Contributors

Andrew D. Dick Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K.

Erich F. Elstner TU-Mu€nchen, Institute of Phytopathology, Freising-

Weihenstephan, Germany

Matthias Elstner Department of Neurology, Ludwig-Maximilian University,

Munich, Germany

Sharon Gordon Human Resources Development and Training, University Office, King’s College, Aberdeen, U.K.

Derick Han Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Regine Heller Department of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich-Schiller-University of Jena, Jena, Germany

Susanne Hippeli TU-Mu€nchen, Institute of Phytopathology, Freising-

Weihenstephan, Germany

Lars-Oliver Klotz Department of Molecular Aging Research, Institut fu€r Umweltmedizinische Forschung (IUF) at Heinrich-Heine-University, Du€sseldorf, Germany

Despina Kokkinou Sektion fu€r Experimentelle Vitreoretinale Chirurgie, Universit€ats-Augenklinik Tu€bingen, Tu€bingen, Germany

J€urgen Kopitz Zentrum fu€r Pathologie, Abt. Angewandte Tumorbiologie, Klinikum der Ruprecht-Karls-Universit€at, Im Neuenheimer Heidelberg, Germany

Santiago Lamas Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain

Janet Liversidge Department of Ophthalmology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, U.K.

Angel Messeguer Department of Biological Organic Chemistry, Centre d’Investigacio i Desenvolupament (CID), CSIC Jordi Girona Salgado, Barcelona, Spain

Maria Miranda Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain

Maria Muriach Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain

Lindsay B. Nicholson Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K.

Cecilia Gonza´lez de Ordu~na Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain

Neville N. Osborne Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, U.K.

Narsing A. Rao Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Morag J. Robertson Department of Ophthalmology, University of Aberdeen, Aberdeen, U.K.

Francisco J. Romero Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain

Tadeusz Sarna Department of Biophysics, Jagiellonian University Krakow,

Krakow, Poland

Harald Schempp TU-Mu€nchen, Institute of Phytopathology, Freising-

Weihenstephan, Germany

Ulrich Schraermeyer Sektion fu€r Experimentelle Vitreoretinale Chirurgie, Universit€ats-Augenklinik Tu€bingen, Tu€bingen, Germany

Tobias Schwarz Sektion fu€r Experimentelle Vitreoretinale Chirurgie, Universit€ats-Augenklinik, Tu€bingen, Tu€bingen, Germany

Grzegorz Szewczyk Department of Biophysics, Jagiellonian University

Krakow, Krakow, Poland

Guey-Shuang Wu Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Li-Peng Yap Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, U.S.A.

Andrzej Zadlo Department of Biophysics, Jagiellonian University Krakow,

Krakow, Poland

oxidants and antioxidants.1,2 This concept of oxidative stress entails a global view of, for example, thiol/disulfide balance –a major determinant of the cell redox state– and fails to recognize discrete redox pathways. Based on this, Jones3 provided a new definition of oxidative stress as a disruption of redox signaling and control, in essence, a mechanistic concept. This is important, for redox regulation of cell signaling occurs in discreet cellular regions that respond differently to oxidative and/or nitrosative stress situations. More recently, Sies and Jones introduced a new definition of oxidative stress in the Encyclopedia of Stress as an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage.4

THE UNIVALENT REDUCTION OF OXYGEN AND

OXIDATION OF NITROGEN

Univalent reduction of oxygen (O2) to superoxide anion (O2 ) is accomplished by various mechanisms. However, the two most significant sources in vivo are the mitochondria and inflammatory cells. Mitochondria are recognized as the major cellular sources of O2 , largely originating from the autoxidation of ubisemiquinone — a mobile carrier that (a) transfers electrons from complex I and II to complex III of the mitochondrial respiratory chain and from

(b) rotenone-sensitive complex I. Another major source of O2 , during inflammatory conditions is the activity of NADPH oxidase, a multi-subunit flavoheme enzyme. The four steps encompassed in the univalent reduction of oxygen yields free radicals and oxidants as shown in Fig. 1: superoxide anion

Figure 1 (See color insert.) Univalent reduction of oxygen and univalent oxidation of nitric oxide.

(O2 ), hydrogen peroxide (H2O2), hydroxyl radical (HO ), and water (H2O), the latter being generated by the four-electron reduction of O2 at the cytochrome c oxidase (complex IV) of the respiratory chain.

The generation of reactive nitrogen species –formation of nitric oxide

( NO)—requires the five electron oxidation of the guanidine group of L-arginine by the nitric oxide synthases (L-arginine þ NADPH þ O2 ? L-citrulline þ NADPþ

þNO). Subsequent one-electron oxidations yield, among others, nitrite (NO2 ),

nitrogen dioxide (NO2 ), and nitrate (NO3 ).

Hence, univalent reduction of oxygen and univalent oxidation of NO yield a variety of oxidants and free radicals that are involved in several aspects of cell function ranging from redox regulation of cell signaling to irreversible damage of cellular constituents.

Of interest, the reaction of O2 and NO yields peroxynitrite (ONOO ), an oxidant with a reduction potential of about þ1.0 volt and is involved in oxidation and nitration reactions. This nonenzymic reaction proceeds at diffusioncontrolled rates (O2 þ NO ? ONOO ; k ¼ 1.9 1010 M 1s 1), slightly faster

than the dismutation of O2 by the enzyme superoxide dismutase (O2 þ O2 þ 2Hþ ? H2O2 þ O2; k ¼ 2.3 109 M 1s 1).

PROTEIN POST-TRANSLATIONAL MODIFICATIONS BY REACTIVE OXYGEN AND NITROGEN SPECIES

H2O2, NO, and ONOO are distinctly involved in different steps of redox cell signaling through specific protein post-translational modifications. H2O2, essential for cell signaling,5 is produced by mammalian cells to mediate several physiological responses such as cell proliferation, differentiation, and migration6 through reductive- oxidative-based mechanisms.5 The signaling properties of H2O2 are exerted in the cytosol, where this oxidant increases protein phosphorylation largely upon inhibition of protein phosphatases;5 it is important to recognize that these H2O2-driven signaling pathways are in unique equilibrium with the activities of peroxiredoxins.5

NO exerts its effects on cell signaling via (a) guanylyl cyclase and cyclic GMP-dependent pathways and (b) cyclic GMP-independent pathways, the latter including post-translational modifications of proteins. Protein S-nitrosylation, a post-translational modification of thiol residues to form S-nitrosothiols, is a major

mechanism of redox signaling by which NO alters cellular function through the modification of protein thiol residues.7,8 NO-mediated S-nitrosylation of proteins appears to be a reversible process and has been identified in a limited subset of proteins in in vitro and in vivo studies.8 Hogg7 lists four major mechanisms of S-nitrosylation that potentially occur in biological systems. As mentioned above, S-nitrosation appears to be a reversible process: the reversible transfer of the nitroso group from an S-nitrosothiol to a thiol (transnitrosation: RSNO þ R0S $ RS þ R0SNO).7 S-Nitrosylation has been compared with phosphorylation as a cellular signaling mechanism.9,10 An interesting concept is that S-nitrosylation is likely to promote S-glutathionylation, that is, the incorporation of glutathione into proteins via mixed disulfide bonds. S-glutathionylation is an important protein

Figure 3 Specific removal of reactive oxygen species.

SPECIFIC REMOVAL OF REACTIVE OXYGEN SPECIES

The intermediates in the univalent reduction of oxygen sequence described above are of free radical or oxidant nature. Mitochondria are endowed with specific antioxidant systems aimed at removing O2 and H2O2 (Fig. 3). The former is specifically reduced to H2O2 by Mn-superoxide dismutase, present in the mitochondrial matrix at a concentration of 0.3 10 5 M16.

Mitochondria also contain a Cu,Zn-superoxide dismutase in the intermembrane space,17 the activation of which seems to require an oxidative modification of its critical thiol groups.18 The presence of Mn-superoxide dismutase in the mitochondrial matrix allowed an estimation of a steady-state concentration of O2 of about 10 10 M18, slightly higher than that in the cytosolic compartment (10 11 M). H2O2 is specifically removed by glutathione peroxidase, with an assumed concentration in the mitochondrial matrix of 1.17 10 6 M: the steady-state level of H2O2 in the matrix is estimated at 0.5 10 8 M16.

MITOCHONDRIAL FEATURES AND CELL FUNCTION

As mentioned above, mitochondria are energy-transducing organelles (the powerhouses of the cell) that generate metabolic energy for cell function and

maintenance; mitochondria are major cellular sources of O2 and H2O2, and also of NO by a mitochondrial nitric oxide synthase.16,17 It appears that mito-

chondrial nitric oxide synthase is a voltage-dependent enzyme, responsible forNO diffusion to cytosol and modulated by the mitochondrial metabolic states.19,20 Another function of mtNOS, at least in brain mitochondria or synaptosome mitochondria, is – in coordination with Mn-superoxide dismutase – to maintain brain redox status and participate in the normal physiology of brain development.21

Hence, mitochondria generate O2 , H2O2, and NO. Formation of O2

during mitochondrial electron transfer along with that of NO by mitochondrial NOS sets the ground for the formation of ONOO , which seems to specifically

Figure 6 Hydrogen peroxide, superoxide anion, and nitric oxide in cell signaling and gene expression.

mitochondria represents the initial step in the executioner phase of mitochondrion driven apoptosis. When released after membrane permeability transition, cytochrome c interacts with Apaf-1 to form the apoptosome and then can recruit and activate pro-caspase-9 in an ATP dependent process. Caspase-9 in turn activates caspase-3 and -7. These effector caspases are then responsible for the biochemical and morphological changes characteristic to apoptosis. It has been recently demonstrated that caspase-2 which is activated by genotoxic stress is directly involved in cytochrome c release. This is important as it represents an important link between DNA damage and mitochondrial apoptotic pathway that is directly engaged by caspase-2.38

CONCLUSION

Mitochondria are the powerhouses of the cell as they do generate energy in the form of ATP to support cellular metabolic processes. During respiration, a fraction of oxygen is reduced univalently to O2 with subsequent dismutation to H2O2; mitochondria are recognized as major cellular sources of these species along with NO by virtue of a mitochondrial nitric oxide synthase, probably attached to the inner mitochondria membrane and in close proximity to complex IV, cytochrome c oxidase. Mitochondrion-generated free radicals are involved in

the redox regulation of cell signaling, for they act as second messengers: H2O2

and NO can easily cross membranes and regulate cytosolic processes. Because of these and other properties, mitochondria became the harbinger of cell death upon the release of factors—most notably cytochrome c—that activate cytosolic apoptotic cascades.

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REACTIVE OXYGEN SPECIES

Reactive oxygen species (ROS) are biologically important O2 derivatives which possess higher reactivity than molecular oxygen.3,15 They include free radicals or one-electron oxidants such as superoxide anion ( O2 ), hydroxyl radical ( OH) or

nitric oxide ( NO) and nonradical two-electron oxidants, for example hydrogen peroxide (H2O2), hypochlorite/hypochlorous acid ( OCl/HOCl) and peroxynitrite (ONOO ). The sources of ROS in mammalian tissues are manifold and involve enzymatic and nonenzymatic intracellular pathways as well as the extracellular milieu. O2 may derive from aerobic respiration in the mitochondria16 or from enzymatic sources including phagocytic and vascular NAD (P)H oxidases,17–19 xanthine oxidase20 and uncoupled endothelial nitric oxide synthase (eNOS), i.e. eNOS deficient in its substrate arginine or its cofactor tetrahydrobiopterin.21,22 H2O2 is generated from dismutation of O2 , ONOO derives from the reaction of O2 with NO and HOCl is produced from Cl and

H2O2 by the phagocyte-derived myeloperoxidase. The most reactive radical,

OH, is produced by high energy irradiation or via the superoxide-driven Fenton reaction using traces of catalytic metal ions such as iron or copper. This radical is not counteracted by specific defence strategies and is probably the major representative of ROS-mediated cell damage.23

Traditionally, ROS were considered as potentially injurious by-products of normal oxidative metabolism or as tools through which phagocytes accomplish antimicrobial activity. Current evidence suggests, however, that ROS participate

in cell signalling pathways leading to changes in gene transcription and cellular functions.15,24,25 Intracellular production of ROS is elicited in response to a host

of stimuli including growth factors, cytokines, vasoactive substances and shear stress. Under physiological conditions ROS are produced in a controlled manner and contribute to the regulation of growth and tissue repair. Dependent on the magnitude of dose, the kinetics and duration of exposure and the type of cells ROS can also lead to transient or permanent growth arrest and finally to apoptotic or necrotic cell death. These responses are coordinated by a large number of signalling pathways including mitogen-activated protein kinases, phosphoinosi- tide-3-kinase/Akt, phospholipase C-g1, Janus protein tyrosine kinases, p53, the transcription factors NFkB, AP-1 and HIF-1 as well as heat shock proteins.

Molecular targets for ROS involve protein thiol groups, methionine residues, iron sulphur clusters and metals.15,24,25

The generation of ROS is usually in balance with antioxidant defence. In pathological settings an increase of ROS production or a reduction of antioxidant reserves may lead to an imbalance between oxidants and antioxidants in favour of the oxidants. This situation is defined as oxidative stress26 and may potentially cause oxidative damage if adaptive responses are not sufficient to compensate. Oxidative stress may involve uncontrolled activation of specific ROS signalling pathways and/or direct oxidation of DNA, lipids, and proteins. These processes have been suggested to contribute to a variety of diseases, including atherosclerosis, neurodegenerative disorders,

Interestingly, some antioxidant enzymes underlie adaptive responses initiated by electrophiles or oxidative stress and mediated by the antioxidant-

response element (ARE)-nuclear factor-erythroid-2-related factor (Nrf2) signalling pathway.39,40 Nrf2 is sequestered in the cytoplasm by the Kelch-like

ECH-associated protein 1 (Keap 1). Oxidation of cysteine thiol groups of Keap 1 results in a conformational change that renders Keap 1 unable to bind to Nrf2 which then translocates to the nucleus, activates ARE and leads to transcriptional regulation of target genes.41 This pathway has been described for drug metabolizing enzymes but also for heme oxgenase, thioredoxin, gastrointestinal glutathione peroxidase, the subunits of g-glutamate cysteine ligase, manganese superoxide dismutase and catalase.39,40 It has been speculated that this adaptive response may contribute to the beneficial health effects of exercise42 since this is known to cause low levels of lipid peroxidation and formation of electrophilic lipids.43

Dismutation and diversion of ROS by antioxidant enzymes is efficiently supported by small molecules (scavengers) which interact with primary ROS, such as O2 or with secondary reactive species such as lipid radicals. These low molecular weight antioxidants include water-soluble compounds (glutathione, ascorbic acid, uric acid, bilirubin, lipoic acid, polyphenols) and lipid-soluble compounds (vitamin E, ubiquinol, carotenoids, polyphenols) and are either of endogenous or dietary origin.30 Interestingly, when these compounds react with free radicals they are transformed into radicals themselves. Antioxidant radicals comprise lower reactivity but still need to be reduced or recycled to avoid damage. This implies an interaction with other antioxidants in a so-called antioxidant network.44 For instance, a-tocopherol, the most active form of vitamin E in human tissues, produces the a-tocopheroxyl radical which can be reduced back by ascorbate, ubiquinols or bilirubin.45–47 Furthermore, glutathione is maintained in a reduced state via reduction of the glutathione thiyl radical by

ascorbate.48 Conversely, glutathione or lipoic acid are able to recycle dehydroascorbic acid back to ascorbic acid.49,50 Through these interactions anti-

oxidants may also spare each other and elicit synergistic effects.

ANTIOXIDANT VITAMINS

Dietary antioxidants seem to play a major role in the antioxidant defence system and to be critical for optimal cellular and systemic health. The best investigated

natural compounds are ascorbic acid (vitamin C) and a-tocopherol, others are carotenoids and polyphenols such as flavonoids.51–55 Ascorbic acid is one of

the most important water-soluble antioxidants with almost ideal properties.56,57 Due to its low reduction potential it is able to react with virtually all physiologically relevant reactive oxygen and nitrogen species including nonradical

oxidants such as HOCl and ONOO although the protection against these oxidants may not be complete.29,58–60 Furthermore, the ascorbyl radical formed

from ascorbate in one-electron oxidations has a low reactivity and ascorbate can

be readily regenerated from its oxidized forms by spontaneous chemical or by

enzymatic reactions.58 a-Tocopherol, on the other hand, is the major natural lipid-soluble antioxidant.61,62 It belongs to the vitamin E group which consists of

two classes of compounds, i.e. tocopherols and tocotrienols, with four structurally related isoforms in each class (a, b, g and d-forms). Due to a selective sorting in the liver by a specific a-tocopherol transfer protein a-tocopherol predominates in human blood and tissues.63 It is located in membranes and lipoproteins and its major antioxidant action is thought to be scavenging of lipid

peroxyl radicals.64 In contrast, a-tocopherol does not seem to protect against HOCl or ONOO .65–67

Interestingly, a-tocopherol has been shown to modulate cellular signalling and transcriptional regulation independent of its antioxidative properties, partially via inhibition of protein kinase C.68,69 Proteins downregulated by a-tocopherol are the scavenger receptors SRA and CD36, interleukins 1b and 4, as well as the adhesion molecules VCAM-1 and CD11b/CD18. Ascorbic acid has also activities in addition to oxidant scavenging which are, however, related to its electron donor abilities. Ascorbate acts as a cofactor for several enzymes

engaged in hydroxylation reactions, for example enzymes involved in the biosynthesis of collagen or carnitin70,71 and it has also been shown to affect the

expression of extracellular matrix proteins and to upregulate antioxidant enzymes.72

Dietary antioxidants have garnered considerable interest during the last years which is mainly based on the observation that diets rich in antioxidants

seem to be associated with a lower risk to develop oxidative stress related diseases.10–12,73,74 In addition, antioxidant vitamins were generally thought to

have few adverse side effects and to be safe in therapeutical trials. In this context, effects of natural antioxidants, especially vitamin C and vitamin E, on cardiovascular diseases were intensively investigated.

OXIDATIVE STRESS AND ATHEROSCLEROSIS

Cardiovascular disease and the underlying pathology of atherosclerosis have been shown to represent a state of increased oxidative stress in the vascular wall.3,4 Moreover, oxidative stress is thought to be a unifying mechanism for many risk factors of atherosclerosis, such as smoking, obesity, diabetes and hypertension.27,75–79 One of the hypotheses of atherogenesis, the oxidative modification hypothesis, proposes that oxidation of LDL converts the native lipoprotein into a particle with proatherogenic activities which is responsible for the formation and development of atherosclerotic lesions.3,80–82 LDL modification may be mediated by radicals which lead to lipid oxidation or by twoelectron oxidants such as HOCl or ONOO which primarily modify apoprotein B.3 Oxidized LDL is susceptible to macrophage uptake via scavenger receptors leading to foam cell formation, and stimulates processes known to be involved in lesion formation. These include monocyte chemotaxis (via direct effects or via

induction of monocyte chemotactic protein-1), endothelial adhesion molecule expression, recruitment of inflammatory cells, smooth muscle cell proliferation, and apoptosis of several cell types.3,83 In support of the oxidation theory oxidized lipids and proteins have been found in atherosclerotic lesions, and oxidized LDL as well as autoantibodies to oxidized LDL have been detected in the plasma of patients.3,84 Furthermore, increased levels of urinary and circulating F2 isoprostanes (chemically stable free-radical-catalysed products of arachidonic acid) have been found in patients with atherosclerosis or with risk factors for atherosclerosis indicating oxidative stress in vivo.75

Oxidative processes, either directly or via oxidation of LDL, may also

contribute to endothelial dysfunction, i.e. to a loss of NO bioavailability, which is thought to be an early step in atherogenesis.85–88 NO is produced in endo-

thelial cells and is known to be a central regulator of vascular homeostasis with vasorelaxing and antiatherogenic properties including inhibition of platelet aggregation, monocyte and leukocyte adhesion to the endothelium and smooth muscle cell proliferation.89 ROS may affect NO bioavailability in several ways.O2 , for example, has been shown to scavenge and inactivate NO directly whereas ONOO is thought to inhibit NO biosynthesis via oxidation of the Znthiolate cluster of eNOS or via oxidation of tetrahydrobiopterin.21,22 Tetrahydrobiopterin is a reducing cofactor of eNOS.90,91 It is responsible for coupling oxygen reduction to arginine oxidation and prevents O2 formation by eNOS. Upon reaction with oxidants tetrahydrobiopterin forms a neutral trihydrobiopterin radical which further disproportionates to the quinonoid 6,7-[8H]- dihydrobiopterin. Both compounds can either be recycled or irreversibly oxidized. The latter leads to tetrahydrobiopterin depletion and, as a consequence,

not only less NO is formed but eNOS is uncoupled, i.e. converted into a O2 generating enzyme.21,22,91,92

ANTIOXIDANT VITAMINS AND ATHEROSCLEROSIS

Experimental and Animal Studies

Based on the observation that oxidative stress is associated with atherogenesis and that plasma levels of ascorbic acid and a-tocopherol are inversely correlated to the mortality from coronary heart disease, antioxidant vitamins were suggested to protect from cardiovascular disease. This assumption has been encouraged by the majority of in vitro and cell culture studies demonstrating inhibitory effects of ascorbic acid or a-tocopherol on key events of atherogenesis.93,94 It has been clearly demonstrated that a-tocopherol acts as a chain-breaking antioxidant by scavenging highly reactive lipid peroxyl and alkoxyl radicals and stopping the propagation of lipid peroxidation and thus LDL oxidation.95 Ascorbate supports a-tocopherol by regenerating the a-tocopheroxyl radical

and by scavenging oxidants that may initiate lipid peroxidation in the aqueous milieu.45,96 Ascorbate and a-tocopherol have both been shown to decrease

adhesion molecule expression on endothelial cells and to reduce leukocyte

adhesion either by antioxidant mechanisms or, in the case of a-tocopherol, by inhibition of protein kinase C.97–101 Finally, both, ascorbic acid and a-tocopherol

have been reported to improve endothelial dysfunction via several mechanisms.88,102

Ascorbate in high concentrations ( 10 mM) protects NO from inactivation by scavenging O2 .103 Furthermore, ascorbate is able to reduce the trihydrobiopterin radical as well as the quinonoid 6,7-[8H]-dihydrobiopterin and

thus, to regenerate oxidized tetrahydrobiopterin and to prevent eNOS uncoupling.104–107 In contrast, the beneficial effect of a-tocopherol on endo-

thelial dysfunction is mainly attributed to its ability to counteract adverse effects of oxidized LDL on NO formation.108 In addition, a-tocopherol has been shown to promote activation of eNOS via effects on eNOS phosphorylation.109 The vasoprotective effects of natural antioxidants described in vitro

have been confirmed in animal models of atherogenesis and atherosclerosis regression although results have not been uniformly positive.94,110,111

Epidemiological and Clinical Studies

The effect of ascorbic acid and a-tocopherol on cardiovascular disease in humans has been investigated in various epidemiological and clinical studies. As a first approach, several large prospective observational studies were performed which compared the development of cardiovascular disease as measured by defined endpoints (for example myocardial infarction or mortality from coronary heart disease) in subjects with a different estimated intake of antioxidant vita-

mins (dietary and supplemental). Many but not all of these studies suggested an inverse association of vitamin E or C intake and cardiovascular disease.9,93,111–113

Subsequently, large-scale randomized clinical trials were carried out to prove a

causal relationship between the increased intake of natural antioxidants and the reduced risk for cardiovascular disease.13,14,111,114–117 Most of these trials were

conducted on patients with established atherosclerosis or with high risk for cardiovascular disease. In most cases vitamin E alone or in combination with other antioxidants was investigated while vitamin C alone was not tested. Vitamin E was used at different doses and pharmaceutical formulations (natural or synthetic a-tocopherol preparations) and for different periods. The majority of controlled interventional trials was not able to demonstrate beneficial effects of antioxidant supplementation despite the fact that observational studies strongly suggested this benefit. Protective effects of vitamin E supplements on the progression of

cardiovascular disease have only been documented in subgroups or in some smaller studies.118–120 On the other hand, there is some evidence of potentially

adverse effects of vitamin E supplements including an increase of overall mortality.121

In addition to interventional trials with endpoint measurements, a large number of studies has examined the effect of natural antioxidants on several clinical markers of cardiovascular disease including flow-mediated vasodilation,

Requirement of a Basal Oxidant Tone

Recent data on regulatory and signalling effects of ROS suggest that a basal or tonal concentration of ROS is essential for maintaining cellular functions.15,24,25

This is especially important at the level of the mitochondria where electron transport to molecular oxygen is coupled to production of ATP (oxidative phosphorylation). ROS are formed in different compartments and concentration gradients are highly important. Some degree of localized oxidation seems to play a role in protein folding in the endoplasmic reticulum to permit disulfide formation,127 in growth factor signalling,128 in activation of several gene transcription factors or in mediating adaptive responses and upregulation of protective systems that render the cells more resistant to a subsequent insult (antioxidant enzymes, ferritin, heat shock proteins).39,129 Low quantities of ROS are known to stimulate cell proliferation. H2O2, for instance, has been shown to inactivate protein tyrosine phosphatases via oxidation of a critical cysteine resi-

due which may be essential for tyrosine phosphorylation of growth factor receptors.130,131 A similar mechanism may play a role in insulin signalling and

thus in the regulation of insulin sensitivity.132 ROS are also involved in the regulation of protein degradation and apoptosis.133 Oxidation of cysteine sulfhydryl groups of thioredoxin, for example, leads to the release of the apoptosis signal-regulating kinase from its complex with thioredoxin and subsequently to stimulation of stress-activated protein kinases and apoptosis.134

The requirement of a basal ROS tone for cell signalling may help to explain why many antioxidant-based therapies failed. Abolishment of ROS by vigorous use of antioxidants may not always be beneficial. Antioxidants may inhibit cell proliferation, prevent adaptation to oxidative stress or even accelerate oxidative damage. Furthermore, inhibition of ROS-induced apoptosis may lead to increased necrotic cell death with release of cell contents such as transition metals that could amplify oxidative processes. Thus, a more subtle approach of antioxidant therapy appears to be required which should consider the type and location of ROS generation. A recent meta-analysis of clinical studies demonstrating that high-dosage vitamin E supplementation (>400 IU/day) increased all-cause mortality seems to support the concept that global suppression of

oxidation may eliminate some beneficial processes121 although this analysis was not uniformly accepted.135,136,137 In contrast, anti-inflammatory activities of

a-tocopherol (inhibition of pro-inflammatory cytokine release, reduction of monocyte adhesion to endothelial cells, decrease of C-reactive protein levels) which are increasingly thought to be implicated in its vasoprotective effects have been shown to require high doses ( 600–800 IU/day).69 Clearly, more data on dose-effect relationships of antioxidants are needed.

Characterization of Oxidative Events as a Cause of Disease

Antioxidants tested in intervention studies so far were selected according to their ability to inhibit free radical-induced LDL oxidation and may not have

sufficiently targeted other relevant oxidants. There is growing evidence that two-

electron oxidants, in particular H2O2, HOCl or ONOO are involved in atherosclerosis.3 HOCl and ONOO have already been shown to modify LDL67,138

and other proteins, and ONOO seems to be a major oxidant reacting with tetrahydrobiopterin.139 Furthermore, H2O2 as a component of cell signalling is increasingly thought to mediate augmented proliferative processes which have

been implicated in lesion formation.140 Importantly, lipid-soluble antioxidants such as vitamin E are not able to affect nonradical oxidants65–67 and as a con-

sequence, processes due to uncontrolled generation of H2O2, HOCl or ONOO may not have been altered in intervention studies with vitamin E supplementation. In the future, a more complete understanding of the oxidative events promoting atherosclerosis will allow a more specific selection of appropriate antioxidants for therapeutic strategies. Additionally, it will be necessary to characterize the stage of disease which is mainly promoted by oxidative stress. It is possible, for example, that ROS generation is more relevant to the initiation of lesion formation and that antioxidant protection is needed at an early age. Accordingly, the beneficial effects seen in dietary studies may reflect a life-long support with dietary antioxidants. In contrast, most antioxidant interventional trials were performed in patients with advanced atherosclerosis.13,14

One must also consider the possibility that oxidative events represent rather a consequence than a cause of cardiovascular disease. Indeed, athero-

sclerotic lesion formation can also be dissociated from the occurrence of lipid peroxidation.141,142 It may be possible that oxidative events are a result of

vascular inflammation and not strictly required for the progression of atherosclerosis (oxidative response to inflammation hypothesis of atherosclerosis3). In this case, antioxidant treatment may not have a major impact on the development of disease since it would not affect the link between inflammation and atherosclerosis. Moreover, antioxidants may even attenuate the healing response to inflammation which may be promoted by ROS at low levels, and, as a consequence, worsen lesion formation. Thus, a clear distinction and characterization of oxidative events as cause of atherosclerosis is requisite for antioxidant strategies.

Antioxidant Supplementation Versus Diet

The antioxidant intake recorded in dietary studies and shown to be inversely associated with cardiovascular disease may be a marker for some other dietary or lifestyle factor that is providing cardiovascular benefit. It is plausible to suppose that persons who select a diet rich in antioxidants have also other health habits that may lower their risk for cardiovascular disease. Furthermore, dietary compounds may act as Nrf-2-Keap1-ARE activators and improve the defence system provided by antioxidant enzymes.143,144 These components include sulforaphane, a metabolite of the glucosinolate glucoraphanin which is found in crucifers (particularly in broccoli),145 diallyl sulphide from allium vegetables146 as well as

the flavonoids kaempferol, epigallocatechin-3-gallate and curcumin.147–149 It may also be that the combination of antioxidants (vitamin C and E, carotenoids, flavonoids and other polyphenols) provided with diet is more efficient than the supplementation of a single compound. In plants, for example, knockout of a single antioxidant may cause a serious injury to the cells despite the presence of many other antioxidants.150 It is known that antioxidants need to recharge each other after they have reacted with free radicals and have been converted into radicals themselves. The function of a-tocopherol as a chain-breaking, for example, requires the presence of a coantioxidant to reduce the a-tocopheroxyl radical which otherwise would mediate further formation of lipid radicals.151 Thus, supplementation with a combination of antioxidants may reduce the potential for a paradoxical increase in oxidant generation. According to their structural features antioxidants may also protect different intracellular compartments, i.e. membrane or cytoplasm, and react with different radical and/or nonradical oxidants. As a consequence, they may exhibit distinct protective effects. Data from our group, for instance, demonstrate that ascorbate and a-tocopherol affect endothelial NO synthesis independently from each other via different mechanisms, i.e. ascorbic acid but not a-tocopherol regenerates oxidized tetrahydrobiopterin and a-tocopherol but not ascorbate promotes eNOS phosphorylation at serine 1177. Additionally, we were able to show that interactions between the two compounds take place, i.e. ascorbate is able to

potentiate the effect of a-tocopherol, most probably by recycling oxidized a-tocopherol.106,109

Selection of Patients

A detailed knowledge about the nature of oxidative processes which trigger atherosclerotic lesion formation is not only important for the selection of specific antioxidants but will also allow selection and monitoring of a population that may respond to antioxidant treatment. It is possible that patients included in previous intervention trials were inappropriate to test the therapeutic efficacy of antioxidants since they were not selected according to a biochemical evidence for elevated ROS formation. It will be important to identify novel biomarkers

which indicate increased HOCl or ONOO generation in addition to the known markers of lipid peroxidation.152–154 Indeed, it has been shown that F2 iso-

prostanes were only linked to some (smoking, obesity, diabetes) but not all risk factors of atherosclerosis.77 Oxidative events other than LDL oxidation, for

example a loss of NO bioavailability which can be measured as endothelialdependent vasodilation may be used to identify patients at risk and to monitor antioxidant action.155–158 Furthermore, evaluation of the endogenous antioxidant

defence system and of oxidant enzymes will be important to characterize the risk to develop oxidative stress as a cause of disease.38,159 In this context, genetic

factors involved in oxidative processes and antioxidant defence will help to identify patients that may respond to an antioxidant treatment.87,160

Selection of Antioxidants

Future studies may better define targets for antioxidant therapy other than LDL oxidation. For example, restoring endothelial function has become an attractive

therapeutic approach86,156 and may be realized by preventing tetrahydrobiopterin oxidation and eNOS uncoupling.21,22,91,92 Furthermore, it may be important to

target specific ROS populations or compartments of ROS generation. Leakage from the mitochondrial electron transport chain, for example, is a significant source of O2 16 and antioxidants which unlike vitamin C and E preferentially accumulate in the mitochondria may be more effective in ameliorating oxidative stress-mediated disease.161,162 Mitochondrial targeting is based on biophysical properties of the mitochondria (high negative internal potential promoting accumulation of lipophilic cations) and on the unique mitochondrial localization of enzymes and transporters.163–165 The mitochondrially targeted compounds described so far have shown promising results in a range of in-vitro systems.162 Finally, the best antioxidants may be those that interfere with the production of ROS. Drugs that influence the expression and activity of NAD(P)H oxidases such as statins, angiotensin-converting enzyme inhibitors or ligands of peroxisome proliferator-activated receptor-gamma have already been shown to attenuate cardiovascular oxidative stress.166–169 The development of specific

inhibitors that interfere with the assembly of NAD(P)H oxidase components170 or compounds that target the myeloperoxidase pathway171–172 may represent

novel antioxidant strategies.

CONCLUSION

Although atherosclerosis represents a state of increased oxidative stress in the vasculature antioxidant strategies have not been proven to limit cardiovascular events based on atherosclerotic processes. It seems, however, to be premature to conclude that the oxidation hypothesis of disease causality has to be rejected and antioxidant modulation of disease is not effective (Table 2). Pharmacological intervention with antioxidants requires a better understanding of ROS signalling pathways and ROS localization as well as a clear definition of oxidants which are involved in disease aetiology. Antioxidants should target the dysregulation rather than interfere with physiological signalling of ROS. An important prerequisite for antioxidant strategies is the development of sensitive and specific biomarkers that can be used to assess the oxidative stress phenotype which underlies a certain vascular pathology and to monitor antioxidant action. Identification of patients at risk may include the characterization of genetic variants of oxidant and antioxidant enzymes. Future antioxidative acting drugs should target specific intracellular compartments of ROS production such as mitochondria or oxidant enzymes such as NAD(P)H oxidase. Furthermore, combinations of different antioxidants or of antioxidative and anti-inflammatory treatments may help in early intervention. In conclusion, the challenge of future

research will be to develop specific antioxidant approaches for specific oxidant phenotypes of patients that are likely to develop atherosclerosis or other oxidative stress-related diseases.

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as nitration, S-gluthathionylation or S-nitrosation. These modifications can have functional consequences.

From a chemical viewpoint these reactive species can be divided into two main groups: those that possess an unpaired electron, called free radicals, and those that are not free radicals but have oxidizing effects. Free radicals include the superoxide anion, the hydroxyl radical, nitric oxide and lipid radicals; non free radicals include hydrogen peroxide, peroxynitrite and hypochlorous acid.

Superoxide Anion

The superoxide anion is produced by the reduction of one electron from molecular oxygen, yielding a negatively charged free radical.

This oxygen species is very unstable and reacts with other species to produce other ROS. However, cells have a detoxifying system to control increased and deleterious levels of superoxide anion. The principal enzymes implicated in this detoxifying action are superoxide dismutases (SODs), which transform the superoxide anion into hydrogen peroxide and molecular oxygen.

The presence of SOD ensures that superoxide anion concentrations do not exceed the picomolar range. When the enzyme is absent the levels of O2 can reach the nanomolar range, favoring the formation of other ROS. It can also react with NO giving rise to the production of peroxynitrite.

This reaction is non enzymatic but occurs at a very fast rate which actually exceeds by 3-fold the capacity of SOD to reduce O2 . Peroxynitrite also reduces the availability of NO, with potential consequences for its physiological actions2; and ONOO is also involved in the formation of hydroxyl radicals by promoting the release of iron.

Hydroxyl Radical

This free radical appears to have a much greater potential for catalyzing reactions that could be involved in signaling processes than does the superoxide anion. It is formed via the Fenton reaction, which consists of the reaction of hydrogen peroxide with ferrous iron.3 It can react with thiols and lipids, generating vasoactive isoprostanes and lipid peroxidation products.4

Nitric Oxide

This labile radical can interact with ferrous heme groups, certain other metal sites, thiol groups, and free radical species. The most potent actions of NO occur above the nanomolar range, and cells can produce this concentration under pathological conditions associated with inflammatory processes, neurotoxicity and ischemia. When it interacts with the superoxide anion, nitrogen dioxide (NO2) may be formed in addition to ONOO . NO2 can be formed from nitrite,

which is a decomposition product of NO, or directly by the reaction of molecular oxygen with NO.

Lipid Radicals (LO AND LOO )

These are able to react with NO to produce LONO and LOONO5,6

Hydrogen Peroxide

This is a relatively stable species compared with the free radicals. It can be formed from the reaction of SOD with superoxide anion or by the action of certain oxidases through a two electron reduction of molecular oxygen. Because of its structural similarities it has comparable diffusion properties to water. Hence it may move freely into the cell and produce alterations such as activation of gluthathione redox cycles,7 oxidation of intracellular sulfhydryls,8 or DNA damage.9 The main enzymes which account for its metabolism are catalase, glutathione peroxidase and the cyclooxygenases Cox 1 and Cox 2.

Hypochlorous Acid

This species is less diffusible than hydrogen peroxide and thus interacts mainly with membrane components. It has toxic properties such as oxidative bleaching of heme groups and iron-sulfur centers,10 and chlorination of amines and unsaturated lipids.

Peroxynitrite

Because O2 and NO are both free radicals and contain unpaired electrons they undergo an extremely rapid reaction, leading to the formation of peroxynitrite, a much stronger oxidant than O2 . The most important effect of ONOO appears to be thiol modification, but it also causes the nitration of tyrosine residues on proteins. The formation of ONOO is associated with the inhibition of several antioxidant systems, such as catalase,11 GSH peroxidase,12 and mitochondrial SOD.13 At high concentrations ONOO promotes formation of NO donors via the modification of alcohols and sugars to nitrated species which release NO in the presence of thiols.14

HOW DO THEY FORM?

To better understand the effects of oxidant stress it is key to identify the sources of ROS and when they are produced. In the vascular context, in particular in endothelial cells, ROS can be derived from several systems such as mitochondrial respiration, enzymes of the arachidonic acid pathways, cytochrome

Nitric oxide synthases18 are hemeproteins that catalyze the oxidation of L- arginine to L-citrulline and nitric oxide. There exist three isoforms of the enzyme in mammals: a constitutive neuronal NOS19 (nNOS or NOS I); an endotoxinand cytokine-inducible NOS20 (iNOS or NOS II); and a constitutive endothelial NOS21 (eNOS or NOS III). NOS contain four redox active prosthetic groups – FAD, FMN, iron protoporphyrin IX (heme), and tetrahydrobiopterin BH4 – and catalyze the flavin mediated electron transport from the donor, NADPH, to the heme group.

In the absence of the cofactor BH4 or the substrate L-arginine, the enzyme can produce superoxide anion and hydrogen peroxide, a phenomenon known as NOS uncoupling. In this uncoupled state the electrons that normally flow from the reductase domain of one subunit to the oxygenase domain of the other are driven to the molecular oxygen rather than to L-arginine, giving rise to the formation of superoxide rather than NO.22

There are several mechanisms whereby NOS can became uncoupled. One of them is the inactivation of the cofactor BH4 by its oxidation with peroxynitrite. BH4 is essential for enzyme activity because it stabilizes the NOS dimer and facilitates its formation, but it also increases the affinity of NOS for L-arginine and affects the spin state of the heme iron, thereby playing an important role in oxygen activation.23 Peroxynitrite is capable of rapidly oxidizing the cofactor BH4, with superoxide formation as the inevitable result. Another mechanism of uncoupling is the absence of L-arginine or mutations in GTP cyclohydrolase I, the enzyme that catalyzes the first step in the biosynthesis of BH4.24

PROTEIN MODIFICATIONS PRODUCED BY ROS AND RNS

When ROS and RNS are produced, the cell needs to sense the changed environment and activate diverse pathways to respond to it. There are several mechanisms for this regulation, including protein-protein interactions, allosteric changes induced by the ligand binding and proteolytic processing. One of the best characterized is postranslational modifications of proteins (Fig. 2). For a protein modification to be physiologically relevant to the modulation of protein function, it must be specific, preferable reversible, and its formation must occur within a physiological concentration range and time frame, (Table 1).

S-Glutathionylation

This protein modification is a reversible covalent addition of glutathione (GSH) to a cysteine residue of a protein, through the formation of a mixed disulfide. The reduced form of the tripeptide GSH is one of the most important antioxidant molecules in mammalian cells and is present in cells at concentrations between 1 and 10 mM.25 GSH provides reducing equivalents for enzymes involved in the metabolism of ROS and RNS, and thus exerts its antioxidant actions by

Figure 2 Species implicated on postranslational modifications.

Table 1 Postranslational modifications related to ROS and RNS

scavenging NO and oxidants.26 The availability of GSH in oxidative situations is ensured by GSH recycling and biosynthetic pathways.27

Apart from providing a reducing environment, GSH plays a role in the regulation of protein function through the formation of mixed disulfides between the protein cysteine residues and GSH.28 This process is called S-glutathionylation or S-glutathiolation; and this modification is implicated in the protection of proteins against irreversible oxidation of critical cysteine residues. Because this modification is reversible, dethiolation of S-glutathionylated proteins occurs, and can take place either by a non-enzymatic reduction or by an enzymatic cleavage of the disulfide bond, involving the action of thioredoxins and glutaredoxins.29 Therefore this modification fulfils the criteria of physiological relevance and S-glutathionylation may confer specificity and regulatory potential to the posttranslational control of protein function.

Nitric Oxide can induce protein S-glutathionylation. This was first proposed in 1988 by J.W. Park.30 Then in 1997 it was demonstrated that GSNO could form a mixed disulfide with aldose reductase;31 and in the following year the role of NO as a mediator of this modification was highlighted by experiments in endothelial cells demonstrating that exogenous NO leads to S-glutathionylation of a number of proteins.32

NO may target the incorporation of GSH into some proteins in the following way. Exposure of cells to NO and other RNS leads to the formation of GSSG by the oxidation of GSH33 and its conversion to GSNO.34 This GSNO may be a source of GSSG through its reaction with superoxide35 or thiols,36 or by the breakdown of nitrosothiol.37 Therefore RNS causes S-glutathionylation indirectly, by forming GSSG.38

S-Nitros(yl)ation

This modification is one of the most extensively studied protein modifications induced by reactive species because it is implicated in all classes of cell signalling, ranging from the regulation of ion channels and G-protein coupled reactions to receptor stimulation and activation of nuclear regulatory proteins.39 S-nitrosylated proteins are formed when a cysteine thiol reacts with NO in the presence of an electron acceptor to form an S-NO bond. In fact the direct reaction of an NO radical with a thiol does not yield nitrosylation:40 previous reaction with molecular oxygen via the formation of higher nitrogen oxides is thought to be necessary.41 However, transnitrosylation can occur, involving the transfer of NO between a nitrosothiol and another thiol.42

S-nitrosylation is a very labile covalent modification under physiological conditions, which makes it difficult to study. The bond can be cleaved by reaction with transition metals, or by transnitrosation, but it is also very sensitive to ultraviolet light. Several enzymes have also been described that help in the

breakdown of nitrosothiols.43 Another problem in the study of S-nitrosylation is the low intracellular concentrations of the nitrosylated proteins, which makes it difficult to detect using current methodologies. The methods to date available for the detection of nitrosylation include electrospray ionization mass spectrometry (ESI-MS44 or ozone chemiluminescence, which can measure NO released from nitrosothiols when the S-NO is broken by photolytic cleavage.45 Nitrosylated proteins can also be identified by the biotin-switch method, which can be combined with immunoprecipitation.46 With the combined use of all these techniques new proteins have been identified as nitrosylated, such as Hsp90, b-actin and anexin II.47

S-nitrosylation reactions cause specific physiological or pathophysio-

logical activities by modifying protein function. S-nitrosylation can promote an increase in protein activity as in the case of p21ras or thioredoxin,48,49 but it can

also inhibit the activity of proteins such as caspases, methionine adenosyl transferase, or Hsp90.50,51

Tyrosine Nitration

Protein tyrosine nitration is a covalent protein modification resulting from the addition of a nitro group to one of the carbons of the aromatic ring of a tyrosine residue.52 It is mediated by reactive nitrogen species such as the peroxynitrite anion; and the presence of nitrotyrosine has been used as a marker of oxidative stress and pathology. The nitration of proteins has been proposed to play a role in diseases such as amyotrophic lateral sclerosis,53 Alzheimer’s disease,54 Parkinson’s disease,55 cancer,56 atherosclerosis,57 and myocardial contractile failure.58

Tyrosine nitration appears to be catalyzed primarily by metalloproteins. Enzymes such as myeloperoxidases or cytochrome P-450 catalyze the oxidation of nitrite to nitrogen dioxide, which is able to nitrate tyrosine residues.59 Other metalloproteins such as manganese superoxide dismutase can catalyze their own nitration from peroxynitrite.60 Other reactive species capable of nitrating tyrosines are the intermediates of the reaction between peroxynitrite with carbon dioxide and the acidification of nitrite to form nitrous acid.61

The level of protein nitration is low: under inflammatory conditions between one and five 3-nitrotyrosine residues per 10,000 tyrosine residues are detected59 This fraction of nitrated protein is very small in the context of total tissue protein and raises questions about its possible biological relevance. Given that the molecular species participating in nitration have short diffusion distances, nitration may be site-specific, resulting in localized foci of nitration in a particular cell or tissue compartment. This would clearly limit the number of proteins that are available as targets for nitration; and in addition to this, only a few specific tyrosines in any particular protein can be nitrated.

When a protein is nitrated its function can be altered in two ways: it can undergo a loss of function or a gain of function. To be biologically significant, a loss of function must affect a large fraction of a specific protein, but for gain of function only a small fraction needs to be nitrated to elicit a substantive biological signal.62 In addition to its direct effects on protein structure and function, tyrosine nitration can also have an significant impact on cell function by altering the availability of tyrosine residues for phosphorylation.63

CONCLUSION

All these posttranslational modifications are biological processes associated with nitric oxide and reactive oxygen biochemistry and biology. The physiological relevance of these processes has begun to emerge, but much more remains to be discovered and understood. The confinement of modifications to restricted subcellar locations will probably prove to be important for greater understanding of their biological relevance, as will their specificity and reversibility. In the future, in vivo experimental models will be required to demonstrate the involvement of these modifications in specific physiological and disease processes.

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Figure 1 Structure of glutathione (GSH) and involvement in peroxide (ROOH) reduction by glutathione peroxidases (GPx). Glutathione disulfide (GSSG) is formed during reduction of peroxides and is reduced back to GSH at the expense of NADPH by glutathione reductase (GR). Cellular sources of NADPH include the pentose phosphate pathway, as well as reactions catalyzed by malic enzyme (malate dehydrogenase, decarboxylating) or NADP+-dependent isocitrate dehydrogenase.

As a thiol (R-SH, or thiolate, R-S ), GSH is readily oxidized under physiological conditions, forming sulfenic acid (R-SOH, or sulfenate, R-SO ) or disulfides (R-S-S-R’). This oxidation may occur both enzymatically (Figure 1) or nonenzymatically by interaction with reactive oxygen species. Even higher glutathione oxidation states, sulfinic acid (R-SO2H) and sulfonic acid (R-SO3H), were observed, but they are usually not reduced under physiological conditions, although the reduction of sulfinates has recently been shown to be feasible in some cases.5 The balance between glutathione (GSH) and glutathione disulfide (GSSG) concentrations is believed to be a determinant in the cellular capability to cope with an oxidative stressful stimulus. In addition to serving as an electron donor in the cellular antioxidative defense, glutathione serves a regulatory role in affecting enzyme activities by glutathiolation, i.e., by the formation of mixed disulfides between GSH and a protein thiol.1,6

In order to analyse a possible role of glutathione in a cellular process of interest, it will have to be tested whether an elevation and/or lowering of cellular glutathione levels affects the investigated process. Thus, a brief introduction to experimental means of modulating cellular glutathione levels and to methods for the determination of glutathione concentrations will be given in this chapter.

EXPERIMENTAL MODULATION OF CELLULAR

GLUTATHIONE CONCENTRATIONS

In order to experimentally elevate cellular GSH levels, glutathione precursors or derivatives need to be applied because glutathione is not taken up by cells to a significant extent. N-acetyl cysteine, a cell permeant derivative of cysteine, may

Figure 2 Reaction of diazenedicarboxylic acid bis(N,N-dimethylamide), or diamide, with glutathione. The reactive agent is the glutathione thiolate, which reacts with diamide to form a sulfenyl hydrazine and, upon reaction with a second glutathione thiolate to release glutathione disulfide (GSSG), the corresponding hydrazine.

be used to feed cellular GSH synthesis by supplying cysteine which is coupled to glutamate by g-glutamylcysteine synthetase (see below), but due to feedbackinhibition of GSH biosynthesis this approach is not always successful. Membrane permeant glutathione esters that are hydrolysed to GSH intracellularly are frequently employed instead.7

Several compounds exist that deplete GSH, including (i) electrophilic compounds nonenzymatically reacting with thiols (or thiolates), such as diamide, (ii) compounds that are coupled to GSH enzymatically, such as diethyl maleate, or (iii) inhibitors of glutathione biosynthesis. Diamide, diazenedicarboxylic acid bis(N, N-dimethylamide), has been employed for decades to deplete cellular GSH following the reaction depicted in Figure 2.8 Although GSH was found to be more reactive towards diamide than other non-protein thiols, the reaction is not specific for GSH, and diamide will thus also deplete several other cellular thiols, including protein-bound cysteinyl residues, to form diamide-SR adducts and/or (mixed) disulfides.8 However, as GSH is the major non-protein thiol in mammalian cells, diamide will usually preferentially react with GSH.

The reaction of diamide with GSH is nonenzymatic. Employing the cellular machinery of specifically coupling GSH to electrophiles, the glutathione S-transferases (GSTs), would thus result in an enhanced specificity in terms of depleting GSH rather than other available thiols. Diethyl maleate (DEM) is an example of a compound that is recognized as a substrate by GSTs and by being coupled to GSH (see Figure 3) causes the depletion of cellular GSH.

Buthionine sulfoximine (BSO) was identified as a specific inhibitor of g-glutamylcysteine synthetase,9 the initial step in GSH biosynthesis. Application of BSO will cause a loss of cellular GSH by preventing its resynthesis when cellular stores are depleted by export or by normal cellular metabolism, e.g. by

Figure 3 Reaction of diethyl maleate with glutathione as catalyzed by glutathione S-transferases (GST).

Figure 4 Glutathione (GSH) is synthesised de novo in two steps from glutamate via g-glutamylcysteine in reactions catalysed by g-glutamylcysteine synthetase (GCS) and glutathione synthetase (GS). GCS is inhibited by the transition state analogue buthionine sulfoximine (BSO).

GST-dependent coupling of GSH or by peroxide reduction (Figure 1) with the production and subsequent export of GSSG. Of all possible enantiomers, L-buthionine (S)-sulfoximine was demonstrated to be the effective form (Figure 4).10

METHODS FOR THE DETERMINATION OF CELLULAR GLUTATHIONE CONCENTRATIONS

In order to experimentally assess cellular GSH levels, essentially the same types of reaction can be exploited that were discussed above as being applied for the more or less specific depletion of GSH. Thiol-reactive substances are used that, upon reaction with GSH, form a product that is detectable photometrically or fluorimetrically. A widely applied reagent is Ellman’s reagent (5,5’-dithiobis-2- nitrobenzoic acid, DTNB),11 a disulfide that reacts with thiols to form mixed disulfides and thionitrobenzoate (TNB), a dianion with an absorption maximum around 412 nm (see below and Figure 6):

DTNB þ RS ! TNB-SR ðTNB=RS-mixed disulfideÞ þ TNB TNB-SR þ R0S ! TNB þ R0SSR

Thiol concentrations can be estimated by either comparing absorptions with those of a standard curve established by reacting DTNB with different thiol concentrations or by calculating concentrations using the published TNB

absorption coefficient at 412 nm, which was recently reevaluated to be 14.15 mM 1cm 1 and 13.8 mM 1cm 1 at 258C and 378C, respectively (pH 7.4).12

A second group of widely employed thiol-reactive compounds is that of bimane (1,5-diazabicyclo[3.3.0]octadienedione) derivatives, most notably the bromobimanes.13 The nonfluorescent monobromobimane (mBBr), upon reaction with a thiol, forms a fluorescent adduct (Figure 5) the concentration of which can be estimated directly by fluorimetry using an appropriate standard.

As with DTNB, mBBr does not exclusively react with GSH, and various non-protein thiols as well as protein-bound cysteines can also be labeled.

Figure 7 1-Chloro-2,4-dinitrobenzene (CDNB) is coupled to glutathione in a nucleophilic substitution catalyzed by glutathione S-transferases (GST). The formation of the adduct can be followed photometrically at 340 nm.

DTNB and increase in absorbance at 412 nm due to formation of TNB. The slope of TNB formation directly correlates with glutathione concentration. As GSSG is continuously recycled and thus introduced into the assay, glutathione concentrations determined actually comprise GSH plus GSSG levels which can be analysed separately with this assay only after derivatization of GSH, e.g., with 2-vinylpyridine.14

Glutathione S-transferases (GSTs) were mentioned before as another group of enzymes specifically recognising GSH. Employing GST and a substrate, 1-chloro-2,4-dinitrobenzoic acid (CDNB), GSH concentrations are determined according to the reaction depicted in Figure 7.15 Different from the DTNB/GR assay, GSH is determined directly.

A comparison of the mentioned glutathione assays with the same array of different GSH concentrations revealed that assay sensitivities are in the following order:16 DTNB/GR & CDNB/GST > DTNB (nonenzymatic) > mBBr/ HPLC. In summary, the two enzymatic assays not only appear to be more specific but also more sensitive than the assays solely based on the direct interaction between reagent (DTNB or mBBr) and thiol.

AN EXAMPLE: MENADIONE AND CELLULAR GLUTATHIONE LEVELS

Menadione (2-methyl-1,4-naphthoquinone, vitamin K3) is a known redox cycler and alkylating agent17 that causes the production of reactive oxygen species (Figure 8) and the depletion of thiols (Figure 9) in cells exposed to the quinone.

Intracellularly, menadione is reduced to the corresponding semior hydroquinone by oneand two-electron reduction, respectively (Figure 8). The semiquinone is oxidized back to the quinone form by molecular oxygen (which is present in physiological systems in high micromolar concentrations) under concomitant generation of superoxide. Similarly, the hydroquinone may be oxidized by oxygen unless it is deactivated in phase II reactions and exported.

Superoxide will dismutate both spontaneously and catalysed by superoxide dismutases to form hydrogen peroxide, which in turn is reduced to water at the expense of GSH by glutathione peroxidases (see Figure 1). Hence, menadione

Figure 8 Redox cycling of menadione (2-methyl-1,4-naphthoquinone). Menadione is reduced intracellularly by one-electron reductases or in a two electron-reduction catalyzed by NAD(P)H:quinone oxidoreductase-1 (NQOR, DT-diaphorase). The resulting semiquinone and, to a lesser extent, also the corresponding hydroquinone, may be oxidized by molecular oxygen which is thereby reduced to superoxide.

affects the cellular balance between GSH and GSSG. Menadione also causes direct depletion of GSH by arylation, i.e. through a Michael-type addition of thiolates at C-3 (Figure 9).

To analyse the effect of menadione and a known glutathione depletor, DEM (see above), on cellular glutathione levels, rat liver epithelial cells were exposed to these agents as described in Figure 10. As expected from the mechanisms outlined above, both menadione and, more so, DEM deplete total glutathione. While total glutathione levels are lowered by approximately 30%, concentrations of GSSG are significantly enhanced and those of GSH strongly diminished in cells exposed to menadione. These data are in line with GSH being lost in at least two ways under the influence of menadione, i.e. by oxidation of GSH to GSSG and most probably by direct interaction with menadione (arylation).

Figure 9 Arylation of thiols by menadione, i.e., Michael addition of thiols/thiolates to menadione.

Figure 10 Glutathione levels in cells exposed to menadione or diethyl maleate (DEM). Rat liver epithelial cells were exposed to menadione (50 mM), DEM (1 mM) or DMSO ("–") as vehicle control for 15 min. Cells were washed and lysed in 10 mM HCl, protein was precipitated from the lysates with 5-sulfosalicylic acid, followed by analysis of glutathione in the protein-free fraction employing the DTNB/GR assay: total glutathione (GSH and GSSG) was analyzed from the acidic lysates, for identification of GSSG thiols were blocked by 2-vinylpyridine prior to the assay. GSH levels were calculated from total glutathione and GSSG concentrations. Data are given as means of 3 independent measurements ± SD (modified and recalculated from Abdelmohsen et al.18).

Different from menadione, the changes in total glutathione levels seen in cells treated with DEM are not due to changes in GSSG concentrations but to a loss of GSH (see Figures 3 and 10).

SUMMARY

Glutathione is an essential component in the cellular line of antioxidative defense. Changes in glutathione concentrations and in glutathione redox state are important parameters for the evaluation of potential susceptibility of cells to oxidative damage. Methods for the experimental analysis of GSH and GSSG concentrations as well as tools for the modulation of cellular glutathione levels were described.

ACKNOWLEDGMENT

Research in the author’s laboratory is funded by Deutsche Forschungsgemeinschaft, Bonn, Germany (SFB 728/B3, SFB 575/B4, GRK 1033), and the Forschungskommission der Medizinischen Fakulta¨t at Heinrich-Heine-University, Du¨sseldorf. Dedicated to my mother, Mrs. Eva-Marie Klotz, on the occasion of her 60th birthday.

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