I

i I

Oxidative Stress: From Basic Research to Clinical Application HELMUT SIES, M.D.,

DEJsseldo~Germany

The occurrence of reactive oxygen species, known as pro-oxidants, is an attribute of normal aerobic life. The steady-state formation of pro-oxidants is balanced by a similar rate of their consumption by antioxidants that are enzymatic and/or nonenzymatic. "Oxidative stress" results from imbalance in this prooxidant-antioxidant equilibrium in favor of the pro-oxidants. A number of diseases are associated with oxidative stress, being the basis of antioxidant therapy. Current evidence in clinical research does not show unequivocal distinction between causal or associative relationships of prooxidants to the disease process.

6 6~

xidative stress" is associated with a disturbanee in the pro-oxidant-antioxidant balance in favor of the pro-oxidant [1]. The occurrenee of reactive oxygen species, known as prooxidants, is an attribute of normal aerobic life. The existence and development of cells in an oxygencontaining environment would not be possible without the presence of defense systems that include powerful enzymes and nonenzymatie antioxidant components. Aerobic life is characterized by a steady formation of pro-oxidants balanced by a similar rate of their consumption by antioxidants. To maintain homeostasis, there is a requirement for the continuous regeneration of antioxidant capacity, and if this is not met, oxidative damage accumulates, resulting in pathophysiologieal events [2]. Many of the pro-oxidants are free radicals, and a study of these provide a new field of interest in biology and medicine [3], including a number of physiological and pathophysiologieal phenomena and proeesses as diverse as inflammation, ageing, carcinogenesis, drug action, drug toxicity, and defense against protozoa.

REACTIVE OXYGEN SPECIES

From the Institute f~Jr Physiologische Chemie I, Universitat Dfisseldorf, DOsseldon', Federal Republic of Germany. Requests for reprints should be addressed to Helmut Sies, M.D., Institut far Physiologische Chemie I, Universit~it DOsseldorf, Moorenstrasse 5, D-4000 DLisseldorf, Federal Republic of Germany.

Reactive oxygen species [4,5], many of which are oxygen free radicals and involved in oxidative damage, are shown in Table I. Some, such as singlet molecular oxygen (102) and hydrogen peroxide (H202), are nonradicals, and their half-lives are different (Table II). The hydroxyl radical (. OH) is the most reactive and short lived. The peroxyl radical (RO0-) is long lived, resulting in a biological diffusion pathlength, and those radicals that may be formed from polyunsaturated fatty acids (PUFAs) could potentially diffuse from their site of generation unless deactivated by antioxidants. The problems faced in the study of reactive oxygen species arise from their short lifetimes, and from their very low steady-state concentrations. Nevertheless, some quantification attempts have been made; for example, the steady-state concentration of H202 in the peroxisome has been determined spectroscopically by titrations of catalase compound I [6]. Packer and Glazer [7] have compiled current methods in the analysis of oxygen radicals.

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TABLE III

Reactive Oxygen Species of Interest in Oxidative Stress

Antioxidant Defense in Biological Systems

Compound

Remarks

System

02 -, superoxideanion

One-electronreductionstate,formedin many autoxidationreactions(forexample,flavoproteins, redoxcycling) H02., perhydroxyradical Protonatedform of 02 ', morelipidsoluble H202,hydrogenperoxide Two-electronreductionstate,formedfrom 0~. (H02') by dismutationor directlyfrom 02 HO. (OH.), hydroxylradical Three-electronreductionstate,formedby Fenton reaction,metal(iron)-catalyzedHaber-Weiss reaction;highlyreactive RO., alkoxyradical Oxygen-centered organic(forexample,lipid)radical RO0., peroxyradical Formallyformedfrom organic(for example,lipid) hydroperoxide,ROOH,by hydrogenabstraction ROOH Organichydroperoxide(forexample,lipid-, thymine-OOH) lag 02(also102) Singletmolecularoxygen,first excitedstate~ 22 kcal/molabovegroundstate (triplet)~02; red (dimol)or infrared(monomol)photoemission RO(alsoRO*) Excitedcarbonyl,blue-greenphotoemission(for example,formedvia dioxetaneas intermediate) Modified from [1].

TABLE II Estimate of the Half-lives of Oxygen Radicals and Related Species Radical HO. RO. RO0. L. § H202

Substrate*

Contce~atet

Half.Life(at3TC)

LH$ LH LH 02

1M 100 mM 1 mM 20 #M

H2 For example, cigarettetar free radical

Solvent

10-9 sec 10-6 sec 7 sec 10-s sec Stable;enzymatic reduction Spontaneousand enzymaticdismutation 10-° sec Days

02. 102 Q-. NO.

5.6 sec 0.1 sec (in heart)**

*Substrate chosenas representativeof typical reactivetarget moleculesfor the species,n the first column. % concentration of the substratethat approximatesthe sum of all reactive species in the :~icinity of the radical and is chosen to reflect the selectivity of the radical. Linoleate. §L. is the linolenyl radical.The reversibility of the L. +02 reactions has been neglected. An oxygen concentration is used that is typical of moderately oxygenatedtissue. **From [5] Adapted from [4].

ANTIOXlDANT DEFENSE Detoxication of reactive oxygen species is one of the prerequisites of aerobic life, and many defenses have evolved, providing an important antioxidant defense system (Table III) of prevention, interception, and repair consisting of nonenzymatic scavengers and quenchers, known as antioxidants, as well as enzymatic systems including superoxide dismutases and hydroxyperoxidases, such as glutathione peroxidase, catalase, and other hemoprotein peroxidases. There are many other important enzyme systems present, including regenerative reactions and the glutathione (GSH) backup system. GSSG reductase 3C-32S

Nonenzymatic e-Tocopherol(vitaminE) Ascorbate(vitaminC) Glutathione(GSH) Flavonoids Chemical

/~-Carotene Urate Plasmaproteins Enzymatic Superoxidedismutases GSHperoxidases Catalase Ancillaryenzymes NADPH-quinone oxidoreductase (DT-diaphoraee) Epoxidehydrelase Conjugationenzymes GSSGreductase NADPHsupply Transportsystems

Remarks Membranebound;receptors?Regenerationfrom chromanoxylradical? Watersoluble Plantantioxidants(rutin,quercetin,etc.) Foodadditives,for example,BHA(butylated hydroxyanisole),and BHT(butylatedhydroxytoluene); thiol compounds(GSHprecursors);enzymemimics (forexample,ebselen,CuDIPS) Singletoxygenquencher Singletoxygenquencher,radicalscavenger? Ceruloplasmin CuZnenyzme,Mn enzyme Selenoenzyme;non-Seenzyme:someGSHS-transferases, for example,isoenzymesB andAAcytosoland mitochondrialmatrix; Hemeenzyme;predominantlyin peroxisomalmatrix Two-electronreduction,dicoumarolsensitive

UDP-glucuronyltransferase Sulfonyltransferase GSHS-transferases Glucose-6-phosphatedehydrogenase,6-phosphogluconatedehydrogenase,isocitratedehydrogenase, malicenzyme,energy-linkedtranshydrogenase GSSGexport,conjugateexport

Modified from [1].

can become pivotal in antioxidant defense. A decrease in the steady-state level of reactive compounds capable of generating reactive oxygen species results in a decreased level of oxidative stress. Hence, the two-electron reduction of quinones by NADPH-quinone oxidoreductase (DT diaphorase) and the conjugation reactions of hydroquinone form part of the antioxidant defense. The export from cells of reactive species in a free or conjugated form also serves as a detoxification function, and the study of the transport of conjugates and of GSSG from cells is of interest [8]. The nonenzymatic systems include lipophilic and hydrophilic antioxidants. There are important interrelationships between these two systems, indicating the need for coupling to nonradical regenerative processes. The initial radical damage in a membrahe needs to be repaired to allow for sustained free radical scavenging activity. Tocopherols (vitamin E) act as biological antioxidants [9,10]. Vitamin E accounts for much of the lipid-soluble chainbreaking antioxidant capacity in the human blood plasma and erythrocyte membranes [11]. Hence, a major biological function of tocopherol as an antioxidant lies in its reactivity as a free radical quencher. The rate constants with peroxyl radicals are in the range 106-10 s M-is -1, depending on the experimen-

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tal conditions [12]. The reaction involves a peroxyl radical and the phenolic hydroxyl group of tocopherol to generate the organic hydroperoxide and the tocopherol radical [13]: RO0. +vit E-OH

> ROOH + v i t E - O .

and transposes the radical function from the reactive organic radical (e.g., of a polyunsaturated fatty acid) into a less reactive chromanoxyl radical. Tocopherol breaks the chain reaction of peroxidation arising from the fact that peroxyl radicals react with other lipids with rate constants of about 50 M-is -1, but with tocopherol 10 4 or 10 5 times faster. There is a consensus hypothesis regarding the reversibility of tocopheryl radical formation that the hydrogen donor in the reaction vit E - O . + AH

>

vit E-OH + A.

will be water soluble, i.e., the radical challenge is removed from the membrane into the aqueous compartment of the cell. A possibility is that vitamin C (ascorbate) interacts with vitamin E, as suggested by Tappel [9], and consistent with this idea is the observation [14-16] that tocopherol must be present in biological membranes for ascorbate to protect it from peroxidation. However, there remains some doubt as to whether this reaction occurs in vivo or even in the model systems [17]. As evidence against a direct ascorbate-tocopheryl reaction in vivo, it has been reported [18] that ascorbate deftciency has no effect on the rate of tocopherol turnover or loss in the guinea pig. Thiols may also react with tocopheryl radicals to regenerate tocopherol and, conversely, tocopherols can also repair thiyl radicals. In the cell, glutathione (GSH) is the major low-molecular weight thiol present. An enzymatic mechanism, "glutathione free radical reductase" has been proposed, evidence for which is indirect, however. Burk et al [19,20] observed that lipid peroxidation in purified rat liver microsomes was inhibited by glutathione but protection was eliminated by heating. The GSH-dependent "factor" has resisted further attempts at purification. Although there is no doubt that GSH has an overall sparing effect on tocopherol in rat liver microsomes, this mechanism in other tissues and other species is still uncertain [21]. Non-GSH thiols, such as dihydrolipoate, a lipid-soluble dithiol and powerful reductant, inhibit microsomal peroxidation [22,23] and spare tocopherol in a manner similar to glutathione, but are not lost after heating or trypsinization [23]. It is possible that GSH enzymatically inhibits

peroxidation in a way that requires but does not regenerate tocopherol, and GSH may act via the membrane-bound GSH peroxidase, which reduces phospholipid hydroperoxides [24]. In the absence of GSH, the hydroperoxides quickly accumulate by rapid and irreversible chain reactions. Tocopherol generates hydroperoxides without formation of further chain reactions, and prevents the peroxidase from being depleted [24]. Alternatively, the protection against autocatalytic peroxidation of lipids by tocopherol may be curtailed if tocopherol is used in reactions with cysteinyl or other radicals associated with proteins, which GSH may be able to intercept. Supporting this idea is the observation [25] that the oxidation of protein thiols parallels the loss of tocopherol in microsomes subjected to a wide range of peroxidation agents. Tocopherols, apart from being radical scavengers, react with singlet molecular oxygen [26,27]. Carotenoids, a class of antioxidants present in a lower concentration than the tocopherols, may provide protection to more hydrophobic compartments not accessible to tocopherols due to their good solubility properties. Krinsky [28] has reviewed the antioxidant functions of carotenoids and Truscott [29], their photophysics and photochemistry. Our own work [30] on carotenoids has shown that lycopene is the most efficient biological singlet oxygen quencher known.

SOME ASPECTS OF ENZYMATIC ANTIOXIDANT DEFENSE Superoxide dismutases and hydroperoxidases, such as glutathione peroxidase, catalase, and other hemoprotein peroxidases, are extensively studied antioxidant enzyme defenses. They are characterized by a high specific cellular content, by specific organ and subcellular localizations that often overlap in a complementary manner, and by a specific form of metal involvement in catalysis, including copper, zinc, manganese, iron (heine), and selenium. These antioxidant systems are widely distributed in nature, underlining their importance in preventing the damaging effects of reactive oxygen metabolites in biological systems. Their distribution is crucial in target organ toxicity. Decrease in Levels of Reactive Compounds A decrease in steady-state level of reactive compounds capable of generating reactive oxygen species results in a decreased oxidative stress; hence, the two-electron reduction of quinones by NADPHquinone oxidoreductase (DT diaphorase) and the conjugation reactions of hydroquinone are part of the antioxidant defense [31,32].

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t -Butyl Hydroperoxide

I

Enzymatic

/

(GSH Peroxidase; Se, non-Se)

Efflux from cells

GS-R

• Mercapturates

Alkylation (GSH S-Transferases) / P h o r o n ! Diethylmaleate

GSSG

GSSG

non-enzymatic

Biosynthesis (Inhibitionby Buthionine Sulfoximine)

\ Amino acids

(Diazene: Diamide) Figure 1. Some experimental ways of affecting intracellular glutathione levels by thiol oxidation and inhibition of GSH synthesis or alkylation of GSH. [Reproducedwith permission from [103]).

Export of ReactiveSpecies:Free or Conjugated

NEW GLUTATHIONE-DEPENDENTACTIVITIES

The export of reactive species in free or conjugated form also serves as a detoxication function, so that transport of conjugates and of GSSG from cells is of interest. The binding of conjugates of GSH to GSSG-binding sites may have metabolic significance. It has been shown in kinetic and x-ray crystallographic studies [33] that GSH conjugates bind to the GSSG-binding site in the active center of GSSG reductase, inhibiting enzymatic activity. An increase in GSSG levels causes metabolic perturbation, including an inhibition of protein synthesis [34]. The transport systems for GSSG and for GSH conjugates have been studied [35]. In liver, there is mutual competition for biliary export between these two types of GSH derivatives [36], indicating that the canalicular carrier system may accept both of these substrates for transport. There appears to be a GSSG activatable by ATPase in the hepatic plasma membrane [37]. Mutual competition for export of GSSG and GSH conjugates was also detected in the heart [38]. Using the creatine kinase reaction as an indicator of the metabolite system, it was found that GSSG transport across the cardiac plasma membrane was half-maximal at (ATP/ADP) free ratios of about 10 in the intact perfused rat heart preparation [39]. A prominent GSH transferase activity is isozyme 4-4, accepting 4-hydroxynonenal as a substrate [40], and, therefore, capable of removing this biologically active product of lipid peroxidation [41].

A novel protein protecting membranes from peroxidation and exhibiting GSH peroxidase activity with phosphatidylcholine hydroperoxides has been identified and characterized as a selenoenzyme [42]. A GSH-dependent, heat-labile factor that inhibits lipid peroxidation in biological membranes has been characterized [19,20,43]. This cytosolic protein represents a new GSH-dependent protein [44]. Some GSH transferase isozymes can catalyze the GSH peroxidase reaction, using organic hydroperoxides as substrate [45]. These non-selenium-dependent activities may become essential during selenium deficiency when the Se enzyme, GSH peroxidase, has a very low cellular activity. Since there is no GSH transferase activity with H202, there is no full substitution for the selenoenzyme. This may explain why in Se deficiency, overt clinical symptoms, such as cardiomyopathy (known as Keshan disease [46]) may arise.

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GSH REGENERATIONFROM GSSG: NADPH REQUIREMENT A number of other defense systems are important; for example, many of the radical or nonradical reactions in cells may lead to oxidation of the thiol to the disulfide (e.g., the oxidation of glutathione to form GSSG) (Figure 1). The regenerative reaction of the reduction to GSH, catalyzed by GSSG reductase, can become pivotal in antioxidant defense. Hence, the provision of reducing equivalents to this

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enzyme is essential and the NADPH-regenerating systems are also of interest.

CONTROL OF ANTIOXIDANT CAPACITY The control of the level of antioxidant enzymes is under genetic control; for example, the induction of catalase and superoxide dismutase (SOD) in microorganisms, such as Escherichia coli or Salmonella typhimurium, during anaerobic shifts [47] or by treatment with H202 [48] has been observed, and adaptation phenomena have been described [4952]. A double mutant of E. coli, devoid of the two SOD activities, was unable to grow on minimal glucose medium [53]. During adaptation of S. typhimurium to H202, 30 proteins were induced, and it was shown that 9 proteins are under positive control of the oxyR regulon for defenses against oxidative stress [54]. The oxyR regulon controls a global response to a DNA-damaging agent, in addition to three other global responses (the SOS response, adapting to alkylating agents, and heat shock). The oxyR protein is directly activated by oxidation [55]. In the cells in which the oxyR regulon was deleted, spontaneous mutagenesis was dramatically increased, and the level of mutagenesis was less than in the controls in cells in which oxyR gene was overexpressed [56]. Similarly, the E. coli mutants lacking SOD showed oxygen-dependent mutagenesis [57]. Although the control of the patterns of antioxidant enzymes, and also the control of the levels of antioxidants such as vitamin E, are not well characterized in mammalian cells, it appears that the adaptation phenomena of this nature may also be iraportant in eukaryotes, and the changes in the biochemical pattern of the hepatic nodules in the cells may be adaptive. These nodules contain clones of hepatocytes in which a new state of liver differentiation is acquired, and is considered a physiological response to environmental perturbations [58], such as the oxyR response. The changes observed in the nodules refer to some of the ancillary antioxidant enzymes, such as increases in the cellular activities of isoenzymes of glucuronyl transferases, glutathione transferase, T-glutamyl transferase, epoxide hydrolase, and NADPH-quinone oxidoreductase. These enzymes belong to the phase II group involved in xenobiotic transformation. The enzymes of phase I, such as cytochromes P450 and bs, are greatly decreased in their cellular activities. These changes in gene expression are related to DNA methylation [59,60]. In experiments to decrease DNA methylation at the cytosine residues by treating animals with the drug analogue 5-azacytidine, a decrease in cytochromes P450 and b5 [61] was observed; NADPH-quinone oxidoreductase was

cloned and found to be hypomethylated in persisting liver nodules [62], showing the relationships between the status of DNA methylation and the importance of some of the enzymes in defense against oxidative challenge [63].

SYNTHETIC ANTIOXIDANTS Numerous drugs useful as antioxidants, ranging from phenolic antioxidants added to foodstuffs to drugs used in medicine, have been synthesized and tested in biological environments. Ebselen, 2-phenyl-l-benzoisoselenazol-3(2H)one, a novel synthetic selenorganic compound, shows antioxidant properties [64]. In an assay of lipid peroxidation using rat liver microsomes, the lag phase preceding the onset of ascorbate/ADPFe-induced lipid peroxidation is increased by the addition of ebselen, whereas the sulfur analog is inactive, being associated with a low-level chemiluminescence and to other parameters of lipid peroxidation, such as the evolution of ethane and npentane or the production of thiobarbiturate-reactive material. Ebselen also acts catalytically in the GSH peroxidase reaction [64-66]: 2 GSH + ROOH

) GSSG + ROH + H20

This activity is thought to be responsible for the protection of isolated hepatocytes against oxidative challenge. Significant protection was afforded against ADP-Fe-induced cell damage in control cells, whereas cells previously made deficient in GSH were not protected by ebselen [66]. Ebselen also inhibits the lipoxygenase pathway [67] and whether this is achieved by the removal of activatory hydroperoxide through the GSH peroxidase reaction, or by another mechanism, is unclear. Selenium has many biological effects [68], with a prominent one being its role as a constituent of the active center of GSH peroxidase as selenocysteine [69-72]. The role of selenium as an antioxidant [73], as an anticarcinogen, and its potential as a carcinogen and as a cytotoxic agent has been reviewed [74].

PHARMACOLOGICALASPECTS OF THIOL GROUPS Many biological and pharmacologic-toxicologic effects in the cell are mediated by thiol-redox changes that are related to signal transmission. The development of pharmacologically reactive compounds, e.g., N-acetylcysteine, penicillamine, mercaptopropionylglycine, dihydrolipoate, and captopril, has shown some specific points of action. Some sulfur-containing compounds with antioxidant properties have been used in therapy [75]; for ex-

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ample, thiols have been used to suppress oxidation of proteinase inhibitors, to prevent tissue damage [76]. The properties of N-acetylcysteine have been discussed [77,78]. Its use as an antidote in acetaminophen (paracetamol) intoxication is an established therapy [79,80]. Similarly, there are literature reports on penicillamine [81], mercaptopropionyl glycine [82], and dihydrolipoate and lipoate [83]. Recently, we were able to show that dihydrolipoate protects membranes against peroxidation [23]. Since sulfhydryl groups lead to a release of calcium [84] when oxidized, a protection by thiol compounds has been studied [85]. The loss of sulfhydryl groups during the course of lipid peroxidation parallels the loss of antioxidants, such as tocopherol [86]. The antioxidant capacity associated with the plasma levels of nonenzymatic antioxidants have epidemiological relationships with several diseases, such as arteriosclerosis [87], ischemic disease [88], and different types of carcinoma [89]. The role of oxygen radicals in degenerative diseases and in tumor development has been discussed [90-92]. Several types of reactions producing free radicals lead to chromosome breaks [93]. Free radicals are involved in skin damage (photo-oxidative stress) [94] by ultraviolet radiation, and also lead to mutations in human cells [95]. The observation [96,97] that traumatic shock in experimental anireals can be prevented by radical scavengers shows a possible use of antioxidants in shock and trauma and in intestinal ischemia and reperfusion. The ageing process seems also to involve free radicals, arising from an increase in the level of oxidized amino acid side chains with age [98]. The respiratory airways during inflammation [99] are also targets for reactive oxygen species. In the lung and in bronchoalveolar lavage fluid, products of lipid peroxidation were detected [100] with antioxidant activities observed in the surfactant layer [101]. The ozone toxicity is an oxidative attack of the lung tissue and has been investigated [102].

ACKNOWLEDGMENT This work has been generously supported by the Deutsche Forschungsgemeinschaft, The National Foundation for Cancer Research, the Fends der Chemischen Industrie, the Ernst Jung-Stiffung for Medizin und Wissenschaft, and the Alexander yon Humboldt-Stiftung.

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