**Tissue Occurrence of Carbonyl Products of Lipid Peroxidation and Their Role in Inflammatory Disease**

Maria Armida Rossi

278 Lipid Peroxidation

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51343

## **1. Introduction**

The lipid peroxidation is a diffuse process which regards the polyunsaturated fatty acids of lipids, when they are exposed to oxygen-derived free radicals.

The process occurs when oils or foods, vegetables or meats, or other materials are exposed to air and causes their alteration at least in part through the peroxidative decomposition of the fatty acids contained in their lipids.

The lipid peroxidation does not need the action of enzymes and brings to the progressive decomposition of the unsaturated fatty acids till to the formation of carbonylic end products, aldehydes and ketones.

Oxygen-derived free radicals can be produced by the effect of sun rays on O2, but an important source of them is the cellular metabolism, too.

The interest and the importance of the lipid peroxidation arise from the fact that the polyunsaturated fatty acids are contained in the phospholipids present in all cellular membranes; their structure and function can be strongly modified by this process.

The cellular effects of the lipid peroxidation change according to its degree. A high lipoperoxidative rate can produce serious damages to the cells and their death; on the contrary, a low degree of it allows cell survival and may modulate tissue metabolism.

## **2. Steps of the lipoperoxidative process**

The lipid peroxidation has many steps,as shown in the Figure 1. The process is started by the attach of free radicals to poly-unsaturated fatty acids of lipids. Free radicals are chemical

species which have a single, unpaired electron in an outer orbit. Their molecular configuration is unstable and so they react with the adjacent molecules to acquire a more stable configuration. The polyunsaturated fatty acids contained in the phospholipids of cell membranes are a good target for their reaction; in the attach of free radicals to the unsaturated fatty acids of lipids a methylen group near a double bond can give the electron required by the free radical to form the electon pair. So the unsaturated fatty acid has become a free radical and reacts with another molecule, starting the propagation phase which characterizes the lipoperoxidative process. In our cells the molecular oxygen is always present and can react with the lipid radical to form a lipoperoxide. This molecule has un unstable configuration too. The formed lipoperoxides react with adjacent membrane molecules, either other lipids or proteins. The reaction of a lipoperoxide with a protein molecule changes it in a reactive free radical; the so activated protein can interact with another protein to give a protein complex or interacts with lipids to form lipofuscin molecules. The presence of lipofuscin is frequent in the tissues of old people; this fact was well known by the anatomists already in the past century; the mechanism of their formation has been clarified with the discovery of the lipoperoxidative process. Beside the reaction with other molecules, the lipoperoxides can break to give more stable end products, aldehydes and ketones. These carbonylic end products of lipid peroxidation are formed above all in the microsomes where the rate of the lipid peroxidation is strong, but they can diffuse and react with various molecular targets both within the cell and outside it.

**Figure 1.** First steps of the lipid peroxidative process. 1) The free radical A abstracts an electron from a near molecule, which becomes a free radical; the target molecule is often a polyunsaturated fatty acid. 2) Its molecular configuration is unstable, so a shift of the double bond occurs. 3) This still unstable free radical binds O2 and becomes a peroxide. 4) The peroxide captures an electron from the molecule B and forms an hydro-peroxide. Now the molecule B is a free radical. The further fate of the hydroperoxide is its fragmentation in small carbonylic compounds (not shown iin the Figure)

The first reports of the actual occurrence of the lipid peroxidation in tissues include the researches separately carried on by Comporti M et al.[1] and by Recknagel RO and Ghoshal AK. [2] to explain the liver damage induced by the rat treatment with CCl4. Both these works used methods of investigation quite modern for those years and brought important findings to understand the structure and the functions of the different cell compartments: nucleus, mithochondria, microsomes, lysosomes. The further experimental studies on the steps and the effects of the lipid peroxidation have been deeply facilitated by Benedetti al. [3] who were able to develop a method to synthetize its carbonylic end-products, above all the aldehyde 4-hydroxy-2,3-trans-nonenal (HNE), whose chemical structure is shown in the Figure 2. HNE was shown to be produced in good amounts when the lipid peroxidation was stimulated; furthermore several experimental researches found that this aldehyde was the more cytotoxic end product of the lipoperoxidative process [4]. The first experimental works on the effects used millimolar concentrations of the aldehyde which were rather high; later the researchers found that it could display several biological effects at concentrations micromolar or less, which can be easily found in tissues even in normal conditions.

**Figure 2.** Structure of 4-hydroxy-2,3-trans-nonenal.

280 Lipid Peroxidation

species which have a single, unpaired electron in an outer orbit. Their molecular configuration is unstable and so they react with the adjacent molecules to acquire a more stable configuration. The polyunsaturated fatty acids contained in the phospholipids of cell membranes are a good target for their reaction; in the attach of free radicals to the unsaturated fatty acids of lipids a methylen group near a double bond can give the electron required by the free radical to form the electon pair. So the unsaturated fatty acid has become a free radical and reacts with another molecule, starting the propagation phase which characterizes the lipoperoxidative process. In our cells the molecular oxygen is always present and can react with the lipid radical to form a lipoperoxide. This molecule has un unstable configuration too. The formed lipoperoxides react with adjacent membrane molecules, either other lipids or proteins. The reaction of a lipoperoxide with a protein molecule changes it in a reactive free radical; the so activated protein can interact with another protein to give a protein complex or interacts with lipids to form lipofuscin molecules. The presence of lipofuscin is frequent in the tissues of old people; this fact was well known by the anatomists already in the past century; the mechanism of their formation has been clarified with the discovery of the lipoperoxidative process. Beside the reaction with other molecules, the lipoperoxides can break to give more stable end products, aldehydes and ketones. These carbonylic end products of lipid peroxidation are formed above all in the microsomes where the rate of the lipid peroxidation is strong, but they can

diffuse and react with various molecular targets both within the cell and outside it.

**Figure 1.** First steps of the lipid peroxidative process. 1) The free radical A

its fragmentation in small carbonylic compounds (not shown iin the Figure)

near molecule, which becomes a free radical; the target molecule is often a polyunsaturated fatty acid. 2) Its molecular configuration is unstable, so a shift of the double bond occurs. 3) This still unstable free radical binds O2 and becomes a peroxide. 4) The peroxide captures an electron from the molecule B and forms an hydro-peroxide. Now the molecule B is a free radical. The further fate of the hydroperoxide is

abstracts an electron from a

#### **3. Lipoperoxidative effects on cell compartments**

The damage to the different cell structures induced by the rat treatment with CCl4 are similar to the alterations found in different pathological processes and are followed by similar changes in the tissue metabolism. In fact all the cell structures, mithocondria, microsomes, lysosomes, nuclei, are delimitated by membranes where the lipid peroxidation can take place and cause damage, bringing to changes in their functions. a) Effects of the lipid peroxidation on microsomes. The action of toxic compounds on cells leads to a quite rapid swelling of rough and smooth endoplasmic reticulum; the ribosomes dissociate from the rough endoplasmic reticulum and the protein synthesis decreases within less than 30 minutes [5,6]. The inhibition of microsomal glucose-6-phosphatase activity is seen very early in the action of toxic compounds; afterwords the activity of several other enzymes (hexokinase, lactate dehydrogenase, alpha and beta polymerases, 5'nucleotidase) has been found to decrease under the effect of the end products of the lipid peroxidation. However the effects of the inhibition of the protein synthesis can be seen only after different hours because the cells have a reserve of preformed proteins which can be used. The microsomes are the site of the drug metabolizing enzyme system (d.m.e.s.) which metabolizes different compounds, either endogenous components, such as different hormones, or various xenobiotics. The result of the changes induced by the d.m.e.s. on a compound can be different: the compound can be inactivated, it can change its functions or it can even acquire a toxic action. . . . CCl4 has solvent properties in high amounts, but much smaller quantities can induce biological toxic effects through its homolitical cleavage catalysed by the d.m.e.s. In fact CCl4 fission generates free radicals able to trigger the lipid peroxidative process, starting from microsomal membranes. In the rats intoxicated with CCl4 an early effect is the decrease of the hepatic content of cytochrome P450, which is part of the d.m.e.s., the enzyme system which metabolizes the haloalkane, generating the free radicals responsible of many of its dangerous effects. The lipid peroxidation increases strongly in the liver of rats treated with this haloalkane; it is started by the free radicals generated by CCl4 fission in the microsomes. The decrease of the cytochrome P450 and the damage to the liver endoplasmic reticulum lead to an apparent and quite interesting "paradox", shown by Ugazio et al. [7], i.e. the pre-treatment with a sublethal dose of CCl4 protects the rats from the subsequent administration of a higher, potentially lethal dose. In fact the pretrearment impairs the hepatic microsome metabolic ability and so the subsequent haloalkane dose is less metabolized and it is unable to cause a serious liver damage and the animal death. A single, non lethal dose, of CCl4 induces fatty liver in rats; if the treatmemt is unrepeated this degenerative process can be reversible and the hepatic tissue returns to a normal anatomic aspect and to its usual physiological functions. The demonstration that the toxicity of small doses of this haloalkane was not due to its solvent properties but was the consequence of its cleavage by the d.m.e.s underlined the importance of the interaction between the various xenobiotics, foods or drugs or air inquinants, and the living organism, human or animal. A different behaviour of the cell metabolism due to genetic factors or to different other causes, such as diseases or the assumption of various substances, can higly modify the response to xenobiotics and their effects on the health. An important step in the studies on CCl4 toxicity was the finding that the pre-treatment of rats with antioxidants (DPPD, GSH, propyl gallate) could prevent them both from liver damage and cell death, suggesting the role of an oxidative mechanism in the development of its toxic action [8,9]. b) Lipoperoxidative effects on mitochondria. The effects of the lipid peroxidation induced by toxic compounds can be seen also in mitochondria which show a swelling and a change in ATP synthesis [9,10]. The studies with the electron microscope revealed the damage to mitochondrial components. In the first phases of the mitochondrial swelling the production of ATP can increase for an easier entry of the substrates in the organelle through the more permeable membrane, then it decreases and stops. In many intoxication both the decrease of the synthesis of ATP and the damage of the plasma membrane contribute to an increase of the Ca2+ influx in cells, which comes before the cell necrosis. [11]. Ca2+ concentration is strictly checked in cells; the cytosolic free calcium is maintained at concentrations < 0.1 μM , which are much lower than the extracellular ones. This control is very important because an increase in Ca2\* activates several enzymes (ATPases, phospholipases, proteases, endonucleases) which can damage the same cell structures. c) Effects on lysosomes. The lysosomes are damaged by the attack of free radicals and by the onset of the lipid peroxidation in their membrane (12,13); so their lytic enzymes can be released in the cytoplasm. In the injured cells the intracellular pH tend to be acid; so the released lysosomal enzymes can be activated and destroy important cell components. The damage of the lysosomal membrane can lead to the enzymatic digestion of proteins, RNA, DNA and the cell dies by necrosis. The occurrence of the lipid peroxidation in lysosomes can also lead to the inactivation of their lytic enzymes if the lipoperoxidative rate is very high; Krohne et al. [14] have shown that the lipid peroxidaction end products, HNE and malonaldehyde (MDA), inactivated lysosomal cysteine proteases by covalent binding to their active center. d) Lipid peroxidation-induced changes in the nucleus. The cell nucleus has a membrane, like the other cell organelles; if the lipid peroxidation occurs in the nuclear membrane, it can cause serious damage. The nuclear importance is due to the presence of the DNA molecule; a damage to the DNA can lead to alterations in the codified proteins. If these changes involve important sites of the molecule, the protein can be no more functional. Some alterations in the DNA molecule are lethal, others lead to vital, but modified cells. Some changes in the DNA molecule can bring to the generation of transformed cells which show different changes in their morphology, metabolism and behaviour toward the near cells. The reaction of the different products of the lipid peroxidation with DNA has been extensively studied [15,16]; it can lead to the formation of adducts to DNA bases, which have profound mutagenic potential. The alterations of DNA molecule are believed to be important in the pathogenesis of cancer; a special attention has been given to the oncogenes and antioncogenes, which play an important role in regulating cell division.

#### **4. Effects of lipid peroxidation in inflammation**

282 Lipid Peroxidation

in the action of toxic compounds; afterwords the activity of several other enzymes (hexokinase, lactate dehydrogenase, alpha and beta polymerases, 5'nucleotidase) has been found to decrease under the effect of the end products of the lipid peroxidation. However the effects of the inhibition of the protein synthesis can be seen only after different hours because the cells have a reserve of preformed proteins which can be used. The microsomes are the site of the drug metabolizing enzyme system (d.m.e.s.) which metabolizes different compounds, either endogenous components, such as different hormones, or various xenobiotics. The result of the changes induced by the d.m.e.s. on a compound can be different: the compound can be inactivated, it can change its functions or it can even acquire a toxic action. . . . CCl4 has solvent properties in high amounts, but much smaller quantities can induce biological toxic effects through its homolitical cleavage catalysed by the d.m.e.s. In fact CCl4 fission generates free radicals able to trigger the lipid peroxidative process, starting from microsomal membranes. In the rats intoxicated with CCl4 an early effect is the decrease of the hepatic content of cytochrome P450, which is part of the d.m.e.s., the enzyme system which metabolizes the haloalkane, generating the free radicals responsible of many of its dangerous effects. The lipid peroxidation increases strongly in the liver of rats treated with this haloalkane; it is started by the free radicals generated by CCl4 fission in the microsomes. The decrease of the cytochrome P450 and the damage to the liver endoplasmic reticulum lead to an apparent and quite interesting "paradox", shown by Ugazio et al. [7], i.e. the pre-treatment with a sublethal dose of CCl4 protects the rats from the subsequent administration of a higher, potentially lethal dose. In fact the pretrearment impairs the hepatic microsome metabolic ability and so the subsequent haloalkane dose is less metabolized and it is unable to cause a serious liver damage and the animal death. A single, non lethal dose, of CCl4 induces fatty liver in rats; if the treatmemt is unrepeated this degenerative process can be reversible and the hepatic tissue returns to a normal anatomic aspect and to its usual physiological functions. The demonstration that the toxicity of small doses of this haloalkane was not due to its solvent properties but was the consequence of its cleavage by the d.m.e.s underlined the importance of the interaction between the various xenobiotics, foods or drugs or air inquinants, and the living organism, human or animal. A different behaviour of the cell metabolism due to genetic factors or to different other causes, such as diseases or the assumption of various substances, can higly modify the response to xenobiotics and their effects on the health. An important step in the studies on CCl4 toxicity was the finding that the pre-treatment of rats with antioxidants (DPPD, GSH, propyl gallate) could prevent them both from liver damage and cell death, suggesting the role of an oxidative mechanism in the development of its toxic action [8,9]. b) Lipoperoxidative effects on mitochondria. The effects of the lipid peroxidation induced by toxic compounds can be seen also in mitochondria which show a swelling and a change in ATP synthesis [9,10]. The studies with the electron microscope revealed the damage to mitochondrial components. In the first phases of the mitochondrial swelling the production of ATP can increase for an easier entry of the substrates in the organelle through the more permeable membrane, then it decreases and stops. In many intoxication both the decrease of the synthesis of ATP and the damage of the plasma membrane contribute to an increase of the Ca2+ influx in cells, which comes before the cell necrosis. [11]. Ca2+ concentration is strictly checked in cells; the

The lipid peroxidation plays an important role in inflammation; in this process its presence is constant and its degree can reach high values.

Inflammation is the local response to any tissue damage. It is characterized by two main events: 1. changes in the blood flow in the microcirculation of the injured site. 2. recruitment of leukocytes, neutrophils and monocyte-macrophages; these cells phagocyte and destroy the agents of the tissue injury: bacteria, virus, parasites, dead cells, tissue debris. The leukocytes which gain the damaged tissue are activated by cytokines (IL-1, IL-6, TNF, MCP-1), which trigger the respiratory or phagocytic burst in them. This process is characterized by a strong increase of the consumption of oxygen, which is used to produce the superoxide anion (O2 ); its synthesis is catalysed by the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase [17]. The NADPH oxidase is formed by a complex of proteins which are located both in the plasma membrane and in the cytoplasm in the resting neutrophil. When the neutrophil is activated by different stimuli (the phagocytosis itself, various cytokines), the components of the NADPH oxidase assemble on the membrane of the phagosome and the enzymatic complex can reduce oxygen to superoxide anion as shown in the following reaction:

**Figure 3.** List of the principal chemotactic compounds.

The superoxide anion is a free radical and so it is highly reactive.Two molecules of superoxide anion can react together and form the hydrogen peroxide (H2O2); this molecule has a low bactericidal power and it is also used as a disinfectant in pharmacology. In the tissues the hydrogen peroxide is used in a reaction catalysed by the myeloperoxidase (MPO) to form hypochlorite (OCl ). The microbial power of the hypochlorite is very strong; furthermore it can oxidase protein and lipids and so it can trigger the lipid peroxidation. The hydrogen peroxide can also be converted to the hydroxyl radical (OH ), a free radical with a very short lifetime; in fact it reacts with the nearest molecule to acquire a more stable configuration. Both the anion superoxide and the hydroxyl radical are able to induce the lipid peroxidation and this fact explains its steady presence in inflammation. The occurrence of the lipoperoxidative process may lead to a worsening of the tissue damage, but it also contributes to the recruitment of leukocytes, both neutrophils, and monocyte-macrophages since some lipid peroxidation end products display a chemotactic power, as shown in the Figure 2. The migration of leukocytes from blood to the inflammed tissue requires several passages [18]. Both the leukocytes and the endothelial cells need the presence of adhesion molecules on their surface to allow the leukocyte binding to the microcirculation of the damaged tissue. At first the binding is not firm and allows the leukocyte rolling on the endothelial surface; afterwards it becomes very strong and this firm adhesion is followed by the leukocyte passage outside the blood vessels to gain the site of the inflammation.

284 Lipid Peroxidation

reaction:

**Figure 3.** List of the principal chemotactic compounds.

to form hypochlorite (OCl

the components of the NADPH oxidase assemble on the membrane of the phagosome and the enzymatic complex can reduce oxygen to superoxide anion as shown in the following

NADPH + O2 NADP+ + O2

The superoxide anion is a free radical and so it is highly reactive.Two molecules of superoxide anion can react together and form the hydrogen peroxide (H2O2); this molecule has a low bactericidal power and it is also used as a disinfectant in pharmacology. In the tissues the hydrogen peroxide is used in a reaction catalysed by the myeloperoxidase (MPO)

furthermore it can oxidase protein and lipids and so it can trigger the lipid peroxidation.

with a very short lifetime; in fact it reacts with the nearest molecule to acquire a more stable configuration. Both the anion superoxide and the hydroxyl radical are able to induce the lipid peroxidation and this fact explains its steady presence in inflammation. The occurrence of the lipoperoxidative process may lead to a worsening of the tissue damage, but it also contributes to the recruitment of leukocytes, both neutrophils, and monocyte-macrophages since some lipid peroxidation end products display a chemotactic power, as shown in the Figure 2. The migration of leukocytes from blood to the inflammed tissue requires several

The hydrogen peroxide can also be converted to the hydroxyl radical (OH

). The microbial power of the hypochlorite is very strong;

), a free radical

 

> The chemotactic compounds or chemotaxins display different actions on the leukocytes. The term "chemotaxis" refers to the ability of a molecule to stimulate the oriented migration of a cell in the presence of a chemical gradient of the chemotactic compound or chemotaxin; the leukocytes have specific receptors for the different chemotaxins and move toward the site where the chemotaxins have the highest concentration. Beside this property, the chemotactic compounds display many other functions on the leukocytes: they induce the phagocyte burst, activate the adhesion molecules which are expressed on the plasmamembrane of the neutrophils, promote the synthesis of different cytokines, expecially by the macrophages.

> a. Chemotactic activity of the products of the lipid peroxidation. The lipid peroxidation end product HNE has been shown to display a chemotactic power toward the polymorphonuclear leukocytes. At first this property was found by Curzio et al. (19) on rat neutrophils; the chemotactic concentrations of this aldehyde ranged from 10 μM to 0.1 μM. These doses are rather low and are devoid of any cytotoxic property. HNE chemotactic activity was initially demonstrated "in vitro" by the use of a Boyden chamber. This chamber has two compartments separated by a filter made of a mixture of cellulose esters with a pore size of 3 μ; the cells are placed in the upper chamber, the solution containing the substances to be tested in the lower one. The so mounted chamber is incubated at 37ºC for 75 min; then the chamber is removed and opened; the filter is removed, fixed in ethanol and stained with haematoxylin. The cell migration can be evaluated under the light microscope by the leading front technique. The first demonstration of HNE chemotactic power was obtained "in vitro", but afterwards it was confirmed by Schaur et al. [20] who carried on "in vivo" experimental researches. They induced an aseptic inflammation in the subcutaneous tissue of a rat leg by injecting in it some polydextrane Sephadex G-200; in control rats they inoculated Sephadex alone, while in the experimental group of rats they inoculated Sephadex together with a solution of preformed HNE. When they examinaed the histological samples obtained from the two groups of rats, they found the migration of neutrophils in both of them, but their number was much more higher around the Sephadex plus HNE. In their experimental researches the authors excluded the presence of any cytotoxic effects by the aldehyde concentrations able to stimulate the oriented migration of the neutrophils. Beside HNE, other 4-hydroxy-alkenals have been shown to display a chemotactic activity toward rat neutrophils: 4-hydroxy-2,3-hexenal(HEE) and 4 hydroxy-2,3-octenal(HOE). HOE was the most active of the lipoperoxidative end products; it could stimulate the oriented migration of neutrophils even at very low concentrations [21] between 10-11 and 10-8 M. Most chemotactic compounds can activate a phosphoinositide specific phospholipase C (PL-C) [22]; their stimulation of PL-C activity is mediated by a regulatory G protein and leads to the production of

diacylglycerol and inositol-1,4,5-tris-phosphate (Ins-P3). The diacylglycerol activates the protein kinase C and the Ins-P3 promotes the mobilization of Ca++ from intracellular stores. The well known chemotaxin N-formylmethionyl-leucyl-phenylalanine (fMLP) increases the PL-C activity of neutrophils and its action is prevented by the cell pretreatment with pertussis toxin, which ADP ribosylates the alpha subunit of some G proteins. The chemotactic 4-hydroxy-alkenals formed by the lipid peroxidation have been found to activate the PL-C [23] of rat neutrophils and a good correspondence could be found between the concentrations able to increase the PL-C activity and those which regulated the cell migration. The pretreatment of neutrophils with pertussis toxin prevented the activation of PL-C by HOE, too; this finding suggested that its mechanism of action was like that of other well known chemotaxins. This discovery of the stimulation of an enzyme activity by very low doses of 4-hydroxyalkenals represented a clean change in the evaluation of the lipoperoxidative process and of the functions of its end-products. The first experimental studies on the biological effects of the lipid peroxidation supplied a lot of proofs about the inhibition of several enzymes in tissues where the lipid peroxidation rate was stimulated [5] or about the decrease of their activity in tissue homogenates or in subcellular fractions incubated in the presence of high concentrations of HNE [24].


arachidonic acid by two cyclooxigenases, COX-1 and COX-2; the COX-1 is constitutively, while the COX-2 is inducible. The COX-2 js present in leukocytes and mastzellen and is induced by different mediators of inflammation. HNE has been shown to induce the synthesis of COX-2 [27] too; this finding underlines the importance of the lipid peroxidation role in inflammation.

## **5. Positive and negative actions of inflammation**

286 Lipid Peroxidation

of high concentrations of HNE [24].

release of IL-8.

diacylglycerol and inositol-1,4,5-tris-phosphate (Ins-P3). The diacylglycerol activates the protein kinase C and the Ins-P3 promotes the mobilization of Ca++ from intracellular stores. The well known chemotaxin N-formylmethionyl-leucyl-phenylalanine (fMLP) increases the PL-C activity of neutrophils and its action is prevented by the cell pretreatment with pertussis toxin, which ADP ribosylates the alpha subunit of some G proteins. The chemotactic 4-hydroxy-alkenals formed by the lipid peroxidation have been found to activate the PL-C [23] of rat neutrophils and a good correspondence could be found between the concentrations able to increase the PL-C activity and those which regulated the cell migration. The pretreatment of neutrophils with pertussis toxin prevented the activation of PL-C by HOE, too; this finding suggested that its mechanism of action was like that of other well known chemotaxins. This discovery of the stimulation of an enzyme activity by very low doses of 4-hydroxyalkenals represented a clean change in the evaluation of the lipoperoxidative process and of the functions of its end-products. The first experimental studies on the biological effects of the lipid peroxidation supplied a lot of proofs about the inhibition of several enzymes in tissues where the lipid peroxidation rate was stimulated [5] or about the decrease of their activity in tissue homogenates or in subcellular fractions incubated in the presence

b. Activation of the exocytosis by 4-hydroxynonenal. HNE was found to induce the exocytosis in DMSO-differentiated HL-60 cells. [25] This human promyelocitic cell line was chosen because it could be induced to differentiate toward the granulocytic cell line and therefore it represented a good in vitro model to study the mechanism of action of a chemotactic compound, like 4-hydroxynonenal. The exocytosis was valued by measuring the secretion of ß-glucuronidase, an enzyme of the azure granules, by the cells incubated in the presence of different HNE concentrations. The exocytosis was triggered by HNE doses between 10-8 and 10-6 M, which are wholly devoid of any cytotoxic power. The lack of any effect on the cell viability was checked by measuring the release of lactate dehydrogenase (LDH) in the cells incubated at 37°C for 1 hour in the presence of different HNE concentrations; the presence of HNE between 0.01 and 1.0 μM failed to induce any

c. Stimulation of IL-8 release by 4-hydroxynonenal. I recently found HNE ability to change the release of the chemokine interleukin-8 (IL-8) in DMSO–differentiated HL-60 cells [26]; the aldeyde failed to modify the intracellular concentration of IL-8, but after 30 min of incubation it began to enhance the chemokine release. The increase of IL-8 level in the cell suspensions incubated in the presence of HNE was quite slow and became remarkable only after 1 h. This fact suggested that the effects shown by the aldehyde both on the chemotaxis and on the exocytosis were not mediated through the

d. 4-hydroxynonenal induced synthesis of cyclooxygenase-2. The vascular reactions of inflammation are regulated by many chemicals mediators; among them the prostaglandins influence several cell functions. The prostaglandins play an important role in inflammation; above all the PGE2 and the PGD2 induce vasodilation and increase the permeability of post-capillary venules. These prostaglandins are produced from

increase of the enzyme loss by the cells in the incubation period [25].

Inflammation has many positive effects and it is considered a defensive response of the organism, but it is followed by negative aspects which may contribute to increase the tissue damage, as shown in Figure 3.

The leukocytes which reach a damaged tissue can remove the injurious agents. They can phagocyte and kill the microrganisms of an infectious disease; they also phagocyte the dead cells or the cell debris which are left in any damaged tissue.

**Figure 4.** Main events in inflammation.

The Figure 3 underlines that the positive functions of the leukocytes are also followed by unpleasant effects, which can be caused both by the phagocytic burst and by the exocytosis. The phagocytic burst leads to the generation of free radicals and reactive oxygen species (ROS) which can diffuse outside the phagocytic cells and amplify the effects of the initial injurious agent. Moreover the induction of the lipid peroxidation by the ROS and the free radicals can worsen the tissue damage. . The exocytosis is a kind of physiological, controlled secretion of lysosomal enzymes by neutrophils and is activated by several chemotactic agent; their azurofil granules fuse with the plasma membrane and release their content in the extracellular space. In this way different lytic enzymes can diffuse in the inflammed tissue; the blood stasis which is always present in the late phases of the inflammatory process favours the lowering of pH in the damaged tissue; this fact leads to the activation of the released lysosomal enzymes and the tissue itself can be damaged.

## **6. Action of lipid peroxidation on atherosclerosis**

Medical progress has brought good successes against many diseases in the past century. Antibiotics can win many infectious agents; the progress in surgery can correct cardiac malformations; the consequences of a vessel obstruction can be obviated by the insertion of a by pass.

In our times, when the life is becoming longer, the atherosclerosis represents a serious problem which can compromise the life of many people. The complications of the atherosclerosis are becoming the main causes of death.

Its pathological lesion is the atheroma, which is localized in the arteries of big and medium calibre: aorta, carotids, coronaries.

The atheroma is characterized by an accumulation of cholesterol and cholesterol esters both inside and outside the cells. It contains lipid-loaded cells, called foam cells. They are thought to derive from monocytes or smooth muscular cells, which have migrated in the arterial intima and have been engulfed by oxidized LDL. The lipid peroxidation plays an important role in the pathogenesis of this diffuse process through its intervent in LDL oxidation.

The modification of LDL by oxidation leads to its unregulated uptake by intimal macrophages to form foam cells[28]. In the oxidation of LDL the lipid peroxidation is stimulated and it contributes to modify their apolipoprotein. HNE, the major lipid peroxidation end product is formed also in the process of LDL oxidation and is present in the oxidized-LDL. Esterbauer et al. found that the aldehyde alone could modify the LDL. He incubated native LDL in the presence of different HNE concentrations and observed its covalent binding to the apolipoprotein B with the blockage of the epsilon-amino groups on lysine residues. Both the modification of LDL by oxidation and its modification by HNE binding were associated with an increased degradation by macrophages and a lipid loading of them.

The migration of macrophages toward the arterial intima is stimulated by chemotactic compounds, like the migration of leukocytes to a site of inflammation. The oxidized LDL have been shown to stimulate the synthesis of the monocyte chemotactic protein-1 (MCP-1) by macrophages [29]. This cytokine has a chemotactic power specific for the monocytemacrophages; however the macrophage recruitment in the atheroma could be also favoured by the 4-hydroxyalkenals which have been found in the oxidized LDL: 4-hydrohexenal (HEE), 4-hydroxyoctenal (HOE) and 4-hydroxynonenal (HNE). These aldehydes are lipid peroxidation end products which display both a cytotoxic and a chemotactic power. They are likely to be produced by the oxidation of the LDL which have reached the intima of arteries and can contribute to the recruitment of monocyte-macrophages[30]; a direct cytotoxic effect on the foam cells of the atheroma was considered unlikely by Muller because it required higher levels of the aldehydes.

## **7. The lipid peroxidation role in ischemia-reperfusion.**

The onset of the lipid peroxidation in a tissue requires the presence of molecular oxygen; however the ischemia can induce changes of the cell metabolism which may increase the tissue damage if the blood supply returns. This unexpected fact happens in the ischemiareperfusion[31]. During ischemia the lack of oxygen causes the catabolism of ATP with an increased production of ipoxantine. which is an oxidable substrate for the xanthine dehydrogenase. Moreover in the ischemic tissue there is the conversion of the native xanthine dehydrogenase to a superoxide producing-oxidase; this conversion is thought to be produced by a calcium triggered protease. In the reperfusion the O2 which reaches the tissue is transformed by the xanthine oxidase in superoxide anion; this free radical contribute to extend the tissue damage induced by the ischemia.

The tissue necrosis triggers an inflammatory process and the leukocytes which can reach the tissue in the reperfusion can worsen the tissue damage through the production of ROS and the release of lytic enzymes.

## **8. Conclusions**

288 Lipid Peroxidation

a by pass.

oxidation.

of them.

**6. Action of lipid peroxidation on atherosclerosis** 

atherosclerosis are becoming the main causes of death.

calibre: aorta, carotids, coronaries.

it required higher levels of the aldehydes.

Medical progress has brought good successes against many diseases in the past century. Antibiotics can win many infectious agents; the progress in surgery can correct cardiac malformations; the consequences of a vessel obstruction can be obviated by the insertion of

In our times, when the life is becoming longer, the atherosclerosis represents a serious problem which can compromise the life of many people. The complications of the

Its pathological lesion is the atheroma, which is localized in the arteries of big and medium

The atheroma is characterized by an accumulation of cholesterol and cholesterol esters both inside and outside the cells. It contains lipid-loaded cells, called foam cells. They are thought to derive from monocytes or smooth muscular cells, which have migrated in the arterial intima and have been engulfed by oxidized LDL. The lipid peroxidation plays an important role in the pathogenesis of this diffuse process through its intervent in LDL

The modification of LDL by oxidation leads to its unregulated uptake by intimal macrophages to form foam cells[28]. In the oxidation of LDL the lipid peroxidation is stimulated and it contributes to modify their apolipoprotein. HNE, the major lipid peroxidation end product is formed also in the process of LDL oxidation and is present in the oxidized-LDL. Esterbauer et al. found that the aldehyde alone could modify the LDL. He incubated native LDL in the presence of different HNE concentrations and observed its covalent binding to the apolipoprotein B with the blockage of the epsilon-amino groups on lysine residues. Both the modification of LDL by oxidation and its modification by HNE binding were associated with an increased degradation by macrophages and a lipid loading

The migration of macrophages toward the arterial intima is stimulated by chemotactic compounds, like the migration of leukocytes to a site of inflammation. The oxidized LDL have been shown to stimulate the synthesis of the monocyte chemotactic protein-1 (MCP-1) by macrophages [29]. This cytokine has a chemotactic power specific for the monocytemacrophages; however the macrophage recruitment in the atheroma could be also favoured by the 4-hydroxyalkenals which have been found in the oxidized LDL: 4-hydrohexenal (HEE), 4-hydroxyoctenal (HOE) and 4-hydroxynonenal (HNE). These aldehydes are lipid peroxidation end products which display both a cytotoxic and a chemotactic power. They are likely to be produced by the oxidation of the LDL which have reached the intima of arteries and can contribute to the recruitment of monocyte-macrophages[30]; a direct cytotoxic effect on the foam cells of the atheroma was considered unlikely by Muller because Tne lipid peroxidation can be regarded as a common process which happens in our cells. In fact low levels of the lipid peroxidation end products have been shown in tissues even in normal conditions [20]. The rate of the lipid peroxidation can be stimulated by the ROS and the free radicals which can arise also from the normal metabolism of cells.

The lipid peroxidation rate can be increased by some xenobiotics; the experimental works about the action of the haloalkane CCl4 have been the source of the first explanations of its effects [1-3].

In any inflammation the leukocytes, above all the neutrophils and the macrophages, produce the superoxide anion and other free radicals, which can increase the lipoperoxidative rate[18]. A high degree of the lipid peroxidation is followed by some unavoidable damages to the tissue; some lesions can be reversible and can be repaired by the normal reparative process or by the aid of a pharmachological support; however a low alteration of the tissue integrity and function can follow any inflammatory event .

The aging is viewed by some authors [32] as the sum of the repeated tissue damages which occur in our life and the lipid peroxidation can take a part in them.

Another aspect of the lipid peroxidation regards its possible modulation of the normal metabolism; low concentrations of HNE, the major lipid peroxidation product, can modulate the activity of some enzymes, like the phosphoinositide-dependent phospholipase C [23] and several other enzymes [24].

#### **Author details**

Maria Armida Rossi *Department of Experimental Medicine and Oncology, University of Turin, Turin, Italy* 

### **Acknowledgement**

The Author is grateful to his teacher, the Prof. Mario Umberto Dianzani (University of Turin, Turin, Italy) and to the late Prof. Hermann Esterbauer (University of Gratz, Gratz, Austria).

#### **9. References**


Austria).

**9. References** 

**Acknowledgement** 

1965;29(3)185-204

Biophys Acta 1980; 620(2), 281-96

triphosphate. Nature 1964; 204,1196-7

rats. Biochem J. 1955; 59(1) 141-5

Biol Chem. 1983; 258(22) 13733-7

Lipids 1973; 8(4)177-82

353-6

22

The Author is grateful to his teacher, the Prof. Mario Umberto Dianzani (University of Turin, Turin, Italy) and to the late Prof. Hermann Esterbauer (University of Gratz, Gratz,

[1] Comporti M, Saccocci C, Dianzani MU. Effect of CCl4 in vitro and in vivo on lipid peroxidation of rat liver homogenates and subcellular fractions. Enzymologia

[2] Recknagel RO, Ghoshal AK. Lipoperoxidation of rat liver microsomal lipids induced by

[3] Benedetti A, Comporti M, Esterbauer H. Identification of 4-hydroxynonenal as a cytotoxic product originating from the production of liver microsomal lipids. Biochim

[4] Esterbauer H, Schaur RJ, Zollner H. Chemistry and bio-chemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11(9) 81-128 [5] Glende EA, Hruszkawycz AM, Recknagel RO. Critical role of lipid peroxidation in carbon tetrachloride-induced loss of aminopyrine demethylase, cytochrome P-450 and

[6] Bidlack WR, Tappel AL. Damage to microsomal membrane by lipid peroxidation.

[7] Ugazio G, Torrielli MV, Burdino E, Sawyer BC, Slater TF. Long range effects of products of carbon tetrachloride-stimulated lipid peroxidation. Biochem Soc Trans. 1976; 4(2)

[8] Dianzani MU, Ugazio G. Lipoperoxidation after carbon tetrachloride poisoning in rats

[9] Christophersen BO. The inhibitory effect of reduced gluthathione on the lipid peroxidation of the microsomal fraction and mitochondria. Biochem J. 1968; 106(2) 515-

[10] Hyams DE, Issebalbacher KJ. Prevention of fatty liver by administration of adenosine

[11] Dianzani MU, Viti I. The content and distribution of cyto-chrome c in the fatty liver of

[12] Yoshikawa T, Yokoe N, Takamura S, Kato A, HottaT, Matsumura N, Ikezaki M, Hosokawa K, Kondo M. Lipid peroxidation and lysosomal enzymes in D-galactosamine

[13] Mak IT. Misra HP,,Weglicki WB. Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes. J

[14] Krohne TU, Kaemmer E, Hotz EG, Kopitz J. Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment. J Exp Eye Res. 2010; 90(2) 261-6

hepatitis and its protection by vitamin E. Gastroenterol Jpn. 1979; 14(1) 31-9

previously treated with antioxidants. Chem Biol Interact. 1973; 6(2) 67-79

carbon tetrachloride. Nature 1966;210(5041) 1162-63

glucose-6-phosphatase. Biochem Pharmacol. 1976;25(19) 2163-70



## **The Role of Physical Exercise on Lipid Peroxidation in Diabetic Complications**

Yaşar Gül Özkaya

292 Lipid Peroxidation

95

[31] McCord JM, Roy RS, Schaffer SW. Free radicals and myocardial ischemia. The role of

[32] Balaban RS, Nemoto S, Finkel T. Mitochondria,oxidants and aging. Cell 2005,120(4) 483-

xanthina oxidase. Adv Myocardiol. 1985;5:183-9

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46182

## **1. Introduction**

Diabetes mellitus is a group of metabolic disorder characterized by hyperglicemia and insufficiency of action or secretion of insulin. More than 346 million people worldwide have diabetes. 80 per cent of diabetes-induced deaths ocur in low- and middle-income countries. Most people with diabetes are above the age of retirement in developed countries, whereas in developing countries those most frequently affected are aged between 35 and 64 [1]. Although the etiology of this disease is not well defined, viral infections, autoimmunity, genetic and environmental factors have been implicated [2-5]. Four major types of diabetes have been defined by the American Diabetes Association (ADA): type 1 diabetes, type 2 diabetes, other spesific types of diabetes and gestational diabetes mellitus (GDM) [6].

Type 1 diabetes (T1D) usually develops in childhood and adolescence and the cause of the disease is an absolute deficiency of insulin secretion. Individuals at increased risk of developing this type of diabetes can often be identified by serological evidence of an autoimmune pathologic process occurring in the pancreatic islets and by genetic markers [6].

Type 2 diabetes (T2D) usually develops in adulthood and is related to obesity, lack of physical activity, and unhealthy diets. This is the more common type of diabetes (representing 90% of diabetic cases worldwide) and the cause is a combination of resistance to insulin action and an inadequate compensatory insulin secretory response. In Type 2 diabetes, a degree of hyperglycemia sufficient to cause pathologic and functional changes in various target tissues, but without clinical symptoms, may be present for a long period of time before diabetes is detected. During this asymptomatic period, it is possible to demonstrate an abnormality in carbohydrate metabolism by measurement of plasma glucose in the fasting state or after a challenge with an oral glucose load.

© 2012 Özkaya, licensee InTech. This is an open access chapter 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. © 2012 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.

The third category "other spesific types of diabetes" includes diabetes caused by a spesific and identified underlying defect, such as genetic syndromes, acquired processes such as pancreatitis, diseases such as cystic fibrosis, exposure to certain drugs, viruses, and unknown causes. Gestational diabetes is a state of hyperglicemia which develops during pregnancy [6].

Currently, ADA recommends the use of any of the following four criteria for diagnosing diabetes: 1) glycated hemoglobin (A1c) value of 6.5% or higher, 2) fasting plasma glucose ≥ 126 mg.dL-1 (7.0 mmol.L-1), 3) 2-h plasma glucose ≥ 200 mg.dL-1 (11.1 mmol.L-1) during an oral glucose tolerance test using 75 g of glucose, and/or 4) classic symptoms of hyperglycemia (e.g., polyuria, polydipsia, and unexplained weight loss) or hyperglycemic crisis with a random plasma glucose of 200 mg.dL-1 (11.1 mmol.L-1) or higher. In the absence of unequivocal hyperglycemia, the first three criteria should be confirmed by repeat testing [6].

Hyperglycaemia and hyperlipidaemia are key promoters of diabetes dysmetabolism, namely, through the formation of reactive oxygen species (ROS) and advanced glycation end products (AGEs), which causes cell damage and insulin resistance [7-9]. Moreover, both of them stimulate proinflammatory cytokines, thus contributing to *β*-cell degradation, particularly due to apoptosis pathways [10].

Increased oxidative stress is a widely accepted participant in the development and progression of diabetes and its complications [11-13]. Diabetes is usually accompanied by increased production of free radicals [14,15] or impaired antioxidant defenses [16]. Mechanisms by which increased oxidative stress is involved in the diabetic complications are partly known, including activation of transcription factors, advanced glycated end products (AGEs) [2], and protein kinase C [17].

Modern medical care uses a vast array of lifestyle and pharmaceutical interventions aimed at preventing and controlling hyperglycemia. In addition to ensuring the adequate delivery of glucose to the tissues of the body, treatment of diabetes attempts to decrease the likelihood that the tissues of the body are harmed by hyperglycemia. The importance of protecting the body from hyperglycemia cannot be overstated; the direct and indirect effects on the human vascular tree are the major source of morbidity and mortality in both type 1 and type 2 diabetes. Generally, the injurious effects of hyperglycemia are separated into macrovascular complications (coronary artery disease, peripheral arterial disease, and stroke) and microvascular complications (diabetic nephropathy, neuropathy, and retinopathy) [11].

Physical activity (PA) and diet are cornerstones of diabetes therapy [19]. Physical activity is a multifaceted behavior of which exercise is just one component. PA is defined as "bodily movement produced by the contraction of skeletal muscle that substantially increases energy expenditure'' and exercise is defined as ''a subset of PA done with the intention of developing physical fitness (i.e., cardiovascular, strength, and flexibility training).'' [19]. In this chapter, PA and exercise is used interchangeably.

In last decades, an impressive body of research has accumulated that demonstrates the varied benefits of regular physical activity for people with type 1 or type 2 diabetes [20]. Notably, exercise has been shown to improve glycemic control, reduce the need for insulin and oral hypoglycemic agents, and improve body weight control. Exercise has been shown to promote beneficial effects on insulin resistance, both in humans and in rodent models of T2DM [21, 22]. Moreover, exercise has myriad benefits for all people beyond those relating to diabetes alone. It can work wonders for the heart, improving the lipid profile, reducing risk for heart disease, restoring function after a heart attack, and moderating blood pressure. It helps in maintaining bone health regardless of age, it can significantly relieve depression and anxiety, and it appears to help maintain cognitive function in old age [23, 24]. A correlation between the effects of acute and chronic aerobic exercise upon oxidative stress and inflammation and the diabetic dysmetabolism has been previously described [25-27].

This chapter focuses on recent clinical and experimental studies of diabetes and exercise interventions done within the context of lipid peroxidation.

## **2. Lipid peroxidation and diabetic complications**

294 Lipid Peroxidation

pregnancy [6].

retinopathy) [11].

particularly due to apoptosis pathways [10].

products (AGEs) [2], and protein kinase C [17].

this chapter, PA and exercise is used interchangeably.

[6].

The third category "other spesific types of diabetes" includes diabetes caused by a spesific and identified underlying defect, such as genetic syndromes, acquired processes such as pancreatitis, diseases such as cystic fibrosis, exposure to certain drugs, viruses, and unknown causes. Gestational diabetes is a state of hyperglicemia which develops during

Currently, ADA recommends the use of any of the following four criteria for diagnosing diabetes: 1) glycated hemoglobin (A1c) value of 6.5% or higher, 2) fasting plasma glucose ≥ 126 mg.dL-1 (7.0 mmol.L-1), 3) 2-h plasma glucose ≥ 200 mg.dL-1 (11.1 mmol.L-1) during an oral glucose tolerance test using 75 g of glucose, and/or 4) classic symptoms of hyperglycemia (e.g., polyuria, polydipsia, and unexplained weight loss) or hyperglycemic crisis with a random plasma glucose of 200 mg.dL-1 (11.1 mmol.L-1) or higher. In the absence of unequivocal hyperglycemia, the first three criteria should be confirmed by repeat testing

Hyperglycaemia and hyperlipidaemia are key promoters of diabetes dysmetabolism, namely, through the formation of reactive oxygen species (ROS) and advanced glycation end products (AGEs), which causes cell damage and insulin resistance [7-9]. Moreover, both of them stimulate proinflammatory cytokines, thus contributing to *β*-cell degradation,

Increased oxidative stress is a widely accepted participant in the development and progression of diabetes and its complications [11-13]. Diabetes is usually accompanied by increased production of free radicals [14,15] or impaired antioxidant defenses [16]. Mechanisms by which increased oxidative stress is involved in the diabetic complications are partly known, including activation of transcription factors, advanced glycated end

Modern medical care uses a vast array of lifestyle and pharmaceutical interventions aimed at preventing and controlling hyperglycemia. In addition to ensuring the adequate delivery of glucose to the tissues of the body, treatment of diabetes attempts to decrease the likelihood that the tissues of the body are harmed by hyperglycemia. The importance of protecting the body from hyperglycemia cannot be overstated; the direct and indirect effects on the human vascular tree are the major source of morbidity and mortality in both type 1 and type 2 diabetes. Generally, the injurious effects of hyperglycemia are separated into macrovascular complications (coronary artery disease, peripheral arterial disease, and stroke) and microvascular complications (diabetic nephropathy, neuropathy, and

Physical activity (PA) and diet are cornerstones of diabetes therapy [19]. Physical activity is a multifaceted behavior of which exercise is just one component. PA is defined as "bodily movement produced by the contraction of skeletal muscle that substantially increases energy expenditure'' and exercise is defined as ''a subset of PA done with the intention of developing physical fitness (i.e., cardiovascular, strength, and flexibility training).'' [19]. In

#### **2.1. Overview of lipid peroxidation and diabetic complications**

Excessively high levels of free radicals cause damage to cellular proteins, membrane lipids and nucleic acids, and eventually cell death [2]. Various mechanisms have been suggested to contribute to the formation of these reactive oxygen-free radicals in diabetic state. Glucose oxidation is believed to be the main source of free radicals. In its enediol form, glucose is oxidized in a transition-metal dependent reaction to an enediol radical anion that is converted into reactive ketoaldehydes and to superoxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide, which if not degraded by catalase or glutathione peroxidase, and in the presence of transition metals, can lead to production of extremely reactive hydroxyl radicals [28, 29]. Superoxide anion radicals can also react with nitric oxide to form reactive peroxynitrite radicals [30, 31]. Hyperglycemia is also found to promote lipid peroxidation of low density lipoprotein (LDL) by a superoxidedependent pathway resulting in the generation of free radicals [32, 33]. Another important source of free radicals in diabetes is the interaction of glucose with proteins leading to the formation of an Amadori product and then advanced glycation endproducts (AGEs) [34, 35]. These AGEs, via their receptors (RAGEs), inactivate enzymes and alter their structures and functions [36], promote free radical formation [37, 38], and quench and block antiproliferative effects of nitric oxide [39, 40]. By increasing intracellular oxidative stress, AGEs activate the transcription factor NF-κB, thus promoting up-regulation of various NFκB controlled target genes [41]. NF-κB enhances production of nitric oxide, which is believed to be a mediator of islet beta cell damage. Considerable evidence also implicates activation of the sorbitol pathway by glucose as a component in the pathogenesis of diabetic complications, for example, in lens cataract formation or peripheral neuropathy [42-44]. Efforts to understand cataract formation have provoked various hypotheses. In the aldose reductase osmotic hypothesis, accumulation of polyols initiates lenticular osmotic changes. In addition, oxidative stress is linked to decreased glutathione levels and depletion of NADPH levels [45, 46]. Alternatively, increased sorbitol dehydrogenase activity is associated with altered NAD+ levels, which results in protein modification by nonenzymatic glycosylation of lens proteins [47, 48]. Mechanisms linking the changes in diabetic neuropathy and induced sorbitol pathway are not well delineated. One possible mechanism, metabolic imbalances in the neural tissues, has been implicated in impaired neurotrophism [49, 50], neurotransmission changes [51, 52], Schwann cell injury [53, 54], and axonopathy [55, 56].

#### **2.2. Overview of antioxidants**

While on the one hand hyperglycemia engenders free radicals, on the other hand it also impairs the endogenous antioxidant defense system in many ways during diabetes [57]. Antioxidant defense mechanisms involve both enzymatic and nonenzymatic strategies. Common antioxidants include the vitamins A, C, and E, glutathione, and the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Other antioxidants include α-lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid, vitamins B1, B2, B6, B12). They work in synergy with each other and against different types of free radicals. Vitamin E suppresses the propagation of lipid peroxidation; vitamin C, with vitamin E, inhibits hydroperoxide formation; metal complexing agents, such as penicillamine, bind transition metals involved in some reactions in lipid peroxidation [58] and inhibit Fenton and Haber- Weiss-type reactions; vitamins A and E scavenge free radicals [30, 37].

Several discrepancies observed in the activities of SOD, catalase, and glutathione peroxidase in experimentally diabetic animals. Decreased levels of glutathione and elevated concentrations of thiobarbituric acid reactants are consistently observed in diabetes [59, 60]. In addition, changes in nitric oxide and glycated proteins are also seen in diabetes.

## **3. Biomarkers of lipid peroxidation**

Since the initial discoveries of Dilliard and colleagues [61], several commercial assay kits have been made available for the measurement of oxidative stress, with many new kits emerging each year. Furthermore, the discovery and utilization of F2-isoprostanes, a prostaglandin like compound, measured via gas chromotomography mass spectrometry has emerged as a substantially more reliable and valid measure of lipid peroxidation [62]. Newly developed ELISA kits for both isoprostanes as well as protein carbonyls are also now available, proving an opportunity for a more widespread use of these biomarkers. In regards to measurement of oxidative stress, due to the high reactivity and relatively short half lives (e.g., 10-5, 10-9 seconds for superoxide radical and hydroxyl radical, respectively) of reactive oxygen and nitrogen species (RONS), direct measurement is extremely difficult to employ. However, direct assessment of free radical production is possible via electron spin resonance spectroscopy (ESR) involving spin traps, as well as two other less common techniques such as radiolysis and laser flash photolysis [63]. ESR works by recording the energy changes that occur as unpaired electrons align in response to a magnetic field [64]. Due to the high cost of such equipment and the high degree of labor associated with each direct method, the majority of free radial research related to exercise has utilized indirect methods for the assessment of resultant oxidative stress. Indirect assessment of oxidative stress involves the measurement of the more stable molecular products formed via the reaction of RONS with certain biomolecules. Common molecular products include stable metabolites (e.g., nitrate/nitrite), and/or concentrations of oxidation target products, including lipid peroxidation end products [isoprostanes, malondialdehyde (MDA), thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), conjugated dienes (CD), oxidized low density lipoprotein (oxLDL)], oxidized proteins [protein carbonyls (PC), individual oxidized amino acids, nitrotyrosine (NT), and nucleic acids [8 hydroxy-2-deoxyguanosine (8-OHdG), oxidized DNA bases (via the Comet Assay), strand breaks] [65]. Additionally, oxidative stress can be measured by observing alterations in the body's antioxidant defense system. This is typically done by measuring the redox changes in the major endogenous antioxidant glutathione, as well as circulating levels of vitamin E, and vitamin C. Moreover, the activity of certain antioxidant enzymes [e.g., superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR)] can be assessed as indicators of the oxidative stress imposed on the tissue. Numerous antioxidant capacity assays also exist and include: Trolox Equivalent Antioxidant Capacity (TEAC), Total Antioxidant Status (TAS), Ferric Reducing Ability of Plasma (FRAP), Total RadicalTrapping Antioxidant Parameter (TRAP), and Oxygen Radical Absorbance Capacity (ORAC) [66].

### **4. Exercise and lipid peroxidation**

296 Lipid Peroxidation

[55, 56].

radicals [30, 37].

**2.2. Overview of antioxidants** 

**3. Biomarkers of lipid peroxidation** 

In addition, oxidative stress is linked to decreased glutathione levels and depletion of NADPH levels [45, 46]. Alternatively, increased sorbitol dehydrogenase activity is associated with altered NAD+ levels, which results in protein modification by nonenzymatic glycosylation of lens proteins [47, 48]. Mechanisms linking the changes in diabetic neuropathy and induced sorbitol pathway are not well delineated. One possible mechanism, metabolic imbalances in the neural tissues, has been implicated in impaired neurotrophism [49, 50], neurotransmission changes [51, 52], Schwann cell injury [53, 54], and axonopathy

While on the one hand hyperglycemia engenders free radicals, on the other hand it also impairs the endogenous antioxidant defense system in many ways during diabetes [57]. Antioxidant defense mechanisms involve both enzymatic and nonenzymatic strategies. Common antioxidants include the vitamins A, C, and E, glutathione, and the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Other antioxidants include α-lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid, vitamins B1, B2, B6, B12). They work in synergy with each other and against different types of free radicals. Vitamin E suppresses the propagation of lipid peroxidation; vitamin C, with vitamin E, inhibits hydroperoxide formation; metal complexing agents, such as penicillamine, bind transition metals involved in some reactions in lipid peroxidation [58] and inhibit Fenton and Haber- Weiss-type reactions; vitamins A and E scavenge free

Several discrepancies observed in the activities of SOD, catalase, and glutathione peroxidase in experimentally diabetic animals. Decreased levels of glutathione and elevated concentrations of thiobarbituric acid reactants are consistently observed in diabetes [59, 60].

Since the initial discoveries of Dilliard and colleagues [61], several commercial assay kits have been made available for the measurement of oxidative stress, with many new kits emerging each year. Furthermore, the discovery and utilization of F2-isoprostanes, a prostaglandin like compound, measured via gas chromotomography mass spectrometry has emerged as a substantially more reliable and valid measure of lipid peroxidation [62]. Newly developed ELISA kits for both isoprostanes as well as protein carbonyls are also now available, proving an opportunity for a more widespread use of these biomarkers. In regards to measurement of oxidative stress, due to the high reactivity and relatively short half lives (e.g., 10-5, 10-9 seconds for superoxide radical and hydroxyl radical, respectively) of reactive oxygen and nitrogen species (RONS), direct measurement is extremely difficult to employ. However, direct assessment of free radical production is possible via electron spin resonance spectroscopy (ESR) involving spin traps, as well as two other less common

In addition, changes in nitric oxide and glycated proteins are also seen in diabetes.

#### **4.1. Lipid peroxidation and antioxidant status in acute exercise research**

Numerous studies have reported an increase in several lipid peroxidation markers following both maximal [67-69] and submaximal [70, 71] exercise. In opposition to these findings, a few studies have reported no increase in lipid peroxidation despite the use of similar maximal [72-74] and submaximal [75, 76] protocols. Increased lipid peroxidation seems to be a result of increased mitochondrial oxidative enyzme activation during aerobic exercise. However, studies reporting conflictiong findings for lipid peroxidation may be partially related to the timing of sampling, in addition to the trained status of the subjects or an insufficient intensity of exercise.

In response to conditions of strenuous physical work the body's antioxidant capacity may be temporarily decreased as its components are used to quench the harmful radicals produced. It appears that the antioxidant capacity may be temporarily reduced during and immediately post exercise [77, 78], after which time levels typically increase above basal conditions during the recovery period [79, 80]. However, conflicting findings have been reported for each of the four main enzymes, with investigators noting increases in GPx [81, 82], SOD [82, 83], and CAT [70, 84, 85], as well as decreases in GPx [86], GR [81], SOD [78]. Furthermore, no change has also been reported for GPx [69], GR [87], SOD (Tauler et al., 2006), CAT [87] activity following exercise. Clearly, these results are mixed and likely depend on the time of sampling, as well as the duration and intensity of exercise, which has varied considerably across studies.

During low-intensity and duration protocols, antioxidant defenses appear sufficient to meet the RONS production, but as intensity and/or duration of exercise increases, these defenses are no longer adequate, potentially resulting in oxidative damage to surrounding tissues [67]. Other factors appear to impact the degree of antioxidant defenses present, including age, training status [81, 88], and dietary intake [80].

It has been shown that anaerobic exercise results in increased RONS production [89]. The mechanisms responsible for the exercise-induced increases in RONS have been suggested to be largely a function of radical generating enzymes (activated in response to ischemia followed by reperfusion) and/or phagocytic immune response following muscle damaging exercise. In the literature, there are fewer data on the markers of lipid peroxidation after anaerobic exercise. It currently remains to be elucidate whether increased RONS formation observed during anaerobic exercise represents a necessary or harmful event.

#### **4.2. Lipid peroxidation and antioxidant status after exercise training**

Regular physical exercise exerts numereous adaptive responses in several tissues. In the context of lipid peroxidation, repeated exposure of RONS production appears to induced to maintain the optimal health. Literature data demonstrated that regular moderate exercise is strengthening the endogenous antioxidant defense system [90, 91,92], and in some animal studies, it has decreased lipid peroxidation. On the other hand, exercise training – both endurance and interval type - appears to protects against exercise induced oxidative stress [93, 94, 95].

## **5. Exercise and diabetes**

The therapeutic use of physical exercise for diabetes treatment has been promoted since 600 B.C. before the discovery of insulin in 1922. Some investigators highlighted the interaction between this hormone and regular physical activity, with possible beneficial results in diabetes treatment [96]. Recent guidelines provide exercise recommendations for people with diabetes based on the strong and convincing epidemiologic association of aerobic exercise with lower cardiovascular disease risk in people with diabetes. The recent 2010 ADA/American College of Sports Medicine (ACSM) exercise guidelines recommend 150 minutes of weekly aerobic exercise (i.e. brisk walking or an equivalent activity with intensity ≥ 40% VO2max); and resistance exercise of major muscle groups two to three times weekly on non-consecutive days (ACSM evidence category B, ADA B level recommendation). The ADA/ACSM guidelines also suggest adding unstructured physical activity as much as possible. Before undertaking exercise more intense than brisk walking, sedentary people with T2D should be evaluated by a physician and an exercise trainer [19].

Exercise has been shown to promote beneficial effects on insulin resistance, both in humans and in rodent models of diabetes [97, 98]. Regular physical exercise may prevent diabetes complications through beneficial effects on glycemic control, insulin sensitivity, blood pressure, lipid profile, and endothelial function. Moderate exercise training has been demonstrated to decrease the plasma glucose concentration in STZ-induced diabetic rats [99]. Hypoglycemic effect of exercise can be explained by exercise induced increase in uptake of glucose of muscle which induces increase of GLUT 4 expression and translocation from intracellular pool [100, 101]. Increase in glucose uptake seems to be related to the increased number of GLUT-4 glucose transporters, although the type of training, strain, age and sex of the animals seem to affect significantly the expression of GLUT-4 [100]. On the other hand, Etgen et al. [101] found that exercise training of normal rats results in an elevated maximal insulin-stimulated hindlimb glucose uptake. They suggested that this increase was only partially explained by an increase in total muscle GLUT-4 protein content. A recent study showed that physical training improves in vivo mitochondrial function concomitantly with increased insulin sensitivity in type 2 diabetes patients and control participants [102].

#### **6. Exercise and chronical complications of diabetes**

#### **6.1. Exercise and cardiovascular disease (CVD)**

298 Lipid Peroxidation

[93, 94, 95].

**5. Exercise and diabetes** 

varied considerably across studies.

age, training status [81, 88], and dietary intake [80].

Furthermore, no change has also been reported for GPx [69], GR [87], SOD (Tauler et al., 2006), CAT [87] activity following exercise. Clearly, these results are mixed and likely depend on the time of sampling, as well as the duration and intensity of exercise, which has

During low-intensity and duration protocols, antioxidant defenses appear sufficient to meet the RONS production, but as intensity and/or duration of exercise increases, these defenses are no longer adequate, potentially resulting in oxidative damage to surrounding tissues [67]. Other factors appear to impact the degree of antioxidant defenses present, including

It has been shown that anaerobic exercise results in increased RONS production [89]. The mechanisms responsible for the exercise-induced increases in RONS have been suggested to be largely a function of radical generating enzymes (activated in response to ischemia followed by reperfusion) and/or phagocytic immune response following muscle damaging exercise. In the literature, there are fewer data on the markers of lipid peroxidation after anaerobic exercise. It currently remains to be elucidate whether increased RONS formation

Regular physical exercise exerts numereous adaptive responses in several tissues. In the context of lipid peroxidation, repeated exposure of RONS production appears to induced to maintain the optimal health. Literature data demonstrated that regular moderate exercise is strengthening the endogenous antioxidant defense system [90, 91,92], and in some animal studies, it has decreased lipid peroxidation. On the other hand, exercise training – both endurance and interval type - appears to protects against exercise induced oxidative stress

The therapeutic use of physical exercise for diabetes treatment has been promoted since 600 B.C. before the discovery of insulin in 1922. Some investigators highlighted the interaction between this hormone and regular physical activity, with possible beneficial results in diabetes treatment [96]. Recent guidelines provide exercise recommendations for people with diabetes based on the strong and convincing epidemiologic association of aerobic exercise with lower cardiovascular disease risk in people with diabetes. The recent 2010 ADA/American College of Sports Medicine (ACSM) exercise guidelines recommend 150 minutes of weekly aerobic exercise (i.e. brisk walking or an equivalent activity with intensity ≥ 40% VO2max); and resistance exercise of major muscle groups two to three times weekly on non-consecutive days (ACSM evidence category B, ADA B level recommendation). The ADA/ACSM guidelines also suggest adding unstructured physical activity as much as possible. Before undertaking exercise more intense than brisk walking, sedentary people

observed during anaerobic exercise represents a necessary or harmful event.

**4.2. Lipid peroxidation and antioxidant status after exercise training** 

with T2D should be evaluated by a physician and an exercise trainer [19].

Regular exercise has beneficial effects on glucose control and cardiovascular disease (CVD) risk factors. Exercise improves and maintains cardiorespiratory fitness, muscular strength, endurance, and body composition [103]. Exercise has a favorable effect on cardiovascular risk factors. In particular, it has specific beneficial effects on the reduction of hypertension, hyperlipidemia, and obesity and the improvement in blood lipid profile [104] even when combined with a rigorous calorie-restricted diet in obese patients with T2DM [105].

The effects of exercise training on abnormal vascular structure and function (including endothelial dysfunction and vascular distensibility) associated with diabetes are yet to be fully understood [106].

Oxidative stress has been suggested to play a role in either the primary or secondary etiology of both congestive heart failure (CHF) and coronary arter disease (CAD) [107, 108] evident by increased oxidative stress biomarkers and/or decreased antioxidant defenses at rest in diseased compared to healthy controls [109]. Increased TBARS [110, 111] and GSSG [110] have been reported following submaximal aerobic exercise in type 1 diabetic subjects. In regards to maximal exercise, direct production of RONS via electron spin resonance spectroscopy has been reported following a graded exercise testing. However, it is important to note that significance was only achieved when data for both type 1 diabetic and healthy control subjects were pooled [112]. Despite the observation of increased levels of exercise-induced oxidative stress biomarkers in studies involving type 1 diabetics, when compared to healthy individuals, the relative magnitude of increase does not differ; rather the group differences at rest are merely maintained during the post exercise period. Other investigators have reported no changes in MDA [112], total glutathione (TGSH), antioxidant enzyme activity or circulating antioxidants [111] in response to acute exercise in type 1 diabetics .

### **6.2. Diabetic nephropathy**

Diabetic nephropathy is the most feared complication of diabetes, due to its substantial comorbidity (need for dialysis, blindness, amputations, etc.), cost, and mortality (the annual mortality rate of diabetic patients with kidney failure on dialysis is about 25%) [113, 114]. The major determinants of kidney disease and its progression to end-stage kidney failure in diabetes are uncontrolled blood glucose, blood pressure and albuminuria [115, 116].

Diabetic nephropathy is an important complication of diabetes since it can lead to end-stage renal failure and also it is a risk factor of cardiovascular disease. The clinical problems caused by diabetic nephropathy are proteinuria and decreased renal function. Diabetic nephropathy is defined by proteinuria > 500 mg in 24 hours in the setting of diabetes, but this is preceded by lower degrees of proteinuria, or "microalbuminuria.". Microalbuminuria is defined as albumin excretion of 30–299 mg/24 hours [11]. Without intervention, diabetic patients with microalbuminuria typically progress to proteinuria and overt diabetic nephropathy.

In vitro studies indicate that hyperglycemia directly enhances oxidative stress in cultured endothelial and mesangial cells, which are targets for injury in diabetes [38, 117]. Several different antioxidants, including vitamin E (VE), vitamin C (VC), taurine, and α-lipoic acid (LA), have been reported to ameliorate renal injury in experimental diabetes [118-120]. In human diabetes, there is evidence that short-term (3 to 4 mo), high-dose (1600 to 1800 IU/d) VE supplementation reduces proteinuria in type 1 and 2 patients with overt nephropathy and decreases hyperfiltration in type 1 patients without overt nephropathy [121].

Urinary albumin excretion occurs normally after exercise [122, 123]. Post-exercise urinary albumin excretion is explained by increased glomerular capillary membrane permeability as a result of increased filtration pressure with increased filtered protein load, and decreased tubular absorption [122, 123]. In normal subjects, proteinuria is better related to exercise intensity and lactate production than to exercise duration [124], it diminishes after 1 h [125] and returns to baseline within 24 h [122]. In diabetes mellitus, the kidneys are more sensitive to the haemodynamic exercise stress [122]. Under exercise, patients with Type 1 diabetes show a partial depletion of negative charges on the glomerular capillary wall [126] that permits the increase of urinary albumin excretion [127]. Reports indicate that urinary albumin excretion increases after exercise, without correlation with glycaemic control, renal function, disease evolution or resting urinary albumin excretion [128, 129]. In contrast, postexercise albuminuria has been found to be associated with HbA1c [127, 129].

On the other hand, recent data demonstrated that exercise might protect the diabetic renal function [130]. Kutlu et al. demonstrated that moderate exercise with combined vitamin E and C supplement was strengthen the antioxidant defense system and reduced the lipid peroxidation in STZ-induced diabetic rat kidney [131].

#### **6.3. Diabetic retinopathy**

300 Lipid Peroxidation

diabetics .

nephropathy.

**6.2. Diabetic nephropathy** 

enzyme activity or circulating antioxidants [111] in response to acute exercise in type 1

Diabetic nephropathy is the most feared complication of diabetes, due to its substantial comorbidity (need for dialysis, blindness, amputations, etc.), cost, and mortality (the annual mortality rate of diabetic patients with kidney failure on dialysis is about 25%) [113, 114]. The major determinants of kidney disease and its progression to end-stage kidney failure in

Diabetic nephropathy is an important complication of diabetes since it can lead to end-stage renal failure and also it is a risk factor of cardiovascular disease. The clinical problems caused by diabetic nephropathy are proteinuria and decreased renal function. Diabetic nephropathy is defined by proteinuria > 500 mg in 24 hours in the setting of diabetes, but this is preceded by lower degrees of proteinuria, or "microalbuminuria.". Microalbuminuria is defined as albumin excretion of 30–299 mg/24 hours [11]. Without intervention, diabetic patients with microalbuminuria typically progress to proteinuria and overt diabetic

In vitro studies indicate that hyperglycemia directly enhances oxidative stress in cultured endothelial and mesangial cells, which are targets for injury in diabetes [38, 117]. Several different antioxidants, including vitamin E (VE), vitamin C (VC), taurine, and α-lipoic acid (LA), have been reported to ameliorate renal injury in experimental diabetes [118-120]. In human diabetes, there is evidence that short-term (3 to 4 mo), high-dose (1600 to 1800 IU/d) VE supplementation reduces proteinuria in type 1 and 2 patients with overt nephropathy

Urinary albumin excretion occurs normally after exercise [122, 123]. Post-exercise urinary albumin excretion is explained by increased glomerular capillary membrane permeability as a result of increased filtration pressure with increased filtered protein load, and decreased tubular absorption [122, 123]. In normal subjects, proteinuria is better related to exercise intensity and lactate production than to exercise duration [124], it diminishes after 1 h [125] and returns to baseline within 24 h [122]. In diabetes mellitus, the kidneys are more sensitive to the haemodynamic exercise stress [122]. Under exercise, patients with Type 1 diabetes show a partial depletion of negative charges on the glomerular capillary wall [126] that permits the increase of urinary albumin excretion [127]. Reports indicate that urinary albumin excretion increases after exercise, without correlation with glycaemic control, renal function, disease evolution or resting urinary albumin excretion [128, 129]. In contrast, post-

On the other hand, recent data demonstrated that exercise might protect the diabetic renal function [130]. Kutlu et al. demonstrated that moderate exercise with combined vitamin E and C supplement was strengthen the antioxidant defense system and reduced the lipid

and decreases hyperfiltration in type 1 patients without overt nephropathy [121].

exercise albuminuria has been found to be associated with HbA1c [127, 129].

peroxidation in STZ-induced diabetic rat kidney [131].

diabetes are uncontrolled blood glucose, blood pressure and albuminuria [115, 116].

Diabetic retinopathy may be the most common microvascular complication of diabetes. The risk of developing diabetic retinopathy or other microvascular complications of diabetes depends on both the duration and the severity of hyperglycemia. Development of diabetic retinopathy in patients with type 2 diabetes was found to be related to both severity of hyperglycemia and presence of hypertension in the U.K Prospective Diabetes Study (UKPDS) [132] and most patients with type 1 diabetes develop evidence of retinopathy within 20 years of diagnosis [133]. Retinopathy may begin to develop as early as 7 years before the diagnosis of diabetes in patients with type 2 diabetes [134]. There are several proposed pathological mechanisms by which diabetes may lead to development of retinopathy.

Oxidative stress may also play an important role in cellular injury from hyperglycemia. High glucose levels can stimulate free radical production and reactive oxygen species formation. Animal studies have suggested that treatment with antioxidants, such as vitamin E, may attenuate some vascular dysfunction associated with diabetes, but treatment with antioxidants has not yet been shown to alter the development or progression of retinopathy or other microvascular complications of diabetes [11].

#### **6.4. Diabetic neuropathy**

Diabetic polyneuropathy affects 30% of the hospital-based population and 20% of community based samples of diabetic patients [135]. There is a growing body of evidence to support the notion that oxidative stress is the biochemical trigger for nerve dysfunction. Various disturbances such as reduced endoneurial blood ow, altered electroconductive properties of the myelin sheath, impaired incorporation of acetate and glucose into the neuron cells should also be mentioned in diabetic condition. It has been shown [136] that superoxide dismutase activity is decreased in nerves from streptozotocin-induced diabetic rats. Glutathione content and glutathione peroxidase activity are also diminished in sciatic nerves from diabetic rats [137, 138]. Nerves of diabetic rats show lower amounts of vitamin E compared to control animals [139]. Lipid peroxidation products such as malondialdehydes or conjugated dienes are elevated in diabetic sciatic nerves [136, 139]. Treatment of diabetic rats with insulin or antioxidants is associated with improved nerve function [51, 140].

Sensory, visual and auditory neural conduction deficits are well documented both in diabetic animals and human studies. As an early marker of visual system deficits observed in diabetic state, visual evoked potential (VEP) latencies were measured in STZ-induced diabetic rats in our laboratories. The results of the previous studies were demonstrated that visual evoked potential (VEP) latencies were prolonged in STZ-induced diabetic rats whereas the latencies were restored by moderate physical exercise [60, 141, 142]. The VEP alterations were found to be accompanied with the increased TBARS concentration in the brain tissues of the diabetic rats.

The impact of diabetes on nervous system is complex and poorly elucidated. The brain is particularly vulnerable to oxidative damage because of its high rate of oxygen consumption, intense production of reactive radicals, and high levels of transition metals, such as iron, that catalyze the production of reactive radicals [143]. Moreover, neuronal membranes are rich in poly unsaturated fatty acids, which are a source of lipid peroxidation [37]. Free radicals are formed disproportionately in diabetes by glucose oxidation, non-enzymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation, and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes mellitus [143].

Previous experimental studies demonstrated that diabetes resulted increased lipid peroxidation and decreased antioxidant enzymes in several brain regions such as hypocampus, striatum and cerebral cortex as well as in whole brain tissue homogenates [59]. The lipids oxidation in the CNS usually demonstrates different concentrations at different regions of the brain, and it can be attributed to regional differences in the O2 consumption [144, 145].

Nervous system complications of diabetes mellitus can become one of the most debilitating complications and affect sensitive and cognitive functions that modulates memory function, resulting in significant functional impairment and dementia. Oxidative stress forms the foundation for the induction of multiple cellular pathways that can ultimately lead to both the onset and subsequent complications of DM [146]. Defects in hippocampal synaptic plasticity and transmission resulting in impairment of learning and memory is one the central nervous system complications of diabetes mellitus [147, 148]. Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a central role in the onset and subsequent complications of diabetes mellitus [149].

Physical exercise has been demonstrated to induce several neurobiological changes in the brain and to prevent diabetes-induced cognitive decline. The neurobiological changes induced by physical exercise have been demonstrated to facilitate the acquisition of a spatial memory task in rats. Exercise has also been demonstrated to increase the cognitive function both in healthy and diabetic people [150, 151, 152]. However, intense exercise has been shown to impaired the cognitive function in murine model that was prevented by vitamin C and E supplementation [153].

### **7. Conclusion**

Literature results emphasize the beneficial role of physical exercise in the promotion of in diabetic complications probably by decreasing hyperglicemia, increasing insulin sensitivity and enhancing antioxidant status of the several systems. The type, duration and the intensity of the exercise as well as the degree of the diabetic complications should be determined before the exercise prescription in diabetic person. For future research, the effects of the different exercise protocols for maintaining the optimum health and stimulating the cellular processes for decreasing the hyperglicemia-induced complications in diabetes in children and older people remains to be explored.

### **Author details**

302 Lipid Peroxidation

consumption [144, 145].

and E supplementation [153].

**7. Conclusion** 

The impact of diabetes on nervous system is complex and poorly elucidated. The brain is particularly vulnerable to oxidative damage because of its high rate of oxygen consumption, intense production of reactive radicals, and high levels of transition metals, such as iron, that catalyze the production of reactive radicals [143]. Moreover, neuronal membranes are rich in poly unsaturated fatty acids, which are a source of lipid peroxidation [37]. Free radicals are formed disproportionately in diabetes by glucose oxidation, non-enzymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation, and development of insulin resistance. These consequences of oxidative stress

Previous experimental studies demonstrated that diabetes resulted increased lipid peroxidation and decreased antioxidant enzymes in several brain regions such as hypocampus, striatum and cerebral cortex as well as in whole brain tissue homogenates [59]. The lipids oxidation in the CNS usually demonstrates different concentrations at different regions of the brain, and it can be attributed to regional differences in the O2

Nervous system complications of diabetes mellitus can become one of the most debilitating complications and affect sensitive and cognitive functions that modulates memory function, resulting in significant functional impairment and dementia. Oxidative stress forms the foundation for the induction of multiple cellular pathways that can ultimately lead to both the onset and subsequent complications of DM [146]. Defects in hippocampal synaptic plasticity and transmission resulting in impairment of learning and memory is one the central nervous system complications of diabetes mellitus [147, 148]. Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a central role in

Physical exercise has been demonstrated to induce several neurobiological changes in the brain and to prevent diabetes-induced cognitive decline. The neurobiological changes induced by physical exercise have been demonstrated to facilitate the acquisition of a spatial memory task in rats. Exercise has also been demonstrated to increase the cognitive function both in healthy and diabetic people [150, 151, 152]. However, intense exercise has been shown to impaired the cognitive function in murine model that was prevented by vitamin C

Literature results emphasize the beneficial role of physical exercise in the promotion of in diabetic complications probably by decreasing hyperglicemia, increasing insulin sensitivity and enhancing antioxidant status of the several systems. The type, duration and the intensity of the exercise as well as the degree of the diabetic complications should be determined before the exercise prescription in diabetic person. For future research, the effects of the different exercise protocols for maintaining the optimum health and

can promote the development of complications of diabetes mellitus [143].

the onset and subsequent complications of diabetes mellitus [149].

Yaşar Gül Özkaya *School of Physical Education and Sports, Akdeniz University, Antalya, Turkey* 

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## **Reactive Oxygen Species Act as Signaling Molecules in Liver Carcinogenesis**

María Cristina Carrillo, María de Luján Alvarez, Juan Pablo Parody, Ariel Darío Quiroga and María Paula Ceballos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45966

## **1. Introduction**

314 Lipid Peroxidation

[151] Cetin E, Top EC, Sahin G, Ozkaya YG, Aydin H, Toraman F. Effect of vitamin e supplementation with exercise on cognitive functions and total antioxidant capacity in

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[153] Rosa EF, Takahashi S, Aboulafia J, Nouailhetas VLA, Oliveira MGM. Oxidative stress induced by intense and exhaustive exercise impairs murine cognitive function. Journal

older people. Journal of Nutrition Health & Aging. 2010;14(9):763-9.

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of Neurophysiology. 2007;98(3):1820-6.

Reactive Oxygen Species (ROS) were viewed as the ''bad'' molecules of cells for a long time, but in the recent years, several lines of evidence indicate the contrary: ROS are essential participants in cell signaling and regulation depending on their concentration.

At present it is well established that ROS signaling is an important factor of many gene- and enzyme-catalyzed processes. ROS signaling is responsible for activation or inhibition of numerous processes catalyzed by protein kinases, phosphatases, and many other enzymes although these reactions proceed by heterolytic (non-free radical) mechanisms [1]. Therefore, ROS signaling can initiate both inhibition and activation of tumor formation. This fact might be of utmost importance for the development of anticancer treatment by the drugs possessing both prooxidant and antioxidant properties.

In this chapter, we summarize a series of experiments that have allowed us to establish the role of oxidative stress in the early development of liver cancer process and the effects of cytokines on the modulation of this process.

Through a series of *in vivo* and *in vitro* experiments we are able to describe:


© 2012 Carrillo et al., licensee InTech. This is an open access chapter 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. © 2012 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.

## **2. Experimental models of liver cancer development**

Hepatocellular carcinoma (HCC) is a malignant solid tumor that arises from the major cell type in the liver: the hepatocyte. HCC is the most common type of primary hepatic tumor; it represents approximately 6% of all malignancies and is the fifth most common tumor worldwide [2].

Nearly all types of primary liver tumors known to occur in humans can be reproduced by chemicals in laboratory animals, especially in rats [3]. In experimental carcinogenesis, preneoplastic foci of altered hepatocytes (AHF) emerge weeks or months before the appearance of hepatocellular adenomas and HCCs [4,5] and this has also been discovered in human with hepatocellular neoplasms and/or cirrhosis [6]. This fact has led to the development of a number of *in vivo* systems for the study of early neoplasia in rat liver [7,8]. The initiation-promotion or two-stage model of cancer development mimics the early events of the latent period of human carcinogenesis. Several two stages models have been developed, including the protocols of Solt and Farber [9], Ito *et al.* [10] and Rao *et al*. [11], that comprise necrogenic doses of carcinogens or other models such as the protocols of Peraino et al. [12] and Pitot et al. [13] that use low, non toxic doses of carcinogens.

In this context, the initiation stage of cancer development can be produced in rat liver by the administration of diethylnitrosamine (DEN) [9–11], a complete carcinogen that produces DNA ethylation and mutagenesis [13]. Necrogenic doses of DEN cause massive hepatic necrosis followed by regeneration [14] and would be expected to cause not only increased gene expression related to regeneration, but also increased expression related to oncogene mutation. Administration of promoting agents causes selective enhancement of the proliferation of initiated cell populations over non-initiated cells in the target tissue [5].

Accordingly, we have developed a two-phase model of liver preneoplasia in rat: basically, the animals are initiated with two necrogenic doses of DEN and subsequently 2 acetylaminofluorene (2-AAF) is administered as promoting agent. The experimental protocol takes six weeks, and at the end of the treatment animals show 5% of liver tissue occupied by microscopic preneoplastic foci. A diagram of the experimental model is shown in Figure 1.

**Figure 1. Two-phase or initiation-promotion (IP) model of rat chemical hepatocarcinogenesis.** Initiation stage is performed by the administration of 2 necrogenic doses of diethylnitrosamine (DEN, 150 mg / kg body weight, intraperitoneal), separated by 2 weeks. A week after the last injection of DEN, the promotion phase begins by the administration of 2-acetylaminofluorene (2-AAF, 20 mg / kg body weight) by gavage, 4 days per week during 3 weeks. At the end of the sixth week, rat livers show microscopic preneoplastic foci.

The presence of preneoplastic foci in this two-phase (initiation-promotion, IP) hepatocarcinogenic model was determined using rat Pi class isoenzyme of glutathione Stransferase (GST) as a foci marker [15]. This isoenzyme has been described as the most effective single marker of hepatic preneoplasia in the rat [16], and immunohistochemical detection of Pi class GST is the most widely used method for identification, quantitation and assessment of rat AHF [17].

## **3. GSTs and liver preneoplasia: Our first studies**

316 Lipid Peroxidation

worldwide [2].

in Figure 1.

microscopic preneoplastic foci.

**2. Experimental models of liver cancer development** 

Hepatocellular carcinoma (HCC) is a malignant solid tumor that arises from the major cell type in the liver: the hepatocyte. HCC is the most common type of primary hepatic tumor; it represents approximately 6% of all malignancies and is the fifth most common tumor

Nearly all types of primary liver tumors known to occur in humans can be reproduced by chemicals in laboratory animals, especially in rats [3]. In experimental carcinogenesis, preneoplastic foci of altered hepatocytes (AHF) emerge weeks or months before the appearance of hepatocellular adenomas and HCCs [4,5] and this has also been discovered in human with hepatocellular neoplasms and/or cirrhosis [6]. This fact has led to the development of a number of *in vivo* systems for the study of early neoplasia in rat liver [7,8]. The initiation-promotion or two-stage model of cancer development mimics the early events of the latent period of human carcinogenesis. Several two stages models have been developed, including the protocols of Solt and Farber [9], Ito *et al.* [10] and Rao *et al*. [11], that comprise necrogenic doses of carcinogens or other models such as the protocols of

Peraino et al. [12] and Pitot et al. [13] that use low, non toxic doses of carcinogens.

In this context, the initiation stage of cancer development can be produced in rat liver by the administration of diethylnitrosamine (DEN) [9–11], a complete carcinogen that produces DNA ethylation and mutagenesis [13]. Necrogenic doses of DEN cause massive hepatic necrosis followed by regeneration [14] and would be expected to cause not only increased gene expression related to regeneration, but also increased expression related to oncogene mutation. Administration of promoting agents causes selective enhancement of the proliferation of initiated cell populations over non-initiated cells in the target tissue [5].

Accordingly, we have developed a two-phase model of liver preneoplasia in rat: basically, the animals are initiated with two necrogenic doses of DEN and subsequently 2 acetylaminofluorene (2-AAF) is administered as promoting agent. The experimental protocol takes six weeks, and at the end of the treatment animals show 5% of liver tissue occupied by microscopic preneoplastic foci. A diagram of the experimental model is shown

**Figure 1. Two-phase or initiation-promotion (IP) model of rat chemical hepatocarcinogenesis.** Initiation stage is performed by the administration of 2 necrogenic doses of diethylnitrosamine (DEN, 150 mg / kg body weight, intraperitoneal), separated by 2 weeks. A week after the last injection of DEN, the promotion phase begins by the administration of 2-acetylaminofluorene (2-AAF, 20 mg / kg body weight) by gavage, 4 days per week during 3 weeks. At the end of the sixth week, rat livers show

GSTs are a family of multifunctional dimeric enzymes with an important role in detoxification processes of several xenobiotics, including anticancer drugs, carcinogens and mutagens [18–20]. These enzymes catalyze the nucleophilic attack of reduced glutathione (GSH) on electrophilic compounds [19,21].

Thus, GSTs are part of a cellular defense system which also includes GSH levels (and enzymes related to its biosynthesis) and proteins involved in the uptake of drugs and in the excretion of glutathione conjugates [22]. In the liver, among the several cytosolic classes of GSTs, Pi class GST (GST P), is particularly interesting because its expression in the adult tissue is associated with preneoplastic and neoplastic development [23]. In addition, increased expression of GST P was found to be associated with resistance of tumor tissues to several cytostatic drugs [24,25].

There is a significative increase of GST P in preneoplastic livers. This enzyme has shown to be the more efficient isoenzyme in the catalysis of conjugation of ethacrynic acid (EA) with GSH. How does this enzyme act in the preneoplastic condition?

EA, an electrophilic loop diuretic drug, causes hepatotoxicity through lipid peroxidation mediated by its oxidative metabolism [26,27]. This drug has a preferential conjugation with GSH either spontaneous or GST catalyzed, reducing its intracellular levels and consequently favoring oxidative stress in isolated hepatocytes [27]. The glutathione conjugate of EA (EA-SG) is a substrate of human multidrug-resistance protein 2 (MRP2) and probably of rat Mrp2 [28]. Thus, it has been suggested that EA-SG is excreted through this active canalicular transport protein into bile [29]. In addition, EA and EA-SG (as well as many others α,αunsaturated carbonyl derivatives and their glutathione conjugates) are important *in vivo* and *in vitro* inhibitors of several human and rat GSTs activities [19,30,31].

As was stated above, at the inactivation step, GSTs are playing a major role by catalyzing the conjugation reaction of GSH with the drug and leading to the inactivation of the therapeutic agent. EA and EA-SG have been proved to be good inhibitors of GSTs activities [19,30,31]. For this reason, we evaluated the enzymatic and cellular *in vitro* response to EA in isolated hepatocytes from preneoplastic rat livers, which present high levels of GST P, and analyzed the role of the GSTs/GSH system and Mrp2 (as a measure of the multidrug resistance) in these cells [15].

Results showed that hepatocytes from IP animals presented higher levels of cell viability than control hepatocytes in the presence of EA. In accordance with this data, IP hepatocytes

showed lower levels of release of lactate dehydrogenase and alanine aminotransferase than control cells throughout the incubation time, indicating higher levels of cellular integrity. This suggests that hepatocytes from IP animals are more resistant to the cytotoxic effects of EA than control cells.

Control hepatocytes suspensions showed increased levels of lipid peroxidation measured through the quantification of TBARS (Thiobarbituric Acid Reactive Substances, [32]) production in a time- and dose-dependent manner in the presence of EA. This data was expected since oxidative metabolism of the drug and a subsequent lipid peroxidation was already described as part of the EA-induced toxicity [27]. However, IP hepatocytes suspensions did not show increased levels of lipid peroxidation during incubation at all times and EA-doses evaluated.

The higher basal levels of TBARS observed in preneoplastic hepatocytes could be attributed to the hepatocarcinogenic treatment, since it was described that lipid hydroperoxides are produced by some promotion regimens [33]. The unchanged levels during EA incubation are probably a consequence of both GST P activities: lipid peroxidase [34] and catalysis of EA conjugation with GSH, avoiding its oxidative metabolism.

Although intracellular total GSH (tGSH) levels decreased in both hepatocytes suspensions without EA, initial tGSH levels showed a mild although significantly higher value in hepatocytes from IP animals. This fact suggests that the small extra pool of tGSH is certainly an advantageous factor to prevent susceptibility to oxidative stress.

GST P has shown to be the more efficient isoenzyme in the catalysis of conjugation of EA with GSH [35,36] and may have a role in the detoxification of lipid hydroperoxides through its selenium-independent peroxidase activity [34]. We observed decreased levels of both Alpha and Mu class GSTs in preneoplastic hepatocytes. This fact, and the presence of GST P in hepatocytes from IP rats, gives to this induced isoenzyme a compensatory role in these cells. Based on the measurement of total GST activity and in data obtained from other publications [37,38], we have proposed that GST P could be playing a major role in the defense system against the cytotoxic effects of EA in our preneoplastic model. At high EA concentrations this resistance is overwhelmed over time, probably as a consequence of inhibition of GST P activity by EA-SG acummulation and depleted levels of intracellular tGSH. In the absence of GST P and GSH, EA may alkylate cell proteins thiols, which might be the major determinants of the cytotoxic effects observed with higher EA concentrations.

It has been demonstrated that MRP2 expression, the canalicular membrane protein reported to be the transporter of GSH and GSH conjugates, is higher in human HCCs than in normal cells [39]. MRP2 increased expression could suggest accelerated GSH depletion and hence, enhanced toxicity of cytotoxic compounds. On the other hand, diminution of MRP2 expression could indicate a preservation of GSH intracellular pool. In spite of the data demonstrated in human HCC, in our preneoplastic model, we observed a 75–85% decrease in the expression of Mrp2 in freshly isolated hepatocytes compared to control cells. Thus, for this reason, reduced levels of Mrp2 in preneoplastic liver cells could contribute to the preservation of intracellular GSH and would result, in addition, in an accumulation of EA-SG and a consequent inhibition of GSTs activities suppressing more GSH consumption.

We also saw increased levels of Mrp2 in intracellular microsomal membrane fraction in a EA-dose dependent manner in both control and IP hepatocytes. This internalization phenomenon was already described [40] and could imply a process which takes place as a feedback mechanism under GSH-consumption conditions at the highest dose of EA. Our results showed that the rate of internalization of Mrp2 with increasing doses of EA was markedly higher in IP hepatocytes, although the initial basal values were significantly lower. To our knowledge, this was the first study evaluating this accelerated internalization process in isolated hepatocytes from preneoplastic rat livers.

In conclusion, hepatocytes of IP rats showed an intrinsic resistance to the cytotoxic effects of low doses of EA and it seems likely that the presence of GST P, the higher levels of GSH, and the lower expression of Mrp2 in the cellular membrane are closely related to this phenotype.

## **4. Interferon α-2b gets into scene**

318 Lipid Peroxidation

EA than control cells.

times and EA-doses evaluated.

showed lower levels of release of lactate dehydrogenase and alanine aminotransferase than control cells throughout the incubation time, indicating higher levels of cellular integrity. This suggests that hepatocytes from IP animals are more resistant to the cytotoxic effects of

Control hepatocytes suspensions showed increased levels of lipid peroxidation measured through the quantification of TBARS (Thiobarbituric Acid Reactive Substances, [32]) production in a time- and dose-dependent manner in the presence of EA. This data was expected since oxidative metabolism of the drug and a subsequent lipid peroxidation was already described as part of the EA-induced toxicity [27]. However, IP hepatocytes suspensions did not show increased levels of lipid peroxidation during incubation at all

The higher basal levels of TBARS observed in preneoplastic hepatocytes could be attributed to the hepatocarcinogenic treatment, since it was described that lipid hydroperoxides are produced by some promotion regimens [33]. The unchanged levels during EA incubation are probably a consequence of both GST P activities: lipid peroxidase [34] and catalysis of

Although intracellular total GSH (tGSH) levels decreased in both hepatocytes suspensions without EA, initial tGSH levels showed a mild although significantly higher value in hepatocytes from IP animals. This fact suggests that the small extra pool of tGSH is certainly

GST P has shown to be the more efficient isoenzyme in the catalysis of conjugation of EA with GSH [35,36] and may have a role in the detoxification of lipid hydroperoxides through its selenium-independent peroxidase activity [34]. We observed decreased levels of both Alpha and Mu class GSTs in preneoplastic hepatocytes. This fact, and the presence of GST P in hepatocytes from IP rats, gives to this induced isoenzyme a compensatory role in these cells. Based on the measurement of total GST activity and in data obtained from other publications [37,38], we have proposed that GST P could be playing a major role in the defense system against the cytotoxic effects of EA in our preneoplastic model. At high EA concentrations this resistance is overwhelmed over time, probably as a consequence of inhibition of GST P activity by EA-SG acummulation and depleted levels of intracellular tGSH. In the absence of GST P and GSH, EA may alkylate cell proteins thiols, which might be the major determinants of the cytotoxic effects observed with higher EA concentrations.

It has been demonstrated that MRP2 expression, the canalicular membrane protein reported to be the transporter of GSH and GSH conjugates, is higher in human HCCs than in normal cells [39]. MRP2 increased expression could suggest accelerated GSH depletion and hence, enhanced toxicity of cytotoxic compounds. On the other hand, diminution of MRP2 expression could indicate a preservation of GSH intracellular pool. In spite of the data demonstrated in human HCC, in our preneoplastic model, we observed a 75–85% decrease in the expression of Mrp2 in freshly isolated hepatocytes compared to control cells. Thus, for this reason, reduced levels of Mrp2 in preneoplastic liver cells could contribute to the

EA conjugation with GSH, avoiding its oxidative metabolism.

an advantageous factor to prevent susceptibility to oxidative stress.

Human lymphoblastoid IFN α has been shown to have a powerful antiproliferative effect on human hepatoma cell line PLC/PRF/5 in a dose-dependent manner, both *in vitro* and *in vivo*, after implantation in nude mice. Moreover, IFN α inhibits liver regeneration by decreasing DNA and total protein synthesis [41,42].

Considerable expectations in reducing the incidence of HCC were connected with the use of IFN α in antiviral treatment of hepatitis B or C. By now, clinical trials have indeed confirmed a reduced incidence of HCC in IFN α–treated patients with chronic hepatitis B or C [43,44]. In contrast, the benefit derived from IFN α treatment of established HCC remains controversial [45,46]. It is important to deepen the understanding of the action of IFN α on HCC cells, because some patients with hepatitis B– or hepatitis C–related liver diseases may already have small, clinically undetectable preneoplastic foci during IFN α therapy. Experimental studies have shown that IFN α exerts its antiproliferative effects against HCC cell lines *in vitro* by inducing apoptosis and inhibiting cell-cycle progression [47–49]. However, the sensitivity of early-stage HCC to IFN α could not be estimated from the sensitivity of the cell lines that have a larger number of gene abnormalities and higher proliferation capability, whereas the activity of IFN is expected to be minimal [50]. However, it was unknown whether IFN α prevents *in vivo* oncogenesis by expressing these effects in the very-early-stage, clinically undetectable cancer cells.

In this context, we have demonstrated that administration of IFN α-2b during the development of rat liver preneoplasia significantly decreased both number and volume percentage of GST P–positive foci [14]. Particularly, these reductions where observed when IFN α-2b was administered during the initiation phase or during the entire experimental protocol. However, when IFN α-2b was administered during the promotion phase no effect on these parameters could be observed. Thus, the use of IFN α-2b as an

antitumor agent was lost when it was administered only at the 2-AAF phase. Nevertheless, we cannot discount that the lack of IFN α-2b effect during the 2-AAF phase reflects some interaction between 2-AAF and the cytokine. Administration of IFN α-2b during the initiation stage seems to be essential to exert inhibitory effects against DENinitiated hepatic carcinogenesis in the rat.

Contrary to our expectations, the proliferation index (measured by immunohistochemical detection of proliferating cell nuclear antigen or PCNA) in preneoplastic foci was not reduced by treatment with IFN α-2b. On the other hand, the apoptotic index (measured by TUNEL technique) in AHF was significantly increased in the groups that received IFN α-2b. The number of apoptotic cells and bodies in AHF after treatment with IFN α-2b was higher than for control rats. Then, the reduction of both number and volume percentage of AHF in IFN α-2b–treated animals is explained by a greater programmed cell death within the foci.

In regard to the effects of IFN α on the cell cycle progression of various normal and tumor cell lines, most studies have observed inhibitory effects on G1 to S phase transition [51,52]; other studies have shown S phase accumulation in response to treatment with IFN α [48,49]. In our studies, the animals with liver preneoplasia that were treated with IFN α-2b showed a diminution in the percentage of preneoplastic hepatocytes in S phase and an accumulation in the G1 phase. Although apoptosis may be initiated in any phase of the cell cycle, most cells undergo apoptosis primarily in the G1 phase of cycling cells, and there is a positive relationship between apoptosis and cell proliferation [53]. This relationship is explained by the presence of many cell cycle regulators/apoptosis inducers such as p53, which operates at the G1/S checkpoint [54].

In this connection, we examined whether p53 and 3 members of the Bcl-2 family (Bax, Bcl-2, and Bcl-xL), which are important regulators of apoptosis [53] were involved in IFN α-2b– mediated programmed cell death. It is known that p53 down-regulates Bcl-2 [55] and upregulates Bax genes [56]. The role of the Bcl-2 family in IFN α–induced apoptosis still remains controversial. For example, IFN α–induced apoptosis in cells of hematopoietic and hepatic origins can occur without involvement of the Bcl-2 family [48,57] whereas transfection of IFN α–sensitive cell lines with a Bcl-2 expression vector conferred partial resistance to cell death mediated by IFN α [58]. Our results showed that members of the Bcl-2 family were involved in the apoptotic elimination of preneoplastic hepatocytes after treatment with IFN α-2b. Specifically, treatment with IFN α-2b increased levels of the proapoptotic protein Bax, in parallel with increases of p53 protein levels. In addition, there were decreases in the levels of Bcl-2 and Bcl-xL proteins, which are known to promote cell survival through homodimerization. Bax protein promotes cell death via homodimerization, whereas heterodimerization with either Bcl-2 or Bcl-xL results in cell survival [59,60]. The relative prevalence of Bax and Bcl-xL protein are critical factors influencing cell fate, promoting either survival or death, whose ultimate outcome largely depends on the Bax/Bcl-xL ratio. Thus, apoptosis pathways can be activated under conditions in which Bax protein expression is elevated and/or Bcl-xL protein expression is decreased.

We also observed increased Bax protein translocation into the mitochondria in the animals that received IFN α-2b. It has been established that subcellular localization of Bax protein is an important regulator of apoptosis. Bax is localized in the cytoplasm and translocates to the mitochondria at the early stage of apoptosis. Bax mediates its proapoptotic effects through a channel-forming activity of the mitochondrial membrane, resulting in disruption of mitochondrial function, release of cytochrome *c*, and apoptosis [61].

In brief, our experimental observations led us conclude that preneoplastic hepatocytes in the IFN α-2b–treated rats are "primed" for apoptosis and undergo programmed cell death as a primary result of a substantial increase in the level of mitochondrial Bax protein, producing a further increase in the Bax/Bcl-xL protein ratio.

## **5. Has TGFβ1 any role in this scenario?**

320 Lipid Peroxidation

within the foci.

the G1/S checkpoint [54].

initiated hepatic carcinogenesis in the rat.

antitumor agent was lost when it was administered only at the 2-AAF phase. Nevertheless, we cannot discount that the lack of IFN α-2b effect during the 2-AAF phase reflects some interaction between 2-AAF and the cytokine. Administration of IFN α-2b during the initiation stage seems to be essential to exert inhibitory effects against DEN-

Contrary to our expectations, the proliferation index (measured by immunohistochemical detection of proliferating cell nuclear antigen or PCNA) in preneoplastic foci was not reduced by treatment with IFN α-2b. On the other hand, the apoptotic index (measured by TUNEL technique) in AHF was significantly increased in the groups that received IFN α-2b. The number of apoptotic cells and bodies in AHF after treatment with IFN α-2b was higher than for control rats. Then, the reduction of both number and volume percentage of AHF in IFN α-2b–treated animals is explained by a greater programmed cell death

In regard to the effects of IFN α on the cell cycle progression of various normal and tumor cell lines, most studies have observed inhibitory effects on G1 to S phase transition [51,52]; other studies have shown S phase accumulation in response to treatment with IFN α [48,49]. In our studies, the animals with liver preneoplasia that were treated with IFN α-2b showed a diminution in the percentage of preneoplastic hepatocytes in S phase and an accumulation in the G1 phase. Although apoptosis may be initiated in any phase of the cell cycle, most cells undergo apoptosis primarily in the G1 phase of cycling cells, and there is a positive relationship between apoptosis and cell proliferation [53]. This relationship is explained by the presence of many cell cycle regulators/apoptosis inducers such as p53, which operates at

In this connection, we examined whether p53 and 3 members of the Bcl-2 family (Bax, Bcl-2, and Bcl-xL), which are important regulators of apoptosis [53] were involved in IFN α-2b– mediated programmed cell death. It is known that p53 down-regulates Bcl-2 [55] and upregulates Bax genes [56]. The role of the Bcl-2 family in IFN α–induced apoptosis still remains controversial. For example, IFN α–induced apoptosis in cells of hematopoietic and hepatic origins can occur without involvement of the Bcl-2 family [48,57] whereas transfection of IFN α–sensitive cell lines with a Bcl-2 expression vector conferred partial resistance to cell death mediated by IFN α [58]. Our results showed that members of the Bcl-2 family were involved in the apoptotic elimination of preneoplastic hepatocytes after treatment with IFN α-2b. Specifically, treatment with IFN α-2b increased levels of the proapoptotic protein Bax, in parallel with increases of p53 protein levels. In addition, there were decreases in the levels of Bcl-2 and Bcl-xL proteins, which are known to promote cell survival through homodimerization. Bax protein promotes cell death via homodimerization, whereas heterodimerization with either Bcl-2 or Bcl-xL results in cell survival [59,60]. The relative prevalence of Bax and Bcl-xL protein are critical factors influencing cell fate, promoting either survival or death, whose ultimate outcome largely depends on the Bax/Bcl-xL ratio. Thus, apoptosis pathways can be activated under conditions in which Bax

protein expression is elevated and/or Bcl-xL protein expression is decreased.

Given its antiproliferative, proapoptotic role in the liver, TGFβ1 could be expected to act as a tumor suppressor. However, various types of neoplastic liver cells respond quite differently to TGFβ1. Whereas some human and rat hepatoma cell lines are sensitive to TGFβ1 [62–64], resistance has been reported for other hepatoma cells [64,65]. In addition, TGFβ1 overexpression seems to be a hallmark of human liver cancer [66]. Thus, the relationship between TGFβ1 and cancer is complex: TGFβ1 may stimulate malignant progression itself; conversely, it can have tumor suppressor activity [67]. The escape of certain hepatoma cells from TGFβ1–induced apoptosis seems to be an important and essential step in malignant progression [68,69]. Moreover, it has been suggested that TGFβ1 overexpression is a late event in human hepatocarcinogenesis [66]. These data indicate that loss of TGFβ1 responsiveness is not an initiating or strongly predisposing event, but rather a late event in carcinogenesis [67,70].

Therefore, it was of interest to study if liver preneoplasia as an early stage of cancer development is still sensitive toward TGFβ1 actions.

Given that the changes of pro- and anti-apoptotic proteins induced by IFN α-2b in rats with liver preneoplasia were similar to those attributed to TGFβ1 in other experimental models [62,63,71], we studied the possibility that TGFβ1 could be involved in the programmed cell death induced by IFN α-2b [72]. Primary, we observed that serum TGFβ1 levels in the animals treated with IFN α-2b were significantly increased. In accordance with this, immunohistochemical studies showed that IFN α-2b treatment significantly augmented the quantity of TGFβ1–positive hepatocytes in preneoplastic livers. At first sight, these findings seemed to indicate that administration of IFN α-2b increased serum TGFβ1 production and the number of TGFβ1–positive hepatocytes. Although the mechanisms by which IFN α-2b treatment induced TGFβ1 in the preneoplastic livers were not completely explored, we observed, using Western blot analysis, that preneoplastic livers expressed higher levels of IFN α receptors than control livers. In addition, IFN α-2b administration in animals subjected to the preneoplastic protocol induced elevated levels of phosphorylated Stat1, indicating activation of the IFN α pathway.

Recent investigations have reported that the induction of apoptosis by endogenous TGFβ1 does not require an overall increase in its hepatic concentration [70]. In view of the fact that TGFβ1 hepatic content may not reflect the induction of apoptosis by this cytokine, we determined the nuclear content of p-Smads-2/3 (critical intracellular transducers of TGFβ1 signaling). We observed high levels of p-Smads-2/3 proteins in the nuclear extracts of IFN α-2b–treated animals. These results corresponded with the increased number of TGFβ1– positive hepatocytes, indicating increased TGFβ1 activation in rats with liver preneoplasia that received IFN α-2b.

Nonparenchymal cells, including Kupffer cells and peritoneal macrophages, are the main source of hepatic TGFβ1 [73,74]. Hepatocytes, however, may synthesize TGFβ1 *in vitro* [75] as well as during hepatocarcinogenesis [66]. During liver preneoplasia, neither peritoneal macrophages nor Kupffer cells secreted detectable levels of TGFβ1 when they were stimulated with IFN α-2b. Conversely, hepatocytes from normal, untreated livers did not secrete TGFβ1 in the absence or presence of IFN α-2b. Nevertheless, hepatocytes from preneoplastic livers produced and secreted detectable levels of TGFβ1 when they were cultured without IFN α-2b stimulus, and IFN α-2b presence in the culture media induced several-fold increases of TGFβ1 production.

*In vitro* studies with isolated hepatocytes have allowed us to demonstrate that IFN α-2b induces apoptosis in hepatocytes from preneoplastic livers, measured by fluorescence microscopy and caspase-3 activity. These cells also had higher nuclear accumulation of p-Smads-2/3, indicating increased TGFβ1 activation. When anti–TGFβ1 was added to the culture media, TGFβ1 activation and apoptosis induced by IFN α-2b were completely blocked. Therefore, the apoptotic effect of IFN α-2b is mediated by the production of TGFβ1 from hepatocytes.

Thus, our work determined for the first time that endogenous TGFβ1 is implicated in the increased apoptosis into the AHF of IFN α-2b-treated rats. Taken together, these data clearly showed that TGFβ1, which is produced and secreted by hepatocytes from preneoplastic liver under IFN α-2b treatment, stimulates hepatocytes apoptotic cell death in an autocrine/paracrine fashion. This postulated mode of action is in agreement with data published previously [70,76,77]. The reduction of preneoplastic foci by endogenous TGFβ1 early in the carcinogenesis process would likewise protect against tumor formation.

## **6. Participation of ROS**

In a new series of *in vitro* experiments, we proved that IFN α-2b induces the production of TGFβ1 in hepatocytes from preneoplastic livers by activation of NADPH oxidase complex (superoxide-producing enzyme consisting of membrane (gp91phox and p22phox) and cytosolic (p47phox, p67phox, and p40phox) components [78]), and TGFβ1 induces apoptosis through a mechanism linked to the production of ROS by the same oxidase [79]. In order to confirm that the induction of NADPH oxidase activity was the main pathway producing ROS, additional experiments were made using IFN α-2b plus an inhibitor of NADPH oxidase activity, diphenyleneiodonium (DPI). Presence of DPI in the culture media totally blocked the activity of NADPH oxidase, the production of ROS and the subsequent apoptosis induced by IFN α-2b.

322 Lipid Peroxidation

that received IFN α-2b.

from hepatocytes.

**6. Participation of ROS** 

several-fold increases of TGFβ1 production.

Recent investigations have reported that the induction of apoptosis by endogenous TGFβ1 does not require an overall increase in its hepatic concentration [70]. In view of the fact that TGFβ1 hepatic content may not reflect the induction of apoptosis by this cytokine, we determined the nuclear content of p-Smads-2/3 (critical intracellular transducers of TGFβ1 signaling). We observed high levels of p-Smads-2/3 proteins in the nuclear extracts of IFN α-2b–treated animals. These results corresponded with the increased number of TGFβ1– positive hepatocytes, indicating increased TGFβ1 activation in rats with liver preneoplasia

Nonparenchymal cells, including Kupffer cells and peritoneal macrophages, are the main source of hepatic TGFβ1 [73,74]. Hepatocytes, however, may synthesize TGFβ1 *in vitro* [75] as well as during hepatocarcinogenesis [66]. During liver preneoplasia, neither peritoneal macrophages nor Kupffer cells secreted detectable levels of TGFβ1 when they were stimulated with IFN α-2b. Conversely, hepatocytes from normal, untreated livers did not secrete TGFβ1 in the absence or presence of IFN α-2b. Nevertheless, hepatocytes from preneoplastic livers produced and secreted detectable levels of TGFβ1 when they were cultured without IFN α-2b stimulus, and IFN α-2b presence in the culture media induced

*In vitro* studies with isolated hepatocytes have allowed us to demonstrate that IFN α-2b induces apoptosis in hepatocytes from preneoplastic livers, measured by fluorescence microscopy and caspase-3 activity. These cells also had higher nuclear accumulation of p-Smads-2/3, indicating increased TGFβ1 activation. When anti–TGFβ1 was added to the culture media, TGFβ1 activation and apoptosis induced by IFN α-2b were completely blocked. Therefore, the apoptotic effect of IFN α-2b is mediated by the production of TGFβ1

Thus, our work determined for the first time that endogenous TGFβ1 is implicated in the increased apoptosis into the AHF of IFN α-2b-treated rats. Taken together, these data clearly showed that TGFβ1, which is produced and secreted by hepatocytes from preneoplastic liver under IFN α-2b treatment, stimulates hepatocytes apoptotic cell death in an autocrine/paracrine fashion. This postulated mode of action is in agreement with data published previously [70,76,77]. The reduction of preneoplastic foci by endogenous TGFβ1

In a new series of *in vitro* experiments, we proved that IFN α-2b induces the production of TGFβ1 in hepatocytes from preneoplastic livers by activation of NADPH oxidase complex (superoxide-producing enzyme consisting of membrane (gp91phox and p22phox) and cytosolic (p47phox, p67phox, and p40phox) components [78]), and TGFβ1 induces apoptosis through a mechanism linked to the production of ROS by the same oxidase [79]. In order to confirm that the induction of NADPH oxidase activity was the main pathway producing ROS, additional experiments were made using IFN α-2b plus an inhibitor of NADPH oxidase activity, diphenyleneiodonium (DPI). Presence of DPI in the culture media totally

early in the carcinogenesis process would likewise protect against tumor formation.

ROS production induced by IFN α-2b showed a singular pattern of two peaks: one peak in ROS generation at 1 hour of culture, and another peak at 9 hours. The addition of anti-TGFβ1 to the culture media did not block the production of the first peak of ROS whereas totally blocked the appearance of the second one. On the other hand, when ASC was added to the culture media the production of both peaks was abolished. Based on these findings, the postulated mechanism by which ROS act as signaling molecules in liver preneoplasia is as follow: IFN α-2b induces, via NADPH oxidase activation, an early ROS production that serves as a messenger, promoting TGFβ1 production and secretion. This growth factor triggers the production of more reactive oxygen intermediates, as a late event, by inducing the same enzyme complex. It was demonstrated that synthesis of new protein is required for NADPH activation and subsequent apoptosis [80]. This event shows an additive response in ROS production and imposes the final onset of the apoptotic effect. The presence of ASC in the culture media totally blocked the increase in the activity of the NADPH oxidase complex, ROS production and the final apoptotic effect induced by IFN α-2b.

Once the source of ROS was assessed, we analyzed the cellular antioxidant defenses and their behavior during the studied times. We observed a reduction in tGSH levels from 7 hours of culture onwards. For that reason we studied if any form of glutathione was being exported out of the cell, and whether the biosynthetic GSH capacity was altered. We found an increase in oxidize glutathione (GSSG) levels probably due to the oxidation of the reduced form within the cytosol, and its exportation to the culture media, possibly in order to protect cells from a shift in the redox equilibrium. IFN α-2b treatment resulted in the loss of GSH biosynthetic capacity since glutamate cysteine ligase (GCL) activity was decreased at 7 hours of culture and a rapid decrease of the mRNA expression of the catalytic subunit of GLC (GCLC) through a mechanism mediated by TGFβ1 was also observed. Moreover, it was found that IFN α-2b-induced apoptosis in hepatocytes from rat preneoplastic livers is accompanied by the cleavage and loss of GCLC protein, through a mechanism mediated by TGFβ1.

A decrease in the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities was observed when hepatocytes were treated with IFN α-2b. On the other hand, treatment with anti-TGFβ1 or ASC totally blocked the decrease in CAT and SOD enzymatic activities. These findings indicate that IFN α-2b induced the decrease in enzymatic CAT and SOD activities by a mechanism mediated by ROS and TGFβ1. These enzymes probably protect hepatocytes from the initial IFN α-2b-induced burst of ROS and this may be the reason for the rapid decrease of the first peak of ROS.

These results confirmed that the perturbation of the redox status produced by the IFN α-2b induction of NADPH oxidase complex triggered TGFβ1 synthesis and secretion and assessed the downregulation of antioxidative systems. Similar data have been reported by Herrera et al. [80] when they treated fetal rat hepatocytes with TGFβ1.

Since ASC abolished all the apoptotic effects induced *in vitro* by IFN α-2b, we determined the relevance of ROS on the onset of the apoptotic process *in vivo*, in the whole preneoplastic liver. IFN α-2b plus ASC treatment of rats with liver preneoplasia abrogated the apoptotic effect induced by IFN α-2b, leading to no reduction on size/number of foci. Interestingly, foci volume was almost twice higher in the animals that received IFN α-2b plus ASC than in IFN α-2b-treated rats. This result highlights the importance of ROS signaling during the beneficial effects of IFN α-2b treatment of hepatic preneoplasia. In this regard, it was found that ASC at low concentrations stimulates growth of malignant cells [81], while inhibits their growth at high doses [82]. At the present time, many cancer patients combine some forms of complementary and alternative medicine therapies with their conventional therapies. The most common choice of these therapies is the use of antioxidants such as vitamin C. It must be assumed that any antioxidant, used to reduce toxicity of tumor therapy on healthy tissue, has the potential to decrease effectiveness of cancer therapy on malignant cells [83]. Some data suggest that antioxidants can ameliorate toxic side effects of therapy without affecting treatment efficacy, whereas other data suggest that antioxidants interfere with radiotherapy or chemotherapy [83].

In summary, we demonstrated that increase in ROS levels turns on the process of programmed hepatocytes death, leading to the elimination of these malignant cells. The inhibition of ROS production with an antioxidant such as ASC in the co-treatment with IFN α-2b may be not a beneficial therapy for the prevention of preneoplastic foci.

## **7. Is p38 MAPK implied in the process?**

p38 MAPK pathway has been implicated in a wide range of cellular functions. However, it is now well established that p38 MAPK activation and its role depends on the cellular context, on the specific stimuli, and on the specific p38 MAPK activated isoform [84]. There are controversies about the role of p38 MAPK in apoptosis. It has been shown that p38 MAPK signaling promotes cell death [85,86], whereas it has also been shown that p38 MAPK cascades enhance survival [87,88], cell growth [89], and differentiation [90]. Furthermore, it has been reported that p38 MAPK participates on the estradiol-mediated inhibition of apoptosis in endothelial cells [91], while participates on the apoptosis induced by thrombospondin-1 [92], or by high leves of D-glucose in the same cells [93]. It is believed that p38 MAPK mediates its apoptotic effects through the phosphorylation of proteins of the apoptotic pathways [94].

Previous reports in hematopoietic cells have shown that IFN α and TGFβ1 play their growth inhibitory effects through activation of the p38 MAPK pathway via phosphorylation (activated p38 MAPK or p-p38 MAPK) [95]. However, these effects are primarily ascribed to G1 cell cycle arrest and not to induction of apoptosis. Others have suggested that during the TGFβ1-induced apoptosis in fetal rat hepatocytes, ROS activates p38 MAPK not by induction of apoptosis, but mediating ROS regulation of TGFβ1-gene expression [96]. On the other hand, it was demonstrated that inactivation of p38 MAPK pathway in cultured mice fibroblasts promotes tumor development [97]. Moreover, it was demonstrated that treatment with an inhibitor of p38 MAPK activation, induced carcinogenesis in mice resistant to tumor development, indicating the leading role of p38 MAPK in the regulation of tumor growth [98].

Using *in vivo* studies we could demonstrate that rats subjected to a 2-phase model of chemical hepatocarcinogenesis have less hepatic p38 MAPK activation than control rats, determined as p-p38 MAPK levels [79]. This is in agreement with Honmo et al. [99] that showed that 2-AAF administration induces a decrement of p38 MAPK activation promoting tumor development.

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or chemotherapy [83].

Since ASC abolished all the apoptotic effects induced *in vitro* by IFN α-2b, we determined the relevance of ROS on the onset of the apoptotic process *in vivo*, in the whole preneoplastic liver. IFN α-2b plus ASC treatment of rats with liver preneoplasia abrogated the apoptotic effect induced by IFN α-2b, leading to no reduction on size/number of foci. Interestingly, foci volume was almost twice higher in the animals that received IFN α-2b plus ASC than in IFN α-2b-treated rats. This result highlights the importance of ROS signaling during the beneficial effects of IFN α-2b treatment of hepatic preneoplasia. In this regard, it was found that ASC at low concentrations stimulates growth of malignant cells [81], while inhibits their growth at high doses [82]. At the present time, many cancer patients combine some forms of complementary and alternative medicine therapies with their conventional therapies. The most common choice of these therapies is the use of antioxidants such as vitamin C. It must be assumed that any antioxidant, used to reduce toxicity of tumor therapy on healthy tissue, has the potential to decrease effectiveness of cancer therapy on malignant cells [83]. Some data suggest that antioxidants can ameliorate toxic side effects of therapy without affecting treatment efficacy, whereas other data suggest that antioxidants interfere with radiotherapy

In summary, we demonstrated that increase in ROS levels turns on the process of programmed hepatocytes death, leading to the elimination of these malignant cells. The inhibition of ROS production with an antioxidant such as ASC in the co-treatment with IFN

p38 MAPK pathway has been implicated in a wide range of cellular functions. However, it is now well established that p38 MAPK activation and its role depends on the cellular context, on the specific stimuli, and on the specific p38 MAPK activated isoform [84]. There are controversies about the role of p38 MAPK in apoptosis. It has been shown that p38 MAPK signaling promotes cell death [85,86], whereas it has also been shown that p38 MAPK cascades enhance survival [87,88], cell growth [89], and differentiation [90]. Furthermore, it has been reported that p38 MAPK participates on the estradiol-mediated inhibition of apoptosis in endothelial cells [91], while participates on the apoptosis induced by thrombospondin-1 [92], or by high leves of D-glucose in the same cells [93]. It is believed that p38 MAPK mediates its apoptotic effects through the phosphorylation of proteins of the apoptotic pathways [94].

Previous reports in hematopoietic cells have shown that IFN α and TGFβ1 play their growth inhibitory effects through activation of the p38 MAPK pathway via phosphorylation (activated p38 MAPK or p-p38 MAPK) [95]. However, these effects are primarily ascribed to G1 cell cycle arrest and not to induction of apoptosis. Others have suggested that during the TGFβ1-induced apoptosis in fetal rat hepatocytes, ROS activates p38 MAPK not by induction of apoptosis, but mediating ROS regulation of TGFβ1-gene expression [96]. On the other hand, it was demonstrated that inactivation of p38 MAPK pathway in cultured mice fibroblasts promotes tumor development [97]. Moreover, it was demonstrated that treatment with an inhibitor of p38 MAPK activation, induced carcinogenesis in mice resistant to tumor development, indicating the leading role of p38 MAPK in the regulation of tumor growth [98].

α-2b may be not a beneficial therapy for the prevention of preneoplastic foci.

**7. Is p38 MAPK implied in the process?** 

Another important finding of the *in vivo* studies was the effect of IFN α-2b on the activation of p38 MAPK in rat preneoplastic livers. Preneoplastic animals treated with IFN α-2b showed similar p-p38 MAPK levels to those in controls. In this connection, cultured hepatocytes from preneoplastic livers treated with IFN α-2b plus SB-203580 (inhibitor of α and β isoforms of p38 MAPK), totally blocked the IFN α-2b-induced apoptosis. It is clear that activation of p38 MAPK pathway plays a key role in promoting apoptosis after IFN α-2b treatment in our model of experimental preneoplasia. It was previously reported that IFN α suppresses the growth of leukemia cell progenitors through activation of p38 MAPK, which leads to cell cycle arrest in different phases [100].

We demonstrated that IFN α-2b induces an early production of ROS (first peak), in hepatocytes from preneoplastic livers. Then, ROS stimulate the production and secretion of TGFβ1 from hepatocytes, which in turn, generates a new burst of ROS (second peak). These oxygen radicals act as signaling mediators of the onset of the IFN α-2b-induced apoptosis.

Activation of p38 MAPK after IFN α-2b stimulus occurred preceding each increment in ROS generation and so, the particular pattern of two peaks was also functioning for p38 MAPK activation. Interestingly, treatment with ASC was able to block only the second peak, indicating that early activation of the pathway was independent of ROS, while late activation depended on ROS produced by endogenous TGF-β1. Treatment with anti-TGFβ1 completely blocked the second p38 MAPK, demonstrating that TGF β1 induces activation of p38 MAPK through ROS, as previously reported in fetal rat hepatocytes [96].

Another relevant issue is the activation of transcription factors by p38 MAPK. Cell signaling pathway activation could be transmitted to the nucleus in different ways, depending on the stimulus. To assess whether activation of p38 MAPK transmitted the IFN α-2b stimulus to the nucleus, we analysed phosphorylation status of specific p38 MAPK transcription factors CREB/ATF-1 and ATF-2. Our findings documented that early p38 MAPK activation under IFN α-2b stimulus mainly activates the transcription of ATF-2-regulated genes, whereas the late signal of p38 MAPK activation is transmitted to the nucleus mainly by the phosphorylation of CREB/ATF-1. Moreover, it can be also inferred that early phosphorylation of ATF-2 may be dependent on activation of p38 MAPK by IFN α-2b, while late phosphorylation of CREB/ATF-1 may be dependent on activation of p38 MAPK by TGFβ1.

## **8. Relationship between p38 and NADPH oxidase**

We inferred that p38 MAPK activation is essential for NADPH oxidase to function in preneoplastic hepatocytes treated with IFN α-2b, because the presence of p38 MAPK inhibitor SB-203580 totally blocked the activation of the enzyme [101]. Cytosolic component of NADPH oxidase complex, p47phox got phosphorylated following the same pattern as p38 MAPK induction and ROS generation: an early, first increment and a late, second increase. The first increase of p47phox phosphorylation by IFN α-2b was independent of ROS, since ASC did not block such phosphorylation. However, it was dependent of p38 MAPK activation, since it was blocked by SB203580. This is a very interesting finding since it suggests that p-p38 MAPK phosphorylates p47phox, initiating the activation of NADPH oxidase in cells from preneoplastic livers. Analysis at higher times demonstrated that late phosphorylation of p47phox was completely blocked by anti-TGFβ1 or ASC, evidencing the participation of TGF β1 and ROS in this process. Studies of p47phox translocation from cytosol to plasma membrane were consistent with the phosphorylation findings.

It is clear that in liver preneoplasia there is a positive cross-talk between IFN α-2b, TGFβ1 and p38 MAPK pathways. Taken altogether, evidence indicates that p38 MAPK pathway plays a critical role in the generation of the suppressive effects of IFN α-2b, as well as TGFβ1 in the very early stages of hepatic neoplasia. There is strong indication that this signaling cascade acts as a converging signaling point for signaling pathways activated by different cytokines to mediate apoptotic or suppressive signals. These findings may have important clinical implications, as improving the pharmacological development of better drugs for the prevention and treatment of hepatic illness such as cancer.

## **9. How are IFN α and TGFβ1 signaling pathways connected?**

Interactions between TGFβ and other cytokines signaling pathways have been extensively studied, particularly the cross-talk between TGFβ/Smad and IFN γ/Stat signaling in their antagonistic role on collagen deposition and fibrosis [102–107]. However, despite the fact that TGFβ plays a crucial role in cancer, little is known about TGFβ signaling interactions during this process. An investigation in hepatoma cells have described a cross-talk between Il-6 and TGFβ signaling [108] and another study in a melanoma cell line normally resistant to IFN α, have demonstrated that co-stimulation with IFN α and TGFβ induces antiproliferative activity [109].

As was stated above, the relationship between TGFβ and cancer is complex: it functions as a tumor suppressor in early epithelial carcinogenesis, but often becomes prooncogenic in late stages of tumor progression [110]. Autocrine TGFβ1 is known to suppress tumorigenesis and tumor progression in normal and early transformed cells, but it can also promote the survival of various cancer cells [111]. Besides, dysregulation of the downstream effectors of TGFβ has been described in late steps of promotion stage, indicating that may contribute to the progression of preneoplastic lesions [112].

We demonstrated that during liver preneoplasia TGFβ1 has a beneficial role, promoting apoptotic death of AHF. Therefore, we attempted to get more insight into the relationship between IFN α-2b and autocrine TGFβ1 in preneoplastic rat livers. Many *in vitro* cell systems are good tools to explain related actions of distinct types of cytokines in various biological signaling pathways, but they are not physiological. However, the study of IFN α-2b and TGFβ1 signals interactions in hepatocytes derived from the whole preneoplastic liver may be relevant for understanding the mechanisms operating in patients with chronic hepatitis B or C treated with IFN α-2b, who already have small, clinically undetectable preneoplastic liver foci during therapy.

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p38 MAPK induction and ROS generation: an early, first increment and a late, second increase. The first increase of p47phox phosphorylation by IFN α-2b was independent of ROS, since ASC did not block such phosphorylation. However, it was dependent of p38 MAPK activation, since it was blocked by SB203580. This is a very interesting finding since it suggests that p-p38 MAPK phosphorylates p47phox, initiating the activation of NADPH oxidase in cells from preneoplastic livers. Analysis at higher times demonstrated that late phosphorylation of p47phox was completely blocked by anti-TGFβ1 or ASC, evidencing the participation of TGF β1 and ROS in this process. Studies of p47phox translocation from

It is clear that in liver preneoplasia there is a positive cross-talk between IFN α-2b, TGFβ1 and p38 MAPK pathways. Taken altogether, evidence indicates that p38 MAPK pathway plays a critical role in the generation of the suppressive effects of IFN α-2b, as well as TGFβ1 in the very early stages of hepatic neoplasia. There is strong indication that this signaling cascade acts as a converging signaling point for signaling pathways activated by different cytokines to mediate apoptotic or suppressive signals. These findings may have important clinical implications, as improving the pharmacological development of better drugs for the

Interactions between TGFβ and other cytokines signaling pathways have been extensively studied, particularly the cross-talk between TGFβ/Smad and IFN γ/Stat signaling in their antagonistic role on collagen deposition and fibrosis [102–107]. However, despite the fact that TGFβ plays a crucial role in cancer, little is known about TGFβ signaling interactions during this process. An investigation in hepatoma cells have described a cross-talk between Il-6 and TGFβ signaling [108] and another study in a melanoma cell line normally resistant to IFN α, have demonstrated that co-stimulation with IFN α and TGFβ induces

As was stated above, the relationship between TGFβ and cancer is complex: it functions as a tumor suppressor in early epithelial carcinogenesis, but often becomes prooncogenic in late stages of tumor progression [110]. Autocrine TGFβ1 is known to suppress tumorigenesis and tumor progression in normal and early transformed cells, but it can also promote the survival of various cancer cells [111]. Besides, dysregulation of the downstream effectors of TGFβ has been described in late steps of promotion stage, indicating that may contribute to

We demonstrated that during liver preneoplasia TGFβ1 has a beneficial role, promoting apoptotic death of AHF. Therefore, we attempted to get more insight into the relationship between IFN α-2b and autocrine TGFβ1 in preneoplastic rat livers. Many *in vitro* cell systems are good tools to explain related actions of distinct types of cytokines in various biological signaling pathways, but they are not physiological. However, the study of IFN α-2b and TGFβ1 signals interactions in hepatocytes derived from the whole preneoplastic liver may be relevant for understanding the mechanisms operating in patients with chronic

cytosol to plasma membrane were consistent with the phosphorylation findings.

**9. How are IFN α and TGFβ1 signaling pathways connected?** 

prevention and treatment of hepatic illness such as cancer.

antiproliferative activity [109].

the progression of preneoplastic lesions [112].

The obtained results provided evidence for the integration of TGFβ1 and IFN α-2b signaling pathways during the development of liver carcinogenesis. IFN α-2b treatment of hepatocytes from preneoplastic livers produced a rapid activation of IFN α signaling, with increased p-Stat1 levels. Subsequently, autocrine TGFβ1 produced under IFN α-2b stimulus was able to induce the activation of TGFβ1/Smad signaling pathway, determined by nuclear content of p-Smad2/3 and confirmed by the use of specific TGF β1 signaling inhibitors (anti-TGFβ1 and SB-431542) [113].

A critical mechanism for regulating the cellular response to cytokines resides at the level of receptor expression. TGFβRII plays a key role in receptor activation and subsequent TGFβ1 signal propagation, functioning both to bind ligand and to activate TGFβRI. Disorders of TGFβRII expression lead to various diseases. For example, reduction of TGFβRII levels contributes to the resistance of tumor cells to TGFβ [114].

We observed that TGFβRII was up-regulated at mRNA and protein levels. This induction was mediated by autocrine TGFβ1, since it was blocked by inhibitors of TGFβ1 signaling. This is an outstanding finding, since TGFβ1-dependent regulation of TGFβRII has not been previously reported.

Inhibitory Smad7 is a key component of TGFβ1 signals. Its expression is not only induced by TGFβ, but also controlled by, for example, IFN γ [102,107]. Therefore, Smad7 is considered as a protein involved in the fine-tuning of the cellular responses to the TGFβ family by integrating various signaling pathways. However, in our model, Smad7 did not show changes in its protein levels, at least during the studied times. Furthermore, Smad7 protein levels in hepatocytes from preneoplastic livers were significantly reduced with respect to their levels in hepatocytes from normal livers. So, additional experiments of Smad7 induction by phorbol 12-myristate 13-acetate (PMA) were performed in order to evaluate if the decreased Smad7 levels showed in preneoplastic livers may contribute in TGFβ1 signaling activation. Results showed that this possibility seems unlikely; provided that Smad7 protein reached similar levels to those in normal hepatocytes, and TGFβ1 signaling continued activated. These experiments indicated that Smad7 protein is not directly related with TGFβ1 and IFN α signals interaction in hepatocytes from preneoplastic livers.

Another decisive aspect in signaling pathways relationships is the availability of certain coactivators for interacting with specific transcription factors. The cofactor p300 is an important component of the transcriptional machinery that integrates TGFβ/ IFN γ-induced signals [115].

In normal fibroblasts exposed to IFN γ and TGFβ simultaneously, activated Stat1 and activated Smad2/3 compete each other for limiting p300. IFN γ-activated Stat1 appears to sequester p300, thereby disrupting TGFβ-induced interaction of p300 with Smad2/3. Ectopic p300 rescues stimulation in the presence of IFN γ, suggesting that p300 acts as an integrator of IFN γ/Stat1 and TGFβ/Smad2/3 signals [103]. In addition, Inagaki et al. [116] have demonstrated that IFN α antagonizes TGFβ/Smad-induced hepatic fibrosis by competition between Stat1 and Smad3 for binding to p300 protein.

In our study, we found that IFN α-2b induced a direct interaction between activated Stat1 and p300 in hepatocytes from preneoplastic livers. Furthermore, activated Smad2/3 induced by autocrine TGFβ1 were able to physically associate with p300. In addition, levels of p300 in hepatocytes from preneoplastic livers were significantly higher than in normal hepatocytes. Together, these findings suggested that in hepatocytes from preneoplastic livers, the intracellular signals triggered by TGFβ1 and IFN α-2b are integrated at the nuclear level, where p-Stat1 and p-Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.

It was recently found that TGFβ signals potentiate Il-6 signaling in hepatoma cells. This cross-talk occurs by physical interactions between Stat3 and Smad3, bridged by p300 [108]. In our model of liver preneoplasia we did not observe physical interaction between Stat1 and Smad3, but it seems to be enough p300 protein available to interact with p-Stat1 on one hand, and with p-Smad2/3 on the other, leading to the activation of TGFβ1 and IFN α signaling simultaneously.

In fact, we have described for the first time a positive cross-talk between IFN α and TGFβ1 signaling.

## **10. Summary # 1**

In these series of experiments, it was demonstrated that NADPH oxidase complex is activated when IFN α-2b binds to type I receptor. This binding produces early amounts of ROS. ROS, in turn, trigger TGFβ1 production and secretion. TGFβ1, when binding to its receptor, also induces NADPH oxidase complex activation, and, besides, decreases the antioxidant defenses of the cell. Moreover, we demonstrated that p38 MAPK activation is essential for NADPH oxidase to function.

Furthermore, ROS initiate mitochondrial apoptosis directly and/or acting by the Bcl-2 family proteins inducing a mitochondrial permeability transition pore (MPTP), releasing cytochrome c and activating caspase 3. TGFβ1 could induce, as a late event, the activation of caspase 8, which, in turn, induces a higher MPTP through activation of Bid, another Bcl-2 family member [117]. A graphic outline of these concerns is shown in Figure 2.

Altogether, our results demonstrate that the oxidative stress induced in preneoplastic liver by IFN α-2b is able to trigger the apoptotic mechanism and brings into the play another key cytokine in the cancer process: TGFβ1.

## **11. Targeting the Wnt/β-catenin signaling pathway**

Among the growth factor signaling cascades dysregulated in HCC, evidences suggest that the Wnt/Frizzled-mediated signaling pathway plays a key role in hepatic carcinogenesis. Aberrant activation of the signaling in HCC is mostly due to dysregulated expression of the Wnt/β-catenin signaling components. This leads to the activation of the β-catenin/TCF dependent target genes, which control cell proliferation, cell cycle, apoptosis or motility. It has been shown that disruption of the Wnt/β-catenin signaling cascade displayed anticancer properties in HCC [118].

For this reason, we determined the status of the Wnt/β-catenin/TCF pathway in the preneoplastic stage and evaluated the possible effects of IFN α-2b on this pathway.

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restrictive cellular amounts.

signaling simultaneously.

**10. Summary # 1** 

essential for NADPH oxidase to function.

cytokine in the cancer process: TGFβ1.

cancer properties in HCC [118].

signaling.

In our study, we found that IFN α-2b induced a direct interaction between activated Stat1 and p300 in hepatocytes from preneoplastic livers. Furthermore, activated Smad2/3 induced by autocrine TGFβ1 were able to physically associate with p300. In addition, levels of p300 in hepatocytes from preneoplastic livers were significantly higher than in normal hepatocytes. Together, these findings suggested that in hepatocytes from preneoplastic livers, the intracellular signals triggered by TGFβ1 and IFN α-2b are integrated at the nuclear level, where p-Stat1 and p-Smad2/3 are capable of interact with p300, present in no

It was recently found that TGFβ signals potentiate Il-6 signaling in hepatoma cells. This cross-talk occurs by physical interactions between Stat3 and Smad3, bridged by p300 [108]. In our model of liver preneoplasia we did not observe physical interaction between Stat1 and Smad3, but it seems to be enough p300 protein available to interact with p-Stat1 on one hand, and with p-Smad2/3 on the other, leading to the activation of TGFβ1 and IFN α

In fact, we have described for the first time a positive cross-talk between IFN α and TGFβ1

In these series of experiments, it was demonstrated that NADPH oxidase complex is activated when IFN α-2b binds to type I receptor. This binding produces early amounts of ROS. ROS, in turn, trigger TGFβ1 production and secretion. TGFβ1, when binding to its receptor, also induces NADPH oxidase complex activation, and, besides, decreases the antioxidant defenses of the cell. Moreover, we demonstrated that p38 MAPK activation is

Furthermore, ROS initiate mitochondrial apoptosis directly and/or acting by the Bcl-2 family proteins inducing a mitochondrial permeability transition pore (MPTP), releasing cytochrome c and activating caspase 3. TGFβ1 could induce, as a late event, the activation of caspase 8, which, in turn, induces a higher MPTP through activation of Bid, another Bcl-2

Altogether, our results demonstrate that the oxidative stress induced in preneoplastic liver by IFN α-2b is able to trigger the apoptotic mechanism and brings into the play another key

Among the growth factor signaling cascades dysregulated in HCC, evidences suggest that the Wnt/Frizzled-mediated signaling pathway plays a key role in hepatic carcinogenesis. Aberrant activation of the signaling in HCC is mostly due to dysregulated expression of the Wnt/β-catenin signaling components. This leads to the activation of the β-catenin/TCF dependent target genes, which control cell proliferation, cell cycle, apoptosis or motility. It has been shown that disruption of the Wnt/β-catenin signaling cascade displayed anti-

family member [117]. A graphic outline of these concerns is shown in Figure 2.

**11. Targeting the Wnt/β-catenin signaling pathway** 

**Figure 2.** Graphic outline of IFN α-2b, TGFβ1, p38 MAPK, NADPH oxidase and ROS interactions in liver preneoplasia.

The major findings of our studies were related to the impairment of the canonical Wnt/βcatenin/TCF pathway in a very early stage of hepatic carcinogenesis. In addition, we demonstrated that *in vivo* IFN α-2b treatment produces an attenuation of TCF transcriptional activity and enhances FOXO transcriptional activity in preneoplastic livers.

The common denominator of an abnormal Wnt signaling is the stabilization and accumulation of unphosphorylated β-catenin in the cytoplasm of a cell. Eventually, this allows entry of unphosphorylated β-catenin into the nucleus where it promotes the transcription of a subset of genes implicated in cellular proliferation. This β-catenin stabilization was demonstrated in our two-phase carcinogenic model, where plasma membrane delocalization and cytoplasmic accumulation of β-catenin were observed [119]. Moreover, significant reductions of phosphorylated β-catenin levels were found in IP animals. Since total β-catenin (phosphorylated and unphosphorylated) protein levels were preserved in all studied groups, these results indicate a lower phosphorylation rate of cytoplasmic β-catenin in IP rats.

We have also found up-regulation of TCF target genes Cyclin D1, MMP-7, Axin 2, and SP5 in preneoplastic livers. Up-regulation of Cyclin D1 was predicted since this protein is an important regulator of cell cycle progression, and its activity is required for G1 to S-phase transition. Overexpression of this gene has been associated with the development and progression of several cancers [120]. In addition, it has been reported that overexpression of Cyclin D1 in tumor cells contributes with their resistance to cytotoxic drugs [121]. In fact, inhibition of Cyclin D1 enhances the effects of several chemotherapeutic agents [121]. In agreement with these results, we have previously described (see *GSTs and liver preneoplasia: our first studies*) a drug-resistance phenotype in isolated hepatocytes obtained from rat preneoplastic livers. Thus, it is possible that the overexpression of Cyclin D1 could play a role in the drug-resistance phenotype of this model. MMP-7, a member of the matrix metalloproteinase family, acts as a specific proteolytic enzyme for degradation of certain components of the extracellular matrix. This protein was already shown to be important for the growth of early adenomas [122] and its function is essential in more advanced stages such as tumor progression and metastasis, where an invasive growth is a highlight of these steps [123,124]. Hence, enhanced MMP-7 expression could be proposed as an indicator of potential tumor progression, invasiveness, and metastatic ability at a very early stage of hepatocarcinogenic development. It has been reported that the tumor suppressor Axin 2 is a target of Wnt signaling [125,126]. The up-regulation of Axin 2 showed in IP rats, which is known to be a negative regulator of free β-catenin [127,128], could be an expression of a feedback preservation mechanism of the preneoplastic tissue, and might not be sufficient to prevent cytoplasmic β-catenin accumulation. SP5, a member of the SP1 transcription factor family and known target of Wnt signaling [129] was also over-expressed. This protein seems to work as a transcriptional repressor, preventing the expression of genes involved in cell cycle G1 phase arrest such as p21 [129].

In order to determine the involvement of a mutated β-catenin protein in the activation of this pathway as was described for HCC [130–133], we performed a direct sequencing of amplicons encoding a region of exon 2 of rat liver β-catenin gene. Our results demonstrated that this sequence had no deletion or point mutations in any of the studied groups.

Even with a wild-type β-catenin, the pathway can also be triggered because of alterations in other components of the cascade signaling. The Frizzled protein family acts as a seven-span transmembrane receptor for Wnt proteins. It was recently reported an up-regulation of the Frizzled-7 receptor in the presence of wild-type β-catenin in four murine transgenic models of hepatocarcinogenesis [134] and in human HCC [135] with activation of the Wnt/βcatenin/TCF pathway. Therefore, it was suggested that overexpression of Frizzled-7 could lead or contribute to activation of Wnt signaling. The obtained data showed a marked increase of this receptor in preneoplastic livers at mRNA and protein levels. Since it was reported that Frizzled-7 is also a target gene of the Wnt/β-catenin/TCF pathway [136], we presume that overexpression is rather a consequence than a cause of abnormal activation of the Wnt/β-catenin/TCF pathway.

Once we demonstrated that the Wnt/β-catenin/TCF pathway is activated in preneoplastic rat livers, we analyzed the effects of IFN α-2b treatment. Results showed that *in vivo* IFN α-2b administration did not prevent β-catenin delocalization and cytoplasmic accumulation; however, it certainly attenuates activation of the canonical Wnt/β-catenin/TCF pathway as measured by four TCF target genes. The transcription levels of these genes were similar to controls in IP animals that received IFN α-2b.

330 Lipid Peroxidation

cycle G1 phase arrest such as p21 [129].

the Wnt/β-catenin/TCF pathway.

We have also found up-regulation of TCF target genes Cyclin D1, MMP-7, Axin 2, and SP5 in preneoplastic livers. Up-regulation of Cyclin D1 was predicted since this protein is an important regulator of cell cycle progression, and its activity is required for G1 to S-phase transition. Overexpression of this gene has been associated with the development and progression of several cancers [120]. In addition, it has been reported that overexpression of Cyclin D1 in tumor cells contributes with their resistance to cytotoxic drugs [121]. In fact, inhibition of Cyclin D1 enhances the effects of several chemotherapeutic agents [121]. In agreement with these results, we have previously described (see *GSTs and liver preneoplasia: our first studies*) a drug-resistance phenotype in isolated hepatocytes obtained from rat preneoplastic livers. Thus, it is possible that the overexpression of Cyclin D1 could play a role in the drug-resistance phenotype of this model. MMP-7, a member of the matrix metalloproteinase family, acts as a specific proteolytic enzyme for degradation of certain components of the extracellular matrix. This protein was already shown to be important for the growth of early adenomas [122] and its function is essential in more advanced stages such as tumor progression and metastasis, where an invasive growth is a highlight of these steps [123,124]. Hence, enhanced MMP-7 expression could be proposed as an indicator of potential tumor progression, invasiveness, and metastatic ability at a very early stage of hepatocarcinogenic development. It has been reported that the tumor suppressor Axin 2 is a target of Wnt signaling [125,126]. The up-regulation of Axin 2 showed in IP rats, which is known to be a negative regulator of free β-catenin [127,128], could be an expression of a feedback preservation mechanism of the preneoplastic tissue, and might not be sufficient to prevent cytoplasmic β-catenin accumulation. SP5, a member of the SP1 transcription factor family and known target of Wnt signaling [129] was also over-expressed. This protein seems to work as a transcriptional repressor, preventing the expression of genes involved in cell

In order to determine the involvement of a mutated β-catenin protein in the activation of this pathway as was described for HCC [130–133], we performed a direct sequencing of amplicons encoding a region of exon 2 of rat liver β-catenin gene. Our results demonstrated

Even with a wild-type β-catenin, the pathway can also be triggered because of alterations in other components of the cascade signaling. The Frizzled protein family acts as a seven-span transmembrane receptor for Wnt proteins. It was recently reported an up-regulation of the Frizzled-7 receptor in the presence of wild-type β-catenin in four murine transgenic models of hepatocarcinogenesis [134] and in human HCC [135] with activation of the Wnt/βcatenin/TCF pathway. Therefore, it was suggested that overexpression of Frizzled-7 could lead or contribute to activation of Wnt signaling. The obtained data showed a marked increase of this receptor in preneoplastic livers at mRNA and protein levels. Since it was reported that Frizzled-7 is also a target gene of the Wnt/β-catenin/TCF pathway [136], we presume that overexpression is rather a consequence than a cause of abnormal activation of

Once we demonstrated that the Wnt/β-catenin/TCF pathway is activated in preneoplastic rat livers, we analyzed the effects of IFN α-2b treatment. Results showed that *in vivo* IFN α-2b

that this sequence had no deletion or point mutations in any of the studied groups.

In addition, IFN α-2b-treated IP rats showed that Frizzled-7 levels remained unchanged compared to control animals. These results reinforced our hypothesis that Frizzled-7 upregulation occurs as a result of the abnormal activation of the studied pathway.

In an attempt to get more insight into the regulation of Wnt/β-catenin/TCF pathway, FOXO transcription family has come into scene. Recent studies reported that FOXO interacts with β-catenin in a competitive manner with TCF, particularly under cellular oxidative stress conditions [137,138]. Taking this into consideration and the fact that *in vivo* IFN α-2b treatment induces endogenous ROS formation in preneoplastic livers, we analyzed interactions between β-catenin with TCF4 and FoxO3a and association of these transcription factors with their corresponding target gene promoters. Co-immunoprecipitation assays showed that β-catenin/TCF4 interaction effectively occurs in preneoplastic livers and administration of IFN α-2b not only attenuates this interaction but also promotes βcatenin/FoxO3a association. Using ChIP assay, we verified that interaction of FoxO3a with the promoter region of its target gene is enhanced in preneoplastic livers treated with IFN α-2b. On the other hand, TCF4 remains associated with SP5 gene promoter region in all studied groups. It is known that TCF4 contains a conserved domain that binds DNA irrespective of its interaction with β-catenin; however, the transcriptional activity is blocked by the presence of a family of transcriptional repressors [139,140]. TCF4 must bind β-catenin for its transactivation and this interaction was verified by co-immunoprecipitation assays. In addition, it has been demonstrated that interaction of β-catenin with FOXO enhances its transcriptional activity [137,138], so we measured the expression of p130, a FOXO target gene whose main function is related to the maintenance of cell cycle arrest. Furthermore, it was suggested that p130 may exert a proapoptotic effect on certain tumor samples [141]. We found up-regulation of p130 transcript in preneoplastic livers treated with IFN α-2b. These findings suggest that IFN α-2b treatment in preneoplastic livers decreases β-catenin/TCF interaction and consequently reduces TCF transcriptional activity probably via ROS induction. Furthermore, IFN α-2b-induced ROS production could stimulate β-catenin/FOXO interaction, thereby favoring cell cycle arrest and apoptosis. In agreement with this proposal, recent unpublished results from our group demonstrate the participation of ROS in these events.

Collectively, our data demonstrate that the canonical Wnt/β-catenin/TCF signaling pathway is activated at a very early stage of the development of the hepatocarcinogenic process, even with a wild-type β-catenin. More importantly, *in vivo* IFN α-2b treatment could be an efficient therapy to attenuate Wnt/β-catenin/TCF signaling promoting diminution of preneoplastic foci by an apoptotic process. A graphic outline of these concerns is shown in Figure 3.

**Figure 3.** Graphic outline of IFN α-2b, Wnt/β-catenin pathway and ROS/FOXO interactions in liver preneoplasia.

## **12. Studies in HCC cell lines**

The elucidation of the signals induced by IFN α and TGFβ in human liver tumor cells, and their possible cross-talks with other intracellular signals, would have relevance in the future design of therapeutic tools to balance the cellular responses in favor of liver tumor suppression. To gain mechanistic insights into these cooperative signals, we analyzed the effects of IFN α-2b and TGFβ1 on Wnt/β-catenin pathway and Smads intermediates in HepG2/C3A and Huh7 HCC cell lines. We could demonstrate that IFN α-2b or TGFβ1 stimulations not only decreased cellular proliferation but also increased apoptotic cell death [142]. The apoptotic and anti-proliferative effects of both cytokines separately have already been reported in HepG2 and Huh7 [143–145]. More interestingly, we demonstrated that the combined treatment increased these effects. Until now, combined treatment with both cytokines has only been used to analyze their impact on proliferation in human melanoma cell lines [109]. Treatments impact on Wnt/β-catenin pathway was analyzed, together with the analysis of the effects of IFN α-2b and TGFβ1 on Smads proteins. Insufficient information is available concerning TCF4/Smads association and their impact on carcinogenesis in HCC cell lines. Labbé et al. [146] and Letamendia et al. [147] reported the interaction between Smads 2, 3 and 4 and TCF/LEF in HepG2. Additionally, treatment with TGFβ1 in HepG2 reduced the amount of Smad4 protein bound to TCF/LEF and this was associated with the capacity of TGFβ1 of inhibiting cell proliferation [148]. To date, no study on IFN α and Smads has been carried out. More insight could be gained by analyzing the amount of each Smad protein inside the β-catenin/TCF4 nuclear complex, since all Smads coexist in this complex and the balance between them could contribute to the overall cell response by differently regulating gene expression as suggested by Edlund et al. [149].

Our findings clearly showed a negative modulation of IFN α-2b and TGFβ1 on Wnt/βcatenin pathway. This attenuation was evidenced by a decrease in β-catenin and Frizzled-7 receptor proteins levels in C3A and Huh7 and by a diminution in the amount of β-catenin bound to TCF4. Stimulation with both cytokines also caused a decrease in Smads protein contents and their association with TCF4. This effect on Smads proteins seems to be linked to the decrease of β-catenin. Finally, the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, since the overall action of IFN α-2b and/or TGFβ1 treatments on both HCC cell lines was the diminution in cellular proliferation and the increase in apoptotic cell death. In conclusion, our results support the efficacy of inhibiting Wnt/β-catenin pathway in HCC cell lines through an IFN α-2b and TGFβ1 combined treatment, proving that is effective against either wild-type or truncated βcatenin. These findings open a wide therapeutic option for patients with HCC.

#### **13. Summary # 2**

332 Lipid Peroxidation

preneoplasia.

**12. Studies in HCC cell lines** 

**Figure 3.** Graphic outline of IFN α-2b, Wnt/β-catenin pathway and ROS/FOXO interactions in liver

The elucidation of the signals induced by IFN α and TGFβ in human liver tumor cells, and their possible cross-talks with other intracellular signals, would have relevance in the future design of therapeutic tools to balance the cellular responses in favor of liver tumor suppression. To gain mechanistic insights into these cooperative signals, we analyzed the effects of IFN α-2b and TGFβ1 on Wnt/β-catenin pathway and Smads intermediates in HepG2/C3A and Huh7 HCC cell lines. We could demonstrate that IFN α-2b or TGFβ1 stimulations not only decreased cellular proliferation but also increased apoptotic cell death [142]. The apoptotic and anti-proliferative effects of both cytokines separately have already been reported in HepG2 and Huh7 [143–145]. More interestingly, we demonstrated that the combined treatment increased these effects. Until now, combined treatment with both cytokines has only been used to analyze their impact on proliferation in human melanoma cell lines [109]. Treatments impact on Wnt/β-catenin pathway was analyzed, together with the analysis of the effects of IFN α-2b and TGFβ1 on Smads proteins. Insufficient The presented data suggest a model in which IFN α-2b provides a link between TGFβ1 and Wnt signaling pathways and the oxidative stress/FOXO pathway. The stress caused by IFN α-2b treatment might strengthen the interaction between FOXO and β-catenin and potentially inhibit the interaction with TCF and Smads. The inhibition of βcatenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis. These findings may have important clinical implications, since β-catenin, Smads, TCF, and FOXO arise as molecular targets for novel therapies that can modify their interactions favoring cellular apoptosis over proliferation in patients that underwent a potential carcinogenic hepatic injury.

## **14. Concluding remarks: Oxidative stress as a critical factor in cancer therapy**

Preneoplastic hepatocytes are more resistant to oxidative stress than normal ones. Nevertheless, we demonstrated that increase in ROS levels triggered by IFN α-2b enhances the process of programmed hepatocytes death, leading to the elimination of malignant cells. The study of the mechanism of IFN α-2b-induced apoptosis led to demonstrate a link between TGFβ1 and Wnt signaling pathways and the oxidative stress/FOXO pathway.

In conclusion, reactive oxygen species emerge as key mediators in the context of using cytokines as therapeutic agents in the treatment of human liver diseases, so the use of antioxidants could have the potential to decrease effectiveness of the therapy.

## **Author details**

María Cristina Carrillo\* , María de Luján Alvarez, Juan Pablo Parody, Ariel Darío Quiroga and María Paula Ceballos *Institute of Experimental Physiology (IFISE-CONICET), Faculty of Biochemistry and Pharmacological Sciences, National University of Rosario, Rosario, Argentina*

## **Acknowledgement**

The authors kindly thank BioSidus Laboratory and Cassará Laboratory for the generous gift of recombinant IFN α-2b.

The work presented in this chapter was supported by research grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT).

#### **15. References**


<sup>\*</sup> Corresponding Author

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**Author details** 

María Cristina Carrillo\*

**Acknowledgement** 

of recombinant IFN α-2b.

**15. References** 

discussion 673–4.

Ariel Darío Quiroga and María Paula Ceballos

de Promoción Científica y Tecnológica (ANPCyT).

In conclusion, reactive oxygen species emerge as key mediators in the context of using cytokines as therapeutic agents in the treatment of human liver diseases, so the use of

, María de Luján Alvarez, Juan Pablo Parody,

The authors kindly thank BioSidus Laboratory and Cassará Laboratory for the generous gift

The work presented in this chapter was supported by research grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and from Agencia Nacional

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## **Lipid Peroxidation and Antioxidants in Arterial Hypertension**

Teresa Sousa, Joana Afonso, Félix Carvalho and António Albino-Teixeira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50346

## **1. Introduction**

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This chapter aims at giving a critical overview of the major oxidant and antioxidant changes in arterial hypertension, summarizing the experimental and clinical evidence about the involvement of oxidative stress in the pathophysiology of hypertension, either as a cause or a consequence of this disease. This review also provides a description of the biomarkers commonly used to evaluate lipid peroxidation and antioxidant defenses in experimental and human hypertension. Finally, we review the strategies (antioxidants, antihypertensive drugs) known to prevent or ameliorate oxidative damage, both in animal models of hypertension and hypertensive patients.

## **2. Pathophysiological role of oxidative stress in arterial hypertension**

## **2.1. ROS sources and oxidative pathways involved in the pathogenesis of hypertension**

In aerobic organisms, the beneficial effects of oxygen come with the price of reactive oxygen species (ROS) formation. These highly bioactive and short-lived molecules can interact with lipids, proteins and nucleic acids, causing severe molecular damage. However, living organisms have evolved specific mechanisms to adapt to the coexistence of ROS. In physiological conditions, there is a delicate balance between oxidants and antioxidants that not only protects our cells from the detrimental effects of reactive oxygen species (ROS), but also allows the existence of redox signaling processes that regulate cellular and organ functions. However, the disruption of redox homeostasis, leading to persistent high levels of ROS, is potentially pathological [1, 2]. Besides ROS, another group of molecules collectively designated as reactive nitrogen species (RNS) also exerts important functions in diverse physiological and pathological redox signaling processes. The excess of RNS is often termed nitrosative stress [3, 4].

© 2012 Sousa et al., licensee InTech. This is an open access chapter 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. © 2012 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.

ROS can be classified into two main categories: free radicals [e.g. superoxide (O2.-), hydroxyl (HO. ), peroxyl (ROO. )], which are highly reactive species due to the presence of one or more unpaired electrons, and non-radical oxidants [e.g. singlet oxygen (1O2) hydrogen peroxide (H2O2), hypochlorous acid (HOCl)] that have generally more specific reactivity and higher stability [3, 5, 6]. RNS include nitric oxide (. NO) and nitrogen dioxide radicals (. NO2 and also non radicals such as nitrous acid (HNO2), peroxynitrite (ONOO- ), peroxynitrous acid (ONOOH) and alkyl peroxynitrites (ROONO) [3]. Among biological ROS and RNS, O2.-, H2O2, . NO and ONOO appear to be especially relevant in neuronal, renal and vascular control of blood pressure [3, 7 ,8] (Table 1). Major sources of ROS (and also RNS) within these systems include, but are not limited to, NADPH oxidases, xanthine oxidase, mitochondrial respiratory chain enzymes, . NOsynthases and myeloperoxidase [3, 8, 9].



NADPH oxidases (Nox) are enzyme complexes that catalyze the reduction of molecular oxygen using NADPH as an electron donor. Generally, the product of the electron transfer reaction is O2.- but H2O2 is also rapidly formed from dismutation of Nox-derived O2.- due to the presence of superoxide dismutase (SOD) in the cells or by spontaneous reaction. Nox-derived ROS have been shown to play a role in host defense and also in diverse signaling processes [10]. The Nox family comprises seven members (Nox1-5 and Duox1-2) with distinct tissue distribution and functions [10, 11]. So far, only Nox1, Nox2 and Nox4 have been shown to play relevant roles in hypertension pathophysiology [5, 8, 10]. These isoforms are localized in major sites of blood pressure control. For example, Nox1, Nox2 and Nox4 are expressed in the central nervous system where they appear to regulate sympathetic nerve activity [8]. Nox2 and Nox4 participate in the regulation of renal functions and contribute to end-organ damage associated with hypertension [8, 12]. In the vasculature, Nox1 controls smooth muscle cell growth and migration, Nox2 contributes to endothelial dysfunction and Nox4 controls vascular smooth muscle cell differentiation and improves endothelial-dependent vasodilatation [8, 13, 14].

Xanthine oxidoreductase has two inter-convertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), that participate in purines metabolism catalyzing the conversion of hypoxanthine to xanthine and xanthine to uric acid [15, 16]. XDH preferentially uses NAD+ as an electron acceptor while the oxidase reduces molecular oxygen in a reaction that generates O2.- and H2O2 [15, 16]. The XO form predominates in oxidative stress conditions and may contribute to endothelium dysfunction due to its localization in the luminal surface of vascular endothelium [16, 17]. Besides the production of ROS by XO, both XDH and XO generate uric acid which possesses antioxidant properties, such as scavenging of ONOO and HO. , prevention of oxidative inactivation of endothelium enzymes and stabilization of Vitamin C [18-22]. On the other hand, uric acid may also have prooxidant and proinflammatory effects [23, 24]. Indeed, high systemic levels of uric acid are associated with increased cardiovascular disease and poor outcome but it is not clear whether these effects reflect deleterious actions of uric acid or the oxidative damage caused by XO-derived ROS [23, 25].

346 Lipid Peroxidation

), peroxyl (ROO.

NO and ONOO-

stability [3, 5, 6]. RNS include nitric oxide (.

mitochondrial respiratory chain enzymes, .

**RNS** .

(HO.

H2O2, .

**ROS** 

ROS can be classified into two main categories: free radicals [e.g. superoxide (O2.-), hydroxyl

unpaired electrons, and non-radical oxidants [e.g. singlet oxygen (1O2) hydrogen peroxide (H2O2), hypochlorous acid (HOCl)] that have generally more specific reactivity and higher

(ONOOH) and alkyl peroxynitrites (ROONO) [3]. Among biological ROS and RNS, O2.-,

control of blood pressure [3, 7 ,8] (Table 1). Major sources of ROS (and also RNS) within these systems include, but are not limited to, NADPH oxidases, xanthine oxidase,

**Table 1.** Reactive oxidant species involved in cardiovascular and renal physiology or pathophysiology

NADPH oxidases (Nox) are enzyme complexes that catalyze the reduction of molecular oxygen using NADPH as an electron donor. Generally, the product of the electron transfer reaction is O2.- but H2O2 is also rapidly formed from dismutation of Nox-derived O2.- due to the presence of superoxide dismutase (SOD) in the cells or by spontaneous reaction. Nox-derived ROS have been shown to play a role in host defense and also in diverse signaling processes [10]. The Nox family comprises seven members (Nox1-5 and Duox1-2) with distinct tissue distribution and functions [10, 11]. So far, only Nox1, Nox2 and Nox4 have been shown to play relevant roles in hypertension pathophysiology [5, 8, 10]. These isoforms are localized in major sites of blood pressure control. For example, Nox1, Nox2 and Nox4 are expressed in the central nervous system where they appear to regulate sympathetic nerve activity [8]. Nox2 and Nox4 participate in the regulation of renal functions and contribute to end-organ damage associated with hypertension [8, 12]. In the vasculature, Nox1 controls smooth muscle cell growth and migration, Nox2 contributes to endothelial dysfunction and Nox4 controls vascular smooth muscle cell differentiation and improves endothelial-dependent vasodilatation [8, 13, 14].

Xanthine oxidoreductase has two inter-convertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), that participate in purines metabolism catalyzing the conversion of hypoxanthine to xanthine and xanthine to uric acid [15, 16]. XDH preferentially uses NAD+ as an electron acceptor while the oxidase reduces molecular oxygen in a reaction that generates O2.- and H2O2 [15, 16]. The XO form predominates in oxidative stress conditions and may contribute to endothelium dysfunction due to its localization in the luminal surface of vascular endothelium [16, 17]. Besides the production of ROS by XO, both XDH and XO generate uric

prevention of oxidative inactivation of endothelium enzymes and stabilization of Vitamin C

acid which possesses antioxidant properties, such as scavenging of ONOO-

non radicals such as nitrous acid (HNO2), peroxynitrite (ONOO-

O2.- HO. ROO.

)], which are highly reactive species due to the presence of one or more

NO) and nitrogen dioxide radicals (.

NOsynthases and myeloperoxidase [3, 8, 9].

H2O2 HClO

appear to be especially relevant in neuronal, renal and vascular

**Free radicals Non radical oxidants** 

NO ONOO\_

NO2 and also

and HO.

,

), peroxynitrous acid

Mitochondrial respiratory chain enzymes are primary intracellular sources of ROS. More than 90% of the total oxygen consumed by aerobic organisms is utilized by mitochondrial oxidases which produce ATP in a process coupled to the reduction of cellular oxygen to water [26]. About 1-4% of the oxygen used in these reactions is converted to O2.- and H2O2 which can be largely detrimental to mitochondrial functions if not adequately detoxified [26- 28]. ROS levels in the mitochondria are regulated by the respiratory rate and manganese SOD [29]. Hypertensive animals have increased mitochondrial ROS production in the vessels, kidney and CNS [30-32].

. NO synthases (NOS) constitute a family of enzyme isoforms (neuronal NOS, nNOS; inducible NOS, iNOS; endothelial NOS, eNOS) that produce . NO in a reaction that converts L-arginine to L-citrulline [28]. However, in conditions of limited bioavailability of the cofactor tetrahydrobiopterin, or the substrate L-arginine, these enzymes become unstable and reduce molecular oxygen to O2.- instead of . NO production (uncoupled NOS) [28, 29]. NOS uncoupling is more often described for eNOS and is triggered by oxidative/nitrosative stress [28, 33]. Numerous experimental studies have shown that arterial hypertension is associated with eNOS dysregulation and endothelial dysfunction [34, 35].

Myeloperoxidase (MPO) is a heme protein secreted by activated neutrophiles and monocytes in inflammatory conditions and produces several oxidizing molecules that can affect lipids and proteins [28, 36]. MPO uses H2O2 to produce ROS such as HOCl, chloramines, tyrosyl radicals and nitrogen dioxides [36, 37]. Although MPO-derived ROS have a primary role in microbial killing, they also cause tissue damage in the heart, vessels, kidney and brain and appear to contribute to endothelial dysfunction [37, 40]. Figure 1 illustrates the major sources of ROS and/or RNS generation.

Of all the putative oxidative pathways involved in the pathogenesis of hypertension, the impairment of endothelial-dependent vasorelaxation by O2.- is by far the most studied [41- 44]. In conditions of increased O2.- bioavailability, this ROS rapidly inactivates endothelialderived . NO leading to endothelial dysfunction [41]. In addition, O2.- may also modulate vascular tone by increasing intracellular Ca2+ concentration in vascular smooth muscle cells and endothelial cells [45]. The imbalance between O2.- and . NO also affects the renal function, leading to enhanced sodium reabsorption and increased ONOO formation, which contributes to tissue damage [12, 46]. In the CNS, elevated O2.- generation also appears to contribute to hypertension by reducing the cardiovascular depressor actions of . NO in the rostral ventrolateral medulla [47]. In recent years H2O2 has also emerged as a pivotal molecule in the pathophysiology of arterial hypertension [48-50]. Of note, H2O2 seems to be even more harmful than O2.- due to its higher life span and diffusibility within and between cells [7, 51]. Furthermore, the conversion of O2.- to H2O2 appears to be favored in cardiovascular diseases since the expression and activity of SOD is enhanced by

inflammatory cytokines in hypertension or in response to the pressor peptide, angiotensin II [7]. Several prohypertensive effects have been described for H2O2, such as increased vasoconstriction, vascular hypertrophy and hyperplasia, decreased diuresis and natriuresis and also increased spinal sympathetic outflow [7, 50, 52-58]. Increasing evidence has also shown that H2O2 amplifies oxidative stress by stimulating ROS generation by NADPH oxidases, XO and eNOS [7, 51]. In addition, H2O2 also appears to enhance the activation of the intrarenal renin-angiotensin system, a major regulator of blood pressure and renal function [49]. Altogether, these effects propagate H2O2 generation and prolong the redox pathologic signaling involved in blood pressure dysregulation. The oxidative mechanisms contributing to hypertension are summarized in Table 2.

**Figure 1.** Sources of ROS and/or RNS generation - In normal cells, 1–2% of electrons carried by the mitochondrial electron transport chain leak from this pathway and pass directly to oxygen generating superoxide radical (O2.-) which can be a source of other ROS. O2.- can also be formed by xanthine oxidase (XO) which catalyzes the oxidation of hypoxanthine and xanthine. All NOX enzymes utilize NADPH as an electron donor and catalyze transfer of electrons to molecular oxygen to generate O2. and/or H2O2. Nitric Oxide synthases (NOS) generate . NO and L-citrulline from arginine and O2. Under pathologic conditions of oxidative stress, or when tetrahydrobiopterin (BH4) or L-arginine are deficient, NOS enzymes become structurally unstable (uncoupled NOS) resulting in production of O2.- rather than . NO. Activated monocytes also secrete a heme enzyme, myeloperoxidase(MPO), that uses H2O2 as a substrate to generate products that can oxidize lipids and proteins. One of these oxidants is hypochlorous acid (HOCI) which plays a critical role in host defenses against invading bacteria, viruses, and tumor cells but may also injure normal tissue. Within cell membranes, ROS can trigger lipid peroxidation, a self-propagating chain-reaction that can result in significant tissue damage.


**Table 2.** Putative oxidative pathways leading to arterial hypertension

348 Lipid Peroxidation

inflammatory cytokines in hypertension or in response to the pressor peptide, angiotensin II [7]. Several prohypertensive effects have been described for H2O2, such as increased vasoconstriction, vascular hypertrophy and hyperplasia, decreased diuresis and natriuresis and also increased spinal sympathetic outflow [7, 50, 52-58]. Increasing evidence has also shown that H2O2 amplifies oxidative stress by stimulating ROS generation by NADPH oxidases, XO and eNOS [7, 51]. In addition, H2O2 also appears to enhance the activation of the intrarenal renin-angiotensin system, a major regulator of blood pressure and renal function [49]. Altogether, these effects propagate H2O2 generation and prolong the redox pathologic signaling involved in blood pressure dysregulation. The oxidative mechanisms

**Figure 1.** Sources of ROS and/or RNS generation - In normal cells, 1–2% of electrons carried by the mitochondrial electron transport chain leak from this pathway and pass directly to oxygen generating superoxide radical (O2.-) which can be a source of other ROS. O2.- can also be formed by xanthine oxidase (XO) which catalyzes the oxidation of hypoxanthine and xanthine. All NOX enzymes utilize NADPH as an electron donor and catalyze transfer of electrons to molecular oxygen to generate O2.-

pathologic conditions of oxidative stress, or when tetrahydrobiopterin (BH4) or L-arginine are deficient, NOS enzymes become structurally unstable (uncoupled NOS) resulting in production of O2.- rather than

hypochlorous acid (HOCI) which plays a critical role in host defenses against invading bacteria, viruses, and tumor cells but may also injure normal tissue. Within cell membranes, ROS can trigger lipid peroxidation, a self-propagating chain-reaction that can result in significant tissue damage.

NO. Activated monocytes also secrete a heme enzyme, myeloperoxidase(MPO), that uses H2O2 as a

substrate to generate products that can oxidize lipids and proteins. One of these oxidants is

NO and L-citrulline from arginine and O2. Under

contributing to hypertension are summarized in Table 2.

and/or H2O2. Nitric Oxide synthases (NOS) generate .

.

#### **2.2. Evidence for redox changes in experimental and human hypertension**

In the last two decades several studies have consistently observed increased oxidative stress in experimental and human arterial hypertension. Studies in diverse experimental models of hypertension have demonstrated raised prooxidant activity and ROS levels, altered antioxidant defenses and increased ROS-mediated damage, both at peripheral and central sites of cardiovascular regulation [8, 33, 59]. In human hypertensive patients there is also evidence of redox dysfunction. O2.- release from peripheral polymorphonuclear leucocytes is higher in hypertensive patients than in normotensive subjects [60]. Plasma H2O2 production is also raised in hypertensive patients. Furthermore, among still normotensive subjects, those with a family history of hypertension have a higher H2O2 production [61, 62]. An elevation of several oxidative stress byproducts, such as malondialdehyde, 8-isoprostanes, 8-oxo-2'-deoxyguanosine, oxidized low density lipoproteins, carbonyl groups and nitrotyrosine has also been observed in plasma or serum, urine or blood cells of hypertensive patients [63-66]. Furthermore, both enzymatic and non-enzymatic antioxidant defenses appear to be significantly reduced in human hypertension [65, 67]. Alterations of redox biomarkers in human hypertension are summarized in Table 3.




NOx- nitrites and nitrates; PMN – Polymorphonuclear leucocytes; GSH – reduced glutathione; GSSG- oxidized glutathione;

**Table 3.** Altered oxidative/nitrosative stress biomarkers in human arterial hypertension

#### **2.3. Oxidative stress as a cause for arterial hypertension**

350 Lipid Peroxidation

**Prooxidant enzymes** 

**Oxidative or nitrosative stress byproducts** 

NADPH oxidase

Malondialdehyde (MDA)/Thiobarbituric

F2-Isoprostane (or 8 isoprostane or 8-epi-

Oxidized low density

**Antioxidants** GSH Mononuclear cells

Total antioxidant status

Ferric reducing activity of plasma (FRAP)

Uric acid Plasma

p22phox (Nox subunit) mRNA and protein

activity

expression

acid reactive substances (TBARS)

PGF2)

8-Oxo-2' deoxyguanosine

lipoproteins

GSH/GSSG

Vitamin C (ascorbic acid)

Vitamin E (-Tocopherol,

(TAS)

**Redox status** GSSG/GSH

**Biomarker Evaluated in: Alteration in** 

Mononuclear cells

Mononuclear cells

Plasma

Plasma

Urine

Erythrocytes

Mononuclear cells and whole blood

Carbonyl groups Serum [64]

3-Nitrotyrosine Plasma [66, 79]

Mononuclear cells and whole blood

Erythrocytes

Erytrocytes

Serum

Plasma Serum

Plasma

Plasma

and whole blood

**hypertensive patients** 

 

 

Erytrhocytes [67]

Plasma [63, 78]

**References** 

[71]

[71]

[67, 72]

[65]

[65]

[65]

[74]

[65]

[64]

[79] [80]

[67] [81]

[63, 82]

[74, 83]

[76, 77]

[64, 73, 74]

[63, 66, 74]

[63, 74, 75]

Whether oxidant imbalance is a cause or a consequence of high blood pressure remains a debatable question. The hypothesis that oxidative stress contributes to arterial hypertension is supported by several lines of evidence: (1) the induction of oxidative stress by the administration of lead or the glutathione synthesis inhibitor, buthionine sulfoximine, or the SOD inhibitor, sodium diethyldithiocarbamate, increases blood pressure in rats [48, 84]; (2) the infusion of H2O2 into the renal medulla leads to hypertension [48]; the treatment of hypertensive animals with antioxidants or inhibitors of ROS production prevents or attenuates hypertension [50, 85-87]; (3) the manipulation of genes related to ROS generation or elimination can alter blood pressure [88, 89]; (4) the *in vitro* exposure of cells and tissues to exogenous oxidants reproduces events involved in the pathophysiology of hypertension [43]; (5) systemic and tissue redox dysfunction appears to precede the blood pressure elevation [90].

#### **2.4. Oxidative stress as a consequence of arterial hypertension**

Arterial hypertension is associated with oscillatory shear stress and vascular stretch caused by increased vascular pressure. These mechanical forces are known to induce oxidative stress and vascular damage [91]. Furthermore, there is evidence that lowering blood pressure *per se* causes reduction of oxidative stress and improvement in endothelial function [92]. Several antihypertensive drugs with distinct mechanisms of action have been shown to decrease oxidant biomarkers in experimental and human hypertension [93-95]. However, there is limited evidence supporting the use of antioxidants to lower blood pressure in human hypertensive patients [5, 92]. Nevertheless, the failure of these studies does not

exclude a role for oxidative stress in human essential hypertension but instead suggests that the antioxidant supplementation approach was not the appropriate therapeutic strategy [96].

## **3. Biomarkers of redox status in arterial hypertension**

The evaluation of redox status may provide valuable information about the pathogenesis and progression of arterial hypertension and related cardiovascular and renal diseases. However, the short lifetime of ROS turns their assessment in animal models and humans a significant challenge, leading to a growing interest in the development and validation of oxidative stress biomarkers. Traditional approaches to evaluate oxidant status have frequently relied on indirect measurements of ROS bioavailability (e.g. evaluation of prooxidant and antioxidant activity, oxidized products from ROS and the GSH/GSSG ratio) as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to therapeutic intervention [9, 96-99].

A biomarker of oxidative stress is classically defined as a biological molecule whose chemical structure has been modified by ROS and that can be used to reliably assess oxidative stress status in animal models and humans [100]. The ideal biomarker of oxidative stress depends on its ability to contribute to an early indication of disease severity and/or its progression, as well as to evaluate therapy efficacy. The measurement of redox status biomarkers may also help to clarify the pathophysiologic mechanisms mediating oxidative injury and may allow the prediction of disease. Ideally, biomarkers of oxidative damage for human studies would be evaluated in specimens that can be collected relatively easily, such as blood or urine. However, to serve these purposes, an ideal biomarker of oxidative damage should fulfill several conditions, such as: a) being a stable product, not susceptible to artifactual induction, oxidation, or loss during sample handling, processing, analysis, and storage; b) having a well-established relationship with the generation of ROS and/or progression of disease; c) allowing direct assessment in a target tissue or being able to generate a valid substitute that quantitatively reflects the oxidative modification of the target tissue; d) being present at concentrations high enough to be a significant detectable product; e) showing high specificity for the reactive species in question and free of erroneous factors from dietary intake; f) being noninvasive; g) being measurable by a specific, sensitive, reproducible and inexpensive assay; h) being measurable across populations; i) being present in concentrations that do not vary widely in the same persons under the same conditions at different times [97].

#### **3.1. Systemic and tissue antioxidant defenses**

ROS are involved in many biological processes including cell growth, differentiation, apoptosis, immunity and defense against micro-organisms [1, 101, 102]. Low or moderate concentrations of ROS are beneficial for living organisms. However, high concentrations of ROS can cause direct damage of macromolecules such as DNA, proteins, carbohydrates, and lipids, or disrupt redox signaling and control pathways, leading to a myriad of human diseases [103]. ROS bioavailability is determined by the balance between their production by prooxidant enzymes and their clearance by various antioxidant compounds and enzymes [1]. As defined by Halliwell and Gutteridge, an antioxidant is any substance that, at low concentration, is able to significantly delay or inhibit the oxidation of an oxidizable substrate [104]. Biological antioxidant defenses have evolved to match the diversity of prooxidants and several enzymatic and non-enzymatic molecules exist in cells and body fluids to control ROS levels within the physiological range [105]. The coordinated action of antioxidants results in the interception and deactivation of the damaging species. For example, the radical chain events initiated by free radicals can be terminated by the interaction of radicals with different non-enzymatic antioxidants [e.g. GSH, ascorbic acid, uric acid, α-tocopherol, etc] or prevented by specialized enzymatic defenses such as SOD, catalase and glutathione peroxidase (GPx) [105, 106]. The reduction of antioxidants bioavailability disrupts redox homeostasis leaving organisms more vulnerable to oxidative damage. Therefore, antioxidants may be useful biomarkers for risk stratification and disease prognostication.

#### **3.2. Enzymatic antioxidants defenses**

352 Lipid Peroxidation

[96].

exclude a role for oxidative stress in human essential hypertension but instead suggests that the antioxidant supplementation approach was not the appropriate therapeutic strategy

The evaluation of redox status may provide valuable information about the pathogenesis and progression of arterial hypertension and related cardiovascular and renal diseases. However, the short lifetime of ROS turns their assessment in animal models and humans a significant challenge, leading to a growing interest in the development and validation of oxidative stress biomarkers. Traditional approaches to evaluate oxidant status have frequently relied on indirect measurements of ROS bioavailability (e.g. evaluation of prooxidant and antioxidant activity, oxidized products from ROS and the GSH/GSSG ratio) as indicators of normal biological processes, pathogenic processes, or pharmacologic

A biomarker of oxidative stress is classically defined as a biological molecule whose chemical structure has been modified by ROS and that can be used to reliably assess oxidative stress status in animal models and humans [100]. The ideal biomarker of oxidative stress depends on its ability to contribute to an early indication of disease severity and/or its progression, as well as to evaluate therapy efficacy. The measurement of redox status biomarkers may also help to clarify the pathophysiologic mechanisms mediating oxidative injury and may allow the prediction of disease. Ideally, biomarkers of oxidative damage for human studies would be evaluated in specimens that can be collected relatively easily, such as blood or urine. However, to serve these purposes, an ideal biomarker of oxidative damage should fulfill several conditions, such as: a) being a stable product, not susceptible to artifactual induction, oxidation, or loss during sample handling, processing, analysis, and storage; b) having a well-established relationship with the generation of ROS and/or progression of disease; c) allowing direct assessment in a target tissue or being able to generate a valid substitute that quantitatively reflects the oxidative modification of the target tissue; d) being present at concentrations high enough to be a significant detectable product; e) showing high specificity for the reactive species in question and free of erroneous factors from dietary intake; f) being noninvasive; g) being measurable by a specific, sensitive, reproducible and inexpensive assay; h) being measurable across populations; i) being present in concentrations that do not vary widely in the same persons

ROS are involved in many biological processes including cell growth, differentiation, apoptosis, immunity and defense against micro-organisms [1, 101, 102]. Low or moderate concentrations of ROS are beneficial for living organisms. However, high concentrations of ROS can cause direct damage of macromolecules such as DNA, proteins, carbohydrates, and lipids, or disrupt redox signaling and control pathways, leading to a myriad of human

**3. Biomarkers of redox status in arterial hypertension** 

responses to therapeutic intervention [9, 96-99].

under the same conditions at different times [97].

**3.1. Systemic and tissue antioxidant defenses** 

All eukaryotic cells possess powerful antioxidant enzymes which are responsible for neutralizing ROS.The first line of defense against ROS is achieved by SOD which is active in catalyzing the detoxication of O2.-. This radical can be readily converted into H2O2 by SOD enzymes present in the cytosol and organelles (Cu,Zn-SOD or SOD-1), mitochondria (Mn-SOD or SOD-2) and extracellular fluids (EC-SOD or SOD-3) [36, 107, 108]. H2O2 generated in this reaction can be further decomposed to water and oxygen. This is achieved primarily by catalase in the peroxisomes and also by GPx enzymes in the cytosol and mitochondria [107, 108]. GPx are selenium-containing enzymes whose activity is dependent on GSH availability [108]. Besides neutralizing H2O2, GPx also degrades lipid hydroperoxides to lipid alcohols [36]. These reactions lead to the oxidation of GSH to GSSG. Catalase and GPx are differentially required for the clearance of high-levels or low-levels of H2O2, respectively [36]. Figure 2 illustrates major antioxidant enzymatic pathways.

In addition to these key antioxidant enzymatic defenses, there are other specialized enzymes with direct and/or indirect antioxidant functions. Glutathione reductase (GR) is responsible for the replenishment of GSH from GSSG disulphide. Glutathione-S-transferase catalyzes the conjugation of GSH with reactive electrophiles and is also involved in the detoxification of some carbonyl-, peroxide- and epoxide-containing metabolites produced within the cell in oxidative stress conditions [109]. Peroxiredoxins are selenium-independent enzymes that decompose H2O2, organic hydroperoxides and peroxynitrite [110]. Thioredoxin (Trx) and glutaredoxin (Grx) systems include several enzymes that regulate the thiol-disulphide state of proteins and influence their structure and function [110]. Trx isoforms reduce disulphide bonds in proteins, especially in peroxiredoxins and Trx reductase regenerates the oxidized Trx. Grx protects proteins SH-groups from irreversible oxidation by catalyzing Sglutathionylation and restores functionally active thiols through catalysis of deglutathionylation [110]. Grx enzymes are functionally coupled to GR which reduces the GSSG produced in the deglutathionylation reaction [110].

**Figure 2.** Major antioxidant enzyme defenses

Hypertensive patients have reduced activity and decreased content of antioxidant enzymes, including SOD, GPx, and catalase [43]. However, several studies have also described an adaptive increase in antioxidant enzyme activities in some experimental models of hypertension [50, 111, 112]. The uncoordinated activity of antioxidant enzymes may aggravate oxidative stress. For example, the increased dismutation of O2.- by SOD significantly increases H2O2 concentration, and may lead to deleterious consequences for the tissue in the absence of compensation of catalase and GPx activities [113]. Examples of altered antioxidant defenses in human and experimental hypertension are shown in Table 3 and Table 4, respectively.



**Figure 2.** Major antioxidant enzyme defenses

SOD Brain expression and activity of Mn-SOD

Cu, Zn-SOD activity

SOD1 and SOD2

expression and activity of

and Table 4, respectively.

**Biomarker Evaluated in:** 

Hypertensive patients have reduced activity and decreased content of antioxidant enzymes, including SOD, GPx, and catalase [43]. However, several studies have also described an adaptive increase in antioxidant enzyme activities in some experimental models of hypertension [50, 111, 112]. The uncoordinated activity of antioxidant enzymes may aggravate oxidative stress. For example, the increased dismutation of O2.- by SOD significantly increases H2O2 concentration, and may lead to deleterious consequences for the tissue in the absence of compensation of catalase and GPx activities [113]. Examples of altered antioxidant defenses in human and experimental hypertension are shown in Table 3

**Alteration Hypertension Model Reference** 

(SHR)

SHR

Spontaneously hypertensive rats

[114]

[115]

[116]

Stroke prone spontaneously hypertensive rats (SHRSP)

> **Table 4.** Alterations in major antioxidant enzyme defenses in the brain, kidney and arteries in experimental models of hypertension

#### **3.3. Non-enzymatic antioxidants defenses**

Non-enzymatic antioxidants such as GSH, ascorbic acid (vitamin C) and α-tocopherol (vitamin E) play an excellent role in protecting the cells from oxidative damage [4]. GSH has a potent electron-donating capacity that renders GSH both a potent antioxidant *per se* and a conventional cofactor for enzymatic reactions that require readily available electron pairs. In physiological conditions, GSH is present inside the cells mainly in its reduced form and less than 10 percent of total GSH exists in the oxidized form, GSSG [127]. Therefore, intracellular GSH status can be used as a sensitive marker of the cell health and resistance to toxicity. Furthermore, it has been demonstrated that GSH depletion can lead to cell apoptosis [128]. The measurements of GSH and GSSG have been considered useful indicators of the status of oxidative stress [4, 129]. Vitamins E and C are among the major dietary antioxidants. The vitamins have received considerable attention in clinical trials of primary and secondary prevention of cardiovascular diseases (CVD) and cancer. Vitamin E is found in lipoproteins, cell membranes and extracellular fluids. It terminates lipid peroxidation processes and converts O2.-and HO. to less reactive forms [130]. Vitamin C, a water soluble antioxidant, is found in high concentrations in the adrenal and pituitary glands, liver, brain, spleen and pancreas. It is hydrophilic and can directly scavenge ROS and lipid hydroperoxides. Vitamin C can also restore oxidized vitamin E and can spare selenium [131]. Carotenoids, such as carotene are lipid soluble antioxidants that function as efficient scavengers of 1O2 but may also quench ROO. radicals [108]. Uric acid is a highly abundant aqueous antioxidant, considered to be the main contributor for the antioxidant capacity in the plasma [96, 132]. It has the ability to quenche HO. and ONOO and may prevent lipid peroxidation [21, 132]. The scavenging of ONOO by uric acid is significantly increased in the presence of Vitamin C and cysteine which regenerate the urate radical formed in these reactions. Uric acid also acts as a chelator of iron in extracellular fluids [16]. However, once inside the cells, uric acid appears to exert prooxidant effects. It is not clear whether the correlation between the raised plasma levels of uric acid and cardiovascular risk are due to increased ROS generation by XO or to the prooxidative effects of uric acid itself. Some authors speculate that the increased concentrations of urate might be an adaptive mechanism that confers protection from oxidative damage [132]. It is likely that uric acid effects have different consequences depending on the surrounding microenvironment [21]. Bilirrubin, the end-product of heme catabolism, also appears to function as a chainbreaking antioxidant [133]. Low circulating bilirrubin levels are considered a risk factor for cardiovascular diseases [134]. Plasma albumin, the predominant plasma protein, is also an antioxidant due to its sulfhydryl groups and is able to scavenge MPO-derived chlorinated reactive species and ROO. radicals [108, 135].

The combined antioxidant activities of aqueous- and lipid-antioxidants, including GSH, vitamins, uric acid, bilirrubin, albumin, etc, can be evaluated in the plasma and serum by several assays that measure the ability of the antioxidants present in the sample to inhibit the oxidation of the cation radical ABTS+ [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid] (Total Antioxidant Status assay), to reduce a ferric-tripyridyltriazine complex (Ferric Reducing Ability of Plasma, FRAP assay) or to trap free radicals (Oxygen Radical Absorbance Capacity, ORAC assay; Total radical Trapping Parameter, TRAP) [50, 136-138]. The measurement of the overall antioxidant capacity may be more representative of the *in vivo* balance between oxidizing and antioxidant compounds than the evaluation of individual antioxidants [139]. Nevertheless, these assays have also some limitations. First, they correlate poorly with each other as the various antioxidants react differently in each assay. Second, in biological fluids, uric acid appears to account for more than 50% of the total antioxidant activity measured in most assays [108]. However, the putative protective effect of uric acid is debatable [140, 141].

Under conditions of high ROS levels it is expected a decrease of non-enzymatic antioxidants defenses in plasma, since the need for neutralization ROS species implies a higher consumption of endogenous antioxidants. For example, decreased levels of antioxidant vitamins C and E have been demonstrated in newly diagnosed untreated hypertensive patients compared with normotensive control subjects [142-144].

#### **3.4. Systemic, urinary and tissue markers of lipid peroxidation**

Measuring oxidative stress in biological systems is complex and requires accurate quantification of ROS or damaged biomolecules. One method to quantify oxidative stress is to measure lipid peroxidation. Lipids that contain unsaturated fatty acids with more than one double bond are particularly susceptible to the action of free radicals. The peroxidation of lipids disrupts biological membranes and is thereby highly deleterious to its structure and function [145]. A large number of by-products are formed during this process and can be measured by different assays. Common biomarkers of lipid peroxidation damage include hydroperoxides, which are primary products generated in the initial stages of lipid peroxidation, and secondary products formed at later lipid peroxidation stages, such as malondialdehyde (MDA) or F2-isoprostanes (Table 5) [146, 147] .The lag time required for the exponential generation of lipid peroxidation products can also be used to evaluate the susceptibility of lipid molecules to free radical damage. Therefore, lipids with higher resistance to oxidative stress exhibit longer lag times than those which are easily attacked by free radicals [147].

#### **3.5. F2-isoprostanes**

356 Lipid Peroxidation

converts O2.-and HO.

quenche HO.

ONOO-

and ONOO-

reactive species and ROO. radicals [108, 135].

**3.3. Non-enzymatic antioxidants defenses** 

Non-enzymatic antioxidants such as GSH, ascorbic acid (vitamin C) and α-tocopherol (vitamin E) play an excellent role in protecting the cells from oxidative damage [4]. GSH has a potent electron-donating capacity that renders GSH both a potent antioxidant *per se* and a conventional cofactor for enzymatic reactions that require readily available electron pairs. In physiological conditions, GSH is present inside the cells mainly in its reduced form and less than 10 percent of total GSH exists in the oxidized form, GSSG [127]. Therefore, intracellular GSH status can be used as a sensitive marker of the cell health and resistance to toxicity. Furthermore, it has been demonstrated that GSH depletion can lead to cell apoptosis [128]. The measurements of GSH and GSSG have been considered useful indicators of the status of oxidative stress [4, 129]. Vitamins E and C are among the major dietary antioxidants. The vitamins have received considerable attention in clinical trials of primary and secondary prevention of cardiovascular diseases (CVD) and cancer. Vitamin E is found in lipoproteins, cell membranes and extracellular fluids. It terminates lipid peroxidation processes and

found in high concentrations in the adrenal and pituitary glands, liver, brain, spleen and pancreas. It is hydrophilic and can directly scavenge ROS and lipid hydroperoxides. Vitamin C can also restore oxidized vitamin E and can spare selenium [131]. Carotenoids, such as carotene are lipid soluble antioxidants that function as efficient scavengers of 1O2 but may also quench ROO. radicals [108]. Uric acid is a highly abundant aqueous antioxidant, considered to be the main contributor for the antioxidant capacity in the plasma [96, 132]. It has the ability to

by uric acid is significantly increased in the presence of Vitamin C and cysteine which

regenerate the urate radical formed in these reactions. Uric acid also acts as a chelator of iron in extracellular fluids [16]. However, once inside the cells, uric acid appears to exert prooxidant effects. It is not clear whether the correlation between the raised plasma levels of uric acid and cardiovascular risk are due to increased ROS generation by XO or to the prooxidative effects of uric acid itself. Some authors speculate that the increased concentrations of urate might be an adaptive mechanism that confers protection from oxidative damage [132]. It is likely that uric acid effects have different consequences depending on the surrounding microenvironment [21]. Bilirrubin, the end-product of heme catabolism, also appears to function as a chainbreaking antioxidant [133]. Low circulating bilirrubin levels are considered a risk factor for cardiovascular diseases [134]. Plasma albumin, the predominant plasma protein, is also an antioxidant due to its sulfhydryl groups and is able to scavenge MPO-derived chlorinated

The combined antioxidant activities of aqueous- and lipid-antioxidants, including GSH, vitamins, uric acid, bilirrubin, albumin, etc, can be evaluated in the plasma and serum by several assays that measure the ability of the antioxidants present in the sample to inhibit the oxidation of the cation radical ABTS+ [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid] (Total Antioxidant Status assay), to reduce a ferric-tripyridyltriazine complex (Ferric Reducing Ability of Plasma, FRAP assay) or to trap free radicals (Oxygen Radical Absorbance Capacity, ORAC assay; Total radical Trapping Parameter, TRAP) [50, 136-138].

to less reactive forms [130]. Vitamin C, a water soluble antioxidant, is

and may prevent lipid peroxidation [21, 132]. The scavenging of

F2-isoprostanes are prostaglandin F2α isomers primarily produced by free radical-catalyzed peroxidation of the polyunsaturated fatty acid (PUFA), arachidonic acid [97]. Although there is also evidence of F2-isoprostane formation by the action of cyclooxygenase, it is currently assumed that systemic and urinary F2-isoprostanes are mostly derived from free radical-induced lipid peroxidation, independently of cyclooxygenase enzymatic activity. Therefore, F2-isoprostanes have been regarded as reliable biomarkers of oxidative stress. Furthermore, F2-isoprostanes have been shown to exert potent vasoconstrictor effects on animal and human vessels, suggesting a pathogenic role in cardiovascular diseases and have been extensively used as markers of lipid peroxidation in human diseases [74, 75,148]. Their high stability and presence in measurable concentrations in many biological tissues and fluids, under physiological and pathological conditions, has also allowed the establishment of reference intervals and the comparison or monitoring of disease states

[97,149,150]. Urine specimens are particularly suited for F2-isoprostanes measurements. First, the ex vivo formation of F2 isoprostanes is minimized in these samples due to the low urinary lipid content, avoiding the need for time-sensitive sample processing [97, 149, 151, 152]. Second, they provide a noninvasive route for systemic oxidative stress evaluation. Although they can also be locally produced in the kidney, many studies have demonstrated that urinary F2-isoprostanes are mainly derived from free F2-isoprostanes filtered from the circulation [97, 149, 151, 152]. Only hydrolyzed isoprostanes are excreted into the urine whereas blood plasma samples contain both free and esterified isoprostanes. Since plasma samples have considerable amounts of arachidonic acid, the addition of preservatives, such as butylated hydroxytoluene (BHT) and indomethacin, and the storage at -80°C, are recommended to avoid degradation and/or ex vivo formation of F2-isoprostanes [97.

#### **3.6. TBARS**

The free radical attack to PUFAs in cellular membranes leads to the disruption of cell structure and function. MDA, one of the end products of these oxidative reactions, can be detected in several biological fluids and tissues and is therefore used as a biomarker of lipid peroxidation and oxidative stress [153]. High MDA levels indicate a high rate of lipid peroxidation [154]. The reaction of MDA with 2-thiobarbituric acid (TBA) is frequently used to estimate oxidative stress [155]. MDA reacts with TBA under conditions of high temperature and acidity generating 2-thiobarbituric acid reactive substances (TBARS) that can be measured either spectrophotometrically or spectrofluorometrically. However, these products can also be formed by sample autooxidation under assay conditions or by cross-reactivity with non-MDA substrates such as bile pigments, proteins, carbohydrates and other aldehydes. Therefore, TBARS measurements often originate doubts due to their limited specificity as markers of lipid peroxidation [156]. Nevertheless, undesirable autooxidation and non-MDA substrates reactivity can be minimized by adding BHT during sample preparation. Plasma TBARS measurements have been reported to correlate with some clinical features of cardiovascular disease, preeclampsia, ischemia/reperfusion, chronic kidney disease and cerebrovascular disorders [157-160]. Since the TBARS assay may overestimate MDA, other methods can be used to evaluate lipid peroxidation products, such as the lipid hydroperoxide (LPO) test. The principle of the LPO test is that in the presence of hemoglobin, lipid hydroperoxides are reduced to hydroxyl derivates with the equimolar production of a methylene blue product, which can be quantified spectrophotometrically [161].

#### **3.7. HNE**

The aldehyde 4-hydroxy-2-nonenal (4-HNE) is one of the most cytotoxic products of free radical attack on ω6-PUFA, namely arachidonic and linoleic acids, being able to react with diverse biological molecules such as proteins, peptides, phospholipids and nucleic acids. It also acts as an important mediator of oxidant-induced signaling, cellular proliferation and apoptosis [97, 162]. 4-HNE can be detected in plasma and several biological tissues under physiological conditions but its generation is significantly raised in pathological states associated with oxidative stress [97, 162-164]. Renovascular hypertensive rats showed increased 4-HNE deposition in the intima of injured mesenteric arteries, suggesting the presence of free radical injury and cytotoxicity induced by 4-HNE [163]. A wide diversity of effects have been demonstrated for 4-HNE depending on its concentration. Concentrations below 0.1 microM are within the physiological range and appear to induce chemotaxis and stimulation of guanylate cyclase and phospholipase C [165]. 4-HNE concentrations between 1-20 microM inhibit DNA and protein synthesis and stimulate phospholipase A2. Higher concentrations (100 microM and above) are cytotoxic and genotoxic leading to cell death [165]. Thus, 4-HNE represents a broad indicator of lipid peroxidation.

#### **3.8. Early stage of lipid peroxidation products**

358 Lipid Peroxidation

**3.6. TBARS** 

**3.7. HNE** 

[97,149,150]. Urine specimens are particularly suited for F2-isoprostanes measurements. First, the ex vivo formation of F2 isoprostanes is minimized in these samples due to the low urinary lipid content, avoiding the need for time-sensitive sample processing [97, 149, 151, 152]. Second, they provide a noninvasive route for systemic oxidative stress evaluation. Although they can also be locally produced in the kidney, many studies have demonstrated that urinary F2-isoprostanes are mainly derived from free F2-isoprostanes filtered from the circulation [97, 149, 151, 152]. Only hydrolyzed isoprostanes are excreted into the urine whereas blood plasma samples contain both free and esterified isoprostanes. Since plasma samples have considerable amounts of arachidonic acid, the addition of preservatives, such as butylated hydroxytoluene (BHT) and indomethacin, and the storage at -80°C, are

recommended to avoid degradation and/or ex vivo formation of F2-isoprostanes [97.

which can be quantified spectrophotometrically [161].

The free radical attack to PUFAs in cellular membranes leads to the disruption of cell structure and function. MDA, one of the end products of these oxidative reactions, can be detected in several biological fluids and tissues and is therefore used as a biomarker of lipid peroxidation and oxidative stress [153]. High MDA levels indicate a high rate of lipid peroxidation [154]. The reaction of MDA with 2-thiobarbituric acid (TBA) is frequently used to estimate oxidative stress [155]. MDA reacts with TBA under conditions of high temperature and acidity generating 2-thiobarbituric acid reactive substances (TBARS) that can be measured either spectrophotometrically or spectrofluorometrically. However, these products can also be formed by sample autooxidation under assay conditions or by cross-reactivity with non-MDA substrates such as bile pigments, proteins, carbohydrates and other aldehydes. Therefore, TBARS measurements often originate doubts due to their limited specificity as markers of lipid peroxidation [156]. Nevertheless, undesirable autooxidation and non-MDA substrates reactivity can be minimized by adding BHT during sample preparation. Plasma TBARS measurements have been reported to correlate with some clinical features of cardiovascular disease, preeclampsia, ischemia/reperfusion, chronic kidney disease and cerebrovascular disorders [157-160]. Since the TBARS assay may overestimate MDA, other methods can be used to evaluate lipid peroxidation products, such as the lipid hydroperoxide (LPO) test. The principle of the LPO test is that in the presence of hemoglobin, lipid hydroperoxides are reduced to hydroxyl derivates with the equimolar production of a methylene blue product,

The aldehyde 4-hydroxy-2-nonenal (4-HNE) is one of the most cytotoxic products of free radical attack on ω6-PUFA, namely arachidonic and linoleic acids, being able to react with diverse biological molecules such as proteins, peptides, phospholipids and nucleic acids. It also acts as an important mediator of oxidant-induced signaling, cellular proliferation and apoptosis [97, 162]. 4-HNE can be detected in plasma and several biological tissues under physiological conditions but its generation is significantly raised in pathological states associated with oxidative stress [97, 162-164]. Renovascular hypertensive rats showed Lipid hydroperoxides are the primary products of lipid peroxidation and can further react to form secondary products including aldehydes such as MDA and 4-HNE [166,167]. Therefore, lipid hydroperoxides may be used to evaluate initial stage or acute lipid peroxidation while MDA and 4-HNE appear to be more representative of chronic oxidative stress. Recent reports described that 13‐hydroperoxyoctadecadienoic acid (13‐HPODE), a precursor to 3‐hydroxyoctadecadienoic acid (13‐HODE) is able to react with proteins forming adducts by covalently binding to specific amino acid residues. The Hexanoyl‐ Lysine (HEL) adduct results from the oxidative modification of ω6‐PUFAs such as linoleic acid, the predominant PUFA in the human diet, and arachidonic acid [168]. HEL may be another useful biomarker for detecting and quantifying the earlier stages of lipid peroxidation. Monoclonal antibodies and ELISA kits have been developped, and HEL can be detected in oxidatively modified LDL, in human atherosclerotic lesions, human urine and serum. It has been also reported that HEL is formed in rat muscle during exercise and that its formation is inhibited by antioxidants such as flavonoids [169].


The lipid peroxidation biomarkers most commonly evaluated in hypertensive patients or experimental hypertension are shown in Table 3 and Table 5, respectively.


**Table 5.** Lipid peroxidation biomarkers in experimental hypertension

#### **3.9. Other prooxidant biomarkers**

Besides antioxidants and lipid peroxidation parameters, there are other important indexes of oxidant status. These include the expression and activity of prooxidant enzymes, ROS concentration, byproducts formed by ROS/RNS interaction with DNA (8-hydroxy-2 deoxyguanosine) or proteins (3-nitrotyrosine, carbonyl groups) and redox-sensitive transcription factors such as nuclear factor kappa B (NF-KB). Major sources of cellular ROS include Nox enzymes, mitochondrial electron transport enzymes, uncoupled NOS, XO and MPO. Table 6 summarizes several prooxidant biomarkers evaluated in experimental models of hypertension.



**Table 6.** Other Prooxidant status biomarkers in experimental hypertension

360 Lipid Peroxidation

**Lipid peroxidation biomarker** 

of hypertension.

**Biomarkers of prooxidant status** 

Mitochondrial electron transport chain enzymes/

mitochondrial ROS production

NADPH oxidase family enzymes (or

NOXs)

F2-**Isoprostanes** Plasma

Plasma

Urine

Aorta

**4-HNE** Mesenteric arteries

**3.9. Other prooxidant biomarkers** 

**Measured in: Alteration Experimental model of** 

Brain Oxidative impairment

Kidney mitochondrial ROS production

Brain NADPH oxidase activity

Kidney Nox4 expression,

Arteries **expression of** 

activity

of mitochondrial enzymes

NADPH oxidase

**NAD(P)H oxidase subunits (p67(phox) and gp91(phox)** 

 **Nox1 and Nox4 expression** 

**Table 5.** Lipid peroxidation biomarkers in experimental hypertension

**4-HNE adducts** Blood SHR [178]

Besides antioxidants and lipid peroxidation parameters, there are other important indexes of oxidant status. These include the expression and activity of prooxidant enzymes, ROS concentration, byproducts formed by ROS/RNS interaction with DNA (8-hydroxy-2 deoxyguanosine) or proteins (3-nitrotyrosine, carbonyl groups) and redox-sensitive transcription factors such as nuclear factor kappa B (NF-KB). Major sources of cellular ROS include Nox enzymes, mitochondrial electron transport enzymes, uncoupled NOS, XO and MPO. Table 6 summarizes several prooxidant biomarkers evaluated in experimental models

**hypertension** 

hypertension

**Evaluated in: Alteration Hypertension model Reference** 

SHR [30]

Salt-loaded SHRSP [181]

[179, 180]

[49]

[182]

[183]

SHR, Mineralocorticoid

hypertension

Ang II–induced hypertension

Ang II-induced hypertension

SHRSP

SHR

SHRSP

Salt-sensitive hypertension

Renovascular hypertension

Glucocorticoid-induced

**References** 

[175]

[87]

[170, 176]

[163]

[177]

## **4. Prophylactic and therapeutic strategies to reduce oxidative damage in arterial hypertension**

A plethora of studies has demonstrated that hypertension is associated with an imbalance between oxidants and antioxidants that leads to altered cell signaling and oxidative damage. Therefore, extensive research has been conducted in order to identify the ROS involved in blood pressure dysregulation, as well as the major prooxidant enzymes and antioxidant defenses that contribute to the loss of redox homeostasis in cardiovascular and renal systems. Furthermore, studies on experimental models of hypertension recognized several important neurohumoral stimuli responsible for ROS overproduction and also the main targets for ROS-induced dysfunction [8, 43]. Therapeutic interventions to reduce oxidative stress in hypertension have mostly relied on the administration of drugs that increase antioxidant capacity or inhibit ROS generation. In addition, other strategies aimed at reducing the activation of neurohumoral pathways that stimulate ROS production (upstream mediators) or at blocking/repairing the downstream targets affected by ROS have also been tested [196, 198, 199].

#### **4.1. Targeting oxidative stress in experimental hypertension**

The pharmacological modulation of ROS bioavailability in animal models of hypertension has been useful to demonstrate a causative role for oxidative stress in the pathophysiology of hypertension [43, 50]. However, the blood pressure lowering efficacy of these strategies appears to differ when comparing distinct experimental models [48, 50, 85, 200, 201]. This is probably because the development of each animal model was based on a particular etiological factor presumably responsible for human hypertension, such as high salt intake, overactivation of the renin-angiotensin system, genetic factors or renal disease. Since these factors may stimulate different redox pathways, the effectiveness of an antioxidant in one model does not necessarily translate to other models or to human essential hypertension which is known to have a multifactorial nature. Another important observation is that treatments with antioxidants or ROS inhibitors are generally more effective in preventing rather than reversing the hypertension [49, 50, 87, 202]. Indeed, there are several studies demonstrating that ROS activate feed-forward mechanisms that amplify the cardiovascular and renal dysfunction [8, 43, 49, 51]. Once triggered, these pathways may be sufficient to sustain the deleterious effects of oxidative stress even after ROS blockade or elimination [49]. *In vivo* drug treatments targeting oxidative stress in experimental models of hypertension are reviewed below and their effects on blood pressure are summarized in Table 7.

#### *4.1.1. Drugs inhibiting ROS production*

**Apocynin** is a methoxy-substituted catechol (4-hydroxy-3-methoxy-acetophenone), originally extracted from the roots of the tradicional medicinal herb *Picrorhiza kurroa* which has antiinflammatory properties [203]. Several experimental studies have used apocynin for its ability to inhibit Nox enzymes. The mechanism of inhibition involves the blockade of translocation of cytosolic protein subunits to the membrane which is crucial for the activation of Nox1 and Nox2 [204]. Thus, the effect of apocynin is restricted to inducible Nox enzymes that require cytosolic activators and it does not seem to affect constitutively active Nox isoforms and their putative physiological actions [204]. However, to be an effective Nox inhibitor, apocynin has to undergo a peroxidase-mediated oxidation to be converted into the metabolically active diapocynin [205-207]. The activation of apocynin occurs in the presence of MPO and H2O2 [205, 207]. This fact suggests that apocynin may function only in conditions of high inflammatory and prooxidant activity. Apocynin has also been shown to have direct antioxidant properties, being able to scavenge H2O2 derived products [205, 207]. However, it can also function as a prooxidant in resting cells [203]. Nevertheless, it is possible that when administered in conditions of enhanced oxidative stress, the protective effect prevails.

**Gp91ds-tat** is a chimeric peptide that specifically inhibits NADPH oxidase by preventing the assembly of its subunits. It is constituted by a segment of gp91phox (*gp91ds*) important for the interaction of this membrane subunit with the cytosolic subunit, p47phox, and by a *tat* peptide from the HIV virus, which allows the uptake of the peptide into the cell [208, 209]. However, since it is a peptide it may have poor oral bioavailability and may induce sensitization reactions. Furthermore, the *tat* segment may have side effects on cellular signaling and activity [204, 208]. Thus, it is not suitable for long treatments or to clinical use in the treatment of human cardiovascular diseases. Although it was designed to block Nox2, it may also inhibit Nox1 given the substantial degree of homology between the two isoforms [204]. As for apocynin, Nox4 is not likely to be affected by gp91ds-tat since it is constitutively active and does not require the activation of cytosolic subunits [204].

**Allopurinol** and its metabolite **oxypurinol** are hypoxanthine and xanthine analogs, respectively, that inhibit XO activity [16]. At low concentrations, allopurinol is a competitive inhibitor of XO, while at higher concentrations it behaves as a non-competitive inhibitor [16]. XO rapidly metabolizes allopurinol into oxypurinol, a noncompetitive inhibitor of the enzyme which has a much higher half-life and is therefore responsible for most of the pharmacological effects of allopurinol [16]. In addition, both allopurinol and oxypurinol have intrinsic antioxidant properties, being able to scavenge ROS such as O2.-, HO. and HClO [210-212]. However, these effects appear to require higher doses than those required for XO inhibition [210]. Allopurinol is approved for the treatment of human patients with gout or hyperuricemia, but it has also potential therapeutic application in cardiovascular diseases. Most common adverse effects are nauseas, diarrheas, hypersensitivity reactions and skin rash [16].

#### *4.1.2. Antioxidants*

362 Lipid Peroxidation

Table 7.

*4.1.1. Drugs inhibiting ROS production* 

also been tested [196, 198, 199].

Therefore, extensive research has been conducted in order to identify the ROS involved in blood pressure dysregulation, as well as the major prooxidant enzymes and antioxidant defenses that contribute to the loss of redox homeostasis in cardiovascular and renal systems. Furthermore, studies on experimental models of hypertension recognized several important neurohumoral stimuli responsible for ROS overproduction and also the main targets for ROS-induced dysfunction [8, 43]. Therapeutic interventions to reduce oxidative stress in hypertension have mostly relied on the administration of drugs that increase antioxidant capacity or inhibit ROS generation. In addition, other strategies aimed at reducing the activation of neurohumoral pathways that stimulate ROS production (upstream mediators) or at blocking/repairing the downstream targets affected by ROS have

The pharmacological modulation of ROS bioavailability in animal models of hypertension has been useful to demonstrate a causative role for oxidative stress in the pathophysiology of hypertension [43, 50]. However, the blood pressure lowering efficacy of these strategies appears to differ when comparing distinct experimental models [48, 50, 85, 200, 201]. This is probably because the development of each animal model was based on a particular etiological factor presumably responsible for human hypertension, such as high salt intake, overactivation of the renin-angiotensin system, genetic factors or renal disease. Since these factors may stimulate different redox pathways, the effectiveness of an antioxidant in one model does not necessarily translate to other models or to human essential hypertension which is known to have a multifactorial nature. Another important observation is that treatments with antioxidants or ROS inhibitors are generally more effective in preventing rather than reversing the hypertension [49, 50, 87, 202]. Indeed, there are several studies demonstrating that ROS activate feed-forward mechanisms that amplify the cardiovascular and renal dysfunction [8, 43, 49, 51]. Once triggered, these pathways may be sufficient to sustain the deleterious effects of oxidative stress even after ROS blockade or elimination [49]. *In vivo* drug treatments targeting oxidative stress in experimental models of hypertension are reviewed below and their effects on blood pressure are summarized in

**Apocynin** is a methoxy-substituted catechol (4-hydroxy-3-methoxy-acetophenone), originally extracted from the roots of the tradicional medicinal herb *Picrorhiza kurroa* which has antiinflammatory properties [203]. Several experimental studies have used apocynin for its ability to inhibit Nox enzymes. The mechanism of inhibition involves the blockade of translocation of cytosolic protein subunits to the membrane which is crucial for the activation of Nox1 and Nox2 [204]. Thus, the effect of apocynin is restricted to inducible Nox enzymes that require cytosolic activators and it does not seem to affect constitutively active Nox isoforms and their putative physiological actions [204]. However, to be an effective Nox inhibitor, apocynin has to undergo a peroxidase-mediated oxidation to be converted into the metabolically active

**4.1. Targeting oxidative stress in experimental hypertension** 

**Tempol** (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a membrane-permeable nitroxide that catalyzes the conversion of O2.- to H2O2 thus functioning as a SOD mimetic [213, 214]. Tempol protects the lipids or proteins from oxidative damage and interacts with other antioxidants to promote the reduction of oxidized lipids [214]. The main antihypertensive effect of this drug is related to the reduction of the O2.- interaction with . NO which improves vasodilation [213, 214]. It also promotes natriuresis by enhancing the vasodilation of renal medullary vessels in a . NO independent manner [214]. Indeed, tempol has been shown to have sympatholytic actions, being able to inhibit afferent, peripheral and central activation of the sympathetic nervous system [214]. These actions are responsible for the rapid fall of blood pressure and heart rate after acute intravenous administration of tempol [214]. Nevertheless, some studies reported that the formation of H2O2 by tempol can

counteract its vasodilator, natriuretic and antihypertensive effects in models of hypertension where H2O2 plays a more prominent role than O2.- [48, 50, 53]. The co-administration of catalase in these conditions restores the protective action of tempol [48, 50].

**N-acetylcysteine** (NAC) is a thiol containing compound. It is the acetylated derivative of the aminoacid L-cysteine and a precursor for reduced glutathione (GSH) [215, 216]. It appears to have direct antioxidant action since its free thiol can interact with the electrophilic groups of ROS [215]. However, this effect does not seem likely to occur in vivo because NAC has poor oral bioavailability being rapidly metabolized into GSH, among other metabolites [216]. Thus, the main protective action of NAC is probably related to its role as a GSH precursor, which then detoxifies reactive species either by enzymatic or non-enzymatic reactions [216]. In humans, NAC is approved as a mucolytic agent because it destroys the disulphide bridges of mucoproteins [215]. It is also used as an antidote for acetaminophen poisoning which dramatically depletes hepatic GSH content causing severe damage [217]. NAC may also have potential therapeutical applications in the treatment of heart diseases [218].

**Polyethylene glycol-catalase** is the conjugated form of the enzyme catalase with polyethylene glycol (PEG) which enhances the stability in aqueous solution, reduces immunogenicity and decreases sensitivity to proteolysis, thus increasing the circulatory half-life of catalase [219]. PEG also enhances the catalase association with cells [219]. The antioxidant effect of PEG-catalase results from the enzymatic degradation of H2O2 to water.

**Ebselen** (2-phenyl-1,2-benzisoselenazol-3[2H]-one) is a lipid-soluble seleno-organic compound that mimics glutathione peroxidase activity, being able to react with H2O2 and organic hydroperoxides including membrane-bound phospholipid and cholesterylester hydroperoxides [220]. It appears to reduce lipid peroxidation initiated by hydroperoxides but not free radicals initiators [221]. In addition, ebselen reacts rapidly with ONOO- . The ebselen selenoxid product yielded in this reaction is regenerated to ebselen by GSH, which allows its reutilization as a defense against ONOO- [222, 223]. Ebselen also directly inhibits inflammation-related enzymes such as 5-lipoxygenase, . NO synthases, protein kinase C, NADPH oxidase and H+/K+-ATPase by reacting with the SH group, leading to the formation of a selenosulphide complex [221]. Some authors have also proposed that the antioxidant and anti-inflammatory actions of ebselen are mediated through interactions with the thioredoxin (Trx) system [220]. Reduced Trx is important for growth and redox regulation by thiol redox control [220]. Ebselen was found to be an excellent substrate for mammalian TrxR and a highly efficient oxidant of reduced Trx. It also seems to function as a Trx peroxidase or peroxiredoxin mimic, thus contributing to the elimination of H2O2 and lipid hydroperoxides [220]. Ebselen has been used in clinical trials for the treatment of patients with acute ischemic stroke or delayed neurological deficit after aneurismal subarachnoid hemorrhage [224, 225].

**Vitamin C** (ascorbic acid) is a water soluble antioxidant found in the body as an ascorbate anion. It acts as a free radical scavenger [226]. Although this effect requires higher concentrations than those achieved in the plasma by oral administration, ascorbate appears to concentrate in tissues in much higher levels than those found in the plasma and can act effectively as a ROS scavenger [227]. In addition, it reduces membrane lipid peroxidation and regenerates Vitamin E [226]. Recent reports also suggest that Vitamin C can suppress NADPH oxidase activity [227].

364 Lipid Peroxidation

counteract its vasodilator, natriuretic and antihypertensive effects in models of hypertension where H2O2 plays a more prominent role than O2.- [48, 50, 53]. The co-administration of

**N-acetylcysteine** (NAC) is a thiol containing compound. It is the acetylated derivative of the aminoacid L-cysteine and a precursor for reduced glutathione (GSH) [215, 216]. It appears to have direct antioxidant action since its free thiol can interact with the electrophilic groups of ROS [215]. However, this effect does not seem likely to occur in vivo because NAC has poor oral bioavailability being rapidly metabolized into GSH, among other metabolites [216]. Thus, the main protective action of NAC is probably related to its role as a GSH precursor, which then detoxifies reactive species either by enzymatic or non-enzymatic reactions [216]. In humans, NAC is approved as a mucolytic agent because it destroys the disulphide bridges of mucoproteins [215]. It is also used as an antidote for acetaminophen poisoning which dramatically depletes hepatic GSH content causing severe damage [217]. NAC may

also have potential therapeutical applications in the treatment of heart diseases [218].

not free radicals initiators [221]. In addition, ebselen reacts rapidly with ONOO-

delayed neurological deficit after aneurismal subarachnoid hemorrhage [224, 225].

reutilization as a defense against ONOO-

inflammation-related enzymes such as 5-lipoxygenase, .

**Polyethylene glycol-catalase** is the conjugated form of the enzyme catalase with polyethylene glycol (PEG) which enhances the stability in aqueous solution, reduces immunogenicity and decreases sensitivity to proteolysis, thus increasing the circulatory half-life of catalase [219]. PEG also enhances the catalase association with cells [219]. The antioxidant effect of PEG-catalase results from the enzymatic degradation of H2O2 to water. **Ebselen** (2-phenyl-1,2-benzisoselenazol-3[2H]-one) is a lipid-soluble seleno-organic compound that mimics glutathione peroxidase activity, being able to react with H2O2 and organic hydroperoxides including membrane-bound phospholipid and cholesterylester hydroperoxides [220]. It appears to reduce lipid peroxidation initiated by hydroperoxides but

selenoxid product yielded in this reaction is regenerated to ebselen by GSH, which allows its

NADPH oxidase and H+/K+-ATPase by reacting with the SH group, leading to the formation of a selenosulphide complex [221]. Some authors have also proposed that the antioxidant and anti-inflammatory actions of ebselen are mediated through interactions with the thioredoxin (Trx) system [220]. Reduced Trx is important for growth and redox regulation by thiol redox control [220]. Ebselen was found to be an excellent substrate for mammalian TrxR and a highly efficient oxidant of reduced Trx. It also seems to function as a Trx peroxidase or peroxiredoxin mimic, thus contributing to the elimination of H2O2 and lipid hydroperoxides [220]. Ebselen has been used in clinical trials for the treatment of patients with acute ischemic stroke or

**Vitamin C** (ascorbic acid) is a water soluble antioxidant found in the body as an ascorbate anion. It acts as a free radical scavenger [226]. Although this effect requires higher concentrations than those achieved in the plasma by oral administration, ascorbate appears to concentrate in tissues in much higher levels than those found in the plasma and can act effectively as a ROS scavenger [227]. In addition, it reduces membrane lipid peroxidation

. The ebselen

[222, 223]. Ebselen also directly inhibits

NO synthases, protein kinase C,

catalase in these conditions restores the protective action of tempol [48, 50].

**Vitamin E** is a generic term for a group of compounds including tocopherols and tocotrienols. The isoform -tocopherol appears to be the most abundant in vivo [227]. Vitamin E terminates the propagation of the free radical chain reaction in lipid membranes and inhibits LDL oxidation [226, 227]. Vitamin E can also have non antioxidant actions primarily through the regulation of enzymes involved in signal transduction. Enzymes inhibited by vitamin E include protein kinases C and B, protein tyrosine kinase, lipoxygenases, mitogen activated protein kinases, phospholipase A2 and cyclooxygenase-2. In contrast, vitamin E has stimulatory effects on protein tyrosine phosphatase and diacylglycerol kinase [228]. Both vitamins C and E have been shown to stimulate the activation of NOS activity and increase . NO synthesis in endothelial cells and thus may contribute to improved endothelial-dependent vasodilation in hypertension [229]. However, although Vitamins C and E are generally considered to be non-toxic, they can undergo oxidation and generate pro-oxidant molecules [226]. Nevertheless, it appears that this is more likely to occur with Vitamin E, especially in the absence of sufficient Vitamin C to regenerate the -tocopherol radical [227, 230, 231].

**Alpha-lipoic acid** (1,2-dithiolane-3-pentanoic acid or thioctic acid) has a wide range of effects on cell functions, acting as an antioxidant, a metal chelator and a signaling mediator [232]. Both lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA), may scavenge HO. and HClO, although neither species is able to neutralize H2O2 [232]. DHLA also regenerates Vitamins C and E and does not become a free radical after reacting with these species. Furthermore, LA and DHLA chelate transition metals, thus reducing the metalcatalyzed free radical damage [232]. LA also contributes to improve antioxidant defenses by increasing the intracellular levels of Vitamin C and GSH. Many of LA protective actions have been attributed to its interference in cell signaling processes [232]. For example, LA effect on GSH appears to be mediated by nuclear factor erythroid 2- related factor 2 (Nrf2), an important transcription factor regulating gene transcription through the Antioxidant Response Element. LA was also shown to interact with several kinases and protein phosphatases [232]. Its interaction with components of the insulin signaling cascade also appears to improve glucose disposal in animal models of diabetes and human diabetic patients [232]. In addition, LA improves endothelial . NO synthesis and endothelialdependent vasodilation and prevents deleterious modifications of thiol groups in Ca2+ channels [232]. It has also important anti-inflammatory effects by inhibiting the activation of NF-KB, a transcription factor that regulates the expression of proinflammatory genes [232].

**Pyrrolidine dithiocarbamate** (PDTC), a low-molecular weight thiol compound, has the ability to scavenge oxygen radicals and to chelate metals [233, 234]. It may also act as a prooxidant and a thiol group modulator [233]. PDTC has been shown to interfere with the activation of several transcription factors, being a potent inhibitor of NF-KB [233, 234]. PDTC can also activate other signaling pathways, such as the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and the transcription factor Heat Shock Factor (HSF) [233, 235].

**5, 6, 7, 8-Tetrahydrobiopterin (BH4)** is a key cofactor of NOS [236, 237]. It is involved in the formation and stabilization of eNOS and iNOS [236, 238]. In the absence of BH4, NOS can become uncoupled and starts producing O2.- instead of . NO [33, 237]. Furthermore, BH4 also possesses direct antioxidant activity, being able to scavenge O2.- and HO. [239]. The protective effects of BH4 on the development of hypertension appear to be due an increase in eNOS activity, a reduction in O2.- production and a decrease in iNOS expression [199].



**5, 6, 7, 8-Tetrahydrobiopterin (BH4)** is a key cofactor of NOS [236, 237]. It is involved in the formation and stabilization of eNOS and iNOS [236, 238]. In the absence of BH4, NOS can

protective effects of BH4 on the development of hypertension appear to be due an increase in eNOS activity, a reduction in O2.- production and a decrease in iNOS expression [199].

**Drug Antihypertensive effect Lack of antihypertensive effect** 

possesses direct antioxidant activity, being able to scavenge O2.- and HO.

NO [33, 237]. Furthermore, BH4 also

Failed to prevent the hypertension induced by chronic infusion of

Failed to prevent hypertension in transgenic mice overexpressing renin or angiotensinogen [247, 248]

Failed to prevent Ang II-induced hypertension in rats [249, 250]

Failed to attenuate salt-sensitive

Failed to prevent or attenuate mineralocorticoid-induced hypertension [254]

Failed to prevent glucocorticoidinduced hypertension [255] Failed to prevent or attenuate adrenocorticotropic-induced

Failed to prevent the development of hypertension induced by the blockade

of nitric oxide synthesis [256] Failed to prevent the progression of hypertension in young SHR [257]

Failed to prevent Ang II-induced

Failed to attenuate hypertension induced by inhibition of superoxide

hypertension [251]

hypertension [242]

hypertension [264]

dismutase [48]

endothelin-1 [200]

[239]. The

become uncoupled and starts producing O2.- instead of .

**Apocynin** Prevented/attenuated mineralocorticoid-induced

Prevented/reversed glucocorticoid-induced

Prevented the development of Ang II-induced

Prevented the development of renovascular

Prevented the development of hypertension

Reduced blood pressure in borderline and

Attenuated salt-sensitive hypertension [245] Normalized blood pressure in a model of hypertension induced by disruption of dopamine

Prevented glucocorticoid-induced hypertension

Prevented the progression of hypertension in salt-

Attenuated mineralocorticoid-induced

Prevented/reversed adrenocorticotropic hormone-induced hypertension [242]

hypertension [86, 240]

hypertension in mice [186]

induced by RAS activation [50]

spontaneous hypertension [244]

**Gp91ds-tat** Attenuated the blood pressure rise induced by

**Allopurinol** Attenuated salt-sensitive hypertension [252]

hypertension [241]

hypertension [243]

D2 receptor [246]

[253]

Ang II in mice [209]

**Oxypurinol** Reduced blood pressure in SHR [258] **Tempol** Attenuated hypertension in SHR [201]

loaded SHRSP [259]

hypertension [260]


**Table 7.** Effect of chronic treatment with antioxidants or inhibitors of ROS production on blood pressure

#### **4.2. Antioxidant approaches in human hypertension**

Although there is considerable evidence of oxidative stress involvement in the pathophysiology of hypertension, the attempts to demonstrate benefits from antioxidant therapy in human cardiovascular diseases have been very disappointing [5, 96, 283]. Most of the large trials regarding the effects of diet supplementation with Vitamin C, Vitamin E and -carotene failed to show significant improvements in blood pressure and other cardiovascular endpoints [5, 283]. Furthermore, some of them also led to the conclusion that antioxidant treatment with Vitamin E or -carotene may even be harmful [283-285]. In contrast, smaller clinical trials have provided some evidence of antioxidant treatment advantages. For example, some studies showed that systemic Vitamin C levels inversely correlates with blood pressure and that Vitamin C supplementation effectively attenuates hypertension [142, 286]. Vitamin E and lipoic acid have also been shown to improve vascular function, though there is not consistent evidence of a blood pressure lowering effect of these agents in human patients [5, 287, 288]. Nevertheless, it has been demonstrated that a high consumption of dietary fruits and vegetables increases plasma antioxidant capacity and reduces blood pressure [289, 290]. Thus, it appears that a diet rich in fruits and vegetables is a better strategy than antioxidant supplementation to improve antioxidant status and cardiovascular health [5]. Overall, the clinical trials with antioxidant supplements have been very unsatisfactory and are in disagreement with the findings obtained in experimental hypertension studies. There are some possible justifications for the disappointing outcomes of these trials. First, the type of the drug used as well as the dose and duration of the therapy might not be adequate [5, 291, 292]. Most trials followed an antioxidant strategy based in the administration of ROS scavengers such as Vitamins C and E. However, these drugs do not neutralize H2O2 which has been shown to play a relevant role in the pathophysiology of hypertension and other cardiovascular diseases [5, 7, 48-50]. Furthermore, it is known that human blood and tissues have plenty antioxidants and that several stimuli induce an adaptive increase of enzymatic antioxidant defenses which can mask the benefits of exogenously administered antioxidants [293]. In addition, the antioxidant doses used in most of the experimental studies have been much higher than those tested in human patients [291]. So, there is the possibility that in humans the antioxidants did not achieve effective concentrations to neutralize ROS. Furthermore, it is not known if orally administered antioxidants can reach the precise sites of increased ROS production as oxidative stress is heterogeneously distributed throughout the organs, tissues and cellular compartments [5, 96, 291]. Indeed, the unspecific scavenging of ROS may even interfere with many important physiological functions in a deleterious manner [29, 96]. Another important limitation of most antioxidants tested is that they can exert themselves prooxidant effects in the absence of a coordinated antioxidant response [5, 96]. For example, Vitamin E needs to be regenerated by Vitamin C otherwise it may cause oxidative damage [231].

368 Lipid Peroxidation

pressure

hypertension [279] **PDTC** Perinatal administration ameliorated

hypertension [260]

196]

SHR [199]

hypertension in SHR offsprings [280]

Attenuated mineralocorticoid-induced

**BH4** Suppressed the development of hypertension in

Reduced hypertension in SHR [281]

**4.2. Antioxidant approaches in human hypertension** 

Prevented /Reduced hypertension in SHR [32,

**Drug Antihypertensive effect Lack of antihypertensive effect** 

**Table 7.** Effect of chronic treatment with antioxidants or inhibitors of ROS production on blood

Although there is considerable evidence of oxidative stress involvement in the pathophysiology of hypertension, the attempts to demonstrate benefits from antioxidant therapy in human cardiovascular diseases have been very disappointing [5, 96, 283]. Most of the large trials regarding the effects of diet supplementation with Vitamin C, Vitamin E and -carotene failed to show significant improvements in blood pressure and other cardiovascular endpoints [5, 283]. Furthermore, some of them also led to the conclusion that antioxidant treatment with Vitamin E or -carotene may even be harmful [283-285]. In contrast, smaller clinical trials have provided some evidence of antioxidant treatment advantages. For example, some studies showed that systemic Vitamin C levels inversely correlates with blood pressure and that Vitamin C supplementation effectively attenuates hypertension [142, 286]. Vitamin E and lipoic acid have also been shown to improve vascular function, though there is not consistent evidence of a blood pressure lowering effect of these agents in human patients [5, 287, 288]. Nevertheless, it has been demonstrated that a high consumption of dietary fruits and vegetables increases plasma antioxidant capacity and reduces blood pressure [289, 290]. Thus, it appears that a diet rich in fruits and vegetables is a better strategy than antioxidant supplementation to improve antioxidant status and cardiovascular health [5]. Overall, the clinical trials with antioxidant supplements have been very unsatisfactory and are in disagreement with the findings obtained in experimental hypertension studies. There are some possible justifications for the disappointing outcomes of these trials. First, the type of the drug used as well as the dose and duration of the therapy might not be adequate [5, 291, 292]. Most trials followed an antioxidant strategy based in the administration of ROS scavengers such as Vitamins C and E. However, these drugs do not neutralize H2O2 which has been shown to play a relevant role in the pathophysiology of hypertension and other cardiovascular diseases [5, 7, 48-50]. Furthermore, it is known that human blood and tissues have plenty antioxidants and that several stimuli induce an adaptive increase of enzymatic antioxidant defenses which can

Failed to attenuate hypertension in

Failed to prevent the development of adrenocorticotropic hormone in rat

castrated SHR [281]

[282]

There are also drawbacks in clinical trials design. In large trials of antioxidant supplementation, patients have not been recruited accordingly to their redox status [5, 294]. It is unlikely that a beneficial effect of antioxidant therapy would be observed in patients without previous evidence of increased oxidative stress. Another important consideration is that these clinical trials often have heterogeneous populations in terms of the etiology of cardiovascular disease [295]. Indeed, most studies have indiscriminately enrolled any patient at cardiovascular risk [294]. This is in obvious contrast to the homogeneous populations analyzed in experimental studies. Furthermore, some of the patients may be at an advanced stage of disease exhibiting irreversible damage insusceptible to antioxidant interventions [5, 29]. It should also be highlighted that many patients enrolled in these studies were already being treated with drugs such as aspirin, lipid-lowering agents and some antihypertensive drugs which can themselves interfere with oxidant status and mask the effects of additional therapy with antioxidants [5, 92, 296, 297].

So far, most interventions aimed at reducing oxidative stress in human hypertension have relied on antioxidant supplementation. However, it is possible that a strategy based on the inhibition of ROS production is more effective than the antioxidant interventions [5, 96]. The disruption of cardiovascular redox status is most likely triggered by an increase in prooxidant activity rather than a reduction in antioxidant defenses. Indeed, many neurohumoral or ambiental prohypertensive stimuli (angiotensin II, aldosterone, high-salt intake) are known to upregulate the expression and activity of prooxidant enzymes [5, 8, 43]. Nevertheless, there are already some studies that investigated the cardiovascular effects of prooxidant enzyme inhibition. Patients treated with allopurinol showed improvements in vascular function [298, 299]. However, a blood pressure lowering effect of this XO inhibitor has been shown only in newly diagnosed hypertensive adolescents and in hyperuricemic patients with normal renal function [300, 301]. Furthermore, the combination of allopurinol with antihypertensive drugs did not provide additional benefits on blood pressure [299]. This is probably because XO is not a major contributor to the development of hypertension, even though its activity may be increased in pathophysiological conditions [5]. Indeed, compelling evidence indicates that NADPH oxidases are the main contributors to ROS overproduction in cardiovascular and renal diseases [5, 8, 302]. Moreover, Nox-derived ROS are known to amplify redox dysfunction by inducing the activation of other prooxidant enzymes, such as XO, mitochondrial enzymes and NOS synthases [7, 51]. Since many antihypertensive drugs block upstream activators of Nox enzymes, it is not surprising that the inhibition of XO by allopurinol does not improve blood pressure control in patients already treated with antihypertensive drugs. To date, no Nox inhibitors have been tested in clinical trials although some specific Nox inhibitors have already been developed and patented [96, 296]. Future strategies to demonstrate the benefits of oxidative stress reduction in cardiovascular diseases should include the testing of specific Nox inhibitors in human patients. Moreover, the development of reliable oxidative stress biomarkers for risk stratification and monitoring of therapy is also highly desirable [96, 296]. Table 8 summarizes the possible reasons for the failure of antioxidants in clinical trials.


**Table 8.** Possible reasons for the failure of clinical trials with antioxidants in cardiovascular diseases

#### **4.3. Antihypertensive treatments with direct and indirect antioxidant effects**

It is known that first-line antihypertensive drugs such as angiotensin II receptor blockers (ARB) and angiotensin converting enzyme inhibitors (ACEi) can reduce oxidative stress due to their inhibitory effect on angiotensin II, which is a major stimulus for the activation or upregulation of Nox enzymes [5, 296]. ROS such as O2.- and H2O2 are widely recognized as important downstream mediators of Ang II physiological and pathological effects [303]. Nevertheless, some of these antihypertensive drugs also possess antioxidant effects independently of RAS inhibition. For example, captoptil, a thiol-containing ACEi, is a ROS scavenger and a metal chelator [304]. The ARBs candesartan and olmesartan also exhibit antioxidant effects independent of AT1 receptor blockade or blood pressure control [305- 307]. In addition, other agents belonging to the beta-blocker or calcium channel blocker drug classes have also been shown to exert antioxidant effects unrelated to their blood pressure lowering action. The beta-blockers carvedilol and nebivolol appear to possess ROS scavenging properties as well as inhibitory effects on ROS production, such as the inhibition of Nox activation [308, 309]. In addition, nebivolol also increases . NO release from the endothelium, thus attenuating oxidative stress effects on endothelium-dependent vasodilation [309, 310]. The calcium channel blocker lacidipine has also been demonstrated to have a potent antioxidant activity and to reduce the intracellular production of ROS induced by oxidized LDL [311, 312]. Therefore, even though convincing evidence is lacking regarding a clinical therapeutic effect of antioxidants, there is extensive data showing that currently approved antihypertensive treatments have the ability to modify oxidative stress status. Lipid Peroxidation and Antioxidants in Arterial Hypertension 371

#### **5. Conclusions**  to have a potent antioxidant activity and to reduce the intracellular production of ROS

370 Lipid Peroxidation

Inadequate dose or duration of therapy

Some antioxidants may themselves become prooxidants in the absence of a coordinated

Unspecific scavenging of ROS may disrupt

Lack of effect on prooxidant activity

of increased ROS production

antioxidant response

physiological functions

Lack of effect on non-radical oxidants such as H2O2

Inaccessibility of ROS scavengers to intracellular sites

are known to amplify redox dysfunction by inducing the activation of other prooxidant enzymes, such as XO, mitochondrial enzymes and NOS synthases [7, 51]. Since many antihypertensive drugs block upstream activators of Nox enzymes, it is not surprising that the inhibition of XO by allopurinol does not improve blood pressure control in patients already treated with antihypertensive drugs. To date, no Nox inhibitors have been tested in clinical trials although some specific Nox inhibitors have already been developed and patented [96, 296]. Future strategies to demonstrate the benefits of oxidative stress reduction in cardiovascular diseases should include the testing of specific Nox inhibitors in human patients. Moreover, the development of reliable oxidative stress biomarkers for risk stratification and monitoring of therapy is also highly desirable [96, 296]. Table 8

**Limitations related to the drug treatment Limitations related to the clinical trials design** 

**Table 8.** Possible reasons for the failure of clinical trials with antioxidants in cardiovascular diseases

**4.3. Antihypertensive treatments with direct and indirect antioxidant effects** 

of Nox activation [308, 309]. In addition, nebivolol also increases .

It is known that first-line antihypertensive drugs such as angiotensin II receptor blockers (ARB) and angiotensin converting enzyme inhibitors (ACEi) can reduce oxidative stress due to their inhibitory effect on angiotensin II, which is a major stimulus for the activation or upregulation of Nox enzymes [5, 296]. ROS such as O2.- and H2O2 are widely recognized as important downstream mediators of Ang II physiological and pathological effects [303]. Nevertheless, some of these antihypertensive drugs also possess antioxidant effects independently of RAS inhibition. For example, captoptil, a thiol-containing ACEi, is a ROS scavenger and a metal chelator [304]. The ARBs candesartan and olmesartan also exhibit antioxidant effects independent of AT1 receptor blockade or blood pressure control [305- 307]. In addition, other agents belonging to the beta-blocker or calcium channel blocker drug classes have also been shown to exert antioxidant effects unrelated to their blood pressure lowering action. The beta-blockers carvedilol and nebivolol appear to possess ROS scavenging properties as well as inhibitory effects on ROS production, such as the inhibition

endothelium, thus attenuating oxidative stress effects on endothelium-dependent vasodilation [309, 310]. The calcium channel blocker lacidipine has also been demonstrated

Lack of previous evidence of increased redox

Heterogeneous populations in terms of the

Some patients may be at an advanced stage of disease exhibiting irreversible damage

Patients treated simultaneously with drugs that interfere with oxidant status (aspirin, lipid lowering agents, antihypertensive drugs) Lack of validated oxidative stress biomarkers for risk stratification and monitoring of therapy

NO release from the

dysfunction in patients analyzed

etiology of cardiovascular disease

summarizes the possible reasons for the failure of antioxidants in clinical trials.

Extensive experimental evidence has shown that unbalanced ROS and/or RNS production can disturb several physiological functions, leading to the genesis and progression of arterial hypertension. Many studies have observed marked alterations in direct and indirect oxidative stress biomarkers, such as lipid peroxidation products, prooxidant enzymes and antioxidant defenses. However, most clinical trials with antioxidants have failed to demonstrate a protective effect on blood pressure and cardiovascular function. This does not necessarily exclude a role for oxidative stress in human cardiovascular diseases but instead suggests that other approaches should be addopted to recover redox homeostasis. The inhibition of Nox enzymes appears to be a promising strategy as these enzymes are major sources of ROS overproduction at cardiovascular and renal sites of blood pressure control. Indeed, several drugs already in use for the treatment of hypertension (e.g. ARBs, ACEi, the -blocker nebivolol) or dyslipidemia (statins) are known to reduce the activation of Nox enzymes. In addition, there is an urgent need to implement universally validated approaches to evaluate oxidative status in human patients. These should cover a broader range of redox biomarkers and would add valuable information for risk stratification and therapeutic monitoring in human patients. induced by oxidized LDL [311, 312]. Therefore, even though convincing evidence is lacking regarding a clinical therapeutic effect of antioxidants, there is extensive data showing that currently approved antihypertensive treatments have the ability to modify oxidative stress status. **5. Conclusions**  Extensive experimental evidence has shown that unbalanced ROS and/or RNS production can disturb several physiological functions, leading to the genesis and progression of arterial hypertension. Many studies have observed marked alterations in direct and indirect oxidative stress biomarkers, such as lipid peroxidation products, prooxidant enzymes and antioxidant defenses. However, most clinical trials with antioxidants have failed to demonstrate a protective effect on blood pressure and cardiovascular function. This does not necessarily exclude a role for oxidative stress in human cardiovascular diseases but instead suggests that other approaches should be addopted to recover redox homeostasis. The inhibition of Nox enzymes appears to be a promising strategy as these enzymes are major sources of ROS overproduction at cardiovascular and renal sites of blood pressure

#### **Author details**  ACEi, the -blocker nebivolol) or dyslipidemia (statins) are known to reduce the activation **Acknowledgments**

Teresa Sousa, Joana Afonso and António Albino-Teixeira *Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Portugal*  Félix Carvalho of Nox enzymes. In addition, there is an urgent need to implement universally validated approaches to evaluate oxidative status in human patients. These should cover a broader range of redox biomarkers and would add valuable information for risk stratification and therapeutic monitoring in human patients. "This work was supported by FEDER funds via COMPETE and by national funds through FCT – Portuguese Foundation for Science and Technology [project grant: PTDC/SAU-TOX/114166/2009]."

control. Indeed, several drugs already in use for the treatment of hypertension (e.g. ARBs,

#### *University of Porto, Portugal*  **Author details**

Rev 54: 619-634.

Rev 54: 619-634.

**6. References** 

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C849-868.

**6. References**  Teresa Sousa, Joana Afonso and António Albino-Teixeira *Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Portugal* 

*REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy,* 

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## **Lipid Peroxidation and Reperfusion Injury in Hypertrophied Hearts**

Juliana C. Fantinelli, Ignacio A. Pérez Núñez, Luisa F. González Arbeláez and Susana M. Mosca

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45913

## **1. Introduction**

392 Lipid Peroxidation

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Maruyama, T., Otagiri, M. (2009) Evaluation for antioxidant and renoprotective activity

[306] Kadowaki, D., Anraku, M., Tasaki, Y., Taguchi, K., Shimoishi, K., Seo, H., Hirata, S.,

[307] Miyata, T., van Ypersele de Strihou, C., Ueda, Y., Ichimori, K., Inagi, R., Onogi, H., Ishikawa, N., Nangaku, M., Kurokawa, K. (2002) Angiotensin II receptor antagonists and angiotensin-converting enzyme inhibitors lower in vitro the formation of advanced glycation end products: biochemical mechanisms. J Am Soc Nephrol 13: 2478-2487.

[308] Dandona, P., Ghanim, H., Brooks, D.P. (2007) Antioxidant activity of carvedilol in

[309] Evangelista, S., Garbin, U., Pasini, A.F., Stranieri, C., Boccioletti, V., Cominacini, L. (2007) Effect of DL-nebivolol, its enantiomers and metabolites on the intracellular production of superoxide and nitric oxide in human endothelial cells. Pharmacol Res 55:

[307] Miyata, T., van Ypersele de Strihou, C., Ueda, Y., Ichimori, K., Inagi, R., Onogi, H., Ishikawa, N., Nangaku, M., Kurokawa, K. (2002) Angiotensin II receptor antagonists and angiotensin-converting enzyme inhibitors lower in vitro the formation of advanced glycation end products: biochemical mechanisms. J Am Soc Nephrol 13: 2478-2487. [308] Dandona, P., Ghanim, H., Brooks, D.P. (2007) Antioxidant activity of carvedilol in

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Oxidative stress is characterized by an imbalance between increased exposure to reactive oxygen species (ROS), and antioxidant defenses, comprised of both small molecular weight antioxidants like glutathione, and antioxidant enzymes like superoxide dismutase. ROS cause direct damage to critical biomolecules including DNA, lipids, and proteins. Oxidative stress has been involved in the genesis of hypertension [1, 2] and implicated in the mechanisms of reversible postischemic contractile dysfunction (myocardial stunning), microvascular dysfunction, arrhythmias and cell death [3-6]. In spontaneously hypertensive rats (SHR) there are few reports showing the protective action of antioxidants against ischemia-reperfusion injury [7-9] and specifically in regard to the effects of the scavenger N- (2-mercaptopropionyl)-glycine (MPG) these have not been yet examined.

Ischemic preconditioning (IP) is acknowledged to be an endogenous mechanism of cardioprotection against ischemia and reperfusion injury [10-11]. This intervention is based in that one or more brief periods of ischemia applied previous to a prolonged ischemic period exert beneficial effects on myocardium attenuating the deleterious effects observed in the reperfused myocardium. Although there are some studies showing the beneficial effects of IP in hypertensive animals [12-15], under certain circumstances the effectiveness of that intervention is questioned [16-18]. A recent investigation performed in our laboratory shows that a single cycle of IP attenuated the myocardial stunning produced by 20-min global ischemia in SHR [19] and decreased the lipid peroxidation. Whether this protective action of IP is operating at more extended ischemic period and involves changes in oxidative stress in this rats strain is a point that needs to be clarified.

Therefore, the aim of the present study was to determine if alterations of lipid peroxidation and endogenous antioxidants are linked to myocardial and vascular postischemic damage in ischemic control, preconditioned and MPG treated hearts from SHR.

© 2012 Fantinelli et al., licensee InTech. This is an open access chapter 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. © 2012 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.

## **2. Material and methods**

### **2.1. Isolated heart preparation**

Experiments were performed in SHR of 5-month-old following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). Beginning at 12 weeks of age, systolic blood pressure (SBP) was measured weekly in all animals by the standard tail-cuff method [20] following the modifications detailed in a recent paper by Fritz and Rinaldi [21]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body wt). The heart was rapidly excised and perfused by the non-recirculating Langendorff technique with Ringer's solution containing (in mmol/L): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.35 CaCl2, 20 NaCO3H and 11.1dextrose. The buffer was saturated with a mixture of 95% O2-5% CO2, had a pH 7.4, and was maintained at 37°C. The conductive tissue in the atrial septum was damaged with a fine needle to achieve atrioventricular block, and the right ventricle was paced at 280 ± 10 beats/min. A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve; the opposite end of the tube was then connected to a Statham P23XL pressure transducer. The balloon was filled with water to provide an enddiastolic pressure (LVEDP) of 8-12 mmHg and this volume remained unchanged for the rest of the experiment. Coronary perfusion pressure (CPP) was monitored at the point of cannulation of the aorta and adjusted to approximately 70 mmHg. Coronary flow (CF), controlled with a peristaltic pump, was 11 ± 2 mL/min. Left ventricular pressure (LVP) and CPP data were acquired by using an analog-to-digital converter and acquisition software (Chart V4.2.3 ADInstruments).

### **2.2. Experimental protocols**

After 10 min of stabilization, hearts from SHR were assigned to the following experimental protocols (Fig. 1):

Non-ischemic control hearts (NIC): Hearts were perfused for 3 hs without any treatment.

Ischemic control hearts (IC): Hearts were subjected to 35 min or 50 min of normothermic global ischemia followed by 2 hours of reperfusion. Global ischemia was induced by stopping the perfusate inflow line and the heart was placed in a saline bath held at 37°C.

Ischemic preconditioning (IP1): A single cycle of 5-min ischemia and 10-min reperfusion was applied previous to the 35-min and 50-min ischemic periods followed by 2-hour reperfusion.

Ischemic preconditioning (IP3): Three cycles of 2-min f ischemia and 5-min reperfusion was applied prior to the 50-min ischemic period followed by 2-hour reperfusion. Previous experiments performed by us showed that three cycles are the fewest for achieving myocardial protection of SHR when global ischemia was extended to 50 min.

MPG: Hearts were treated 10 min before ischemia and during the first 10 min of reperfusion with N-(2-mercaptopropionyl)-glycine (MPG) 2 mM. The administration time for MPG was chosen to attenuate the ROS production during ischemia and reperfusion. The dose was selected according previous experiments performed in our laboratory [22].

Additional experiments were performed (n = 6 for each protocol) to assess the biochemical parameters.

**Figure 1.** Scheme of the experimental protocols.

#### **2.3. Infarct size determination**

394 Lipid Peroxidation

**2. Material and methods** 

(Chart V4.2.3 ADInstruments).

**2.2. Experimental protocols** 

protocols (Fig. 1):

reperfusion.

**2.1. Isolated heart preparation** 

Experiments were performed in SHR of 5-month-old following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). Beginning at 12 weeks of age, systolic blood pressure (SBP) was measured weekly in all animals by the standard tail-cuff method [20] following the modifications detailed in a recent paper by Fritz and Rinaldi [21]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body wt). The heart was rapidly excised and perfused by the non-recirculating Langendorff technique with Ringer's solution containing (in mmol/L): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.35 CaCl2, 20 NaCO3H and 11.1dextrose. The buffer was saturated with a mixture of 95% O2-5% CO2, had a pH 7.4, and was maintained at 37°C. The conductive tissue in the atrial septum was damaged with a fine needle to achieve atrioventricular block, and the right ventricle was paced at 280 ± 10 beats/min. A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve; the opposite end of the tube was then connected to a Statham P23XL pressure transducer. The balloon was filled with water to provide an enddiastolic pressure (LVEDP) of 8-12 mmHg and this volume remained unchanged for the rest of the experiment. Coronary perfusion pressure (CPP) was monitored at the point of cannulation of the aorta and adjusted to approximately 70 mmHg. Coronary flow (CF), controlled with a peristaltic pump, was 11 ± 2 mL/min. Left ventricular pressure (LVP) and CPP data were acquired by using an analog-to-digital converter and acquisition software

After 10 min of stabilization, hearts from SHR were assigned to the following experimental

Ischemic control hearts (IC): Hearts were subjected to 35 min or 50 min of normothermic global ischemia followed by 2 hours of reperfusion. Global ischemia was induced by stopping the perfusate inflow line and the heart was placed in a saline bath held at 37°C.

Ischemic preconditioning (IP1): A single cycle of 5-min ischemia and 10-min reperfusion was applied previous to the 35-min and 50-min ischemic periods followed by 2-hour

Ischemic preconditioning (IP3): Three cycles of 2-min f ischemia and 5-min reperfusion was applied prior to the 50-min ischemic period followed by 2-hour reperfusion. Previous experiments performed by us showed that three cycles are the fewest for achieving

MPG: Hearts were treated 10 min before ischemia and during the first 10 min of reperfusion with N-(2-mercaptopropionyl)-glycine (MPG) 2 mM. The administration time for MPG was

myocardial protection of SHR when global ischemia was extended to 50 min.

Non-ischemic control hearts (NIC): Hearts were perfused for 3 hs without any treatment.

Infarct size was assessed by the widely validated triphenyltetrazolium chloride (TTC) staining technique [23]. At the end of reperfusion, atrial and right ventricular tissues were excised and left ventricle (VI) was frozen. The freeze VI was cut into six transverse slices, which were incubated for 5 minutes at 37°C in a 1% solution of triphenyltetrazolium chloride (TTC). To measure myocardial infarction, the slices were weighed and scanned. The infarcted (pale) and viable ischemic/reperfused (red) areas were measured by computed planimetry (Scion Image 1.62; Scion Corp., Frederick, Maryland, USA). Infarct weights were calculated as (A1 × W1) + (A2 × W2) + (A3 × W3) + (A4 × W4) + (A5 × W5) + (A6 × W6), where A is the infarct area for the slice and W is the weight of the respective section. Infarct size was expressed as a percentage of the total area (area at risk, AAR) [24].

#### **2.4. Systolic and diastolic function**

Myocardial contractility was assessed by the left ventricular developed pressure (LVDP), obtained by subtracting LVEDP to LVP peak, and maximal velocity of contraction (+dP/dtmax). The diastolic function was evaluated through LVEDP.

#### **2.5. Assessment of coronary resistance (CR)**

CR was calculated as a quotient between CPP and CF and expressed as difference between the values obtained at the end of reperfusion period and that observed in the preischemic period.

#### **2.6. Preparation of tissue homogenate**

At the end of reperfusion a portion of VI was homogenized in 5 volume of 25 mM PO4KH2 - 140 mM ClK at pH = 7.4 with a Polytron homogenizer. Aliquots of homogenate were used to assess reduced glutathione content (GSH) and thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. The remaining homogenate was centrifuged at 12,000 g for 5 min at 4º C and the supernatant stored at -70 ºC until superoxide dismutase (SOD) activity was assayed.

#### *2.6.1. Assessment of reduced glutathione (GSH)*

GSH was determined by Ellman's method [25]. This method was based on the reaction of GSH with 5, 5′ dithiobis (2-nitrobenzoic acid) to give a compound that absorbs at 412 nm. GSH levels were expressed as μg/mg of protein.

#### *2.6.2. Assessment of lipid peroxidation*

TBARS concentration was determined in the supernatant following the Buege and Aust method´s [26]. Absorbance at 535nm was measured and TBARS expressed in nmol/g of tissue using an extinction coefficient of 1.56x105 M-1 cm-1.

#### *2.6.3. Measurement of SOD cytosolic activity*

SOD activity was measured by means of the nitroblue tetrazolium (NBT) method [27]. Briefly, the supernatant was added to the reaction mixture of NBT with xanthine-xanthine oxidase, and the SOD activity measured colorimetrically in the form of inhibitory activity toward blue formazan formation by SOD in the reaction mixture.

#### *2.6.4. Protein determination*

The protein concentration was evaluated by the Bradford method [28] using bovine serum albumin as a standard.

#### *2.6.5. Correlations*

The relationships between TBARS, GSH and infarct size and CR were determined by linear regression (equation y = a + b . x).

#### **2.7. Statistical analysis**

Data are presented as mean ± SE and repeated measures of two-way analysis of variance (ANOVA) with Newman-Keuls test were used for multiple comparisons among groups. Relationships were tested for significance using the Pearson correlation coefficient (r). A P value < 0.05 was considered significant.

#### **3. Results**

396 Lipid Peroxidation

**2.6. Preparation of tissue homogenate** 

*2.6.1. Assessment of reduced glutathione (GSH)* 

GSH levels were expressed as μg/mg of protein.

*2.6.3. Measurement of SOD cytosolic activity* 

*2.6.4. Protein determination* 

regression (equation y = a + b . x).

value < 0.05 was considered significant.

**2.7. Statistical analysis** 

albumin as a standard.

*2.6.5. Correlations* 

tissue using an extinction coefficient of 1.56x105 M-1 cm-1.

toward blue formazan formation by SOD in the reaction mixture.

*2.6.2. Assessment of lipid peroxidation* 

(SOD) activity was assayed.

At the end of reperfusion a portion of VI was homogenized in 5 volume of 25 mM PO4KH2 - 140 mM ClK at pH = 7.4 with a Polytron homogenizer. Aliquots of homogenate were used to assess reduced glutathione content (GSH) and thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. The remaining homogenate was centrifuged at 12,000 g for 5 min at 4º C and the supernatant stored at -70 ºC until superoxide dismutase

GSH was determined by Ellman's method [25]. This method was based on the reaction of GSH with 5, 5′ dithiobis (2-nitrobenzoic acid) to give a compound that absorbs at 412 nm.

TBARS concentration was determined in the supernatant following the Buege and Aust method´s [26]. Absorbance at 535nm was measured and TBARS expressed in nmol/g of

SOD activity was measured by means of the nitroblue tetrazolium (NBT) method [27]. Briefly, the supernatant was added to the reaction mixture of NBT with xanthine-xanthine oxidase, and the SOD activity measured colorimetrically in the form of inhibitory activity

The protein concentration was evaluated by the Bradford method [28] using bovine serum

The relationships between TBARS, GSH and infarct size and CR were determined by linear

Data are presented as mean ± SE and repeated measures of two-way analysis of variance (ANOVA) with Newman-Keuls test were used for multiple comparisons among groups. Relationships were tested for significance using the Pearson correlation coefficient (r). A P Fig. 2 shows the infarct size in ischemic control and preconditioned hearts from SHR. In non-ischemic control hearts at the end of the 3-hour perfusion the infarct size was approximately 1 % of risk area. After 35-min global ischemia and 2-hour reperfusion, the infarct size was 35 ± 5 %, which was significantly decreased by one cycle of IP (IP1). When ischemia was extended to 50 min, the infarct size (58 ± 5 %) was not reduced by IP1 indicating that this preconditioning protocol is not adequate for protecting that rat strain against reperfusion injury. However, when a larger number of cycles (three in our case) were applied the hearts were protected and the infarct size diminished. A significant reduction of infarct size was also obtained when MPG was added to the perfusate during 10 min before 50-min ischemia and during the first 10 min of reperfusion.

**Figure 2.** Infarct size (IS), expressed as percentage of risk area, in ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia), preconditioned hearts (IP1= one cycle; IP3= three cycles) and MPG treatment. Note that hearts from SHR showed a higher IS at 50-min compared to 35-min GI. IP1 diminished the IS at 35-min ischemia but it was necessary to apply three cycles (IP3) to protect the hearts when the prolonged ischemia was extended to 50 min and that MPG decreased the IS at a similar value to IP3. \* P < 0.05 with respect to GI; # P < 0.05 with respect to 35-min GI.

At the end of 3-hour non-ischemic hearts exhibited a decrease in contractility of approximately 10 %. After 35-min ischemia and 2-hour reperfusion contractility decreased approximately 90 % with respect to preischemic values. As it is depicted in Fig. 3 the recovery of systolic function was improved by both IP protocols. At the end of the reperfusion period, LVDP and +dP/dtmax reached higher values than those obtained in ischemic control hearts. When ischemia was more prolonged (50 min) the postischemic recovery of contractility was scarce (LVDP and +dP/dtmax reached values of approximately 2 %) and it was significantly improved by IP3 and MPG treatment.

The diastolic stiffness characterized by LVEDP increased during 35-min and 50-min global ischemia and acquired greater values during reperfusion. These increases were attenuated by both IP protocols and MPG treatment. Fig. 4 shows the changes of LVEDP occurring at 50-min global ischemia in ischemic control and intervened hearts.

The increase in perfusion pressure at constant coronary flow resulted in an increase of coronary resistance. The increases (4.2 ± 0.4 and 7.0 ± 0.9 mmHg/ml x min-1 after 35-min and 50-min ischemia, respectively) were significantly attenuated by both IP protocols and MPG treatment (Fig. 5).

**Figure 3.** Values of left ventricular developed pressure (LVDP) and maximal velocity of contraction (+dP/dtmax) at the end of reperfusion period expressed as percentage of preischemic values, in ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia), preconditioned hearts (IP) and MPG treatment. Observe that IP and MPG significantly improved the postischemic recovery of myocardial systolic function at 35-min and 50-min GI. \* P < 0.05 with respect to IC

treatment (Fig. 5).

0

10

20

Postischemic recovery (%)

30

\*

40

recovery of contractility was scarce (LVDP and +dP/dtmax reached values of approximately 2

The diastolic stiffness characterized by LVEDP increased during 35-min and 50-min global ischemia and acquired greater values during reperfusion. These increases were attenuated by both IP protocols and MPG treatment. Fig. 4 shows the changes of LVEDP occurring at

The increase in perfusion pressure at constant coronary flow resulted in an increase of coronary resistance. The increases (4.2 ± 0.4 and 7.0 ± 0.9 mmHg/ml x min-1 after 35-min and 50-min ischemia, respectively) were significantly attenuated by both IP protocols and MPG

**Figure 3.** Values of left ventricular developed pressure (LVDP) and maximal velocity of contraction (+dP/dtmax) at the end of reperfusion period expressed as percentage of preischemic values, in ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia), preconditioned hearts (IP) and MPG treatment. Observe that IP and MPG significantly improved the postischemic recovery of

GI35 GI50

\* \* \* \*

LVDP +dP/dtmax

 IC IP MPG

myocardial systolic function at 35-min and 50-min GI. \* P < 0.05 with respect to IC

LVDP +dP/dtmax

%) and it was significantly improved by IP3 and MPG treatment.

50-min global ischemia in ischemic control and intervened hearts.

\*

**Figure 4.** Time course of left ventricular end diastolic pressure (LVEDP) in ischemic control (IC = GI50: 50-min global ischemia), preconditioned hearts (IP) and MPG treatment. The three cycles of IP (IP3) and MPG attenuated in a similar manner the increase of LVEDP detected in IC hearts. \* P < 0.05 with respect to IC.

**Figure 5.** Changes of coronary resistance (CR) at the end of reperfusion in ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia), preconditioned hearts (IP) and MPG treatment. The interventions attenuated the increase of CR detected in IC hearts being MPG the most effective. \* P < 0.05 with respect to IC; # P < 0.05 with respect to GI35.

Given that an increase of ROS generation accompanied by a diminution of antioxidants may be responsible for myocardial reperfusion injury [29, 30], we next determined the impact of IP and MPG on myocardial GSH content, a marker of oxidative stress. Fig. 6 shows that GSH content in non-ischemic hearts (2 ± 0.3 μg/mg prot) was significantly reduced by ischemia and reperfusion. A single or three cycles of IP and MPG treatment were able to preserve part of the GSH content.

**Figure 6.** Myocardial reduced glutathione content (GSH, μg/mg protein) in non-ischemic control (NIC), ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia) and preconditioned (IP) and MPG treated hearts. Observe that GSH levels decreased after ischemia and reperfusion in both ischemic periods and were partially preserved by IP and MPG. \* P < 0.05 with respect to NIC; # P < 0.05 with respect to IC.

Moreover, the SOD cytosolic activity increased in ischemic controls hearts and significantly decreased in all intervened hearts (Fig. 7). Both parameters (GSH and SOD) are indicating the presence of oxidative stress caused by ischemia-reperfusion which may be attenuated by IP and MPG treatment.

Since ROS induce membrane lipid peroxidation [29], we determined TBARS content of untreated and treated ischemic-reperfused hearts. Although TBARS determination suffers from potential artifacts associated with sampling, storage and problems caused by the complexity of the biological systems, being easy and reproducible, it is one of the most widely used indexes for assessing oxidative stress. There was an increase in myocardial TBARS content in hearts submitted to ischemia and reperfusion detecting a higher value at 50-min compared to 35-min global ischemia. Preconditioned and MPG treated hearts exhibited lower TBARS levels (Fig. 8).

The analysis of data of the different interventions showed the presence of significant positive correlations TBARS vs IS (Fig. 9, A panel; r = 0,47) and TBARS vs CR (Fig. 9, B panel; n = 0,45) and negative correlations GSH vs IS (Fig. 10, A panel; n = 0,41) and GSH vs CR (Fig. 10, B panel; n = 0,40) in isolated hearts from SHR.

with respect to IC.

IP and MPG treatment.

0,0

0,5

1,0

1,5

2,0

**GSH (g/mg protein)**

2,5

3,0

3,5

exhibited lower TBARS levels (Fig. 8).

CR (Fig. 10, B panel; n = 0,40) in isolated hearts from SHR.

preserve part of the GSH content.

GSH content in non-ischemic hearts (2 ± 0.3 μg/mg prot) was significantly reduced by ischemia and reperfusion. A single or three cycles of IP and MPG treatment were able to

**Figure 6.** Myocardial reduced glutathione content (GSH, μg/mg protein) in non-ischemic control (NIC), ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia) and preconditioned (IP) and MPG treated hearts. Observe that GSH levels decreased after ischemia and reperfusion in both ischemic periods and were partially preserved by IP and MPG. \* P < 0.05 with respect to NIC; # P < 0.05

**GI35 GI50**

\*

#

 NIC IC IP MPG 

#

\*

#

\*

**NIC**

Moreover, the SOD cytosolic activity increased in ischemic controls hearts and significantly decreased in all intervened hearts (Fig. 7). Both parameters (GSH and SOD) are indicating the presence of oxidative stress caused by ischemia-reperfusion which may be attenuated by

Since ROS induce membrane lipid peroxidation [29], we determined TBARS content of untreated and treated ischemic-reperfused hearts. Although TBARS determination suffers from potential artifacts associated with sampling, storage and problems caused by the complexity of the biological systems, being easy and reproducible, it is one of the most widely used indexes for assessing oxidative stress. There was an increase in myocardial TBARS content in hearts submitted to ischemia and reperfusion detecting a higher value at 50-min compared to 35-min global ischemia. Preconditioned and MPG treated hearts

The analysis of data of the different interventions showed the presence of significant positive correlations TBARS vs IS (Fig. 9, A panel; r = 0,47) and TBARS vs CR (Fig. 9, B panel; n = 0,45) and negative correlations GSH vs IS (Fig. 10, A panel; n = 0,41) and GSH vs

**Figure 7.** Myocardial SOD cytosolic activity (SOD, % inhibition/mg protein) in non-ischemic control (NIC), ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia), preconditioned (IP) and MPG treated hearts. Note that SOD cytosolic activity increased after 35-min or 50-min GI in comparison to NIC. These increases were attenuated by both interventions (IP and MPG). \* P < 0.05 with respect to NIC; # P < 0.05 with respect to IC.

**Figure 8.** Myocardial thiobarbituric acid reactive substances (TBARS) concentration, expressed in nmol/mg protein in non-ischemic control (NIC), ischemic control (IC = GI35: 35-min global ischemia; GI50: 50-min global ischemia), preconditioned (IP) and MPG treated hearts. An increase of TBARS occurred at the end of reperfusion after the two ischemic periods which were attenuated by IP and MPG. \* P < 0.05 with respect to NIC ; # P < 0.05 with respect to IC; P < 0.05 with respect to GI35.

**Figure 9.** Relationship between TBARS and infarct size (IS, A panel) and TBARS and coronary resistance (CR, B panel) in all experimental situations. The resulting data were fitted to straight line by linear regression. Significant positive correlations between TBARS and IS and CR were found.

**Figure 10.** Relationship between GSH and infarct size (IS, A panel) and TBARS and coronary resistance (CR, B panel) in all experimental situations. The resulting data were fitted to straight line by linear regression. Significant negative correlations between GSH and IS and CR were found.

#### **4. Discussion**

402 Lipid Peroxidation

**Figure 9.** Relationship between TBARS and infarct size (IS, A panel) and TBARS and coronary resistance (CR, B panel) in all experimental situations. The resulting data were fitted to straight line by linear regression. Significant positive correlations between TBARS and IS and CR were found.

To our knowledge, this is the first demonstration that the beneficial action of ischemic preconditioning and MPG against ischemia-reperfusion injury is similar in hearts from SHR and is associated with a mitigation of oxidative stress. Thus, our data show the existence of a positive correlation between TBARS concentration-used as an index of lipid peroxidationand infarct size indicating that it will be found more infarct size when TBARS acquire higher values.

Simultaneously an inverse correlation was detected between GSH content and infarct size indicating that higher levels of GSH are associated to minor infarct size. Both variables (TBARS and GSH) suffered opposite changes due to a possible cause-effect relationship.

These results were also accompanied by changes of SOD cytosolic activity which showed lesser values in preconditioned and MPG treated hearts. Taken together, these data provide evidence to suggest that formation of lipoperoxides is a significant cause of ischemia and reperfusion injury and that the mechanism whereby IP and MPG confer cardioprotection involves, at least in part, an attenuation of those nocive products through a diminution of ROS release and/or production and an improvement of the endogenous antioxidants.

This study clearly shows that hearts from SHR suffer higher irreversible damage at 50-min compared to 35-min global ischemia accompanied with greater impairment of postichemic myocardial function. Thus, at the end of reperfusion the recovery of systolic function was scarce and diastolic stiffness significantly increased in ischemic control hearts. These alterations were attenuated by IP being one cycle of IP (IP1) effective when the ischemic period was 35 min and three cycles (IP3) when the ischemia was extended to 50 min. Thus, although the cardioprotective action of IP in hypertrophied hearts was previously reported [12-15] our study demonstrates that the optimum protocol of IP to protect SHR hearts must be selected according to the duration time of prolonged ischemia. Then, it seems to be possible that the number of IP cycles appears as other key factor for determining the efficacy of IP. Moreover MPG treated hearts in the same way that the preconditioned showed lesser infarct size and improved postischemic recovery of myocardial function in comparison to ischemic control hearts.

Hypertension is associated with an elevation of ROS and frequently with an impairment of endogenous antioxidant mechanisms [30]. These alterations have also been described during ischemia and reperfusion [3, 4, 31-33]. In this study, at the end of reperfusion after ischemic period cardiac tissue showed lesser GSH content, higher TBARS concentration and SOD cytosolic activity in comparison to non-ischemic control hearts. Major changes of GSH and TBARS were detected at 50-min compared to 35-min global ischemia. However, SOD cytosolic activity showed higher increase at 35 min of ischemia. This result may explain the lesser lipid peroxidation found in this experimental group. All these changes were partially reversed by both IP protocols and MPG treatment. Thus, GSH content was higher and SOD cytosolic activity was lower than the values observed in untreated hearts. The favorable changes in GSH and SOD cytosolic activity were reflected in the lower lipid peroxidation (decreased TBARS concentration) observed in preconditioned hearts and in those treated with MPG in comparison to ischemic control hearts. In other words the improvement of the antioxidant systems (SOD and GSH) by IP and MPG treatment were enough to attenuate the oxidative damage detected in untreated hearts. These results suggest that changes of lipid peroxidation and antioxidant systems would be sufficient to promote differences in the cell death and the attenuation of oxidative stress would be considered as a factor contributing to the cardioprotection by IP and MPG treatment in hearts from SHR.

On the other hand, a balance between the production of nitric oxide (NO) and ROS controls the endothelial function [34, 35]. When the NO production is normal its bioavailability may be reduced because of the oxidative inactivation by an excessive production of superoxide (O2−. ) in the vascular wall. The available data on the NO system in SHR are limited and apparently contradictory. Increased ROS in SHR have been demonstrated to enhance NO inactivation and reduce NO bioavailability [36], which contributes to the maintenance of hypertension. According to a previous study the peroxynitrite- product of NO and O2−. combination- may also be involved in maintenance of the high levels of blood pressure in SHR [37. Furthermore in this rats strain was reported that the activity and/or expression of the different nitric oxide synthase (NOS) isoforms would be altered [38-40] which might act as a compensatory mechanism to maintain the production of bioactive NO in the face of increased oxidant stress [41]. In our study, ischemic control hearts showed an increase of coronary resistance at the end of reperfusion compared to pre-ischemic period which was greater after 50-min than 35-min global ischemia. These increases were attenuated by IP and MPG treatment being this last intervention the most effective. Thus, the beneficial effect of IP and MPG on coronary resistance would be attributed to a greater NO availability mediated by an attenuation of oxidative stress. This mechanism could explain the significant correlations between TBARS, GSH and coronary resistance found in this study and reinforces the idea that changes of oxidative stress constitute the basis of myocardial and vascular postischemic alterations.

## **5. Conclusions**

404 Lipid Peroxidation

ischemic control hearts.

Simultaneously an inverse correlation was detected between GSH content and infarct size indicating that higher levels of GSH are associated to minor infarct size. Both variables (TBARS and GSH) suffered opposite changes due to a possible cause-effect relationship.

These results were also accompanied by changes of SOD cytosolic activity which showed lesser values in preconditioned and MPG treated hearts. Taken together, these data provide evidence to suggest that formation of lipoperoxides is a significant cause of ischemia and reperfusion injury and that the mechanism whereby IP and MPG confer cardioprotection involves, at least in part, an attenuation of those nocive products through a diminution of

This study clearly shows that hearts from SHR suffer higher irreversible damage at 50-min compared to 35-min global ischemia accompanied with greater impairment of postichemic myocardial function. Thus, at the end of reperfusion the recovery of systolic function was scarce and diastolic stiffness significantly increased in ischemic control hearts. These alterations were attenuated by IP being one cycle of IP (IP1) effective when the ischemic period was 35 min and three cycles (IP3) when the ischemia was extended to 50 min. Thus, although the cardioprotective action of IP in hypertrophied hearts was previously reported [12-15] our study demonstrates that the optimum protocol of IP to protect SHR hearts must be selected according to the duration time of prolonged ischemia. Then, it seems to be possible that the number of IP cycles appears as other key factor for determining the efficacy of IP. Moreover MPG treated hearts in the same way that the preconditioned showed lesser infarct size and improved postischemic recovery of myocardial function in comparison to

Hypertension is associated with an elevation of ROS and frequently with an impairment of endogenous antioxidant mechanisms [30]. These alterations have also been described during ischemia and reperfusion [3, 4, 31-33]. In this study, at the end of reperfusion after ischemic period cardiac tissue showed lesser GSH content, higher TBARS concentration and SOD cytosolic activity in comparison to non-ischemic control hearts. Major changes of GSH and TBARS were detected at 50-min compared to 35-min global ischemia. However, SOD cytosolic activity showed higher increase at 35 min of ischemia. This result may explain the lesser lipid peroxidation found in this experimental group. All these changes were partially reversed by both IP protocols and MPG treatment. Thus, GSH content was higher and SOD cytosolic activity was lower than the values observed in untreated hearts. The favorable changes in GSH and SOD cytosolic activity were reflected in the lower lipid peroxidation (decreased TBARS concentration) observed in preconditioned hearts and in those treated with MPG in comparison to ischemic control hearts. In other words the improvement of the antioxidant systems (SOD and GSH) by IP and MPG treatment were enough to attenuate the oxidative damage detected in untreated hearts. These results suggest that changes of lipid peroxidation and antioxidant systems would be sufficient to promote differences in the cell death and the attenuation of oxidative stress would be considered as a factor contributing to

On the other hand, a balance between the production of nitric oxide (NO) and ROS controls the endothelial function [34, 35]. When the NO production is normal its bioavailability may

the cardioprotection by IP and MPG treatment in hearts from SHR.

ROS release and/or production and an improvement of the endogenous antioxidants.

We can conclude that the level of lipid peroxidation and antioxidant defenses are linked to reperfusion injury in hypertrophied hearts from SHR. The finding that IP and MPG reduce the postischemic myocardial and vascular injury as well as levels of TBARS and improve the endogenous antioxidants suggest that the decrease in ROS levels would be the common mechanism of cardioprotection of both interventions.

## **Author details**

Juliana C. Fantinelli, Ignacio A. Pérez Núñez, Luisa F. González Arbeláez and Susana M. Mosca *Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina* 

## **Acknowledgement**

This work was supported in part by the grant PICT 1046 from Agencia Nacional de Promoción Científica y Técnica of Argentina to Dr Susana M Mosca.

#### **6. References**

[1] Newaz MA, Nawal NN (1999) Effect of gamma-tocotrienol on blood pressure, lipid peroxidation and total antioxidant status in spontaneously hypertensive rats (SHR). Clin. Exp. Hypertens. 21: 1297-1313.


[17] Ebrahim Z, Yellon DM, Baxter GF (2007) Attenuated cardioprotective response to bradykinin, but not classical ischaemic preconditioning, in DOCA-salt hypertensive left ventricular hypertrophy. Pharmacol. Res. 55(1): 42-48.

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animal and human studies. Circulation 108: 2034-2040.

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isolated rat heart. Life Sci. 65: 1927-1930.

Endocrinol. Metab. 297(3): 685-694.

Clin. Exp. Hyperten. 30: 673-681.

1148-1156.

Cardiol. 92: 410-416.

Exp. Gerontol. 42(8): 807-814.

hypertensive rats. Pharmacol. Rep. 62: 674-682.

[2] Vaziri ND, Sica DA (2004) Lead-induced hypertension: role of oxidative stress. Curr.

[3] Griendling KK, FitzGerald GA (2003) Oxidative stress and cardiovascular injury: Part II:

[4] Papaharalambus CA, Griendling KK (2007) Basic mechanisms of oxidative stress and reactive oxygen species in cardiovascular injury. Trends Cardiovasc. Med. 17: 48-54. [5] Ravingerová T, Slezák J, Tribulová J, Džurba A, Uhrík B, Ziegelhöffer A (1999) Reactive oxygen species contribute to high incidence of reperfusion-induced arrhythmias in

[6] Kalaycioglu S, Sinci V, Imren Y, Oz E (1999) Metoprolol prevents ischemia–reperfusion

[7] Abebe W, Liu JY, Wimborne H, Mozaffari MS (2010) Effects of chromium picolinate on vascular reactivity and cardiac ischemia-reperfusion injury in spontaneously

[8] Potenza MA, Gagliardi S, De Benedictis L, Zigrino A, Tiravanti E, Colantuono G, Federici A, Lorusso L, Benagiano V, Quon MJ, Montagnani M (2009) Treatment of spontaneously hypertensive rats with rosiglitazone ameliorates cardiovascular pathophysiology via antioxidant mechanisms in the vasculature. Am. J. Physiol.

[9] Sahna E, Deniz E, Bay-Karabulut A, Burma O (2008) Melatonin protects myocardium from ischemia-reperfusion injury in hypertensive rats: role of myeloperoxidase activity.

[10] Mosca SM, Gelpi RJ, Milei J, Fernandez Alonso G, Cingolani HE (1998) Is stunning

[11] Yellon DM, Downey JM (2003) Preconditioning the myocardium: from cellular

[12] Speechly-Dick ME, Baxter GF, Yellon DM (1994) Ischaemic preconditioning protects

[13] Boutros A, Wang J (1995) Ischaemic preconditioning, adenosine and bethanechol protect spontaneously hypertensive isolated rat hearts. J. Pharmacol. Exp. Ther. 275:

[14] Pantos CI, Davos CH, Carageorgiou HC, Varonos DV, Cokkinos DV (1996) Ischaemic preconditioning protects against myocardial dysfunction caused by ischaemia in

[15] Nakagawa C, Asayama J, Katamura M, Matoba S, Keira N, Kawahara A, Tsuruyama K, Tanaka T, Kobara M, Akashi K, Ohta B, Tatsumi T, Nakagawa M (1997) Myocardial strech induced by increased left ventricular diastolic pressure preconditions isolated perfused hearts of normotensive and spontaneously hypertensive rats. Basic Res.

[16] Ebrahim Z, Yellon DM, Baxter GF (2007) Ischemic preconditioning is lost in aging hypertensive rat heart: independent effects of aging and longstanding hypertension.

prevented by ischemic preconditioning? Mol . Cell. Biochem. 186: 123-129.

physiology to clinical cardiology. Physiol. Rev. 83: 1113–1151.

hypertrophied myocardium. Cardiovasc. Res. 28(7):1025-1029.

isolated hypertrophied rat hearts. Basic Res. Cardiol. 91: 444-449.


## **Lipid Peroxidation by-Products and the Metabolic Syndrome**

Nicolas J. Pillon and Christophe O. Soulage

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46019

## **1. Introduction**

408 Lipid Peroxidation

2313.

[33] Haramaki N, Stewart DB, Aggarwal S, Ikeda H, Reznick AZ, Packer L (1998) Networking antioxidants in the isolated rat heart are selectively depleted by ischemia-

[34] Kodja G, Harrison D (1999) Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart

[35] Cai , Harrison DG (2000) Endothelial dysfunction in cardiovascular diseases: the role of

[36] Shah AM, MacCarthy PA (2000) Paracrine and autocrine effects of nitric oxide in myocardial function. Pharmacol. Ther. 86: 49-86. [37] Kagota S, Tada Y, Kubota Y, Nejime N, Yamaguchi Y, Nakamura K, Kunitomo M, Shinozuka K (2007) Peroxynitrite is involved in the dysfunction of vasorelaxation in SHR/NDmcr-cp rats, spontaneously

[38] Piech A, Dessy C, Havaux X, Feron O, Balligand JL (2003) Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive

[39] Vaziri ND, Ni Z, Oveisi F (1998) Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension 31: 1248-1254. [40] Nava E, Noll G, Luscher TF (1995) Increased activity of constitutive nitric oxide synthase in cardiac endothelium in spontaneous hypertension. Circulation 91: 2310-

[41] Vapaatalo H, Mervaala E, Nurminen ML (2000) Role of endothelium and nitric oxide in

reperfusion. Free Rad. Biol. Med. 25(3): 329-339.

hypertensive rats. J. Cardiovasc. Pharmacol. 50: 677-685.

experimental hypertension. Physiol. Res. 49: 1–10.

failure. Cardiovasc. Res. 43: 562-571.

oxidant stress. Circ. Res. 87: 840-844.

rats. Cardiovasc. Res. 57: 456-467.

About twenty-one percent of the air we breathe is composed of oxygen, and our life will not be possible without it. Oxygen is however a toxic, highly reactive molecule which was originally released in atmosphere as a waste product of the first photosynthetic organisms. Its accumulation on Earth indeed led to a massive extinction of living species. Few organisms survived and some developed the ability to use this toxic oxygen to improve the production of energy from carbohydrates. This "oxidative metabolism" was however a double-edge sword as the use of intracellular oxygen generates deleterious oxidative damages. To protect themselves toward this toxicity, those organisms consequently developed several "antioxidants" protection mechanisms which helped them maintain a balance between oxidative damage and efficient use of oxygen to produce energy.

When antioxidant defences are reduced and/or oxidative mechanisms increased, uncontrolled oxidation of cell targets leads to the accumulation of reactive oxygen species (ROS) and a state of "oxidative stress", often deleterious for the cells. This stress is involved in the pathophysiology of several human diseases, and especially in the development of metabolic diseases, even if its causative role remains questionable. A definite increase in oxidative stress biomarkers can be found in obese and diabetic humans as well as in animal model of these diseases. Accumulation of ROS can be deleterious by itself or can induce the oxidation of proteins, nucleic acids and lipids, generating secondary by-products. The specific reactivity of ROS towards polyunsaturated fatty acids (PUFAs) present in cell membranes induces lipid peroxidation, a noxious mechanism producing toxic aldehydes. Among them, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been extensively studied. Originally simple markers of lipid peroxidation, these aldehydes have demonstrated causative roles in the impairment of cellular functions: activation of signalling pathways, apoptosis, and modification of enzyme function. In addition to being hallmarks

© 2012 Pillon and Soulage, licensee InTech. This is an open access chapter 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. © 2012 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.

of oxidative damage, lipid aldehydes could be mediators of oxidative insults, propagating tissue injury and activating cellular stress signalling pathways. Several studies demonstrated the association of obesity and diabetes with lipid peroxidation by-products, and the role of aldehydes in impairment of insulin function and signalling was recently pointed out.

This chapter aims to review the diverse implications of lipid peroxidation by-products in the pathophysiology of metabolic diseases, from evidence of their production during obesity and diabetes to the cellular mechanisms of their toxicity and protection against their deleterious effects.

## **2. Lipid peroxidation by-products**

Under conditions of oxidative stress, excessive production of reactive oxygen species promotes the peroxidation of polyunsaturated fatty acids (PUFA). The resulting accumulation of hydroperoxides, unstable molecules, leads to their non-enzymatic degradation in many compounds, including aldehydes. Quantification of lipid peroxidation in biological samples has been extensively performed with the thiobarbituric acid (TBA) test. TBA detects malondialdehyde (MDA), an end-product of nonenzymatic PUFA oxidative degradation, which has therefore been used for decades as a marker of lipid peroxidation (Gutteridge, 1982). Another aldehyde: acrolein, first attracted attention because of its formation during tobacco combustion and its ubiquitous presence in the environment (Dong & Moldoveanu, 2004). Because of its carcinogenic potential, its role in smoking-related diseases has received extensive attention; however, acrolein is also produced endogenously though lipid peroxidation and its link with oxidative-associated pathologies is now well established. 4-hydroxy-2-alkenals are specific by-products of the oxidation of omega-3 and omega-6 fatty acids. 4-hydroxy-2-nonenal (HNE) is derived from the oxidation of polyunsaturated fatty acids of the n-6 series, mainly linoleic and arachidonic acids, while 4 hydroxy-2-hexenal (HHE) results from the peroxidation of polyunsaturated fatty acids of the n-3 series (mainly docosahexaenoic, eicosapentaenoic and linolenic acid). The peroxidation of arachidonic acid via 12-lipoxygenase leads to the formation of 4-hydroxy-2 dodecadienal (HDDE) (Guichardant *et al.*, 2006).


**Table 1.** α,β-Unsaturated aldehydes produced during polyunsaturated fatty acids oxidation

#### **2.1. Chemistry and reactivity**

410 Lipid Peroxidation

pointed out.

**Name** 

deleterious effects.

**2. Lipid peroxidation by-products** 

dodecadienal (HDDE) (Guichardant *et al.*, 2006).

Acrolein (prop-2-enal) 56.1 C3H4O Malondialdehyde (propanedial, MDA) 72.1 C3H4O2 4-hydroxy-2-hexenal (HHE) 114.1 C6H10O2 4-hydroxy-2-nonenal (HNE) 156.2 C9H16O2

4-hydroxy-2-dodecadienal (HDDE) 196.0 C12H20O2

**Table 1.** α,β-Unsaturated aldehydes produced during polyunsaturated fatty acids oxidation

of oxidative damage, lipid aldehydes could be mediators of oxidative insults, propagating tissue injury and activating cellular stress signalling pathways. Several studies demonstrated the association of obesity and diabetes with lipid peroxidation by-products, and the role of aldehydes in impairment of insulin function and signalling was recently

This chapter aims to review the diverse implications of lipid peroxidation by-products in the pathophysiology of metabolic diseases, from evidence of their production during obesity and diabetes to the cellular mechanisms of their toxicity and protection against their

Under conditions of oxidative stress, excessive production of reactive oxygen species promotes the peroxidation of polyunsaturated fatty acids (PUFA). The resulting accumulation of hydroperoxides, unstable molecules, leads to their non-enzymatic degradation in many compounds, including aldehydes. Quantification of lipid peroxidation in biological samples has been extensively performed with the thiobarbituric acid (TBA) test. TBA detects malondialdehyde (MDA), an end-product of nonenzymatic PUFA oxidative degradation, which has therefore been used for decades as a marker of lipid peroxidation (Gutteridge, 1982). Another aldehyde: acrolein, first attracted attention because of its formation during tobacco combustion and its ubiquitous presence in the environment (Dong & Moldoveanu, 2004). Because of its carcinogenic potential, its role in smoking-related diseases has received extensive attention; however, acrolein is also produced endogenously though lipid peroxidation and its link with oxidative-associated pathologies is now well established. 4-hydroxy-2-alkenals are specific by-products of the oxidation of omega-3 and omega-6 fatty acids. 4-hydroxy-2-nonenal (HNE) is derived from the oxidation of polyunsaturated fatty acids of the n-6 series, mainly linoleic and arachidonic acids, while 4 hydroxy-2-hexenal (HHE) results from the peroxidation of polyunsaturated fatty acids of the n-3 series (mainly docosahexaenoic, eicosapentaenoic and linolenic acid). The peroxidation of arachidonic acid via 12-lipoxygenase leads to the formation of 4-hydroxy-2-

> **Molecular Weight (***Da***)**

**Molecular**

**Formula Skeletal Formula** 

Acrolein, MDA and 4-hydroxy-alkenals are α,β-unsaturated aldehydes, a class of compounds sharing the general structure C=C−C=O. They are characterised by an aldehyde group (C=O) on carbon 1 and a conjugated double bond (C=C) between carbons 2 and 3 (Table 1). In this structure, the oxygen atom of the carbonyl group increases the polarity of the double bond, which makes α,β-unsaturated aldehydes potent electrophiles. Acrolein has the simplest structure composed of 3 carbons, MDA is a dicarbonyl compound and 4-hydroxy-2-alkenals are characterized by the presence of a hydroxyl group on carbon 4. HHE, HNE and HDDE only differ by the length of their carbon chain and the presence of an additional double bond for the HDDE. In the case of MDA and 4 hydroxy-alkenals, the presence of a second oxygen atom makes the double bond even more reactive.

These aldehydes are part of the "*reactive electrophile species*" able to form covalent adducts with the nucleophilic groups present in DNA, proteins and phospholipids. In physiological conditions, they spontaneously react with the thiol group of glutathione to form Michael adduct by attack of the nucleophilic group of glutathione to the double bond of aldehydes. They can also react with thiol groups present on cysteine residues of certain proteins, leading to impairment of their biological activity. Under certain conditions, especially alkaline pH, aldehydes react with the amine groups present in proteins, nucleic acids and aminophospholipids, leading to Michael adducts. On the other hand, the reaction between a primary amine group and the carbonyl group of the aldehyde leads to the formation of Schiff bases (Schaur, 2003).

Aldehydes produced during lipid peroxidation are precursors of Advanced Lipoxidation End products (ALEs). Together with Advanced Glycation End products (AGEs) generated during glycoxidation, they accumulate in cells and tissues. The "carbonyl stress" is a result of this adduct accumulation, which induces protein dysfunctions and consequent pathological events such as inflammation and apoptosis (Negre-Salvayre *et al.*, 2008).

#### **2.2. Cellular effects**

#### *2.2.1. Cytotoxicity*

Since α,β-unsaturated compounds are strong electrophiles, they exhibit a high cytotoxic and mutagenic potential and have consequently been extensively studied for their effects on cell viability. HNE, HHE and acrolein indeed induce cell death, but the lethal concentration 50 (LC50), concentration that induces the death of 50% of the cells, is subject to variation, depending on the aldehyde, exposure duration and cell type (Table 2). The LC50 for a long exposure (>16 hours) to HNE or HHE is however consistently found 20-60 µM in several cell types, including human lymphoma Jurkat cells, lens epithelial cells, hamster V79-4 cells and muscle cells (Table 2). For acrolein, the same treatment gives a range of LC50 of 5-100 µM in human fibroblasts, human neuroblastoma cells, PC12 chromaffin cells and lymphocytes. MDA-induced cell death is less documented, even if

the LC50 range for MDA is found around 1 mM in cortical, endothelial cells and fibroblasts. MDA was also reported to induce cell cycle arrest, which is to relate to cell damage and death (Ji *et al.*, 1998). HDDE appears to be the most toxic lipid aldehyde with a LC50 in endothelial cells in the submicromolar range.


**Table 2.** Range of lethal concentration 50 for long-term treatment (>16 hours) with α,β-unsaturated aldehydes. LC50 values were calculated from the indicated references.

On the opposite, very little cell death is detectable for short term treatments (<4 hours), likely because aldehyde-induced cell death involves apoptosis mechanisms not yet occurring during this short period of time. Several studies indeed reported that cell death is induced by aldehydes through apoptosis for low concentration and both apoptosis and necrosis for high doses (Luo *et al.*, 2005; Liu *et al.*, 2010). Acrolein-induced necrosis was described in few studies (Luo *et al.*, 2005), but mitochondrial-driven cell death seems to be the canonical road and was widely studied. Acrolein-induced apoptosis was indeed confirmed in several cell types through DNA fragmentation, phosphatidylserine externalization, poly(ADP-ribose) polymerase cleavage and activation of caspases (Pan *et al.*, 2009; Roy *et al.*, 2010). Last but not least, hydroxyalkenals are potent activators of apoptosis. They both induce DNA fragmentation and activation of caspases in very different cell types (Choudhary *et al.*, 2002; Vaillancourt *et al.*, 2008). In addition, HHE has been shown to decrease the expression/phosphorylation of Bcl-2, while increasing that of Bax, leading to apoptosis of human renal epithelial cells (Bae *et al.*, 2011; Bodur *et al.*, 2012).

Interestingly, the toxicity of aldehydes is highly correlated to their ability to form covalent adducts on proteins. In muscle cells, the lethal concentration 50 (LC50) for 12 different aldehydes was calculated, including HHE and HNE (Pillon *et al.*, 2010). This LC50 was strongly correlated with their respective potency to form covalent adducts on albumin *in vitro* (Figure 1). This demonstrates that the cytotoxicity and likely other biological effects of aldehydes mainly occur through chemical adduction of other biomolecules.

**Figure 1.** Toxicity is correlated to the adduction ability. Viability of muscle cells was measured in response to 12 different aldehydes1. The calculated LC50 was then correlated to their ability to form covalent adducts on bovine serumalbumin (Pillon et al., 2010).

#### *2.2.2. Oxidative stress and ROS production*

412 Lipid Peroxidation

Acrolein

Malondialdehyde

4-hydroxy-2-hexenal

4-hydroxy-2-nonenal

4-hydroxy-2-dodecadienal

the LC50 range for MDA is found around 1 mM in cortical, endothelial cells and fibroblasts. MDA was also reported to induce cell cycle arrest, which is to relate to cell damage and death (Ji *et al.*, 1998). HDDE appears to be the most toxic lipid aldehyde with

Jia *et al.*, 2009*b*, 2009*a*

Hipkiss *et al.*, 1997; Cheng *et al.*, 2011

Pillon *et al.*, 2010; Li *et al.*, 2011

Pillon *et al.*, 2010; Li *et al.*, 2011

(prop-2-enal) 5-100 Poirier *et al.*, 2002; Luo *et al.*, 2005;

(HHE) 20-60 Liu *et al.*, 2000; Choudhary *et al.*, 2002;

(HNE) 20-60 Liu *et al.*, 2000; Choudhary *et al.*, 2002;

**Table 2.** Range of lethal concentration 50 for long-term treatment (>16 hours) with α,β-unsaturated

On the opposite, very little cell death is detectable for short term treatments (<4 hours), likely because aldehyde-induced cell death involves apoptosis mechanisms not yet occurring during this short period of time. Several studies indeed reported that cell death is induced by aldehydes through apoptosis for low concentration and both apoptosis and necrosis for high doses (Luo *et al.*, 2005; Liu *et al.*, 2010). Acrolein-induced necrosis was described in few studies (Luo *et al.*, 2005), but mitochondrial-driven cell death seems to be the canonical road and was widely studied. Acrolein-induced apoptosis was indeed confirmed in several cell types through DNA fragmentation, phosphatidylserine externalization, poly(ADP-ribose) polymerase cleavage and activation of caspases (Pan *et al.*, 2009; Roy *et al.*, 2010). Last but not least, hydroxyalkenals are potent activators of apoptosis. They both induce DNA fragmentation and activation of caspases in very different cell types (Choudhary *et al.*, 2002; Vaillancourt *et al.*, 2008). In addition, HHE has been shown to decrease the expression/phosphorylation of Bcl-2, while increasing that of Bax, leading to

Interestingly, the toxicity of aldehydes is highly correlated to their ability to form covalent adducts on proteins. In muscle cells, the lethal concentration 50 (LC50) for 12 different aldehydes was calculated, including HHE and HNE (Pillon *et al.*, 2010). This LC50 was strongly correlated with their respective potency to form covalent adducts on albumin *in vitro* (Figure 1). This demonstrates that the cytotoxicity and likely other biological effects of

(propanedial, MDA) 600-3000 Michiels & Remacle, 1991;

apoptosis of human renal epithelial cells (Bae *et al.*, 2011; Bodur *et al.*, 2012).

aldehydes mainly occur through chemical adduction of other biomolecules.

a LC50 in endothelial cells in the submicromolar range.

**Name LC50, µM References**

(HDDE) 0.22 Riahi *et al.*, 2010

aldehydes. LC50 values were calculated from the indicated references.

The classical sequence of events is that oxidative stress triggers lipid peroxidation which in turns produces aldehydes by-products. However, an interesting paradigm was pointed out by several groups: lipid aldehydes are able to induce the production of ROS, and this is thought to be of importance in their deleterious effects. For instance, acrolein treatment produces nitric oxide (Misonou *et al.*, 2006) and induces generation of intracellular oxidants (Luo *et al.*, 2005; Wang *et al.*, 2011). Similarly, accumulation of intracellular ROS was described in cells treated with MDA (Cheng *et al.*, 2011); and unsurprisingly, the 4 hydroxyalkenals HHE and HNE share the same ability. HHE induces ROS in neurons and tubular epithelial cells (Long *et al.*, 2008; Bae *et al.*, 2011) and HNE induces mitochondrial oxidative stress in neurons, vascular muscle, liver and skeletal muscle cells (Uchida *et al.*, 1999; Lee *et al.*, 2006; Pillon *et al.*, 2012). The source of ROS was suggested to be mitochondria, as several aldehydes have been shown to induce a significant decrease in mitochondrial membrane potential (Uchida *et al.*, 1999; Luo *et al.*, 2005).

**Figure 2.** The vicious circle linking ROS and lipid aldehyde production

<sup>1 4-</sup>hydroxy-2-hexenal, 4-hydroxy-2-nonenal, 4-hydroxy-2-dodecenal, 4-hydroxy-2-hexenal

dimethylacetal, 4-hydroxy-2-nonenal dimethylacetal, 4-hydroxy-2-dodecenal dimethylacetal, trans-2 hexenal, trans-2-nonenal, trans-2-dodecenal, hexanal, nonanal, dodecanal

This concept is reinforced by the fact that an increase in glutathione pool can prevent the deleterious effects of HNE on both adduct formation and ROS production (Pillon *et al.*, 2012) and that dysfunction of glutathione S-transferase, a major enzyme for aldehyde detoxification, leads to excess 4-hydroxy-2-nonenal and oxidative stress (Kostyuk *et al.*, 2010; Curtis *et al.*, 2010). Based on this body of evidence, lipid by-products can be seen as parts of a vicious circle in which increased ROS production generates aldehydes which further amplify the generation of oxidative species and so on (Figure 2).

#### *2.2.3. Signalling pathways and transcription factors*

Consistent with the extensive work done on cytotoxicity, the stress signalling pathways have been primarily pointed out as the main intracellular route activated by aldehydes. The mitogen-activated protein kinases (MAPKs) are indeed activated by several aldehydes including MDA, acrolein and HNE. MDA activates c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK) (Cheng *et al.*, 2011) and acrolein-induced apoptosis occurs through activation of p38 and ERK (Tanel & Averill-Bates, 2007). The activation of these three MAPK has also been described following treatment with HNE (Uchida *et al.*, 1999; Zarrouki *et al.*, 2007; Pillon *et al.*, 2012) and HHE (Je *et al.*, 2004; Bae *et al.*, 2011). Overall, most studies investigated the cytotoxic effects of aldehydes and thus focused on cellular stress pathways; therefore, very little data is available regarding their potential effects on other pathways. Only very recent work shows that HNE interfere with insulin signalling pathway through oxidative stress and adduction of IRS1 and Akt (Demozay *et al.*, 2008; Shearn *et al.*, 2011; Pillon *et al.*, 2012).

Aldehydes regulate gene expression by activating the signalling pathways described above, or by direct modification of transcription factors. Unsurprisingly, aldehyde production regulates the expression of several antioxidant enzymes such as NAD(P)H quinone oxidoreductase-1, Heme oxygenase-1 and glutathione S-transferase (GST). This occurs through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which drives the expression of these antioxidant enzymes. Aldehyde-induced Heme Oxygenase-1 expression is indeed mediated by the Nrf2 pathway in HUVECs (Lee *et al.*, 2011) and Nrf2 silencing significantly attenuates the induction of this same gene by acrolein (Zhang & Forman, 2008).

Beyond the expression of antioxidant enzymes, cellular growth, apoptosis and inflammatory responses can be induced by aldehydes, involving the activation of the nuclear factor kappa B (NF-κB) family. The effects of acrolein on NF-κB activation are controversial, but its activation by HNE and HHE is well documented. HHE and HNE induce NF-kappaB activation through IKK/NIK pathway, leading to IκB phosphorylation and subsequent proteolysis (Page *et al.*, 1999; Je *et al.*, 2004; Lee *et al.*, 2004). HNE has moreover been shown to induce DNA-binding of NF-κB in vascular smooth muscle cells (Ruef *et al.*, 2001).

The peroxisome proliferator-activated receptor (PPAR) family regulate the expression of genes that encode proteins involved in energy balance. They act as ligand-activated transcription factors and are responsive to the lipid status of the cell, therefore important during high fat diets and obesity. HNE is an intracellular agonist of PPARβ/δ while HHE do not activate this receptor (Coleman *et al.*, 2007). Through this pathway, HNE significantly elevates adiponectin gene expression, concomitant with increased PPAR-γ gene expression and transactivity. Meanwhile, HDDE acts through PPARδ signalling pathways to regulate glucose transport in vascular endothelial cells subjected to hyperglycemia (Riahi *et al.*, 2010) and HNE stimulates insulin secretion from Beta cells through interaction with PPARδ (Cohen *et al.*, 2011).

#### **2.3. Concentration in plasma and tissues**

414 Lipid Peroxidation

This concept is reinforced by the fact that an increase in glutathione pool can prevent the deleterious effects of HNE on both adduct formation and ROS production (Pillon *et al.*, 2012) and that dysfunction of glutathione S-transferase, a major enzyme for aldehyde detoxification, leads to excess 4-hydroxy-2-nonenal and oxidative stress (Kostyuk *et al.*, 2010; Curtis *et al.*, 2010). Based on this body of evidence, lipid by-products can be seen as parts of a vicious circle in which increased ROS production generates aldehydes which further

Consistent with the extensive work done on cytotoxicity, the stress signalling pathways have been primarily pointed out as the main intracellular route activated by aldehydes. The mitogen-activated protein kinases (MAPKs) are indeed activated by several aldehydes including MDA, acrolein and HNE. MDA activates c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK) (Cheng *et al.*, 2011) and acrolein-induced apoptosis occurs through activation of p38 and ERK (Tanel & Averill-Bates, 2007). The activation of these three MAPK has also been described following treatment with HNE (Uchida *et al.*, 1999; Zarrouki *et al.*, 2007; Pillon *et al.*, 2012) and HHE (Je *et al.*, 2004; Bae *et al.*, 2011). Overall, most studies investigated the cytotoxic effects of aldehydes and thus focused on cellular stress pathways; therefore, very little data is available regarding their potential effects on other pathways. Only very recent work shows that HNE interfere with insulin signalling pathway through oxidative stress and adduction of IRS1 and Akt (Demozay *et al.*,

Aldehydes regulate gene expression by activating the signalling pathways described above, or by direct modification of transcription factors. Unsurprisingly, aldehyde production regulates the expression of several antioxidant enzymes such as NAD(P)H quinone oxidoreductase-1, Heme oxygenase-1 and glutathione S-transferase (GST). This occurs through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which drives the expression of these antioxidant enzymes. Aldehyde-induced Heme Oxygenase-1 expression is indeed mediated by the Nrf2 pathway in HUVECs (Lee *et al.*, 2011) and Nrf2 silencing significantly attenuates the induction of this same gene by acrolein (Zhang &

Beyond the expression of antioxidant enzymes, cellular growth, apoptosis and inflammatory responses can be induced by aldehydes, involving the activation of the nuclear factor kappa B (NF-κB) family. The effects of acrolein on NF-κB activation are controversial, but its activation by HNE and HHE is well documented. HHE and HNE induce NF-kappaB activation through IKK/NIK pathway, leading to IκB phosphorylation and subsequent proteolysis (Page *et al.*, 1999; Je *et al.*, 2004; Lee *et al.*, 2004). HNE has moreover been shown

The peroxisome proliferator-activated receptor (PPAR) family regulate the expression of genes that encode proteins involved in energy balance. They act as ligand-activated

to induce DNA-binding of NF-κB in vascular smooth muscle cells (Ruef *et al.*, 2001).

amplify the generation of oxidative species and so on (Figure 2).

*2.2.3. Signalling pathways and transcription factors* 

2008; Shearn *et al.*, 2011; Pillon *et al.*, 2012).

Forman, 2008).

Little data is available concerning aldehydes levels in biological fluids, except for MDA and HNE which have been widely used as lipid peroxidation markers. The concentration of MDA in the plasma of healthy subjects is around 2-5 µM and increases up to 2-fold in type 2 diabetic patients (Figure 4). In the specific context of metabolic diseases, MDA is positively correlated with BMI and waist circumference in obese patients (Furukawa *et al.*, 2004). Depending on the study, HNE concentration has been found to range from 50 nM to 10 µM under normal conditions. This significant variability in concentration according to the authors could be explained partly by the method used (LC/MS, GC/MS ...) and also by the difficulty to measure such reactive derivatives. If HNE has been widely studied, there is however scarce data in the literature regarding the pathophysiological concentrations of HHE and HDDE. Plasma HHE concentration was however found to be around 9 nM in human, and dramatically increases to reach 90 nM after several weeks of a diet rich in omega-3 fatty acids (Calzada *et al.*, 2010). Our group recently showed that HHE concentration was 20 nM in humans and 7 nM in rats and that it increases in both type-2 diabetes patients and type-1 diabetic rats, reinforcing existing evidence for a role of lipid aldehydes in metabolic diseases.


**Table 3.** Concentration range of aldehydes in healthy plasma and tissues

One should keep in mind that all current quantification methods (HPLC, GC) only assay the free fraction (*i.e.* unreacted) of lipid aldehydes present in samples. Being very reactive, this is certainly not representative of the amount indeed produced from lipid peroxidation, which rapidly react with neighbour targets to form covalent adducts and thus, other nonquantitative methods estimating the amount of HNE have been used. For example, detection of protein adducts by immunohistochemistry has shown a significant increase in HHE and HNE proteins adducts in Parkinson's disease and in chronic liver disease (Yoritaka *et al.*, 1996; Paradis *et al.*, 1997). Of particular interest for this chapter, HNE adducts on plasma albumin are increased in type 2 diabetes (Toyokuni *et al.*, 2000).

#### **3. Insulin secretion and type-1 diabetes**

#### **3.1. Lipid aldehydes and insulin secretion**

Oxidative stress associated with hyperglycemia is suspected to participate in beta cell dysfunction in terms of insulin synthesis and/or secretion. Diabetic animals display increased levels of lipoperoxidation in pancreas, and HNE concentration was reported to reach up to 35 µM in pancreatic islets of diabetic rats (Miwa *et al.*, 2000). In addition, HNEmodified proteins are increased in the pancreatic beta-cells of Goto-Kakizaki rats, a genetic model of non obese type 2 diabetes (Ihara *et al.*, 1999), as well as in type 2 diabetic patients (Sakuraba *et al.*, 2002). HNE and other lipid peroxidation by-products such as 2-hexenal and 2-butenal inhibit glucose-induced insulin secretion in isolated rat islets. Both glucose utilization and glucose oxidation are blunted in islets after treatment with aldehydes suggesting that they impair glucose-induced insulin secretion through an interference with glycolytic pathway and citric acid cycle (Miwa *et al.*, 2000). Another piece of evidence comes from the exposure of beta cells to NO donors and to interleukin-1 beta, which leads to generation of oxidative stress and lipoperoxidation by-products. MDA and HNE produced under this condition are involved in the activation of an apoptotic program, contributing to the reduction in the beta cell mass (Cahuana *et al.*, 2003). Peroxynitrite indeed triggers lipoperoxidation in the beta-cell line RIN-5-F, and the resulting protein carbonylation is a key factor linking NO-dependent lipoperoxidation and apoptosis (Cahuana *et al.*, 2003). Alloxan, a toxic glucose analogue, has been widely used to generate rodent models of type-1 diabetes, as it selectively destroys insulin-producing cells in the pancreas. Alloxan-induced diabetic rats exhibit increased lipid peroxidation associated with defects in insulin secretion, which can be prevented by the antioxidant S-allyl cysteine therefore restoring insulin secretion and ameliorating the glycaemic control (Augusti & Sheela, 1996).

#### **3.2. Lipid aldehydes and the beta cells: Doctor Jeckyll or Mr Hyde?**

HNE was shown to elicit various physiological or physiopathological responses: high concentrations elicit beta cell death and defect in insulin secretion, while lower concentrations act as signalling mediators. In INS-1E beta-cells, elevated glucose levels increase the release of arachidonic acid and linoleic acid from membrane phospholipids and promote their peroxidation to HNE. At non cytotoxic concentrations, HNE behaves as an endogenous ligand for nuclear receptor PPAR-δ, stimulating insulin secretion in beta-cells (Cohen *et al.*, 2011). In rat islet beta-cell-derived RINm5F cells, a recent report demonstrates the involvement of the transient receptor potential (TRP) cation channels in the HNEinduced insulin secretion. Short-term (1h) exposure to HNE induces a transient increase in intracellular calcium concentration and triggers insulin secretion. HNE induces calcium influx through activation of TRP channels (amongst which TRPA1) which appears to be coupled with the L-type voltage-dependent calcium channel, and ultimately insulin secretion (Numazawa *et al.*, 2012). Lipid aldehydes should therefore be considered either as detrimental (>10 µM) or as beneficial (sub micromolar range) depending on their actual tissue concentration.

#### **3.3. Direct adduction of the insulin polypeptide**

416 Lipid Peroxidation

One should keep in mind that all current quantification methods (HPLC, GC) only assay the free fraction (*i.e.* unreacted) of lipid aldehydes present in samples. Being very reactive, this is certainly not representative of the amount indeed produced from lipid peroxidation, which rapidly react with neighbour targets to form covalent adducts and thus, other nonquantitative methods estimating the amount of HNE have been used. For example, detection of protein adducts by immunohistochemistry has shown a significant increase in HHE and HNE proteins adducts in Parkinson's disease and in chronic liver disease (Yoritaka *et al.*, 1996; Paradis *et al.*, 1997). Of particular interest for this chapter, HNE adducts

Oxidative stress associated with hyperglycemia is suspected to participate in beta cell dysfunction in terms of insulin synthesis and/or secretion. Diabetic animals display increased levels of lipoperoxidation in pancreas, and HNE concentration was reported to reach up to 35 µM in pancreatic islets of diabetic rats (Miwa *et al.*, 2000). In addition, HNEmodified proteins are increased in the pancreatic beta-cells of Goto-Kakizaki rats, a genetic model of non obese type 2 diabetes (Ihara *et al.*, 1999), as well as in type 2 diabetic patients (Sakuraba *et al.*, 2002). HNE and other lipid peroxidation by-products such as 2-hexenal and 2-butenal inhibit glucose-induced insulin secretion in isolated rat islets. Both glucose utilization and glucose oxidation are blunted in islets after treatment with aldehydes suggesting that they impair glucose-induced insulin secretion through an interference with glycolytic pathway and citric acid cycle (Miwa *et al.*, 2000). Another piece of evidence comes from the exposure of beta cells to NO donors and to interleukin-1 beta, which leads to generation of oxidative stress and lipoperoxidation by-products. MDA and HNE produced under this condition are involved in the activation of an apoptotic program, contributing to the reduction in the beta cell mass (Cahuana *et al.*, 2003). Peroxynitrite indeed triggers lipoperoxidation in the beta-cell line RIN-5-F, and the resulting protein carbonylation is a key factor linking NO-dependent lipoperoxidation and apoptosis (Cahuana *et al.*, 2003). Alloxan, a toxic glucose analogue, has been widely used to generate rodent models of type-1 diabetes, as it selectively destroys insulin-producing cells in the pancreas. Alloxan-induced diabetic rats exhibit increased lipid peroxidation associated with defects in insulin secretion, which can be prevented by the antioxidant S-allyl cysteine therefore restoring insulin

on plasma albumin are increased in type 2 diabetes (Toyokuni *et al.*, 2000).

secretion and ameliorating the glycaemic control (Augusti & Sheela, 1996).

**3.2. Lipid aldehydes and the beta cells: Doctor Jeckyll or Mr Hyde?** 

HNE was shown to elicit various physiological or physiopathological responses: high concentrations elicit beta cell death and defect in insulin secretion, while lower concentrations act as signalling mediators. In INS-1E beta-cells, elevated glucose levels increase the release of arachidonic acid and linoleic acid from membrane phospholipids and

**3. Insulin secretion and type-1 diabetes** 

**3.1. Lipid aldehydes and insulin secretion** 

Under conditions of oxidative stress, insulin, a polypeptidic hormone composed of 51 amino acid residues, is exposed to direct oxidative insult or to modification by lipoperoxidation byproducts. Several amino acids are putative sites of adduction, and thus, covalent binding of lipid aldehydes affect the biological actions of this hormone. This applies to acrolein and methylglyoxal, whose fixation on insulin has been shown to reduce both hypoglycemic effects in rats and glucose uptake in 3T3-L1 adipocytes (Jia *et al.*, 2006; Medina-Navarro *et al.*, 2007). HHE and HNE, toxic aldehydes generated during lipid peroxidation, also modify the B-chain of human insulin *in vitro*, predominantly at the His B5 and His B10 residues via Michael adduction (Figure 3). Adduct formation affects the biological activity of insulin *in vivo*, decreasing its hypoglycemic effect in mice and stimulation of glucose uptake in adipose and muscle cells (Pillon *et al.*, 2011).

**Figure 3.** Structure of insulin monomer displaying two HHE adducts on histidine residues. From Pillon et al, 2011.

#### **4. Obesity, insulin resistance and type-2 diabetes**

#### **4.1. Lipid peroxidation by-products association with obesity**

Obesity is a major factor in the development of metabolic syndrome. After consumption of an energy-dense (i.e. high-fat) diet, plasma HNE levels increase rapidly and significantly within minutes (Devaraj *et al.*, 2008). When consumed regularly, this diet promotes obesity, which suggests a role for HNE very early in the development of obesity. On the other hand, levels of circulating HNE tend to decrease when obese people are maintained on calorie restriction (Johnson *et al.*, 2007), demonstrating that lipid peroxidation is tightly linked to high fat diet and obesity. Furukawa et al. reported that increased oxidative stress in accumulated fat is an important pathogenic mechanism of obesity-associated metabolic syndrome. Production of ROS is indeed selectively increased in white adipose tissue of obese mice and associated with a blunted expression of antioxidant enzymes. In good agreement, fat accumulation correlates with systemic lipid peroxidation in humans (Furukawa *et al.*, 2004), and the plasma concentration of MDA is 1.8 fold higher in subjects with a BMI above 40 kg/m2 compared to lean individuals (Olusi, 2002). Diet-induced obesity increases tissue and plasma accumulation of ALEs (protein–acrolein and protein–HNE adducts for example), suggesting that obesity is associated with an increase in the formation of lipid peroxidation-derived aldehydes (Baba *et al.*, 2011). A significant accumulation of HNE was noticed in the white adipose tissue of obese mice, where the adipocyte fatty acid binding protein (AFABP also known as aP2) is the soluble protein most highly modified by HNE in this tissue (Grimsrud *et al.*, 2007). In obese mice roughly 7% of the AFABP in adipose tissue is covalently modified by HNE resulting in a decreased binding affinity for fatty acids. Lipid peroxidation is however not restricted to adipose tissue since HNE is also elevated in skeletal muscles of Otsuka Long-Evans Tokushima fatty (OLETF) rat, a model for hyperphagic obesity (Morris *et al.*, 2008). Intracellular triglyacylglycerols accumulate in the muscle of obese humans where it is considered as a pathogenic factor in the development of insulin resistance. In obese compared to endurance-trained subjects, the lipid peroxidation to intracellular triacylglycerols ratio was 4-fold higher suggesting that obesity is associated with increased muscle lipid peroxidation (Russell *et al.*, 2003; Vincent *et al.*, 2006).

Chronic, low grade inflammation of white adipose tissue is a hallmark of obesity and a major contributor to oxidative stress and lipid peroxidation (Wellen & Hotamisligil, 2003). In the expanding adipose tissue, hypertrophied adipocytes contribute to the inflammation by up-regulating the expression and release of pro-inflammatory cytokines. In 3T3-L1 adipocytes, HNE can dose-dependently increase the expression of the inducible cyclooxygenase (COX-2) (Zarrouki *et al.*, 2007) and that of the plasminogen activator inhibitor-1 (PAI-1). In the meantime, HNE decreases the expression of the antiinflammatory, insulin-sensitizing hormone adiponectin (Soares *et al.*, 2005; Wang *et al.*, 2012), therefore linking lipid peroxidation by-products and chronic inflammation.

#### **4.2. Lipid peroxidation by-products association with type-2 diabetes**

Epidemiological studies demonstrates that fasting glycemia is positively correlated with oxidative stress markers such as 8-epi-PGF2α and TBARs and negatively correlated with plasma glutathione (Trevisan *et al.*, 2001; Menon *et al.*, 2004). In type-2 diabetic individuals, 8-epi-PGF2α is positively correlated to the HOMA index for insulin resistance (Gopaul *et al.*, 2001) and urinary acrolein correlates with glycated haemoglobin HbA1c (Daimon *et al.*, 2003). This was confirmed in animal models of insulin resistance which exhibit increased markers of oxidative stress, such as plasma F2-isoprostanes (Laight *et al.*, 1999*a*). In parallel, antioxidant defences are reduced during an oral glucose tolerance test in normal and noninsulin-dependent diabetic subjects (Ceriello *et al.*, 1998), and diabetes is associated with decreased vitamin C and glutathione (Maxwell *et al.*, 1997; Dierckx *et al.*, 2003). Reciprocally, an intensive treatment of diabetes improves circulating levels of H2O2 and MDA (Wierusz-Wysocka *et al.*, 1995); and improved insulin sensitivity resulting from exercise and/or dietary restriction is associated with reduced levels of lipid peroxidation products (Reviewed by Vincent *et al.*, 2007). On the other hand, insulin sensitivity can be improved through antioxidant or carbonyl scavenging treatment (Kamenova, 2006; Vincent *et al.*, 2009), demonstrating the tight link existing between oxidative stress, oxidation by-products and insulin resistance.

418 Lipid Peroxidation

*al.*, 2006).

restriction (Johnson *et al.*, 2007), demonstrating that lipid peroxidation is tightly linked to high fat diet and obesity. Furukawa et al. reported that increased oxidative stress in accumulated fat is an important pathogenic mechanism of obesity-associated metabolic syndrome. Production of ROS is indeed selectively increased in white adipose tissue of obese mice and associated with a blunted expression of antioxidant enzymes. In good agreement, fat accumulation correlates with systemic lipid peroxidation in humans (Furukawa *et al.*, 2004), and the plasma concentration of MDA is 1.8 fold higher in subjects with a BMI above 40 kg/m2 compared to lean individuals (Olusi, 2002). Diet-induced obesity increases tissue and plasma accumulation of ALEs (protein–acrolein and protein–HNE adducts for example), suggesting that obesity is associated with an increase in the formation of lipid peroxidation-derived aldehydes (Baba *et al.*, 2011). A significant accumulation of HNE was noticed in the white adipose tissue of obese mice, where the adipocyte fatty acid binding protein (AFABP also known as aP2) is the soluble protein most highly modified by HNE in this tissue (Grimsrud *et al.*, 2007). In obese mice roughly 7% of the AFABP in adipose tissue is covalently modified by HNE resulting in a decreased binding affinity for fatty acids. Lipid peroxidation is however not restricted to adipose tissue since HNE is also elevated in skeletal muscles of Otsuka Long-Evans Tokushima fatty (OLETF) rat, a model for hyperphagic obesity (Morris *et al.*, 2008). Intracellular triglyacylglycerols accumulate in the muscle of obese humans where it is considered as a pathogenic factor in the development of insulin resistance. In obese compared to endurance-trained subjects, the lipid peroxidation to intracellular triacylglycerols ratio was 4-fold higher suggesting that obesity is associated with increased muscle lipid peroxidation (Russell *et al.*, 2003; Vincent *et* 

Chronic, low grade inflammation of white adipose tissue is a hallmark of obesity and a major contributor to oxidative stress and lipid peroxidation (Wellen & Hotamisligil, 2003). In the expanding adipose tissue, hypertrophied adipocytes contribute to the inflammation by up-regulating the expression and release of pro-inflammatory cytokines. In 3T3-L1 adipocytes, HNE can dose-dependently increase the expression of the inducible cyclooxygenase (COX-2) (Zarrouki *et al.*, 2007) and that of the plasminogen activator inhibitor-1 (PAI-1). In the meantime, HNE decreases the expression of the antiinflammatory, insulin-sensitizing hormone adiponectin (Soares *et al.*, 2005; Wang *et al.*,

Epidemiological studies demonstrates that fasting glycemia is positively correlated with oxidative stress markers such as 8-epi-PGF2α and TBARs and negatively correlated with plasma glutathione (Trevisan *et al.*, 2001; Menon *et al.*, 2004). In type-2 diabetic individuals, 8-epi-PGF2α is positively correlated to the HOMA index for insulin resistance (Gopaul *et al.*, 2001) and urinary acrolein correlates with glycated haemoglobin HbA1c (Daimon *et al.*, 2003). This was confirmed in animal models of insulin resistance which exhibit increased markers of oxidative stress, such as plasma F2-isoprostanes (Laight *et al.*, 1999*a*). In parallel,

2012), therefore linking lipid peroxidation by-products and chronic inflammation.

**4.2. Lipid peroxidation by-products association with type-2 diabetes** 

**Figure 4.** Plasma MDA concentration (µM) and blood GSH content (mmol/g haemoglobin) in healthy and type-2 diabetic (T2D) individuals. Results are a meta-analysis from 23 different publications; one dot represents the mean value obtained in one study2. In average, MDA is significantly increased by 60% while GSH is decreased by 25% in T2D compared to healthy subjects (paired student t-test, n=21).

Among α,β-unsaturated aldehydes, only MDA and to some extend HNE have been studied as oxidative stress biomarkers in diabetes, and they are indeed both increased up to 2-fold in both human (Figure 4) and animal models of type-2 diabetes (Wierusz-Wysocka *et al.*, 1995; Dierckx *et al.*, 2003). It has moreover been demonstrated that Type-2 diabetes duration is independently associated with increased levels of lipid peroxidation (Nakhjavani *et al.*, 2010), and our group recently showed an increase in HHE concentration in type-2 diabetes patients, reinforcing existing evidence for the specific role of lipid aldehydes in insulin resistance.

<sup>2</sup> Results from Wierusz-Wysocka et al., 1995; Vijayalingam et al., 1996; Feillet-Coudray et al., 1999; Rábago-Velasco et al., 2000; Rizvi & Zaid, 2001; Seghrouchni et al., 2002; Dinçer et al., 2002; Dierckx et al., 2003; Duman et al., 2003; Pasaoglu et al., 2004; Memişoğullari & Bakan, 2004; Skrha et al., 2005; Kurtul et al., 2005; Ozdemir et al., 2005; Mahboob et al., 2005; Saxena et al., 2005; Sampathkumar et al., 2005; Kuppusamy et al., 2005; Lapolla et al., 2007; Sathiyapriya et al., 2007; Singhania et al., 2008; Jain et al., 2009; Nakhjavani et al., 2010; Narasimhan et al., 2010; Shinde et al., 2011; Huang et al., 2011; Bahadoran et al., 2011; Zhang et al., 2011; Pácal et al., 2011; Calabrese et al., 2011 and Rasic-Milutinovic et al., 2012

Aldehydes concentration is increased in several tissues during diabetes, as revealed by increased levels of HNE in pancreas, liver, brain and heart. Pancreatic islets from type-2 diabetic patients are positively stained with HNE, suggesting that oxidative stress through lipid peroxidation could contribute to the reduced beta-cell mass and islet-cell injury (Sakuraba *et al.*, 2002). An increased level of HNE-modified proteins is reported in the pancreatic beta cells of Goto Kakizaki rats as a result of hyperglycaemia (Ihara *et al.*, 1999); and accumulation of HNE is observed in liver of diabetic rats due to the impairment of HNE-metabolizing enzymes (Traverso *et al.*, 1998, 2002). Diabetic mice under a high fat diet exhibit increased HNE adducts levels in temporal lobes relative to control (Lyn-Cook *et al.*, 2009), and HNE conjugation of GLUT3, the glucose transporter present in neurons, is increased in the hippocampus of diabetic rats subjected to stress (Reagan *et al.*, 2000). When db/db obese mice are fed a Western diet containing 21% fat and 0.15% cholesterol, they develop obesity, hyperglycemia, and insulin resistance. In this situation, HNE is significantly elevated in the left ventricular myocardium of diabetic mice compared to their lean littermates (Yamashita *et al.*, 2010). Finally, hyperglycemic Zucker Diabetic Fatty (ZDF) rats exhibited a 8-fold increase in plasma HDDE concentration compared to their lean non diabetic counterparts (Riahi *et al.*, 2010).

#### **4.3. Causative role for lipid peroxidation by-products in the metabolic syndrome**

This body of evidence linking oxidative stress with the metabolic syndrome is however only based on correlations and do not decipher the mechanisms and/or the causative role of oxidative stress in diabetes. There are consequently two main hypotheses:


We will focus in this chapter on the second hypothesis for which evidences have been recently accumulating from human, animal and cell culture studies.

Oxidative stress, through reactive oxygen species can positively and negatively regulate insulin signalling, depending on time, dose, model and free radical used (for review, see Bashan *et al.*, 2009). It is however admitted that prolonged oxidative stress impairs insulin signalling, insulin-induced GLUT4 translocation and glucose uptake. This occurs through several mechanisms including, but not limited to IRS inhibitory phosphorylation, MAPK activation and endoplasmic reticulum stress. This body of evidence for the role of oxidative stress in insulin resistance has been demonstrated in adipocytes, muscle, liver and cardiac cells (Rudich *et al.*, 1998; Bloch-Damti *et al.*, 2006; JeBailey *et al.*, 2007; Singh *et al.*, 2008*a*; Shibata *et al.*, 2010; Tan *et al.*, 2011). Even *in vivo*, a pro-oxidant challenge provokes the onset of type-2 diabetes in insulin resistant rats (Laight *et al.*, 1999*b*) and chronic methylglyoxal infusion by minipump causes pancreatic beta-cell dysfunction and induces type-2 diabetes in Sprague-Dawley rats (Dhar *et al.*, 2011).

The role of lipid aldehydes *in vivo* during metabolic diseases is often indirectly assessed, and the most compelling evidence for a causative role of aldehydes comes from polymorphisms in the glutathione-S-transferase (GST) gene. This family of enzymes is responsible for the detoxification of aldehydes through conjugation to glutathione; and several deletion polymorphisms leading to blunted enzyme activity have been described. Patients carrying certain null GST polymorphisms had up to 3-fold increased incidence of type-2 diabetes mellitus compared to those with normal genotypes (Amer *et al.*, 2011). Accordingly, the expression of the GST4A is blunted in the adipose tissue of obese insulin resistant subjects (Curtis *et al.*, 2010).

**Figure 5.** The obesity – lipid peroxidation vicious circle

420 Lipid Peroxidation

diabetic counterparts (Riahi *et al.*, 2010).

Aldehydes concentration is increased in several tissues during diabetes, as revealed by increased levels of HNE in pancreas, liver, brain and heart. Pancreatic islets from type-2 diabetic patients are positively stained with HNE, suggesting that oxidative stress through lipid peroxidation could contribute to the reduced beta-cell mass and islet-cell injury (Sakuraba *et al.*, 2002). An increased level of HNE-modified proteins is reported in the pancreatic beta cells of Goto Kakizaki rats as a result of hyperglycaemia (Ihara *et al.*, 1999); and accumulation of HNE is observed in liver of diabetic rats due to the impairment of HNE-metabolizing enzymes (Traverso *et al.*, 1998, 2002). Diabetic mice under a high fat diet exhibit increased HNE adducts levels in temporal lobes relative to control (Lyn-Cook *et al.*, 2009), and HNE conjugation of GLUT3, the glucose transporter present in neurons, is increased in the hippocampus of diabetic rats subjected to stress (Reagan *et al.*, 2000). When db/db obese mice are fed a Western diet containing 21% fat and 0.15% cholesterol, they develop obesity, hyperglycemia, and insulin resistance. In this situation, HNE is significantly elevated in the left ventricular myocardium of diabetic mice compared to their lean littermates (Yamashita *et al.*, 2010). Finally, hyperglycemic Zucker Diabetic Fatty (ZDF) rats exhibited a 8-fold increase in plasma HDDE concentration compared to their lean non

**4.3. Causative role for lipid peroxidation by-products in the metabolic syndrome** 

This body of evidence linking oxidative stress with the metabolic syndrome is however only based on correlations and do not decipher the mechanisms and/or the causative role of

1. First is chronic hyperglycemia which then leads to oxidative stress. In this case, lipid peroxidation products would be by-products of this oxidative stress and a consequence of diabetes, even if contributing to the progression of the disease and its complications. 2. First is oxidative stress which precedes the development of diabetes and plays a causative role in its development. In this hypothesis, deregulation of antioxidant defences and increased oxidative stress would lead to accumulation of secondary by-

We will focus in this chapter on the second hypothesis for which evidences have been

Oxidative stress, through reactive oxygen species can positively and negatively regulate insulin signalling, depending on time, dose, model and free radical used (for review, see Bashan *et al.*, 2009). It is however admitted that prolonged oxidative stress impairs insulin signalling, insulin-induced GLUT4 translocation and glucose uptake. This occurs through several mechanisms including, but not limited to IRS inhibitory phosphorylation, MAPK activation and endoplasmic reticulum stress. This body of evidence for the role of oxidative stress in insulin resistance has been demonstrated in adipocytes, muscle, liver and cardiac cells (Rudich *et al.*, 1998; Bloch-Damti *et al.*, 2006; JeBailey *et al.*, 2007; Singh *et al.*, 2008*a*; Shibata *et al.*, 2010; Tan *et al.*, 2011). Even *in vivo*, a pro-oxidant challenge provokes the onset of type-2 diabetes in insulin resistant rats (Laight *et al.*, 1999*b*) and chronic methylglyoxal

oxidative stress in diabetes. There are consequently two main hypotheses:

products consequently inducing insulin resistance.

recently accumulating from human, animal and cell culture studies.

This same observation was made in animals model, were it has been suggested that excessive production of HNE might be sufficient to cause obesity and the metabolic syndrome. Mice lacking the gene encoding the HNE-conjugating enzyme mGSTA4-4 develop obesity and insulin resistance, unsurprisingly associated with HNE accumulation in multiple tissues (Singh *et al.*, 2008*b*). Cell culture work further shows that dysfunction of glutathione S-transferase or its ablation by siRNA leads to excess HNE accumulation, increased protein carbonylation, oxidative stress, and mitochondrial dysfunction (Kostyuk *et al.*, 2010; Curtis *et al.*, 2010). On the other hand, overexpression of glutathione-Stransferase A4-4 protects against oxidative stress and HNE-induced apoptosis (Vaillancourt *et al.*, 2008). Additional findings suggest that HNE causes fat accumulation by promoting fatty acid synthesis and suppressing fatty acid beta-oxidation. Interestingly, the phenotype of mGSTA4-4 null mice is strain dependent: mGSTA4-4 null mice with 129/sv genetic background exhibit both increased accumulation of HNE and obesity while those with C57Bl6 genetic background are lean and HNE is unchanged (Singh *et al.*, 2008*b*). In good agreement, silencing of the mGSTA4-4 gene in the nematode Caenorhabditis elegans also results in an accumulation of lipid peroxidation by-products and a fatty phenotype (Singh *et* 

*al.*, 2009). When HNE is experimentally increased in the nematode, either by genetic deletion or through direct exposure, it promotes fat accumulation. The mechanism involves HNE inhibition of mitochondrial aconitase leading to an accumulation of malonyl CoA, precursor of fatty acid synthesis (Zimniak, 2010). Taken together, these data suggest that accumulation of lipid aldehydes and fat deposition could be mutually inductive leading to a vicious circle promoting fat accretion (Figure 5).

The direct causative role of lipid peroxidation by-products in insulin resistance has also been recently evidenced in cell cultures studies. A first study identified that methylglyoxal, an aldehyde by-product of glucose oxidation, can impair the insulin signalling pathways independently of the formation of intracellular reactive oxygen species (Riboulet-Chavey *et al.*, 2006). Then, focusing on lipid peroxidation products, two studies demonstrated that HNE can induce insulin resistance in adipocytes and muscle cell through inhibition of IRS and Akt signalling, as well as insulin-induced glucose uptake (Demozay *et al.*, 2008; Pillon *et al.*, 2012). These two studies identified carbonyl stress (notably IRS1 adduction) and ROS production as the possible mechanisms responsible for HNE effects; a third one being possibly the adduction of Akt2, which inhibits insulin-dependent Akt signalling in HepG2 cells (Shearn *et al.*, 2011). Altogether, these studies strongly suggest that excessive production of aldehydes might be sufficient to cause obesity, diabetes and the metabolic syndrome.

## **5. Preventing the deleterious effects of aldehydes**

It has been known for decades that supplementation in α-lipoic acid in type-2 diabetic subject improves glucose tolerance and insulin sensitivity (Jacob et al., 1996). Similarly, increased intake of Vitamin E in obese and insulin resistant patients reduces fasting glycemia and the HOMA index for insulin resistance, and this is correlated with a decrease in the concentration of peroxides in plasma (Manning et al., 2004). This adds further evidence for a causative role of oxidative stress in the metabolic syndrome, and opens new therapeutic perspectives using antioxidant and/or scavenging of toxic aldehydes.

#### **5.1. Glutathione and enzymatic detoxification**

Glutathione (GSH) is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) which is present in high concentration in the cytoplasm of living cells: 5-50 nmol/mg proteins (Jungas *et al.*, 2002; Dominy *et al.*, 2007). GSH is an important coenzyme of several enzymatic reactions, and exerts its antioxidant activity mainly through regeneration of vitamin E, and also through direct interaction with free radical and aldehydes. GSH is obviously necessary for the activity of glutathione-S-transferases (GSTs), a family of enzymes responsible for detoxification of electrophile by-products, such as the ones derived from lipid peroxidation, and GSH exhibits indeed a high reactivity for HNE. Though HNE is electrophile enough to spontaneously react with GSH, this reaction is dramatically accelerated via the conjugation process catalyzed by GSTs (Alin *et al.*, 1985). As described above, mice lacking GSTs develop obesity and insulin resistance (Singh *et al.*, 2008*b*), and humans carrying null polymorphisms for GST have a 3-fold increased risk of having type-2 diabetes (Amer *et al.*, 2011), pointing out the important role of GSH in the metabolic syndrome. As addition reaction to GSH contributes to the detoxification of aldehydes, pharmacological strategies to increase glutathione pools or GST activity should be protective against aldehydes. Several studies were indeed successful in protecting cultured cells from the deleterious effects of oxidative stress through an increase in intracellular pools of reduced glutathione. This strategy was particularly efficient to protect the cells against the deleterious effects of HNE (Yadav *et al.*, 2008; Jia *et al.*, 2009*b*; Pillon *et al.*, 2012).

In addition to glutathione-S-transferases, several enzymes are also responsible for the detoxification of aldehydes: aldehyde dehydrogenase, alcohol dehydrogenase (Hartley *et al.*, 1995), aldose reductase (Srivastava *et al.*, 2000) and fatty aldehyde dehydrogenase (Demozay *et al.*, 2008). These enzymes however participate to a lower extent in the metabolism of aldehydes, compared to glutathione and GST. Recent data indicate that if HHE and HNE are both metabolized via glutathione, the effectiveness of detoxification differs for these two molecules. By extension, aldehydes may be metabolized with different affinities and efficiencies by detoxification enzymes, which could explain some differences in their respective toxicities (Long *et al.*, 2010).

#### **5.2. Scavenging**

422 Lipid Peroxidation

syndrome.

aldehydes.

promoting fat accretion (Figure 5).

**5. Preventing the deleterious effects of aldehydes** 

**5.1. Glutathione and enzymatic detoxification** 

*al.*, 2009). When HNE is experimentally increased in the nematode, either by genetic deletion or through direct exposure, it promotes fat accumulation. The mechanism involves HNE inhibition of mitochondrial aconitase leading to an accumulation of malonyl CoA, precursor of fatty acid synthesis (Zimniak, 2010). Taken together, these data suggest that accumulation of lipid aldehydes and fat deposition could be mutually inductive leading to a vicious circle

The direct causative role of lipid peroxidation by-products in insulin resistance has also been recently evidenced in cell cultures studies. A first study identified that methylglyoxal, an aldehyde by-product of glucose oxidation, can impair the insulin signalling pathways independently of the formation of intracellular reactive oxygen species (Riboulet-Chavey *et al.*, 2006). Then, focusing on lipid peroxidation products, two studies demonstrated that HNE can induce insulin resistance in adipocytes and muscle cell through inhibition of IRS and Akt signalling, as well as insulin-induced glucose uptake (Demozay *et al.*, 2008; Pillon *et al.*, 2012). These two studies identified carbonyl stress (notably IRS1 adduction) and ROS production as the possible mechanisms responsible for HNE effects; a third one being possibly the adduction of Akt2, which inhibits insulin-dependent Akt signalling in HepG2 cells (Shearn *et al.*, 2011). Altogether, these studies strongly suggest that excessive production of aldehydes might be sufficient to cause obesity, diabetes and the metabolic

It has been known for decades that supplementation in α-lipoic acid in type-2 diabetic subject improves glucose tolerance and insulin sensitivity (Jacob et al., 1996). Similarly, increased intake of Vitamin E in obese and insulin resistant patients reduces fasting glycemia and the HOMA index for insulin resistance, and this is correlated with a decrease in the concentration of peroxides in plasma (Manning et al., 2004). This adds further evidence for a causative role of oxidative stress in the metabolic syndrome, and opens new therapeutic perspectives using antioxidant and/or scavenging of toxic

Glutathione (GSH) is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) which is present in high concentration in the cytoplasm of living cells: 5-50 nmol/mg proteins (Jungas *et al.*, 2002; Dominy *et al.*, 2007). GSH is an important coenzyme of several enzymatic reactions, and exerts its antioxidant activity mainly through regeneration of vitamin E, and also through direct interaction with free radical and aldehydes. GSH is obviously necessary for the activity of glutathione-S-transferases (GSTs), a family of enzymes responsible for detoxification of electrophile by-products, such as the ones derived from lipid peroxidation, and GSH exhibits indeed a high reactivity for HNE. Though HNE is electrophile enough to spontaneously react with GSH, this reaction is dramatically accelerated via the conjugation process catalyzed by GSTs (Alin *et al.*, 1985). As described above, mice lacking GSTs develop In chemistry, a scavenger is a chemical substance able to remove or inactivate impurities or unwanted reaction products. In living cells, a scavenger is, by extension, a molecule able to inactivate toxic compounds such as ROS and aldehydes, therefore preventing their deleterious effects. In the case of aldehydes, a scavenger would be a strong nucleophile molecule on which HHE, HNE or any other aldehyde would form a covalent adduct. Consequently, most of the aldehyde scavengers are amino- or sulphur-containing drugs such as N-acetyl-cysteine (NAC), hydralazine, S-adenosyl-methionine (SAM) aminoguanidine (AGD) and α-lipoic acid, the latter being tested in several human studies for treatment of type-2 diabetes.

#### *5.2.1. α-lipoic acid*

α-lipoic acid is a natural compound found in many foodstuffs (such as potatoes, broccoli and meat), but in rather low amount. The effect of lipoic acid was demonstrated in animal models, where it enhances insulin-stimulated glucose metabolism in skeletal muscle from insulin-resistant rat (Jacob et al., 1996). It was rapidly tested in several clinical studies which demonstrated its beneficial effects in type-2 diabetes through a decrease in fasted blood glucose, enhancement of glucose disposal, improved insulin sensitivity and decreased insulin resistance (Jacob *et al.*, 1995). These results were confirmed *in vitro*, where α-lipoic acid prevents the development of glucose-induced insulin resistance in adipocytes (Greene et al., 2001) and were reproduced by many independent studies. Treatment with lipoic acid decreases oxidative stress in both adipocytes and muscle cells (Rudich et al., 1999; Maddux

et al., 2001) and also decreases lipid peroxidation markers in insulin resistant rats (Thirunavukkarasu & Anuradha, 2004), suggesting that the improvement of insulin sensitivity is due to its antioxidant properties. Hence, the current literature supports the use of alpha lipoic acid for the treatment of diabetes complications and it consequently became the first antioxidant supplement used for the treatment of diabetes complications, being already approved in Germany for the treatment of diabetic neuropathy.

#### *5.2.2. N-acetylcysteine (NAC)*

N-acetylcysteine is a cysteine derivative and a potent antioxidant. Its properties are mainly due to its thiol group able to reduce free radicals as well as its role as a precursor in the formation of glutathione (Zafarullah *et al.*, 2003). NAC exhibits highly protective scavenging properties against aldehydes and protects against MDA increase and GSH decrease in animal models of insulin resistance. NAC is able to improve insulin sensitivity in healthy rats (Figure 6) and reverses insulin resistance and aldehyde-induced hypertension in rats (Haber *et al.*, 2003). In cell culture studies, NAC can prevent the insulin resistance induced by HNE in muscle cells (Pillon *et al.*, 2012), as well as the one induced by advanced glycation end products in adipocytes (Unoki *et al.*, 2007), thus confirming the important role NAC can play in improving both oxidative stress parameters and insulin resistance.

**Figure 6.** Insulin sensitizing effect of N-acetylcysteine. Wistar rats were given NAC in drinking water for one week (total intake was 225 mg.kg-1.day-1). Insulin sensitivity was calculated using a standard insulin tolerance test3. Results are average ± SEM from 5 different animals per group, expressed as percent of basal glycemia. From Pillon et al. unpublished results.

<sup>3</sup> Animals fasted overnight were then injected intraperitoneally with 0.5 UI/kg body weight of insulin. Plasma glucose was measured from tail vein blood using a glucometer at 0, 20, 40, 60, and 120 min following the injection. Glucose disappearance rate for ITT (KITT; %/min) was calculated as: KITT=(0.693x100)/t1/2, where t1/2 was calculated from the slope of the plasma glucose concentration, considering an exponential decrement of glucose concentration during the 20 min after insulin administration. Higher insulin sensitivity index (KITT) scores mean higher response of tissues to insulin.

Results from clinical trials in type-2 diabetic subjects show that NAC is able to decrease oxidative stress parameters, increase GSH and decrease plasma VCAM-1 (De Mattia et al., 1998); moreover, long-term N-acetylcysteine administration reduces endothelial activation and is proposed as a potential antiatherogenic therapy (Martina et al., 2008). Despite the fact that NAC can improve insulin sensitivity in women with polycystic ovary syndrome (Fulghesu *et al.*, 2002) and this body of evidence suggesting that NAC may slow down the progression of diabetic complications, to date no clinical trial demonstrated any significant benefit of its supplementation in diabetes.

#### *5.2.3. Aminoguanidine (AGD)*

424 Lipid Peroxidation

*5.2.2. N-acetylcysteine (NAC)* 

et al., 2001) and also decreases lipid peroxidation markers in insulin resistant rats (Thirunavukkarasu & Anuradha, 2004), suggesting that the improvement of insulin sensitivity is due to its antioxidant properties. Hence, the current literature supports the use of alpha lipoic acid for the treatment of diabetes complications and it consequently became the first antioxidant supplement used for the treatment of diabetes complications, being

N-acetylcysteine is a cysteine derivative and a potent antioxidant. Its properties are mainly due to its thiol group able to reduce free radicals as well as its role as a precursor in the formation of glutathione (Zafarullah *et al.*, 2003). NAC exhibits highly protective scavenging properties against aldehydes and protects against MDA increase and GSH decrease in animal models of insulin resistance. NAC is able to improve insulin sensitivity in healthy rats (Figure 6) and reverses insulin resistance and aldehyde-induced hypertension in rats (Haber *et al.*, 2003). In cell culture studies, NAC can prevent the insulin resistance induced by HNE in muscle cells (Pillon *et al.*, 2012), as well as the one induced by advanced glycation end products in adipocytes (Unoki *et al.*, 2007), thus confirming the important role NAC can

**Figure 6.** Insulin sensitizing effect of N-acetylcysteine. Wistar rats were given NAC in drinking water for one week (total intake was 225 mg.kg-1.day-1). Insulin sensitivity was calculated using a standard insulin tolerance test3. Results are average ± SEM from 5 different animals per group, expressed as

**Control NAC**

\*\*

**0**

**2**

**KITT (%.min-1)**

**4**

**6**

NAC

**0 20 40 60 80 100 120**

**Time (min)**

3 Animals fasted overnight were then injected intraperitoneally with 0.5 UI/kg body weight of insulin. Plasma glucose was measured from tail vein blood using a glucometer at 0, 20, 40, 60, and 120 min following the injection. Glucose disappearance rate for ITT (KITT; %/min) was calculated as: KITT=(0.693x100)/t1/2, where t1/2 was calculated from the slope of the plasma glucose concentration, considering an exponential decrement of glucose concentration during the 20 min after insulin

administration. Higher insulin sensitivity index (KITT) scores mean higher response of tissues to insulin.

percent of basal glycemia. From Pillon et al. unpublished results.

**Glycemia (% basal)**

already approved in Germany for the treatment of diabetic neuropathy.

play in improving both oxidative stress parameters and insulin resistance.

**<sup>120</sup>** Control

Aminoguanidine is a highly nucleophilic agent which reacts *in vitro* and *in vivo* with aldehydes, therefore protecting against the deleterious effects of ALE precursors (Peyroux & Sternberg, 2006). It is also an antioxidant able to quench hydroxyl radicals and *in vivo*. AGD in drinking water decreases lipid peroxidation in type-1 diabetic rats and rabbits (Ihm *et al.*, 1999). In experimental animal models of diabetes, AGD demonstrates significant effects in protecting against pathological complications, such as diabetic nephropathies, atherosclerosis and neurovascular complications (El Shazly *et al.*, 2009). Consequently, several clinical trials in humans have been designed to evaluate AGD efficiency but they demonstrate only mild effects and were not conclusive, partly because of side-effects, and of weak carbonyl scavenger effects in human vascular tissues (Bolton *et al.*, 2004).

#### *5.2.4. Hydralazine*

Primarily used as an antihypertensive drug, hydralazine exhibits a pronounced nucleophilicity and is consequently very efficient in scavenging several aldehydes (acrolein, HNE) and ketones, as well as aldehyde-adducted proteins (Burcham et al., 2002). It is also a powerful antioxidant, able to inhibit the generation of ROS (Münzel *et al.*, 1996). Its scavenging activity *in vivo* was demonstrated by its ability to reverse the formation of HNE and acrolein adducts on tissue proteins in atherosclerotic aortas of hypercholesterolemic animals (Vindis et al., 2006), but its effects on insulin resistance and diabetes are to date uncharacterized.

#### *5.2.5. S-adenosyl-methionine (SAM)*

In living organisms, SAM is endogenously synthesized from methionine in every cell, but the liver is the major site of its synthesis and degradation. SAM is an important precursor for cysteine and glutathione production (Lu, 2000) and its involvement in several metabolic pathways makes it essential for a wide spectrum of cellular processes. SAM inhibits both HNE production and adducts formation and efficiently prevents high-fat diet-induced nonalcoholic steatohepatitis in rats (Lieber et al., 2007), even if its direct effects on insulin resistance are still unknown.

#### **6. Conclusion**

Lipid peroxidation by-products are associated with metabolic diseases, but their primary role during obesity and diabetes is subject to debate. *In vitro* and *in vivo* animal studies highlighted that an aldehyde challenge, through a state of carbonyl stress, affects several steps involved in the development of obesity and type-2 diabetes (Figure 7). On insulinsensitive tissues (muscle, adipose tissue), aldehydes lead to insulin resistance, while in pancreas, aldehydes impair insulin secretion. Together with the carbonylation of the insulin peptide itself, aldehydes could contribute to the defects in insulin action, leading to the metabolic syndrome.

**Figure 7.** Lipid aldehydes effects leading to the metabolic syndrome.

Despite this evidence, the role of aldehydes is mitigated by the mild effect obtained with antioxidants and/or aldehyde scavengers in the treatment of diabetic complications in human. "Oxidative stress" and its derivatives are nevertheless important in the metabolic syndrome, and prevention or treatment of some of its associated complications could be accessed through reduction of both ROS and toxic aldehydes by-products, as it is the case with lipoic acid.

#### **Author details**

426 Lipid Peroxidation

**6. Conclusion** 

metabolic syndrome.

**Figure 7.** Lipid aldehydes effects leading to the metabolic syndrome.

Despite this evidence, the role of aldehydes is mitigated by the mild effect obtained with antioxidants and/or aldehyde scavengers in the treatment of diabetic complications in

Lipid peroxidation by-products are associated with metabolic diseases, but their primary role during obesity and diabetes is subject to debate. *In vitro* and *in vivo* animal studies highlighted that an aldehyde challenge, through a state of carbonyl stress, affects several steps involved in the development of obesity and type-2 diabetes (Figure 7). On insulinsensitive tissues (muscle, adipose tissue), aldehydes lead to insulin resistance, while in pancreas, aldehydes impair insulin secretion. Together with the carbonylation of the insulin peptide itself, aldehydes could contribute to the defects in insulin action, leading to the

Nicolas J. Pillon and Christophe O. Soulage *Université de Lyon, INSA de Lyon, CarMeN, INSERM U1060, Univ Lyon-1, F-69621, Villeurbanne, France* 

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