Implication of free radical mechanisms in ethanol-induced cellular injury

Free Radical Biology & Medicine, Vol. 12, pp. 219-240, 1992 Printed in the USA. All fights reserved.

0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press pic

Review Article '

IMPLICATION OF FREE RADICAL MECHANISMS IN ETHANOL-INDUCED CELLULAR INJURY

ROGER NORDMANN, CATHERINE RIBIF.RE, and H~.I&NER O U A C H Department of Biomedical Research on Alcoholism, University Ren~ Descartes (Pads V), 45 Rue des Saints-P&es, 75270 Pads Cedex 06, France (Received 10 July 1991; Revised and Accepted 15 November 1991) Abstract--Numerous experimental data reviewed in the present article indicate that free radical mechanisms contribute to ethanol-induced liver injury. Increased generation of oxygen- and ethanol-derived free radicals has been observed at the microsomal level, especially through the intervention of the ethanol-inducible cytochrome P450 isoform (CYP2E 1). Furthermore, an ethanol-linked enhancement in free radical generation can occur through the cytosolic xanthine and/or aldehyde oxidases, as well as through the mitochondrial respiratory chain. Ethanol administration also elicits hepatic disturbances in the availability of non-safely-sequestered iron derivatives and in the antioxidant defense. The resulting oxidative stress leads, in some experimental conditions, to enhanced lipid peroxidation and can also affect other important cellular components, such as proteins or DNA. The reported production ofa chemoattractant for human neutrophils may be of special importance in the pathogenesis of alcoholic hepatitis. Free radical mechanisms also appear to be implicated in the toxicity of ethanol on various extrahepatic tissues. Most of the experimental data available concern the gastric mucosa, the central nervous system, the heart, and the testes. Clinical studies have not yet demonstrated the role of free radical mechanisms in the pathogenesis of ethanol-induced cellular injury in alcoholics. However, many data support the involvement of such mechanisms and suggest that dietary and/or pharmacological agents able to prevent an ethanol-induced oxidative stress may reduce the incidence of ethanol toxicity in humans. KeywordswFree radicals, Ethanol, Alcoholism, Antioxidants, Lipid peroxidation, Oxidative stress, Liver, Extrahepatic tissues

finding that an enhanced lipid peroxidation occurred both in rat liver homogenates following the in vitro addition of ethanol and in liver homogenates obtained from rats after oral ethanol administration. 2 Di Luzio and Hartman furthermore reported that the increase in lipid peroxidation resulting from the addition of ethanol to normal rat liver homogenates could be prevented by the simultaneous addition ofantioxidants such as N-N'-diphenyl-p-phenylenediamine.2 These results allowed them to suggest that ethanol or its metabolites can stress the balance in the liver toward autooxidation, either acting as prooxidants or reducing the antioxidant level.2 The protective action ofantioxidants would then probably be due to an inhibition of free-radical-induced chain reactions, with the resulting prevention of peroxidative deterioration of structural lipids in membranous organelles.2 An acute ethanol load was also reported by Comporti and colleagues3 to increase liver lipid peroxidation 1 to 12 hours after oral intubation. However, a controversy about the onset and role of enhanced liver lipid peroxidation started when Hashi-

INTRODUCTION

Having observed that the acute ethanol-induced fatty liver can be prevented in rats by the administration of some antioxidants, Di Luzio first suggested that ethanol could affect the antioxidant balance of the hepatic cell. 1 This hypothesis was strengthened by the

Address correspondence to Professor R. Nordmann, 45 Rue des Saints-P~res, 75270 Paris Cedex 06, France. Roger Nordmann obtained his M.D. degree from the Medical Faculty, Paris, in 1954 and his Ph.D. from the Faculty of Sciences, Pads, in 1958. After his clinical training as interne and chef de clinique (H6pitaux de Pads), he was a research fellow at the Institut National d'Hygi~ne (1955-1958) and was appointed Professor of Biochemistry in 1958. He is presently Professor and Head of the Department of Biochemical Research on Alcoholism at the University Ren6 Descartes (Paris V) and President of ESBRA (European Society for Biomedical Research on Alcoholism). Catherine Ribi~re received her Ph.D. from the University Paris V in 1980 and has been a research associate at INSERM (Institut National de la Sant6 et de la Recherche M&tieale) since 1980. H61~ne Rouach obtained her Ph.D. from the same university in 1980 and was an assistant professor from 1980 to 1987. She has been an associate professor (maitre de conf6renees) since 1988. 219

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moto and Recknagel 4 reported no increased conjugated dienes in rat liver mitochondrial or microsomal lipids following administration of a single dose of ethanol. Other investigators questioned the ethanol-induced occurrence of lipid peroxidation since they did not observe any increase in thiobarbituric acid reactive substances in the liver after acute or chronic ethanol administration. 5'6 The controversial results concerning the onset and putative importance of hepatic lipid peroxidation during ethanol intoxication have been reviewed by Dianzani. 7'8 As stated by Dicker and Cederbaum, 9 these controversies may not be surprising in view of the numerous variables and the varying reaction conditions utilized by investigators. The discrepancies concern the conditions of ethanol administration as well as the techniques used for assessing lipid peroxidation. The sole determination of thiobarbituric acid reactive substances as an index of lipid peroxidation has specifically been subjected to criticism. Although controversial, the findings concerning liver lipid peroxidation were of primary importance since they led to considering the possible involvement of free radicals in ethanol metabolism and toxicity. As early as 1972, Slater devoted a whole chapter to the hepatotoxic effect of alcohol in his well-known book Free Radical M e c h a n i s m s in Tissue Injury. lO Shortly after, Cohen suggested that a novel route for the metabolism of ethanol may be represented by its oxidation by hydroxyl free radicals.l~ As shown by comparison with earlier reviews that we have devoted to the involvement of free radicals in the metabolism and toxicity of ethanol] 2-~4 this review demonstrates that considerable advances have been achieved in this area very recently. Recent experiments have allowed a better insight into the relationship between alcohol intake and hepatic lipid peroxidation. The development of noninvasive methods, such as low-level chemiluminescence assay or determination of alkane generation, was of primary importance since it enabled researchers to ascertain the occurrence of enhanced lipid peroxidation during the metabolism of ethanol by perfused rat livers. ~5 More recently, spin-trapping procedures (as previously developed by Albano et al. ~6 to study the free radical products formed by metabolic activation of carbon tetrachloride) have also been used to ascertain ethanol-induced lipid peroxidation in vivo. Using such procedures, Reinke and colleagues ~7 could detect by electron spin resonance (ESR) spectroscopy ESR signals corresponding most likely to lipid carbon-centered radicals resulting from peroxidative events in the liver of chronically ethanol-fed rats receiving a spin-trapping agent. These data deafly show

that ethanol administration elicits an enhanced liver lipid peroxidation in certain experimental conditions. However, this is far from constant, as discussed in the section of the present review on ethanol-induced oxidative stress in the liver. Numerous experimental studies were conducted to ascertain the mechanism(s) responsible for this ethanol effect. They emphasized the role of the ethanolinducible cytochrome P450 in the microsomes, as well as of the molybdo-flavoenzymes xanthine oxidase and aldehyde oxidase in the cytosol. They also showed that ethanol administration may affect the mitochondrial free radical generation. Special attention has been given to the putative role of ethanol-induced disturbances in iron metabolism in relation to iron as a prooxidant factor. Since an oxidative stress affects cellular integrity only when the antioxidant mechanisms are no longer able to cope with the free radical generation, many experimental studies were conducted to ascertain the effects of acute and/or chronic ethanol administration on the main liver antioxidant substrates and enzymes. Therefore, we review successively the effects of ethanol administration on the free radical production in the various liver subcellular compartments and on the main liver antioxidants. We then consider the targets of the disturbed free radical mechanisms, which may be represented in the liver not only by polyunsaturated fatty acids but also by proteins and nucleic acids. We also evoke the strong enhancement in liver toxicity resulting from ethanol administration associated with conditions favoring free-radical-mediated disturbances, such as hypoxia or administration of various xenobiotics. Together with the numerous experimental studies designed to define the free-radical-mediated mechanisms involved in ethanol-induced liver injury, other studies have been dedicated to extrahepatic tissues. Contrary to the initial proposal suggesting that the ethanol-induced enhancement in lipid peroxidation could affect the liver specifically, many experimental data have indeed shown that free-radical-mediated mechanisms also appear to be involved in some of the ethanol effects on other tissues, t8 Since acetaldehyde resulting from ethanol oxidation has been shown to play a prominent role in ethanol-induced liver lipid peroxidation, ~9'1° it is not surprising that such a lipid peroxidation has also been detected in extrahepatic tissues actively oxidizing ethanol (i.e., gastric mucosa). Many recent data show, however, that free radical mechanisms could be involved in the adverse ethanol effects even in tissues poorly metabolizing ethanol, such as the heart or the central nervous system.

Ethanol and free radicals

Following the sections devoted to experimental studies, we review the recent clinical studies designed to ascertain the role of free radical mechanisms in the pathogenesis of human alcohol-related diseases. Since it is questionable whether free radical disturbances are the cause or consequence of tissue injury,2t,22 it is not surprising that no clear-cut evidence is available concerning the role that such disturbances play following alcohol abuse in humans.

ETHANOL-INDUCED FREE RADICAL MECHANISMS IN THE LIVER (Animal Studies)

Generation offree radical species in the liver Microsomes Reactive oxygen derivatives. Chronic alcohol intake has been shown in the rat to enhance the production of reactive oxygen species by isolated microsomes. This holds true for the production of superoxide, 2a,24 hydrogen peroxide, 2.'26 and hydroxyl radiC4¢llS,27'28

These radicals can be generated by nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase, a flavoenzyme that can produce superoxide, which may lead to hydroxyl radicals ( ' O H ) through an iron-catalyzed Haber-Weiss reaction. An enhanced activity of this reductase has been reported after chronic ethanol intake. 29'3° However, the extent of this enhancement appears small compared to the changes in cytochrome P450. 3° Most of the oxygen derivatives produced in excess in microsomes from ethanol-fed rats appear to be generated through the intervention of cytochrome P450. Following the initial report by Lieber and De Carli 25 of the inducibility by chronic alcohol feeding ofa microsomal ethanol oxidizing system involving cytochrome P450, a unique form of this enzyme has been isolated from ethanol-treated rabbits 3t and subsequently from liver microsomes from rats 32,33 and humans. 34,3s This isoenzyme (CYP2E l in the recent international nomenclature36) has been shown to be inducible by many structurally unrelated compounds (e.g., ethanol, 3~'37'3s as well as other alcohols, 39 acetone, 33'4°'4~ imidazole, 37,41,42 isoniazid, 32'3s,4° benzene,43 dimethylsulfoxide,39 and pyrazole37). It can also metabolize various substrates (e.g., ethanol, as well as other alcohols, 3~,44,45acetaminophen,46 nitrosamines, 3s'45'47 diethylether, 4s acetone, 49 and carbon tetrachlorideS°). This ethanol-inducible cytochrome P450 is likely to play an important role in the increased generation of reactive oxygen species by microsomes isolated from ethanol-fed rats, since it reduces dioxygen to ox-

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ygen-derived radicals and H202 e v e n in the absence of hydroxylatable substrates. 5~,52A correlation between the production of these oxygen derivatives (02-, H202) and the amount of CYP2E 1 was actually 013served in microsomal samples of variously treated rats.24 Furthermore, immunological evidence using specific antibodies demonstrated the participation of this unusual cytochrome in the microsomal NADPHdependent formation of H202 .24

Ethanol-derived free radicals. Rat liver microsomes incubated with ethanol and a spin-trapping agent were shown to generate a free radical that could be identified by ESR spectroscopy as the adduct of the l-hydroxyethyl radical with the spin trap. 17'53-56Furthermore, it was reported that l-hydroxyethyl radical formation increased with the concentration of ethanol added to the microsomal suspension and that this production was much higher in microsomes isolated from ethanol-fed rats than in those prepared from rats not previously fed ethanol. Hydroxyl radicals generated in a Fenton-type reaction from endogenously formed hydrogen peroxide in presence of trace amounts of iron could represent the agent oxidizing ethanol to l-hydroxyethyl radicals. 51,57,5s The proposed mechanism is that a hydroxyl radical abstracts a hydrogen from carbon l in the ethanol molecule yielding the 1-hydroxyethylradical. This would represent the basis of the previously reported activity of ethanol as "OH scavenger in some experimental conditions. Such a mechanism is also consistent with the reported inhibition of microsomal NADPH cytochrome-P450-dependent ethanol oxidation by adding either iron chelators, such as desferrioxamine, or competing "OH scavengers, such as 2-keto-4-thiomethylbutyric acid. 57 This "OH-dependent mechanism represents, however, only a minor contribution toward microsomal ethanol oxidation under normal conditions. 59 Its importance increases under experimental conditions promoting microsomal "OH production, such as adding ferric ethylenediaminetetraacetic acid (EDTA), whereas it decreases under conditions that minimize production of "OH.59 Besides this "OH-dependent mechanism, many experimental data show that ethanol can be oxidized by liver microsomes through a reaction catalyzed by cytochrome P450 that is not affected by "OH scavengers. 6°'6~ This holds especially true in microsomes isolated from ethanol-fed rats. EkstrOm and colleagues3° have demonstrated that, in the absence of contaminant iron, the ethanol-inducible isoenzymatic form of cytochrome P450 exhibits a high specificity for ethanol oxidation, but not for the production of "OH.

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Moreover, antibodies prepared against the ethanolinducible cytochrome P450 were found to decrease by 75% the oxidation of ethanol in such microsomal preparations not contaminated by iron. 62 That this oxidation is not inhibited by "OH scavengers could be linked to the intervention of oxidizing species different from "OH. The 1-hydroxyethyl radical could thus be generated from ethanol by oxidizing intermediates, such as perferryl iron complexes, possibly involving the haem-iron of P450. Another possibility suggested by Ingelman-Sundberg and Johansson would be represented by the intervention of" OH radicals produced at the active site of the enzyme itself and therefore not accessible to "OH scavengers. 5~ Thus it appears that at least two pathways are involved in the generation of l-hydroxyethyl radicals from ethanol, the one involving "OH radicals produced in a Fenton-type reaction from endogenously formed H202, and the other being cytochrome-P450mediated (as shown by its strong depression by P450 inhibitors) and apparently independent from "OH radicals. $3.54,56 Besides CYP2E 1, other cytochrome P450 enzymes could be involved in the microsomal free-radical generation in ethanol-treated rats. These other forms of cytochrome P450 might be responsible, at least partly, for the fraction of the microsomal free-radical generation remaining after inhibition of CYP2E1 by antibodies specifically directed against this isoenzyme. 63 Whereas l-hydroxyethyl radicals have been detected by several investigators in liver microsomes oxidizing ethanol, the formation of such radicals has been ascertained only recently in vivo after administration of ethanol together with a spin trap. Reinke and colleagues64'65 detected this radical in liver extracts from rats administered ethanol and a spin trap. Furthermore, Knecht and colleagues66 identified the l-hydroxyethyl radical in the bile from alcohol-dehydrogenase-deficient deermice administered ethanol together with a spin trap. As emphasized by Slater, 67 hydrogen-atom abstraction from ethanol can lead to radicals different from the 1-hydroxyethyl one (CH3C" HOH), namely the 2-hydroxyethyl radical (C" H2CH2OH) and the ethoxyl radical (CH3CH20"). They may be produced in small amounts during ethanol oxidation. Furthermore, vitamin C has been reported to mediate the formation of ethoxy radicals in the presence of ethanol .68 Ethanol-derived free radicals could contribute, together with the enhanced production of oxygen derivatives, to injury of important microsomal constituents. A further contribution could be represented by a free

radical derivative generated during the microsomal oxidation of acetaldehyde, which has been tentatively identified as the acetyl radical. 69 This radical is able to induce chemiluminescence7° and to reduce ferricytochrome c.7~

Lipid-derived .free radicals. Using ESR spectroscopy, Reinke and colleagues observed radical adducts that were most likely carbon-centered adducts of membrane lipids in liver microsomes prepared from chronically ethanol-fed rats administered a spin trap. ~7 This finding confirmed the enhancement in lipid peroxidation previously detected by the determination ofthiobarbituric acid reactive substances24'72'73 or by chemiluminescence assayTM in liver microsomes after chronic ethanol administration. Since this lipid peroxidation can be almost completely inhibited by anti-CYP2El-IgG, the involvement of the ethanol-inducible cytochrome P450 appears prominent. 24 One may recall that CYP2E1 efficiently reduces oxygen even in the absence of substrates, with the resulting generation of oxygen species capable of initiating lipid peroxidation. 24 Since it has been shown that chronic ethanol administration elicits a selective centrilobular induction of CYP2E 1 in rat 75'76and human 76liver, it appears likely that lipid peroxidation linked to this enzyme is partly responsible for the centrilobular localization of ethanol-induced liver injury. The prominent role of CYP2E1 does not, however, exclude the contribution of the peroxidase activities of other forms of P450. 24 Membranous lipids are not the sole target of free radical attack. 77 The increased microsomal generation of active oxygen derivatives could also contribute to ethanol toxicity through radical-mediated inactivation of metabolic enzymes. 78 Mitochondria. It is well known that superoxide radicals can result from the activity of the mitochondrial respiratory chain, which represents one of the main physiological sources of 02-. 79 Since mitochondria contain an active superoxide dismutase, 02- can generate H2Oz, which is destroyed through mitochondrial glutathione peroxidase. However, in the presence of iron, a part of H202 escaping this destruction could generate aggressive radicals that may result in mitochondrial structural and functional disturbances. We have reported that an acute ethanol load significantly increases liver mitochondrial superoxide generation in submitochondrial particles. 8° The decrease in the NAD+/NADH ratio induced by acute ethanol administration 8~ could furthermore favor mitochondrial 02- generation by increasing electron flow along the respiratory electron transport chain. This may re-

Ethanol and free radicals suit in increased electron leakage giving oxygen derived reactive species. The enhanced 02- generation could itself contribute to the increased susceptibility to lipid peroxidation that was apparent in isolated rat liver mitochondria following acute ethanol administration, s2 Chronic ethanol consumption by experimental animals induces morphological abnormalities in mitochondrial structure, s3,s4 These abnormalities represent one of the earliest manifestations of the effects of prolonged alcohol intake on the liver. Free radical mechanisms could be involved, at least partly, in the pathogenesis of mitochondrial injury. We observed an enhanced liver mitochondrial sensitivity to lipid peroxidation in rats during chronic exposure to ethanol vapor inhalation as well as during the early stage of ethanol withdrawal, as We therefore suggested that these alterations of lipid peroxidation are initiated by the presence and/or the metabolism of ethanol, whereas changes in the membrane structure would be responsible for the maintenance of the disturbances after ethanol withdrawal. However, chronic ethanol feeding to rats elicits a decreased liver mitochondrial production of both superoxide s6 and hydrogen peroxide, s7 It also results in a decrease in the rate and efficiency of adenosine 5'triphosphate (ATP) synthesis via oxidative phosphorylation. Decreased levels of mitochondria-derived polypeptide components of the oxidative phosphorylation systems may be responsible for this depression. 88

Peroxisomes. Studying hydrogen peroxide generation in perfused livers from fasted rats, Handler and Thurman 89 reported that this generation was strongly increased by the addition of fatty acid-albumin complexes, which are substrates for H202 generation via the peroxisomal r-oxidation system. They further suggested that ethanol oxidation is mediated predominantly via catalase in the fasted state, where concentrations of fatty acids in the circulation and liver are e l e v a t e d . 9°'91 It may be hypothesized that a part of the hydrogen peroxide produced inside the peroxisomes is not used to oxidize ethanol but may, in the presence of iron, contribute to the generation of aggressive species and the enhancement of liver lipid peroxidation induced by acute ethanol administration in fasted animals. Chronic ethanol administration was shown, on the other hand, not to affect the activity of liver peroxisomal oxidases, such as acyl-CoA oxidase, L-a-hydroxyacid oxidase, D-amino acid oxidase, and urate oxidase. 92

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Cytosol. The possibility that acetaldehyde may contribute to the ethanol-induced increase in free radical generation has been based on studies demonstrating that acetaldehyde increases the production ofalkanes by isolated perfused rat liver, 19'93 promotes lipid peroxidation in isolated hepatocytes, 2° and causes an increase in the antioxidant-sensitive respiration in the perfused liver. 94 In vivo administration of acetaldehyde to rats has also been shown to enhance hepatic lipid peroxidation and to decrease hepatic glutathione levels. 95 Such a peroxidation is inhibited by 4-methyl pyrazole, an alcohol dehydrogenase inhibitor. 96'97 This reflects the putative importance of acetaldehyde in ethanol-induced liver lipid peroxidation. Scavenging of "OH radicals by 4-methyl pyrazole 98 could, however, contribute to its inhibitory effect on ethanol-induced lipid peroxidation. The ability of acetaldehyde to generate free radicals has often been ascribed to its oxidation by xanthine oxidase, a superoxide-radical-generating enzyme. Two conditions must be fulfilled for such an oxidation to occur efficiently: (1) Xanthine dehydrogenase (XD), which represents the main form of the enzyme, must be largely converted into its oxidase form (XO), and (2) acetaldehyde should be a substrate for the enzyme. The first condition is realized following ethanol administration, since Oei et al. 99'1°° and Sultatos ~°1 have shown that acute ethanol exposure favors the conversion of XD into XO. This effect of ethanol may be linked to hypoxia, which has been reported to be produced by ethanol in centrilobular hepatocytes. 1°2 Such a hypoxia might lead to activation of the calcium-dependent protease(s) within pericentral hepatocytes, thereby converting XD into XO. Interestingly, the specific activity of XO in rat liver has recently been shown to be much higher in perivenous than in periportal hepatocytes. 1°3 Other mechanisms able to favor the conversion of XD into XO can be considered after acute ethanol administration. Thus, acetaldehyde itself may cause a reversible conversion of XD into XO by its well-known reaction with free sulfhydryl groups. 104Since NADH has been shown to inhibit XD activity, l°s the decrease in the NAD+/ NADH resulting from acute ethanol administration 81 may also favor the XO pathway.I°5 Studying the effects of chronic ethanol intoxication on the rat liver xanthine oxidase status, Abbondanza et al. 1°6 reported an increase in the XO form after repeated ethanol administration and an increase in the intermediate XD/XO form (able to react with 02 as well as with NAD ÷ as electron acceptor) after prolonged ethanol feeding. Whereas several mechanisms may contribute to an

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ethanol-linked conversion of XD into XO, the in vivo contribution of this enzyme to an enhanced production of free radicals following ethanol administration remains controversial. To generate such radicals, the enzyme needs not only to be present in its oxidase form, but also to be given appropriate substrates. Acetaldehyde itself could act as a substrate of XO. However, one must consider that the acetaldehyde concentration achieved in the liver following ethanol administration (0.1-0.2 mM) ~°7 is far below the concentration required for half-maximal velocity (Km) of XO with acetaldehyde as substrate (> 30 mM).l°8 Puntarulo and Cederbaum 7° have nevertheless suggested that relatively "low" concentrations of acetaldehyde (e.g., 0.1 mM), as found in the liver during ethanol metabolism, may react at the active site of XO with generated "OH to produce longer-lived acetaldehyde radicals. This putative mechanism of acetaldehyde contribution to the generation of an oxidative stress requires further evaluation. An overproduction of free radicals through the intervention of XO after ethanol administration could also result from substrates different from acetaldehyde and having greater affinities for XO, such as hypoxanthine and xanthine. An increase in purine degradation after ethanol administration has been documented by enhanced hepatic adenosine 5'-monophosphate (AMP), hypoxanthine, and xanthine levels, as well as by an increased urinary output ofallantoin and uric acid. ~°5 Acetate resulting from ethanol metabolism may represent the mediator of this increased adenine nucleotide degradation, 1°9 which may contribute to ethanol-induced hyperuricemia in alcoholics. ~o Whatever the nature of its substrates, the contribution of XO to ethanol-induced hepatic lipid peroxidation is suggested by our finding that such a peroxidation is significantly inhibited in rats pretreated with allopurinol, an inhibitor of XO. ~ ~A preventive activity of allopurinol in ethanol-induced lipid peroxidation has also been reported by Kato and colleagues, l°5 in contrast to the data from Kera and colleagues. 96 Besides XO, another closely related molybdo-flavoenzyme (i.e., aldehyde oxidase) could be involved in the ethanol-induced hepatic oxidative stress. The Km of aldehyde oxidase for acetaldehyde ( 1 mM) ~12is much lower than that of XO (> 30 mM) ~°8 with this substrate, and its role in the production of free radicals with acetaldehyde as substrate should be considered. Evidence of a contribution of aldehyde oxidase in ethanol-induced lipid peroxidation is supported by the finding that ethanol-induced alkane production is reduced to a much larger extent in rat hepatocytes isolated after chronic feeding of tungstate than in heI

patocytes isolated from ailopurinol-pretreated rats. ~3 Tungstate feeding results in decreased activity of both aldehyde oxidase and XO, whereas allopurinol inhibits only XO.

Role of iron availability in the generation of free radicals in the liver of ethanol-treated rats. It is well established that non-safely-sequestered iron plays an important role in initiating and catalyzing a variety of free radical reactions that contribute to oxygen-dependent tissue injury. It" The most widely proposed mechanism is the iron-mediated Haber-Weiss reaction leading to the generation of" OH from 02 • However, other oxidants can be produced by reaction of "free" iron with 02- o r H z O 2. Moreover, iron plays an additive role in lipid peroxidation by accelerating the decomposition of lipid peroxides, resulting in peroxyl and alkoxyl radicals that can abstract H" and stimulate lipid peroxidation. The "free" iron complexes able to promote free radical reactions in vivo appear to be present as a small pool ofchelatable low-molecular-weight iron derivatives that may consist of non-protein-bound "free" iron attached to such metal-binding agents as ATP, adenosine 5'-diphosphate (ADP), guanosine 5'triphosphate (GTP), or citrate. Other nonheme nonferritin iron derivatives may be present in microsomal membranes and be involved in the catalysis of free radical generation. ~is The role of nonheme iron and more specifically of its "free" components led to investigations of the influence of ethanol administration on iron distribution in the liver. Few reports have been dedicated to the effects of acute ethanol on the nonheme iron content in the liver, and conflicting results have been reported/16.117 We observed a significant increase in the liver nonheme iron content after an ethanol load. 118 This increase was apparent in the light mitochondrial, microsomal, and cytosolic fractions and was maximal in the microsomal fraction, ~18 where it could contribute to the ethanol-induced microsomal lipid peroxidation. 9v We furthermore reported an increase in the percentage of total nonheme iron present in the cytosol as low-molecular-weight iron derivatives. ''8 This suggests than ethanol administration facilitates iron release from iron stores. Because superoxide radicals are able to release iron from ferritin, 1~4"~19an increased xanthine oxidase O2- production dependent on ethanol ingestion could be involved. Since NADH may also favor the release of iron from ferritin, ~2° another contributing factor may be represented by the increase in the NADH/NAD + ratio resulting from ethanol oxidation in the liver. Furthermore, NADH may

Ethanol and freeradicals also represent the reductant involved in the labilization and reduction of iron bound to transferrin, t2~ The cellular uptake of iron from transferrin has been shown in various reports to involve, in addition to receptor-mediated endocytosis, a system involving reduction of iron. At the microsomal level, iron may be released in its ferrous form from nonheme, nonferritin iron via direct reduction by NADPH P450 reductase.115 Whereas the exact mechanisms responsible for the changes in iron distribution in the liver following an acute ethanol load need to be clarified by further studies, their importance is underlined by the report that administration of allopurinol prior to an acute ethanol load prevents the increase in liver lipid peroxidation 111 and the changes in nonheme iron, 122 suggesting a link between both disturbances. Furthermore, it has been reported that the extent of liver lipid peroxidation resulting from acute ethanol administration is significantly exacerbated in rats acutely t ~7 or chronically82 iron overloaded. Chronic ethanol feeding has generally been reported to increase the total nonheme liver iron content in rodents. 116,123-126 However, this increase is not always apparent, and the discrepancies are probably related to differences in diets. 127 In our study concerning long-term ethanol administration to rats, 126 we observed that this administration elicits an increase in the low-molecular-weight iron derivatives in the liver cytosolic fraction. This increase may contribute to enhanced free radical generation in the liver.

Effects of ethanol on liver antioxidants Glutathione. Whereas most of the investigators reported that acute ethanol administration elicits a decrease in the hepatic glutathione content, the mechanism(s) involved in this lowering are highly controversial.128-137 The formation of an adduct between acetaldehyde resulting from ethanol oxidation and glutathione may represent one of these mechanisms. 13a However, it could at most play a minor role, since acetaldehyde does not easily form a stable conjugate with glutathio n e . 139'14° Unphysiologically high acetaldehyde concentrations are therefore required to lead to a significant adduct formation./36 Another mechanism could be represented by increased glutathione oxidation resulting from the enhanced generation of oxidizing free radicals? 32 The preventive effect of desferrioxamine administration on the ethanol-induced decrease in liver glutathione that we have previously reported TM argues in favor of

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such a mechanism. However, some investigators did not report any changes in the hepatic oxidized glutathione (GSSG) content and the ratio of reduced glutathione (GSH) to GSSG ~29'135and did not observe any increase in the biliary excretion of GSH and GSSG 133 after an ethanol load. Further mechanisms that appear to play a prominent role in liver glutathione depletion induced by acute ethanol administration are reduced hepatic glutathione synthesis as well as increased efflux ofglutathione from the liver. 133"135 There is no agreement about the effects of chronic ethanol administration on the hepatic glutathione content. Some investigators have reported no change in this content, ~42 while others found it either decreased 129-132'143'144 or increased. 127'145-148 The decrease observed in some experimental conditions has been related to an enhanced oxidation of GSH into GSSG as a consequence of increased generation ofprooxidant free radicals and lipid peroxides.132 The reported increased biliary GSSG release in ethanol-fed rats argues in favor of such a mechanism./49 As observed by some authors,142'144'~50 chronic ethanol feeding also increased hepatic turnover and sinusoidal eftlux of glutathione in rats. These effects appeared linked to a direct effect of ethanol on hepatic glutathione transport and not to an alteration in extrahepatic disposition of glutathione. Fernandez-Checa and colleagues, 144 studying the effects of chronic ethanol feeding on rat hepatocytes, reported a modest fall in cytosolic and a marked fall in mitochondrial glutathione together with an increased glutathione ettlux. These changes could be reevaluated by the recently described use of hepatic couplets. TM On the other hand, as already pointed out, increased hepatic glutathione levels as a consequence of chronic exposure to ethanol have been reported by several investigators./27'~4s-148 This unexpected enhancement of an important cellular antioxidant may result from an adaptive increase in hepatic glutathione synthesis. It has thus been observed that the activity of the glutathione synthetizing system (3,-glutamyl-cysteine synthetase + glutathione synthetase) progressively increases during chronic exposure to ethanol./29 The wide disparity in the reported effects of chronic ethanol feeding on liver glutathione appears related to the duration of the exposure to ethanol as well as to nutritional influences or diurnal variations. These effects show that liver glutathione depletion is not a constant feature of alcohol intoxication.

a-Tocopherol. An acute ethanol load does not alter significantly the total liver a-tocopherol concentra-

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tion in rats. 152,153However, it induces a decrease in the a-tocopherol level of liver mitochondria. 8zThis lowering could result from increased mitochondrial a-tocopherol demand secondary to the ethanol-induced enhancement of free radical reactions. Feeding ethanol (35% of total caloric intake) for 5 to 6 weeks elicits in the rat a lowering in the liver a-tocopherol content. 153 This lowering is particularly noticeable in the liver light mitochondrial fraction. 154A significant increase in the microsomal a-tocopheryl quinone/a-tocopherol ratio (aTQ/aToc) after chronic ethanol feeding has been reported by Kawase and co-workers. 127 An enhanced generation of free radicals is probably responsible for the enhanced conversion of a-tocopherol into a-tocopheryl quinone, a conversion that appears mediated by free radical mechanisms and irreversible under physiological conditions. Such an increased a-TQ/a-Toc ratio was especially noticeable in rats fed a low-vitamin-E diet (a group in which hepatic lipid peroxidation was apparent after chronic ethanol feeding), whereas no peroxidation was evident in rats receiving an adequate-vitamin-E diet. 127 A lowered a-tocopherol level (expressed per gram wet weight) was also reported in rat liver after chronic ethanol inhalation. 155 However, this decrease was no more apparent when the hepatic a-tocopherol concentration was expressed per milligram of protein. Using milder conditions of chronic ethanol administration, Tyopponen and Lindros 1s6 did not observe any effect on the liver vitamin E content. In conclusion, the effects of chronic ethanol administration on rat liver a-tocopherol appear to depend largely on the ethanol dose administered and on the vitamin E content of the diet. The reported data indicate that free radical mechanisms are involved in these disturbances and that a poor vitamin E status may potentiate the toxicity of ethanol on the liver. ~56 One may add that the disturbances in the a-tocopherol distribution in the liver from ethanol-fed rats are apparently not linked to a decreased absorption of vitamin E. 157

Hepatic antioxidant enzymes. We have previously reported 158'159 that an acute ethanol load elicits a decrease in the activity of extraperoxisomal catalase, which precedes a reduction in the cytosolic Cu,Zn-superoxide dismutase (SOD) activity. Since superoxide radicals can inactivate catalase 16° while H202 can inactivate Cu,Zn-SOD, 161 an overproduction of 02- could represent the initiator of the observed disturbances in both enzymes. Acute ethanol administration does not alter the activity of glutathione peroxidase 137'141A62-164but lowers that of glutathione-S-transferase.97'137,148,163,165

Whereas decreased activities of catalase and Cu,ZnSOD have been reported by most investigators in the liver of rodents after chronic ethanol administration, 146"j66-168Rikans and Gonzalez 155did not observe any effect of chronic ethanol inhalation on the activity of these enzymes. The reported changes in glutathione peroxidase activity after chronic ethanol administration appear highly contradictory. 147,149,168-172 Increased hepatic cytosolic glutathione-S-transferase activity has been often reported to be associated with chronic ethanol feeding. 147'17°,173-175 This enzyme shares the characteristic of inducibility with the microsomal drug-metabolizing enzymes. 176 Ethanol may therefore enhance glutathione-S-transferase activity by a mechanism similar to the other inducers of cytochrome P450. This enhancement could represent an adaptive response protecting against lipid peroxidation. It may enhance the formation of adducts between glutathione and cytotoxic aldehydes, such as 4-hydroxynonenal, giving less toxic conjugates. It may furthermore favor the reduction of lipid hydroperoxides to stable lipid alcohols. In conclusion, no ethanol-induced constant defect in the main liver antioxidants has yet been reported. Several reports show, however, that some lines of defense against free radical attack are frequently altered following an acute ethanol load. The situation is less clear-cut following chronic ethanol intake. The reported discrepancies may be due to variations in experimental design (mode, dose, and period of ethanol administration). Furthermore, adaptive processes may reduce the extent of the disturbances in some antioxidants, whereas, on the contrary, these disturbances may be enhanced by feeding the animals diets poor in essential antioxidants.

Ethanol-induced oxidative stress in the liver An oxidative stress, which can be defined as an increase in the cellular ratio between prooxidants and antioxidants,177 is often characterized by an enhanced lipid peroxidation. As stated in the introduction of the present review, such an enhancement has often, but not constantly, been reported after acute ethanol administration. The discrepancies in the reported data are likely linked to differences in the dose and route of ethanol administration, as well as in the diet previously fed to the experimental animals. The decrease in the hepatic NAD+/NADH ratio induced by an acute ethanol load 1°,81 may contribute to the oxidative stress, since NADH may favor mitochondrial and cytosolic freeradical generation, as already discussed. NADH could furthermore promote microsomal generation of reac-

Ethanol and free radicals tive species since it has been shown to be effective in reducing ferric chelates in microsomal preparations. 9 Although chronic ethanol administration induces CYP2E l and favors the microsomal free-radical generation, enhanced liver lipid peroxidation is not a constant feature after long-term alcohol consumption. This may be due to adaptative processes resulting in an enhanced antioxidant defense. One of the main adaptative mechanisms may be represented by the enhanced hepatic glutathione level often reported in such conditions. Other not yet identified cytosolic low-molecular-weight components involved in the rat liver antioxidant defense are also apparently increased after chronic ethanol administration. 178However, long-term alcohol feeding has been reported to potentiate liver lipid peroxidation resulting from an acute ethanol load in baboons. TM The reported discrepancies concerning the onset of enhanced hepatic lipid peroxidation after ethanol administration appear linked, at least partly, to varying conditions of the lipid and vitamin E content of the diet. Lipid peroxidation is largely favored by fat-rich diets 17as well as by dietary vitamin E deficiency) 79'18° In a recent report,~27 enhanced hepatic lipid peroxidation was observed after long-term ethanol feeding only in rats fed a low-vitamin E diet. Vitamin E supplementation was found to provide protection against the ethanol-induced oxidative stress in the liver/s° Such an oxidative stress is potentiated when various xenobiotics are administered together with ethanol. As emphasized by Lieber, Isl this effect appears largely linked to the unique capacity of the ethanol-inducible CYP2E 1 to activate xenobiotic agents to toxic metabolites. For example, it has been shown in rats that longterm ethanol feeding preceding an oral dose of carbon tetrachloride along with a spin-trapping agent significantly enhances the intensity of the ESR signal due to the trichloromethyl radical (" CC13) adduct of the spin trap in the hepatic lipid extracts.~S2 The stimulation of the trichloromethyl radical generation from carbon tetrachloride in ethanol-fed rats is consistent with the major role attributed to CYP2E 1 in carbon tetrachloride-induced lipid peroxidation. The potentiation of the CC14 effects by chronic alcohol administration is also demonstrated by the development of severe fibrotic liver injury in rats exposed to a combination of ethanol and CCI4 vapor, wherein neither agent alone, in the same quantities used, produced significant liver damage.183 This synergy can also occur without prior induction of the P450 system, as shown by the enhancement of CCl4-induced liver injury by a single dose of ethanol. 184 The contribution of the disturbances in hepatic lipid peroxidation to the evolution of ethanol-in-

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duced liver injury remains controversial. However, the recently reported taS-~ssgeneration of a neutrophil chemoattractant may play a crucial role. Roll and colleagues ~as,t86 reported that cultured rat hepatocytes, when exposed to ethanol, generate a lipid factor that may be an arachidonic acid metabolite distinct from leukotriene B4 and that possesses potent chemotactic activity for human neutrophils. A similar activity was generated in a cytosolic cell-free system derived from rat liver. The generation of this chemoattractant was inhibited by "OH scavengers, whereas it was considerably increased when rat liver cytosol deficient in both glutathione and glutathione peroxidase was incubated with ethanol, t87 The chemoattractant appeared to be formed via a lipid peroxide (acetaldehyde was a key intermediate, possibly as a generator of oxygen-derived radicals~88). It was established furthermore that the chemoattractant production was almost undetectable in cultures of hepatocytes prepared from rats fed an iron-deficient diet. The same held true when the iron chelator desferrioxamine mesylate was added to cultures of iron-loaded cells. These data provided evidence for the importance of hepatocellular iron in the production of this alcohol-related lipid chemoattractant. This chemoattractant may play an important role in the initiation of inflammation associated with alcoholic hepatitis in humans. Whereas both rat and human hepatocytes produce the factor in response to ethanol, rat leukocytes, unlike human cells, do not respond chemotactically to the factor. ~s8 The lack of hepatic inflammation in long-term ethanol-fed rats may therefore be related to the lack of response of rat leukocytes to this chemoattractant. Furthermore, the chemoattractive properties of a lipid peroxidation product, 4-hydroxy nonenal, on human neutrophils ~s9as well as the enhanced neutrophil free-radical production in response to acetaldehyde-altered hepatocyte membranes 19° could facilitate alcoholic hepatitis in humans. Membranous lipids are not the sole target of an ethanol-induced oxidative stress in the liver. It is well documented that cellular proteins may also be disturbed by free radicals. Fraga and co-workers, ~9, studying protein synthesis and lipid peroxidation in rat liver slices incubated in the presence of oxidants and protein synthesis inhibitors, reported that oxidant-induced lipid peroxidation and protein synthesis damage occurred concurrently and that protein synthesis inhibition may be involved in cell injury mediated by free radicals. Conditions of enhanced radical flux may also accelerate proteolysis, directly by fragmenting proteins and indirectly by rendering proteins more susceptible to proteinases. 192 Remmer and colleagues ~93 thus reported that the

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primary target of the oxygen-radical attack promoted by ethanol is represented by cellular proteins and not by lipids. They suggested that ethanol stimulates the formation of 02- radicals that attack not phospholipids, but proteins at the lipophilic side chain of aminoacids. They furthermore pointed out that thiobarbituric acid reactive substances can represent scission products of several aminoacids and that the increased formation of these substances often reported after ethanol administration should therefore not be viewed as a consequence of lipid peroxidation only. Key metabolic enzymes may be involved among the proteins damaged by free radical attack. Fucci and co-workers ~94 showed that microsomal mixed-function oxidation reactions involving eytochrome P450 inactivate 10 enzymes playing a key role in metabolism. The enhanced generation of free radicals during ethanol metabolism has been shown by Rajasinghe and co-workers wS to cause DNA cleavage in isolated rat hepatocytes. The authors suggested that this damage may contribute to alcohol-induced injury and carcinogenesis. The potentiation by ethanol of aflatoxin Bl hepatotoxicity appears linked to the enhancement of both lipid peroxidation 196 and covalent binding of 8,9-epoxide-aflatoxin B~ to DNA) 64 The depletion of glutathione may be the main factor responsible for this enhancement. Still other cellular constituents may be damaged by ethanol-generated free radicals. The experimental data of Shaw and colleagues ~97 suggest that an ethanol-induced enhancement of free-radical-mediated folate cleavage contributes to the folate deficiency resulting from excessive alcohol intake. Reactive oxygen radicals also appear to be involved in the enhancement of ethanol-induced hepatotoxicity under conditions of low oxygen supply. Younes and colleagues 198't99 reported that marked liver damage as evidenced by a strong release ofglutathione and various hepatic enzymes is apparent in ethanol-perfused rat livers in both models of partial as well as total ischemia and reperfusion. On the contrary, ethanol perfused under normotoxic conditions leads only to a moderate hepatotoxicity. The strong potentiation of ethanol toxicity under hypoxic conditions was prevented when inhibiting ethanol metabolism by 4-methyl-pyrazole. It was also partially inhibited by desferrioxamine, allopurinol, catalase, and superoxide dismutase. These findings indicate that oxygen radicals resulting from xanthine oxidase acting on purine metabolites and acetaldehyde may be involved in this potentiation. They could act through direct toxic actions and/or by inhibiting glycolysis, the main source of energy-rich phosphates under hypoxic conditions, z°°

ETHANOL-INDUCED FREE RADICAL MECHANISMS IN EXTRAHEPATIC TISSUES (ANIMAL STUDIES)

As stated earlier, numerous studies show that ethanol administration to rodents, in certain experimental conditions, elicits enhanced free radical mechanisms such as lipid peroxidation in various extrahepatic tissues. Most of these studies concern tissues that are highly sensitive to ethanol-induced damage (i.e., stomach, central nervous system, heart, and testes). Before considering the ethanol effects in these tissues that are likely related to free radical mechanisms, we note that some reports have shown that ethanol administration may also disturb the antioxidant defense in other tissues, such as kidney ~29'~3°'2°~ or lung.~55

Implication of[ree radical mechanisms in ethanol-induced gastric mucosal damage Oral administration of absolute ethanol in rats resulted in enhanced lipid peroxide levels and decreased nonprotein sulfhydryl levels in the gastric mucosa. 2°2 Since antiperoxidative drugs were able to reduce the severity of the mucosal injury, lipid peroxidation was suggested to contribute to the ethanol-induced gastric i n j u r y . 2°3,2°4

The involvement of oxygen radicals in ethanol-induced gastric injury was confirmed in cultured mucosal cells. 2°5 Exposure to ethanol increased, in a dosedependent manner, the generation of superoxide anions and the extent of cellular damage. On the other hand, pretreatment with desferrioxamine decreased the ethanol-induced cellular injury in the cells. 2°5 Gastric mucosa represents a site of ethanol metabolism and is rich in XO, so the increased free radical production observed in the presence of ethanol could be related to the oxidation through XO of locally produced acetaldehydefl°6 It was shown furthermore that in vivo administration of ethanol disturbs the gastric intrinsic factor, as well as glutathione, and that these effects are reversed by inactivation of molybdenumdependent oxidases (such as XO) through tungsten feeding. This suggests that ethanol-generated free radicals due to the metabolism of acetaldehyde by such oxidases may contribute to the gastric injury observed after ethanolfl°6 Another report suggests, however, that the ethanol-induced gastric mucosal injury is neutrophil-mediated and does not involve oxy radicals, z°7

Implication offree radical mechanisms in the effects of ethanol on the central nervous system Nervous tissue is characterized by its high oxygen consumption and its high content in easily oxidizable

Ethanol and free radicals substrates (mainly polyunsaturated fatty acids and catecholamines), contrasting with the low activity of some enzymes involved in the cellular antioxidant defense. 2°8 It could therefore be highly susceptible to injury by a lipid peroxidative process. It is thus not surprising that recent studies have provided support for the occurrence of free radical and lipid peroxidative reactions in the injured or ischemic central nervous system.2°9 Whereas the initial reports showed no ethanol-induced increase in the peroxide level of rat brain homogenates, 21°it was later shown that ethanol administration can favor lipid peroxidation in nervous tissue in some experimental conditions. Lipid peroxidation was found enhanced in the spinal cord following minimal trauma in ethanol-pretreated cats but not in naive animals. 211 Our group reported later that acute ethanol administration without any associated trauma can induce an oxidative stress at the cerebellar level in r a t s . 212-214 The cerebellum was selected in these studies because of its particularly well-known sensitivity to ethanol-induced dysfunction. Furthermore, among all the brain regions, the cerebellum shows the lowest concentration of a-tocophero1215 and is especially sensitive to a-tocopherol deftciency.2~6 The mechanisms involved in the induction ofcerebellar lipid peroxidation following an acute ethanol load are not yet fully ascertained. However, an important factor may be an increase in the low-molecularweight chelatable iron content observed in the cerebellar cytosolic fraction of ethanol-treated animals. 118'217 As discussed when considering this non-safely-sequestered iron fraction in the liver, its increase can be mediated by an enhanced production of reducing equivalents, such as NADH or superoxide radicals, that could be produced during the metabolism of ethanol or acetaldehyde. It is interesting, therefore, to note that recent results have shown that such a metabolism can occur in the central nervous system through various pathways previously described in the liver. Kerr and colleagues218 have thus detected alcohol dehydrogenase in certain neurons and suggested that intraneuronal ethanol metabolism may lead to focal accumulation of acetaldehyde. At the cerebellar level, alcohol dehydrogenase was detected in Purkinje cell cytoplasm. At the microsomal level, an ethanolinducible cytochrome P450 has recently been characterized in various parts of the rat central nervous system, such as the cerebellum. 219 Ethanol oxidation by the rat brain has furthermore been reported to involve catalase. 22° Recent data have confirmed the generation o f H 2 0 2 in the rat brain in vivo and suggested that acetaldehyde formed via brain catalase may be respon-

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sible for some of the psychopharmacological actions of ethanol.221,222 Whatever the mechanism(s) involved in the increase in the low-molecular-weight chelatable iron content and their putative links to local ethanol metabolism, this increase may favor the biosynthesis of aggressive prooxidant radicals in the cerebellum. The occurrence of an ethanol-induced cerebellar oxidative stress following acute ethanol administration could be furthermore favored by a decrease in the main antioxidant elements. We observed that this administration elicits a decrease in the cerebellar concentrations of a-tocopherol and ascorbate 214as well as of selenium, zinc, and copper.122 The role ofa-tocopherol as an antioxidant in the brain has been confirmed in studies on the effects of vitamin E deficiency on postischemic cerebral lipid peroxidation. 223 Ascorbate, which can act as a prooxidant at low concentrations, behaves as an antioxidant at higher concentrations. Its concentration in the brain is the highest of any tissue next to the adrenal g l a n d , 224 and ascorbate together with a-tocopherol appears to have a protective role against brain lipid peroxidation. Although we did not observe disturbances in the mitochondrial glutathione peroxidase (GSH-Px) activity, 122 the reported decrease in selenium may contribute to the oxidative stress by affecting cerebellar selenoproteins involved in the antioxidant defense but that are different from GSH-Px. The decrease in zinc and copper concentrations is also likely a contributing factor, since zinc itself has an antioxidant function, whereas copper is involved in the activity ofcytosolic superoxide dismutase. The activity of the latter enzyme is decreased in the brain following acute ethanol administration. 225 Besides in the cerebellum, lipid peroxidation may also be enhanced by an acute ethanol load in other parts of the central nervous system. Acute ethanol administration elicits an increase in the lipid peroxide level and a decrease in the glutathione level in whole brain homogenates without the cerebellum. 226 Increased levels of thiobarbituric acid reactive substances have also been reported in the cerebellum, cerebral cortex, and brain stem following acute ethanol administration to rats fed a normal laboratory d i e t . 227 However, increased lipid peroxidation was not observed in these brain areas when a single large dose of ethanol was administered to rats fed a vitamin-E-supplemented diet resulting in a daily vitamin E intake higher than the normal rat requirement. 227 Chronic ethanol administration is also able to induce an oxidative stress in the central nervous system, at least in certain experimental conditions. Increased lipid peroxidation has thus been reported after long-

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term ethanol feeding in various regions of the rat brain, 227 but not in whole rat brain homogenates.228 Chronic ethanol intake also affects the antioxidant defense in the central nervous system, as shown by a decrease in the cerebellar a-tocopherol content ~26,229 as well as in the brain glutathione level 129and the cytosolic superoxide dismutase activity.22s,23° Furthermore, it elicits, like acute ethanol administration, an increased concentration of low-molecular-weight iron species in the cerebellar cytosolic fraction. 126.229 Further research is necessary to ascertain the conditions required for chronic ethanol intoxication to elicit an oxidative stress and enhance lipid peroxidation in the central nervous system. The question is of special importance, since oxidative stress could represent a biochemical link between ethanol ingestion and some of its effects on the central nervous system. Since an enhanced lipid peroxidation disturbs synaptosomal membrane fluidity as well as the metabolism of some neurotransmitters,TM it could contribute to the development of ethanol tolerance and dependence. If this holds true, pharmacological agents able to prevent the induction of an oxidative stress could be valuable for the prevention of these major disturbances linked to alcohol misuse. Such agents may be represented by iron chelators. The reduction in the severity of physical dependence on ethanol in mice caused by desferrioxamine administration supports this hypothesis.232 Another pharmacological agent of putative interest is allopurinol, which we reported to prevent the enhanced cerebellar lipid peroxidation as well as the disturbances in pro- and anti-oxidant elements induced by acute ethanol administration to rats. t22 Besides their possible involvement in the pathogenesis of ethanol-induced brain injury in adult animals, free-radical-mediated mechanisms could also play an important role in the fetal alcohol syndrome. Ethanol added to neural crest cell cultures severely depressed cell viability while simultaneously inducing the generation ofsuperoxide, hydrogen peroxide, and hydroxyl radicals. 233 Ethanol cytotoxicity was furthermore significantly reduced by the addition of superoxide dismutase. It was therefore suggested that reactive free radicals are, at least partly, involved in the ethanol-induced injury of neural crest cells that can lead to developmental craniofacial anomalies associated with the fetal alcohol syndrome.233

Implication offree radical mechanisms in ethanol-induced myocardial injury An oxidative stress may represent a fundamental mechanism in the production of myocardial in-

jury. 234-236 The pathogenesis of the myocardial distur-

bances linked to chronic ethanol consumption are not yet clearly established, and it is not surprising that the intervention of lipid peroxidation has been suggested. 237 Several reports indicate the occurrence of myocardial enhanced lipid peroxidation during chronic alcohol intake. 92'238'239 More directly, carboncentered free radicals that were most likely lipid radicals have been detected in the hearts of rats chronically fed a high-fat, ethanol-containing diet and receiving an additional acute ethanol load along with a spin-trapping agent. 17 Such free radicals have also been detected in heart extracts after an acute dose of ethanol or acetaldehyde.69 The increased conversion of XD into XO that has been detected in the heart after administration of a single ethanol dose, 1°° as well as in chronically ethanol-treated rats, 99 may contribute to this lipid peroxidation. Since chronic ethanol treatment elicits an enhanced activity of peroxisomal acyl CoA-oxidase and catalase in rat myocardium,92increased production of hydrogen peroxide resulting from the activation of acyl CoA-oxidase together with enhanced acetaldehyde generation through the catalase pathway may also play a role in the ethanol-induced enhancement in myocardial lipid peroxidation.239 Ethanol-induced disturbances in the antioxidant defense are also likely to be involved in the myocardial oxidative stress. 24° The myocardial glutathione level is thus decreased after acute and chronic alcohol intoxication) 29 On the other hand, Choy et al. TM reported that the ethanol-induced disturbances in phospholipid metabolism observed in isolated rat heart could be prevented by adding a-tocopherol to the perfusion. Prevention of some of the cardiotoxic effects of ethanol by vitamin E had been previously reported in mice. 242

Implication offree radical mechanisms in ethanol-induced testicular injury Rosenblum and colleagues243,244 reported that rats fed alcohol chronically had an increase in testicular lipid peroxidation accompanied by a decrease in peroxidizable polyunsaturated fatty acid and glutathione contents. The apparent direct correlation between the gross testicular atrophy observed and the reduced glutathione levels supports the concept that enhanced lipid peroxidation may be responsible, at least in part, for the toxic effects of ethanol on testes. This hypothesis is strengthened by the reported attenuation of ethanol-induced testicular lipid peroxidation and of testicular atrophy when feeding diets supplemented with

Ethanol and free radicals

vitamin A, which potentially can function as a free radical scavenger. 245 CLINICAL STUDIES

The experimental data have shown the importance of the liver as the site of ethanol-induced free radical attack. The clinical studies have been mostly dedicated to the search of evidence for free radical mechanisms in human alcoholic liver disease. Disturbances linked to lipid peroxidation, such as changes in the thiobarbituric acid reactive substances or diene conjugation, have been considered in liver and/or blood plasma in some of these studies, whereas others are concerned with ethanol-induced antioxidant disturbances. Suematsu and colleagues246 reported that heavy drinkers had higher levels of hepatic lipoperoxides than did nonalcoholic individuals. Of these heavy drinkers, cases with a remarkable high level of liver lipoperoxides showed a high incidence of hepatic cell necrosis. These results indicate a possible involvement of lipid peroxidation in alcohol-induced liver damage in humans. This suggestion was strengthened by the report of Shaw and co-workers, 247who observed in liver biopsies from alcoholics decreased hepatic glutathione and increased diene conjugates in hepatic lipids. These disturbances were present at all stages of alcoholic liver damage, including fatty liver, but unrelated to the nutritional status in these patients. An important and controversial question is the specific involvement of ethanol abuse in such disturbances. A decrease of hepatic glutathione248 and an increase in liver thiobarbituric acid reactive s u b s t a n c e s 249 w e r e reported in patients with alcoholic as well as nonalcoholic liver diseases. The severity of the liver disease may also be of crucial importance, since Peters and colleagues25°reported decreased hepatic phospholipid polyunsaturated fatty acid contents and enhanced breath alkane exhalation in patients suffering from alcoholic liver cirrhosis but not from milder forms of alcoholic liver diseases. The concentration of octadeca-9,1 1-dienoic acid [18:2(9, 1 1)] in serum phospholipids was reported to be significantly increased in 75% of chronic alcoholics and to return to normal on alcohol withdrawal.251,252 This nonperoxide isomer of linoleic acid has been identified in esterified form as the predominant diene conjugated acyl residue in the main serum lipid classes of human serum. 253 The mechanism of its formation is unclear, and elevations of its serum level have been reported in a variety of inflammatory processes. It is therefore probably not a marker specifi-

231

cally linked to ethanol intake. The proportion of this linoleic acid isomer in lipid extracts from liver biopsy specimens obtained from alcoholic subjects correlates significantly with inflammatory histological features and inversely with hepatic glutathione.T M Besides the disturbances in glutathione, many reports were dedicated to changes in other liver and blood antioxidants in alcoholics. Direct measurements of a-tocopherol in the liver of alcoholics have been reported by a few authors. In a limited group of patients, a lowered hepatic a-tocopherol level expressed per milligram of total lipids was apparent in alcoholic as well as in nonalcoholic liver diseases. 255A lowered a-tocopherol/total lipid ratio was also reported in 35 of 39 liver biopsy specimens from alcoholic subjects. However, the decrease in this ratio was unrelated to the severity of the cellular disturbances, as judged by histological criteria. 2s4 Majumdar and co-workers256observed that 30% of chronic alcoholics had reduced plasma vitamin E levels on admission. Bjorneboe and colleagues257,25s reported that about half of the alcoholic patients they considered had a serum concentration of a-tocopherol below the limit of reference ( 14 #tool/L). Furthermore, the mean serum concentration ofa-tocopherol was reduced by 37% as compared to controls. Tanner et al. 259 compared the vitamin E level in two groups of alcoholics, one with established alcoholic liver disease and the other comprising alcoholic patients without clinical and biological evidence of liver disease. They observed that the serum vitamin E level was lowered to a similar extent in both groups. No correlation between the serum a-tocopherol level and the nutritional status could be established. On the contrary, Ward and co-workers26° reported no disturbances in the serum ~-tocopherol concentration in the fatty and fibrotic group of alcoholic patients, whereas only in the cirrhotic group did the concentration of this vitamin decrease significantly. In contradiction with this report, Girre and colleagues26t reported that the vitamin E levels were significantly depressed in both plasma and erythrocytes in chronic alcoholic patients without liver cirrhosis. The plasma vitamin E levels were furthermore still low after 14 days of abstinence and not significantly different from the levels measured before abstinence. Whereas most reported data show a decreased plasma a-tocopherol level in alcoholic patients, at least in those who suffer from severe liver alcoholic disease, the mechanisms involved in this decrease are controversial. They may comprise malabsorption of vitamin E, decreased hepatic secretion of very low density lipoproteins involved in the transport of the vitamin in the plasma, as well as increased consump-

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tion of a-tocopherol linked to the scavenging of free radicals produced in excess, mainly in the liver. Another important antioxidant that has often been reported to be decreased by excessive alcohol intake is selenium. Whereas acute alcohol intake does not modify the serum selenium content in nonalcoholic volunteers, 262 a decrease in this content is generally observed in alcoholics. 258'259'261-267 Whereas this decrease has been attributed to the deterioration of liver function, irrespective of its etiology,262 other investigators reported decreased serum selenium in alcoholics with normal liver function.265 They observed, however, that the extent of this decrease is enhanced in the presence of overt liver dysfunction and appears correlated with the severity of liver disease. 264,265It has been reported to occur even in the absence of malnutrition 264but to correlate closely with poor nutritional status in other studies.259 Dworkin and co-workers268 measured the selenium content in autopsy livers from alcoholics and reported significantly decreased hepatic selenium stores in cases of severe alcoholic cirrhosis. Fewer data report disturbances of other blood serum antioxidants in alcoholics. Ward and colleagues26°'269 observed a decrease in the mean plasma concentration of/~-carotene that was progressive with increasing severity of liver damage. They also reported a decrease in the mean retinol concentration that only reached statistical significance in patients with cirrhosis. Since liver dysfunction and nutritional disorders appear to contribute largely to the described changes in antioxidant substrates among alcoholics, some investigators studied in such patients the status of antioxidant enzymes, which may more directly reflect disturbed free radical mechanisms. Most of these studies were dedicated to antioxidant enzymes in the erythrocytes. The report by Del Villano and colleagues27° of increased superoxide dismutase (SOD) levels in the erythrocytes of black alcoholics indicates that the determination of this enzyme may be a useful biological marker for alcohol abuse. Further reports, however, have been disappointing. Thus, Mongiat et al. TM as well as Bjorneboe et al. 272 did not find any significant change in the mean erythrocyte SOD activity in alcoholics. Emerit and co-workers reported either enhanced 273 or reduced 274 activities, depending on the method used to assay erythrocyte SOD activity. Ledig et al.275 reported that SOD activity in erythrocytes was at the control level in alcoholics without liver injury but increased significantly during the evolution of liver injury. Studying the activity of this enzyme in acute alcoholics on admission, Rooprai and co-

al.

workers276observed that it does not form a single population but clusters in two groups, either above or below the normal range. The values in both groups reverted toward the normal after a week ofdetoxification. Rooprai and colleagues suggested that the differences observed on admission are acute responses, possibly to diverse drinking patterns in the period immediately preceding admission. Erythrocyte catalase and glutathione peroxidase activities have been reported to be unchanged in alcoholics. 272 However, a decreased activity of both enzymes was found in another study in alcoholic patients, but only in those with liver cirrhosis. TM Finally, a reduced activity of glutathione peroxidase together with a rapid trend toward normalization during abstinence from alcohol have been reported in chronic alcoholics. 261 Another enzymatic disturbance has been reported by Harrison and co-workers, 277who observed that hepatocytes coexpressed both alpha and pi class glutathione-S-transferase during severe alcoholic liver disease. This coexpression had been previously described in humans only in the fetal liver.

CONCLUSION Numerous experimental data indicate that free radical mechanisms contribute to ethanol-induced liver injury. Increased generation of oxygen- and ethanolderived free radicals has been observed at the microsomal level, especially through the intervention of the ethanol-inducible cytochrome P450 isoform (CYP2E 1). Furthermore, an ethanol-linked enhancement in free radical generation can occur at the level of the cytosolic molybdo-flavoenzymes xanthine oxidase and aldehyde oxidase. The preferential localization ofCYP2E 1 and xanthine oxidase within perivenous hepatocytes is likely playing a role in the higher incidence of ethanol toxicity on these hepatocytes as compared to the periportal ones. Mitochondria and peroxisomes also appear to be implicated in the disturbed free radical generation in the liver following ethanol administration. The disturbances in nonheine iron availability, especially in its "free" components, may also contribute to the enhanced hepatic free-radical generation. The detection of l-hydroxyethyl radicals during ethanol metabolism in microsomal preparations as well as in vivo suggests that free radical mechanisms contribute to the oxidation of ethanol. The importance of this contribution to the overall ethanol elimination is likely not negligible27s and dependent on iron availability, as shown by the decrease in the ethanol elimination rate following des-

Ethanol and free radicals

ferrioxamine administration 278 and, on the contrary, its increase in iron-overloaded rats. 279 Besides increasing the generation of free radical species in the liver, ethanol administration elicits disturbances in the hepatic antioxidant defense, thus leading in many experimental conditions to an oxidative stress. This oxidative stress may result in enhanced hepatic lipid peroxidation. However, this is not constantly observed. Therefore, the controversy persists over the role of lipid peroxidation resulting from ethanol administration. The discrepancies between the reported data appear largely linked to the varying conditions of ethanol administration and of dietary intake of fat and antioxidants, such as vitamin E. Recent studies have emphasized the intervention of adaptative processes able to increase the antioxidant defense during chronic ethanol intake. They demonstrated an ethanol-induced generation ofa neutrophil chemoattractant, which likely results from peroxidative events and may contribute to the initiation of human alcoholic hepatitis. Other important cellular components, such as enzymes or nucleic acids, may also represent targets of the ethanol-induced oxidative stress. Free radical mechanisms also appear to be implicated in the toxicity of ethanol on various extrahepatic tissues. Most of the available data concern the gastric mucosa, the central nervous system, the heart, and the testes. The clinical studies have not yet demonstrated clearly the role of free radical mechanisms in the pathogenesis of ethanol-induced cellular injury. However, some data support the involvement of such mechanisms. The most consistent data concern changes in diene conjugates and in important antioxidants, such as a-tocopherol and selenium, in the blood serum of alcoholics. They suggest that dietary and/or pharmacological means to prevent an ethanolinduced oxidative stress may reduce the incidence of ethanol toxicity in humans. Well-controlled clinical trials are needed, however, to demonstrate clearly that such means are beneficial in alcoholic patients. Acknowledgments--Work from the authors' laboratory that is mentioned in this review was supported by INSERM (CRE 900207) and Haut Comit6 d'Etude et d'Information sur l'Alcoolisme. The authors wish to thank Maryse Dehauteur for typing the manuscript.

REFERENCES

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