**Region Specific Vulnerability to Lipid Peroxidation in the Human Central Nervous System**

Alba Naudí, Mariona Jové, Victòria Ayala, Omar Ramírez, Rosanna Cabré, Joan Prat, Manuel Portero-Otin, Isidre Ferrer and Reinald Pamplona

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46022

### **1. Introduction**

436 Lipid Peroxidation

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Zhang H & Forman HJ (2008). Acrolein induces heme oxygenase-1 through PKC-delta and PI3K in human bronchial epithelial cells. *Am J Respir Cell Mol Biol* 38, 483–490. Zimniak P (2010). 4-Hydroxynonenal and fat storage: A paradoxical pro-obesity Around 100 billion neurons in the human nervous system orchestrate an exceptionally wide range of motor, sensory, regulatory, behavioural, and executive functions. Such diverse functional output is the product of different molecular events occurring in nervous cells and particularly, neurons. Morphologically, central nervous system (CNS) neurons differ in size, number and complexity of dendrites, number of synaptic connections, length of axons and distance across which synaptic connections are established, extent of axonal myelination, and other cellular characteristics. Neuronal diversity is also amplified by the inclusion of chemical specificity on the basis of the neurotransmitters, which they use for chemical transmission or neuromodulation. This great diversity among neuronal populations is a strong indication that although all neurons contain the same genetic code in their genome, each neuronal population has their own gene expression profile. While the diversity of neuronal structures and functions are well documented, what is less appreciated is the diverse response of neurons to stresses and adverse factors during aging or as a result of neurodegenerative diseases. Furthermore, to add complexity to an already heterogeneous landscape, non-neuronal populations, often described as a 'supporting matrix' are recently being recognized as active, information-rich, cellular counterpart in CNS function.

In this scenario of cellular diversity emerges the concept of selective neuronal vulnerability (SNV). SNV is described as the differential sensitivity of neuronal populations in the nervous system to stresses that cause cell damage or death and can lead to neurodegeneration [1,2]. The fact that specific regions of the nervous system exhibit differential vulnerabilities to aging and various neurodegenerative diseases is a reflection of

© 2012 Naudí 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.

both the specificity in the aetiology of each disease and of the heterogeneity in neuronal (and non-neuronal) responses to cell-damaging processes associated with each of the diseases [3]. The appearance of SNV is not limited to cross-regional differences in the nervous system, as within a single e.g. brain region, such as the hippocampus or the entorhinal cortex, where SNV is manifested as internal, sub-regional differences in relative sensitivities to stress and disease [2,3]. Among these cell-damaging processes one could count inflammatory, proteotoxicity, vascular and many other pathophysiological processes, including an excess of lipid peroxidation (see later).

Oxidative stress (OS) is involved in the basic mechanisms of nervous system aging; whilst an excessive oxidation has been invoked as an etiopathogenic or physiopathologic mechanism for neurodegeneration. Oxidative stress, the result of an imbalance between production of free radicals and the enzymatic or non-enzymatic detoxification of these highly reactive species, is detrimental to cells because free radicals chemically modify lipids, proteins, and nucleic acids. So, it is very important to define the vulnerability of the different neuronal populations in terms of susceptibility to oxidative stress in physiological conditions in order to extent this knowledge to improve our understanding of how this particular form of cell vulnerability causes selective neuronal losses in nervous system, as well as reveal potential molecular and cellular mechanisms that bring about relative resistance or sensitivity of neurons to stresses.

## **2. Mitochondrial free radical generation and membrane fatty acid composition in the CNS**

#### **2.1. Mitochondrial free radical production**

Chemical reactions in cells of the nervous system are under strict enzyme control and conform to a tightly regulated metabolic program in order to minimize unnecessary side reactions. Nevertheless, apparently uncontrolled and potentially deleterious reactions occur, even under physiological conditions. Reactive oxygen species (ROS) express a variety of molecules and free radicals (chemical species with one unpaired electron) physiologically generated from the metabolism of molecular oxygen [4]. They are extremely reactive and have damaging effects. Superoxide anion, the product of a one-electron reduction of oxygen, is the precursor of most ROS and a mediator in oxidative chain reactions [4] (**Figure 1**). The character of radical is not circumscribed to oxygen containing species, as nitrogen, chloride and sulphide containing molecules could also play a significant role. Globally, in cells of the CNS the major sites of physiological ROS generation are the complex I and III of the mitochondrial electron transport chain, which contains several redox centers (flavins, iron– sulphur clusters, and ubisemiquinone) capable of transferring one electron to oxygen to form superoxide anion [5,6]. Oxidative damage is a broad term used to cover the attack upon biological molecules by free radicals. ROS attack/damage all cellular constituents [6], but especially biological membranes.

Region Specific Vulnerability to Lipid Peroxidation in the Human Central Nervous System 439

**Figure 1.** The three main physiological ROS. CI, mitochondrial complex I; CIII, mitochondrial complex III; MnSOD, Manganese superoxide dismutase; Gpx, glutathione peroxidase. MnSOD and Gpx are antioxidants enzymes.

#### **2.2. Membrane fatty acid composition in neural cells**

438 Lipid Peroxidation

both the specificity in the aetiology of each disease and of the heterogeneity in neuronal (and non-neuronal) responses to cell-damaging processes associated with each of the diseases [3]. The appearance of SNV is not limited to cross-regional differences in the nervous system, as within a single e.g. brain region, such as the hippocampus or the entorhinal cortex, where SNV is manifested as internal, sub-regional differences in relative sensitivities to stress and disease [2,3]. Among these cell-damaging processes one could count inflammatory, proteotoxicity, vascular and many other pathophysiological processes,

Oxidative stress (OS) is involved in the basic mechanisms of nervous system aging; whilst an excessive oxidation has been invoked as an etiopathogenic or physiopathologic mechanism for neurodegeneration. Oxidative stress, the result of an imbalance between production of free radicals and the enzymatic or non-enzymatic detoxification of these highly reactive species, is detrimental to cells because free radicals chemically modify lipids, proteins, and nucleic acids. So, it is very important to define the vulnerability of the different neuronal populations in terms of susceptibility to oxidative stress in physiological conditions in order to extent this knowledge to improve our understanding of how this particular form of cell vulnerability causes selective neuronal losses in nervous system, as well as reveal potential molecular and cellular mechanisms that bring about relative

**2. Mitochondrial free radical generation and membrane fatty acid** 

Chemical reactions in cells of the nervous system are under strict enzyme control and conform to a tightly regulated metabolic program in order to minimize unnecessary side reactions. Nevertheless, apparently uncontrolled and potentially deleterious reactions occur, even under physiological conditions. Reactive oxygen species (ROS) express a variety of molecules and free radicals (chemical species with one unpaired electron) physiologically generated from the metabolism of molecular oxygen [4]. They are extremely reactive and have damaging effects. Superoxide anion, the product of a one-electron reduction of oxygen, is the precursor of most ROS and a mediator in oxidative chain reactions [4] (**Figure 1**). The character of radical is not circumscribed to oxygen containing species, as nitrogen, chloride and sulphide containing molecules could also play a significant role. Globally, in cells of the CNS the major sites of physiological ROS generation are the complex I and III of the mitochondrial electron transport chain, which contains several redox centers (flavins, iron– sulphur clusters, and ubisemiquinone) capable of transferring one electron to oxygen to form superoxide anion [5,6]. Oxidative damage is a broad term used to cover the attack upon biological molecules by free radicals. ROS attack/damage all cellular constituents [6],

including an excess of lipid peroxidation (see later).

resistance or sensitivity of neurons to stresses.

**2.1. Mitochondrial free radical production** 

**composition in the CNS** 

but especially biological membranes.

All living organisms have lipid membranes. Biological membranes are dynamic structures that generally consist of bilayers of amphipathic molecules hold together by non-covalent bonds [7]. Phospholipids, consisting of a hydrophilic head group with attached hydrophobic acyl chains, are the predominant membrane lipids and are, from a topographic point of view, asymmetrically distributed across the bilayer. The variation in head groups and aliphatic chains allows the existence of a huge range of different phospholipid species [8,9]. The acyl chains are either saturated, monounsaturated or polyunsaturated hydrocarbon chains that normally vary from 14 to 22 carbons in length (**Figure 2**), with an average chain length strictly maintained around 18 carbon atoms, and a relative distribution between saturated and unsaturated fatty acids of 40:60 (SFA and UFA, respectively) [10]. Polyunsaturated fatty acids (PUFAs) are essential components of cellular membranes that strongly affect their fluidity, flexibility and selective permeability, as well as many cellular and physiological processes [11].

Long-chain polyunsaturated fatty acids are highly enriched in the nervous system. Docosahexaenoic acid (DHA; 22:6n-3; 4,7,10,13,16,19-C22:6), in particular, is the most abundant PUFA in the brain and is concentrated in aminophospholipids of cell membranes. Numerous studies have indicated that this concentration of DHA in the nervous system is essential for optimal neuronal functions. Although the underlying mechanisms of its essential function are still not clearly understood, emerging evidence suggests that unique metabolism of DHA in relation to its incorporation into neuronal membrane phospholipids plays an important role.

Accretion of DHA in the CNS actively occurs during the developmental period, primarily relying on circulating plasma DHA derived from diet or from biosynthesis in the liver [12]. However, local biosynthesis of DHA also occurs in the brain, providing an alternative source of DHA for its accumulation in the brain [13]. It is well established that DHA can be biosynthesized from α-linolenic acid (18:3n-3; 9,12,15-C18:3), a shorter chain n-3 fatty acid precursor, through chain elongation and desaturation processes [14] (**Figure 3**). Linolenic

**Figure 2.** Fatty acid nomenclature. As an example, the structure of linoleic acid (C18:2n-6) is given.

acid is desaturated to 18:4n-3 (6,9,12,15-C18:4) by Δ6-desaturase, chain-elongated to 20:4n-3 (8,11,14,17-C20:4), and subsequently converted to eicosapentaenoic acid (20:5n-3; 5,8,11,14,17-C20:5) by Δ5-desaturase in the endoplasmic reticulum (ER). Mammalian Δ5 and Δ6-desaturases have been identified and cloned [15]. However, Δ4-desaturase, responsible for making 22:6n-3 directly from 22:5n-3, an elongation product of 20:5n-3, has been identified only in microalgae [16]. In mammals, 22:5n-3 is further elongated to 24:5n-3 (9,12,15,18,21-C24:5) followed by desaturation by Δ6-desaturase to 24:6n-3 (6,9,12,15,18,21- C24:6). Subsequently, 24:6n-3 is transferred to peroxisomes and converted to 22:6n-3 by removing two carbon chains by β-oxidation. DHA thus formed is transferred back to the ER and quickly incorporated into membrane phospholipids by esterification during de novo synthesis or by a deacylation-reacylation reaction. Because biosynthesis of both fatty acids and phospholipids occurs in ER, a particular fatty acid intermediate can be either incorporated into phospholipids or further chain-elongated/desaturated, although the regulation of these processes is still poorly understood. Long-chain n-6 fatty acids are biosynthesized from linoleic acid (18:2n-6; 9,12-C18:2) using the analogous pathway and the same enzyme system (**Figure 3**). In most tissues, the commonly observed long-chain n-6 fatty acid is arachidonic acid (AA; 20:4n-6; 5,8,11,14-C20:4). Docosapentaenoic acid (DPAn-6; 22:5n-6; 4,7,10,13,16-C22:5) produced by further elongation and desaturation of AA and subsequent peroxisomal β-oxidation, is rather a minor component, and yet it accumulates in the brain in place of DHA when the DHA supply is inadequate, especially during developmental periods. The distinctive fatty acid profile in the brain enriched with DHA or DPAn-6 may reflect the brain-specific uptake and/or regulation of fatty acid synthesis and esterification into membrane phospholipids. The liver is considered to be the primary site for biosynthesis of DHA, which becomes available to brain uptake through subsequent secretion into the circulating blood stream. Among neural cells, consisting of neurons, astrocytes, microglia, and oligodendrocytes, the capacity to synthesize DHA has been demonstrated only in astrocytes [13]. Despite the fact that neurons are major targets for DHA accumulation, they cannot produce DHA because of lack of desaturase activity. Cerebromicrovascular endothelia can also elongate and desaturate shorter carbon chain fatty acids. However, they cannot perform the final desaturation step to produce either 22:5n-6 or 22:6n-3 [17].

**Figure 2.** Fatty acid nomenclature. As an example, the structure of linoleic acid (C18:2n-6) is given.

cannot perform the final desaturation step to produce either 22:5n-6 or 22:6n-3 [17].

acid is desaturated to 18:4n-3 (6,9,12,15-C18:4) by Δ6-desaturase, chain-elongated to 20:4n-3 (8,11,14,17-C20:4), and subsequently converted to eicosapentaenoic acid (20:5n-3; 5,8,11,14,17-C20:5) by Δ5-desaturase in the endoplasmic reticulum (ER). Mammalian Δ5 and Δ6-desaturases have been identified and cloned [15]. However, Δ4-desaturase, responsible for making 22:6n-3 directly from 22:5n-3, an elongation product of 20:5n-3, has been identified only in microalgae [16]. In mammals, 22:5n-3 is further elongated to 24:5n-3 (9,12,15,18,21-C24:5) followed by desaturation by Δ6-desaturase to 24:6n-3 (6,9,12,15,18,21- C24:6). Subsequently, 24:6n-3 is transferred to peroxisomes and converted to 22:6n-3 by removing two carbon chains by β-oxidation. DHA thus formed is transferred back to the ER and quickly incorporated into membrane phospholipids by esterification during de novo synthesis or by a deacylation-reacylation reaction. Because biosynthesis of both fatty acids and phospholipids occurs in ER, a particular fatty acid intermediate can be either incorporated into phospholipids or further chain-elongated/desaturated, although the regulation of these processes is still poorly understood. Long-chain n-6 fatty acids are biosynthesized from linoleic acid (18:2n-6; 9,12-C18:2) using the analogous pathway and the same enzyme system (**Figure 3**). In most tissues, the commonly observed long-chain n-6 fatty acid is arachidonic acid (AA; 20:4n-6; 5,8,11,14-C20:4). Docosapentaenoic acid (DPAn-6; 22:5n-6; 4,7,10,13,16-C22:5) produced by further elongation and desaturation of AA and subsequent peroxisomal β-oxidation, is rather a minor component, and yet it accumulates in the brain in place of DHA when the DHA supply is inadequate, especially during developmental periods. The distinctive fatty acid profile in the brain enriched with DHA or DPAn-6 may reflect the brain-specific uptake and/or regulation of fatty acid synthesis and esterification into membrane phospholipids. The liver is considered to be the primary site for biosynthesis of DHA, which becomes available to brain uptake through subsequent secretion into the circulating blood stream. Among neural cells, consisting of neurons, astrocytes, microglia, and oligodendrocytes, the capacity to synthesize DHA has been demonstrated only in astrocytes [13]. Despite the fact that neurons are major targets for DHA accumulation, they cannot produce DHA because of lack of desaturase activity. Cerebromicrovascular endothelia can also elongate and desaturate shorter carbon chain fatty acids. However, they

**Figure 3.** Long chain and very long-chain fatty acid biosynthesis in mammals. The long chain saturated fatty acids and unsaturated fatty acids of the n-10, n-7 and n -9 series can be synthesized from palmitic acid (C16:0) produced by the fatty acid synthase (FAS). Long-chain fatty acids of the n-6 and n-3 series can only be synthesized from precursors obtained from dietary precursors (DIET). Elovl, elongation of very long chain fatty acids (fatty acid elongase); Fads, fatty acid desaturases.

DHA synthesis in astrocytes is negatively influenced by the availability of preformed DHA [18] and thus may represent a quantitatively minor source for the neural DHA accretion when the circulating DHA supply is adequate. Incorporation of circulating DHA across the blood brain barrier appears to be an important route for maintaining adequate levels of DHA in the brain. In agreement with this notion, it has been shown that constant basal turnover of esterified DHA in the brain with unesterified DHA in plasma occurs at an estimated rate of 2–8% per day in adult rats [19]. Generally, it is difficult to deplete DHA from the neural membranes of adult mammals even with a DHA low diet, presumably because of preferential uptake of DHA into the brain to support the basal turnover. In the case of insufficient supply of n-3 fatty acids during development, the loss of DHA does occur but is compensated with DPAn-6 through reciprocal replacement, suggesting a requirement of very long-chain, highly unsaturated fatty acids in neural membranes.

Whether brain DHA is derived from the circulating plasma pool or biosynthesized locally, in the astrocytes, which are situated in close contact with neurons, appear to play an important role in supplying DHA to neurons. DHA can be released readily from astroglial membranes under basal and stimulated conditions, and supplied to neurons. Despite its high abundance in neuronal membranes, DHA is not easily released but is tenaciously retained in the neuronal membranes under the conditions in which AA can be released. Considering the fact that astroglia support neurons by providing neurotrophic factors, DHA supplied by astroglia may also be trophic. Indeed, DHA has been shown to promote neuronal survival [20] and differentiation [21] in both transformed and primary neuronal cells in culture.

## **3. Membrane unsaturation and lipoxidation-derived molecular damage**

The susceptibility of membrane phospholipids to oxidative demise is related to two inherent traits, the physico-chemical properties of the membrane bilayer and the intrinsic chemical reactivity of the fatty acids composing the membrane [10,22]. The first property is related to the fact that oxygen and free radicals are more soluble in the fluid lipid bilayer than in the aqueous solution. Thus, membranes contain an interior organic phase in which the oxygen may tend to concentrate. Therefore, these differences in solubility are important when considering the availability of oxygen/free radicals for chemical reactions inside living systems: organic regions may contain more free radicals than aqueous regions [8] and, consequently, membrane lipids become primary targets of oxidative damage. The second property is related to the fact that PUFA residues of phospholipids are extremely sensitive to oxidation. Every membrane phospholipid contains an unsaturated fatty acid residue esterified to the 2-hydroxyl group of its glycerol moiety. Many of these are polyunsaturated and the presence of a methylene group between two double bonds renders the fatty acid more sensitive to ROS-induced damage. Therefore, the sensitivity of these molecules to oxidation increase exponentially as a function of the number of double bonds per fatty acid molecule [22,23]. Consequently, the high concentration of PUFAs in phospholipids not only makes them prime targets for reaction with oxidizing agents but also enables them to participate in long free radical chain reactions. Reactive free radicals can pull off hydrogen atoms from PUFA side chains. A hydrogen atom (H•) has only one electron. This hydrogen is bonded to a carbon in the fatty acid backbone by a covalent bond. Hence, the carbon from which H• is abstracted now has an unpaired electron (i.e it is a free radical). PUFA side chains (two or more double bonds) are more sensitive to attack by radicals than are SFAs (no double bonds) or monounsaturated fatty acids (MUFA,one double bond) side chains. When C• radicals are generated in the hydrophobic interior of membranes, their most likely fate is combination with oxygen dissolved in the membrane. The resulting peroxyl radical is highly reactive: it can attack membrane proteins and oxidize adjacent PUFA side chains. So, the reaction is repeated and the whole process continues in a free radical chain reaction, generating lipid hydroperoxides [4]. Lipid hydroperoxides are more hydrophilic than unperoxidized fatty acid side chains. They try to migrate to the membrane surface to interact with water, thus disrupting the membrane structure, altering fluidity and other functional properties and making the membrane leaky.

Lipid peroxidation generates hydroperoxides as well as endoperoxides, which undergo fragmentation to produce a broad range of reactive intermediates called reactive carbonyl species (RCS) (**Figure 4**) with three to nine carbons in length, the most reactive being α,βunsaturated aldehydes [4-hydroxy-trans-2-nonenal (HNE) and acrolein], di-aldehydes [malondialdehyde (MDA) and glyoxal], and keto-aldehydes [4-oxo-trans-2-nonenal (ONE) and isoketals] [24]. 2-Hydroxyheptanal (2-HH) and 4-hydroxyhexenal (4-HHE) are aldehydic product of lipid peroxidation of PUFAn-6. Additionally, a number of other short chain aldehydes are produced during lipid peroxidation through poorly understood mechanisms. These carbonyl compounds, ubiquitously generated in biological systems, have unique properties contrasted with free radicals. Thus, compared with reactive oxygen and nitrogen species, reactive aldehydes have a much longer half-life (i.e., minutes to hours instead of microseconds to nanoseconds for most free radicals). Further, the non-charged structure of aldehydes allows them to migrate relatively ease through hydrophobic membranes and hydrophilic cytosolic media, thereby extending the migration distance far from the production site. Based on these features alone, these carbonyl compounds can be more destructive than ROS and may have far-reaching damaging effects on target sites within or outside membranes. For the same reason, their long half-life allows them to subserve as second-messenger, signalling for important cellular responses in a mostly unknown fashion.

442 Lipid Peroxidation

cells in culture.

high abundance in neuronal membranes, DHA is not easily released but is tenaciously retained in the neuronal membranes under the conditions in which AA can be released. Considering the fact that astroglia support neurons by providing neurotrophic factors, DHA supplied by astroglia may also be trophic. Indeed, DHA has been shown to promote neuronal survival [20] and differentiation [21] in both transformed and primary neuronal

**3. Membrane unsaturation and lipoxidation-derived molecular damage** 

functional properties and making the membrane leaky.

The susceptibility of membrane phospholipids to oxidative demise is related to two inherent traits, the physico-chemical properties of the membrane bilayer and the intrinsic chemical reactivity of the fatty acids composing the membrane [10,22]. The first property is related to the fact that oxygen and free radicals are more soluble in the fluid lipid bilayer than in the aqueous solution. Thus, membranes contain an interior organic phase in which the oxygen may tend to concentrate. Therefore, these differences in solubility are important when considering the availability of oxygen/free radicals for chemical reactions inside living systems: organic regions may contain more free radicals than aqueous regions [8] and, consequently, membrane lipids become primary targets of oxidative damage. The second property is related to the fact that PUFA residues of phospholipids are extremely sensitive to oxidation. Every membrane phospholipid contains an unsaturated fatty acid residue esterified to the 2-hydroxyl group of its glycerol moiety. Many of these are polyunsaturated and the presence of a methylene group between two double bonds renders the fatty acid more sensitive to ROS-induced damage. Therefore, the sensitivity of these molecules to oxidation increase exponentially as a function of the number of double bonds per fatty acid molecule [22,23]. Consequently, the high concentration of PUFAs in phospholipids not only makes them prime targets for reaction with oxidizing agents but also enables them to participate in long free radical chain reactions. Reactive free radicals can pull off hydrogen atoms from PUFA side chains. A hydrogen atom (H•) has only one electron. This hydrogen is bonded to a carbon in the fatty acid backbone by a covalent bond. Hence, the carbon from which H• is abstracted now has an unpaired electron (i.e it is a free radical). PUFA side chains (two or more double bonds) are more sensitive to attack by radicals than are SFAs (no double bonds) or monounsaturated fatty acids (MUFA,one double bond) side chains. When C• radicals are generated in the hydrophobic interior of membranes, their most likely fate is combination with oxygen dissolved in the membrane. The resulting peroxyl radical is highly reactive: it can attack membrane proteins and oxidize adjacent PUFA side chains. So, the reaction is repeated and the whole process continues in a free radical chain reaction, generating lipid hydroperoxides [4]. Lipid hydroperoxides are more hydrophilic than unperoxidized fatty acid side chains. They try to migrate to the membrane surface to interact with water, thus disrupting the membrane structure, altering fluidity and other

Lipid peroxidation generates hydroperoxides as well as endoperoxides, which undergo fragmentation to produce a broad range of reactive intermediates called reactive carbonyl species (RCS) (**Figure 4**) with three to nine carbons in length, the most reactive being α,β-

**Figure 4.** General structures of principal lipoxidative reactive carbonyl species detected in biological systems.

Carbonyl compounds react with nucleophilic groups in macromolecules like proteins, DNA, and aminophospholipids, among other, resulting in their chemical, nonenzymatic, and irreversible modification and formation of a variety of adducts and crosslinks collectively named Advanced Lipoxidation Endproducts (ALEs) [10,25,26] (**Figure 5**) . Thus, by reacting with nucleophilic sites in proteins (belonging basically to Cys, Lys, Arg, and His residues), carbonyl compounds generate ALE adducts such as MDA-Lys, HNE-Lys, FDP-Lys, carboxymethyl-lysine (CML) and S-carboxymethyl-cysteine; and the crosslinks glyoxallysine dimer (GOLD), and methylglyoxal-lysine dimer (MOLD), among several others. The accumulation of MDA adducts on proteins is also involved in the formation of lipofuscin (a nondegradable intralysosomal fluorescent pigment formed through lipoxidative reactions). Lipid peroxidation-derived endproducts can also react at the exocyclic amino groups of deoxyguanosine, deoxyadenosine, and deoxycytosine to form various alkylated products. Guanine is, however, the most commonly modified DNA base because of its high nucleophilicity. Some common enals that cause DNA damage, analogously to proteins, are

**Figure 5.** Reactive carbonyl species react with nucleophilic groups in macromolecules (A, proteins; B, DNA) resulting in their chemical, nonenzymatic, and irreversible modification and formation of a variety of adducts and crosslinks collectively named Advanced Lipoxidation Endproducts (ALEs).\

MDA, HNE, and acrolein, among others. Thus, the most common adducts arising from enals are exocyclic adducts such as etheno adducts, and MDA-deoxyguanosine (M1dG). Finally, the amino group of aminophospholipids can also react with carbonyl compounds and initiate some of the reactions ocurring in proteins and DNA, leading to the formation of adducts like MDA-phosphatidylethanolamine, and carboxymethylphosphatidylethanolamine [10].

## **4. ALEs: Molecular and cellular effects**

Reactive carbonyl species (RCS) generated during lipid peroxidation reactions exhibit a wide range of molecular and biological effects, ranging from protein, DNA, and phospholipid damage to signaling pathway activation and/or alteration. The detailed mechanisms of 'toxicity' are, however, mostly unknown.

#### **4.1. Molecular damage**

444 Lipid Peroxidation

**Figure 5.** Reactive carbonyl species react with nucleophilic groups in macromolecules (A, proteins; B, DNA) resulting in their chemical, nonenzymatic, and irreversible modification and formation of a variety of adducts and crosslinks collectively named Advanced Lipoxidation Endproducts (ALEs).\

Lipoxidation reactions lead to structural and functional changes in proteins [25-27] such as i) alterations in physico-chemical properties (conformation, charge, hydrophobicity, elasticity, solubility, and electrophoretic mobility, among others); ii) decrease/inhibition in enzyme activity and growth factors; iii) alteration of protein degradation; iv) alteration in traffic and processing of proteins; and v) formation of intra- and inter-molecular protein cross-links and aggregates.

DNA lipoxidative damage is present in the genome of healthy humans and other animal species at biologically significant levels similar or even higher that oxidation markers *sensu stricto*. DNA damage is mutagenic, carcinogenic, and have powerful effects on signal transduction pathways [28].

Finally, the amino group of aminophospholipids can also react with carbonyl compounds and initiate some of the reactions occurring in proteins [29]. Biological processes involving aminophospholipids could be potentially affected by this process. Among these processes, it may be highlighted i) asymmetrical distribution of aminophospholpids in cellular and different subcellular membranes; ii) translocation between and lateral diffusion in the membrane; iii) membrane physical properties; iv) biosynthesis and turnover of membrane phospholipids; and v) activity of membrane-bound proteins that require aminophospholipids for their function.

#### **4.2. Cellular adaptive responses**

The peroxidation of the PUFA chains of phospholipids generates a complex mixture of carbonyl compounds. Initially, these aldehydes were believed to produce only "cytotoxic" effects associated with oxidative stress (by inducing molecular damage and damaging cellular responses based on inflammatory responses, changes in gene expression and apoptosis) [10], but as depicted above, evidence is increasing in the sense that these compounds can also have specific signaling roles inducing adaptive responses driven to decrease oxidative damage and improve antioxidant defences.

Thus, available studies support the notion that superoxide radical produced by the mitochondrial electron transport chain can cause mild uncoupling of mitochondria by activating the membrane proton conductance by uncoupling proteins (UCPs). Insight into the molecular mechanism by which superoxide radical activates UCPs comes from the finding that the lipid peroxidation product 4-HNE and its homologs induce uncoupling of mitochondria through UCPs and also through the adenine nucleotide translocase [30]. This and other observations support a model in which endogenous superoxide production generates carbon-centred radicals that initiate lipid peroxidation, producing alkenals like 4- HNE that activate UCPs and adenine nucleotide translocase. So, UCPs respond to overproduction of matrix superoxide by catalyzing mild uncoupling, which lower proton motive force and would decrease superoxide production by the electron transport chain. This negative feedback loop will protect cells from ROS-induced damage and might represent the ancestral function of all UCPs.

In addition, RCS can also activate the 'antioxidant response' likely to prevent their accumulation to toxic levels [31]. This signaling cascade culminates in the nuclear translocation of and transactivation by the transcription factor Nrf2, the master regulator of the response [32]. Nrf2 activity is repressed by an inhibitory binding protein, Keap1. Keap1 retains Nrf2 in the cytosol, closely associated with the actin cytoskeleton, and promotes proteasomal degradation of Nrf2 through Cullin3-dependent polyubiquitination. Following exposure to RCS, Keap1 can be directly modified on several cysteine residues, and this modification can promote release of Nrf2. Nrf2 contains a C-terminal basic leucine zipper structure that facilitates dimerization and DNA binding, specifically to the antioxidant response element (ARE). The binding of Nrf2 to the ARE stimulates transcription of downstream cytoprotective genes [32].

### **5. The selective neuronal vulnerability**

The idea that oxygen radicals, especially those of mitochondrial origin, are causally related to the basic aging process is increasingly receiving support from several independent sources [reviewed in 6,33]. Accordingly, the mitochondrial oxygen radical theory of aging apparently fulfils the main characteristics of this natural process: reactive oxygen species (ROS) are endogenously produced at mitochondria under normal physiological conditions, they are produced continuously throughout life (and can thus lead to progressive aging changes), and their deleterious effects on macromolecules may inflict irreversible damage during aging in post-mitotic tissues. The detrimental effects of aging are best observed in postmitotic tissues because cells that are irreversibly damaged or lost cannot be replaced by mitosis of intact ones. Nervous system is considered a postmitotic tissue, and therefore highly susceptible to aging. As this process is involved as a risk factor in most neurodegenerative diseases, and oxidative modifications play a key role in aging, it is often accepted that these diseases should have increased oxidative damage.

In this context, and from an inter-organ comparative approach, two main properties emerge as characteristics that render nervous system as especially sensible to oxidative modification: i) the % free radical leak, and ii) the membrane unsaturation. The % free radical leak (%FRL) refers the fraction (%) of electrons out of sequence which reduce oxygen to oxygen radicals (instead of reducing oxygen to water at cytochrome oxidase) in the mitochondrial respiratory chain. Since two electrons are needed to reduce one molecule of oxygen to H2O2, whereas four electrons are needed to reduce one molecule of oxygen to water, the free radical leak is easily calculated by dividing the rate of ROS production by 2 times the rate of oxygen consumption, the result being multiplied by 100. Results show that the higher %FRL corresponds to brain, suggesting that mitochondria are more inefficient in this tissue than in other organs (**Tables 1** and **2,** and **Figure 6**).

446 Lipid Peroxidation

compounds can also have specific signaling roles inducing adaptive responses driven to

Thus, available studies support the notion that superoxide radical produced by the mitochondrial electron transport chain can cause mild uncoupling of mitochondria by activating the membrane proton conductance by uncoupling proteins (UCPs). Insight into the molecular mechanism by which superoxide radical activates UCPs comes from the finding that the lipid peroxidation product 4-HNE and its homologs induce uncoupling of mitochondria through UCPs and also through the adenine nucleotide translocase [30]. This and other observations support a model in which endogenous superoxide production generates carbon-centred radicals that initiate lipid peroxidation, producing alkenals like 4- HNE that activate UCPs and adenine nucleotide translocase. So, UCPs respond to overproduction of matrix superoxide by catalyzing mild uncoupling, which lower proton motive force and would decrease superoxide production by the electron transport chain. This negative feedback loop will protect cells from ROS-induced damage and might

In addition, RCS can also activate the 'antioxidant response' likely to prevent their accumulation to toxic levels [31]. This signaling cascade culminates in the nuclear translocation of and transactivation by the transcription factor Nrf2, the master regulator of the response [32]. Nrf2 activity is repressed by an inhibitory binding protein, Keap1. Keap1 retains Nrf2 in the cytosol, closely associated with the actin cytoskeleton, and promotes proteasomal degradation of Nrf2 through Cullin3-dependent polyubiquitination. Following exposure to RCS, Keap1 can be directly modified on several cysteine residues, and this modification can promote release of Nrf2. Nrf2 contains a C-terminal basic leucine zipper structure that facilitates dimerization and DNA binding, specifically to the antioxidant response element (ARE). The binding of Nrf2 to the ARE stimulates transcription of

The idea that oxygen radicals, especially those of mitochondrial origin, are causally related to the basic aging process is increasingly receiving support from several independent sources [reviewed in 6,33]. Accordingly, the mitochondrial oxygen radical theory of aging apparently fulfils the main characteristics of this natural process: reactive oxygen species (ROS) are endogenously produced at mitochondria under normal physiological conditions, they are produced continuously throughout life (and can thus lead to progressive aging changes), and their deleterious effects on macromolecules may inflict irreversible damage during aging in post-mitotic tissues. The detrimental effects of aging are best observed in postmitotic tissues because cells that are irreversibly damaged or lost cannot be replaced by mitosis of intact ones. Nervous system is considered a postmitotic tissue, and therefore highly susceptible to aging. As this process is involved as a risk factor in most neurodegenerative diseases, and oxidative modifications play a key role in aging, it is often

accepted that these diseases should have increased oxidative damage.

decrease oxidative damage and improve antioxidant defences.

represent the ancestral function of all UCPs.

downstream cytoprotective genes [32].

**5. The selective neuronal vulnerability** 


Values are mean ± SEM from 8 different animals. State 4, oxygen consumption in the absence of ADP; State 3, oxygen consumption in the presence of ADP. Data from references: 34-36 and unpublished results.



Values are means ± SEM from 8 different animals. Pyr/mal = pyruvate/malate; Glu/mal = glutamate/malate; Succ, succinate; Rot, rotenone; AA, antimycin A. Data from references: 34-36 and unpublished results.

**Table 2.** Rates of mitochondrial H2O2 production (nanomoles H2O2/min mg protein) of different organs from male adult rats.

**Figure 6.** % Free radical leak (% FRL) of different organs from male adult rats. Data from references: 34- 36 and unpublished results.

As mentioned above, the susceptibility of biological membranes to oxidative alterations is related to two inherent traits, the physico-chemical properties of the lipid bilayer and the chemical reactivity of the fatty acids which make up the membrane. **Table 3** shows the mitochondrial fatty acid composition (mol%) of different organs. Data clearly indicate that average chain length is maintained around 18 carbon atoms and that the % saturated:unsaturated follows the ratio around 40:60. By contrast, the more relevant

**Figure 7.** Steady-state level of mitochondrial lipoxidative-derived protein damage in different organs from male adult rats. MDAL, malondialdehyde-lysine. Units: μmol/mol lysine.

differences are droved to the distribution of the different types of PUFAs. Thus, brain is characterized by the presence of the higher content of monounsaturated fatty acids, as well as PUFAn-3, and particularly, the docosahexaenoic acid (22:6n-3) compared to the other postmitotic organs (**Table 3**). This higher PUFAs content which are more susceptible to oxidative damage leads to a higher steady-state level of lipoxidation-derived molecular damage at least at mitochondrial level (**Figure 7**).

448 Lipid Peroxidation

36 and unpublished results.

**Figure 6.** % Free radical leak (% FRL) of different organs from male adult rats. Data from references: 34-

As mentioned above, the susceptibility of biological membranes to oxidative alterations is related to two inherent traits, the physico-chemical properties of the lipid bilayer and the chemical reactivity of the fatty acids which make up the membrane. **Table 3** shows the mitochondrial fatty acid composition (mol%) of different organs. Data clearly indicate that average chain length is maintained around 18 carbon atoms and that the % saturated:unsaturated follows the ratio around 40:60. By contrast, the more relevant

**Figure 7.** Steady-state level of mitochondrial lipoxidative-derived protein damage in different organs

from male adult rats. MDAL, malondialdehyde-lysine. Units: μmol/mol lysine.


Values: mean±SEM. N x group: 8. ACL, average chain length; SFA, saturated faaty acids; UFA, unsaturated fatty acids; PUFA n-6/n-3, polyunsaturated fatty acids n-6 or n-3 series; MUFA, monounsaturated fatty acids. Data from references: 34-36 and unpublished results.

**Table 3.** Mitochondrial fatty acid composition (mol%) of different organs from male adult rats.

Compared to other postmitotic organs, brain seems to be the tissue more susceptible to oxidative damage. However, are there cross-regional differences in the nervous system? Available data seems to indicate that this is the case. Thus, **Table 4** reflects the fatty acid composition in seven different regions from human nervous system samples. The data are conclusive: despite to maintain a stable average chain length (around 18 carbon atoms) and a ratio saurated:unsaturated practically identical (40:60), there is a cross-regional difference with respect to the type of PUFA distribution affecting very specially to both monounsaturated and polyunsaturated fatty acids that seems to be inversely related. In other words, the higher the presence of monounsaturated fatty acids for a given region, the lower the PUFA content. The meaning of this differential distribution remains to be elucidated, but it is evident that determine a differential susceptibility to oxidative damage.


Values: mean±SEM. N x group: 3-9. ACL, average chain length; SFA, saturated faaty acids; UFA, unsaturated fatty acids; PUFA n-6/n-3, polyunsaturated fatty acids n-6 or n-3 series; MUFA, monounsaturated fatty acids. (#) Unpublished results.

**Table 4.** Fatty acid composition (mol%) in the human nervous system: a cross-regional comparative approach #.

This means that saturated and monounsaturated fatty acyl chains (SFA and MUFA) are essentially resistant to peroxidation while polyunsaturates (PUFA) are damaged. Furthermore the greater the degree of polyunsaturation of PUFA the more prone it is to peroxidative damage. Indeed Holman [23] empirically determined (by measurement of oxygen consumption) the relative susceptibilities the different acyl chains (see **Figure 8**): Docosahexaenoic acid (DHA), the highly polyunsaturated omega-3 PUFA with six double bonds is extremely susceptible to peroxidative attack and is eight-times more prone to peroxidation than linoleic acid (LA) which has only two double bonds. DHA is 320-times more susceptible to peroxidation than the monounsaturated oleic acid (OA) [22,23].

450 Lipid Peroxidation

**Frontal Cortex** 

(#) Unpublished results.

approach #.

**Occipital**

Compared to other postmitotic organs, brain seems to be the tissue more susceptible to oxidative damage. However, are there cross-regional differences in the nervous system? Available data seems to indicate that this is the case. Thus, **Table 4** reflects the fatty acid composition in seven different regions from human nervous system samples. The data are conclusive: despite to maintain a stable average chain length (around 18 carbon atoms) and a ratio saurated:unsaturated practically identical (40:60), there is a cross-regional difference with respect to the type of PUFA distribution affecting very specially to both monounsaturated and polyunsaturated fatty acids that seems to be inversely related. In other words, the higher the presence of monounsaturated fatty acids for a given region, the lower the PUFA content. The meaning of this differential distribution remains to be elucidated, but it is evident that determine a differential susceptibility to oxidative damage.

**Cortex Hippocampus Amygdala Substantia**

**14:0** 0.51±0.04 0.54±0.05 0.42±0.05 0.56±0.05 0.54±0.04 0.50±0.04 0.48±0.05 **16:0** 21.13±0.51 20.30±1.70 19.54±0.27 20.69±0.67 12.68±0.39 13.57±0.38 14.75±0.25 **16:1n-7** 0.98±0.12 0.76±0.19 0.89±0.10 1.48±0.12 1.68±0.18 1.21±0.03 0.94±0.06 **18:0** 21.35±1.23 19.23±0.41 21.89±0.20 20.19±0.67 24.61±0.27 25.64±0.61 22.07±1.72 **18:1n-9** 23.78±1.51 28.57±1.16 26.12±0.50 28.40±0.77 33.57±0.19 30.13±0.87 33.51±1.43 **18:2n-6** 0.77±0.12 0.57±0.07 0.59±0.04 0.53±0.09 0.33±0.06 0.56±0.01 0.96±0.61 **18:3n-3** 0.14±0.01 0.21±0.04 0.23±0.02 0.08±0.006 0.17±0.003 0.23±0.01 0.34±0.05 **20:0** 1.25±0.23 2.40±0.52 1.44±0.09 1.71±0.12 4.95±0.12 5.58±0.23 7.62±0.53 **20:1** 0.19±0.04 0.32±0.05 0.37±0.01 0.28±0.02 0.22±0.02 0.45±0.02 0.75±0.09 **20:2n-6** 0.23±0.01 0.22±0.01 0.23±0.02 0.20±0.02 0.23±0.03 0.31±0.06 0.28±0.01 **20:3n-6** 0.63±0.07 0.64±0.04 0.90±0.10 0.60±0.08 0.61±0.02 0.99±0.03 0.87±0.16 **20:4n-6** 8.28±0.19 6.07±0.35 8.05±0.24 7.42±0.42 3.70±0.29 3.95±0.33 3.13±0.09 **22:4n-6** 4.77±0.14 5.06±0.69 6.24±0.20 4.79±0.27 4.32±0.15 4.07±0.08 3.16±0.17 **22:5n-6** 0.67±0.09 0.59±0.09 0.92±0.08 0.94±0.11 0.12±0.007 0.40±0.02 0.24±0.04 **22:5n-3** 0.26±0.03 0.19±0.02 0.33±0.02 0.13±0.04 0.06±0.006 0.09±0.01 0.38±0.14 **22:6n-3** 13.68±0.17 13.71±0.71 10.97±0.31 9.99±0.58 9.74±0.28 9.28±0.45 8.84±0.71 **24:0** 0.38±0.06 0.31±0.05 0.45±0.04 0.47±0.03 0.64±0.06 1.19±0.22 1.00±0.08 **24:1n-9** 0.93±0.23 0.21±0.03 0.33±0.04 1.46±0.32 1.73±0.18 1.75±0.11 0.59±0.10

**ACL** 18.60±0.01 18.56±0.04 18.58±0.008 18.48±0.04 18.59±0.005 18.64±0.03 18.52±0.01 **SFA** 44.64±1.33 42.80±0.74 43.77±0.37 43.65±0.94 43.43±0.34 46.51±0.47 45.95±1.81 **UFA** 55.35±1.33 57.19±0.74 56.22±0.37 56.34±0.94 56.56±0.34 53.48±0.47 54.04±1.81 **MUFA** 25.90±1.70 29.88±1.11 27.74±0.52 31.62±1.05 37.22±0.24 33.56±0.92 35.80±1.53 **PUFA** 29.45±0.46 27.31±0.38 28.48±0.25 24.71±1.02 19.33±0.42 19.92±0.45 18.24±0.43 **PUFAn-6** 15.36±0.36 13.18±0.25 16.95±0.36 14.49±0.66 9.34±0.26 10.30±0.30 8.67±0.81 **PUFAn-3** 14.08±0.18 14.12±0.63 11.53±0.34 10.21±0.59 9.99±0.28 9.61±0.46 9.57±0.57 Values: mean±SEM. N x group: 3-9. ACL, average chain length; SFA, saturated faaty acids; UFA, unsaturated fatty

acids; PUFA n-6/n-3, polyunsaturated fatty acids n-6 or n-3 series; MUFA, monounsaturated fatty acids.

**Table 4.** Fatty acid composition (mol%) in the human nervous system: a cross-regional comparative

**Nigra** 

**Medulla Oblongata**  **Spinal Cord** 

**Figure 8.** The relative susceptibilities of selected unsaturated fatty acids to peroxidation. Data are from [23], and all were empirically determined as rates of oxygen consumption. They are expressed relative to the rate for linoleic acid (18:2n-6) which is arbitrarily given a value of 1.

Combining the relative susceptibilities of different fatty acids with the fatty acid composition of membrane lipids it is possible to calculate a peroxidizability index1 (a measure of the susceptibility to peroxidation) for any particular membrane. The peroxidation index of a membrane is not the same as its unsaturation index (sometimes also called its "double bond index") which is a measure of the density of double bonds in the membrane. For example, a membrane bilayer consisting solely of MUFA will have an unsaturation index of 100 and a peroxidation index of 2.5, while a membrane bilayer consisting of 95% SFA and 5% DHA will have an unsaturation index of 30 and a peroxidation index of 40. This means that although the 5% DHA-containing membrane has only 30% the density of double bonds of the monounsaturated bilayer, it is 16-times more susceptible to peroxidative damage. In this context, data clearly show the existence of very important cross-regional differences in peroxidizability index in human central nervous system (**Figure 9**).

<sup>1</sup> *[Peroxidizability Index (PI) = 0.025 x (% monoenoics) + 1 x (% dienoics) + 2 x (% trienoics) + 4 x (% tetraenoics) + 6 x (% pentaenoics) + 8 x (% hexaenoics), while Unsaturation index (UI) = 1 x (% monoenoics) + 2 x (% dienoics) + 3 x (% trienoics) + 4 x (% tetraenoics) + 5 x (% pentaenoics) + 6 x (% hexaenoics) ]* 

The different cross-regionally PIs observed are due to changes in the type of unsaturated fatty acid that participates in membrane composition. So, there is a systematic redistribution between the types of PUFAs present from highly unsaturated fatty acids to the less unsaturated that is region-specific. Surprisingly, the change shows a gradient that follows the cranio-caudal axis considering the structural organization of the CNS. The mechanism(s) responsible for the cross-regional-related differences in fatty acid profile can be related, in principle, to the fatty acid desaturation pathway, and the deacylation-reacylation cycle. The available delta-5 and delta-6 estimated desaturase activities indicate that they are several folds higher in frontal cortex than in spinal cord (**Figure 10**). The mechanism underlying to the membrane unsaturation regulation could explain the differences in membrane fatty acid composition and, in turn, the peroxidizability index, and suggest a regulatory mechanism region-specific that is expressed differentially in a cranio-caudal axis likely associated to the development process and even the evolution of the central nervous system.

**Figure 9.** Cross-regional differences in the peroxidizability index in the human central nervous system. FC, frontal cortex; OC, occipital cortex; HC, hippocampus; AM, amygdala; SN, substantia nigra; MO, medulla oblongata; SC, spinal cord.

**Figure 10.** Delta-5 and delta-6 desaturase activities in different regions of the human central nervous system. FC, frontal cortex; OC, occipital cortex; HC, hippocampus; AM, amygdala; SN, substantia nigra; MO, medulla oblongata; SC, spinal cord.

In summary, membrane unsaturation is a key characteristic able to define the selective neuronal vulnerability. In this context, it is plausible to postulate that membrane unsaturation could be a main determinant factor in determining differences in the rate of aging for different regions of the CNS, and in the occurrence of neurodegenerative disorders [e.g., Alzheimer's disease (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS), among others] during the sixth, seventh and eighth decades of life. Interestingly, this property is also causally related to the aging process and the lifespan of animal species [6].

#### **6. Conclusions and perspectives**

452 Lipid Peroxidation

medulla oblongata; SC, spinal cord.

MO, medulla oblongata; SC, spinal cord.

The different cross-regionally PIs observed are due to changes in the type of unsaturated fatty acid that participates in membrane composition. So, there is a systematic redistribution between the types of PUFAs present from highly unsaturated fatty acids to the less unsaturated that is region-specific. Surprisingly, the change shows a gradient that follows the cranio-caudal axis considering the structural organization of the CNS. The mechanism(s) responsible for the cross-regional-related differences in fatty acid profile can be related, in principle, to the fatty acid desaturation pathway, and the deacylation-reacylation cycle. The available delta-5 and delta-6 estimated desaturase activities indicate that they are several folds higher in frontal cortex than in spinal cord (**Figure 10**). The mechanism underlying to the membrane unsaturation regulation could explain the differences in membrane fatty acid composition and, in turn, the peroxidizability index, and suggest a regulatory mechanism region-specific that is expressed differentially in a cranio-caudal axis likely associated to the

**Figure 9.** Cross-regional differences in the peroxidizability index in the human central nervous system. FC, frontal cortex; OC, occipital cortex; HC, hippocampus; AM, amygdala; SN, substantia nigra; MO,

**Figure 10.** Delta-5 and delta-6 desaturase activities in different regions of the human central nervous system. FC, frontal cortex; OC, occipital cortex; HC, hippocampus; AM, amygdala; SN, substantia nigra;

development process and even the evolution of the central nervous system.

A major goal of research into aging is to extend 'healthspan' by identifying approaches for delaying or preventing age-related diseases. The fact that many individuals maintain a wellfunctioning nervous system and continue productive lives through their seventies, eighties and even nineties is encouraging. The implication is that if the cellular and molecular mechanisms that determine whether nervous systems adapt positively or develop a disease during aging can be identified, then disease processes can be averted. In this regard, oxidative and metabolic stress and impaired cellular stress adaptation, are mechanisms of aging that render neurons vulnerable to degeneration. On this background of age-related endangerment, genetic and environmental factors likely determine whether a disease process develops. These include causal mutations, more subtle genetic risk factors and environmental factors, including aspects of diet and lifestyle. Because of the cellular and molecular complexity of the nervous system, and the signalling mechanisms that influence neuronal plasticity and survival, the basis of SNV remains elusive. However, available evidence from both an inter-organ comparative approach and cross-regional differences seems to confirm this idea, highlighting membrane unsaturation as a key trait associated with selective neuronal vulnerability. Interestingly, this property is also causally related to the aging process and the lifespan of animal species [6,10], and it is apparently operative in multiple neurodegenerative disorders [3,37]. Currently, most efforts to prevent and treat neurodegenerative disorders are focusing on diet, on lifestyle modification, and on drugs that target disease processes [3,6,33]. Although data on humans is still limited, the emerging evidence that dietary restriction (along with exercise and cognitive stimulation) can bolster neuroprotective mechanisms suggests that diet and lifestyle changes could reduce the risk of neurodegenerative disorders [6,26,33]. Therefore, it seems likely that extension of neural healthspan is possible for most individuals.

#### **Author details**

Alba Naudí, Mariona Jové, Victòria Ayala, Omar Ramírez, Rosanna Cabré, Joan Prat, Manuel Portero-Otin, Isidre Ferrer and Reinald Pamplona *Department of Experimental Medicine, University of Lleida-Biomedical Research Institute of Lleida, Lleida, Spain* 

## **Acknowledgement**

Work carried out at the Department of Experimental Medicine was supported in part by R+D grants from the Spanish Ministry of Science and Innovation [BFU2009-11879/BFI], and the Autonomous Government of Catalonia [2009SGR735]. The authors declare no conflict of interest.

### **7. References**


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**Acknowledgement** 

Work carried out at the Department of Experimental Medicine was supported in part by R+D grants from the Spanish Ministry of Science and Innovation [BFU2009-11879/BFI], and the Autonomous Government of Catalonia [2009SGR735]. The authors declare no conflict of

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## **Role of Lipid Peroxidation in the Pathogenesis of Age-Related Cataract**

Bojana Kisic, Dijana Miric, Lepsa Zoric and Aleksandra Ilic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45942

## **1. Introduction**

456 Lipid Peroxidation

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[35] Caro P, Gomez J, Sanchez I, Naudí A, Ayala V, López-Torres M, Pamplona R, Barja G (2009) Forty percent methionine restriction decreases mitochondrial oxygen radical production and leak at complex I during forward electron flow and lowers oxidative damage to proteins and mitochondrial DNA in rat kidney and brain mitochondria. Rej.

[36] Gomez J, Caro P, Sanchez I, Naudi A, Jove M, Portero-Otin M, Lopez-Torres M, Pamplona R, Barja G (2009) Effect of methionine dietary supplementation on mitochondrial oxygen radical generation and oxidative DNA damage in rat liver and

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intact? Antioxid. Redox Signal. 8(3-4):582-99.

heart. J. Bioenerg. Biomembr. 41(3):309-21.

The occurrence and development of cataract affect the decline of visual, working and living comfort. Cataract is the leading cause of blindness, accounting for 50% of blindness worldwide [1]. Cataract is progressive lens opacity in humans of 45 years or more, occurring without any known cause such as trauma, inflammation, hypocalcemia, medications or congenital factors. Risk factors for the occurrence of cataract are numerous: aging, diabetes mellitus, UV radiation, malnutrition, smoking, hypertension, renal disease, and others. Free oxygen radicals and oxidative stress are considered to be an important factor contributing to age-related cataract 1,2. Oxidative stress has been shown to cause cataract in in vitro models 3. This hypothesis is supported by studies that examined the anticatarogenic effect of different nutritional and physiological antioxidants 4.

Oxygen does not manifest toxic effects on cells of aerobic organisms in molecular form, but in the form of free oxygen radicals. Free radicals occur in univalent transfer of electrons to molecular oxygen. Due to its biochemical nature, and the low activation energy, they are able to react with biomolecules of all cellular structures, thereby carrying out their chemical and physiological modification. Under physiological conditions, the level of free radicals is controlled by mechanism of antioxidant protection. The balance between the production and catabolism of oxidants by cells and tissue is essential for maintenance of the biologic integrity of the tissue. Ocular tissues contain antioxidants that prevent damage from excessive oxygen metabolites: antioxidant enzymes, proteins, ascorbic acid, glutathione, amino acids cysteine and tyrosine, and other.

Changes in the oxidation of biomolecules can be found in many human diseases of the body, but the cataract is one of the most common diseases, where oxidative modifications of proteins 5 and lipids 1,2 is a dominant metabolic substrate of pathological disorders. Oxidative modification of lens proteins, loss of protein function and the creation of protein

© 2012 Kisic 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.

aggregates of high molecular mass, which increases the scattering of light, are the main features of age-related cataract 6. These protein modifications might be caused by oxidative stress resulting in higher levels of reactive oxygen radicals.

## **2. Sources of reactive oxygen species (ROS) in the lens**

#### **2.1. Reactive oxygen species generated in the lens by the UV irradiation**

Human lens has several systems of defense from ROS and oxidative stress, which are together responsible for the maintenance of lens transparency and prevention of cataract. But during the life the lens is exposed to multiple sources of oxidative stress, endogenous (altered mitochondrial respiration, respiratory burst of phagocytes, viral infection) and exogenous (UV light, metals,

drugs, cigarette smoke), which can lead to production of reactive oxygen species: superoxide anion (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (HO ) and others. The lens has a protective role for the other eye structures also, because light and oxygen are synergistically involved in the pathogenesis of cataract. By absorbing the part of the ultraviolet spectrum the lens protects deeper structures of the eye from the harmful effects of the solar radiation, whereby it is only subject to photooxidative damage. Photooxidative stress and the formation of reactive oxygen species by photosensitizing mechanisms are due to absorption of light by the biomolecules of the lens. Specifically, UV irradiation can mediate damage of lens structures, due to: direct absorption of the incident light by the cellular components, resulting in excited state formation and subsequent chemical reaction, and photosensitization mechanisms, where the light is absorbed by endogenous photosensitizers that are excited to their triplet states 6. The excited photosensitizers can induce cellular damage by electron transfer and hydrogen abstraction processes to yield free radicals or energy transfer with O2 to yield the reactive excited state, singlet oxygen.

Experiments in organ culture have shown that cataract can be caused by photochemical production of superoxide radicals, hydroxyl radicals and H2O2 7. Other researchers 8,9 indicate that photochemical generation of reactive species of oxygen in the lens and aqueous and consequent damage to the tissue has been implicated in the genesis of age-related cataract. The fact that the incidence of cataract is higher in the population that is more exposed to sunlight 10 imposes the assumption that photocatalytic conversion of molecular oxygen from ground state to excitatory states, which are highly reactive (O2 , H2O2, HO and others) occurs. High concentration of ascorbate in the aqueous humor is assumed to represent a kind of filter that prevents the penetration of UV light in the lens and thus protects tissue from oxidative damage, particularly photoinduced damage 11.

Photosensible substance, that absorbs certain wavelengths of light, activates and subtracts hydrogen or electrons from the substrate by converting them into free radicals. In the presence of O2 , the energy is transferred from excitatory substance and produce 1O2, which can initiate the process of lipid peroxidation. In the ocular tissues numerous substances can initiate photodynamic reactions. These substances are riboflavin, heme derivatives, tryptophan and its oxidation product N-formylkynurenine, lipofuscin, visible pigments (retinol), and photosensible substances of exogenous origin, such as drugs 12. Key link between photo-oxidation and cataract is that photo-oxidation of thiol groups on lens crystallyne produces disulfide bridges between molecules and, the build-up of these will lead to protein aggregation and hence cataract.

#### **3. Mitochondria as a source of reactive oxygen species in the lens**

458 Lipid Peroxidation

exogenous (UV light, metals,

superoxide anion (O2

H2O2, HO

aggregates of high molecular mass, which increases the scattering of light, are the main features of age-related cataract 6. These protein modifications might be caused by

Human lens has several systems of defense from ROS and oxidative stress, which are together responsible for the maintenance of lens transparency and prevention of cataract. But during the life the lens is exposed to multiple sources of oxidative stress, endogenous (altered mitochondrial respiration, respiratory burst of phagocytes, viral infection) and

drugs, cigarette smoke), which can lead to production of reactive oxygen species:

lens has a protective role for the other eye structures also, because light and oxygen are synergistically involved in the pathogenesis of cataract. By absorbing the part of the ultraviolet spectrum the lens protects deeper structures of the eye from the harmful effects of the solar radiation, whereby it is only subject to photooxidative damage. Photooxidative stress and the formation of reactive oxygen species by photosensitizing mechanisms are due to absorption of light by the biomolecules of the lens. Specifically, UV irradiation can mediate damage of lens structures, due to: direct absorption of the incident light by the cellular components, resulting in excited state formation and subsequent chemical reaction, and photosensitization mechanisms, where the light is absorbed by endogenous photosensitizers that are excited to their triplet states 6. The excited photosensitizers can induce cellular damage by electron transfer and hydrogen abstraction processes to yield free

radicals or energy transfer with O2 to yield the reactive excited state, singlet oxygen.

Experiments in organ culture have shown that cataract can be caused by photochemical production of superoxide radicals, hydroxyl radicals and H2O2 7. Other researchers 8,9 indicate that photochemical generation of reactive species of oxygen in the lens and aqueous and consequent damage to the tissue has been implicated in the genesis of age-related cataract. The fact that the incidence of cataract is higher in the population that is more exposed to sunlight 10 imposes the assumption that photocatalytic conversion of molecular oxygen from ground state to excitatory states, which are highly reactive (O2

assumed to represent a kind of filter that prevents the penetration of UV light in the lens and thus protects tissue from oxidative damage, particularly photoinduced damage 11.

Photosensible substance, that absorbs certain wavelengths of light, activates and subtracts hydrogen or electrons from the substrate by converting them into free radicals. In the presence of O2 , the energy is transferred from excitatory substance and produce 1O2, which can initiate the process of lipid peroxidation. In the ocular tissues numerous substances can initiate photodynamic reactions. These substances are riboflavin, heme derivatives,

and others) occurs. High concentration of ascorbate in the aqueous humor is

), hydrogen peroxide (H2O2), hydroxyl radical (HO

) and others. The

,

oxidative stress resulting in higher levels of reactive oxygen radicals.

**2. Sources of reactive oxygen species (ROS) in the lens** 

**2.1. Reactive oxygen species generated in the lens by the UV irradiation** 

In the ocular tissues, including the lens, as in other organ systems, ROS are formed in the mitochondria via the electron transport chain where inefficient electron coupling leads to the formation of superoxide anion. Molecular oxygen is tightly bound to the enzyme complex cytochrome C oxidase. However, the bond on the vectors of electrons in the respiratory chain in front of the system cytochrome C oxidase, on the level of NADHcoenzyme-Q reductase and the reduced forms of coenzyme Q, is not that strong and some of transferred electrons can "leak" from the system on molecular oxygen, forming O2 . Superoxide production is significantly increased during reperfusion of tissues, when the availability of oxygen is increased.

The human lens consists of three metabolically different zones: the epithelium, the cortex and lens nucleus. Epithelial cells and superficial cortical fibers are metabolically most active, and the greatest part of mitochondrion respiration and aerobic glycolysis in the lens occurs in them 13. One third of total energy produced (ATP) in the lens is produced in epithelial cells under aerobic conditions, while the metabolic activity of nuclear part of the lens is at much lower level. Intense metabolic activity makes epithelial cells susceptible to oxidative damage, especially their membrane pump systems and DNA. Oxidation of unsaturated lipids in epithelial cells could be the initial step that leads to generation of oxidation products. If reactive oxygen species or secondary products of lipid oxidation from the epithelium were to migrate to the fiber cells, it is possible that prolonged accumulation of lipid oxidative products could eventually lead to alterations in fiber cell structure and increased opacity, which leads to the development of cataract.

Thiol (-SH) groups of membrane proteins, the lens epithelial cells, which are significant for regulation of ion transport, are very susceptible to oxidative attack, especially when the concentration if intracellular GSH is reduced. The optimal membrane function of lens epithelial cells depends on reduced state of protein-SH groups. The oxidation of membrane thiol (-SH) groups of the lens cells leads to breakdown of active transport through the membrane, to the increase of membrane permeability and consequently intracellular alternations, which is involved in the development of cataract. The consequence of impaired active transport is also the reduced level of ascorbic acid in the lens. Studies have confirmed that ascorbic acid (AA) levels in human lenses with the development of cataract are reduced 34, and concentration of dehydroascorbic acid (DHA) is increased 14,35. Timely removal of dehydroascorbic acid from the lens is important because of its potential toxicity as oxidant. Increase of the current concentration of DHA/AA redox balance can be an indicator of oxidative stress in the lens 35.

#### **4. The importance of ascorbic acid in lens**

The role of ascorbic acid is important, as a strong reductant and effective scavenger of hydroxyl and superoxide anion radical. Vitamin C has antioxidant, but also prooxidant properties. In which direction will vitamin C work, depends on the concentration of vitamin C, oxygen and the presence of metal ions. Oxidation is the cause of modification of lens proteins which accumulate over a lifetime. Some believe that ascorbate can contribute to protein modifications, react as prooxidant and participate in reactions that generate radicals 14.

These reactions may be caused by light or metal-catalyzed oxidation of endogenous ascorbic acid. It is known that copper and iron are present in micromolar concentrations and that autooxidative processes can occur in the lens. Fenton-type reactions, where H2O2 reacts with free metal ions, iron (Fe2+) or copper (Cu2+) to produce the HO radical, are a major source of oxidative stress initiated by transition metals 15 and are thought to be involved in the formation of cataract 16. In the presence of metals, especially iron and copper, and oxygen, ascorbic acid is oxidized to dehydroascorbate, which produces hydrogen peroxide and metal is reduced. Hydrogen peroxide can react with reduced metal, generating hydroxyl radical and other reactive oxygen radicals 17. When copper and protein-bound iron is included in this reaction, the radicals cause oxidative modification of amino-acids that are near the metal. In this way ascorbate can actually become a prooxidant and lead to protein damage via both H2O2 and Fenton production of HO . These reactions become important when cells lose their ability to remove metals, making it available for reaction and/or when cells lose their ability to maintain their vitamin C in a reduced form. It is noted that during the aging of lens, as in cataract lenses the concentration of copper and iron increases 18,19. The data that confirm the level of iron and copper ions is lower in non-cataract lenses and study that compared cortical nuclear and mature cataracts found higher iron levels in the mature cataract 20 suggest that metal ions that mediate the production of HO , may be important in the development of age-related cataract 16.

Experiments on isolated proteins showed that oxidation products of ascorbate (dehydroascorbic acid) can form cross-link with crystalline lens, producing molecules of high molecular weight, which cause light scattering typical for cataract 21. It is assumed that similar modification of lens proteins occurs in vivo during the development of agerelated cataract 22.

#### **5. Lipid peroxidation in the lens**

In physiological/controlled conditions the process of lipid peroxidation affects the permeability of cell membranes, the metabolism of membrane lipids and proteins, provides control of cell proliferation, but the adverse effects of this process occurring under conditions of oxidation stress, ie. in conditions of impaired balance of prooxidative and antioxidative factors of the cell. Lipid peroxidation (LPO) is considered a pathogenetic factor of cataractogenesis 1,2,23,24,25. LPO in the lens may be induced by endogenous or exogenous factors: enzymes, reactive oxygen species, metal ions, UV irradiations, heat, radical-initiating chemicals, drugs. Cell membrane lipids (phospholipids, glycolipids) are the most common substrates of oxidative attacks, and since the cell membranes have lipoprotein structure, the structure of membrane proteins is disturbed at the same time. That causes the disturbance of cell membrane barrier function, leading to a larger entry of calcium and other ions 26. Structural changes of the cell membrane and its increased permeability change the cell volume and the configuration of the lens, leading to refractory changes that are associated with the early cataract.

460 Lipid Peroxidation

14.

**4. The importance of ascorbic acid in lens** 

free metal ions, iron (Fe2+) or copper (Cu2+) to produce the HO

damage via both H2O2 and Fenton production of HO

important in the development of age-related cataract 16.

related cataract 22.

**5. Lipid peroxidation in the lens** 

The role of ascorbic acid is important, as a strong reductant and effective scavenger of hydroxyl and superoxide anion radical. Vitamin C has antioxidant, but also prooxidant properties. In which direction will vitamin C work, depends on the concentration of vitamin C, oxygen and the presence of metal ions. Oxidation is the cause of modification of lens proteins which accumulate over a lifetime. Some believe that ascorbate can contribute to protein modifications, react as prooxidant and participate in reactions that generate radicals

These reactions may be caused by light or metal-catalyzed oxidation of endogenous ascorbic acid. It is known that copper and iron are present in micromolar concentrations and that autooxidative processes can occur in the lens. Fenton-type reactions, where H2O2 reacts with

oxidative stress initiated by transition metals 15 and are thought to be involved in the formation of cataract 16. In the presence of metals, especially iron and copper, and oxygen, ascorbic acid is oxidized to dehydroascorbate, which produces hydrogen peroxide and metal is reduced. Hydrogen peroxide can react with reduced metal, generating hydroxyl radical and other reactive oxygen radicals 17. When copper and protein-bound iron is included in this reaction, the radicals cause oxidative modification of amino-acids that are near the metal. In this way ascorbate can actually become a prooxidant and lead to protein

when cells lose their ability to remove metals, making it available for reaction and/or when cells lose their ability to maintain their vitamin C in a reduced form. It is noted that during the aging of lens, as in cataract lenses the concentration of copper and iron increases 18,19. The data that confirm the level of iron and copper ions is lower in non-cataract lenses and study that compared cortical nuclear and mature cataracts found higher iron levels in the

Experiments on isolated proteins showed that oxidation products of ascorbate (dehydroascorbic acid) can form cross-link with crystalline lens, producing molecules of high molecular weight, which cause light scattering typical for cataract 21. It is assumed that similar modification of lens proteins occurs in vivo during the development of age-

In physiological/controlled conditions the process of lipid peroxidation affects the permeability of cell membranes, the metabolism of membrane lipids and proteins, provides control of cell proliferation, but the adverse effects of this process occurring under conditions of oxidation stress, ie. in conditions of impaired balance of prooxidative and antioxidative factors of the cell. Lipid peroxidation (LPO) is considered a pathogenetic factor of cataractogenesis 1,2,23,24,25. LPO in the lens may be induced by endogenous or

mature cataract 20 suggest that metal ions that mediate the production of HO

radical, are a major source of

. These reactions become important

, may be

Cell membranes are very sensitive to the effects of oxygen radicals, due to the presence of polyunsaturated fatty acids in lipids. Fatty acids in lipids of cell membranes contain a different number of carbon atoms (14 to 24), and the present double bonds are in cis configuration. The presence of double bond in the neighborhood destabilize the bond between the carbon and the hydrogen of methylene group in the chain of polyunsaturated fatty acid, and by subtracting hydrogen from such a methylene group by reactive oxidant, begins the process of oxidative modification of fatty acids - lipid peroxidation 27. Nonenzymatic peroxidation of polyunsaturated fatty acids is a process that takes place in three stages: initiation, propagation and termination. The intensity of this process, as well as the ability of partial or complete repair of damage, depends on pro/anti-oxidative environment in which this process occurs.

Free radicals formed during lipid peroxidation have a local effect due to short life, but the degradation products of lipid peroxidation can be second messengers of oxidative stress, because of their longer half-life and ability to diffuse from the place of formation. These degradation products, mainly aldehydes, such as malonaldehyde, hexanal, 4 hydroxynonenal or acrolein, have biological roles in cellular signaling, in normal and pathological conditions and in regulation of cell cycle 28. Due to their chemical reactivity these products can covalently modify macromolecules as nucleic acids, proteins and lipids and consequently exhibit different biological effects. They are also biomarkers of lipid peroxidation and oxidative stress.

During the development of cataract, non-enzymatic lipid peroxidation occurs. It is a nonspecific and uncontrolled process in which the resulting reactive oxygen species readily react with surrounding molecules, leading to intensification of the process, and the damage of the cell membrane. The process LPO in the lens can be initiated by hydroxyl radical, singlet oxygen, peroxyl radical. These radicals seize from the unsaturated fatty acids H+ from the methyl group (-CH2) in the α- position of the double bond, to form unsaturated fatty acid lipid radical (L•).

By intramolecular rearrangement of double bonds in lipid radicals, conjugated dienes are formed. By adding the molecular oxygen to conjugated dienes, lipoperoxyl radical (LOO•) is formed. Lipoperoxyl radicals have significant oxidative potential, they can further initiate the seizure of hydrogen from the neighbouring unsaturated fatty acid, by which lipid peroxidation enters the phase of propagation and autooxidaton, resulting in formation of lipid hydroperoxyde (LOOH) and new lipid radicals (L•).

Lipid hidroperoxydes (LOOH), as primary molecular products of lipid peroxidation process, are well-soluble, have ability to migrate from the site of development and are important potential sources for the formation of reactive hydroxyl radical (HO•). In the presence of metals with variable valence they may reopen a cascade of lipid peroxidation 27. In the presence of Fe+2 in the classical Fenton-type reactions, by decomposing of LOOH, HO and alkoxyl radical (LO ) occur. In the presence of Fe+3, LOOH is decomposed to peroxyl radical (LOO ). Alkoxyl and peroxyl radical are responsible for initiation and propagation of LPO processes.

By removing hydroperoxides (LOOH), which occur during the first phase of lipid peroxidation, by activity of glutathione-dependent peroxidase which catalyzes reduction of hydroperoxide into the corresponding alcohol, the reactions of lipid peroxidation propagation can be prevented.

Compounds that react with reactive oxygen species produced during the chain reaction, such as peroxyl and alkoxyl radicals (ROO and RO ), and lead to the formation of the species that are unable to remove the hydrogen atom from unsaturated fatty acids, are considered to be antioxidant switches of chain reactions, of which the most important is liposoluble αtocopherol. In series of complex degradation reactions of hydro- and dihydro-peroxides of polyunsaturated fatty acids, many other aldehydes are produced: 4-hydroxy-alkenals, 4 hydroperoxy-alkenals, 4-oxo-alkenals and bis-aldehyde malonaldehyde (MDA) 28. These aldehydes are highly reactive compounds because they have electrophilic properties, they are less volatile than hydroperoxides, and can diffuse from the place of origin and express their reactivity through biotransformation and adduction to biomolecules (proteins, DNA).

Consequences of peroxidative damage of lens cell membranes are multiple. On the one hand, a number of membrane functions may be altered due to a direct attack by reactive oxygen species on the membrane components responsible for these functions. Indirect consequences of peroxidative damage of membranes are important. Lipid peroxidation modifies the environment of not only membrane proteins and may in this way influence their functional efficiency. The consequences of adduct formation at the protein level is associated with numerous cytotoxic consequences including the disruption of cell signalling, altered gene regulation, inhibition of enzyme activity, mitochondrial dysfunction, impaired energy metabolism. Two different ways of oxidative modifications of cellular constituents have to be considered in cataractogenesis: the direct modification by reactive oxygen species and the indirect modification via reactive products of lipid peroxidation.

Lipid peroxidation products can diffuse across membranes, allowing the reactive aldehyde to covalently modify proteins localized throughout the cell and relatively far away from the initial site of reactive oxygen species (ROS) formation. LPO is implicated in human cataractogenesis because the toxic peroxidation products induce fragmentation of soluble lens proteins and damage vital membrane structures, correlating with an increase in lens opacity and changes in the refractive properties of the lens 23. The results obtained by the authors, that caused posterior subcapsular cataract in the rabbit, by application of peroxidative products in the vitreous, were published 29.

The resulting lipid peroxidation products, such as, 4-hydroxynonenal (HNE), can form protein-HNE adducts that may result in altered protein functions and can mediate oxidative stress-induced cell death in the lens epithelial cells 30. Also, the resulting MDA, can react with amino-groups of proteins, forming intra-molecular cross bonds and bind two distinct proteins by forming the inter-molecular bonds 31 and thus affect the structural and functional properties of proteins. MDA is linked to the Lys residues of proteins and enzymes, and forms Schiff bases, for phospholipids, nucleic acids (shown mutagenic properties), and also MDA inhibits a number of thiol dependent enzymes: glucose-6 phosphatase, Na+-K+-ATP-ase, Ca++-ATP-ase.

462 Lipid Peroxidation

HO

and alkoxyl radical (LO

propagation of LPO processes.

propagation can be prevented.

as peroxyl and alkoxyl radicals (ROO

peroxyl radical (LOO

Lipid hidroperoxydes (LOOH), as primary molecular products of lipid peroxidation process, are well-soluble, have ability to migrate from the site of development and are important potential sources for the formation of reactive hydroxyl radical (HO•). In the presence of metals with variable valence they may reopen a cascade of lipid peroxidation 27. In the presence of Fe+2 in the classical Fenton-type reactions, by decomposing of LOOH,

By removing hydroperoxides (LOOH), which occur during the first phase of lipid peroxidation, by activity of glutathione-dependent peroxidase which catalyzes reduction of hydroperoxide into the corresponding alcohol, the reactions of lipid peroxidation

Compounds that react with reactive oxygen species produced during the chain reaction, such

are unable to remove the hydrogen atom from unsaturated fatty acids, are considered to be antioxidant switches of chain reactions, of which the most important is liposoluble αtocopherol. In series of complex degradation reactions of hydro- and dihydro-peroxides of polyunsaturated fatty acids, many other aldehydes are produced: 4-hydroxy-alkenals, 4 hydroperoxy-alkenals, 4-oxo-alkenals and bis-aldehyde malonaldehyde (MDA) 28. These aldehydes are highly reactive compounds because they have electrophilic properties, they are less volatile than hydroperoxides, and can diffuse from the place of origin and express their

Consequences of peroxidative damage of lens cell membranes are multiple. On the one hand, a number of membrane functions may be altered due to a direct attack by reactive oxygen species on the membrane components responsible for these functions. Indirect consequences of peroxidative damage of membranes are important. Lipid peroxidation modifies the environment of not only membrane proteins and may in this way influence their functional efficiency. The consequences of adduct formation at the protein level is associated with numerous cytotoxic consequences including the disruption of cell signalling, altered gene regulation, inhibition of enzyme activity, mitochondrial dysfunction, impaired energy metabolism. Two different ways of oxidative modifications of cellular constituents have to be considered in cataractogenesis: the direct modification by reactive oxygen species

Lipid peroxidation products can diffuse across membranes, allowing the reactive aldehyde to covalently modify proteins localized throughout the cell and relatively far away from the initial site of reactive oxygen species (ROS) formation. LPO is implicated in human cataractogenesis because the toxic peroxidation products induce fragmentation of soluble lens proteins and damage vital membrane structures, correlating with an increase in lens opacity and changes in the refractive properties of the lens 23. The results obtained by the authors, that caused posterior subcapsular cataract in the rabbit, by application of

and RO

reactivity through biotransformation and adduction to biomolecules (proteins, DNA).

and the indirect modification via reactive products of lipid peroxidation.

peroxidative products in the vitreous, were published 29.

) occur. In the presence of Fe+3, LOOH is decomposed to

), and lead to the formation of the species that

). Alkoxyl and peroxyl radical are responsible for initiation and

At the level of cell membranes in the lens lipid hydroperoxides induce changes of the lipoprotein structure and permeability 13, and oxidation inhibition of membrane enzymes Na+/K+-ATP-ase and Ca++-ATP-ase, which are responsible for osmotic regulation and transport of metabolites. Disturbance of ion transport through the cell membrane is associated with ATP hydrolysis, which increases membrane permeability to protons, as the result of LPO. The Na+/K+-ATP- and Ca++-ATP-ases would be directly affected because of their regulation by proton concentration. Other transport proteins would be affected in a secondary sense because they are coupled to ions whose intracellular concentrations are regulated by the ATP-ase. For example, glucose is the main source of energy for lens cells and is typically co-transported with Na+, and similar transport are known for the import of amino acids across cell membranes. Formation of the lens opacity follows from either osmotic imbalance or a cytoplasmic imbalance of specific cations, in particular Ca+2 32. Disturbance of function Ca++-ATP-ase and the consequent increase of Ca+2 in the lens leads to electrostatic changes in the crystalline, which can disrupt the protein conformation and interaction. Maintenance of calcium homeostasis is critical to lens clarity and cataractous lens has elevated calcium levels. An in vitro binding study indicates that human lens lipids have the capacity to bind nearly all the calcium present in the human lens and that age and cataract diminished the capacity of lens lipids to bind calcium. It is possible that the increased concentration of intracellular Ca+2 and reduced ability of lens lipids to bind calcium, to initiate further disturbances that lead to an increase of light scattering from proteins and lipids 33. Disturbance of the lens cell membrane permeability during the development of cataract, and reduced ability of active transport of substances against the concentration gradient lead to changes in concentration of intracellular compounds and metabolic changes within the cells. This is manifested by lowering the content of GSH, ATP, and other intracellular compounds and electrolytes in lens cells.

Lipid peroxides as potential causes of cataracts, lead to the changes of not only the lens cell membrane, but also of the cytosol, because it serves to reduce concentration of glutathione and cause the change of redox relationship GSH/GSSG 1.

#### **6. Defense against lipid peroxidation in the lens**

Low molecular mass compounds which act primarily against peroxyl radicals involved in radical propagation, provide first line of defense against lipid peroxidation in the lens. These compounds (GSH, ascorbic acid, - tocopherol) can terminate the propagation of free radical mediated reactions and interrupt the autocatalytic chain reaction of lipid peroxidation. GSH is a major antioxidant in the lens, and helps to reduce proteins, contains a side chain of sulfhydryl (-SH) residue that enables it to protect cells against oxidants. GSH can directly scavenge ROS or enzymatically via two major antioxidant enzyme systems, glutathione peroxidases (GPx) and glutathione S-transferases (GST). Enzymes such as superoxide dismuatase (SOD), catalase (CAT) and GPx can decompose ROS and prevent the damage to cellular constituents and initiation of lipid peroxidation. In the event of ROS induced lipid peroxidation, secondary defense enzymes are involved in the removal of LOOH to terminate the autocatalytic chain of lipid peroxidation and protect membranes. GPx and GST which catalyze GSH-dependent reduction of LOOH through their peroxidase activity are the major secondary defenses in the lens against ROS induced lipid peroxidation. Resynthesis of GSH from the oxidized form is catalyzed by glutathione reductase, where the necessary NADPH++H+ is produced in pentose pathway of carbohydrates [36]. Glutathione reductase (GR) is a control enzyme glutathione-redox cycle and by maintaining of intracellular levels of GSH can affect cation transport systems, lens hydration, sulfhydryl groups of proteins, and membrane integrity. Detoxifying role of GST is reflected in its ability to catalyze reactions of conjugation of reduced glutathione with endogenous electrophiles, mostly by the products of oxidative stress, lipid hydroperoxides and final products of lipid peroxidation 37. Class and GST isoenzymes are expressed in the human lens, with the isoenzyme predominating. The highest GST activity occurs in the peripheral and the equatorial cortexes, with the lowest activity in the nucleus 38. Glutathione peroxidase activity is shown by the enzymes that catalyze the reduction of hydrogen peroxide, organic hydroperoxide and phospholipid hydroperoxide using GSH as a hydrogen donor. Superoxide dismutase (SOD) catalyzes the reaction of dismutation of superoxide anion radicals (О2–•) in the presence of hydrogen donor to hydrogen peroxide and molecular oxygen. By removing the O2–•, SOD prevents the formation of 1О2 which can initiate the process of lipid peroxidation.

**Aim.** Our studies have focused on measuring the products of lipid peroxidation in corticonuclear lens blocks, with different type and different degrees of maturity of agerelated cataract. In addition to measuring products of lipid peroxidation in cataract lenses, our study included the determination of activities and ability of lens glutathione peroxidase and glutathione S-transferase to remove hydroperoxides, which are probably involved in the early stages of cataractogenesis and development of mature cataract through oxidative stress.

#### **7. Material and methods – patients**

Clinical and biochemical researches were carried out in 101 patients with age-related cataract, 46 women and 55 men. The average age of the group was 72.5 (SD 7.9). According to the cataract maturity degree the patients were classified into two groups as follows: age-related cataract incipient (N=41) and matura (N=60). In the group age-related cataract incipient there were 23 patients with posterior subcapsular (PS), 9 patients with nuclear subcapsular (NP) and 9 patients with cortical nuclear (CN) cataract. In the group age-related catataract matura there were 19 patients with cataract which started as a posterior subcapsular, 15 patients which started as a nuclear subcapsular, 16 patients with matura, which started as cortical nuclear and 10 patients diagnosed with matura, which started as cortical cataract.

Samples corticonuclear blocks/parts of lens (without epithelial cells) were obtained from patients undergoing extracapsular extraction of cataracts and used as the test material. Types of cataract were estimated during ophthalmologic examination and confirmed during its extraction. Immediately after sample acquisition, samples were closed in individual capsules and deeply frozen. This research has been conducted following the tenets of the Declaration of Helsinki and approved by the ethics committee of Medical Faculty. Informed consent was provided from all patients after a careful explanation of the aims of the study.

Homogenate of lenses from each group was prepared in 0.2 mol/L potassium phosphate buffer (pH 7.2). For analysis we used supernatant obtained by centrifugation of homogenates at 5000 rpm for 15 min at 4°C.

The concentration of conjugated dienes was measured spectrophotometrically at 233 nm 39.

Lens MDA concentrations were measured as the product of the reaction with thiobarbituric acid (TBA) using a modification of the method Ledwozyw et al 40.

Fluorescent products (lipid- and water-soluble) of lipid peroxidation were determined by spectrofluorimetric analysis at 360/430 (excitation/emission) nm 41.

The concentration of GSH in the sample was determined in the reaction 5,5'-dithiobis-2 nitrobenzoic acid (DTNB) (Ellman's reagent), after removal of proteins by perchloric acid [42.

### **8. Enzyme assays**

464 Lipid Peroxidation

initiate the process of lipid peroxidation.

**7. Material and methods – patients** 

stress.

These compounds (GSH, ascorbic acid, - tocopherol) can terminate the propagation of free radical mediated reactions and interrupt the autocatalytic chain reaction of lipid peroxidation. GSH is a major antioxidant in the lens, and helps to reduce proteins, contains a side chain of sulfhydryl (-SH) residue that enables it to protect cells against oxidants. GSH can directly scavenge ROS or enzymatically via two major antioxidant enzyme systems, glutathione peroxidases (GPx) and glutathione S-transferases (GST). Enzymes such as superoxide dismuatase (SOD), catalase (CAT) and GPx can decompose ROS and prevent the damage to cellular constituents and initiation of lipid peroxidation. In the event of ROS induced lipid peroxidation, secondary defense enzymes are involved in the removal of LOOH to terminate the autocatalytic chain of lipid peroxidation and protect membranes. GPx and GST which catalyze GSH-dependent reduction of LOOH through their peroxidase activity are the major secondary defenses in the lens against ROS induced lipid peroxidation. Resynthesis of GSH from the oxidized form is catalyzed by glutathione reductase, where the necessary NADPH++H+ is produced in pentose pathway of carbohydrates [36]. Glutathione reductase (GR) is a control enzyme glutathione-redox cycle and by maintaining of intracellular levels of GSH can affect cation transport systems, lens hydration, sulfhydryl groups of proteins, and membrane integrity. Detoxifying role of GST is reflected in its ability to catalyze reactions of conjugation of reduced glutathione with endogenous electrophiles, mostly by the products of oxidative stress, lipid hydroperoxides and final products of lipid peroxidation 37. Class and GST isoenzymes are expressed in the human lens, with the isoenzyme predominating. The highest GST activity occurs in the peripheral and the equatorial cortexes, with the lowest activity in the nucleus 38. Glutathione peroxidase activity is shown by the enzymes that catalyze the reduction of hydrogen peroxide, organic hydroperoxide and phospholipid hydroperoxide using GSH as a hydrogen donor. Superoxide dismutase (SOD) catalyzes the reaction of dismutation of superoxide anion radicals (О2–•) in the presence of hydrogen donor to hydrogen peroxide and molecular oxygen. By removing the O2–•, SOD prevents the formation of 1О2 which can

**Aim.** Our studies have focused on measuring the products of lipid peroxidation in corticonuclear lens blocks, with different type and different degrees of maturity of agerelated cataract. In addition to measuring products of lipid peroxidation in cataract lenses, our study included the determination of activities and ability of lens glutathione peroxidase and glutathione S-transferase to remove hydroperoxides, which are probably involved in the early stages of cataractogenesis and development of mature cataract through oxidative

Clinical and biochemical researches were carried out in 101 patients with age-related cataract, 46 women and 55 men. The average age of the group was 72.5 (SD 7.9). According to the cataract maturity degree the patients were classified into two groups as follows: age-related cataract incipient (N=41) and matura (N=60). In the group age-related The activity of glutathione peroxidase was determined at 412 nm, by the method Chin et al. [43]. The conjugation of GSH with 1-chloro,2-4 dinitrobenzene (CDNB), a hydrophilic substrate, was examined spectrophotometrically at 340 nm to measure glutathione Stransferase activity 44. One unit of GST was defined as the amount of enzyme required to conjugate 1mol of CDNB with GSH/min. The activity of glutathione reductase was assayed by the procedure of Glatzle et al. 45]. Superoxide dismutase (SOD) activity was determined by the method Misra and Fridovich [47], based on the inhibition of the adrenochrome during the spontaneous oxidation of adrenaline in basic conditions. The change in absorbance was read at 480 nm on a spectrophotometer. The SOD activity was expressed as kU/g protein (one unit was considered to be the amount of enzyme that inhibited adrenaline auto-oxidation by 50%).

To calculate the specific enzyme activity, protein in each sample was estimated by the method of Lowry et al 46.

## **9. Results and discussion**

Senile cataract is manifested in the later years of life, so it is estimated that the costs of operation would be reduced by 45% if the incidence of age-related cataract could be delayed for ten years 48. By studying the oxidation changes of lens structures during the development of cataract, we attempted to contribute to clearing up the process of cataractogenesis, of which even today there are many unknowns.

Lipid peroxidation is one of the possible mechanisms of cataractogenesis, caused by excessive production of reactive oxygen species in aqueous environment and reduced antioxidant defense of the lens. By studying the corticonuclear lens block of the patients with age-related cataract were detected increased concentrations in primary molecular products LPO (diene conjugates and lipid hydroperoxides) and end fluorescent LPO products (table 1).


Data is presented as means SD \*p<0.001

**Table 1.** Lipid peroxidation products in cataractous lenses

In the group of patients with the incipient cataract, we obtained significantly higher concentration of diene conjugates in the lenses compared to matura cataract (p<0.001) (table 1). This can be explained by the fact that at the early stages of the development of cataract the most intense is the proces of lipid peroxidation, which is either the initiator of cataractogenesis proces or initiated by creation of reactive oxigen types, and continues to affect changes in the lens by its propagation.

In the lenses with the incipient cataract, the concentration of conjugated diens is significantly higher in cortical nuclear cataract (CN) compared with the lenses with posterior subcapsular cataract (PS) (p=0.001) (table 2). Also, in matura cataract the concentration of conjugated diens is the highest in the lenses diagnosed with CN cataract (p<0.05) (table 3).


Data is presented as means SD \*p<0.001, ‡p<0.05, †p<0.01.

466 Lipid Peroxidation

products (table 1).

Conjugated diens

Fluorescent products/

Fluorescent products/

Data is presented as means SD \*p<0.001

**Table 1.** Lipid peroxidation products in cataractous lenses

affect changes in the lens by its propagation.

MDA

(p<0.05) (table 3).

**9. Results and discussion** 

Senile cataract is manifested in the later years of life, so it is estimated that the costs of operation would be reduced by 45% if the incidence of age-related cataract could be delayed for ten years 48. By studying the oxidation changes of lens structures during the development of cataract, we attempted to contribute to clearing up the process of

Lipid peroxidation is one of the possible mechanisms of cataractogenesis, caused by excessive production of reactive oxygen species in aqueous environment and reduced antioxidant defense of the lens. By studying the corticonuclear lens block of the patients with age-related cataract were detected increased concentrations in primary molecular products LPO (diene conjugates and lipid hydroperoxides) and end fluorescent LPO

> Age-related cataract incipient (n=41)

(nmol/g weight of lens) 2.48 0.84\* 1.57 0.49

g protein (lipid soluble) 46.59 14.40 70.94 13.21\*

g protein (water soluble) 57.53 18.23 103.08 27.81\*

(nmol/g weight of lens) 1.81 0.67 3.17 0.78\*

In the group of patients with the incipient cataract, we obtained significantly higher concentration of diene conjugates in the lenses compared to matura cataract (p<0.001) (table 1). This can be explained by the fact that at the early stages of the development of cataract the most intense is the proces of lipid peroxidation, which is either the initiator of cataractogenesis proces or initiated by creation of reactive oxigen types, and continues to

In the lenses with the incipient cataract, the concentration of conjugated diens is significantly higher in cortical nuclear cataract (CN) compared with the lenses with posterior subcapsular cataract (PS) (p=0.001) (table 2). Also, in matura cataract the concentration of conjugated diens is the highest in the lenses diagnosed with CN cataract

Age-related cataract matura (n=60)

cataractogenesis, of which even today there are many unknowns.

PS - posterior subcapsular, NP - nuclear subcapsular, CN - cortical nuclear



Data is presented as means SD †p<0.01, ‡p<0.05.

**Table 3.** Products LPO in lenses with cataract matura

In the presence of free metal ions with variable valence (Fe+2 or Cu2+) hydrogen peroxide in Fenton's reaction is translated into highly reactive hydroxyl radical, while the lipid peroxides are translated into peroxyl and alkoxyl radicals. Because of the longer half-life time compared to the alkoxyl radical, peroxyl radical is ideal for propagation of oxidative chain reactions, while the oxidation of alkoxyl radicals produce dihydroperoxide, which is degraded to toxic aldehydes, such as 4-hydroxy-2,3-trans-nonenal, 4-hydroxy-pentanal, short chain malondialdehyde. Such conditions may exist in humane senile lens 49. MDA and 4-hydroxy-2,3-trans-nonenal by forming of Schiff's bases with amino groups amino acid residues of protein contribute to increase of carbonyl groups content and produce fluorescent products of lipid peroxidation. We have measured significantly higher concentration of these fluorescent products of LPO in the lenses with mature cataract (table 1), and the highest concentration was measured in the lenses with the NP and CN cataract in relation to posterior subcapsular cataract (PS) (p<0.05) (table 2,3). Some authors have identified fluorescent Schiff bases in higher concentration in human cataract lenses compared to healthy lenses, resulting from the interaction of reactive carbonyl groups MDA with amino groups of lens membrane phospholipids 50.

By measuring the concentration of MDA in the homogenate of cataract lenses, we obtained significantly higher concentration in the group of patients with matura compared to incipient cataract (p<0.001) (table 1). This can be explained by the fact that the malondialdehyde is one of the final products of LPO, which accumulates in the lens during the process of lipid peroxidation and the development of cataract. Author's results with the experimentally induced cataract that have measured significantly higher concentration of MDA in cataract lenses compared to the control group were published 51, as well as author's works that obtained significantly higher concentration of MDA in cataract lenses of diabetics 2, myopic lenses and senile cataract 52. Reduced activity of glutathione peroxidase enzyme and glutathione S-transferase, that are important for the removal of malondialdehyde, contribute to the increase of its concentration in the lens.

The reasons that cause significantly higher concentration of lipid peroxidation products in lenses with cortical nuclear (CN) and nuclear subcapsular (NP) cataract, in relation to the posterior subcupsular (PS) are numerous. The results of other researchers 49 indicate that during the life time in the lens some kind of "internal" lens barrier is developed between nuclear and corticular parts, which hinders the diffusion of molecules to the nucleus. This barrier prevents the diffusion of antioxidative molecules to the nuclear part, which increases the sensitivity of central part of the lens to oxidative damage. Also, it is possible that unstable prooxidant molecules have longer residency in the central part of the lens. The endogenous lens hromofore, tryptophan metabolites (kynurenine, 3-hydroxykynurenine, 3 hydroxykynurenine glucoside), which are relatively inert photochemically, during oxidative stress and/or aging are formed photochemically active tryptophan metabolite (N-formylkynurenine, xanthurenic acid) that have photochemical properties, and also act as an endogenous photosensitizers in the lens 53.

Through photosensible reactions tryptophan products transfer absorbed energy to oxygen, which further leads to a series of cellular changes through the oxidation. With age, the level of free components of UV filters ie. tryptophan derivatives in the lens are reduced, and their binding to lens proteins increases 54. Tryptophan products are subject non-enzyme deamination, at physiological pH, resulting in ,-unsaturated ketone, reactive intermediates 55, which can covalently bind to amino acids, usually His, Cys, or Lys residues in proteins of human lenses, or react with the Cys residue of glutathione (GSH) to form GSH-3OHKynG 54. GSH that is present in the lens in relatively high concentrations may compete with the amino acid residues for the unsaturated ketone derivative of kynurenine, thereby protecting the crystalline from modification. This covalent modification is particularly expressed in the nucleus of the lens containing the older proteins, causing altered transport/diffusion of small molecules in the lens. Specifically, it develops a barrier in to the movement of molecules between metabolically active cortex and inert nucleus. The barrier also restricts the flow of GSH from the cortex, which reduces the concentration of GSH in the nucleus of the lens, so the response to oxidative damages in this part of the lens is also reduced 49. During the life time, lens fibers are very compactly arranged in nuclear part, with minimal presence of extracellular space. Nuclear plasma of the membrane undergo oxidation damage, whereas the phospholipid molecules modified by oxygen accumulate in the lipid layer, leading to changes in the structure and violate lipid-lipid and protein-lipid interactions in membranes of the lens fibers. This probably contributes to concentration of the lipid peroxidation products to be the highest in the lenses with early nuclear cataract.

468 Lipid Peroxidation

1), and the highest concentration was measured in the lenses with the NP and CN cataract in relation to posterior subcapsular cataract (PS) (p<0.05) (table 2,3). Some authors have identified fluorescent Schiff bases in higher concentration in human cataract lenses compared to healthy lenses, resulting from the interaction of reactive carbonyl groups MDA

By measuring the concentration of MDA in the homogenate of cataract lenses, we obtained significantly higher concentration in the group of patients with matura compared to incipient cataract (p<0.001) (table 1). This can be explained by the fact that the malondialdehyde is one of the final products of LPO, which accumulates in the lens during the process of lipid peroxidation and the development of cataract. Author's results with the experimentally induced cataract that have measured significantly higher concentration of MDA in cataract lenses compared to the control group were published 51, as well as author's works that obtained significantly higher concentration of MDA in cataract lenses of diabetics 2, myopic lenses and senile cataract 52. Reduced activity of glutathione peroxidase enzyme and glutathione S-transferase, that are important for the removal of

The reasons that cause significantly higher concentration of lipid peroxidation products in lenses with cortical nuclear (CN) and nuclear subcapsular (NP) cataract, in relation to the posterior subcupsular (PS) are numerous. The results of other researchers 49 indicate that during the life time in the lens some kind of "internal" lens barrier is developed between nuclear and corticular parts, which hinders the diffusion of molecules to the nucleus. This barrier prevents the diffusion of antioxidative molecules to the nuclear part, which increases the sensitivity of central part of the lens to oxidative damage. Also, it is possible that unstable prooxidant molecules have longer residency in the central part of the lens. The endogenous lens hromofore, tryptophan metabolites (kynurenine, 3-hydroxykynurenine, 3 hydroxykynurenine glucoside), which are relatively inert photochemically, during oxidative stress and/or aging are formed photochemically active tryptophan metabolite (N-formylkynurenine, xanthurenic acid) that have photochemical properties, and also act as an

Through photosensible reactions tryptophan products transfer absorbed energy to oxygen, which further leads to a series of cellular changes through the oxidation. With age, the level of free components of UV filters ie. tryptophan derivatives in the lens are reduced, and their binding to lens proteins increases 54. Tryptophan products are subject non-enzyme deamination, at physiological pH, resulting in ,-unsaturated ketone, reactive intermediates 55, which can covalently bind to amino acids, usually His, Cys, or Lys residues in proteins of human lenses, or react with the Cys residue of glutathione (GSH) to form GSH-3OHKynG 54. GSH that is present in the lens in relatively high concentrations may compete with the amino acid residues for the unsaturated ketone derivative of kynurenine, thereby protecting the crystalline from modification. This covalent modification is particularly expressed in the nucleus of the lens containing the older proteins, causing

malondialdehyde, contribute to the increase of its concentration in the lens.

with amino groups of lens membrane phospholipids 50.

endogenous photosensitizers in the lens 53.

Reduced glutathione (GSH) and GSH-dependent enzymes, glutathione peroxidase (GPx) and glutathione S-transferase (GST), are very important in defending the lens structures of the products of lipid peroxidation. One reason for increased production and accumulation of lipid hydroperoxides in the lens with cataract may also be reduced activity of GPx and GST.

The primary biological role of superoxide dismutase (SOD) is to catalyze reaction of dismutations of superoxide anion radicals (О2–•) in the presence of hydrogen donor to hydrogen peroxide and molecular oxygen. Superoxides can first be degraded into H2O2 by SOD, and subsequently, catalyzed into ground-state oxygen and water by catalase and enzymes of the glutathione redox cycle, including glutathione reductase and glutathione peroxidase. At low levels of hydrogen peroxide, the glutathione redox cycle is responsible for protecting against H2O2-induced damage and maintaining high levels of GSH in the lens, whereas, at a higher concentration, the principal mechanism for the removal of hydrogen peroxide is catalase. The study of the lens cell culture where the expression of DNK for superoxide dismutase was performed on intact lens cells, showed that the cells with higher activity of SOD resistant to oxidative damage, caused by hydrogen peroxide, superoxide anion radical and UV radiation. Expression of the superoxide dismutase enzyme prevented the beginning of the cataract in lens cells 56.

GSH and other sulfhydryls are particularly important in the protection of thiol (-SH) groups of crystalline and prevent the formation of aggregates which reduce transparency of the lens 57. The oxidized glutathione (GSSG) is reduced back to GSH by a NADPH-dependent glutathione reductase, which in physiological conditions maintains a high ratio of GSH/GSSG in the lens and other ocular tissues. Oxidative stress, induced by accumulation of LPO products in the lens during the development of cataract, causes consumption of GSH and disruption of redox balance in the lens, so the age-related cataract is associated with progressive reduction of GSH concentration in the lens. Probably with the progression of the cataract than the consumption of GSH against toxic compounds, the synthesis of GSH is reduced, as the result of deficient availability of substrates and reduced activity of enzymes for its synthesis (-glutamyl-cysteine syntethase). This reduces the amount of available GSH for optimal function GPx and GST, which causes the peroxide metabolism disorder.

**Figure 1.** Lens glutathione and glutathione reductase in cataract.

**Figure 2.** Lens glutathione and glutathione peroxidase in cataract.

The significance of GSH, as an important compound for the function of GPx and GST in the tested lenses, is confirmed also by the positive correlations between the concentration of GSH and glutathione reductase (r= 0.7011, p< 0.001) (figure 1), between the concentration of GSH and glutathione peroxidase activity (r= 0.6749, p< 0.001) (figure 2), and between activities of glutathione S-transferase and the concentration of GSH (r= 0.6379, p< 0.001) (figure 3).

**Figure 3.** Lens glutathione and glutathione S-transferase in cataract.

Experiments showed that applying the injections of buthionine-sulphoximine as inhibitors of reduced glutathione synthesis, on the newborn rats, causes the development of cataract 58.


Data is presented as means SD \*p<0.001

470 Lipid Peroxidation

(figure 3).

**Figure 1.** Lens glutathione and glutathione reductase in cataract.

**Figure 2.** Lens glutathione and glutathione peroxidase in cataract.

The significance of GSH, as an important compound for the function of GPx and GST in the tested lenses, is confirmed also by the positive correlations between the concentration of GSH and glutathione reductase (r= 0.7011, p< 0.001) (figure 1), between the concentration of GSH and glutathione peroxidase activity (r= 0.6749, p< 0.001) (figure 2), and between activities of glutathione S-transferase and the concentration of GSH (r= 0.6379, p< 0.001) GSH (µmol/g weight of lens), GPx, GR, GST (U/g protein), SOD (kU/g protein).

**Table 4.** Antioxidative defense factors in cataractous lenses

In tested corticonuclear lens blocks with mature cataract, we measured lower activity of GR, GPx and GST enzymes in relation to the incipient (p<0.001) (table 4).


Data is presented as means SD \*p<0.001, †p<0.01.

GSH (µmol/g weight of lens), GPx, GR, GST (U/g protein), SOD (kU/g protein)

**Table 5.** Antioxidative defense factors in lenses with cataract incipient

The lowest activity of these enzymes was measured in the lenses with the CN and NP in relation to the PS cataract (table 5). Reduced activity of GR, GPx and GST enzymes is followed by a significant decrease in the concentration of GSH in lens homogenates with matura (0.88 ± 0.26 µmo/g weight), compared to the initial cataract (2.55 ± 0.9 µmo/g weight) (p<0.001) (table 4), with lower concentrations of GSH measured in CN and NP compared to the PS cataract (table 5,6).


Data is presented as means SD \*p<0.001.

GSH (µmol/g weight of lens), GPx, GR, GST (U/g protein), SOD (kU/g protein)

**Table 6.** Antioxidative defense factors in lenses with cataract matura

Also the results of other researchers show that the level of reduced glutathione in the lens decreases with the development of the cataract [24]. Such changes are probably a reflection of oxidative processes, increased by the formation of toxic products of lipid peroxidation, compared to a weakened antioxidative capacity of cataract lenses. In cataract lenses the concentration of GSH is being reduced, since, as a main representative of non-protein thiols, it is included and consumed in oxido-reduction processes in terms of excess oxidized substrates. A possible reason for the consumption of GSH during oxidative stress is its conversion into oxidized form, which can be conjugated with protein thiol groups to form mixed disulphides (PSSG), via a process called protein-S-thiolation 59.

Results of other researchers also show that the human lens with age-related cataract glutathione peroxidase activity was significantly reduced compared with normal lenses. The kinetic study of GPx showed that lipid hydroperoxides achieve saturation of enzymes at a concentration that is approximately 1 mmol ie. that Km GPx is achieved at a concentration of lipid hydroperoxides of 0.434 mmol [60]. Because of these kinetic properties, GPx activity was probably inhibited in age-related cataract by products of lipid peroxidation, using noncompetitive inhibition principle. GPx activity, aside from availability of GSH, is affected by other factors such as glutathione reductase activity, the amount of produced NADPH++H+ in the pentose pathway and availability of selenium. The authors who have examined the activity of GPx in the lenses of experimental mice and compared the degree of lens blur, and age of mice with specific activity of GPx, showed significant correlation between decreased activity of GPx and the level of lens blur, as well as the age of mice 61.

Considering the function of GST to catalyze reactions of conjugation of lipid peroxidation products with GSH 62, thereby reducing the toxicity of electrophylic compounds and their reactivity towards nucleophilic groups in biomolecules, it is logical that the activity of the GST measured in incipient cataract is higher (2.46 1.14 U/g protein), because at the beginning of the development of the cataract the most intense is the process of lipid peroxidation. With advancing of the process of cataractogenesis, the amount of GSH is reduced for glutathione-S-transferase, and the enzyme activity is significantly decreased in the lenses with mature cataract (1.5 0.67 U/g protein) (p<0.001) (table 4).

472 Lipid Peroxidation

the PS cataract (table 5,6).

Cataract matura

Data is presented as means SD \*p<0.001.

The lowest activity of these enzymes was measured in the lenses with the CN and NP in relation to the PS cataract (table 5). Reduced activity of GR, GPx and GST enzymes is followed by a significant decrease in the concentration of GSH in lens homogenates with matura (0.88 ± 0.26 µmo/g weight), compared to the initial cataract (2.55 ± 0.9 µmo/g weight) (p<0.001) (table 4), with lower concentrations of GSH measured in CN and NP compared to

GSH 0.98 ± 0.19 0.78 ± 0.20 0.81 ± 0.35 GPx 2.96 ± 0.99 1.89 ± 0.57\* 1.44 ± 0.31\* GR 2.26 0.60 1.31 0.49\* 1.06 0.47\* GST 2.13 0.50 1.13 0.43\* 1.07 0.49\* SOD 2.96 0.65 1.77 0.52\* 1.25 0.36\*

Also the results of other researchers show that the level of reduced glutathione in the lens decreases with the development of the cataract [24]. Such changes are probably a reflection of oxidative processes, increased by the formation of toxic products of lipid peroxidation, compared to a weakened antioxidative capacity of cataract lenses. In cataract lenses the concentration of GSH is being reduced, since, as a main representative of non-protein thiols, it is included and consumed in oxido-reduction processes in terms of excess oxidized substrates. A possible reason for the consumption of GSH during oxidative stress is its conversion into oxidized form, which can be conjugated with protein thiol groups to form

Results of other researchers also show that the human lens with age-related cataract glutathione peroxidase activity was significantly reduced compared with normal lenses. The kinetic study of GPx showed that lipid hydroperoxides achieve saturation of enzymes at a concentration that is approximately 1 mmol ie. that Km GPx is achieved at a concentration of lipid hydroperoxides of 0.434 mmol [60]. Because of these kinetic properties, GPx activity was probably inhibited in age-related cataract by products of lipid peroxidation, using noncompetitive inhibition principle. GPx activity, aside from availability of GSH, is affected by other factors such as glutathione reductase activity, the amount of produced NADPH++H+ in the pentose pathway and availability of selenium. The authors who have examined the activity of GPx in the lenses of experimental mice and compared the degree of lens blur, and age of mice with specific activity of GPx, showed significant correlation between decreased

Cataract matura began as NP (N=15)

Cataract matura began as CN (N=16)

Cataract matura began as PS (N=19)

GSH (µmol/g weight of lens), GPx, GR, GST (U/g protein), SOD (kU/g protein) **Table 6.** Antioxidative defense factors in lenses with cataract matura

mixed disulphides (PSSG), via a process called protein-S-thiolation 59.

activity of GPx and the level of lens blur, as well as the age of mice 61.

In the study of GST activity in epithelial cells of operated cataract lenses, the group of authors showed that lens epithelial cells with cortical nuclear and cortical cataract show complete loss of activity of glutathione-S-transferase [63].

By analyzing the activity of glutathione reductase, significantly higher activity was found in the lenses with the incipient cataract (3.03 1.29 U/g protein) compared to the lenses with mature cataract (1.61 0.71 U/g protein) (p<0.001) (table 4). Other researchers have obtained similar results after comparing the activities of GR in cataract lenses and intact lenses of older persons [64]. Glutathione reductase plays a key role in maintaining thiol (-SH) groups in the lens, and this is probably the most important role of this enzyme in maintaining lens transparency. It is possible that lower GR activity in comparison to normal activities, can be one of the causes of lens blur. In addition to its predominantly cortical distribution within lens fiber cells, high susceptibility of GR to post-translational modifications 65 could also be of critical importance for the early dysfunction of GPx and GST under oxidative stress.

Based on the fact that the activity of GPx and GST is focused on degradation of lipid peroxidation products, we tested the relationship between the activity of the antioxidant defense enzyme and products of LPO in cataract lenses.

**Figure 4.** Lens conjugated diens and glutathione peroxidase relationship in cataract.

Reduced specific GPx activity (U/g protein) in the lenses shows significant correlation with the increased concentration of conjugated diens (r= -0.476, p<0.01) (figure 4), lipid-soluble fluorescent products (r= -0.429, p<0.01) (figure 5), water-soluble fluorescent products (r= - 0.367, p<0.05) (figure 6) and MDA (r= -0.328, p<0.05) (figure 7).

**Figure 5.** Lens lipid-soluble fluorescent products and GPx relationship in cataract.

**Figure 6.** Lens water-soluble fluorescent products and GPx relationship in cataract.

**Figure 7.** Lens malondialdehide and glutathione peroxidase relationship in cataract.

Reduced specific GPx activity (U/g protein) in the lenses shows significant correlation with the increased concentration of conjugated diens (r= -0.476, p<0.01) (figure 4), lipid-soluble fluorescent products (r= -0.429, p<0.01) (figure 5), water-soluble fluorescent products (r= -

0.367, p<0.05) (figure 6) and MDA (r= -0.328, p<0.05) (figure 7).

**Figure 5.** Lens lipid-soluble fluorescent products and GPx relationship in cataract.

**Figure 6.** Lens water-soluble fluorescent products and GPx relationship in cataract.

Also, the reduced GST activity (U/g protein) shows the significant correlation with the increased concentration of total hydroperoxides (r= -0.313, p<0.05), conjugated diens (r= - 0.465, p<0.01) (figure 8), lipid-soluble fluorescent products (r= -0.398, p=0.01) (figure 9), water-soluble fluorescent products (r= -0.347, p<0.05) (figure 10) and MDA (r= -0.345, p<0.05) (figure 11) in homogenates of the lenses with the incipient cataract.

**Figure 8.** Lens conjugated diens and glutathione S-transferase relationship in cataract.

Probably, the consumption of GSH and other antioxidants in reactions of degradation of lipid peroxidation products, affects the decrease of GPx and GST enzymes activity, and as the process of cataractogenesis progresses towards mature cataract, all lens structures become affected by the changes, which probably leads to the change of enzyme molecules themselves.

**Figure 9.** Lens lipid-soluble fluorescent products and GST relationship in cataract.

**Figure 10.** Lens water-soluble fluorescent products and GST relationship in cataract.

**Figure 11.** Lens malondialdehide and glutathione S-transferase in cataract.

We measured higher activity of SOD (4.13 2.14 kU/g protein) in lenses with incipient cataract in comparison to mature cataract (2.14 0.91 kU/g protein) (p<0.001) (table 2). Such changes are probably a reflection of oxidative processes, increased by formation of toxic products of lipid peroxidation in relation to the weakened antioxidant capacity of cataract lenses. Also, other researchers have measured decreased SOD activity in the lenses of patients with senile and diabetic cataract 66. The decreased activity of superoxide dismutase in the lenses with age-related cataract may be due to denaturation of enzyme molecules, and/or slow enzyme synthesis. Reduced SOD activity and consequently, the increase of concentration of H2O2 lead to the formation of hydroxyl radical from Fentons type reaction. Subsequently, hydroxyl radical induces the formation of superoxide radical, which may initiate the process of lipid peroxidation in the lens.

#### **10. Conclusion**

476 Lipid Peroxidation

**Figure 9.** Lens lipid-soluble fluorescent products and GST relationship in cataract.

**Figure 10.** Lens water-soluble fluorescent products and GST relationship in cataract.

Based on the results of measuring products of lipid peroxidation and antioxidant enzyme activity in corticonuclear lens blocks with age-related cataract, we can say that the lens structure changes induced by lipid peroxidation may with other risk factors present, affect the beginning or the development of cataract. The changes in redox system are particularly pronounced in cortical nuclear cataract, but are reflected in all parts of the lens, regardless of the initial localization of the lens blur. The lowest level of oxidative stress was detected in posterior subcapsular cataract, so it is possible that it has less importance in the

development of PS cataract. The development of cataract can probably be prevented/slowed down by preventing the accumulation of products of lipid peroxidation in the lens and maintaining adequate level of GSH and function of GSH-dependent antioxidant enzymes.

## **Author details**

Bojana Kisic and Dijana Miric *Institute of Biochemistry, Serbia* 

Lepsa Zoric *Clinic for Eye Diseases, Serbia* 

Aleksandra Ilic *Institute of Preventive Medicine, Faculty of Medicine, Kosovska Mitrovica, Serbia* 

## **11. References**


[11] Kannan R, Stolz A, Ji Q, Prasad PD, Ganapathy V (2001) Vitamin C Transport in Human Lens Epithelial Cells: Evidence for the Presence of SVCT2. Exp Eye Res. 73(2):159-165.

478 Lipid Peroxidation

**Author details** 

Lepsa Zoric

Aleksandra Ilic

**11. References** 

Bojana Kisic and Dijana Miric *Institute of Biochemistry, Serbia* 

*Clinic for Eye Diseases, Serbia* 

Fundam Clin Pharmacol. 26(1):86-117.

cataract. Curr Eye Res. 12 (2):163–179.

Arch of Ophthalmol.118(3):385–392.

Curr Eye Res. 25(1): 9-16.

31(10): 845-850.

development of PS cataract. The development of cataract can probably be prevented/slowed down by preventing the accumulation of products of lipid peroxidation in the lens and maintaining adequate level of GSH and function of GSH-dependent antioxidant enzymes.

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**Chapter 22** 

## **Lipid Peroxidation in Hepatic Fibrosis**

Ichiro Shimizu, Noriko Shimamoto, Katsumi Saiki, Mai Furujo and Keiko Osawa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46180

## **1. Introduction**

482 Lipid Peroxidation

Curr Eye Res. 12(5): 433-437.

[62] Srivastava SK, Singhal SS, Awasthi S, Pikula S, Ansari NH, Awasthi YC (1996) A glutathione S-transferases isozyme (bGST 5.8) involved in the metabolism of 4-hydroxy-2-trans-nonenal is localized in bovine lens epithelium. Exp Eye Res. 63(3):329-337. [63] Huang QL, Lou MF, Straatsma BR, Horwitz J (1993) Distribution and activity of glutathione-S-transferse in normal human lenses and in cataractous human epithelia.

[64] Yan H, Harding JJ, Xing K, Lou MF (2007) Revival of Glutathione in Human Cataractous and Clear Lens Extracts by Thioredoxin and Thioredoxin Reductase, in

Conjunction with α-Crystallin or Thioltransferase. Curr Eye Res. 32(5): 455-463. [65] Linetsky M, Chemoganskiy VG, Hu F, Ortwerth BJ (2003) Effect of UVA light on the activity of several aged human lens enzymes. Invest Ophthalmol Vis Sci. 44(1):264-74. [66] Ozmen B, Ozmen D, Erkin E, Habif S, Bayindir O (2002) Lens speroxide dismutase and

catalase activities in diabetic cataract. Clin Biochem. 35(1):69-72.

Hepatic fibrosis is a complex dynamic process which is mediated by death of hepatocytes and activation of hepatic stellate cells (HSCs). Lipid peroxidation including the generation of reactive oxygen species (ROS), transforming growth factor-β, and tumor necrosis factor-α can be implicated as a cause of hepatic fibrosis.

Damage of any etiology, such as infection with hepatitis C virus (HCV) or hepatitis B virus (HBV), heavy alcohol intake, and iron overload, to hepatocytes can produce oxygen-derived free radicals and other ROS derived from lipid peroxidative processes. Persistent production of ROS constitutes a general feature of a sustained inflammatory response and liver injury, once antioxidant mechanisms have been depleted. The major source of ROS production in hepatocytes is NADH and NADPH oxidases localized in mitochondria (Figure 1). NADH and NADPH oxidases leak ROS as part of its operation. Kupffer cells (hepatic resident macrophages), infiltrating inflammatory cells such as macrophages and neutrophils, and HSCs also produce ROS in the injured liver.

## **2. Oxidative stress in liver injury**

ROS include the free radicals superoxide (O2- ) and hydroxyl radical (HO- ) and non-radicals such as hydrogen peroxide (H2O2). A number of reactive nitrogen species including nitric oxide (NO) and peroxynitrite (ONOO- ) are also ROS. Superoxide production is mediated mainly by NADH oxidase. Hydrogen peroxide is more stable and membrane permeable in comparison to other ROS. Thus, hydrogen peroxide plays an important role in the intracellular signaling under physiological conditions. With respect to pathological actions, ROS participate in the development of liver disease. In this regard, hydrogen peroxide is converted into the hydroxyl radical, a harmful and highly reactive ROS, in the presence of transition metals such as iron (Figure 1). The hydroxyl radical is able to induce not only lipid peroxidation in the structure of membrane phospholipids, which results in irreversible

© 2012 Shimizu 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.

modifications of cell membrane structure and function (membrane injury), but DNA cleavage (DNA injury) as well. Such a chain of events due to increased ROS production exceeding cellular antioxidant defense systems are called oxidative stress, inducing cell death.

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) (Figure 1), end products of lipid peroxidation, are discharged from destroyed hepatocytes into the space of Disse (Figure 2). Cells are well equipped to neutralize the effects of ROS by virtue of a series of the antioxidant protective systems, including superoxide dismutase (SOD), glutathione peroxidase, glutathione (GSH), and thioredoxin. Upon oxidation, GSH forms glutathione disulfide (GSSG).

**Figure 1.** Oxidative stress and hepatocyte damage (Shimizu et al., 2012). A primary source of reactive oxygen species (ROS) production is mitochondrial NADPH/NADH oxidase. Hydrogen peroxide (H2O2) is converted to a highly reactive ROS, the hydroxyl radical, in the presence of transition metals such as iron (+Fe) and copper. The hydroxyl radical induces DNA cleavage and lipid peroxidation in the structure of membrane phospholipids, leading to cell death and discharge of products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE) into the space of Disse. Cells have comprehensive antioxidant protective systems, including SOD, glutathione peroxidase and glutathione (GSH). Upon oxidation, GSH forms glutathione disulfide (GSSG).

A single liver injury eventually results in an almost complete resolution, but the persistence of the original insult causes a prolonged activation of tissue repair mechanisms, thereby leading to hepatic fibrosis rather than to effective tissue repair. Hepatic fibrosis, or the excessive collagen deposition in the liver (see next section), is associated with oxidative stimuli and cell death. Cell death is a consequence of severe liver damage that occurs in many patients with chronic liver disease, regardless of the etiology such as HCV/HBV infection, heavy alcohol intake, and iron overload.

#### **3. What is hepatic fibrosis?**

At the cellular levels, origin of hepatic fibrosis is initiated by the damage of hepatocytes, followed by the accumulation of neutrophils and macrophages including Kupffer cells on the sites of injury and inflammation in the liver. When hepatocytes are continuously damaged, leading to cell death, production of extracellular matrix proteins such as collagens predominates over hepatocellular regeneration. Overproduced collagens are deposited in injured areas instead of destroyed hepatocytes. In other words, hepatic fibrosis is fibrous scarring of the liver in which excessive collagens build up along with the duration and extent of persistence of liver injury. Hepatic fibrosis itself causes no symptoms but can lead to the end-stage cirrhosis. In cirrhosis the failure to properly replace destroyed hepatocytes and the excessive collagen deposition to distort blood flow through the liver (portal hypertension) result in severe liver dysfunction. Cirrhosis is an important host-related risk factor for the development of hepatocellular carcinoma (HCC) in chronic hepatitis C and B, as well as a major factor predicting a poor response to interferon-based antiviral therapy in chronic hepatitis C. Staging of chronic liver disease by assessment of hepatic fibrosis always is a major function of prognostic interpretation of individual data including liver biopsy. Of the commonly used staging systems, the METAVIR fibrosis score has been widely used (Huwart et al., 2008). The stages are determined by both the quantity and location of the fibrosis. With this score, F0 represents no fibrosis; F1 (mild fibrosis), portal fibrosis without septa; F2 (moderate fibrosis), portal fibrous and few septa; F3 (severe fibrosis), numerous septa without cirrhosis; and F4, cirrhosis (Figure 3), the tissue is eventually composed of nodules surrounded completely by fibrosis.

**Figure 2.** Schema of the sinusoidal wall of the liver (Shimizu et al., 2012). Schematic representation of hepatic stellate cells (HSCs) was based on the studies by Wake (Wake, 1999). Kupffer cells (hepatic resident macrophages) rest on fenestrated endothelial cells. HSCs are located in the space of Disse in close contact with endothelial cells and hepatocytes, functioning as the primary retinoid storage area. Collagen fibrils course through the space of Disse between endothelial cells and the cords of hepatocytes.

## **4. Activation of HSCs**

484 Lipid Peroxidation

disulfide (GSSG).

modifications of cell membrane structure and function (membrane injury), but DNA cleavage (DNA injury) as well. Such a chain of events due to increased ROS production exceeding

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) (Figure 1), end products of lipid peroxidation, are discharged from destroyed hepatocytes into the space of Disse (Figure 2). Cells are well equipped to neutralize the effects of ROS by virtue of a series of the antioxidant protective systems, including superoxide dismutase (SOD), glutathione peroxidase, glutathione (GSH), and thioredoxin. Upon oxidation, GSH forms glutathione

**Figure 1.** Oxidative stress and hepatocyte damage (Shimizu et al., 2012). A primary source of reactive oxygen species (ROS) production is mitochondrial NADPH/NADH oxidase. Hydrogen peroxide (H2O2) is converted to a highly reactive ROS, the hydroxyl radical, in the presence of transition metals such as iron (+Fe) and copper. The hydroxyl radical induces DNA cleavage and lipid peroxidation in the structure of membrane phospholipids, leading to cell death and discharge of products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE) into the space of Disse. Cells have comprehensive antioxidant protective systems, including SOD, glutathione peroxidase and

A single liver injury eventually results in an almost complete resolution, but the persistence of the original insult causes a prolonged activation of tissue repair mechanisms, thereby leading to hepatic fibrosis rather than to effective tissue repair. Hepatic fibrosis, or the excessive collagen deposition in the liver (see next section), is associated with oxidative stimuli and cell death. Cell death is a consequence of severe liver damage that occurs in many patients with chronic liver disease, regardless of the etiology such as HCV/HBV

At the cellular levels, origin of hepatic fibrosis is initiated by the damage of hepatocytes, followed by the accumulation of neutrophils and macrophages including Kupffer cells on the sites of injury and inflammation in the liver. When hepatocytes are continuously damaged, leading to cell death, production of extracellular matrix proteins such as collagens

glutathione (GSH). Upon oxidation, GSH forms glutathione disulfide (GSSG).

infection, heavy alcohol intake, and iron overload.

**3. What is hepatic fibrosis?** 

cellular antioxidant defense systems are called oxidative stress, inducing cell death.

Normal liver has a connective tissue matrix which includes collagen type IV (non-fibrillary), glycoproteins such as fibronectin and laminin, and proteoglycans such as heparan sulphate. These comprise the low density basement membrane in the space of Disse. Following liver injury there is a 3- to 8-fold increase in the extracellular matrix which is of a high density interstitial type, containing fibril-forming collagens (types 1 and III) as well as fibronectin, hyaluronic acid and proteoglycans. Collagen types 1 and III are major components of the extracellular matrix, which is principally produced by cells known as HSCs. HSCs are located in the space of Disse in close contact with hepatocytes and sinusoidal endothelial cells (Figure 2). Their three-dimensional structure consists of the cell body and several long and branching cytoplasmic processes (Wake, 1999). In the resting liver, HSCs have intracellular droplets containing retinoids. Retinoids refer to a group of chemical compound associated with vitamin A. HSCs contain approximately 50-80% of the whole body stores of retinoids (Blomhoff et al., 1990). In contrast, in the injured liver, HSCs are regarded as the primary target cells for inflammatory and oxidative stimuli, and they are proliferated, enlarged and transformed into myofibroblast-like cells. These HSCs are referred to as activated cells and are responsible for the overproduction of collagens during hepatic fibrosis to cirrhosis. This activation is accompanied by a loss of cellular retinoids, and the synthesis of α-smooth muscle actin (α-SMA), and large quantities of the major components of the extracellular matrix including collagen types I, III, and IV, fibronectin, laminin and proteoglycans. α-SMA is produced by activated HSCs (myofibroblast-like cells) but not by resting (quiescent) HSCs, thereby a marker of HSC activation. Moreover, activated HSCs produce ROS and transforming growth factor-β (TGF-β) (Figure 4). TGF-β is a major fibrogenic cytokine, regulating the production, degradation and accumulation of the extracellular matrix in hepatic fibrosis. TGF-β expression correlates with the extent of hepatic fibrosis (Castilla et al., 1991). This cytokine induces its own expression in activated HSCs, thereby creating a self-perpetuating cycle of events, referred to as an autocrine loop. TGF-β is also released in a paracrine manner from Kupffer cells, endothelial cells, and infiltrating inflammatory cells following liver injury. Similarly, ROS are produced by activated HSCs in response to ROS released from adjacent cells such as destroyed hepatocytes and activated Kupffer cells.

**Figure 3.** Stages of hepatic fibrosis in chronic hepatitis according to the five stages (0-4) of the METAVIR scoring system (1994). With this score, F0 represents no fibrosis; F1 (mild fibrosis), fibrous expansion of portal areas without septa; F2 (moderate fibrosis), fibrous septa extend to form bridges between adjacent vascular structures, both portal to portal and portal to central, occasional bridges; F3 (severe fibrosis), numerous bridges or septa without cirrhosis; and F4 (cirrhosis), the tissue is eventually composed of nodules surrounded completely by fibrosis.

hepatocytes and activated Kupffer cells.

composed of nodules surrounded completely by fibrosis.

hyaluronic acid and proteoglycans. Collagen types 1 and III are major components of the extracellular matrix, which is principally produced by cells known as HSCs. HSCs are located in the space of Disse in close contact with hepatocytes and sinusoidal endothelial cells (Figure 2). Their three-dimensional structure consists of the cell body and several long and branching cytoplasmic processes (Wake, 1999). In the resting liver, HSCs have intracellular droplets containing retinoids. Retinoids refer to a group of chemical compound associated with vitamin A. HSCs contain approximately 50-80% of the whole body stores of retinoids (Blomhoff et al., 1990). In contrast, in the injured liver, HSCs are regarded as the primary target cells for inflammatory and oxidative stimuli, and they are proliferated, enlarged and transformed into myofibroblast-like cells. These HSCs are referred to as activated cells and are responsible for the overproduction of collagens during hepatic fibrosis to cirrhosis. This activation is accompanied by a loss of cellular retinoids, and the synthesis of α-smooth muscle actin (α-SMA), and large quantities of the major components of the extracellular matrix including collagen types I, III, and IV, fibronectin, laminin and proteoglycans. α-SMA is produced by activated HSCs (myofibroblast-like cells) but not by resting (quiescent) HSCs, thereby a marker of HSC activation. Moreover, activated HSCs produce ROS and transforming growth factor-β (TGF-β) (Figure 4). TGF-β is a major fibrogenic cytokine, regulating the production, degradation and accumulation of the extracellular matrix in hepatic fibrosis. TGF-β expression correlates with the extent of hepatic fibrosis (Castilla et al., 1991). This cytokine induces its own expression in activated HSCs, thereby creating a self-perpetuating cycle of events, referred to as an autocrine loop. TGF-β is also released in a paracrine manner from Kupffer cells, endothelial cells, and infiltrating inflammatory cells following liver injury. Similarly, ROS are produced by activated HSCs in response to ROS released from adjacent cells such as destroyed

**Figure 3.** Stages of hepatic fibrosis in chronic hepatitis according to the five stages (0-4) of the METAVIR scoring system (1994). With this score, F0 represents no fibrosis; F1 (mild fibrosis), fibrous expansion of portal areas without septa; F2 (moderate fibrosis), fibrous septa extend to form bridges between adjacent vascular structures, both portal to portal and portal to central, occasional bridges; F3 (severe fibrosis), numerous bridges or septa without cirrhosis; and F4 (cirrhosis), the tissue is eventually

**Figure 4.** During liver injury, HSCs are proliferated, enlarged and transformed into myofibroblast-like cells (Shimizu, 2001). These activated HSCs produce large quantities of collagens, α-smooth muscle actin (α-SMA), ROS, and transforming growth factor-β (TGF-β), and lose cellular retinoids.

HSCs are activated mainly by ROS, products of lipid peroxidation (MDA and HNE) (Lee et al., 1995; Parola et al., 1993), and TGF-β, which are released from destroyed hepatocytes, activated Kupffer cells and infiltrating macrophages and neutrophils in the injured liver (Figure 5). In addition to ROS, exogenous TGF-β increases the production of ROS, particularly hydrogen peroxide, by HSCs, whereas the addition of hydrogen peroxide induces ROS and TGF-β production and secretion by HSCs (De Bleser et al., 1999). This socalled autocrine loop of ROS by HSCs is regarded as mechanism corresponding to the autocrine loop of TGF-β which HSCs produce in response to this cytokine with an increased collagen expression in the injured liver (Itagaki et al., 2005).

**Figure 5.** Activation of HSCs. HSCs are activated by such factors as ROS, lipid peroxidation products (MDA and HNE), and TGF-β released when adjacent cells including hepatocyte, Kupffer cells, and endothelial cells are injured. ROS and TGF-β are also produced by HSCs in response to exogenous ROS and TGF-β in an autocrine manner.

Other important factors for HSC activation are platelet-derived growth factor (PDGF) released from platelets, and endothelin-1 from endothelial cells. PDGF is the most potent mitogen. HSCs congregate in the area of injury, through proliferation and migration from elsewhere, in response to the release of PDGF and monocyte chemotactic peptide-1 (MCP-1). MCP-1 is produced by activated Kupffer cells and infiltrating macrophages and neutrophils. The number of activated HSCs also increases after liver injury (Enzan et al., 1994).

At the molecular levels, HSCs express the genes which encode for enzymes such as matrix metalloproteinase (MMP)-1 (interstitial collagenase) (Casini et al., 1994), which digests native fibrillar collagen types I and III, and MMP-2 (Milani et al., 1994), which digests denatured collagen types I and III and native collagen type IV, as well as tissue inhibitors of MMPs (TIMP)-1 and TIMP-2 (Iredale et al., 1992). Imbalance between matrix synthesis and degradation plays a major role in hepatic fibrosis (Shimizu, 2001). Matrix degradation depends upon the balance between MMPs, TIMPs and converting enzymes (MT1-MMP and stromelysin) (Li and Friedman, 1999). Collagen types I and III constitute the main framework of the so-called "fibrillar matrix". The space of Disse is a virtual space constituted by an extracellular matrix network composed of collagen type IV and noncollagenous components such as laminin. The large majority of collagen types III and IV, and laminin are synthesized by HSCs and endothelial cells, whereas all cell types synthesize small amounts of collagen type I. During hepatic fibrosis, however, HSCs become the major extracellular matrix producing cell type, with a predominant production of collagen type I (Maher and McGuire, 1990). In the resting liver, a balance between matrix synthesis and degradation exists, whereas, in the injured liver, the balance is disrupted. The net result of the changes during hepatocyte damage is increased degradation of the normal basement membrane collagen, and reduced degradation of interstitial-type collagens. The latter can be explained by increased TIMP-1 and TIMP-2 expressions relative to MMP-1. The degradative portion of the remodeling process is coordinated by MMPs and TIMPs.

#### **5. Oxidative stress and intracellular pathway**

Origin of hepatic fibrosis is initiated by the damage of hepatocytes, resulting in the recruitment of inflammatory cells and platelets, and activation of kupffer cells, with subsequent release of cytokines and growth factors. HSCs are the primary target cells for these inflammatory and oxidative stimuli, because during hepatic fibrosis, HSCs undergo an activation process to a myofibroblast-like cell, which represents the major matrix-producing cell. In the injured liver, hydrogen peroxide seems to act as a second messenger to regulate signaling events including mitogen activated protein kinase (MAPK) activation. The MAPK family includes three major subgroups, extracellular signal regulated kinase (ERK), p38 MAPK (p38), and c-Jun N-terminal kinase/stress activated protein kinase (JNK). MAPK participates in the intracellular signaling to: (1) induce the gene expression of redox sensitive transcription factors, such as activator protein-1 (AP-1) and nuclear factor κB (NFκB) (Pinkus et al., 1996), (2) stimulate apoptosis (Clement and Pervaiz, 1999), and (3) modulate cell proliferation (Lundberg et al., 2000). ERK and JNK lie upstream of AP-1. JNK and p38 activation are more important in stress responses such as inflammation, which can also activate NF-κB. AP-1 and NF-κB induce the expression of multiple genes involved in inflammation and oxidative stress response, cell death and fibrosis, including proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 and interleukin-6 and growth factors such as PDGF and TGF-β. TGF-β is a major fibrogenic cytokine, acting as a paracrine and autocrine (from HSCs) mediator as already noted. TGF-β

triggers and activates the proliferation, enlargement and transformation of HSCs, but it exerts its inhibitory effect on hepatocyte proliferation (Nakamura et al., 1985).

Since many cytokines exert growth factor like activity, in addition to their specific proinflammatory effects, the distinction between cytokines and growth factors is somewhat artificial. No growth factor or cytokine acts independently. The injured liver, predominantly Kupffer cells and infiltrating macrophages and neutrophils, produces TNF-α, interleukin-1 and interleukin-6. These proinflammatory cytokines may also also inhibit hepatic regeneration. In particular, TNF-α plays a dichotomous role in the liver, where it not only induces hepatocyte proliferation and liver regeneration but also acts as a mediator of cell death (Schwabe and Brenner, 2006). During TNF-α-induced apoptosis in hepatocytes, hydrogen peroxide is an important mediator of cell death (Bohler et al., 2000).

In liver injury of hepatitis virus infection, transgenic mice expressing HBsAg exhibit the generation of oxidative stress and DNA damage, leading to the progression of hepatic fibrosis and carcinogenesis (Hagen et al., 1994; Nakamoto et al., 2004). In addition, HBV X protein changes the mitochondrial transmembrane potential and increases ROS production in the liver (Waris et al., 2001). Moreover, structural and non-structural (NS) proteins of HCV are involved in the production of ROS in an infected liver. HCV core protein is associated with increased ROS, decreased intracellular and/or mitochondrial glutathione content, and increased levels of lipid peroxidation products (Moriya et al., 2001). Glutathione is an antioxidant. NS3 protein of HCV activates NADPH oxidase in Kupffer cells to increase production of ROS, which can exert oxidative stress on nearby cells (Thoren et al., 2004).

## **6. Conclusion**

488 Lipid Peroxidation

coordinated by MMPs and TIMPs.

**5. Oxidative stress and intracellular pathway** 

At the molecular levels, HSCs express the genes which encode for enzymes such as matrix metalloproteinase (MMP)-1 (interstitial collagenase) (Casini et al., 1994), which digests native fibrillar collagen types I and III, and MMP-2 (Milani et al., 1994), which digests denatured collagen types I and III and native collagen type IV, as well as tissue inhibitors of MMPs (TIMP)-1 and TIMP-2 (Iredale et al., 1992). Imbalance between matrix synthesis and degradation plays a major role in hepatic fibrosis (Shimizu, 2001). Matrix degradation depends upon the balance between MMPs, TIMPs and converting enzymes (MT1-MMP and stromelysin) (Li and Friedman, 1999). Collagen types I and III constitute the main framework of the so-called "fibrillar matrix". The space of Disse is a virtual space constituted by an extracellular matrix network composed of collagen type IV and noncollagenous components such as laminin. The large majority of collagen types III and IV, and laminin are synthesized by HSCs and endothelial cells, whereas all cell types synthesize small amounts of collagen type I. During hepatic fibrosis, however, HSCs become the major extracellular matrix producing cell type, with a predominant production of collagen type I (Maher and McGuire, 1990). In the resting liver, a balance between matrix synthesis and degradation exists, whereas, in the injured liver, the balance is disrupted. The net result of the changes during hepatocyte damage is increased degradation of the normal basement membrane collagen, and reduced degradation of interstitial-type collagens. The latter can be explained by increased TIMP-1 and TIMP-2 expressions relative to MMP-1. The degradative portion of the remodeling process is

Origin of hepatic fibrosis is initiated by the damage of hepatocytes, resulting in the recruitment of inflammatory cells and platelets, and activation of kupffer cells, with subsequent release of cytokines and growth factors. HSCs are the primary target cells for these inflammatory and oxidative stimuli, because during hepatic fibrosis, HSCs undergo an activation process to a myofibroblast-like cell, which represents the major matrix-producing cell. In the injured liver, hydrogen peroxide seems to act as a second messenger to regulate signaling events including mitogen activated protein kinase (MAPK) activation. The MAPK family includes three major subgroups, extracellular signal regulated kinase (ERK), p38 MAPK (p38), and c-Jun N-terminal kinase/stress activated protein kinase (JNK). MAPK participates in the intracellular signaling to: (1) induce the gene expression of redox sensitive transcription factors, such as activator protein-1 (AP-1) and nuclear factor κB (NFκB) (Pinkus et al., 1996), (2) stimulate apoptosis (Clement and Pervaiz, 1999), and (3) modulate cell proliferation (Lundberg et al., 2000). ERK and JNK lie upstream of AP-1. JNK and p38 activation are more important in stress responses such as inflammation, which can also activate NF-κB. AP-1 and NF-κB induce the expression of multiple genes involved in inflammation and oxidative stress response, cell death and fibrosis, including proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 and interleukin-6 and growth factors such as PDGF and TGF-β. TGF-β is a major fibrogenic cytokine, acting as a paracrine and autocrine (from HSCs) mediator as already noted. TGF-β

Hepatic fibrosis is a complex dynamic process which is mediated by death of hepatocytes and activation of HSCs. Lipid peroxidation including the generation of ROS, TGF-β, and TNF-α can be implicated as a cause of hepatic fibrosis. HSCs are regarded as the primary target cells for inflammatory stimuli, and produce extracellular matrix components. HSCs are activated by such factors as ROS, lipid peroxidation products (MDA and HNE), and TGF-β released when adjacent cells including hepatocyte, Kupffer cells, and endothelial cells are injured. ROS and TGF-β are also produced by HSCs in response to exogenous ROS and TGF-β in an autocrine manner. During TNF-α-induced death in hepatocytes, ROS is an important mediator of cell death. The most common cause of hepatic fibrosis is currently chronic HCV/HBV infection.

Understanding the basic mechanisms underlying the ROS-mediated fibrogenesis provides valuable information on the search for effective antifibrogenic therapies.

## **Author details**

Ichiro Shimizu\* , Noriko Shimamoto, Katsumi Saiki, Mai Furujo and Keiko Osawa *Showa Clinic, Shin Yokohama, Kohoku-ku, Yokohama, Kanagawa, Japan* 

<sup>\*</sup> Corresponding Author

#### **7. References**


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