>5</sup> and Cleva Villanueva<sup>5</sup> \*


### **References**


[4] Newsholme P, Cruzat VF, Keane KN, Carlessi R, de Bittencourt PI Jr. Molecular mechanisms of ROS production and oxidative stress in diabetes. The Biochemical Journal. 2016;**473**(24):4527-4550

reason for the better results in diabetic rather than in non-diabetic animals. The mechanisms of the protective effects in non-diabetic rats were not clear. It is necessary to explore other

This work was supported by Consejo Nacional de Ciencia y Tecnologia, Mexico (Grant CONACYT202483), Instituto Politecnico Nacional (Grant SIP20162031), Secretaria de la Defensa

, Robert David Kross2

5 Instituto Politecnico Nacional, Escuela Superior de Medicina, Mexico City, Mexico

[1] Feigin VL, Forouzanfar MH, Krishnamurthi R, Mensah GA, Connor M, Bennett DA, Moran AE, Sacco RL, Anderson L, Truelsen T, O'Donnell M, Venketasubramanian N, Barker-Collo S, Lawes CM, Wang W, Shinohara Y, Witt E, Ezzati M, Naghavi M, Murray C, Global burden of diseases, injuries, and risk factors study, GBD Stroke Experts Group. Global and regional burden of stroke during 1990-2010: Findings from the global burden

[2] Gill D, Baheerathan A, Aravind A, Veltkamp R, Kar A. Severe hemorrhagic transformation after thrombolysis for acute ischemic stroke prevents early neurological improvement. Journal of Stroke and Cerebrovascular Diseases: The Official Journal of National

[3] Peters SA, Huxley RR, Woodward M. Diabetes as a risk factor for stroke in women compared with men: A systematic review and meta-analysis of 65 cohorts, including 775 385

, Maria Angeles Moro3

, Arturo Alva-Félix-Díaz<sup>5</sup>

,

and

biomarkers and longer periods to elucidate those effects.

Nacional, Mexico, and Kross-Link Laboratories (NY, USA).

, Luis Humberto Pérez-Astudillo<sup>4</sup>

\*Address all correspondence to: villanuevacleva3@gmail.com

of disease study 2010. Lancet. 2014;**383**(9913):245-254

individuals and 12 539 strokes. Lancet. 2014;**383**:1973-1980

Stroke Association. 2016;**25**(9):2232-2236

**Acknowledgements**

84 Free Radicals, Antioxidants and Diseases

**Author details**

Ignacio Lizasoain3

Cleva Villanueva<sup>5</sup>

**References**

Julio Amadeo Mendoza-Betancourt<sup>1</sup>

\*

4 Biotest SC, Hidalgo, Mexico

1 Hospital Central Militar, Mexico City, Mexico

3 Universidad Complutense de Madrid, Madrid, Spain

2 Kross-Link Laboratories, New York, USA


[18] Massmann W. Toxicological investigations on dimethylformamide. British Journal of Industrial Medicine. 1956;**13**(1):51-54

**Chapter 7**

Provisional chapter

**Cardioprotective Effects of S-Nitrosothiols in Ischemia-**

DOI: 10.5772/intechopen.77003

The most important clinical consequence of coronary disease is acute myocardial infarction caused by an occlusion that limits the irrigation to the heart. Although the gold standard treatment is to restore blood flow, this reperfusion causes inherent damage by increasing the size of the infarcted area primarily through the opening of the mitochondrial permeability transition pore (MPTP). The cardioprotective effect of nitric oxide (NO) has been described to operate through S-nitrosylation of several important proteins in the cardiomyocytes such as the calcium channels RyR2 and the L-type Ca2+ channel and mitochondrial proteins, including the MPTP. In this sense, an attractive strategy to prevent the ischemia-reperfusion damage is to increase the bioavailability of endogenous Snitrosothiols. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in the metabolism of NO through denitrosylation, which would limit the cardioprotective effect of NO. Although inhibition of GSNOR has been studied in different organs, its effects on myocardial reperfusion have not yet been fully elucidated. In this chapter, we review the pathophysiology underlying myocardial reperfusion injury and the opening of the MPTP along with the cardioprotective role of S-nitrosothiols and the potential role for GSNOR. Keywords: permeability transition pore, heart, GSNOR, nitric oxide, S-nitrosylation, Ca2+

Coronary heart disease is the leading cause of death worldwide [1]. According to the World Health Organization, in 2008, more than 7 million of deaths worldwide were the result of this

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

Cardioprotective Effects of S-Nitrosothiols in Ischemia-

**Reperfusion: Role for Mitochondria and Calcium**

Reperfusion: Role for Mitochondria and Calcium

**Channels**

Channels

Daniel R. González

Daniel R. González

Abstract

1. Introduction

Oscar Arriagada, Adriana V. Treuer and

Oscar Arriagada, Adriana V. Treuer and

http://dx.doi.org/10.5772/intechopen.77003

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium Channels** Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium Channels

DOI: 10.5772/intechopen.77003

Oscar Arriagada, Adriana V. Treuer and Daniel R. González Oscar Arriagada, Adriana V. Treuer and Daniel R. González

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77003

#### Abstract

[18] Massmann W. Toxicological investigations on dimethylformamide. British Journal of

[19] Ceriello A, Testa R, Genovese S. Clinical implications of oxidative stress and potential role of natural antioxidants in diabetic vascular complications. Nutrition, Metabolism,

[20] Fukami K, Yamagishi S, Coughlan MT, Harcourt BE, Kantharidis P, Thallas-Bonke V, Okuda S, Cooper ME, Forbes JM. Ramipril inhibits AGE-RAGE-induced matrix metalloproteinase-2 activation in experimental diabetic nephropathy. Diabetology & Metabolic

[21] Salum E, Butlin M, Kals J, Zilmer M, Eha J, Avolio AP, Arend A, Aunapuu M, Kampus P. Angiotensin II receptor blocker telmisartan attenuates aortic stiffening and remodel-

[22] Sleem M, Taye A, El-Moselhy MA, Mangoura SA. Combination therapy with losartan and L-carnitine protects against endothelial dysfunction of streptozotocin-induced dia-

[23] Chaturvedi M, Kaczmarek L. Mmp-9 inhibition: A therapeutic strategy in ischemic

[24] Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. The American Journal of Physiology. 1996;**271**(5 Pt 1):C1424-C1437 [25] Mastrocola R, Restivo F, Vercellinatto I, Danni O, Brignardello E, Aragno M, Boccuzzi G. Oxidative and nitrosative stress in brain mitochondria of diabetic rats. The Journal of

[26] Chao XD, Ma YH, Luo P, Cao L, Lau WB, Zhao BC, Han F, Liu W, Ning WD, Su N, Zhang L, Zhu J, Fei Z, Qu Y. Up-regulation of heme oxygenase-1 attenuates brain damage after cerebral ischemia via simultaneous inhibition of superoxide production and

[27] Greco R, Demartini C, Zanaboni AM, Blandini F, Amantea D, Tassorelli C. Modulation of cerebral RAGE expression following nitric oxide synthase inhibition in rats subjected

preservation of NO bioavailability. Experimental Neurology. 2013;**239**:163-169

to focal cerebral ischemia. European Journal of Pharmacology. 2017;**800**:16-22

ling in STZ-diabetic rats. Diabetology & Metabolic Syndrome. 2014;**6**:57

betic rats. European Journal of Pharmacology. 2014;**744**:10-17

stroke. Molecular Neurobiology. 2014;**49**(1):563-573

Endocrinology. 2005;**187**(1):37-44

Industrial Medicine. 1956;**13**(1):51-54

Syndrome. 2014;**6**(1):86

86 Free Radicals, Antioxidants and Diseases

and Cardiovascular Diseases. 2016;**26**(4):285-292

The most important clinical consequence of coronary disease is acute myocardial infarction caused by an occlusion that limits the irrigation to the heart. Although the gold standard treatment is to restore blood flow, this reperfusion causes inherent damage by increasing the size of the infarcted area primarily through the opening of the mitochondrial permeability transition pore (MPTP). The cardioprotective effect of nitric oxide (NO) has been described to operate through S-nitrosylation of several important proteins in the cardiomyocytes such as the calcium channels RyR2 and the L-type Ca2+ channel and mitochondrial proteins, including the MPTP. In this sense, an attractive strategy to prevent the ischemia-reperfusion damage is to increase the bioavailability of endogenous Snitrosothiols. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in the metabolism of NO through denitrosylation, which would limit the cardioprotective effect of NO. Although inhibition of GSNOR has been studied in different organs, its effects on myocardial reperfusion have not yet been fully elucidated. In this chapter, we review the pathophysiology underlying myocardial reperfusion injury and the opening of the MPTP along with the cardioprotective role of S-nitrosothiols and the potential role for GSNOR.

Keywords: permeability transition pore, heart, GSNOR, nitric oxide, S-nitrosylation, Ca2+

#### 1. Introduction

Coronary heart disease is the leading cause of death worldwide [1]. According to the World Health Organization, in 2008, more than 7 million of deaths worldwide were the result of this

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

disease (12.8% of all deaths) [2]. The main clinical manifestation of this condition is acute myocardial infarction, which is characterized by a coronary occlusion, most frequently produced by the rupture of an unstable atherosclerotic plaque [3]. Ischemic damage in the myocardium produced by this pathology requires immediate restoration of blood flow to the affected heart area. The injury size of the resulting infarction depends on: (1) the ischemic area at risk, (2) the duration and intermittency of the coronary occlusion and (3) the magnitude of the residual collateral flow and the degree of microvascular dysfunction [4].

thiol, has showed remarkable cardioprotective effects altering the function of different proteins

Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium…

http://dx.doi.org/10.5772/intechopen.77003

89

One of the main cardioprotective effects of S-nitrosylation is the avoidance of the opening of MPTP. The opening of the MPTP triggers a proapoptotic response mediated by the release of cytochrome C and other factors, as well as necrotic cell death. Agents that induce mitochondrial S-nitrosylation of this pore may reduce the infarcted area after ischemia and to main-

Myocardial reperfusion injury was first described by Jennings et al. in 1960, using a model of coronary occlusion in dogs [11]. They observed alterations in the sarcolemma and in mitochondria, which accelerated the process of cardiomyocyte necrosis. This reperfusion injury in animal models of acute myocardial infarction can result in up a 50% of infarcted area [1]. During reperfusion, inflammatory mediators are released, such as cytokines, which recruit neutrophils to the affected endothelium, permeabilizing it for the entrance to the injured myocardium of more inflammatory cells. In fact, decreasing this influx of neutrophils confer cardioprotection in animal models [12]. In addition, during the period of ischemia, molecules called "danger signals" are released from the extracellular matrix and damaged cells such as fragments of fibronectin, hyaluronic acid, heat shock proteins and high-mobility group box-1 (HMBG1). HMBG1 is able to interact with toll-like receptors (TLR) such as TLR2, TLR4, TLR9 and the receptor for advanced glycation end products (RAGE). These ligand-receptor interactions lead to the nuclear

translocation of NF-kappa B, triggering the transcription of proinflammatory genes [13].

One of the main organelles affected by reperfusion injury is mitochondria, which in pathological conditions may mediate detrimental effects to the cell such as apoptosis or cell necrosis [14].

After ischemia-reperfusion, mitochondria undergo structural as well as functional alterations. One of the most important alterations is the formation of MPTP, a pore of nonselective permeability, stimulated by high concentrations of mitochondrial Ca2+, adenosine and reactive oxygen species (ROS) [15]. The function of this pore is thought to be the regulation of Ca2+ concentrations, preventing the overload of this ion at the mitochondrial level [16]. In fact, inhibition of the mitochondrial Ca2+ exchanger showed cardioprotective effects in isolated

In situations such as Ca2+ overload, alterations in ATP production, mitochondrial membrane depolarization, or inhibition of respiration, mitochondria may undergo a swelling process that represents a permeabilization of the internal membrane, product of the opening of nonspecific channels known as the mitochondrial permeability pore [19]. This pore, which allows the passage of molecules up to 1.5 kDa, mediates the rupture of the outer mitochondrial membrane

important for the regulation of cell function and viability.

tain the functionality of the heart (Figure 1).

hearts of newborn [17] as well as in adult rabbits [18].

3. Mitochondrial permeability transition pore

2. Reperfusion damage

The preservation of myocardial functionality after ischemia is critical for the viability of the damaged heart, and although reperfusion is necessary for the survival of the myocardium, paradoxically this reperfusion itself can generate injury or even induce the death of cardiomyocytes. The damage can range from a reversible effect such as "myocardial stunning" to an irreversible one, increasing the size of the infarcted area or increasing microvascular alterations [5]. This phenomenon is known as reperfusion myocardial damage [6, 7].

To combat this condition, most of the current pharmacological strategies of cardioprotection converge to the mitochondria, particularly, to prevent the opening of mitochondrial permeability transition pore (MPTP), which is a large conductance channels that forms during stressing conditions for the heart such as reduction in ATP synthesis, increased Ca2+ and increased ROS production [8]. Nitric oxide (NO), a potent signaling molecule with pleiotropic effects in the heart, has long been studied to evaluate its cardioprotective effects. Agents that promote continuous NO releases are among the most commonly used drugs to treat cardiovascular disease [9]. Although its effects on vascular tone and cyclic GMP are well studied [10], S-nitrosylation, a posttranslational modification in which an NO group is added to a cysteine

Figure 1. S-nitrosylation in mitochondria. The figure depicts two important targets where S-nitrosothiols (SNO) exert cardioprotective effects within the mitochondria. S-nitrosylation of complex I prevents the burst of reactive oxygen species during reperfusion. CyclophylinD, which interacts with mitochondrial permeability transition pore is also Snitrosylated, reducing its opening.

thiol, has showed remarkable cardioprotective effects altering the function of different proteins important for the regulation of cell function and viability.

One of the main cardioprotective effects of S-nitrosylation is the avoidance of the opening of MPTP. The opening of the MPTP triggers a proapoptotic response mediated by the release of cytochrome C and other factors, as well as necrotic cell death. Agents that induce mitochondrial S-nitrosylation of this pore may reduce the infarcted area after ischemia and to maintain the functionality of the heart (Figure 1).

### 2. Reperfusion damage

disease (12.8% of all deaths) [2]. The main clinical manifestation of this condition is acute myocardial infarction, which is characterized by a coronary occlusion, most frequently produced by the rupture of an unstable atherosclerotic plaque [3]. Ischemic damage in the myocardium produced by this pathology requires immediate restoration of blood flow to the affected heart area. The injury size of the resulting infarction depends on: (1) the ischemic area at risk, (2) the duration and intermittency of the coronary occlusion and (3) the magnitude of

The preservation of myocardial functionality after ischemia is critical for the viability of the damaged heart, and although reperfusion is necessary for the survival of the myocardium, paradoxically this reperfusion itself can generate injury or even induce the death of cardiomyocytes. The damage can range from a reversible effect such as "myocardial stunning" to an irreversible one, increasing the size of the infarcted area or increasing microvascular alterations [5]. This

To combat this condition, most of the current pharmacological strategies of cardioprotection converge to the mitochondria, particularly, to prevent the opening of mitochondrial permeability transition pore (MPTP), which is a large conductance channels that forms during stressing conditions for the heart such as reduction in ATP synthesis, increased Ca2+ and increased ROS production [8]. Nitric oxide (NO), a potent signaling molecule with pleiotropic effects in the heart, has long been studied to evaluate its cardioprotective effects. Agents that promote continuous NO releases are among the most commonly used drugs to treat cardiovascular disease [9]. Although its effects on vascular tone and cyclic GMP are well studied [10], S-nitrosylation, a posttranslational modification in which an NO group is added to a cysteine

Figure 1. S-nitrosylation in mitochondria. The figure depicts two important targets where S-nitrosothiols (SNO) exert cardioprotective effects within the mitochondria. S-nitrosylation of complex I prevents the burst of reactive oxygen species during reperfusion. CyclophylinD, which interacts with mitochondrial permeability transition pore is also S-

nitrosylated, reducing its opening.

the residual collateral flow and the degree of microvascular dysfunction [4].

phenomenon is known as reperfusion myocardial damage [6, 7].

88 Free Radicals, Antioxidants and Diseases

Myocardial reperfusion injury was first described by Jennings et al. in 1960, using a model of coronary occlusion in dogs [11]. They observed alterations in the sarcolemma and in mitochondria, which accelerated the process of cardiomyocyte necrosis. This reperfusion injury in animal models of acute myocardial infarction can result in up a 50% of infarcted area [1]. During reperfusion, inflammatory mediators are released, such as cytokines, which recruit neutrophils to the affected endothelium, permeabilizing it for the entrance to the injured myocardium of more inflammatory cells. In fact, decreasing this influx of neutrophils confer cardioprotection in animal models [12]. In addition, during the period of ischemia, molecules called "danger signals" are released from the extracellular matrix and damaged cells such as fragments of fibronectin, hyaluronic acid, heat shock proteins and high-mobility group box-1 (HMBG1). HMBG1 is able to interact with toll-like receptors (TLR) such as TLR2, TLR4, TLR9 and the receptor for advanced glycation end products (RAGE). These ligand-receptor interactions lead to the nuclear translocation of NF-kappa B, triggering the transcription of proinflammatory genes [13].

One of the main organelles affected by reperfusion injury is mitochondria, which in pathological conditions may mediate detrimental effects to the cell such as apoptosis or cell necrosis [14].

After ischemia-reperfusion, mitochondria undergo structural as well as functional alterations. One of the most important alterations is the formation of MPTP, a pore of nonselective permeability, stimulated by high concentrations of mitochondrial Ca2+, adenosine and reactive oxygen species (ROS) [15]. The function of this pore is thought to be the regulation of Ca2+ concentrations, preventing the overload of this ion at the mitochondrial level [16]. In fact, inhibition of the mitochondrial Ca2+ exchanger showed cardioprotective effects in isolated hearts of newborn [17] as well as in adult rabbits [18].

### 3. Mitochondrial permeability transition pore

In situations such as Ca2+ overload, alterations in ATP production, mitochondrial membrane depolarization, or inhibition of respiration, mitochondria may undergo a swelling process that represents a permeabilization of the internal membrane, product of the opening of nonspecific channels known as the mitochondrial permeability pore [19]. This pore, which allows the passage of molecules up to 1.5 kDa, mediates the rupture of the outer mitochondrial membrane and the release of proapoptotic substances such as endonuclease G and the mitochondrial apoptosis inducing factor (AIF) allowing the exit of elements such as the cytochrome C [20].

3.1.3. Mitochondrial phosphate carrier

3.1.4. Voltage-dependent anion channel

decreasing the apoptotic process [31].

3.2. MPTP opening in ischemia/reperfusion damage

pH. This inhibits phosphofructokinase and activates the H+

3.1.5. F1F0-ATP synthase

the mPTP [20].

3.2.1. Ischemia

such as Na+

because the Na+

is promoted [33].

The mitochondrial phosphate carrier is encoded by the gene SLC25A3 and is a primary transport system of inorganic phosphate (Pi) by a proton transporter or an exchanger with hydroxyl ions [16]. It has been described that Pi is a potent modulator for the MPTP opening. In an energized condition, Pi can induce the opening of the pore. This contrasts with other studies postulating Pi as an inhibitor of pore opening [30]. This carrier could be considered as a structural component

Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium…

This is a channel present in the external mitochondrial membrane that serves as a pore for different substances in or out of the mitochondria. Like ANT, voltage-dependent anion channel (VDAC) can be considered a structural element in the conformation of the MPTP. Inhibiting VDAC activity, inhibits Bax (a proapoptotic protein) function and the release of cytochrome C,

More recent investigations have shown that a dimer of the mitochondrial F1F0-ATP-synthase is essential to form the core of the mPTP. F1F0-ATP-synthase interacts with CyPD and can reversibly undergo a Ca2+-dependent transition to form a channel with the characteristics of

During the period of ischemia, the hypoxic condition makes the mitochondria unable to produce ATP through oxidative phosphorylation, leading to a rapid depletion of cellular ATP reserves, with the concomitant increase of ADP and Pi [15]. There is a change in ATP production from oxidation of fatty acids (most common) to glucose metabolism, but through an anaerobic route, a less efficient mechanism, which leads to increased lactate, NADH and H+ [32] with a decrease in

mechanism is unable to deliver enough ATP to supply cardiomyocytes, and specially pumps

sarcoplasmic reticulum, releasing Ca2+, further contributing to the overload of this ion [1]. The small amount of oxygen that remains serves for the oxidation of xanthine by the enzyme xanthine oxidase, mainly in the endothelium but also in the cardiomyocyte, which produces superoxide and other ROS. The duration of the ischemia period is an important factor; in short periods of ischemia (less than 15 min), reperfusion manages to restore homeostatic values with respect to mitochondrial membrane potential, cardiomyocyte contracture and Ca2+ concentration. However, in periods longer than 25 min, all these values are altered in a way that cell death

/K+ ATPase. The accumulation of Na+ facilitates the accumulation of Ca2+ in the cell

/Ca2+ exchanger works in a reverse mode [15]. Then, oxidative damage alters the

/Na+ symporter. This compensatory

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91

of the MPTP, but studies are still lacking to confirm its actual function.

What is the function of this mitochondrial pore? The pathological effects of the presence of the pore have been well described and usually associated with long-term openings since short-term openings do not have a major impact on cell viability [21]. On the contrary, it is believed that its transient opening can play a very important role both in the physiological regulation of Ca2+ and redox homeostasis in the generation of ROS [22].

#### 3.1. Constituents of the transition pore of mitochondrial permeability

#### 3.1.1. Cyclophilin D

Cyclophilin D (Cyp-D) is an 18 kDa protein encoded by the Ppif gene, synthesized in the cytosol and transported to the mitochondria to form part of the pore. It is inhibited by cyclosporine A, an immunosuppressant, decreasing its sensitivity to elevations in Ca2+ concentration [23]. CypD is a member of a family of cyclophilin proteins that have peptidylprolyl isomerase (PPIase) activity, catalyzing the cis-trans isomerization of peptidylprolyl bonds. In studies in knockout mice for the Ppif gene, mitochondria were more resistant to swelling and decreased the ability to open the pore [24]. It was also observed that overexpression of this gene caused an increase in swelling and cell death after ischemia-reperfusion [25]. Canceling the expression of this protein would, in theory, desensitize to the mitochondria for Ca2+ and will be protective. Nevertheless, it was described that when Ca2+ concentration is high enough, the pore can be opened independent of the presence of CypD [26]. It has been postulated that both CypD and the mitochondrial Ca2+ pore opening participate in the regulation of mitochondrial Ca2+ homeostasis, since the pore opening directly depends on the Ca2+ concentrations reached and its opening would try to reinforce the effect of the Na+ /Ca2+ through an efflux of Ca2+ to prevent the overload of this cation [27].

#### 3.1.2. Adenine nucleotide traslocase

Adenine nucleotide traslocase (ANT) is a member of the long list of mitochondrial carriers. It shows a tripartite conformation of repeated sequences of 100 amino acids, with ANT1 being the isoform present in the heart [28]. Given the importance of the production of ATP by the mitochondria and its use mainly in the cytosol, a mechanism of fast and effective transport between the two compartments is necessary: the exit of ATP to the cytosol and transport of the ADP to the mitochondria. Therefore, ANT1 is one of the most abundant proteins inside the mitochondria and more than 10% of the energy used by the cardiomyocyte is used to maintain this transport [28]. Regarding to the role of ANT in the pore formation, it has been observed that inhibition of binding of ATP and ADP to ANT increases the sensitivity of MPTP in response to variations in [Ca2+]i [19]. Evidence shows that ANT has the ability to bind CypD and that this binding is important for pore opening, with ANT acting as a sensor of [Ca2+]i. Although this protein has been categorized as a component of the MPTP, its importance in the pore gating is controversial since inhibition of both ANT 1 and 2 in mouse liver could still constitute the mitochondrial pore, although with a higher increase in the [Ca2+]i [29].

#### 3.1.3. Mitochondrial phosphate carrier

and the release of proapoptotic substances such as endonuclease G and the mitochondrial apoptosis inducing factor (AIF) allowing the exit of elements such as the cytochrome C [20].

What is the function of this mitochondrial pore? The pathological effects of the presence of the pore have been well described and usually associated with long-term openings since short-term openings do not have a major impact on cell viability [21]. On the contrary, it is believed that its transient opening can play a very important role both in the physiological regulation of Ca2+ and

Cyclophilin D (Cyp-D) is an 18 kDa protein encoded by the Ppif gene, synthesized in the cytosol and transported to the mitochondria to form part of the pore. It is inhibited by cyclosporine A, an immunosuppressant, decreasing its sensitivity to elevations in Ca2+ concentration [23]. CypD is a member of a family of cyclophilin proteins that have peptidylprolyl isomerase (PPIase) activity, catalyzing the cis-trans isomerization of peptidylprolyl bonds. In studies in knockout mice for the Ppif gene, mitochondria were more resistant to swelling and decreased the ability to open the pore [24]. It was also observed that overexpression of this gene caused an increase in swelling and cell death after ischemia-reperfusion [25]. Canceling the expression of this protein would, in theory, desensitize to the mitochondria for Ca2+ and will be protective. Nevertheless, it was described that when Ca2+ concentration is high enough, the pore can be opened independent of the presence of CypD [26]. It has been postulated that both CypD and the mitochondrial Ca2+ pore opening participate in the regulation of mitochondrial Ca2+ homeostasis, since the pore opening directly depends on the Ca2+ concentrations

Adenine nucleotide traslocase (ANT) is a member of the long list of mitochondrial carriers. It shows a tripartite conformation of repeated sequences of 100 amino acids, with ANT1 being the isoform present in the heart [28]. Given the importance of the production of ATP by the mitochondria and its use mainly in the cytosol, a mechanism of fast and effective transport between the two compartments is necessary: the exit of ATP to the cytosol and transport of the ADP to the mitochondria. Therefore, ANT1 is one of the most abundant proteins inside the mitochondria and more than 10% of the energy used by the cardiomyocyte is used to maintain this transport [28]. Regarding to the role of ANT in the pore formation, it has been observed that inhibition of binding of ATP and ADP to ANT increases the sensitivity of MPTP in response to variations in [Ca2+]i [19]. Evidence shows that ANT has the ability to bind CypD and that this binding is important for pore opening, with ANT acting as a sensor of [Ca2+]i. Although this protein has been categorized as a component of the MPTP, its importance in the pore gating is controversial since inhibition of both ANT 1 and 2 in mouse liver could still

constitute the mitochondrial pore, although with a higher increase in the [Ca2+]i [29].

/Ca2+ through an efflux of

redox homeostasis in the generation of ROS [22].

3.1.1. Cyclophilin D

90 Free Radicals, Antioxidants and Diseases

3.1. Constituents of the transition pore of mitochondrial permeability

reached and its opening would try to reinforce the effect of the Na+

Ca2+ to prevent the overload of this cation [27].

3.1.2. Adenine nucleotide traslocase

The mitochondrial phosphate carrier is encoded by the gene SLC25A3 and is a primary transport system of inorganic phosphate (Pi) by a proton transporter or an exchanger with hydroxyl ions [16]. It has been described that Pi is a potent modulator for the MPTP opening. In an energized condition, Pi can induce the opening of the pore. This contrasts with other studies postulating Pi as an inhibitor of pore opening [30]. This carrier could be considered as a structural component of the MPTP, but studies are still lacking to confirm its actual function.

#### 3.1.4. Voltage-dependent anion channel

This is a channel present in the external mitochondrial membrane that serves as a pore for different substances in or out of the mitochondria. Like ANT, voltage-dependent anion channel (VDAC) can be considered a structural element in the conformation of the MPTP. Inhibiting VDAC activity, inhibits Bax (a proapoptotic protein) function and the release of cytochrome C, decreasing the apoptotic process [31].

### 3.1.5. F1F0-ATP synthase

More recent investigations have shown that a dimer of the mitochondrial F1F0-ATP-synthase is essential to form the core of the mPTP. F1F0-ATP-synthase interacts with CyPD and can reversibly undergo a Ca2+-dependent transition to form a channel with the characteristics of the mPTP [20].

#### 3.2. MPTP opening in ischemia/reperfusion damage

#### 3.2.1. Ischemia

During the period of ischemia, the hypoxic condition makes the mitochondria unable to produce ATP through oxidative phosphorylation, leading to a rapid depletion of cellular ATP reserves, with the concomitant increase of ADP and Pi [15]. There is a change in ATP production from oxidation of fatty acids (most common) to glucose metabolism, but through an anaerobic route, a less efficient mechanism, which leads to increased lactate, NADH and H+ [32] with a decrease in pH. This inhibits phosphofructokinase and activates the H+ /Na+ symporter. This compensatory mechanism is unable to deliver enough ATP to supply cardiomyocytes, and specially pumps such as Na+ /K+ ATPase. The accumulation of Na+ facilitates the accumulation of Ca2+ in the cell because the Na+ /Ca2+ exchanger works in a reverse mode [15]. Then, oxidative damage alters the sarcoplasmic reticulum, releasing Ca2+, further contributing to the overload of this ion [1]. The small amount of oxygen that remains serves for the oxidation of xanthine by the enzyme xanthine oxidase, mainly in the endothelium but also in the cardiomyocyte, which produces superoxide and other ROS. The duration of the ischemia period is an important factor; in short periods of ischemia (less than 15 min), reperfusion manages to restore homeostatic values with respect to mitochondrial membrane potential, cardiomyocyte contracture and Ca2+ concentration. However, in periods longer than 25 min, all these values are altered in a way that cell death is promoted [33].

#### 3.2.2. Reperfusion

In reperfusion, other factors beside an increase in Pi, ROS and Ca2+ and the decrease in ATP come into play. Although all of them contribute to the opening of the mitochondrial pore, the diminished pH would be a "protective" factor in the conformation of this pore [34]. In fact, a decrease in pH inhibits the H+ /Na+ symporter, which would prevent accumulation of Ca2+ in the cell [35].

protective effects in ischemia-reperfusion [42]. A decrease in infarcted area was also demon-

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93

Interestingly, CypD can be S-nitrosylated on cysteine 203. This S-nitrosylation reduces the

There are other constituents of the mitochondrial pore that, being inhibited, contribute to avoid the gating of the pore. Unfortunately pore elements such as ANT and the carrier of mitochondrial phosphate are indispensable for other vital cellular functions, and therefore their inhibition would add the cell an additional risk after ischemia-reperfusion. The opening of the pore can also be avoided indirectly. By decreasing the influx of Ca2+ from the extracellu-

Nitric oxide is a molecule that participates in different processes, including neurotransmission, vasodilatation, defense against pathogens and depending on cellular conditions, in apoptosis [44–46]. This molecule is produced by the conversion of L-arginine to L-citrulline, a reaction catalyzed by one of three types of enzyme nitric oxide synthase (NOS) [47]. Neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) are constitutively expressed enzymes and inflammatory nitric oxide synthase (iNOS) is inducible. The activity of NO as a cell signaling molecule via cyclic GMP is well known. The cGMP formed by NO has three targets: protein kinase G (PKG), cyclic nucleotide-dependent channels and phosphodiesterase (PDE) [48] achieving well-studied vasoactive effects. However, recent studies suggest that NO can modify proteins posttranslationally, via a nitrosative pathway, known as S-nitrosylation [49]. S-nitrosylation consists in the addition a NO group to a cysteine residue generating nitrosothiols (SNO). Different studies have shown that this nitrosative modification of proteins alters their function, reversibly [49]. In addition, the S-nitrosylated cysteines of proteins are in a chemical equilibrium with S-nitrosoglutathione (GSNO), a low molecular weight nitrosothiol. In this manner, GSNO functions as a reserve of NO that can be exchanged with GSH in a reaction termed transnitro-

Although the cardioprotective effects of NO by their dependent cGMP pathway are known, recent research suggests a relationship between increased SNO formation and cardioprotection

In ischemia-reperfusion studies, S-nitrosylation has been associated with an increase in the recovery of developed ventricular pressure and ventricular work [54]. This has been observed in both preconditioning and ischemic postconditioning studies [55], resulting in an increase in Snitrosylation associated with an improvement in contractile activity. This recovery of functionality has also been appreciated with the use of S-nitrosothiols donors such as Mito-SNO (targeted to mitochondria) or S-nitroso-N-acetylpenicillamine (SNAP) [56]. Using GSNO to generate a preconditioning effect in an ischemia-reperfusion experiment in mice resulted in a greater

[52]. In cardiomyocytes, S-nitrosylation occurs in a large number of proteins [53].

strated in knockout mice for CypD compared control mice [43].

lar space, the overload of this cation is avoided and thus the pore opening.

sylation and in this way participates in part of NO bioactivity [50, 51].

5.1. S-nitrosylation and cardioprotection

5. Cardioprotective function of nitric oxide

activity of the MPTP [23].

Recovery of oxygen levels re-energizes the mitochondria to restore its role in the production of ATP via fatty acids metabolism. An alteration of the membrane potential causes Ca2+ to enter the mitochondria, while the oxygen entering it contributes to the formation of more ROS due to the reduced respiratory chain [19]. Reperfusion favors the recovery of pH in a period close to 3 min, time in which lactate diffuses. During the first few minutes, the damage caused by reperfusion is small; nevertheless, as reperfusion is delayed, the infarcted area grows. Once the pore has been established as a result of the abovementioned alterations, mitochondria may become swollen and the rupture of the outer membrane occurs, which will trigger the release of cytochrome C and other proapoptotic substances leading to cell death [15, 36–38].

### 4. Mitochondrial ROS release and reperfusion injury

ROS release by mitochondria is one of the major events that trigger ischemia-reperfusion injury. Although this event is thought to be produced by an alteration in the mitochondrial electron transport chain during reperfusion, it is further believed that these reactive species can be produced by an altered metabolic process during ischemia, whose consequences appear in the reperfusion period. For example, in a study by Couchani et al., they observed the accumulation of succinate, a metabolic intermediate of the Krebs cycle, during ischemia, as product of the reversal activity of succinate dehydrogenase. During reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I [39].

#### 4.1. Inhibition of mitochondrial pore opening protects the heart against ischemia-reperfusion injury

The importance of MPTP opening in myocardial damage makes it an excellent target for therapies aimed to reduce reperfusion injury. In studies in animal models in which the mitochondrial pore opening was inhibited, a reduction of the infarcted area was observed in more than 50% [1, 40], highlighting the impact of the presence of this pore on the magnitude of myocardial damage. These types of treatments are currently in development and in the search for safe and specific pore opening inhibitors [1, 38, 41].

In this context, different mechanisms have been developed to mitigate cell damage induced by reperfusion, and although improvements have been achieved with regard to the response of the infarcted region, there is no effective therapy yet to completely avoid reperfusion injury [2].

An interesting target to achieve inhibition of the opening of the pore is cyclophilin D. As mentioned earlier, the inhibition of CypD with cyclosporine in isolated hearts has shown protective effects in ischemia-reperfusion [42]. A decrease in infarcted area was also demonstrated in knockout mice for CypD compared control mice [43].

Interestingly, CypD can be S-nitrosylated on cysteine 203. This S-nitrosylation reduces the activity of the MPTP [23].

There are other constituents of the mitochondrial pore that, being inhibited, contribute to avoid the gating of the pore. Unfortunately pore elements such as ANT and the carrier of mitochondrial phosphate are indispensable for other vital cellular functions, and therefore their inhibition would add the cell an additional risk after ischemia-reperfusion. The opening of the pore can also be avoided indirectly. By decreasing the influx of Ca2+ from the extracellular space, the overload of this cation is avoided and thus the pore opening.

### 5. Cardioprotective function of nitric oxide

3.2.2. Reperfusion

92 Free Radicals, Antioxidants and Diseases

in pH inhibits the H+

injury

pore opening inhibitors [1, 38, 41].

In reperfusion, other factors beside an increase in Pi, ROS and Ca2+ and the decrease in ATP come into play. Although all of them contribute to the opening of the mitochondrial pore, the diminished pH would be a "protective" factor in the conformation of this pore [34]. In fact, a decrease

Recovery of oxygen levels re-energizes the mitochondria to restore its role in the production of ATP via fatty acids metabolism. An alteration of the membrane potential causes Ca2+ to enter the mitochondria, while the oxygen entering it contributes to the formation of more ROS due to the reduced respiratory chain [19]. Reperfusion favors the recovery of pH in a period close to 3 min, time in which lactate diffuses. During the first few minutes, the damage caused by reperfusion is small; nevertheless, as reperfusion is delayed, the infarcted area grows. Once the pore has been established as a result of the abovementioned alterations, mitochondria may become swollen and the rupture of the outer membrane occurs, which will trigger the release

of cytochrome C and other proapoptotic substances leading to cell death [15, 36–38].

ROS release by mitochondria is one of the major events that trigger ischemia-reperfusion injury. Although this event is thought to be produced by an alteration in the mitochondrial electron transport chain during reperfusion, it is further believed that these reactive species can be produced by an altered metabolic process during ischemia, whose consequences appear in the reperfusion period. For example, in a study by Couchani et al., they observed the accumulation of succinate, a metabolic intermediate of the Krebs cycle, during ischemia, as product of the reversal activity of succinate dehydrogenase. During reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by

4.1. Inhibition of mitochondrial pore opening protects the heart against ischemia-reperfusion

The importance of MPTP opening in myocardial damage makes it an excellent target for therapies aimed to reduce reperfusion injury. In studies in animal models in which the mitochondrial pore opening was inhibited, a reduction of the infarcted area was observed in more than 50% [1, 40], highlighting the impact of the presence of this pore on the magnitude of myocardial damage. These types of treatments are currently in development and in the search for safe and specific

In this context, different mechanisms have been developed to mitigate cell damage induced by reperfusion, and although improvements have been achieved with regard to the response of the infarcted region, there is no effective therapy yet to completely avoid reperfusion injury [2]. An interesting target to achieve inhibition of the opening of the pore is cyclophilin D. As mentioned earlier, the inhibition of CypD with cyclosporine in isolated hearts has shown

4. Mitochondrial ROS release and reperfusion injury

reverse electron transport at mitochondrial complex I [39].

/Na+ symporter, which would prevent accumulation of Ca2+ in the cell [35].

Nitric oxide is a molecule that participates in different processes, including neurotransmission, vasodilatation, defense against pathogens and depending on cellular conditions, in apoptosis [44–46]. This molecule is produced by the conversion of L-arginine to L-citrulline, a reaction catalyzed by one of three types of enzyme nitric oxide synthase (NOS) [47]. Neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) are constitutively expressed enzymes and inflammatory nitric oxide synthase (iNOS) is inducible. The activity of NO as a cell signaling molecule via cyclic GMP is well known. The cGMP formed by NO has three targets: protein kinase G (PKG), cyclic nucleotide-dependent channels and phosphodiesterase (PDE) [48] achieving well-studied vasoactive effects. However, recent studies suggest that NO can modify proteins posttranslationally, via a nitrosative pathway, known as S-nitrosylation [49]. S-nitrosylation consists in the addition a NO group to a cysteine residue generating nitrosothiols (SNO). Different studies have shown that this nitrosative modification of proteins alters their function, reversibly [49]. In addition, the S-nitrosylated cysteines of proteins are in a chemical equilibrium with S-nitrosoglutathione (GSNO), a low molecular weight nitrosothiol. In this manner, GSNO functions as a reserve of NO that can be exchanged with GSH in a reaction termed transnitrosylation and in this way participates in part of NO bioactivity [50, 51].

#### 5.1. S-nitrosylation and cardioprotection

Although the cardioprotective effects of NO by their dependent cGMP pathway are known, recent research suggests a relationship between increased SNO formation and cardioprotection [52]. In cardiomyocytes, S-nitrosylation occurs in a large number of proteins [53].

In ischemia-reperfusion studies, S-nitrosylation has been associated with an increase in the recovery of developed ventricular pressure and ventricular work [54]. This has been observed in both preconditioning and ischemic postconditioning studies [55], resulting in an increase in Snitrosylation associated with an improvement in contractile activity. This recovery of functionality has also been appreciated with the use of S-nitrosothiols donors such as Mito-SNO (targeted to mitochondria) or S-nitroso-N-acetylpenicillamine (SNAP) [56]. Using GSNO to generate a preconditioning effect in an ischemia-reperfusion experiment in mice resulted in a greater recovery of developed ventricular pressure and a smaller infarcted area compared to controls [54]. Furthermore, it was observed that the inhibition of caspase-3 by S-nitrosylation decreases its proapoptotic activity [57]. In a study in mice undergoing cardiac ischemia-reperfusion, it was observed that an increase in S-nitrosylation of the cardiac L-type Ca2+ channel reduced Ca2+ entry, thereby reducing Ca2+ overload, subsequently reducing cardiac damage by reperfusion [58]. On the other hand, in transgenic mice with iNOS overexpression submitted to cardiac ischemia-reperfusion, a smaller infarcted area was observed in addition to a lower Ca2+ overload and a smaller opening of the mitochondrial pore [59]. Mito-SNO (5-(2-acetylamino-3-methyl-3 nitrosothiobutyrylamino)-pentyl]-triphenylphosphonium methanesulfonate) was used in a study in mice and it was observed that the application of this donor was able to improve cardiac function and also to decrease the infarcted area compared to controls [60]. This cardioprotective effect was due to the reversible S-nitrosylation of the mitochondrial complex I. This effect would slow mitochondrial activation in the first few minutes of reperfusion, a crucial moment in ROS release, decreasing it and limiting reperfusion injury [61]. Similar effects were found in ischemiareperfusion in rat hearts, where another S-nitrosothiol donor, S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was used, resulting in inhibition of the complex I, mainly during the late stage of ischemia and early reperfusion, thereby reducing ROS production by mitochondria, similar to the previous case. In addition, it was observed that in hearts treated with the nitrosylating agent they were more resistant to the opening of the transition pore [62]. In studies of ischemic preconditioning in mouse hearts, a relationship between increased S-nitrosylation and cardioprotection was observed, where an elevation of the nitrosothiols with GSNO treatment, developed positive ventricular pressure and reduced total infarcted area [54].

increasing levels of glutathione (GSH), thereby reducing nitrosylation [69]. This enzyme is highly conserved from bacteria to mammals and has the function of modulating the S-nitrosylating function of NO and thus avoiding for example nitrosative stress in the presence of

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Although this enzyme does not act directly on SNO-protein substrates, its deficiency causes an increase in the intracellular concentrations of nitrosylated proteins (Figure 2). This means that there is an equilibrium based on the transfer of SNO groups between low molecular weight nitrosothiols and protein cysteines. It is also thought that the role of GSNO is not only a transitrosilation (from one thiol to another) but also function as a source of NO independent

Figure 2. GSNOR in the cardiac cell. Immunocytochemical analysis of S-nitrosoglutathione reductase in cardiac myocytes. (A) The pictures depicts the localization in a mouse cardiomyocytes of GSNOR (green) compared to that of the ryanodine receptor (RyR2, red) using confocal microscopy. The sections demarked by dotted lines are amplified below. Notice that GSNOR partially co-localizes with RyR2, suggesting association in the sarcoplasmic reticulum. (B) Co-localization analysis for RyR2 (red) and S-nitrosylated thiols (Cys-NO, green) using a specific antibody against Cys-NO in wild type (WT) and GSNOR-deficient (GSNOR�/�) mice. The bar graph shows the Pearson analysis for colocalization of RyR2 and CysNO. \*\* indicates p < 0.001 using T test. Number of cells analyzed is indicated between

excessive NO.

of the action of NOS.

parentheses. The scale bar indicates 10 μm.

In mitochondria, complex I is the primary site of ROS production, with the generation of superoxide anion. S-nitrosylation of complex I was shown to inhibit its activity, which reduces ROS production during reperfusion [63]. However, SNAP reversibly inactivated the mitochondrial complex I, resulting in an increase in H2O2 production [64]. On the other hand, S- nitrosylation decreased the Ca2+ elevation produced during reperfusion through the modification of the L-type Ca2+ channel [54], which prevented the opening of the mitochondrial pore.

An interesting question arises as whether nitrosylation occurs equally in all mitochondria. Sun et al. found higher S-nitrosylation in the sub sarcolemmal mitochondria than in the interfibrillar in mice hearts subjected to ischemic preconditioning. Both types of mitochondria differ in protein and lipid composition, in addition to the capacity for protein synthesis and oxygenation [65]. This suggests that subsarcolemmal mitochondria are more susceptible to ischemia-reperfusion injury, with the MPTP more prone to ischemia-reperfusion injury and also being exposed to a higher oxygen gradient during reperfusion [66]. This difference in mitochondrial response has already been observed with the use of other cardioprotective agents such as diazoxide [67].

#### 5.2. S-nitrosoglutathione reductase

The cellular levels of S-nitrosothiols are governed by the enzyme S-nitrosoglutathione reductase (GSNOR), an alcohol dehydrogenase type III enzyme, whose function is to remove NO groups from the cysteine thiols in proteins [68] through denitrosylation, degrading GSNO and increasing levels of glutathione (GSH), thereby reducing nitrosylation [69]. This enzyme is highly conserved from bacteria to mammals and has the function of modulating the S-nitrosylating function of NO and thus avoiding for example nitrosative stress in the presence of excessive NO.

recovery of developed ventricular pressure and a smaller infarcted area compared to controls [54]. Furthermore, it was observed that the inhibition of caspase-3 by S-nitrosylation decreases its proapoptotic activity [57]. In a study in mice undergoing cardiac ischemia-reperfusion, it was observed that an increase in S-nitrosylation of the cardiac L-type Ca2+ channel reduced Ca2+ entry, thereby reducing Ca2+ overload, subsequently reducing cardiac damage by reperfusion [58]. On the other hand, in transgenic mice with iNOS overexpression submitted to cardiac ischemia-reperfusion, a smaller infarcted area was observed in addition to a lower Ca2+ overload and a smaller opening of the mitochondrial pore [59]. Mito-SNO (5-(2-acetylamino-3-methyl-3 nitrosothiobutyrylamino)-pentyl]-triphenylphosphonium methanesulfonate) was used in a study in mice and it was observed that the application of this donor was able to improve cardiac function and also to decrease the infarcted area compared to controls [60]. This cardioprotective effect was due to the reversible S-nitrosylation of the mitochondrial complex I. This effect would slow mitochondrial activation in the first few minutes of reperfusion, a crucial moment in ROS release, decreasing it and limiting reperfusion injury [61]. Similar effects were found in ischemiareperfusion in rat hearts, where another S-nitrosothiol donor, S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was used, resulting in inhibition of the complex I, mainly during the late stage of ischemia and early reperfusion, thereby reducing ROS production by mitochondria, similar to the previous case. In addition, it was observed that in hearts treated with the nitrosylating agent they were more resistant to the opening of the transition pore [62]. In studies of ischemic preconditioning in mouse hearts, a relationship between increased S-nitrosylation and cardioprotection was observed, where an elevation of the nitrosothiols with GSNO treat-

ment, developed positive ventricular pressure and reduced total infarcted area [54].

Ca2+ channel [54], which prevented the opening of the mitochondrial pore.

agents such as diazoxide [67].

94 Free Radicals, Antioxidants and Diseases

5.2. S-nitrosoglutathione reductase

In mitochondria, complex I is the primary site of ROS production, with the generation of superoxide anion. S-nitrosylation of complex I was shown to inhibit its activity, which reduces ROS production during reperfusion [63]. However, SNAP reversibly inactivated the mitochondrial complex I, resulting in an increase in H2O2 production [64]. On the other hand, S- nitrosylation decreased the Ca2+ elevation produced during reperfusion through the modification of the L-type

An interesting question arises as whether nitrosylation occurs equally in all mitochondria. Sun et al. found higher S-nitrosylation in the sub sarcolemmal mitochondria than in the interfibrillar in mice hearts subjected to ischemic preconditioning. Both types of mitochondria differ in protein and lipid composition, in addition to the capacity for protein synthesis and oxygenation [65]. This suggests that subsarcolemmal mitochondria are more susceptible to ischemia-reperfusion injury, with the MPTP more prone to ischemia-reperfusion injury and also being exposed to a higher oxygen gradient during reperfusion [66]. This difference in mitochondrial response has already been observed with the use of other cardioprotective

The cellular levels of S-nitrosothiols are governed by the enzyme S-nitrosoglutathione reductase (GSNOR), an alcohol dehydrogenase type III enzyme, whose function is to remove NO groups from the cysteine thiols in proteins [68] through denitrosylation, degrading GSNO and Although this enzyme does not act directly on SNO-protein substrates, its deficiency causes an increase in the intracellular concentrations of nitrosylated proteins (Figure 2). This means that there is an equilibrium based on the transfer of SNO groups between low molecular weight nitrosothiols and protein cysteines. It is also thought that the role of GSNO is not only a transitrosilation (from one thiol to another) but also function as a source of NO independent of the action of NOS.

Figure 2. GSNOR in the cardiac cell. Immunocytochemical analysis of S-nitrosoglutathione reductase in cardiac myocytes. (A) The pictures depicts the localization in a mouse cardiomyocytes of GSNOR (green) compared to that of the ryanodine receptor (RyR2, red) using confocal microscopy. The sections demarked by dotted lines are amplified below. Notice that GSNOR partially co-localizes with RyR2, suggesting association in the sarcoplasmic reticulum. (B) Co-localization analysis for RyR2 (red) and S-nitrosylated thiols (Cys-NO, green) using a specific antibody against Cys-NO in wild type (WT) and GSNOR-deficient (GSNOR�/�) mice. The bar graph shows the Pearson analysis for colocalization of RyR2 and CysNO. \*\* indicates p < 0.001 using T test. Number of cells analyzed is indicated between parentheses. The scale bar indicates 10 μm.

While most proteins have cysteine residues and NOS is expressed in almost all cells, substrate specificity is an important feature of endogenous S-nitrosylation of proteins. Moreover, in all proteins possessing more than one S-nitrosylation site, the single modification of one cysteine thiol can modify their function under physiological conditions [70].

that the absence of GSNOR may be harmful and that this increase in S-nitrosylation that would cause its absence would be pathological. Rizza et al. observed that hepatocytes from GSNORdeficient mice had mitochondrial alterations, characterized by an increase in the levels and activity of the enzyme succinate dehydrogenase, mediating this alteration in a greater proclivity to the development of hepatocarcinoma [82]. This relationship had already been studied previously by Wei et al. who found that human hepatocarcinoma cells showed less GSNOR activity than noncancerous liver cells [83], inferring that the absence of GSNOR can alter DNA repair and thus induce tumor growth. In lung cancer, studies show that GSNOR is reduced in lung cancer samples relative to normal lung tissue [84]. This emphasizes the relationship between nitrosative stress and the development of neoplasia and how this enzyme can partic-

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97

GSNOR also appears to be important in the immune system. A study in GSNOR-deficient mice shows that the absence of this enzyme would cause a pathological nitrosylation causing apoptosis in thymus cells and a reduction of B and T lymphocytes. All this was mediated by

Given the large number of benefits delivered by S-nitrosylation in the heart, there is currently a search for therapies that pursue a greater availability of S-nitrosothiols in order to reduce cardiac damage and improve ventricular performance after ischemia. Therefore, NO donor agents, ischemic pre- and postconditioning, and modulation of GSNOR activity have

In the cardiomyocyte of knockout mice for GSNOR, a decrease in the response to a β-adrenergic agonist and a decrease in cytosolic Ca2+ concentration were observed [86], highlighting the role of endogenous S-nitrosothiols in cardiac function. In these GSNOR it was observed S-nitrosylation of β-arrestin and G protein receptor kinase 2, two proteins involved in the regulation of the β-adrenergic receptors pathway [87], besides the nitrosylation of different Ca2+ channels, such as the L-type Ca2+ channel, ryanodine receptor RyR2 and the pump SERCA [88] (Figure 3). Lima et al., using a model of myocardial infarction in GSNOR-deficient mice, observed a reduction in the infarcted area, maintenance of ventricular function, with an increase in vascular density compared to wild type mice. This proangiogenic effect of GSNOR inhibition was explained as a result of increased activity of transcription factor hypoxia inducible factor-1 (HIF-1 α) due to increased S-nitrosylation in normoxic conditions, which resulted in increased binding to the

In a similar study in GSNOR-deficient mice that underwent myocardial infarction, the improved contractile function and increased angiogenesis were confirmed. Importantly, these authors reported that after myocardial infarction, the GSNOR knockout mice hearts showed increased proliferation of endogenous cardiac stem cells and increased mitosis in cardiac myocytes com-

On the other hand, pharmacological inhibition of GSNOR has been shown to improve endothelial function in rats. In a study by Chen et al. it was observed that using the GSNOR

ipate in its development.

been tested.

VEGF gene promoter [50].

pared to wild type mice [89].

the uncontrolled activity of iNOS [85].

5.4. GSNOR and cardioprotection

Apparently, the activity of GSNOR is increased lung endothelial cell of females compared to males [71]. In the heart the results are similar. Females possess a higher activity of GSNOR than males [72], without differences in the activity of this enzyme in sexually immature mice. Although there are no significant differences in the expression of GSNOR for either sex, estrogens stimulate the activity of eNOS, which would lead to increased S-nitrosylation, an important factor that would stimulate GSNOR activity. On the other hand androgens may play a role, since in neutered male mice the activity of the GSNOR seems to be similar to that of the females. This mechanism of GSNOR activation is not known [71].

#### 5.3. The complex role of GSNOR

Given the anti-inflammatory and muscle relaxant properties of NO, inhibition of GSNOR is already being tested for the treatment of associated pathologies, mainly respiratory [73]. For example, in bronchoscopy samples from different subjects, GSNOR activity was increased in certain types of asthma [74], which makes it a good target for the treatment of this pathology. In a mice model of asthma, the GSNOR inhibitor SPL-334 was evaluated, resulting in a reduction of bronchial inflammation, airway hyperreactivity, mucus production and eosinophil accumulation and allergen-specific T [75]. Also, in mice lung smooth muscle, inhibition of GSNOR decreased the contractile response in the methacholine test, in addition to anti-inflammatory effects [76]. On the other hand, GSNOR appears to protect lung immune cells from nitrosative stress, since its deficiency tends to increase lung susceptibility for Klebsiella pneumoniae infection [77].

Skeletal muscle tissue has also been evaluated for GSNOR function. With the use of GSNOR knockout mice, the tibialis anterior muscle of GSNOR�/� mice was found to be more resistant to fatigue, with no alterations in mitochondrial function or in capillary density, associated with hypernitrosylation ryanodine receptor (RyR1), which could increase skeletal muscle contractility without altering mitochondrial function [78]. However, another study using GSNOR�/� mice showed the opposite effect, since the silencing of GSNOR revealed a muscular atrophy and loss of muscle mass, associated with increased S-nitrosothiols, along with mitochondrial fragmentation and depolarization [79].

GSNOR also appears to have a role in breast cancer. The study by Cañas et al. indicates that the antiproliferative action exerted by trastuzumab on breast cancer cells overexpressing HER2 is suppressed when GSNOR is inhibited. This indicates that an increase in SNOs would provide a survival advantage for cancer in HER2 + tumors and may constitute a mechanism of resistance to this targeted treatment in breast cancer [80]. On the other hand, other studies have shown that inhibition of GSNOR can be detrimental. Rizza et al. observed that the silencing of GSNOR was shown to protect SH-SY5Y cells from toxins characteristic of Parkinson's disease. However, it was also observed that overexpression of this enzyme is a resistance factor for the treatment of amyotrophic lateral sclerosis [81]. It has also been shown in other investigations

that the absence of GSNOR may be harmful and that this increase in S-nitrosylation that would cause its absence would be pathological. Rizza et al. observed that hepatocytes from GSNORdeficient mice had mitochondrial alterations, characterized by an increase in the levels and activity of the enzyme succinate dehydrogenase, mediating this alteration in a greater proclivity to the development of hepatocarcinoma [82]. This relationship had already been studied previously by Wei et al. who found that human hepatocarcinoma cells showed less GSNOR activity than noncancerous liver cells [83], inferring that the absence of GSNOR can alter DNA repair and thus induce tumor growth. In lung cancer, studies show that GSNOR is reduced in lung cancer samples relative to normal lung tissue [84]. This emphasizes the relationship between nitrosative stress and the development of neoplasia and how this enzyme can participate in its development.

GSNOR also appears to be important in the immune system. A study in GSNOR-deficient mice shows that the absence of this enzyme would cause a pathological nitrosylation causing apoptosis in thymus cells and a reduction of B and T lymphocytes. All this was mediated by the uncontrolled activity of iNOS [85].

#### 5.4. GSNOR and cardioprotection

While most proteins have cysteine residues and NOS is expressed in almost all cells, substrate specificity is an important feature of endogenous S-nitrosylation of proteins. Moreover, in all proteins possessing more than one S-nitrosylation site, the single modification of one cysteine

Apparently, the activity of GSNOR is increased lung endothelial cell of females compared to males [71]. In the heart the results are similar. Females possess a higher activity of GSNOR than males [72], without differences in the activity of this enzyme in sexually immature mice. Although there are no significant differences in the expression of GSNOR for either sex, estrogens stimulate the activity of eNOS, which would lead to increased S-nitrosylation, an important factor that would stimulate GSNOR activity. On the other hand androgens may play a role, since in neutered male mice the activity of the GSNOR seems to be similar to that of the

Given the anti-inflammatory and muscle relaxant properties of NO, inhibition of GSNOR is already being tested for the treatment of associated pathologies, mainly respiratory [73]. For example, in bronchoscopy samples from different subjects, GSNOR activity was increased in certain types of asthma [74], which makes it a good target for the treatment of this pathology. In a mice model of asthma, the GSNOR inhibitor SPL-334 was evaluated, resulting in a reduction of bronchial inflammation, airway hyperreactivity, mucus production and eosinophil accumulation and allergen-specific T [75]. Also, in mice lung smooth muscle, inhibition of GSNOR decreased the contractile response in the methacholine test, in addition to anti-inflammatory effects [76]. On the other hand, GSNOR appears to protect lung immune cells from nitrosative stress, since its

deficiency tends to increase lung susceptibility for Klebsiella pneumoniae infection [77].

Skeletal muscle tissue has also been evaluated for GSNOR function. With the use of GSNOR knockout mice, the tibialis anterior muscle of GSNOR�/� mice was found to be more resistant to fatigue, with no alterations in mitochondrial function or in capillary density, associated with hypernitrosylation ryanodine receptor (RyR1), which could increase skeletal muscle contractility without altering mitochondrial function [78]. However, another study using GSNOR�/� mice showed the opposite effect, since the silencing of GSNOR revealed a muscular atrophy and loss of muscle mass, associated with increased S-nitrosothiols, along with mitochondrial

GSNOR also appears to have a role in breast cancer. The study by Cañas et al. indicates that the antiproliferative action exerted by trastuzumab on breast cancer cells overexpressing HER2 is suppressed when GSNOR is inhibited. This indicates that an increase in SNOs would provide a survival advantage for cancer in HER2 + tumors and may constitute a mechanism of resistance to this targeted treatment in breast cancer [80]. On the other hand, other studies have shown that inhibition of GSNOR can be detrimental. Rizza et al. observed that the silencing of GSNOR was shown to protect SH-SY5Y cells from toxins characteristic of Parkinson's disease. However, it was also observed that overexpression of this enzyme is a resistance factor for the treatment of amyotrophic lateral sclerosis [81]. It has also been shown in other investigations

thiol can modify their function under physiological conditions [70].

females. This mechanism of GSNOR activation is not known [71].

5.3. The complex role of GSNOR

96 Free Radicals, Antioxidants and Diseases

fragmentation and depolarization [79].

Given the large number of benefits delivered by S-nitrosylation in the heart, there is currently a search for therapies that pursue a greater availability of S-nitrosothiols in order to reduce cardiac damage and improve ventricular performance after ischemia. Therefore, NO donor agents, ischemic pre- and postconditioning, and modulation of GSNOR activity have been tested.

In the cardiomyocyte of knockout mice for GSNOR, a decrease in the response to a β-adrenergic agonist and a decrease in cytosolic Ca2+ concentration were observed [86], highlighting the role of endogenous S-nitrosothiols in cardiac function. In these GSNOR it was observed S-nitrosylation of β-arrestin and G protein receptor kinase 2, two proteins involved in the regulation of the β-adrenergic receptors pathway [87], besides the nitrosylation of different Ca2+ channels, such as the L-type Ca2+ channel, ryanodine receptor RyR2 and the pump SERCA [88] (Figure 3). Lima et al., using a model of myocardial infarction in GSNOR-deficient mice, observed a reduction in the infarcted area, maintenance of ventricular function, with an increase in vascular density compared to wild type mice. This proangiogenic effect of GSNOR inhibition was explained as a result of increased activity of transcription factor hypoxia inducible factor-1 (HIF-1 α) due to increased S-nitrosylation in normoxic conditions, which resulted in increased binding to the VEGF gene promoter [50].

In a similar study in GSNOR-deficient mice that underwent myocardial infarction, the improved contractile function and increased angiogenesis were confirmed. Importantly, these authors reported that after myocardial infarction, the GSNOR knockout mice hearts showed increased proliferation of endogenous cardiac stem cells and increased mitosis in cardiac myocytes compared to wild type mice [89].

On the other hand, pharmacological inhibition of GSNOR has been shown to improve endothelial function in rats. In a study by Chen et al. it was observed that using the GSNOR

6. Conclusions

prevention of myocardial diseases.

Acknowledgements

Author details

de Talca, Talca, Chile

icine. 2007;357:1121-1135

References

Talca.

Although reperfusion is essential for the recovery of the heart after ischemia, it can itself initiate myocardial damage and cause death of the cardiomyocyte. A key event in this cellular disorder of this alteration is the opening of the MPTP, which induces mitochondrial dysfunction and ultimately leads to cardiomyocyte death. For this reason, therapies are being developed in order to limit the opening of the pore and reduce the damage by reperfusion. One important mechanisms of cardioprotection is S-nitrosylation, which consists of adding NO groups to cysteine residues of proteins, modifying their function. S-nitrosylation of mitochondrial complex I and MPTP using NO donors and S-nitrosylating agents has shown to induce cardioprotection. It is known that agents or genetic alterations that increase SNO are cardioprotective and in this context the modulation of GSNOR is of relevance, since this protein is a critical regulator of endogenous SNOs. This enzyme participates in the redox balance, regulating the levels of nitrosylation/denitrosylation and its inhibition can increase the bioavailability of S-nitrosothiols, being this a potential cardioprotection element. So far, in the heart, genetic cancelation of GSNOR has indeed shown positive results in the context of damage by infarction. For this reason, more studies are needed in order to assess the impact of inhibition of GSNOR on global cellular functioning. This will indeed help in the development of new knowledge for the treatment and

Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium…

http://dx.doi.org/10.5772/intechopen.77003

99

This work was supported by Interdisciplinary Program PIEI QUIMBIO of the Universidad de

Departamento de Ciencias Básicas Biomédicas, Facultad de Ciencias de la Salud, Universidad

[1] Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. New England Journal of Med-

[2] Hausenloy DJ, Yellon DM. Myocardial ischemia-reperfusion injury: A neglected thera-

peutic target. The Journal of Clinical Investigation. 2013;123:92-100

Oscar Arriagada, Adriana V. Treuer and Daniel R. González\*

\*Address all correspondence to: dagonzalez@utalca.cl

Figure 3. Regulation of intracardiac S-nitrosothiols by GSNOR. Schematic model of the regulation of S-nitrosylation within the cardiac cell by GSNOR. S-nitrosoglutathione reductase is able to metabolize endogenous S-nitrosothiols by controlling the fate of S-nitrosoglutathione (GSNOR). The RyR2 and the L-type (I Ca) calcium channels are nitrosylated (SNO) by the activity of the nitric oxide synthases 1 (NOS1) and 3 (NOS3) located in the sarcoplasmic reticulum and plasmalemma, respectively. SNO are in equilibrium with nitrosoglutathione (GSNO). Thereby, by controlling the fate of GSNO, GSNOR regulates the levels of S-nitrosylation of these channels, important for the reperfusion damage. GSSH, oxidized glutathione, NH3, ammonia.

inhibitor N6338 at a single dose maintained flow-mediated arterial dilation versus inhibition of NOS. In hypertensive rats, application of N6338 was shown to decrease blood pressure and vascular resistance, as well as to restore altered flow-mediated dilatation [90].

Then, the question arises whether inhibition of GSNOR is cardioprotective. Much of the evidence suggests that this would be the case. However, the protective role of GSNOR in preventing pathological S-nitrosylation should be considered, as there are studies that suggest that the enzyme is essential for cardiovascular homeostasis. For example, Sips et al. used mice that overexpressed GSNOR specifically in cardiomyocytes and observed an improvement in myocardial properties post induction of myocardial damage after sepsis, a condition that is associated with nitrosative stress derived from the activity of iNOS [91]. Nevertheless, in conditions where increased bioavailability of S-nitrosothiols is required, such after ischemia, the inhibition of GSNOR appears as an attractive therapeutic strategy. This would increase the S-nitrosylation for example, of calcium-handling proteins, which, in a setting of ischemiareperfusion would prevent the calcium overload. In addition, increased S-nitrosylation of mitochondrial proteins may prevent the generation of ROS and a reduction in mitochondrial permeability transition pore activity, reducing cell death. Nevertheless, these effects remain to be probed experimentally.

### 6. Conclusions

Although reperfusion is essential for the recovery of the heart after ischemia, it can itself initiate myocardial damage and cause death of the cardiomyocyte. A key event in this cellular disorder of this alteration is the opening of the MPTP, which induces mitochondrial dysfunction and ultimately leads to cardiomyocyte death. For this reason, therapies are being developed in order to limit the opening of the pore and reduce the damage by reperfusion. One important mechanisms of cardioprotection is S-nitrosylation, which consists of adding NO groups to cysteine residues of proteins, modifying their function. S-nitrosylation of mitochondrial complex I and MPTP using NO donors and S-nitrosylating agents has shown to induce cardioprotection. It is known that agents or genetic alterations that increase SNO are cardioprotective and in this context the modulation of GSNOR is of relevance, since this protein is a critical regulator of endogenous SNOs. This enzyme participates in the redox balance, regulating the levels of nitrosylation/denitrosylation and its inhibition can increase the bioavailability of S-nitrosothiols, being this a potential cardioprotection element. So far, in the heart, genetic cancelation of GSNOR has indeed shown positive results in the context of damage by infarction. For this reason, more studies are needed in order to assess the impact of inhibition of GSNOR on global cellular functioning. This will indeed help in the development of new knowledge for the treatment and prevention of myocardial diseases.

### Acknowledgements

This work was supported by Interdisciplinary Program PIEI QUIMBIO of the Universidad de Talca.

## Author details

inhibitor N6338 at a single dose maintained flow-mediated arterial dilation versus inhibition of NOS. In hypertensive rats, application of N6338 was shown to decrease blood pressure and

Figure 3. Regulation of intracardiac S-nitrosothiols by GSNOR. Schematic model of the regulation of S-nitrosylation within the cardiac cell by GSNOR. S-nitrosoglutathione reductase is able to metabolize endogenous S-nitrosothiols by controlling the fate of S-nitrosoglutathione (GSNOR). The RyR2 and the L-type (I Ca) calcium channels are nitrosylated (SNO) by the activity of the nitric oxide synthases 1 (NOS1) and 3 (NOS3) located in the sarcoplasmic reticulum and plasmalemma, respectively. SNO are in equilibrium with nitrosoglutathione (GSNO). Thereby, by controlling the fate of GSNO, GSNOR regulates the levels of S-nitrosylation of these channels, important for the reperfusion damage. GSSH,

Then, the question arises whether inhibition of GSNOR is cardioprotective. Much of the evidence suggests that this would be the case. However, the protective role of GSNOR in preventing pathological S-nitrosylation should be considered, as there are studies that suggest that the enzyme is essential for cardiovascular homeostasis. For example, Sips et al. used mice that overexpressed GSNOR specifically in cardiomyocytes and observed an improvement in myocardial properties post induction of myocardial damage after sepsis, a condition that is associated with nitrosative stress derived from the activity of iNOS [91]. Nevertheless, in conditions where increased bioavailability of S-nitrosothiols is required, such after ischemia, the inhibition of GSNOR appears as an attractive therapeutic strategy. This would increase the S-nitrosylation for example, of calcium-handling proteins, which, in a setting of ischemiareperfusion would prevent the calcium overload. In addition, increased S-nitrosylation of mitochondrial proteins may prevent the generation of ROS and a reduction in mitochondrial permeability transition pore activity, reducing cell death. Nevertheless, these effects remain to

vascular resistance, as well as to restore altered flow-mediated dilatation [90].

be probed experimentally.

oxidized glutathione, NH3, ammonia.

98 Free Radicals, Antioxidants and Diseases

Oscar Arriagada, Adriana V. Treuer and Daniel R. González\*

\*Address all correspondence to: dagonzalez@utalca.cl

Departamento de Ciencias Básicas Biomédicas, Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile

### References


[3] Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: Underlying mechanisms and clinical application. Atherosclerosis. 2009;204:334-341

[19] Halestrap AP. What is the mitochondrial permeability transition pore? Journal of Molec-

Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium…

http://dx.doi.org/10.5772/intechopen.77003

101

[20] Bernardi P, Di Lisa F. The mitochondrial permeability transition pore: Molecular nature and role as a target in cardioprotection. Journal of Molecular and Cellular Cardiology.

[21] Petronilli V, Penzo D, Scorrano L, Bernardi P, Di Lisa F. The mitochondrial permeability transition, release of cytochrome c and cell death: Correlation with the duration of pore

[22] Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-

[23] Ju W-K, Kim SY, Sim MS, Kim K-Y, Weinreb RN, Wheeler LA. Inhibition of cyclophilin D by cyclosporin A promotes retinal ganglion cell survival by preventing mitochondrial

[24] Nguyen TT, Stevens MV, Kohr M, Steenbergen C, Sack MN, Murphy E. Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability

[25] Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death.

[26] Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P. Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. Journal of Biological

[27] Bernardi P, von Stockum S. The permeability transition pore as a Ca(2+) release channel:

[28] Halestrap AP, Brenner C. The adenine nucleotide translocase: A central component of the mitochondrial permeability transition pore and key player in cell death. Current Medici-

[29] Leung AW, Halestrap AP. Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore. Biochimica et Biophysica Acta. 1777;2008:946-952

[30] Varanyuwatana P, Halestrap AP. The roles of phosphate and the phosphate carrier in the

[31] Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y. Essential role of voltagedependent anion channel in various forms of apoptosis in mammalian cells. The Journal of

[32] Omar SA, Webb AJ. Nitrite reduction and cardiovascular protection. Journal of Molecular

mitochondrial permeability transition pore. Mitochondrion. 2012;12:120-125

openings in situ. Journal of Biological Chemistry. 2001;276:12030-12034

alteration in ischemic injury. ARVO Meeting Abstracts. 2014;55:2431

transition pore. The Journal of Biological Chemistry. 2011;286:40184-40192

induced ROS release. Physiological Reviews. 2014;94:909-950

New answers to an old question. Cell Calcium. 2012;52:22-27

ular and Cellular Cardiology. 2009;46:821-831

2015;78:100-106

Nature. 2005;434:658-662

Chemistry. 2005;280:18558-18561

nal Chemistry. 2003;10:1507-1525

Cell Biology. 2001;152:237-250

and Cellular Cardiology. 2014;73:57-69


[19] Halestrap AP. What is the mitochondrial permeability transition pore? Journal of Molecular and Cellular Cardiology. 2009;46:821-831

[3] Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: Underlying mecha-

[4] Heusch G, Gersh BJ. The pathophysiology of acute myocardial infarction and strategies of protection beyond reperfusion: A continual challenge. European Heart Journal. 2016

[6] Frohlich GM, Meier P, White SK, Yellon DM, Hausenloy DJ. Myocardial reperfusion injury: Looking beyond primary PCI. European Heart Journal. 2013;34:1714-1722

[7] Hausenloy DJ, Yellon DM. The therapeutic potential of ischemic conditioning: An update.

[8] Heusch G, Boengler K, Schulz R. Inhibition of mitochondrial permeability transition pore opening: The holy grail of cardioprotection. Basic Research in Cardiology. 2010;105:151-154

[9] Jones SP, Bolli R. The ubiquitous role of nitric oxide in cardioprotection. Journal of Molec-

[10] Rastaldo R, Pagliaro P, Cappello S, Penna C, Mancardi D, Westerhof N, et al. Nitric oxide

[11] Jennings RB, Sommers HM, Smyth GA, Flack HA, Linn H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. Archives of Pathology. 1960;70:68-78

[12] Ma XL, Weyrich AS, Lefer DJ, Buerke M, Albertine KH, Kishimoto TK, et al. Monoclonal antibody to L-selectin attenuates neutrophil accumulation and protects ischemic

[13] Timmers L, Pasterkamp G, de Hoog VC, Arslan F, Appelman Y, de Kleijn DP. The innate immune response in reperfused myocardium. Cardiovascular Research. 2012;94:276-283

[14] Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. The Journal of Clinical Investigation. 1994;94:1621-1628

[15] Halestrap AP, Pasdois P. The role of the mitochondrial permeability transition pore in

[16] Elrod JW, Molkentin JD. Physiologic functions of cyclophilin D and the mitochondrial

[17] Liu H, Cala PM, Anderson SE. Na/H exchange inhibition protects newborn heart from

[18] Hendrikx M, Mubagwa K, Verdonck F, Overloop K, Van Hecke P, Vanstapel F, et al. New Na(+)-H+ exchange inhibitor HOE 694 improves postischemic function and high-energy phosphate resynthesis and reduces Ca2+ overload in isolated perfused rabbit heart. Circu-


nisms and clinical application. Atherosclerosis. 2009;204:334-341

[5] Bolli R. Myocardial 'stunning' in man. Circulation. 1992;86:1671-1691

Nature Reviews. Cardiology. 2011;8:619-629

100 Free Radicals, Antioxidants and Diseases

ular and Cellular Cardiology. 2006;40:16-23

and cardiac function. Life Sciences. 2007;81:779-793

reperfused cat myocardium. Circulation. 1993;88:649-658

heart disease. Biochimica et Biophysica Acta. 2009;1787:1402-1415

permeability transition pore. Circulation Journal. 2013;77:1111-1122

ischemia/reperfusion injury by limiting Na<sup>+</sup>

vascular Pharmacology. 2010;55:227-233

lation. 1994;89:2787-2798


[33] Ruiz-Meana M, Inserte J, Fernandez-Sanz C, Hernando V, Miro-Casas E, Barba I, et al. The role of mitochondrial permeability transition in reperfusion-induced cardiomyocyte death depends on the duration of ischemia. Basic Research in Cardiology. 2011;106:1259-1268

[47] Joshua M, Thomas AC. Nitric oxide synthase: Non-canonical expression patterns. Fron-

Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium…

[48] Tani M, Neely JR. Role of intracellular Na<sup>+</sup> in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H<sup>+</sup>

[49] Penna C, Angotti C, Pagliaro P. Protein S-nitrosylation in preconditioning and postcondi-

[50] Lima B, Lam GKW, Xie L, Diesen DL, Villamizar N, Nienaber J, et al. Endogenous Snitrosothiols protect against myocardial injury. Proceedings of the National Academy of

[51] Balazy M, Kaminski PM, Mao K, Tan J, Wolin MS. S-nitroglutathione, a product of the reaction between peroxynitrite and glutathione that generates nitric oxide. Journal of

[52] Kohr MJ, Aponte AM, Sun J, Wang G, Murphy E, Gucek M, et al. Characterization of potential S-nitrosylation sites in the myocardium. American Journal of Physiology. Heart

[53] Ladilov YV, Siegmund B, Piper HM. Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange. The American Journal of Physiology.

[54] Sun J, Morgan M, Shen R-F, Steenbergen C, Murphy E. Preconditioning results in S-nitrosylation of proteins involved in regulation of mitochondrial energetics and calcium transport.

[55] Tong G, Aponte AM, Kohr MJ, Steenbergen C, Murphy E, Sun J. Postconditioning leads to an increase in protein S-nitrosylation. American Journal of Physiology - Heart and Circu-

[56] Sun J, Aponte AM, Menazza S, Gucek M, Steenbergen C, Murphy E. Additive cardioprotection by pharmacological postconditioning with hydrogen sulfide and nitric oxide donors in mouse heart: S-sulfhydration vs. S-nitrosylation. Cardiovascular Research. 2016;110:96-106

[57] Mannick JB, Hausladen A, Liu L, Hess DT, Zeng M, Miao QX, et al. Fas-induced caspase

[58] Schäfer C, Ladilov Y, Inserte J, Schäfer M, Haffner S, Garcia-Dorado D, et al. Role of the reverse mode of the Na+/Ca2+ exchanger in reoxygenation-induced cardiomyocyte injury.

[59] West MB, Rokosh G, Obal D, Velayutham M, Xuan YT, Hill BG, et al. Cardiac myocytespecific expression of inducible nitric oxide synthase protects against ischemia/reperfusion injury by preventing mitochondrial permeability transition. Circulation. 2008;118:


tioning. Experimental Biology and Medicine. 2014;239:647-662


103

http://dx.doi.org/10.5772/intechopen.77003

tiers in Immunology. 2014;5:478

Sciences. 2009;106:6297-6302

1995;268:H1531-H1539

Circulation Research. 2007;101:1155

latory Physiology. 2014;306:H825

denitrosylation. Science. 1999;284:651-654

Cardiovascular Research. 2001;51:241-250

1970-1978

Biological Chemistry. 1998;273:32009-32015

and Circulatory Physiology. 2011;300:H1327-H1335

and Na+


[47] Joshua M, Thomas AC. Nitric oxide synthase: Non-canonical expression patterns. Frontiers in Immunology. 2014;5:478

[33] Ruiz-Meana M, Inserte J, Fernandez-Sanz C, Hernando V, Miro-Casas E, Barba I, et al. The role of mitochondrial permeability transition in reperfusion-induced cardiomyocyte death depends on the duration of ischemia. Basic Research in Cardiology. 2011;106:1259-1268

[34] Halestrap AP. Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH values below 7. Implications for the protective effect of low pH against chemical and hypoxic cell damage. The Biochemical Journal. 1991;278(Pt 3):715-719

[36] Crompton M. The mitochondrial permeability transition pore and its role in cell death.

[37] Borutaite V, Budriunaite A, Morkuniene R, Brown GC. Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia. Biochimica et Bio-

[38] Javadov S, Karmazyn M, Escobales N. Mitochondrial permeability transition pore opening as a promising therapeutic target in cardiac diseases. The Journal of Pharmacology

[39] Chouchani ET, Pell VR, Gaude E, Aksentijevic D, Sundier SY, Robb EL, et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature

[40] Hausenloy DJ, Duchen MR, Yellon DM. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovascular

[41] Leiper J, Nandi M. The therapeutic potential of targeting endogenous inhibitors of nitric

[42] Griffiths EJ, Halestrap AP. Protection by Cyclosporin a of ischemia/reperfusion-induced damage in isolated rat hearts. Journal of Molecular and Cellular Cardiology. 1993;25:

[43] Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic

[44] Balakirev MY, Khramtsov VV, Zimmer G. Modulation of the mitochondrial permeability transition by nitric oxide. European Journal of Biochemistry. 1997;246:710-718

[45] Jekabsone A, Ivanoviene L, Brown GC, Borutaite V. Nitric oxide and calcium together inactivate mitochondrial complex I and induce cytochrome c release. Journal of Molecular

[46] Brune B. Nitric oxide: NO apoptosis or turning it ON? Cell Death and Differentiation.

oxide synthesis. Nature Reviews. Drug Discovery. 2011;10:277-291

but not apoptotic cell death. Nature. 2005;434:652-658

and Cellular Cardiology. 2003;35:803-809

/H<sup>+</sup> exchanger during ischemia and reperfu-

[35] Allen DG, Xiao X-H. Role of the cardiac Na<sup>+</sup>

102 Free Radicals, Antioxidants and Diseases

physica Acta. 2001;1537:101-109

(England). 2014;515(7527):431-435

Research. 2003;60:617-625

1461-1469

2003;10:864-869

sion. Cardiovascular Research. 2003;57:934-941

The Biochemical Journal. 1999;341(Pt 2):233-249

and Experimental Therapeutics. 2009;330:670-678


[60] Methner C, Chouchani ET, Buonincontri G, Pell VR, Sawiak SJ, Murphy MP, et al. Mitochondria selective S-nitrosation by mitochondria-targeted S-nitrosothiol protects against post-infarct heart failure in mouse hearts. European Journal of Heart Failure. 2014;16(7):712

[74] Marozkina NV, Wang XQ, Stsiapura V, Fitzpatrick A, Carraro S, Hawkins GA, et al. Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity.

Cardioprotective Effects of S-Nitrosothiols in Ischemia-Reperfusion: Role for Mitochondria and Calcium…

http://dx.doi.org/10.5772/intechopen.77003

105

[75] Ferrini ME, Simons BJ, Bassett DJP, Bradley MO, Roberts K, Jaffar Z. S-nitrosoglutathione reductase inhibition regulates allergen-induced lung inflammation and airway hyperreac-

[76] Blonder J, Mutka S, Sun X, Qiu J, Green L, Mehra N, et al. Pharmacologic inhibition of Snitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation. BMC Pulmonary Medicine.

[77] Tang CH, Seeley EJ, Huang X, Wolters PJ, Liu L. Increased susceptibility to Klebsiella pneumonia and mortality in GSNOR-deficient mice. Biochemical and Biophysical Research Com-

[78] Moon Y, Cao Y, Zhu J, Xu Y, Balkan W, Buys ES, et al. GSNOR deficiency enhances in situ skeletal muscle strength, fatigue resistance, and RyR1 S-nitrosylation without impacting mitochondrial content and activity. Antioxidants & Redox Signaling. 2017;26:165-181

[79] Montagna C, Di Giacomo G, Rizza S, Cardaci S, Ferraro E, Grumati P, et al. S-nitrosogluathione reductase deficiency-induced S-nitrosylation results in neuromuscular dysfunction.

[80] Canas A, Lopez-Sanchez LM, Penarando J, Valverde A, Conde F, Hernandez V, et al. Altered S-nitrosothiol homeostasis provides a survival advantage to breast cancer cells in HER2 tumors and reduces their sensitivity to trastuzumab. Biochimica et Biophysica Acta.

[81] Rizza S, Cirotti C, Montagna C, Cardaci S, Consales C, Cozzolino M, et al. S-nitrosoglutathione reductase plays opposite roles in SH-SY5Y models of Parkinson's disease and amyo-

[82] Rizza S, Montagna C, Cardaci S, Maiani E, Di Giacomo G, Sanchez-Quiles V, et al. Snitrosylation of the mitochondrial chaperone TRAP1 sensitizes hepatocellular carcinoma cells to inhibitors of succinate dehydrogenase. Cancer Research. 2016;76:4170-4182

[83] Wei W, Li B, Hanes MA, Kakar S, Chen X, Liu L. S-nitrosylation from GSNOR deficiency impairs DNA repair and promotes hepatocarcinogenesis. Science Translational Medicine.

[84] Marozkina NV, Wei C, Yemen S, Wallrabe H, Nagji AS, Liu L, et al. S-nitrosoglutathione reductase in human lung cancer. American Journal of Respiratory Cell and Molecular

[85] Yang Z, Wang ZE, Doulias PT, Wei W, Ischiropoulos H, Locksley RM, et al. Lymphocyte development requires S-nitrosoglutathione reductase. Journal of Immunology. 2010;185:

trophic lateral sclerosis. Mediators of Inflammation. 2015;2015:536238

The European Respiratory Journal. 2015;45:87-97

Antioxidants & Redox Signaling. 2014;21:570-587

tivity. PLoS One. 2013;8:e70351

munications. 2013;442:122-126

2016;1862:601-610

2010;2:19ra3-19ra3

Biology. 2012;46:63-70

6664-6669

2014;14:3


[74] Marozkina NV, Wang XQ, Stsiapura V, Fitzpatrick A, Carraro S, Hawkins GA, et al. Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity. The European Respiratory Journal. 2015;45:87-97

[60] Methner C, Chouchani ET, Buonincontri G, Pell VR, Sawiak SJ, Murphy MP, et al. Mitochondria selective S-nitrosation by mitochondria-targeted S-nitrosothiol protects against post-infarct heart failure in mouse hearts. European Journal of Heart Failure. 2014;16(7):712

[61] Chouchani ET, Methner C, Nadtochiy SM, Logan A, Pell VR, Ding S, et al. Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I. Nature Medicine. 2013;19:

[62] Nadtochiy SM, Burwell LS, Brookes PS. Cardioprotection and mitochondrial S-nitrosation: Effects of S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) in cardiac ischemia-

[63] Shiva S, Sack MN, Greer JJ, Duranski M, Ringwood LA, Burwell L, et al. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron

[64] Borutaite V, Brown GC. S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in mitochondrial hydrogen peroxide production. Biochimica et Biophysica

[65] Riva A, Tandler B, Loffredo F, Vazquez E, Hoppel C. Structural differences in two biochemically defined populations of cardiac mitochondria. American Journal of Physiology.

[66] Sun J, Nguyen T, Aponte AM, Menazza S, Kohr MJ, Roth DM, et al. Ischaemic preconditioning preferentially increases protein S-nitrosylation in subsarcolemmal mitochondria.

[67] Holmuhamedov EL, Oberlin A, Short K, Terzic A, Jahangir A. Cardiac subsarcolemmal and interfibrillar mitochondria display distinct responsiveness to protection by diazoxide.

[68] Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS. A metabolic enzyme for S-

[69] Foster MW, Hess DT, Stamler JS. Protein S-nitrosylation in health and disease: A current

[70] Broniowska KA, Diers AR, Hogg N. S-nitrosoglutathione. Biochimica et Biophysica Acta.

[71] Brown-Steinke K, deRonde K, Yemen S, Palmer LA. Gender differences in S-nitrosogl-

[72] Shao Q, Fallica J, Casin KM, Murphy E, Steenbergen C, Kohr MJ. Characterization of the sex-dependent myocardial S-nitrosothiol proteome. American Journal of Physiology.

[73] Sun X, Wasley JW, Qiu J, Blonder JP, Stout AM, Green LS, et al. Discovery of snitrosoglutathione reductase inhibitors: Potential agents for the treatment of asthma and

other inflammatory diseases. ACS Medicinal Chemistry Letters. 2011;2:402-406

nitrosothiol conserved from bacteria to humans. Nature. 2001;410:490-494

perspective. Trends in Molecular Medicine. 2009;15:391-404

utathione reductase activity in the lung. PLoS One. 2010;5:e14007

Heart and Circulatory Physiology. 2016;310:H505-H515

reperfusion injury. Journal of Molecular and Cellular Cardiology. 2007;42:812-825

transfer. The Journal of Experimental Medicine. 2007;204:2089-2102

Acta (BBA) – Bioenergetics. 2006;1757:562-566

Cardiovascular Research. 2015;106(2):227-236

PLoS One. 2012;7:e44667

1830;2013:3173-3181

Heart and Circulatory Physiology. 2005;289:H868-H872

753-759

104 Free Radicals, Antioxidants and Diseases


[86] Beigi F, Gonzalez DR, Minhas KM, Sun QA, Foster MW, Khan SA, et al. Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109:4314-4319

**Chapter 8**

**Provisional chapter**

**Antimicrobial and Antioxidant Properties of Essential**

**Antimicrobial and Antioxidant Properties of Essential** 

DOI: 10.5772/intechopen.73966

**Oil Isolated from** *Coleus zeylanicus* **under Normal and**

**Oil Isolated from** *Coleus zeylanicus* **under Normal and** 

Essential oils can be used as antibacterial additives and are generally recognized as safe. *Coleus zeylanicus* is one of the medicinal aromatic plant serves as a source of essential oils. Antimicrobial and antioxidant activities of essential oils obtained from the control and salinity stressed *Coleus zeylanicus* plant was investigated in the present study. Essential oils from the control and salinity stressed *Coleus zeylanicus* plant was extracted using Clevenger apparatus. The composition of essential oils was identified using gas chromatography mass spectrometry, which showed a few compounds expressed differentially. The antibacterial activity of the isolated essential oils was studied by using the agar well diffusion method, showing potent inhibitory activity against *Staphylococcus aureus*, *Escherichia coli* and *Pseudomonas aeruginosa*. The antioxidant and antimicrobial constituents of the essential oils were spotted using the bioautography method, revealing that the antioxidant and antimicrobial properties in the essential oils of *Coleus zeylanicus* were

**Keywords:** agar well diffusion method, salinity stress, essential oils, bioautography

In the recent years, the increasing resistance and the wide spread of multi-drug resistant microbes are leading to a serious health problem to the human population. The emergence of resistant microbes is due to the indiscriminate use of antibiotics [1]. Hence there is a need

> © 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

**Salinity Stress Conditions**

**Salinity Stress Conditions**

Viswanatha Chaitanya Kolluru

Viswanatha Chaitanya Kolluru

http://dx.doi.org/10.5772/intechopen.73966

**Abstract**

**1. Introduction**

Divya Kotagiri, Khasim Beebi Shaik and

Divya Kotagiri, Khasim Beebi Shaik and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

increased upon exposure to salinity stress.

method, thin layer chromatography


#### **Antimicrobial and Antioxidant Properties of Essential Oil Isolated from** *Coleus zeylanicus* **under Normal and Salinity Stress Conditions Antimicrobial and Antioxidant Properties of Essential Oil Isolated from** *Coleus zeylanicus* **under Normal and Salinity Stress Conditions**

DOI: 10.5772/intechopen.73966

Divya Kotagiri, Khasim Beebi Shaik and Viswanatha Chaitanya Kolluru Divya Kotagiri, Khasim Beebi Shaik and Viswanatha Chaitanya Kolluru

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73966

#### **Abstract**

[86] Beigi F, Gonzalez DR, Minhas KM, Sun QA, Foster MW, Khan SA, et al. Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function. Proceedings of the National Academy of Sciences of the United States of America. 2012;

[87] Ozawa K, Whalen EJ, Nelson CD, Mu Y, Hess DT, Lefkowitz RJ, et al. S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking. Molecular Cell. 2008;31:395-405

[88] Hare JM. Nitric oxide and excitation-contraction coupling. Journal of Molecular and

[89] Hatzistergos KE, Paulino EC, Dulce RA, Takeuchi LM, Bellio MA, Kulandavelu S, et al. Snitrosoglutathione reductase deficiency enhances the proliferative expansion of adult heart progenitors and myocytes post myocardial infarction. Journal of the American Heart

[90] Chen Q, Sievers RE, Varga M, Kharait S, Haddad DJ, Patton AK, et al. Pharmacological inhibition of S-nitrosoglutathione reductase improves endothelial vasodilatory function in

[91] Sips PY, Irie T, Zou L, Shinozaki S, Sakai M, Shimizu N, et al. Reduction of cardiomyocyte S-nitrosylation by S-nitrosoglutathione reductase protects against sepsis-induced myocardial depression. American Journal of Physiology. Heart and Circulatory Physiology. 2013;

rats in vivo. Journal of Applied Physiology. 2013;114:752-760

109:4314-4319

106 Free Radicals, Antioxidants and Diseases

Cellular Cardiology. 2003;35:719-729

Association. 2015;4:e001974

304:H1134-H1146

Essential oils can be used as antibacterial additives and are generally recognized as safe. *Coleus zeylanicus* is one of the medicinal aromatic plant serves as a source of essential oils. Antimicrobial and antioxidant activities of essential oils obtained from the control and salinity stressed *Coleus zeylanicus* plant was investigated in the present study. Essential oils from the control and salinity stressed *Coleus zeylanicus* plant was extracted using Clevenger apparatus. The composition of essential oils was identified using gas chromatography mass spectrometry, which showed a few compounds expressed differentially. The antibacterial activity of the isolated essential oils was studied by using the agar well diffusion method, showing potent inhibitory activity against *Staphylococcus aureus*, *Escherichia coli* and *Pseudomonas aeruginosa*. The antioxidant and antimicrobial constituents of the essential oils were spotted using the bioautography method, revealing that the antioxidant and antimicrobial properties in the essential oils of *Coleus zeylanicus* were increased upon exposure to salinity stress.

**Keywords:** agar well diffusion method, salinity stress, essential oils, bioautography method, thin layer chromatography

#### **1. Introduction**

In the recent years, the increasing resistance and the wide spread of multi-drug resistant microbes are leading to a serious health problem to the human population. The emergence of resistant microbes is due to the indiscriminate use of antibiotics [1]. Hence there is a need

© 2016 The Author(s). Licensee InTech. This chapter is 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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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 identify new drugs with effective antimicrobial and antioxidant properties to overcome this problem and to replace the usage of synthetic drugs responsible for the cause of side effects in patients. The resistance to antibiotics can be reduced by the use of resistance inhibitors isolated from plants. Salt stress is a vital abiotic stress factor that affects the growth and productivity of plants. In general, salinity refers to the presence of different salts like sodium chloride, calcium sulfate, magnesium, and bicarbonates in water and soil [2]. The uptake of water and absorption of essential nutrients by plants are restricted due to the presence of soluble salts exerting high osmotic pressure which ultimately affects the growth of plants [3]. Plants have adopted a mechanism to tolerate salt stress by the accumulation of solutes such as glycine, betaine, proline, sugar alcohols, polyols, and soluble sugars and by eliminating the toxic Na+ ions in the cytoplasm [4]. Plant bioactive compounds are low molecular weight secondary metabolites distributed largely in plants that play a major role in the adaptation of plants to different environmental changes and in overcoming stress constraints. Medicinal plants exhibit pharmacological properties as they are known to possess various bioactive compounds called secondary metabolites like tannins, terpenes, alkaloids, steroids, flavonoids, glycosides, saponins, etc. These compounds play a major role in protecting the plants from various stress factors. Secondary metabolites that act as active components exhibit a wide range of antimicrobial activity [5]. The target sites shown by plant extracts are active against drug resistant microbes than those used by the antibiotics. It was discovered that the non-antibiotic substances such as essential oils have shown good fighting potential against drug resistant microbes. Essential oils change the rate of an enzyme reaction by interfering with the metabolism of microbes; thereby influencing the uptake of nutrients from the medium and affect the synthesis of enzymes or by changing the membrane structures that leads to the death of microbes. Thus, with the discovery of many natural products from plant species due to advancements in science and technology made a remarkable progress in the field of medicine. Now-a-days, many researchers are interested in isolating the biologically active compounds from plant species for developing the novel drugs in order to combat the microbes responsible for dreadful diseases [6].

role in neutralizing free radicals benefiting human health. Essential oils also showed a potent inhibitory effect against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and yeast. The highest inhibitory activity of *C. zeylanicus* oil against a wide spectrum of bac-

Antimicrobial and Antioxidant Properties of Essential Oil Isolated from *Coleus zeylanicus*…

http://dx.doi.org/10.5772/intechopen.73966

109

*Coleus zeylanicus* is a perennial aromatic herb belonging to the family of *Lamiaceae*. It has astringent and stomachic properties used in the treatment of fever, common cold, asthma, dysentery, diarrhea, vomiting, burning sensation, small pox, eye diseases, worm diseases, chronic ulcers, dental diseases, and thirst [12]. The juice obtained from the stem and leaves of *C. zeylanicus* are used to treat diarrhea when taken along with honey [13]. It acts as diuretic, diaphoretic, and cholagogue which are useful for chronic and acute congestion of the liver. *C. zeylanicus* is also used to develop potential biodegradable micro-biocides [14]. The medicinal properties of this plant were moderately understood, but not much work was done on the properties of the essential oils isolated from the leaves exposed to salinity stress. In the present study, essential oils were isolated from the control and salinity stressed leaves of *Coleus zeylanicus* and their compositions were determined using gas chromatography mass spectrometry. Antibacterial and antioxidant properties of these essential oils were studied.

*Coleus zeylanicus* plants (Herbarium specimen no. 21904) were propagated in the GITAM University botanical garden in 12 inch pots under 720 min natural photoperiod [irradiance (400–700 nm) of 1600–1800 μmol m−2 s−1] with day/night temperatures of 30/23°C with an approximate air humidity of 60%. The pots were arranged in rows 1 m apart and the plants were irrigated daily and fertilized weekly with Hoagland solution. Three month old plants with uniform growth were selected for this study. Plants were then separated into two groups. Control plants were watered daily and salt stressed plants were treated with 250 ml of 300 mM NaCl solution twice a day for a period of 1 week. The plant materials were dried separately in shade and powdered, which were used for the extraction of essential oils.

The essential oils were extracted using Clevenger apparatus. Distillation is carried out for a period of 4 h by immersing the dried leaf powder directly into a round bottom flask filled with water. The contents were boiled and the vapors were condensed, allowing the essential oils to separate based on their difference in immiscibility and density. After extraction, the organic

. The percentage of the oils was calculated using

SO4

Oil (% v/w) = observed volume of oil (ml)/weight of sample (g) × 100.

teria and fungi was reported [11].

**2. Material and methods**

**2.2. Extraction of essential oil**

the following formula:

phase was separated and dried over Na2

**2.1. Plant material and salt stress treatment**

Essential oils are a heterogeneous group of complex mixture of organic compounds synthesized within plants as secondary metabolites with characteristic flavor and odor. The quantity and quality of oil vary depending on the ecological and growth conditions of plant chosen for extraction. The various other factors also influence the yield of essential oils. The different parts of the plant such as leaves, root, stem, seeds, bark, woods, twigs, buds, fruits, and flowers can be used for the extraction of aromatic oily liquids called essential oils [7]. Essential oils are a mixture of compounds principally terpenoids like (C10) monoterpenes, (C15) sesquiterpenes, and (C20) diterpenes, also contains lactones or acyclic esters, low molecular weight aliphatic hydrocarbons, aldehydes, alcohols, acids and rarely may contain coumarins, nitrogen-, and sulfur-containing compounds and homologs of phenylpropanoids [8]. Essential oils are produced commercially by the method of steam distillation; whereas the other methods of extraction, fermentation, and expression can also be performed to obtain oils [9]. Essential oils possess antibacterial, antifungal, antiviral, anticancer, antioxidant, and insecticidal properties [10]. Essential oils from medicinal plants possess antioxidant activity, which plays an important role in neutralizing free radicals benefiting human health. Essential oils also showed a potent inhibitory effect against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and yeast. The highest inhibitory activity of *C. zeylanicus* oil against a wide spectrum of bacteria and fungi was reported [11].

*Coleus zeylanicus* is a perennial aromatic herb belonging to the family of *Lamiaceae*. It has astringent and stomachic properties used in the treatment of fever, common cold, asthma, dysentery, diarrhea, vomiting, burning sensation, small pox, eye diseases, worm diseases, chronic ulcers, dental diseases, and thirst [12]. The juice obtained from the stem and leaves of *C. zeylanicus* are used to treat diarrhea when taken along with honey [13]. It acts as diuretic, diaphoretic, and cholagogue which are useful for chronic and acute congestion of the liver. *C. zeylanicus* is also used to develop potential biodegradable micro-biocides [14]. The medicinal properties of this plant were moderately understood, but not much work was done on the properties of the essential oils isolated from the leaves exposed to salinity stress. In the present study, essential oils were isolated from the control and salinity stressed leaves of *Coleus zeylanicus* and their compositions were determined using gas chromatography mass spectrometry. Antibacterial and antioxidant properties of these essential oils were studied.

### **2. Material and methods**

to identify new drugs with effective antimicrobial and antioxidant properties to overcome this problem and to replace the usage of synthetic drugs responsible for the cause of side effects in patients. The resistance to antibiotics can be reduced by the use of resistance inhibitors isolated from plants. Salt stress is a vital abiotic stress factor that affects the growth and productivity of plants. In general, salinity refers to the presence of different salts like sodium chloride, calcium sulfate, magnesium, and bicarbonates in water and soil [2]. The uptake of water and absorption of essential nutrients by plants are restricted due to the presence of soluble salts exerting high osmotic pressure which ultimately affects the growth of plants [3]. Plants have adopted a mechanism to tolerate salt stress by the accumulation of solutes such as glycine, betaine, proline, sugar alcohols, polyols, and soluble sugars and by eliminating

secondary metabolites distributed largely in plants that play a major role in the adaptation of plants to different environmental changes and in overcoming stress constraints. Medicinal plants exhibit pharmacological properties as they are known to possess various bioactive compounds called secondary metabolites like tannins, terpenes, alkaloids, steroids, flavonoids, glycosides, saponins, etc. These compounds play a major role in protecting the plants from various stress factors. Secondary metabolites that act as active components exhibit a wide range of antimicrobial activity [5]. The target sites shown by plant extracts are active against drug resistant microbes than those used by the antibiotics. It was discovered that the non-antibiotic substances such as essential oils have shown good fighting potential against drug resistant microbes. Essential oils change the rate of an enzyme reaction by interfering with the metabolism of microbes; thereby influencing the uptake of nutrients from the medium and affect the synthesis of enzymes or by changing the membrane structures that leads to the death of microbes. Thus, with the discovery of many natural products from plant species due to advancements in science and technology made a remarkable progress in the field of medicine. Now-a-days, many researchers are interested in isolating the biologically active compounds from plant species for developing the novel drugs in order to combat the

Essential oils are a heterogeneous group of complex mixture of organic compounds synthesized within plants as secondary metabolites with characteristic flavor and odor. The quantity and quality of oil vary depending on the ecological and growth conditions of plant chosen for extraction. The various other factors also influence the yield of essential oils. The different parts of the plant such as leaves, root, stem, seeds, bark, woods, twigs, buds, fruits, and flowers can be used for the extraction of aromatic oily liquids called essential oils [7]. Essential oils are a mixture of compounds principally terpenoids like (C10) monoterpenes, (C15) sesquiterpenes, and (C20) diterpenes, also contains lactones or acyclic esters, low molecular weight aliphatic hydrocarbons, aldehydes, alcohols, acids and rarely may contain coumarins, nitrogen-, and sulfur-containing compounds and homologs of phenylpropanoids [8]. Essential oils are produced commercially by the method of steam distillation; whereas the other methods of extraction, fermentation, and expression can also be performed to obtain oils [9]. Essential oils possess antibacterial, antifungal, antiviral, anticancer, antioxidant, and insecticidal properties [10]. Essential oils from medicinal plants possess antioxidant activity, which plays an important

ions in the cytoplasm [4]. Plant bioactive compounds are low molecular weight

the toxic Na+

108 Free Radicals, Antioxidants and Diseases

microbes responsible for dreadful diseases [6].

#### **2.1. Plant material and salt stress treatment**

*Coleus zeylanicus* plants (Herbarium specimen no. 21904) were propagated in the GITAM University botanical garden in 12 inch pots under 720 min natural photoperiod [irradiance (400–700 nm) of 1600–1800 μmol m−2 s−1] with day/night temperatures of 30/23°C with an approximate air humidity of 60%. The pots were arranged in rows 1 m apart and the plants were irrigated daily and fertilized weekly with Hoagland solution. Three month old plants with uniform growth were selected for this study. Plants were then separated into two groups. Control plants were watered daily and salt stressed plants were treated with 250 ml of 300 mM NaCl solution twice a day for a period of 1 week. The plant materials were dried separately in shade and powdered, which were used for the extraction of essential oils.

#### **2.2. Extraction of essential oil**

The essential oils were extracted using Clevenger apparatus. Distillation is carried out for a period of 4 h by immersing the dried leaf powder directly into a round bottom flask filled with water. The contents were boiled and the vapors were condensed, allowing the essential oils to separate based on their difference in immiscibility and density. After extraction, the organic phase was separated and dried over Na2 SO4 . The percentage of the oils was calculated using the following formula:

Oil (% v/w) = observed volume of oil (ml)/weight of sample (g) × 100.

### **2.3. Identification of compounds using GC-MS**

The identification of the major chemical composition of the isolated essential oil was performed by GC-MS (Sathyabama University, Chennai). The extracted solution containing essential oil was separated using diethyl ether and the injected sample volume was 1.0 μl for control and 0.5 μl for the salt stress sample as it was more concentrated. A Shimadzu GC-MS QP2010, a polyethylene glycol (Carbowax), and model Rtx-Wax (RESTEC) (30 m to 0.25 mm i.d., film thickness 0.25 lm) capillary column were used for the analysis. The temperature was first held at 40°C and then raised to 250°C (10 min, 2°C/min). The carrier gas was helium at a flow rate of 3 ml min−1. The components of the oil were identified based on the comparison of their retention indices and mass spectra with the fragmentation patterns for computer matching with the NIST (National Institute of Standards and Technology) library.

control and salt stress. Both the control and salt stress samples were applied to the TLC plate separately. The solvent system used in the TLC analysis was toluene:ethyl acetate in the ratio of 93:7. TLC was carried out using TLC silica gel 60 F254 aluminum sheets (Merck). After com-

Several TLC techniques have been developed successfully for the analysis of antioxidants both quantitatively and qualitatively. The use of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical reagent for the analysis of antioxidant activity by the TLC method is one among them. TLC bioautography assay for screening of antioxidants possess several advantages that include high throughput, simplicity, and flexibility. In the present study, the antioxidant activity of essential oils is evaluated using the method of TLC bioassay. The components of essential oils were separated on the TLC plate and sprayed with DPPH solution. Antioxidant reduces the

To screen the antibacterial activity of essential oils from control and salt stress, the method of direct bioautography is performed. Initially, the components were separated on the TLC plate and allowed to air dry. The test organisms were allowed to grow on the TLC plate by dipping the plate into the respective medium containing the organism, followed by incubation at 37°C for 24 h. After incubation, the plates were sprayed with 2 mg/ml solution of p-iodonitrotetra-

Results mentioned are reported as the mean ± standard error (SE) values of five independent experiments, conducted on five different plants in each experiment. SE values were calculated directly from the data according to standard methods. Data analyses were carried out using the SPSS package. Mean values were compared by Duncan's multiple range test and P-values

The essential oils obtained by the hydrodistillation of *Coleus zeylanicus* leaf powder under control as well as salinity stress conditions was 1% (v/w) and 0.93% (v/w), respectively, based on the gram dry weight of the leaf powder. Analysis of the essential oil was carried out using GC-MS, which identified a total of 14 compounds in the essential oil isolated from the control *Coleus zeylanicus* plants and 7 compounds from the oil isolated from the *Coleus zeylanicus* leaves exposed to 300 mM salinity stress (**Figures 1** and **2**). Majority of the compounds present

zolium violet dye. A clear zone indicated the inhibition of growth of the organism.

which are less or equal to 0.05 were considered as statistically significant.

values were calculated for each spot.

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Antimicrobial and Antioxidant Properties of Essential Oil Isolated from *Coleus zeylanicus*…

plete elution, the spots were identified and R<sup>f</sup>

radical and produces creamy spots against a purple background.

**2.6. Screening of antioxidant activity**

**2.7. Screening of antimicrobial activity**

**2.8. Statistical analysis**

**3. Results and discussion**

**3.1. Essential oil analysis**

### **2.4. Antimicrobial studies of the isolated essential oils**

#### *2.4.1. Microorganisms used*

Three bacterial strains, *Escherichia coli* (MTCC 1652), *Staphylococcus aureus* (MTCC 3160), and *Pseudomonas aeruginosa* (MTCC 1688), were used in the present study obtained from Microbial Type Culture Collection Centre, Institute of Microbial Technology (IMTECH), Chandigarh, India.

#### *2.4.2. Preparation of inoculum*

The colonies of test organisms were inoculated into 0.85% normal saline and the turbidity adjusted to 0.5 McFarland using the standard, which is equal to 1.5 × 10<sup>8</sup> CFU/ml.

#### *2.4.3. Antibacterial activity by the agar well diffusion method*

The antibacterial activity of the essential oils isolated from *Coleus zeylanicus* control and salinity stressed plants was analyzed by an agar well diffusion method. The essential oil was tested against the selected bacterial strains *E. coli*, *S. aureus*, and *P. aeruginosa*. Sterile Muller Hinton agar medium was poured into each Petri dish and allowed to solidify. After solidification, culture was spread over the plate by a spread plate technique using sterile cotton swab. Wells of 5 mm size were made in the agar plates with the help of sterile cork borer; the wells were then loaded with 200 μl of sample (essential oil dissolved in solvent), solvent alone as negative control and antibiotic as positive control. All the plates were incubated at 37°C for 24–48 h. After incubation, the plates were observed for the zone of inhibition around the well [15]. The zone of inhibition was calculated by measuring the diameters of the inhibition zone around the well.

#### **2.5. Identification and separation of compounds using TLC**

The essential oils obtained by the method of steam distillation are subjected to thin layer chromatography to identify and separate the bioactive compounds present in both the samples of control and salt stress. Both the control and salt stress samples were applied to the TLC plate separately. The solvent system used in the TLC analysis was toluene:ethyl acetate in the ratio of 93:7. TLC was carried out using TLC silica gel 60 F254 aluminum sheets (Merck). After complete elution, the spots were identified and R<sup>f</sup> values were calculated for each spot.

#### **2.6. Screening of antioxidant activity**

**2.3. Identification of compounds using GC-MS**

The identification of the major chemical composition of the isolated essential oil was performed by GC-MS (Sathyabama University, Chennai). The extracted solution containing essential oil was separated using diethyl ether and the injected sample volume was 1.0 μl for control and 0.5 μl for the salt stress sample as it was more concentrated. A Shimadzu GC-MS QP2010, a polyethylene glycol (Carbowax), and model Rtx-Wax (RESTEC) (30 m to 0.25 mm i.d., film thickness 0.25 lm) capillary column were used for the analysis. The temperature was first held at 40°C and then raised to 250°C (10 min, 2°C/min). The carrier gas was helium at a flow rate of 3 ml min−1. The components of the oil were identified based on the comparison of their retention indices and mass spectra with the fragmentation patterns for computer match-

Three bacterial strains, *Escherichia coli* (MTCC 1652), *Staphylococcus aureus* (MTCC 3160), and *Pseudomonas aeruginosa* (MTCC 1688), were used in the present study obtained from Microbial Type Culture Collection Centre, Institute of Microbial Technology (IMTECH), Chandigarh,

The colonies of test organisms were inoculated into 0.85% normal saline and the turbidity

The antibacterial activity of the essential oils isolated from *Coleus zeylanicus* control and salinity stressed plants was analyzed by an agar well diffusion method. The essential oil was tested against the selected bacterial strains *E. coli*, *S. aureus*, and *P. aeruginosa*. Sterile Muller Hinton agar medium was poured into each Petri dish and allowed to solidify. After solidification, culture was spread over the plate by a spread plate technique using sterile cotton swab. Wells of 5 mm size were made in the agar plates with the help of sterile cork borer; the wells were then loaded with 200 μl of sample (essential oil dissolved in solvent), solvent alone as negative control and antibiotic as positive control. All the plates were incubated at 37°C for 24–48 h. After incubation, the plates were observed for the zone of inhibition around the well [15]. The zone of inhibition was calculated by measuring the diameters of the inhibition zone around

The essential oils obtained by the method of steam distillation are subjected to thin layer chromatography to identify and separate the bioactive compounds present in both the samples of

adjusted to 0.5 McFarland using the standard, which is equal to 1.5 × 10<sup>8</sup> CFU/ml.

ing with the NIST (National Institute of Standards and Technology) library.

**2.4. Antimicrobial studies of the isolated essential oils**

*2.4.3. Antibacterial activity by the agar well diffusion method*

**2.5. Identification and separation of compounds using TLC**

*2.4.1. Microorganisms used*

110 Free Radicals, Antioxidants and Diseases

*2.4.2. Preparation of inoculum*

India.

the well.

Several TLC techniques have been developed successfully for the analysis of antioxidants both quantitatively and qualitatively. The use of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical reagent for the analysis of antioxidant activity by the TLC method is one among them. TLC bioautography assay for screening of antioxidants possess several advantages that include high throughput, simplicity, and flexibility. In the present study, the antioxidant activity of essential oils is evaluated using the method of TLC bioassay. The components of essential oils were separated on the TLC plate and sprayed with DPPH solution. Antioxidant reduces the radical and produces creamy spots against a purple background.

#### **2.7. Screening of antimicrobial activity**

To screen the antibacterial activity of essential oils from control and salt stress, the method of direct bioautography is performed. Initially, the components were separated on the TLC plate and allowed to air dry. The test organisms were allowed to grow on the TLC plate by dipping the plate into the respective medium containing the organism, followed by incubation at 37°C for 24 h. After incubation, the plates were sprayed with 2 mg/ml solution of p-iodonitrotetrazolium violet dye. A clear zone indicated the inhibition of growth of the organism.

#### **2.8. Statistical analysis**

Results mentioned are reported as the mean ± standard error (SE) values of five independent experiments, conducted on five different plants in each experiment. SE values were calculated directly from the data according to standard methods. Data analyses were carried out using the SPSS package. Mean values were compared by Duncan's multiple range test and P-values which are less or equal to 0.05 were considered as statistically significant.

### **3. Results and discussion**

#### **3.1. Essential oil analysis**

The essential oils obtained by the hydrodistillation of *Coleus zeylanicus* leaf powder under control as well as salinity stress conditions was 1% (v/w) and 0.93% (v/w), respectively, based on the gram dry weight of the leaf powder. Analysis of the essential oil was carried out using GC-MS, which identified a total of 14 compounds in the essential oil isolated from the control *Coleus zeylanicus* plants and 7 compounds from the oil isolated from the *Coleus zeylanicus* leaves exposed to 300 mM salinity stress (**Figures 1** and **2**). Majority of the compounds present

*zeylanicus*. The difference in the composition of essential oils might be due to salinity stress [17]. Long chain fatty acids 1-heptadecene and 1-nonadecene isolated from the root ethanol extracts of *S. longepedunculata* possess antifungal and antibiotic properties [18, 19]. In the present study, the essential oils extracted from the *Coleus zeylanicus* leaves during control and salinity stress had a different chemical composition, which were tested for their antimicrobial and antioxidant

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113

The antibacterial activities of essential oils extracted from the control and salinity stressed *Coleus zeylanicus* leaves were tested against *E. coli*, *S. aureus* and *P. aeruginosa*, which have shown moderately high inhibition (**Table 3**). The antimicrobial susceptibility testing was done using the disc diffusion method. Essential oils showed effective inhibitory activity against *E. coli* than that of *S. aureus* and *P. aeruginosa* with zone of inhibition increasing from 12 to 16 mm, respectively. The essential oil of *C. furcata* showed inhibition zones of 14 and 13 mm against the Gram-negative strains of *E. coli* and *P. aeruginosa*; 14 mm against the Gram-positive strain *S. aureus* [20]. The oxygenated monoterpenes exhibit strong antimicrobial activity than hydrocarbon derivatives as they are less water soluble limiting the diffusion through medium. Essential oils containing hydroxyl group exhibits strong antimicrobial activity which might be due to the capability of binding easily to the enzyme at the active site and altering their metabolism. The combination of electrolyzed NaCl and essential oil with 0.5% carvacrol and 0.5% thymol reported to be effective in controlling microbial population and chemical deterioration

**Peak No. R. time Area Area (%) Name of the compound**

6 15.840 353,293 10.27 2,4-Di-tert-butylphenol 7 17.725 69,757 2.03 (−)-Caryophyllene oxide

9 20.680 180,412 5.25 Dibenzo[a,h]cyclotetradecene 10 21.642 48,642 1.41 6-Methyl-5-(1-methylethylidene)

12 24.257 143,316 4.17 (−)-Isolongifolol, methyl ether

**Table 1.** Composition of the essential oil extracted from the *Coleus zeylanicus* control plant.

8 17.926 296,236 8.61 1-Heptadecene

11 22.366 235,008 6.83 Z-5-Nonadecene

13 26.413 159,508 4.64 Behenic alcohol 14 30.113 87,997 2.56 1-Heptacosanol

 4.035 44,267 1.29 1-Decene 7.724 45,372 1.32 endo-Borneol 8.105 147,076 4.28 1-Dodecene 10.700 1,366,605 39.74 Thymol 13.085 261,154 7.59 9-Octadecene

properties.

**3.2. Antimicrobial activity**

**Figure 1.** GC-MS result of essential oil obtained from *coleus zeylanicus* control.

**Figure 2.** GC-MS result of essential oil obtained from *coleus zeylanicus* salinity stress plant.

in the essential oil of *Coleus zeylanicus* under control conditions was not identified in the essential oil extracted from the leaves exposed to the salinity stress, which made the essential oil composition of the salinity stressed *Coleus zeylanicus* different from that of *Coleus zeylanicus* plant growing under control conditions (**Tables 1** and **2**). Only compounds that were identified to be common in both the essential oils were Z-5-nonadecene and 2,4-di-tert-butylphenol. Thymol and carvone are found to be present in higher percentages of control and salinity stressed essential oils of *Coleus zeylanicus*. These two terpenoids were the major components of the essential oil extracted from *Lippia* species showing antiprotozoan properties [16]. The percentage of Z-5-nonadecene and 1-nonadecene components was decreased in the salinity stress treated essential oil of *Coleus*  *zeylanicus*. The difference in the composition of essential oils might be due to salinity stress [17]. Long chain fatty acids 1-heptadecene and 1-nonadecene isolated from the root ethanol extracts of *S. longepedunculata* possess antifungal and antibiotic properties [18, 19]. In the present study, the essential oils extracted from the *Coleus zeylanicus* leaves during control and salinity stress had a different chemical composition, which were tested for their antimicrobial and antioxidant properties.

#### **3.2. Antimicrobial activity**

in the essential oil of *Coleus zeylanicus* under control conditions was not identified in the essential oil extracted from the leaves exposed to the salinity stress, which made the essential oil composition of the salinity stressed *Coleus zeylanicus* different from that of *Coleus zeylanicus* plant growing under control conditions (**Tables 1** and **2**). Only compounds that were identified to be common in both the essential oils were Z-5-nonadecene and 2,4-di-tert-butylphenol. Thymol and carvone are found to be present in higher percentages of control and salinity stressed essential oils of *Coleus zeylanicus*. These two terpenoids were the major components of the essential oil extracted from *Lippia* species showing antiprotozoan properties [16]. The percentage of Z-5-nonadecene and 1-nonadecene components was decreased in the salinity stress treated essential oil of *Coleus* 

**Figure 2.** GC-MS result of essential oil obtained from *coleus zeylanicus* salinity stress plant.

**Figure 1.** GC-MS result of essential oil obtained from *coleus zeylanicus* control.

112 Free Radicals, Antioxidants and Diseases

The antibacterial activities of essential oils extracted from the control and salinity stressed *Coleus zeylanicus* leaves were tested against *E. coli*, *S. aureus* and *P. aeruginosa*, which have shown moderately high inhibition (**Table 3**). The antimicrobial susceptibility testing was done using the disc diffusion method. Essential oils showed effective inhibitory activity against *E. coli* than that of *S. aureus* and *P. aeruginosa* with zone of inhibition increasing from 12 to 16 mm, respectively. The essential oil of *C. furcata* showed inhibition zones of 14 and 13 mm against the Gram-negative strains of *E. coli* and *P. aeruginosa*; 14 mm against the Gram-positive strain *S. aureus* [20]. The oxygenated monoterpenes exhibit strong antimicrobial activity than hydrocarbon derivatives as they are less water soluble limiting the diffusion through medium. Essential oils containing hydroxyl group exhibits strong antimicrobial activity which might be due to the capability of binding easily to the enzyme at the active site and altering their metabolism. The combination of electrolyzed NaCl and essential oil with 0.5% carvacrol and 0.5% thymol reported to be effective in controlling microbial population and chemical deterioration


**Table 1.** Composition of the essential oil extracted from the *Coleus zeylanicus* control plant.


antimicrobial properties *in vitro*. Plant phytochemicals serve in defense mechanisms against predation by herbivores, insects and microorganisms. Quinones and tannins were responsible for plant pigment, whereas terpenoids responsible for plant odor and flavor. Highly oxygenated phenols were found to be highly toxic to microorganisms. The search for new compounds with antimicrobial property derived from natural plant sources has gained much attention to replace synthetic drugs. The growth of microbes can be controlled with the use of phytochemicals derived from plant source which are more effective and less toxic [28, 29].

**3.3. Screening of antioxidant and antimicrobial activity by the TLC bioautography** 

characterized by the presence of three antimicrobial compounds with the R<sup>f</sup>

fied and isolated from the husk, cotyledons and tubers of *Tylosema esculentum* [32].

Bioautography technique was employed to detect the antimicrobial and antioxidant activity [30]. In our study, the two essential oils spotted on the TLC plate were separated into distinct

0.54 and 0.72. The developed TLC plates were used separately for determining the presence of antimicrobial and antioxidant compounds present in the essential oils of *Coleus zeylanicus.* The antioxidant activity of the compounds present in the essential oil was also determined by developing the TLC plate with 20 μg/ml of DPPH solution after the separation of compounds (**Figure 3**). Similar results were reported with the essential oil of *Eucalyptus lanceolatus* [31]. By using the agar overlay bioautography method, several antimicrobial compounds were identi-

**Figure 3.** The antioxidant activity of the compounds present in the essential oil determined by developing the TLC plate with 20 μg/ml−1 of DPPH. Lane 1: essential oil isolated from control leaves of *C. zeylanicus*. Lane 2: essential oil isolated

values. Essential oil from control and salt stressed *C. zeylanicus* have

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values of 0.34,

115

**method**

bands with different R<sup>f</sup>

from salinity stressed leaves of *C. zeylanicus*.

**Table 2.** Composition of the essential oil extracted from the salinity stressed *Coleus zeylanicus* plant.


**Table 3.** Antimicrobial activity of both the essential oils by agar well diffusion method.

[21]. Essential oils act indirectly on membranes by secreting toxins which play an important role in controlling the microbial population of *S. aureus* and *B. cereus*. These essential oils can also be used in combination with other antibacterial agents to enhance their activity. Addition of lysozyme enhanced the synergistic activity between carvone and nisin [22]. The antimicrobial activity of essential oils depends on the chemical composition and the amount of single compound present. These compounds occur in the active form in plants or can be activated by specific enzymes when subjected to biotic or abiotic stress. Mechanism of antimicrobial activity of the essential oils includes the damage of the cytoplasmic membrane, degradation of cell wall, decreased ATP synthesis, membrane protein damage, reducing the proton motive force, and increasing the membrane permeability by reducing the membrane potential [23]. In the present study, the antibacterial activity of the essential oil of *C. zeylanicus* under both the conditions of control and salinity stress was effective against the tested strains. It was also observed that the activity was enhanced when the plant is subjected to salinity stress.

Majority of the essential oils has shown effective inhibitory activity against Gram-positive strains [24], might be due to the presence of hydrophilic outer membrane, which prevents the entry of hydrophobic compounds into the target cell membrane, thereby acquiring resistance to the antimicrobial drugs [25]. Another possible reason could be the inhibition of microbial respiration and increased membrane permeability by essential oils resulting in the death of microbes after massive ion leakage [26, 27]. Therapy with traditional herbs is practiced with the plant species containing medicinal properties. Secondary metabolites such as terpenoids, tannins, alkaloids, phenols, and flavonoids rich in plants are found to be responsible for antimicrobial properties *in vitro*. Plant phytochemicals serve in defense mechanisms against predation by herbivores, insects and microorganisms. Quinones and tannins were responsible for plant pigment, whereas terpenoids responsible for plant odor and flavor. Highly oxygenated phenols were found to be highly toxic to microorganisms. The search for new compounds with antimicrobial property derived from natural plant sources has gained much attention to replace synthetic drugs. The growth of microbes can be controlled with the use of phytochemicals derived from plant source which are more effective and less toxic [28, 29].

#### **3.3. Screening of antioxidant and antimicrobial activity by the TLC bioautography method**

Bioautography technique was employed to detect the antimicrobial and antioxidant activity [30]. In our study, the two essential oils spotted on the TLC plate were separated into distinct bands with different R<sup>f</sup> values. Essential oil from control and salt stressed *C. zeylanicus* have characterized by the presence of three antimicrobial compounds with the R<sup>f</sup> values of 0.34, 0.54 and 0.72. The developed TLC plates were used separately for determining the presence of antimicrobial and antioxidant compounds present in the essential oils of *Coleus zeylanicus.* The antioxidant activity of the compounds present in the essential oil was also determined by developing the TLC plate with 20 μg/ml of DPPH solution after the separation of compounds (**Figure 3**). Similar results were reported with the essential oil of *Eucalyptus lanceolatus* [31]. By using the agar overlay bioautography method, several antimicrobial compounds were identified and isolated from the husk, cotyledons and tubers of *Tylosema esculentum* [32].

[21]. Essential oils act indirectly on membranes by secreting toxins which play an important role in controlling the microbial population of *S. aureus* and *B. cereus*. These essential oils can also be used in combination with other antibacterial agents to enhance their activity. Addition of lysozyme enhanced the synergistic activity between carvone and nisin [22]. The antimicrobial activity of essential oils depends on the chemical composition and the amount of single compound present. These compounds occur in the active form in plants or can be activated by specific enzymes when subjected to biotic or abiotic stress. Mechanism of antimicrobial activity of the essential oils includes the damage of the cytoplasmic membrane, degradation of cell wall, decreased ATP synthesis, membrane protein damage, reducing the proton motive force, and increasing the membrane permeability by reducing the membrane potential [23]. In the present study, the antibacterial activity of the essential oil of *C. zeylanicus* under both the conditions of control and salinity stress was effective against the tested strains. It was also

**Positive control (ampicillin) (mm)** **Negative control (solvent) (mm)**

*C. zeylanicus* **salt stress (mm)**

**Table 2.** Composition of the essential oil extracted from the salinity stressed *Coleus zeylanicus* plant.

**Peak No. R. Time Area Area (%) Name of the compound**

 15.842 147,692 5.70 2,4-Di-tert-butylphenol 17.925 119,179 4.60 E-14-Hexadecenal 22.371 82,567 3.19 Z-5-Nonadecene 26.413 55,879 2.16 1-Nonadecene

1 8.110 55,347 2.14 3-Tetradecene 2 10.705 2,000,267 77.23 Carvone 3 13.086 129,091 4.98 1-Pentadecene

*S. aureus* 17 ± 2.87 17 ± 2.82 32 ± 2.65 No zone *E. coli* 12 ± 2.10 16 ± 3.12 No zone No zone *P. aeruginosa* 17 ± 2.75 17 ± 1.80 No zone No zone

**Table 3.** Antimicrobial activity of both the essential oils by agar well diffusion method.

**Strain name** *C. zeylanicus* **control (mm)**

114 Free Radicals, Antioxidants and Diseases

observed that the activity was enhanced when the plant is subjected to salinity stress.

Majority of the essential oils has shown effective inhibitory activity against Gram-positive strains [24], might be due to the presence of hydrophilic outer membrane, which prevents the entry of hydrophobic compounds into the target cell membrane, thereby acquiring resistance to the antimicrobial drugs [25]. Another possible reason could be the inhibition of microbial respiration and increased membrane permeability by essential oils resulting in the death of microbes after massive ion leakage [26, 27]. Therapy with traditional herbs is practiced with the plant species containing medicinal properties. Secondary metabolites such as terpenoids, tannins, alkaloids, phenols, and flavonoids rich in plants are found to be responsible for

**Figure 3.** The antioxidant activity of the compounds present in the essential oil determined by developing the TLC plate with 20 μg/ml−1 of DPPH. Lane 1: essential oil isolated from control leaves of *C. zeylanicus*. Lane 2: essential oil isolated from salinity stressed leaves of *C. zeylanicus*.

In this method of TLC bioautography, a developed TLC plate is dipped into the respective broth containing microbes of pure culture and incubated under humid conditions. The microbes grow directly on the TLC plates except in the regions where the bioactive compounds exhibit the antimicrobial property. The zones of inhibition with creamy spots against purple background are visualized after spraying the plates with INT (p-iodonitrotetrazolium violet) dye. The tetrazolium salts are converted into a purple colored compound called formazan by the dehydrogenase activity of living microbes. Once the activity is located on the TLC plate, the samples can be analyzed by GC-MS to identify the presence of known or unknown compounds responsible for the activity [33]. This method is considered to be convenient for obtaining the reliable information on the activity of single compounds as the plant extracts possess numerous bioactive compounds. The analytical determination of compounds present in plant extracts and the characterization of their biological properties are made possible with the optimized antimicrobial assays. Separation of compounds in plant extracts is necessary to avoid study on fractions with no biological activity. Detection of antimicrobial compounds by this method is rapid, uncomplicated and effective in saving money and time [34]. Apart from the search of bioactive compounds, this method is also used to find out best solvent for the extraction of compounds and for the selection of mobile phase to separate compounds. It was reported that the thymol and carvacrol were responsible for the antimicrobial property present in the essential oils of *T. vulgaris* L. using the dot blot test [35].

**Acknowledgements**

**Author details**

**References**

Visakhapatnam, Andhra Pradesh, India

Sciences. 2008;**2**:57-62

Botany. 2003;**91**:503-527

Research. 2010;**5**:477-482

ties. Molecules. 2012;**17**:3989-4006

Current Medicinal Chemistry. 2004;**11**:1451-1460

2006;**8**:89-101

Research of KVC lab is funded by the University grants commission, Govt. of India,

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117

42-197/2013. Divya K is thankful for the UGC research fellowship.

Divya Kotagiri, Khasim Beebi Shaik and Viswanatha Chaitanya Kolluru\*

Department of Biotechnology, GITAM Institute of Technology, GITAM University,

[1] Stephen TO, Kennedy KA. Bacteria resistance to antibiotics: Recent trends and challenges. International Journal of Biological and Medical Research. 2011;**2**:1204-1210

[2] Ouda SAE, Mohamed SG. Modelling the effect of different stress conditions on maize productivity using yield-stress model. International Journal of Natural and Engineering

[3] Tester M, Davenport R. Na+ tolerant and Na+ transport in higher plants. Annals of

[4] Wuyts N, Swennen R, De Waele D. Effects of plant phenylpropanoid pathway products and selected terpenoids and alkaloids on the behaviour of the plant-parasitic nematodes *Radopholus similis*. *Pratylenchus penetrans* and *Meloidogyne incognita*. Nematology.

[5] Lai PK, Roy J. Antimicrobial and chemo preventive properties of herbs and spices.

[6] Periyasam A, Mahalingam RK. Phytochemical screening and antimicrobial activity from five Indian medicinal plants against human pathogens. Middle-East Journal of Scientific

[7] Bassole IHN, Juliani HR. Essential oils in combination and their antimicrobial proper-

[8] Benchaar C, Calsamiglia S, Chaves AV, Fraser GR, Colombatto D, McAllister TA, Beauchemin KA. A review of plant-derived essential oils in ruminant nutrition and pro-

duction. Animal Feed Science and Technology. 2008;**145**:209-228

\*Address all correspondence to: viswanatha.chaitanya@gmail.com

The TLC bioautography method also used to detect the compounds exhibiting antioxidant activity. The developed TLC plate sprayed with DPPH (2,2-diphenyl-1-picrylhydrazyl radical) solution produces clear creamy yellow spots against a purple background. The DPPH decreases upon the reduction reaction with a radical scavenger leading to the color change which can be observed in a TLC bioassay. The reaction has been depicted in **Figure 3**. The assay depends on the measurement of antioxidants scavenging activity, where the DPPH is characterized as a stable free radical. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants present in the plant extracts to the corresponding hydrazine. Rosmarinic acid, luteolin, chrysoeriol and apigenin were the four different antioxidant compounds isolated from the fruit of *Perilla frutescens var. acuta* by a TLC bioautography method using DPPH as a detection reagent [36].

### **4. Conclusion**

The results of this study showed that the essential oils of *C. zeylanicus* showed good antibacterial activity against the tested pathogenic strains. The essential oils containing compounds with antimicrobial and antioxidant activity were identified by the method of bioautography technique. Chemical profiling by GC-MS showed thymol and carvone as major components in control and salt stressed essential oil. The present study mainly focused on to observe the change in the level of bioactive compounds in the essential oils of *coleus zeylanicus* when the plant is subjected to salinity stress for determining its commercial value. Our study concludes that the antimicrobial and antioxidant activity remained to be effective even under stress conditions.

### **Acknowledgements**

In this method of TLC bioautography, a developed TLC plate is dipped into the respective broth containing microbes of pure culture and incubated under humid conditions. The microbes grow directly on the TLC plates except in the regions where the bioactive compounds exhibit the antimicrobial property. The zones of inhibition with creamy spots against purple background are visualized after spraying the plates with INT (p-iodonitrotetrazolium violet) dye. The tetrazolium salts are converted into a purple colored compound called formazan by the dehydrogenase activity of living microbes. Once the activity is located on the TLC plate, the samples can be analyzed by GC-MS to identify the presence of known or unknown compounds responsible for the activity [33]. This method is considered to be convenient for obtaining the reliable information on the activity of single compounds as the plant extracts possess numerous bioactive compounds. The analytical determination of compounds present in plant extracts and the characterization of their biological properties are made possible with the optimized antimicrobial assays. Separation of compounds in plant extracts is necessary to avoid study on fractions with no biological activity. Detection of antimicrobial compounds by this method is rapid, uncomplicated and effective in saving money and time [34]. Apart from the search of bioactive compounds, this method is also used to find out best solvent for the extraction of compounds and for the selection of mobile phase to separate compounds. It was reported that the thymol and carvacrol were responsible for the antimicrobial property

The TLC bioautography method also used to detect the compounds exhibiting antioxidant activity. The developed TLC plate sprayed with DPPH (2,2-diphenyl-1-picrylhydrazyl radical) solution produces clear creamy yellow spots against a purple background. The DPPH decreases upon the reduction reaction with a radical scavenger leading to the color change which can be observed in a TLC bioassay. The reaction has been depicted in **Figure 3**. The assay depends on the measurement of antioxidants scavenging activity, where the DPPH is characterized as a stable free radical. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants present in the plant extracts to the corresponding hydrazine. Rosmarinic acid, luteolin, chrysoeriol and apigenin were the four different antioxidant compounds isolated from the fruit of *Perilla frutescens var. acuta* by a TLC

The results of this study showed that the essential oils of *C. zeylanicus* showed good antibacterial activity against the tested pathogenic strains. The essential oils containing compounds with antimicrobial and antioxidant activity were identified by the method of bioautography technique. Chemical profiling by GC-MS showed thymol and carvone as major components in control and salt stressed essential oil. The present study mainly focused on to observe the change in the level of bioactive compounds in the essential oils of *coleus zeylanicus* when the plant is subjected to salinity stress for determining its commercial value. Our study concludes that the antimicrobial and antioxidant activity remained to be effective even under stress conditions.

present in the essential oils of *T. vulgaris* L. using the dot blot test [35].

bioautography method using DPPH as a detection reagent [36].

**4. Conclusion**

116 Free Radicals, Antioxidants and Diseases

Research of KVC lab is funded by the University grants commission, Govt. of India, 42-197/2013. Divya K is thankful for the UGC research fellowship.

### **Author details**

Divya Kotagiri, Khasim Beebi Shaik and Viswanatha Chaitanya Kolluru\*

\*Address all correspondence to: viswanatha.chaitanya@gmail.com

Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam, Andhra Pradesh, India

### **References**


[9] Burt S. Essential oils: Their antibacterial properties and potential applications in foods: A review. International Journal of Food Microbiology. 2004;**94**:223-253

[23] Nazzaro F, Fratianni F, De Martino L, Coppola R, De Feo V. Effect of essential oils on

Antimicrobial and Antioxidant Properties of Essential Oil Isolated from *Coleus zeylanicus*…

http://dx.doi.org/10.5772/intechopen.73966

119

[24] Wan J, Wilcock A, Coventry MJ. The effect of essential oils of basil on the growth of *Aeromonas hydrophila* and *Pseudomonas fluorescens*. Applied Microbiology. 1998;**84**:152-158

[25] Inouye S, Yamaguchi H, Takizawa T. Screening of the antibacterial effects of variety of essential oils on respiratory tract pathogens, using a modified dilution assay method.

[26] Lambert RJ, Skandamis PN, Coote PJ, Nycas GJ. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal

[27] Walsh SE, Maillard JY, Russel AD, Catrenich CE, Charbonneau DL, Bartolo RJ. Activity and mechanism of action of selected biocidal agents on gram-positive and negative bac-

[28] Guleria S, Kumar A. Antifungal activity of some Himalayan medicinal plants using

[29] Zakaria Z, Sreenivasan S, Mohamad M. Antimicrobial activity of *Piper ribesoides* root extract against *Staphylococcus aureus*. Journal of Applied Biological Sciences. 2007;**1**:87-90

[30] Marston A, Maillard M, Hostettmann K. The Role of TLC in the Investigations of Medicinal Plants of Africa, South America and Other Tropical Regions. GIT Laboratory

[31] Madhulika B, Sahil G, Vishawdeep Singh J, Sharma S, Meenakshi K, Dawa S, Devi R, Bindu K. Comparative study on chemical profiling and antimicrobial properties of essential oils from different parts of *Eucalyptus lanceolatus*. Indian Journal of Traditional

[32] Mazimba O, Majinda RRT, Modibedi C. *Tylosema esculentum* extractives and their bioac-

[33] Dewanjeea S, Gangopadhyayb M, Bhattacharyaa N, Khanraa R, Dua TK. Bioautography and its scope in the field of natural product chemistry. Journal of Pharmaceutical

[34] Demetrio L, Valle JR, Juliana Janet MP, Esperanza CC, Windell LR. Thin layer chromatography-bioautography and gas chromatography-mass spectrometry of antimicrobial leaf extracts from Philippine *Piper betle* L. against multidrug-resistant bacteria. Evidence-

[35] Jesionek W, Majer-Dziedzic B, Choma IM. TLC-direct bioautography as a method for evaluation of antibacterial properties of *Thymus vulgaris* L. and *Salvia officinalis* L. essential oils of different origin. Journal of Liquid Chromatography and Related Technologies. 2017:1-5

[36] Gu L, Wua T, Wang Z. TLC bioautography-guided isolation of antioxidants from fruit of *Perilla frutescens var. acuta*. LWT- Food Science and Technology. 2009;**42**:131-136

tivity. Bioorganic and Medicinal Chemistry. 2011;**19**:5225-5230

based Complementary and Alternative Medicine. 2016;**2016**:7

Journal. Darmstadt: Rosslerstrasse: GIT Verlag Publishing Ltd.; 1997. pp. 36-39

direct bioautography. Journal of Cell and Molecular Biology. 2006;**5**:95-98

pathogenic bacteria. Pharmaceuticals. 2013;**6**:1451-1474

Journal of Infection and Chemotherapy. 2001;**7**:251-254

teria. Journal of Applied Microbiology. 2003;**94**:240-247

of Applied Microbiology. 2001;**91**:453-462

Knowledge. 2016;**15**:425-432

Analysis. 2015;**5**:75-84


[23] Nazzaro F, Fratianni F, De Martino L, Coppola R, De Feo V. Effect of essential oils on pathogenic bacteria. Pharmaceuticals. 2013;**6**:1451-1474

[9] Burt S. Essential oils: Their antibacterial properties and potential applications in foods: A

[10] Sao Pedro A, Espirito Santo I, Silva CV, Detoni C, Albuquerque E. The use of nanotechnology as an approach for essential oil-based formulations with antimicrobial activity. In: Méndez-Vilas A, editor. In-Microbial Pathogens and Strategies for Combating them: Science, Technology and Education. Formatex research center, Zurbaran, 06002 Badajoz.

[11] Deena MJ, Sreeranjini K, Thoppil JE. Antimicrobial screening of essential oils of *Coleus aromaticus* and *Coleus zeylanicus*. International Journal of Aromatherapy. 2002;**12**:105-107

[13] Dissanayaka MD, Fosberg FR. A Revised Hand Book to the Flora of Ceylon. Smithosan Institute and National Science Foundation. Washington D.C. New Delhi: Amerind

[14] Janssen AM, Schffer JJ, Sveden BA. Antimicrobial activity of essential oils: 1976-1986 literature review. Aspects of the test methods. Planta Medica. 1987;**53**:395-398

[15] Suganya S, Bharathidasan R, Senthilkumar G, Panneerselvam A. Antibacterial activity of essential oil extracted from *Coriandrum sativam* (L.) and GC-MS analysis. Journal of

[16] Patricia E, Sandra Milena L, Laura Viviana H, Jairo Rene M, Elena S. Chemical composition and antiprotozoal activities of Colombian *Lippia* spp. essential oils and their major

[17] Konan N, Kouame BA, Mamyrbekova-Bekro JA, Nemlin J, Yves-Alain B. Chemical composition and antioxidant activities of essential oils of *Xylopia aethiopica*. European Journal

[18] Kalaivani MK, Bhavana J, Sumathy A. GC-MS analysis of chloroform extract of *Croton Bonplandianum*. International Journal of Pharma and Bio Sciences. 2013;**4**:613-617

[19] Gayathri G, Vijayalakshmi K, Ariamuthu S. GC-MS and HPTLC fingerprinting of *Bauhinia Variegata* leaves for anticancer activity. World Journal of Pharmaceutical

[20] Joshi RK. *In vitro* antimicrobial and antioxidant activities of the essential oil of *Craniotome* 

[21] Mahmoud BSM, Yamazaki K, Miyasita R, Rawai Y, Shin IS, Suzuki T. Preservative effect of combined treatment with electrolyzed NaCl solutions and essential oil compounds on carp fillets during convectional air-drying. International Journal of Food Microbiology.

[22] Yamazaki K, Yamamoto T, Kawai Y, Inoue N. Enhancement of antilisterial activity of essential oil constituents by nisin and diglycerol fatty acid ester. Food Microbiology.

[12] Jayaweera DMA. Medicinal plants. National Science Council of Sri Lanka. 1982;**4**:109

review. International Journal of Food Microbiology. 2004;**94**:223-253

Spain; 2013. pp. 1364-1374

118 Free Radicals, Antioxidants and Diseases

Publishing Co.Pvt.Ltd; 1981. pp. l50-l151

of Scientific Research. 2009;**37**:311-318

*furcate*. J Appl. Natural Science. 2010;**2**:57-62

Research. 2014;**3**:1313-1336

2006;**106**:331-337

2004;**21**:283-289

Chemical and Pharmaceutical Research. 2012;**4**:1846-1850

components. Memórias do Instituto Oswaldo Cruz. 2010;**105**:184-190


**Chapter 9**

**Provisional chapter**

**Vascular Calcification and Oxidative DNA Damage as**

**Nontraditional Cardiovascular Risk Factors in Chronic** 

**Vascular Calcification and Oxidative DNA Damage as** 

DOI: 10.5772/intechopen.73095

**Nontraditional Cardiovascular Risk Factors in Chronic**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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 reproduction in any medium, provided the original work is properly cited.

**Keywords:** end-stage renal disease, vascular calcification, oxidative stress, antioxidants,

and decreased catalase activity and greater oxidative DNA damage.

The number of CKD sufferers that require renal replacement techniques (RRTs) is increasing. The severity of cardiovascular disease (CVD) is disproportionate in these kinds of patients and contributes considerably to mortality in CKD patients. We evaluated the association between oxidative DNA damage, antioxidant activity and vascular calcification (VC) in CKD. An analytical cross-sectional study was performed. Two simple plaques were taken for each patient (pelvis-hip, and hands-wrists). The presence of VC was scored as presence (1) and absence (0). Oxidative stress was determined by activity of catalase, superoxide dismutase (SOD) and oxidative DNA damage by determination of 8-OHdG marker. Eighty-one patients were included. The RRT type was similar for hemodialysis (HD) and peritoneal dialysis (PD). Thirty-eight patients (47%) presented VC (p < 0.01); in 61%, the VC was severe (≥3 points). VC prevalence in women was significantly higher, (67%) (p < 0.001), and (29%) men. Sixty four percent of the patients submitted to HD presented VC and 27% to PD (p < 0.001). The activity of the catalase enzyme was significantly decreased in CKD vs. the healthy control (HC) (p < 0.0001). The oxidative DNA damage in CKD was greater vs. HC (p < 0.0001). In conclusion, the VC was frequent (47%) in CKD,

**Renal Disease**

**Renal Disease**

Edith Viridiana Alatorre-Moreno, José Ignacio Cerrillos-Gutiérrez,

Edith Viridiana Alatorre-Moreno, José Ignacio Cerrillos-Gutiérrez,

Alejandra Guillermina Miranda-Díaz

Alejandra Guillermina Miranda-Díaz

http://dx.doi.org/10.5772/intechopen.73095

oxidative DNA damage

Andrés García-Sánchez and

Sandra Carrillo-Ibarra,

**Abstract**

Jorge Andrade-Sierra, Enrique Rojas-Campos, Sandra Carrillo-Ibarra, Sonia Sifuentes-Franco,

Sonia Sifuentes-Franco, Andrés García-Sánchez and

Jorge Andrade-Sierra, Enrique Rojas-Campos,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

#### **Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors in Chronic Renal Disease Nontraditional Cardiovascular Risk Factors in Chronic Renal Disease**

**Vascular Calcification and Oxidative DNA Damage as** 

DOI: 10.5772/intechopen.73095

Edith Viridiana Alatorre-Moreno, José Ignacio Cerrillos-Gutiérrez, Jorge Andrade-Sierra, Enrique Rojas-Campos, Sandra Carrillo-Ibarra, Sonia Sifuentes-Franco, Andrés García-Sánchez and Alejandra Guillermina Miranda-Díaz José Ignacio Cerrillos-Gutiérrez, Jorge Andrade-Sierra, Enrique Rojas-Campos, Sandra Carrillo-Ibarra, Sonia Sifuentes-Franco, Andrés García-Sánchez and Alejandra Guillermina Miranda-Díaz Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73095

Edith Viridiana Alatorre-Moreno,

#### **Abstract**

The number of CKD sufferers that require renal replacement techniques (RRTs) is increasing. The severity of cardiovascular disease (CVD) is disproportionate in these kinds of patients and contributes considerably to mortality in CKD patients. We evaluated the association between oxidative DNA damage, antioxidant activity and vascular calcification (VC) in CKD. An analytical cross-sectional study was performed. Two simple plaques were taken for each patient (pelvis-hip, and hands-wrists). The presence of VC was scored as presence (1) and absence (0). Oxidative stress was determined by activity of catalase, superoxide dismutase (SOD) and oxidative DNA damage by determination of 8-OHdG marker. Eighty-one patients were included. The RRT type was similar for hemodialysis (HD) and peritoneal dialysis (PD). Thirty-eight patients (47%) presented VC (p < 0.01); in 61%, the VC was severe (≥3 points). VC prevalence in women was significantly higher, (67%) (p < 0.001), and (29%) men. Sixty four percent of the patients submitted to HD presented VC and 27% to PD (p < 0.001). The activity of the catalase enzyme was significantly decreased in CKD vs. the healthy control (HC) (p < 0.0001). The oxidative DNA damage in CKD was greater vs. HC (p < 0.0001). In conclusion, the VC was frequent (47%) in CKD, and decreased catalase activity and greater oxidative DNA damage.

**Keywords:** end-stage renal disease, vascular calcification, oxidative stress, antioxidants, oxidative DNA damage

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. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

### **1. Introduction**

#### **1.1. Chronic kidney disease**

Diabetes mellitus (DM) and systemic arterial hypertension are the predominant risk factors for chronic kidney disease (CKD). CKD has become a public health problem. In recent years there has been a progressive increase in the incidence and prevalence of CKD, as well as the number of sufferers who reach the most advanced stage and require renal replacement therapy (RRT); all of which has led to CKD being considered a real epidemic [1]. CKD has the capacity to produce cardiovascular disease (CVD), increasing the risk of hospitalization, morbidity, and mortality [2]. Renal patients are extremely vulnerable to cardiovascular pathology. The mortality is 10–30 times greater in patients with end-stage renal disease (ESRD) compared to the general population. Despite the prevalence of the traditional risk factors being very high, the broadness and severity of cardiovascular complications are clearly disproportionate to the risk profile that these patients have [3]. CVD contributes considerably to the mortality of patients with CKD, including patients with renal transplant (RT) [4]. Therefore, in the past two decades, the interest in nontraditional or emerging risk factors has increased in mineral bone disorders (MBD), inflammation, malnutrition, and oxidative stress. There is evidence that MBD in CKD plays an important role in the increase of cardiovascular morbidity and mortality by favoring the development of vascular calcifications (VC) [5]. MBD increases the risk of CKD-associated CVD with the onset of hyperphosphatemia, VC, and increased levels of fibroblast growth factor 23 [6–8]. When renal function is impaired, it increases the potential for aggravating traditional risk factors (hypertension, dyslipidemia, inflammation, and oxidative stress). The concomitant deterioration of mineral homeostasis and bone metabolism is probably the key factor leading to accelerated CVD [9]. In CKD, MBD alterations occur from the early stages of the disease (stage 2) [10]. MBD alterations favor vascular rigidity by increasing systolic blood pressure, pulse wave velocity, and left ventricular mass in patients with CKD [11]. The structural and functional abnormalities that occur in the vasculature by early CKD cause endothelial dysfunction progressing to VC.

the damping function [15]. Although initial events leading to calcification of the tunica media or the tunica intima differ, both calcifications reflect an active and highly regulated process

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

The oxidative stress is characterized by imbalance between the productions of reactive oxygen species (ROS) that surpasses the capacities of the antioxidants. CKD has been shown to be a prooxidant disease [17]. Atherosclerosis, CVD, and CKD are associated with oxidative stress, inflammation, and reduced availability of nitric oxide (NO). Oxidative stress and inflammation are considered as nontraditional risk factors [18]. The oxidizing compounds have physiological defense mechanisms in the organism, but when there is an imbalance in the generation of the oxidants, it results in damage to the tissues. Oxidative stress has the ability to induce endothelial dysfunction by promoting the progression of atherosclerosis and reducing the availability of NO [19]. There is growing evidence in the general population that indicates correlation between oxidative stress and VC development [20]. Macrophages, endothelial cells, and smooth muscle

that closely resembles endochondral and/or intramembranous bone formation [16].

O2

tive DNA damage, antioxidant activity, and VC in patients with CKD.

stimuli. The free radicals of (NO) are generated in the vascular endothelium from L-arginine by the NO synthase enzyme with the capacity to produce hydroxyl or peroxyl radicals [21].

The most important cellular targets of ROS are DNA damage characterized by rupture of DNA strands, point mutations, and compromise of telomere integrity [22]. Shortening of telomeres and mitochondrial DNA damage is related to the rapid onset of atherosclerosis [23]. Increased oxidative stress is a prominent factor in the pathogenesis of VC, while some evidence suggests that increased DNA damage affects contractility of the vascular tunica media [24]. The ROS are capable of damaging macromolecules and DNA bases (purine, adenine, guanine, cytosine, and thymine). Other DNA bases may also be oxidized in a similar manner by the hydroxyl radical. The important consequences of oxidative damage to DNA bases are produced by mutations by the AT↔GC transition and by GC↔TA transversion. These mutations, if not repaired, can lead to changes in the gene expression of proteins [25]. Oxidative DNA damage can be measured by the 8-hydroxy-2′-deoxyguanosine (8-OHdG) marker. Antioxidants play an important role in the proper balance of oxidative stress. Monitoring the levels of antioxidants and oxidative DNA damage products in patients with CKD and the interpretation of the relationship between these markers could contribute better management of patients with CKD [26]. For the above, we set out to evaluate the association between oxida-

An analytical cross-sectional study was performed. Patient population > 16 years of age who were undergoing RRT and meet the selection criteria while being attended to at the Division of Nephrology-Transplants of the Subspecialty Medical Unit at the Specialties Hospital of the National Occidental Medical Centre, Mexican Social Security Institute (*División de Nefrología-Trasplantes de la Unidad Médica de Alta Especialidad (UMAE) del Hospital de Especialidades, CMNO, del Instituto Mexicano del Seguro Social (IMSS)*), in Guadalajara, Jalisco, Mexico. Patient

) and superoxide anion (O−2)) in response to diverse

http://dx.doi.org/10.5772/intechopen.73095

123

cells produce ROS (hydrogen peroxide (H2

**2. Materials and methods**

The majority of the vascular pathologies that are present in patients with CKD are secondary to the development of VC. They are characterized as being a multifactorial pathological process with an increase in calcium phosphate deposits in a form of hydroxyapatite. The aforementioned is a result of the imbalance in mineral metabolism that kidney patients have [12]. VC is a common phenomenon in the aging of the general population; however, in CKD, the onset is rapidly accelerated, promoting the development of left ventricular hypertrophy, increasing cardiovascular risk with increased cardiac mortality in patients with CKD [13]. VC can present in the tunica media or in the tunica intima (atherosclerosis or sclerosis of Mönckeberg) of the arterial vessels. VC causes thickening of the intima-media with the formation of atherosclerotic plaques and lesions of the tunica intima, producing local inflammation, dyslipidemia, and accumulation of foam cells [14]. Atherosclerosis is restricted to medium or large arteries and is related to other risk factors such as smoking, obesity, dyslipidemia, hypertension, and aging. Calcification of the tunica media can occur in arteries of any size, is a typical process related to age, and is associated with greater arterial stiffness and reduction of the damping function [15]. Although initial events leading to calcification of the tunica media or the tunica intima differ, both calcifications reflect an active and highly regulated process that closely resembles endochondral and/or intramembranous bone formation [16].

The oxidative stress is characterized by imbalance between the productions of reactive oxygen species (ROS) that surpasses the capacities of the antioxidants. CKD has been shown to be a prooxidant disease [17]. Atherosclerosis, CVD, and CKD are associated with oxidative stress, inflammation, and reduced availability of nitric oxide (NO). Oxidative stress and inflammation are considered as nontraditional risk factors [18]. The oxidizing compounds have physiological defense mechanisms in the organism, but when there is an imbalance in the generation of the oxidants, it results in damage to the tissues. Oxidative stress has the ability to induce endothelial dysfunction by promoting the progression of atherosclerosis and reducing the availability of NO [19]. There is growing evidence in the general population that indicates correlation between oxidative stress and VC development [20]. Macrophages, endothelial cells, and smooth muscle cells produce ROS (hydrogen peroxide (H2 O2 ) and superoxide anion (O−2)) in response to diverse stimuli. The free radicals of (NO) are generated in the vascular endothelium from L-arginine by the NO synthase enzyme with the capacity to produce hydroxyl or peroxyl radicals [21].

The most important cellular targets of ROS are DNA damage characterized by rupture of DNA strands, point mutations, and compromise of telomere integrity [22]. Shortening of telomeres and mitochondrial DNA damage is related to the rapid onset of atherosclerosis [23]. Increased oxidative stress is a prominent factor in the pathogenesis of VC, while some evidence suggests that increased DNA damage affects contractility of the vascular tunica media [24]. The ROS are capable of damaging macromolecules and DNA bases (purine, adenine, guanine, cytosine, and thymine). Other DNA bases may also be oxidized in a similar manner by the hydroxyl radical. The important consequences of oxidative damage to DNA bases are produced by mutations by the AT↔GC transition and by GC↔TA transversion. These mutations, if not repaired, can lead to changes in the gene expression of proteins [25]. Oxidative DNA damage can be measured by the 8-hydroxy-2′-deoxyguanosine (8-OHdG) marker. Antioxidants play an important role in the proper balance of oxidative stress. Monitoring the levels of antioxidants and oxidative DNA damage products in patients with CKD and the interpretation of the relationship between these markers could contribute better management of patients with CKD [26]. For the above, we set out to evaluate the association between oxidative DNA damage, antioxidant activity, and VC in patients with CKD.

### **2. Materials and methods**

**1. Introduction**

**1.1. Chronic kidney disease**

122 Free Radicals, Antioxidants and Diseases

Diabetes mellitus (DM) and systemic arterial hypertension are the predominant risk factors for chronic kidney disease (CKD). CKD has become a public health problem. In recent years there has been a progressive increase in the incidence and prevalence of CKD, as well as the number of sufferers who reach the most advanced stage and require renal replacement therapy (RRT); all of which has led to CKD being considered a real epidemic [1]. CKD has the capacity to produce cardiovascular disease (CVD), increasing the risk of hospitalization, morbidity, and mortality [2]. Renal patients are extremely vulnerable to cardiovascular pathology. The mortality is 10–30 times greater in patients with end-stage renal disease (ESRD) compared to the general population. Despite the prevalence of the traditional risk factors being very high, the broadness and severity of cardiovascular complications are clearly disproportionate to the risk profile that these patients have [3]. CVD contributes considerably to the mortality of patients with CKD, including patients with renal transplant (RT) [4]. Therefore, in the past two decades, the interest in nontraditional or emerging risk factors has increased in mineral bone disorders (MBD), inflammation, malnutrition, and oxidative stress. There is evidence that MBD in CKD plays an important role in the increase of cardiovascular morbidity and mortality by favoring the development of vascular calcifications (VC) [5]. MBD increases the risk of CKD-associated CVD with the onset of hyperphosphatemia, VC, and increased levels of fibroblast growth factor 23 [6–8]. When renal function is impaired, it increases the potential for aggravating traditional risk factors (hypertension, dyslipidemia, inflammation, and oxidative stress). The concomitant deterioration of mineral homeostasis and bone metabolism is probably the key factor leading to accelerated CVD [9]. In CKD, MBD alterations occur from the early stages of the disease (stage 2) [10]. MBD alterations favor vascular rigidity by increasing systolic blood pressure, pulse wave velocity, and left ventricular mass in patients with CKD [11]. The structural and functional abnormalities that occur in the vasculature by

The majority of the vascular pathologies that are present in patients with CKD are secondary to the development of VC. They are characterized as being a multifactorial pathological process with an increase in calcium phosphate deposits in a form of hydroxyapatite. The aforementioned is a result of the imbalance in mineral metabolism that kidney patients have [12]. VC is a common phenomenon in the aging of the general population; however, in CKD, the onset is rapidly accelerated, promoting the development of left ventricular hypertrophy, increasing cardiovascular risk with increased cardiac mortality in patients with CKD [13]. VC can present in the tunica media or in the tunica intima (atherosclerosis or sclerosis of Mönckeberg) of the arterial vessels. VC causes thickening of the intima-media with the formation of atherosclerotic plaques and lesions of the tunica intima, producing local inflammation, dyslipidemia, and accumulation of foam cells [14]. Atherosclerosis is restricted to medium or large arteries and is related to other risk factors such as smoking, obesity, dyslipidemia, hypertension, and aging. Calcification of the tunica media can occur in arteries of any size, is a typical process related to age, and is associated with greater arterial stiffness and reduction of

early CKD cause endothelial dysfunction progressing to VC.

An analytical cross-sectional study was performed. Patient population > 16 years of age who were undergoing RRT and meet the selection criteria while being attended to at the Division of Nephrology-Transplants of the Subspecialty Medical Unit at the Specialties Hospital of the National Occidental Medical Centre, Mexican Social Security Institute (*División de Nefrología-Trasplantes de la Unidad Médica de Alta Especialidad (UMAE) del Hospital de Especialidades, CMNO, del Instituto Mexicano del Seguro Social (IMSS)*), in Guadalajara, Jalisco, Mexico. Patient demographics, biochemical date, and time of RRT were recorded. Results were determined: hemoglobin (Hb), hematocrit (Hto), sodium (Na), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), chlorine (Cl), alkaline phosphatase, intact parato-hormone (iPTH), creatinine (Cr), urea, albumin, vitamin D3, and specific C-reactive protein (CRP). VC was determined using simple x-ray plaques according to the method of Dr. Adragao [27]. Two simple plaques were taken for each patient (one of the pelvis and hip and one of hands and wrists); the presence or absence of VC was evaluated in the anatomical territories of the iliac-femoral and radial-digital arteries. VC was scored as 1 (presence) and 0 (absence). The highest score to obtain could be 8 and minimum 0. All plaques were checked by the same radiologist blinded to the clinical characteristics of the patients.

**2.4. Statistical analysis**

with a confidence interval of 95%.

Guadalajara, Jalisco, with folio R-2016-1301-97.

**2.5. Ethical considerations**

**3. Results**

decreased (**Table 1**).

**3.1. Vascular calcification**

The data were analyzed using SPSS software (Statistical Package for the Social Sciences, v. 20, SPSS Inc., Chicago, IL). The Kolmogorov–Smirnov test was used to determine the distribution of the study variables. The quantitative variables are expressed in mean ± standard deviation/ error or median (percentile 25–75%) and Spearman's correlation test. The qualitative variables

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

test for the intragroup analysis. A value of p ≤ 0.05 was considered statistically significant,

The study was carried out according to the General Law of Health of Mexico in the Field of Research for Health. The study was classified as category III, that is to say, research with a risk greater than the minimum, for which it was necessary to sign the consent under information. Confidentiality of data and patient outcomes was respected. The project was evaluated and accepted by the Local Committee of Research and Ethics in Health of the UMAE, HE, CMNO,

We included 81 patients with CKD candidates for TR who had previously received dialysis. The patients were young adults, barely older than 30 years of age. The male–female ratio was 1:1. The RRT type was similar for hemodialysis (HD) and peritoneal dialysis (PD). Seventy-five of patients had at least 1.5 years on dialysis. In general, patients had regular control of azoles, serum electrolytes, lipid profile, and albumin. PCR and iPTH were elevated without clinical evidence of infection. Vitamin D3 levels were found to be

Of the 81 patients evaluated, 38 (47%) had VC, and in 61% of them VC was severe (≥3 points). Women had a significantly higher prevalence of VC: 39 women (67%) had VC, while 42 (29%) of male had VC, although age, dialysis time, lipid profile, urea, creatinine, and albumin were similar in patients with and without VC. iPTH, P, and alkaline phosphatase were significantly higher in patients with VC. Vitamin D3 and calcium levels showed no significant difference (**Table 2**). Patients were compared according to the type of RRT to which they were subjected since the onset of CKD. Sixty-four percent of patients submitted to HD presented VC, and 27% of those submitted to PD. The HD patients were older and were predominantly women,

Catalase was found to be significantly decreased (p < 0.0001) in CKD. The SOD activity was not found to be decreased in CKD (p = 0.58). Regarding the type of RRT and oxidative

had better control of azoles, and had higher levels of vitamin D3.

**3.2. The activity of the antioxidant enzyme**

or Fisher's exact

125

http://dx.doi.org/10.5772/intechopen.73095

are expressed in frequencies and percentages and were analyzed with Chi2

Oxidative stress was determined by the concentrations of antioxidants, and catalase and superoxide dismutase (SOD) and the oxidative DNA damage by determination of the 8-OHdG marker (ELISA technique). Because there were no normal parameters for the reagents, 10 mL extra blood from 20 healthy volunteers (healthy control group) (blood donors) with similar age and gender was used to determine the normal concentrations of the reagents.

#### **2.1. Superoxide dismutase**

The kit manufacturer's instructions were followed (SOD No. 706002, Cayman Chemical Company®, USA). The detection of O2 − generated by the xanthine oxidase and hypoxanthine enzymes was through the reaction of tetrazolium salts. The serum samples were diluted in sample buffer 1:5 in sample buffer: 200 μL of the radicals' detector, diluted 1:400, was placed, and 10 μL of the sample was then added. After slow agitation, 20 μL of xanthine oxidase was added to the wells. The microplate was incubated for 20 min at room temperature, and the absorbency was read at a wavelength of 440 nm.

#### **2.2. Catalase-520**

The determination of the antioxidant activity of catalase was performed according to the manufacturer's commercial kit (Bioxytech® Catalase-520™, USA). The spectrophotometric assay was performed by adding 30 μL of the diluted standards or samples into the corresponding tubes. Five hundred microliter of the substrate (10 mM H2 O2 ) was added to each tube and then incubated for 1 min at room temperature, and 500 μL of the stop reagent was added to each tube. The tubes were capped and mixed by inversion, and 20 μL of the mixture was added. Two milliliter of the HRP chromogenic reagent was added to each tube, mixed inversely, and incubated for 10 min at room temperature. The absorbance was obtained at 520 nm of optical density.

#### **2.3. 8-Hydroxy-2′-deoxyguanosine**

Instructions for the ELISA kit were followed (8-hydroxy-2-deoxyguanosine No. ab10124 Abcam®, Cambridge, United Kingdom). The plasma sample, the EIA buffer, the standards, and the 8-OHdG-AChE tracer were added to all the wells except the blank. The monoclonal antibody 8-OHdG was added, and the plate was incubated for 18 h at 4°C and washed with buffer for the recommended times, and 200 μL of Ellman's reagent was added to each well. The optical density was read at 405 nm.

#### **2.4. Statistical analysis**

demographics, biochemical date, and time of RRT were recorded. Results were determined: hemoglobin (Hb), hematocrit (Hto), sodium (Na), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), chlorine (Cl), alkaline phosphatase, intact parato-hormone (iPTH), creatinine (Cr), urea, albumin, vitamin D3, and specific C-reactive protein (CRP). VC was determined using simple x-ray plaques according to the method of Dr. Adragao [27]. Two simple plaques were taken for each patient (one of the pelvis and hip and one of hands and wrists); the presence or absence of VC was evaluated in the anatomical territories of the iliac-femoral and radial-digital arteries. VC was scored as 1 (presence) and 0 (absence). The highest score to obtain could be 8 and minimum 0. All plaques were checked by the same radiologist blinded

Oxidative stress was determined by the concentrations of antioxidants, and catalase and superoxide dismutase (SOD) and the oxidative DNA damage by determination of the 8-OHdG marker (ELISA technique). Because there were no normal parameters for the reagents, 10 mL extra blood from 20 healthy volunteers (healthy control group) (blood donors) with similar

The kit manufacturer's instructions were followed (SOD No. 706002, Cayman Chemical

enzymes was through the reaction of tetrazolium salts. The serum samples were diluted in sample buffer 1:5 in sample buffer: 200 μL of the radicals' detector, diluted 1:400, was placed, and 10 μL of the sample was then added. After slow agitation, 20 μL of xanthine oxidase was added to the wells. The microplate was incubated for 20 min at room temperature, and the

The determination of the antioxidant activity of catalase was performed according to the manufacturer's commercial kit (Bioxytech® Catalase-520™, USA). The spectrophotometric assay was performed by adding 30 μL of the diluted standards or samples into the corresponding tubes.

bated for 1 min at room temperature, and 500 μL of the stop reagent was added to each tube. The tubes were capped and mixed by inversion, and 20 μL of the mixture was added. Two milliliter of the HRP chromogenic reagent was added to each tube, mixed inversely, and incubated for 10 min at room temperature. The absorbance was obtained at 520 nm of optical density.

Instructions for the ELISA kit were followed (8-hydroxy-2-deoxyguanosine No. ab10124 Abcam®, Cambridge, United Kingdom). The plasma sample, the EIA buffer, the standards, and the 8-OHdG-AChE tracer were added to all the wells except the blank. The monoclonal antibody 8-OHdG was added, and the plate was incubated for 18 h at 4°C and washed with buffer for the recommended times, and 200 μL of Ellman's reagent was added to each well.

O2

generated by the xanthine oxidase and hypoxanthine

) was added to each tube and then incu-

age and gender was used to determine the normal concentrations of the reagents.

−

to the clinical characteristics of the patients.

**2.1. Superoxide dismutase**

124 Free Radicals, Antioxidants and Diseases

**2.2. Catalase-520**

Company®, USA). The detection of O2

absorbency was read at a wavelength of 440 nm.

Five hundred microliter of the substrate (10 mM H2

**2.3. 8-Hydroxy-2′-deoxyguanosine**

The optical density was read at 405 nm.

The data were analyzed using SPSS software (Statistical Package for the Social Sciences, v. 20, SPSS Inc., Chicago, IL). The Kolmogorov–Smirnov test was used to determine the distribution of the study variables. The quantitative variables are expressed in mean ± standard deviation/ error or median (percentile 25–75%) and Spearman's correlation test. The qualitative variables are expressed in frequencies and percentages and were analyzed with Chi2 or Fisher's exact test for the intragroup analysis. A value of p ≤ 0.05 was considered statistically significant, with a confidence interval of 95%.

### **2.5. Ethical considerations**

The study was carried out according to the General Law of Health of Mexico in the Field of Research for Health. The study was classified as category III, that is to say, research with a risk greater than the minimum, for which it was necessary to sign the consent under information. Confidentiality of data and patient outcomes was respected. The project was evaluated and accepted by the Local Committee of Research and Ethics in Health of the UMAE, HE, CMNO, Guadalajara, Jalisco, with folio R-2016-1301-97.

### **3. Results**

We included 81 patients with CKD candidates for TR who had previously received dialysis. The patients were young adults, barely older than 30 years of age. The male–female ratio was 1:1. The RRT type was similar for hemodialysis (HD) and peritoneal dialysis (PD). Seventy-five of patients had at least 1.5 years on dialysis. In general, patients had regular control of azoles, serum electrolytes, lipid profile, and albumin. PCR and iPTH were elevated without clinical evidence of infection. Vitamin D3 levels were found to be decreased (**Table 1**).

#### **3.1. Vascular calcification**

Of the 81 patients evaluated, 38 (47%) had VC, and in 61% of them VC was severe (≥3 points). Women had a significantly higher prevalence of VC: 39 women (67%) had VC, while 42 (29%) of male had VC, although age, dialysis time, lipid profile, urea, creatinine, and albumin were similar in patients with and without VC. iPTH, P, and alkaline phosphatase were significantly higher in patients with VC. Vitamin D3 and calcium levels showed no significant difference (**Table 2**). Patients were compared according to the type of RRT to which they were subjected since the onset of CKD. Sixty-four percent of patients submitted to HD presented VC, and 27% of those submitted to PD. The HD patients were older and were predominantly women, had better control of azoles, and had higher levels of vitamin D3.

#### **3.2. The activity of the antioxidant enzyme**

Catalase was found to be significantly decreased (p < 0.0001) in CKD. The SOD activity was not found to be decreased in CKD (p = 0.58). Regarding the type of RRT and oxidative


**Table 1.** Demographic and biochemical characteristics of the patients.

stress, we evidenced significantly higher levels of the marker of oxidative damage to DNA (8-OHdG) in patients submitted to PD and more consumed SOD activity (p < 0.0001). Serum catalase concentrations were similar between HD and DP (**Table 3**). However, when comparing the results between the antioxidants and the enzymes, we found to decrease significantly the activity of the catalase in CKD vs. the healthy controls (p < 0.0001). The activity of SOD was similar between CKD and healthy controls. We found greater oxidative damage to DNA in CKD vs. healthy controls (p < 0.0001) (**Table 4**).

and VC score showed a positive correlation with alkaline phosphatase and P. iPTH correlated positively marginally. 8-OHdG was negatively correlated with VC, alkaline phosphatase, and P (**Table 5**). The strength of association between VC and oxidative stress was performed through uni- and multivariate logistic regression analysis. Being female and being ≥30 years

BMI, body mass index; LDL, low-density lipoproteins; ALP, alkaline phosphatase; iPTH, intact parato-hormone (data

**CV n-38**

**Age** (years) 29.9 ± 9.2 31.3 ± 10.2 0.51

Female (n (%)) 26 (67) 13 (33) **0.001 Time in dialysis** (months) 18.5 (14.8–30) 22 (13–34) 0.76

**Urea** (mg/dL) 124 ± 47 120 ± 45 0.73 **Creatinine** (mg/dL) 12.8 ± 4.2 13.2 ± 4.2 0.70 **Albumin** (g/dL) 3.7 ± 0.6 3.8 ± 0.7 0.79 **PCR** (mg/mL) 3.8 (3.0–9.8) 3.0 (3.0–13.9) 0.66 **Total cholesterol** (mg/dL) 160 ± 43 159 ± 35 0.95 **Triglyceride** (mg/dL) 160 ± 100 139 ± 69 0.29 **LDL** (mg/dL) 86 ± 36 85 ± 33 0.94

**Vitamin D3** (ng/mL) 21.0 (16.2–26.8) 21.4 (16.2–29.2) 0.91 **iPTH** (pg/mL) 675 (237–980) 428 (114–650) **0.04 Phosphorus (P)** (mg/dL) 4.2 ± 2.3 2.7 ± 1.7 **0.001 Calcium** (mg/dL) 9.0 ± 1.0 9.2 ± 1.1 0.53 **Alkaline phosphatase** (UI/L) 141 (106–317) 100 (77–152) **0.01**

) 24.2 ± 3.7 23.5 ± 3.0 0.50

12 (29) 30 (71)

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

**No CV n-43**

*p*

127

http://dx.doi.org/10.5772/intechopen.73095

Demographic, biochemical, and gender-specific differences in MBD in CKD are shown in age (**Table 7**). Women had significantly higher VC frequency (67%) and were predominantly found in HD, although they had significantly lower levels of creatinine and higher concentrations of P compared to men. Age was similar in patients with and without VC. Significantly,

RRT (HD) predominated in patients aged ≥30 years with less time in dialysis.

of age confer greater risk for VC (**Table 6**).

shown as average ± SD or median (percentile 25–75%)).

**Table 2.** Demographic and biochemical data with and without CV.

**Gender** Male (n (%))

**BMI** (Kg/m2

**MBD in CKD**

To determine the association between oxidative stress data with the MBD results, we used the Spearman correlation test. Catalase showed a negative trend with alkaline phosphatase levels. SOD correlated negatively with the marker of oxidative damage to DNA (8-OHdG), Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors… http://dx.doi.org/10.5772/intechopen.73095 127


BMI, body mass index; LDL, low-density lipoproteins; ALP, alkaline phosphatase; iPTH, intact parato-hormone (data shown as average ± SD or median (percentile 25–75%)).

**Table 2.** Demographic and biochemical data with and without CV.

stress, we evidenced significantly higher levels of the marker of oxidative damage to DNA (8-OHdG) in patients submitted to PD and more consumed SOD activity (p < 0.0001). Serum catalase concentrations were similar between HD and DP (**Table 3**). However, when comparing the results between the antioxidants and the enzymes, we found to decrease significantly the activity of the catalase in CKD vs. the healthy controls (p < 0.0001). The activity of SOD was similar between CKD and healthy controls. We found greater oxidative damage to DNA

**n-81**

**Age** (years) 30.6 ± 9.7

 Female 39 (48) Male 42 (52) **Time of dialysis** (months) 19 (15–30)

Hemodialysis 44 (54)

**Urea** (mg/dL) 121 ± 45 **Creatinine** (mg/dL) 13 ± 4.1 **Phosphorus** (mg/dL) 3.5 ± 2.2 **Calcium** (mg/dL) 9.1 ± 1.1 **Sodium** (mg/dL) 139 ± 4.0 **Total cholesterol** (mg/dL) 160 ± 39 **Triglycerides** (mg/dL) 149 ± 86 **LDL** (mg/dL) 85 ± 35 **Albumin** (g/dL) 3.5 ± 0.7 **Alkaline phosphatase** (U/I) 125 (91–240) **iPTH** (pg/mL) 557 (173–911) **Vitamin D3** (ng/mL) 21.1 (16.2–27.4) **PCR** (mg/mL) 3.5 (3.0–9.6)

Peritoneal dialysis 37 (46)

**Gender** (%)

**Type of dialysis** (n (%))

126 Free Radicals, Antioxidants and Diseases

To determine the association between oxidative stress data with the MBD results, we used the Spearman correlation test. Catalase showed a negative trend with alkaline phosphatase levels. SOD correlated negatively with the marker of oxidative damage to DNA (8-OHdG),

in CKD vs. healthy controls (p < 0.0001) (**Table 4**).

**Table 1.** Demographic and biochemical characteristics of the patients.

Mean ± SD, median (percentile 25–75%).

and VC score showed a positive correlation with alkaline phosphatase and P. iPTH correlated positively marginally. 8-OHdG was negatively correlated with VC, alkaline phosphatase, and P (**Table 5**). The strength of association between VC and oxidative stress was performed through uni- and multivariate logistic regression analysis. Being female and being ≥30 years of age confer greater risk for VC (**Table 6**).

Demographic, biochemical, and gender-specific differences in MBD in CKD are shown in age (**Table 7**). Women had significantly higher VC frequency (67%) and were predominantly found in HD, although they had significantly lower levels of creatinine and higher concentrations of P compared to men. Age was similar in patients with and without VC. Significantly, RRT (HD) predominated in patients aged ≥30 years with less time in dialysis.


**r p**

−0.40 −0.27 0.24 0.30 0.50

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

−0.52 −0.37 −0.55

**OR IC 95%** *p*

**Male (n-42) Female (n-39)** *p*

SOD, superoxide dismutase; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; iPTH, intact parato-hormone.

**SOD** (UI/mL) 1.08 0.99–1.20 0.09 **Age** (≥30 years) 1.1 1.02–1.20 **0.02 Gender** (female) 20.8 2.5–173.70 **0.00**

**VC** (n (%)) 12 (29%) 26 (67%) **0.001 Age** (years) 26.0 (23.5–31.5) 30.0 (25.0–38.0) 0.13 **RRT (HD/DP (%))** 12/30 (29/71) 32/7 (82/18) **<0.0001 Time in dialysis** (months) 21.5 (15.3–30.0) 15.0 (4.5–67.5) 0.37

**Urea** (mg/dL) 114 (96–144) 105 (79–165) 0.54 **Creatinine** (mg/dL) 13.4 (11.3–16.7) 9.7 (7.3–10.9) **0.001 Total cholesterol** (mg/dL) 149 (129–184) 155 (131–183) 0.9 **Triglycerides** (mg/dL) 134 (81–194) 130 (98–179) 0.76

) 22.9 (21.9–25.2) 23.6 (20.7–26.9) 0.96

SOD, superoxide dismutase. Oxidative DNA damage markers: 8-OHdG, PCR, vitamin D3, iPTH, age, gender,

−0.29 0.08

**0.002 0.03** 0.06 **0.02 <0.0001**

http://dx.doi.org/10.5772/intechopen.73095

129

**<0.0001 0.003 <0.001**

**Catalase** (U/mg)

**SOD** (UI/mL) **8-OHdG** (ng/mL) **Score of VC iPTH** (pg/mL)

**8-OHdG** (ng/mL) **Score of VC**

**BMI** (Kg/m2

**Alkaline phosphatase (mg/dL)**

**Alkaline phosphatase** (mg/dL) **Phosphorus (P)** (mg/dL)

**Alkaline phosphatase** (mg/dL) **Phosphorus (P)** (mg/dL)

**Table 5.** Correlation between MBD and CKD.

phosphorus, LDL, albumin, alkaline phosphatase.

**Table 6.** Predictors of VC (Chi2 = 13.38; *p =* 0.004).

MBD, metabolic body disease; iPTH, intact parato-hormone; SOD, superoxide dismutase; 8-OHdG, 8-hydroxi-2′ deoxiguanosina; mean ± SD or median (percentile 25–75%).

**Table 3.** Type of dialysis with and without vascular calcification.


SOD, superoxide dismutase; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; mean ± SD or median (percentile 25–75%).

**Table 4.** Oxidative stress in CKD vs. healthy controls.


SOD, superoxide dismutase; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; iPTH, intact parato-hormone.

**Table 5.** Correlation between MBD and CKD.


SOD, superoxide dismutase. Oxidative DNA damage markers: 8-OHdG, PCR, vitamin D3, iPTH, age, gender, phosphorus, LDL, albumin, alkaline phosphatase.

**Table 6.** Predictors of VC (Chi2 = 13.38; *p =* 0.004).

**CKD Healthy control** *p*

**DP n-37** *p*

**Catalase** (U/mg) 17.7 ± 9.0 94.17 ± 1.58 *<0.0001* **SOD** (UI/mL) 8.2 ± 7.7 10.2 ± 1.9 0.58

**HD n-44**

**Vascular calcification** (n (%)) 28 (64) 10 (27) **0.001 Age** (years) 30.0 (25.0–37.0) 26.0 (23.3–30.8) **0.05 Gender (male)** (n (%)) 12 (27%) 30 (82%) **<0.0001**

**Time in dialysis** (months) 17.0 (6.8–23.5) 21.5 (15.3–33.0) 0.30 **Urea** (mg/dL) 108 (78–120) 120(102–157) **0.03 Creatinine** (mg/dL) 11.0 (9.0–12.1) 13.9 (10.1–16.8) **0.005 Albumin** (g/dL) 3.9 (3.4–4.2) 3.6 (3.0–4.1) 0.18 **Total cholesterol** (mg/dL) 149 (128–174) 160 (142–198) 0.07 **Triglycerides** (mg/dL) 130 (98–179) 134 (82–190) 0.84 **LDL** (mg/dL) 72 (57–87) 92 (73–120) **0.002**

**iPTH** (pg/mL) 593 (173–911) 509 (126–918) 0.66

**Catalase** (U/mg) *13.5 (8.5–19.0) 18.7 (9.5–25.7) 0.19* **SOD** (UI/mL) 7.9 (4.3–13.3) 3.3 (2.3–4.6) **<0.0001**

**8-OHdG** (ng/mL) 4.4 (3.1–6.4) 9.4 (7.3–10.8) **<0.0001**

MBD, metabolic body disease; iPTH, intact parato-hormone; SOD, superoxide dismutase; 8-OHdG, 8-hydroxi-2′-

) 22.3 (21.4–24.3) 23.6 (21.6–26.6) 0.29

**8-OHdG** (ng/mL) 9.4 ± 11.1 4.7 ± 1.0 *<0.0001* SOD, superoxide dismutase; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; mean ± SD or median (percentile 25–75%).

**Antioxidants**

**BMI** (Kg/m2

128 Free Radicals, Antioxidants and Diseases

**MBD and CKD**

**Antioxidants**

**Oxidative DNA damage**

**Oxidative DNA damage**

**Table 4.** Oxidative stress in CKD vs. healthy controls.

deoxiguanosina; mean ± SD or median (percentile 25–75%).

**Table 3.** Type of dialysis with and without vascular calcification.



ERRβ, and ERRγ (NR3B1–NR3B3), which bind to the classical estrogen response elements and to the extended middle site sequences (ERNA TNAAGGTCA; ERR) as monomers or dimers [31]. ERRα is strongly expressed throughout the differentiation of osteoblasts and regulates the expression of osteopontin through a noncanonical ERRα response element [32]. ERRγ is expressed in osteoblast progenitors and negatively regulates osteoblast differentiation induced by BMP2 and bone formation [33]. ERRγ plays a key role in VC through the positive regulation of the BMP2 signaling pathway, suggesting that inhibition of ERRγ could be a potential therapeutic strategy for VC prevention [34]. Previously, the influence of hormonal *status* in patients with ESRD prevalent in HD was evidenced by the association between follicle-stimulating hormone and MBD in CKD, particularly with bone mineral density. This association could also reflect alterations in the appearance of VC [35]. The majority of patients who suffer VC tend to be of advanced ages and have multiple metabolic, renal, and inflammatory complications. Although in recent years knowledge of the pathophysiology of arteriosclerosis and its close relationship to the mechanisms of oxidative stress has increased considerably, there is still little known on the influence these inflammatory processes have on the development of VC, but it is thought that

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

http://dx.doi.org/10.5772/intechopen.73095

131

The secondary hyperparathyroidism, hyperphosphatemia, hypercalcemia, and other inherent factors of ESRD, like the state of uremia, inflammation, and the oxidative stress, all of them play a relevant role in the pathophysiology of these vascular alterations. Patients with ESRD present with a chronic inflammatory state that is associated with VC, increased morbidity, and cardiovascular mortality. As well as promoting simultaneous changes in mineral metabolism, the inflammation favors vascular damage that in the long term could lead to the

Over time, VC has been considered a passive or degenerative illness where minerals spontaneously deposited in the vascular tissue. However, a large number of studies in recent years have contributed to the understanding of the underlying mechanisms of VC and have demonstrated that they form part of a regulated process that carries with it the phenotypical transformation of the vascular smooth muscle cells in osteogenic cells, in response to diverse calcifying stimuli. Thus, it is thought that VC in renal patients is not only due to disturbances in mineral metabolism but that there are other risk factors involved like oxidative stress [38]. The inflammatory mediators must be considered because they are often increased with capacity to activate the renin-angiotensin system in CKD, which probably contributes to increase ROS production and accelerates atherosclerosis. Therefore, promoters of VC increase and inhibitors are reduced, which favors metastatic VC in ESRD. Accelerated atherosclerosis will lead to a higher prevalence of coronary artery disease, heart failure, stroke, and peripheral arterial disease. Consequently, subjects with CKD are exposed to increased morbidity and

In our study, the predictors of VC were significantly the woman being aged ≥30 years. Patients in HD had a higher age and a higher percentage of VC compared to patients undergoing PD, which to some extent was expected due to the higher degree of atherosclerosis and secondary vascular damage that occurs in patients with HD. On the other hand, MBD variables in CKD were evaluated in patients with and without VC; iPTH and alkaline phosphatase levels were elevated in patients with VC (no significant difference) as expected.

there is a close link between them [36].

development of VC [37].

mortality as a result of CVD [39].

VC, vascular calcification; RRT, renal replacement therapy; BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PCR, C-reactive protein; iPTH, intact parato-hormone.

**Table 7.** Demographic, biochemicals, MBD, and CKD data.

#### **4. Discussion**

VC appears as an independent cardiovascular risk factor of the state of hypercholesterolemia and atherosclerosis that present the patient [28]. In the general population, this previously described the protective effect of estrogens against VC [29]. The estrogen-related receptors (ERR) are closely related to estrogen receptors, sharing high homology in the DNA-binding domain, although they do not bind to estrogen [30]. The ERR subfamily consists of three members, ERRα, ERRβ, and ERRγ (NR3B1–NR3B3), which bind to the classical estrogen response elements and to the extended middle site sequences (ERNA TNAAGGTCA; ERR) as monomers or dimers [31]. ERRα is strongly expressed throughout the differentiation of osteoblasts and regulates the expression of osteopontin through a noncanonical ERRα response element [32]. ERRγ is expressed in osteoblast progenitors and negatively regulates osteoblast differentiation induced by BMP2 and bone formation [33]. ERRγ plays a key role in VC through the positive regulation of the BMP2 signaling pathway, suggesting that inhibition of ERRγ could be a potential therapeutic strategy for VC prevention [34]. Previously, the influence of hormonal *status* in patients with ESRD prevalent in HD was evidenced by the association between follicle-stimulating hormone and MBD in CKD, particularly with bone mineral density. This association could also reflect alterations in the appearance of VC [35]. The majority of patients who suffer VC tend to be of advanced ages and have multiple metabolic, renal, and inflammatory complications. Although in recent years knowledge of the pathophysiology of arteriosclerosis and its close relationship to the mechanisms of oxidative stress has increased considerably, there is still little known on the influence these inflammatory processes have on the development of VC, but it is thought that there is a close link between them [36].

The secondary hyperparathyroidism, hyperphosphatemia, hypercalcemia, and other inherent factors of ESRD, like the state of uremia, inflammation, and the oxidative stress, all of them play a relevant role in the pathophysiology of these vascular alterations. Patients with ESRD present with a chronic inflammatory state that is associated with VC, increased morbidity, and cardiovascular mortality. As well as promoting simultaneous changes in mineral metabolism, the inflammation favors vascular damage that in the long term could lead to the development of VC [37].

Over time, VC has been considered a passive or degenerative illness where minerals spontaneously deposited in the vascular tissue. However, a large number of studies in recent years have contributed to the understanding of the underlying mechanisms of VC and have demonstrated that they form part of a regulated process that carries with it the phenotypical transformation of the vascular smooth muscle cells in osteogenic cells, in response to diverse calcifying stimuli. Thus, it is thought that VC in renal patients is not only due to disturbances in mineral metabolism but that there are other risk factors involved like oxidative stress [38]. The inflammatory mediators must be considered because they are often increased with capacity to activate the renin-angiotensin system in CKD, which probably contributes to increase ROS production and accelerates atherosclerosis. Therefore, promoters of VC increase and inhibitors are reduced, which favors metastatic VC in ESRD. Accelerated atherosclerosis will lead to a higher prevalence of coronary artery disease, heart failure, stroke, and peripheral arterial disease. Consequently, subjects with CKD are exposed to increased morbidity and mortality as a result of CVD [39].

In our study, the predictors of VC were significantly the woman being aged ≥30 years. Patients in HD had a higher age and a higher percentage of VC compared to patients undergoing PD, which to some extent was expected due to the higher degree of atherosclerosis and secondary vascular damage that occurs in patients with HD. On the other hand, MBD variables in CKD were evaluated in patients with and without VC; iPTH and alkaline phosphatase levels were elevated in patients with VC (no significant difference) as expected.

**4. Discussion**

**BMI** (Kg/m2

VC appears as an independent cardiovascular risk factor of the state of hypercholesterolemia and atherosclerosis that present the patient [28]. In the general population, this previously described the protective effect of estrogens against VC [29]. The estrogen-related receptors (ERR) are closely related to estrogen receptors, sharing high homology in the DNA-binding domain, although they do not bind to estrogen [30]. The ERR subfamily consists of three members, ERRα,

VC, vascular calcification; RRT, renal replacement therapy; BMI, body mass index; HDL, high-density lipoprotein; LDL,

low-density lipoprotein; PCR, C-reactive protein; iPTH, intact parato-hormone.

**Table 7.** Demographic, biochemicals, MBD, and CKD data.

**Male (n-42) Female (n-39)** *p*

**<30 years (n-48) ≥30 years (n-33)** *p*

**LDL** (mg/dL) 79 (62–112) 81 (68–88) 0.43 **PCR** (mg/mL) 3.1 (3.0–17.3) 3.9 (3.0–8.9) 0.94 **Vitamin D3** (ng/mL) 21.2 (16.2–31.1) 21.0 (15.9–26.4) 0.51 **Calcium** (mg/dL) 9.2 (8.5–9.9) 8.9 (8.4–9.4) 0.33 **Phosphorus (P)** (mg/dL) 2.1 (1.6–2.5) 5.3 (3.2–6.5) **<0.0001 iPTH** (pg/mL) 369 (137–615) 650 (203–950) 0.09 **Alkaline phosphatase** (U/I) 106 (78–138) 150 (95–329) **0.02**

**VC** (n (%)) 24 (50) 14 (42) 0.45 **RRT (HD/DP** (%)) 21/27 (45/55) 23/10 (70/30) **0.03 Time in dialysis** (months) 24 (17–32) 12 (2–15) **0.02**

**Urea** (mg/dL) 108 (93–144) 123 (94–144) 0.46 **Creatinine** (mg/dL) 13.4 (10.8–15.8) 12.0 (9.2–15.7) 0.15 **Total cholesterol** (mg/dL) 153 (129–187) 152 (133–182) 0.86 **Triglycerides** (mg/dL) 131 (98–187) 127 (88–176) 0.45 **LDL** (mg/dL) 81 (68–98) 74 (60–102) 0.52 **PCR** (mg/mL) 4.3 (3.0–13.6) 3.0 (3.0–7.2) 0.12 **Vitamin D3** (ng/mL) 20.5 (15.7–28.0) 21.7 (16.4–27.2) 0.73 **Calcium** (mg/dL) 9.0 (8.4–9.8) 9.2 (8.6–9.8) 0.37 **Phosphorus (P)** (mg/dL) 2.3 (1.8–4.1) 3.7 (2.1–5.9) 0.06 **iPTH** (pg/mL) 557 (222–770) 526 (125–955) 0.64 **Alkaline phosphatase** (U/I) 117 (89–179) 145 (94–320) *0.33*

) 23.2 (21.5–25.2) 22.6 (21.6–28.0) 0.44

Demographic, biochemicals, and MBD in CKD by age data

130 Free Radicals, Antioxidants and Diseases

Vitamin D 3 levels were slightly decreased in patients with VC. This behavior reflects changes of secondary hyperparathyroidism in CKD. The VC is a complex phenomenon, with many causal associations yet to be fully elucidated, despite the fact that current therapeutic strategies still fail to improve the impact on cardiovascular morbidity and mortality in patients with CKD [40].

repaired [44]. If the abasic sites are not repaired, they determine breaks in the DNA strands,

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

http://dx.doi.org/10.5772/intechopen.73095

133

When performing the correlation tests between oxidative stress and MBD results in patients with CKD, we found that SOD were correlated negatively with the 8-0HdG marker and the VC score and the oxidative stress correlated positively with alkaline phosphatase and phosphorus. The 8-OHdG correlated negatively with VC, alkaline phosphatase, and P. Based on the findings, the importance of the association between oxidative stress and VC in patients with ESRD, and the recent studies evidencing the persistence of alterations in oxidative status even after TR, it is necessary to evaluate the usefulness of antioxidant therapeutic strategies in this population of patients [46]. The results of this study, being the first in our country to describe the association between oxidative stress behavior and VC, contribute to the future evaluation of the oxidative

The VC frequency was 47%. Compared with healthy subjects, patients with ESRD had decreased catalase activity and increased marker levels of oxidative DNA damage (8-OHdG). The correlations of the oxidative stress markers were significant weak; in the case of SOD, it had negative significance with the VC score and the 8-OHdG score. There was a positive correlation with alkaline phosphatase and P. The 8-OHdG marker was negatively correlated with VC, alkaline phosphatase, and P. The significant predictors of VC were being female aging ≥30 years.

, José Ignacio Cerrillos-Gutiérrez1

and Alejandra Guillermina Miranda-Díaz3

1 Department of Nephrology and Transplants Hospital of Specialties, National Medical Center of the West, Mexican Institute of Social Security, Guadalajara, Jalisco, México

2 Unit of Medical Research in Renal Diseases, Hospital of Specialties, National Medical Center of the West, Mexican Institute of Social Security, Guadalajara, Jalisco, México

3 Department of Physiology, University Center of Health Sciences, University of

, Sonia Sifuentes-Franco3

, Sandra Carrillo-Ibarra3

\*Address all correspondence to: kindalex1@outlook.com

, Jorge Andrade-Sierra1

,

\*

,

and the induction of cellular apoptosis occurs [45].

status of the ESRD for prognostic and therapeutic purposes.

**5. Conclusions**

**Conflict of interest**

No conflict of interest.

Enrique Rojas-Campos2

Andrés García-Sánchez3

Edith Viridiana Alatorre-Moreno1

Guadalajara, Guadalajara, Jalisco, México

**Author details**

Alkaline phosphatase and P were significantly increased in patients with HD. These results agree with previous studies reporting a lower prevalence of VC in patients submitted to PD, which could be a reflection of factors such as lower age, lower anterior vascular damage, and better residual renal function [35]. The prooxidant status of the ESRD was evaluated; our results were consistent with what was previously reported by other authors [41]. The marker levels of oxidative DNA damage (8-OHdG) were significantly increased in patients with ESRD compared to healthy controls. The antioxidant catalase was found to be significantly decreased in patients with ESRD. We cannot explain why the levels of SOD enzyme activity showed no significant difference, since like catalase reflects the activity of the antioxidant system. This could be a result of the magnitude of the defense system; that is to say that large amounts of SOD are required and therefore small changes are not enough showing mathematical differences. We also do not know if the members of the antioxidant defense system operate jointly or independently in CKD, which could explain such results. However, more specific studies are needed to elucidate such findings. Theoretically, there is a link between oxidative stress and VC in ESRD, since both conditions converge in the uremic environment; however, so far there is insufficient evidence in the available literature in human beings about this association. Among their main experimental theories in the study, the possible role of ROS as promoters of VC in patients with ESRD is postulated [42].

With the obtained information, we cannot explain these results, and we have some questions: The levels of the 8-OHdG marker appear to be a compensatory consequence of the SOD activity in the presence of VC. These findings could be due to the limitation of the type of study performed, because no causal association can be determined. However, it would be logical to think that in those patients with VC that showed better antioxidant activity (SOD); its effect with 8-OHdG levels is reflected, although there are no cellular and/or tissue evaluations to prove it. In addition, we cannot know if those patients with VC and 8-OHdG levels previously had them before the presence of VC. In addition, we wonder if there is another associated determinant in patients with VC, which influences such findings. Regarding this, one of the most studied theories is the presence of repair systems of oxidative damage to DNA. Of these systems, it is known that eukaryotic cells can activate mechanisms to repair damage, modify transcriptional activity, and stop the cell cycle [43]. These mechanisms may act independently but frequently act together. If the level of damage exceeds the mechanisms intended to repair DNA, the cell can activate mechanisms of cell death by apoptosis. Among the major DNA repair enzymes are endonucleases, exonucleases, ligases, and DNA glycosylase. In mammals, an important mechanism of DNA repair is the base cleavage repair system which operates as follows: a DNA glycosylase removes the damaged base, generating an apurinic/apirimidic site or abasic site. Subsequently, the sugar/phosphate residue remaining in the abasic site is eliminated by the activity of the APE1 enzyme. Through the activity of DNA polymerase B and ligase III, the correct nucleotide is incorporated and the 3 'to 5' ends of the DNA strand is repaired [44]. If the abasic sites are not repaired, they determine breaks in the DNA strands, and the induction of cellular apoptosis occurs [45].

When performing the correlation tests between oxidative stress and MBD results in patients with CKD, we found that SOD were correlated negatively with the 8-0HdG marker and the VC score and the oxidative stress correlated positively with alkaline phosphatase and phosphorus. The 8-OHdG correlated negatively with VC, alkaline phosphatase, and P. Based on the findings, the importance of the association between oxidative stress and VC in patients with ESRD, and the recent studies evidencing the persistence of alterations in oxidative status even after TR, it is necessary to evaluate the usefulness of antioxidant therapeutic strategies in this population of patients [46]. The results of this study, being the first in our country to describe the association between oxidative stress behavior and VC, contribute to the future evaluation of the oxidative status of the ESRD for prognostic and therapeutic purposes.

## **5. Conclusions**

Vitamin D 3 levels were slightly decreased in patients with VC. This behavior reflects changes of secondary hyperparathyroidism in CKD. The VC is a complex phenomenon, with many causal associations yet to be fully elucidated, despite the fact that current therapeutic strategies still fail to improve the impact on cardiovascular morbidity and mortality

Alkaline phosphatase and P were significantly increased in patients with HD. These results agree with previous studies reporting a lower prevalence of VC in patients submitted to PD, which could be a reflection of factors such as lower age, lower anterior vascular damage, and better residual renal function [35]. The prooxidant status of the ESRD was evaluated; our results were consistent with what was previously reported by other authors [41]. The marker levels of oxidative DNA damage (8-OHdG) were significantly increased in patients with ESRD compared to healthy controls. The antioxidant catalase was found to be significantly decreased in patients with ESRD. We cannot explain why the levels of SOD enzyme activity showed no significant difference, since like catalase reflects the activity of the antioxidant system. This could be a result of the magnitude of the defense system; that is to say that large amounts of SOD are required and therefore small changes are not enough showing mathematical differences. We also do not know if the members of the antioxidant defense system operate jointly or independently in CKD, which could explain such results. However, more specific studies are needed to elucidate such findings. Theoretically, there is a link between oxidative stress and VC in ESRD, since both conditions converge in the uremic environment; however, so far there is insufficient evidence in the available literature in human beings about this association. Among their main experimental theories in the study, the possible role of

With the obtained information, we cannot explain these results, and we have some questions: The levels of the 8-OHdG marker appear to be a compensatory consequence of the SOD activity in the presence of VC. These findings could be due to the limitation of the type of study performed, because no causal association can be determined. However, it would be logical to think that in those patients with VC that showed better antioxidant activity (SOD); its effect with 8-OHdG levels is reflected, although there are no cellular and/or tissue evaluations to prove it. In addition, we cannot know if those patients with VC and 8-OHdG levels previously had them before the presence of VC. In addition, we wonder if there is another associated determinant in patients with VC, which influences such findings. Regarding this, one of the most studied theories is the presence of repair systems of oxidative damage to DNA. Of these systems, it is known that eukaryotic cells can activate mechanisms to repair damage, modify transcriptional activity, and stop the cell cycle [43]. These mechanisms may act independently but frequently act together. If the level of damage exceeds the mechanisms intended to repair DNA, the cell can activate mechanisms of cell death by apoptosis. Among the major DNA repair enzymes are endonucleases, exonucleases, ligases, and DNA glycosylase. In mammals, an important mechanism of DNA repair is the base cleavage repair system which operates as follows: a DNA glycosylase removes the damaged base, generating an apurinic/apirimidic site or abasic site. Subsequently, the sugar/phosphate residue remaining in the abasic site is eliminated by the activity of the APE1 enzyme. Through the activity of DNA polymerase B and ligase III, the correct nucleotide is incorporated and the 3 'to 5' ends of the DNA strand is

ROS as promoters of VC in patients with ESRD is postulated [42].

in patients with CKD [40].

132 Free Radicals, Antioxidants and Diseases

The VC frequency was 47%. Compared with healthy subjects, patients with ESRD had decreased catalase activity and increased marker levels of oxidative DNA damage (8-OHdG). The correlations of the oxidative stress markers were significant weak; in the case of SOD, it had negative significance with the VC score and the 8-OHdG score. There was a positive correlation with alkaline phosphatase and P. The 8-OHdG marker was negatively correlated with VC, alkaline phosphatase, and P. The significant predictors of VC were being female aging ≥30 years.

### **Conflict of interest**

No conflict of interest.

### **Author details**

Edith Viridiana Alatorre-Moreno1 , José Ignacio Cerrillos-Gutiérrez1 , Jorge Andrade-Sierra1 , Enrique Rojas-Campos2 , Sandra Carrillo-Ibarra3 , Sonia Sifuentes-Franco3 , Andrés García-Sánchez3 and Alejandra Guillermina Miranda-Díaz3 \*

\*Address all correspondence to: kindalex1@outlook.com

1 Department of Nephrology and Transplants Hospital of Specialties, National Medical Center of the West, Mexican Institute of Social Security, Guadalajara, Jalisco, México

2 Unit of Medical Research in Renal Diseases, Hospital of Specialties, National Medical Center of the West, Mexican Institute of Social Security, Guadalajara, Jalisco, México

3 Department of Physiology, University Center of Health Sciences, University of Guadalajara, Guadalajara, Jalisco, México

## **References**

[1] Brück K, Stel VS, Gambaro G, Hallan S, Völzke H, Ärnlöv J, Kastarinen M, Guessous I, Vinhas J, Stengel B, Brenner H, Chudek J, Romundstad S, Tomson C, Gonzalez AO, Bello AK, Ferrieres J, Palmieri L, Browne G, Capuano V, Van Biesen W, Zoccali C, Gansevoort R, Navis G, Rothenbacher D, Ferraro PM, Nitsch D, Wanner C, Jager KJ, European CKD Burden Consortium. CKD prevalence varies across the European general population. Journal of the American Society of Nephrology. 2016;**27**(7):2135-2147

[11] Ix JH, Katz R, De Boer IH, Kestenbaum BR, Allison MA, Siscovick DS, Newman AB, Sarnak MJ, Shlipak MG, Criqui MH. Association of chronic kidney disease with the spectrum of ankle brachial index: The cardiovascular health study. Journal of the American

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

http://dx.doi.org/10.5772/intechopen.73095

135

[12] Lu KC, Wu CC, Yen JF, Liu WC. Vascular calcification and renal bone disorders. The

[13] Hruska KA, Seifert M, Sugatani T. Pathophysiology of the chronic kidney disease-mineral bone disorder. Current Opinion in Nephrology and Hypertension. 2015;**24**(4):303-309

[14] Ross R.Atherosclerosis-an inflammatory disease. The New England Journal of Medicine.

[15] O'Neill WC, Lomashvili KA. Recent progress in the treatment of vascular calcification.

[16] Persy V, D'Haese P. Vascular calcification and bone disease: The calcification paradox.

[17] Bonomini F, Rodella LF, Rezzani R. Metabolic syndrome, aging and involvement of oxi-

[18] Himmelfarb J. Linking oxidative stress and inflammation in kidney disease: Which is the

[19] Locatelli F, Canaud B, Eckardt KU, Stenvinkel P, Wanner C, Zoccali C. Oxidative stress in end-stage renal disease: An emerging threat to patient outcome. Nephrology, Dialysis,

[20] You H, Yang H, Zhu Q, Li M, Xue J, Gu Y, Lin S, Ding F. Advanced oxidation protein products induce vascular calcification by promoting osteoblastic trans-differentiation of smooth muscle cells via oxidative stress and ERK pathway. Renal Failure.

[21] Byon CH, Javed A, Dai Q, Kappes JC, Clemens TL, Darley-Usmar VM, JM MD, Chen Y. Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling. The Journal of Biological Chemistry. 2008;

[22] Fyhrquist F, Saijonmaa O, Strandberg T. The roles of senescence and telomere shortening in cardiovascular disease. Nature Reviews. Cardiology. 2013;**10**:274-283

[23] Salpea KD, Humphries SE. Telomere length in atherosclerosis and diabetes. Athero-

[24] Durik M, Kavousi M, van der Pluijm I, Isaacs A, Cheng C, Verdonk K, Loot AE, Oeseburg H, Bhaggoe UM, Leijten F, van Veghel R, de Vries R, Rudez G, Brandt R, Ridwan YR, van Deel ED, de Boer M, Tempel D, Fleming I, Mitchell GF, Verwoert GC, Tarasov KV, Uitterlinden AG, Hofman A, Duckers HJ, van Duijn CM, Oostra BA, Witteman JC,

chicken and which is the egg. Seminars in Dialysis. 2004;**17**:449-454

College of Cardiology. 2009;**54**:1176-1184

Scientific World Journal. 2014;**2014**(637065)

Kidney International. 2010;**78**:1232-1239

Trends in Molecular Medicine. 2009;**15**:405-416

dative stress. Aging Diseases. 2015;**6**:109-120

Transplantation. 2003;**18**:1272-1280

2009;**31**(4):313-319

**283**(22):15319-15327

sclerosis. 2010;**209**:35-38

1999;**340**:115-126


[11] Ix JH, Katz R, De Boer IH, Kestenbaum BR, Allison MA, Siscovick DS, Newman AB, Sarnak MJ, Shlipak MG, Criqui MH. Association of chronic kidney disease with the spectrum of ankle brachial index: The cardiovascular health study. Journal of the American College of Cardiology. 2009;**54**:1176-1184

**References**

134 Free Radicals, Antioxidants and Diseases

[1] Brück K, Stel VS, Gambaro G, Hallan S, Völzke H, Ärnlöv J, Kastarinen M, Guessous I, Vinhas J, Stengel B, Brenner H, Chudek J, Romundstad S, Tomson C, Gonzalez AO, Bello AK, Ferrieres J, Palmieri L, Browne G, Capuano V, Van Biesen W, Zoccali C, Gansevoort R, Navis G, Rothenbacher D, Ferraro PM, Nitsch D, Wanner C, Jager KJ, European CKD Burden Consortium. CKD prevalence varies across the European general population.

[2] Chien KL, Lin HJ, Lee BC, Hsu HC, Lee YT, Chen MF. A prediction model for the risk of incident chronic kidney disease. The American Journal of Medicine. 2010;**123**:836-846

[3] Liu M, Li XC, Lu L, Cao Y, Sun RR, Chen S, Zhang PY. Cardiovascular disease and its relationship with chronic kidney disease. European Review for Medical and Pharma-

[4] Fliser D, Wiecek A, Suleymanlar G, Ortiz A, Massy Z, Lindholm B, Martinez-Castelao A, Agarwal R, Jager KJ, Dekker FW, Blankestijn PJ, Goldsmith D, Covic A, London G, Zoccali C, for European Renal and Cardiovascular Medicine working group of the European Renal Association–European Dialysis and Transplant Association (ERA– EDTA). The dysfunctional endothelium in CKD and in cardiovascular disease: Mapping the origin(s) of cardio vascular problems in CKD and of kidney disease in cardiovascular

condition for a research agenda. Kidney International. Supplement. 2011;**1**(1):6-9

[5] Staude H, Jeske S, Schmitz K, Warncke G, Fischer DC. Cardiovascular risk and mineral bone disorder in patients with chronic kidney disease. Kidney & Blood Pressure

[6] Moe S, Drüeke T, Cunningham J, Goodman W, Martin K, Olgaard K, Ott S, Sprague S, Lameire N. Eknoyan G; kidney disease: Improving global outcomes(KDIGO). Definition, evaluation, and classification of renal osteodystrophy: A position statement from kidney disease: Improving global outcomes (KDIGO). Kidney International. 2006;**69**:1945-1953

[7] Gutiérrez OM, Mannstadt M, Isakova T, Rauh-Hain JA, Tamez H, Shah A, Smith K, Lee H, Thadhani R, Jüppner H, Wolf M. Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. New England Journal of Medicine. 2008;**359**:584-592

[8] Matsushita K, Sang Y, Ballew SH, Shlipak M, Katz R, Rosas SE, Peralta CA, Woodward M, Kramer HJ, Jacobs DR, Sarnak MJ, Coresh J. Subclinical atherosclerosis measures for cardiovascular prediction in CKD. Journal of the American Society of Nephrology.

[9] Drüeke TB, Massy ZA. Atherosclerosis in CKD: Differences from the general population.

[10] Fang Y, Ginsberg C, Sugatani T, Monier-Faugere MC, Malluche H, Hruska KA. Early chronic kidney disease-mineral bone disorder stimulates vascular calcification. Kidney

Journal of the American Society of Nephrology. 2016;**27**(7):2135-2147

cological Sciences. 2014;**18**(19):2918-2926

Research. 2013;**37**(1):68-83

2014;**26**:439-447

Nature Reviews. Nephrology. 2010;**6**:723-735

International. 2014;**85**:142-150


Duncker DJ, Danser AH, Hoeijmakers JH, Roks AJ. Nucleotide excision DNA repair is associated with age-related vascular dysfunction. Circulation 2012;**126**:468-478

[36] Chen NX, Moe SM. Vascular calcification: Pathophysiology and risk factors. Current

Vascular Calcification and Oxidative DNA Damage as Nontraditional Cardiovascular Risk Factors…

http://dx.doi.org/10.5772/intechopen.73095

137

[37] Nikodimopoulou M, Liakos S. Secondary hyperparathyroidism and target organs in

[38] Leopold JA. Vascular calcification: Mechanisms of vascular smooth muscle cell calcifica-

[39] Schiffrin EL, Lipman ML, Mann JF. Chronic kidney disease: Effects on the cardiovascu-

[40] Rojas-Campos E, Herrera-Llamas R, Montañez Fernandez JL, Martınez-Martınez P, Andrade-Sierra J, Avila-Baray AA, Cueto-Manzano AM. Vascular calcification in mexican hemodialysis patients. Elsevier. Archives of Medical Research. 2013;**44**:628-637 [41] Puchades Montesa MJ, González Rico MA, Solís Salguero MA, Torregrosa Maicas I, Tormos Muñoz MC, Saez Tormo G, Juan Garcia I, Miguel Carrasco A. Estudio del estrés

[42] Al-Aly Z. Phosphate, oxidative stress, and nuclear factor-kB activation in vascular calci-

[43] Norbury CJ, Hickson ID. Cellular responses to DNA damage. Pharmacology and. Toxi-

[44] Hegde ML, Hazra TK, Mitra S. Early steps in the DNA base excision / single strand inter-

[45] Loeb LA, Preston BD. Mutagenesis by apurinic/apyrimidinic sites. Annual Review of

[46] Cerrillos-Gutiérrez JI, Miranda-Díaz AG, Preciado-Rojas P, Gómez-Navarro B, Sifuentes-Franco S, Carrillo-Ibarra S, Andrade-Sierra J, Rojas-Campos E, Cueto-Manzano AM. The beneficial effects of renal transplantation on altered oxidative status of ESRD patients.

oxidativo en enfermedad renal avanzada. Nefrología. 2009;**29**(5):464-473

ruption repair pathway in mammalian cells. Cell Research. 2008;**18**:27-47

Hypertension Reports. 2012;**14**(3):228-237

lar system. Circulation. 2007;**116**(1):85-97

fication. Kidney International. 2011;**79**:1044-1047

Oxidative Medicine and Cellular Longevity. 2016;**2016**

cology. 2001;**41**:367-401

Genetics. 1986;**20**:201-230

chronic kidney disease. Hippokratia. 2011;**15**(1):33-38

tion. Trends in Cardiovascular Medicine. 2015;**25**(4):267-274


[36] Chen NX, Moe SM. Vascular calcification: Pathophysiology and risk factors. Current Hypertension Reports. 2012;**14**(3):228-237

Duncker DJ, Danser AH, Hoeijmakers JH, Roks AJ. Nucleotide excision DNA repair is

associated with age-related vascular dysfunction. Circulation 2012;**126**:468-478

Oxford University Press; 2007

**10**(4):654-666

136 Free Radicals, Antioxidants and Diseases

1999;**100**:2168-2217

2002;**13**:220-225

Circulation Research. 2010;**107**(4):466-475

nition. Gene. 2004;**340**:275-282

2015;**35**(11):2384-2390

Médica MD. 2015;**5**(3):151-160

[25] Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 4th ed. Oxford:

[26] Vostálová J, Galandáková A, Strebl P, Zadražil J, Kajabová M, Schneiderka P. Oxidative

[27] Górriz JL, Molina P, Cerverón MJ, Vila R, Bover J, Nieto J, Barril G, Martínez-Castelao A, Fernández E, Escudero V, Piñera C, Adragao T, Navarro-Gonzalez JF, Molinero LM, Castro-Alonso C, Pallardó LM, Jamal SA. Vascular calcification in patients with nondialysis CKD over 3 years. Clinical Journal of the American Society of Nephrology. 2015;

[28] Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: Evidence for smooth muscle cell-mediated vascular calcification. Circulation.

[29] Osako MK, Nakagami H, Koibuchi N, Shimizu H, Nakagami F, Koriyama H, Shimamura M, Miyake T, Rakugi H, Morishita R. Estrogen inhibits vascular calcification via vascular RANKL system. Common mechanism of osteoporosis and vascular calcification.

[30] Giguère V. To ERR in the estrogen pathway. Trends in Endocrinology and Metabolism.

[31] Razzaque MA, Masuda N, Maeda Y, Endo Y, Tsukamoto T, Osumi T. Estrogen receptorrelated receptor gamma has an exceptionally broad specificity of DNA sequence recog-

[32] Zirngibl RA, Chan JS, Aubin JE. Estrogen receptor-related receptor alpha (ERRalpha) regulates osteopontin expression through a non-canonical ERRalpha response element in a cell context-dependent manner. Journal of Molecular Endocrinology. 2008;**40**:61-73

[33] Jeong BC, Lee YS, Park YY, Bae IH, Kim DK, Koo SH, Choi HR, Kim SH, Franceschi RT, Koh JT, Choi HS. The orphan nuclear receptor estrogen receptor-related receptor gamma negatively regulates BMP2-induced osteoblast differentiation and bone forma-

[34] Kim JH, Choi YK, Do JY, Choi YK, Ha CM, Lee SJ, Jeon JH, Lee WK, Choi HS, Park KG, Lee IK. Estrogen-related receptor γ plays a key role in vascular calcification through the Upregulation of BMP2 expression. Arteriosclerosis, Thrombosis, and Vascular Biology.

[35] Trujillo-Cuellar Hernando, Sierra-Lara Martínez J Daniel y Osorno-Solís Lucía. Alteraciones del metabolismo mineral y óseo asociadas a la enfermedad renal crónica. Revista

tion. The Journal of Biological Chemistry. 2009;**284**:14211-14218

stress in kidney disease patients. Vnitr̆ní Lékar̆ství. 2012;**58**(3):202-207

