Pathophysiology of alcoholic liver disease

Mo/ec. Aspects Med. Vol. 10, pp. 107-146, 1988

0098-2997/88 $0.00 + .50 Copyright © 1988 Pergamon Press plc.

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PATHOPHYSlOLOGY OF ALCOHOLIC LIVER DISEASE Charles S. Lieber Section of Liver Disease and Nutrition, Alcohol Research and Treatment Center, Bronx Veterans Administration Medical Center and Mount Sinai School of Medicine, New York, N.Y., U.S.A.

Mechanism of alcoholic liver disease include either "direct" toxic effects or malnutrition (Fig. I). Malnutrition includes primary and secondary varieties. Secondary malnutrition in turn is subdivided into consequences of a poor supply (inadequate diets and/or malabsorptlon) on the one hand and alterations of either the activation or degradation of key nutrients on the other. The latter "nutritional" effects do not differ fundamentally from various other "toxic" effects (Fig. 2).

Fig. I. Interaction of direct toxicity of ethanol with malnutrition due to primary or secondary deficiencies. Secondary malnutrition may'be caused either by maldlgestlon and malabsorptlon, or by impaired utilization (decreased activation and/or increased inactivation) of nutrients. Both "direct" toxicity of ethanol and malnutrition (whether primary o r secondary) may affect function and structure of liver and gut (Lieber, 1982). 107

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ETHANOL Physical l Dependence? I. 41, t ~ Toxicity ~ ~ Secondary Malnutrition ('vitamins | ~ /~ ~b-2 [ ~ I ~prote ns Organelle ,q~.~l~ Immunologic .J \ ~.j~ ~/ ~. Dysfurcliofl? ~ ' Stimulation? ~ - ~ ~ ]','~, ,= ,,', J O" (1. I "¢ • • MEMBRANE ALTERATIONS a,e--~a.s HypOXC~age "~ UsableEnecgy f Hypedupemla ~ .... iMICROSOMAL ...... ~ . . . . la-2 ADH ~ ~ Metabolic ]H"y~lly"-ce--mi"-aCYTOCHROME P-450 INDUCTION ~ O= =__ ~ Derangementa~Hypedactacidemia Accelerated f a-3 I B-1 r |Denrused Fat Oxidation Metabolism of D~gs ~ ~ ~ / ~IncreMed Collagen " ~ MEOS-~ACETALDEHYDE NADH ~ . Synb'leal$

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Fig. 2. Hepatic nutritional and metabolic abnormalities after ethanol abuse. Malnutrition (b), whether primary (b-l) or secondary (b-2), with direct toxic effects (a). The latter have been attributed, In part, to redox changes (a-i), hypoxla (a-2), acetaldehyde (a-3), direct membrane alterations (a-4), or effects secondary to mlcrosomal induction (a-5) (Lieber, 1988). Respective Roles of~Nutrltlonal and Ethanol-Induced Alterations in Disturbances of the Energy Balance and of Hepatic Integrity. Whereas traditionally, the disorders affecting the liver had been attributed exclusively to nutritional deficiencies accompanying alcoholism (Best et al., 1949) (Fig. i), studies carried out over the last three decades indicate that in addition to the role of dietary deficiencies, alcohol per se can be incriminated as a direct etiological factor in the production of alcoholic liver disease. Indeed it was demonstrated that even in the absence of dietary deficiencies, alcohol can lead to the development of fatty liver in man (Lieber et al., 1963, 1965). An experimental model for this toxic effect was created by overcoming the natural aversion of the rodent for alcohol by incorporating the ethanol in a totally liquid dlet (Lieber et al., 1963, 1965; DeCarll and Lieher, 1968, Lleber and DeCarli 1982, 1986). Application of this liquid diet feeding technique to the baboon resulted in the demonstration of a direct casual role of ethanol in the pathogenesls of liver cirrhosis (Lieber and DeCarll, 1974). While alcohol depresses appetite, the liquid diets still meet nutritional requirements (Lieher and DeCarli, 1982, 1986; Editorial, 1988; Preedy et al., 1988). Some objections raised concerning the diet (Rao and Larkln, 1987) have been refuted (Lieber and DeCarll, 1987). Tampering with the energy density of ethanol in the liquid diet resulted in a decrease of blood alcohol to negligible levels (Rao et al., 1987), which defeats the purpose of the model, namely to mimic, in the rat, the high blood alcohol concentrations and some aspects of liver injury observed in human alcoholics. By raising the alcohol intake to clinically meaningful levels while facilitating the manipulation of dietary constituents one by one, and maintaining the same nutrient intake in control animals, the original liquid diet feeding technique has allowed for the definition not only of the various toxic effects of ethanol, but also of the interactions of ethanol wlth nutritional factors (Lieber and DeCarli, 1970a, 1982, 1986; Wilson et al., 1986a,b, 1988). Alcohol is rich in energy and in many societies, alcoholic beverages are considered part of the basic food supply. In healthy nonalcoholic volunteers, ethanol is utilized as efficiently as fat or carbohydrate as a source of energy (Atwater and Benedict, 1902), but alcoholics given additional calories as alcohol

Pathophysiology of Alcoholic Liver Disease

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under controlled conditions failed to gain weight (Lleber et al., 1965). Despite higher caloric intakes, drinkers are not more obese than nondrinkers (Gruchow et al., 1985). Under metabolic ward conditions, isocalorlc substitution of ethanol for carbohydrate, up to 50% of total energy In a balanced diet, resulted in a decline in body weight and, when given as additional calories, ethanol caused less weight galn than calorically equivalent carbohydrate or fat (Plrola and Lleber, 1972) or no weight galn in lean individuals (Crouse and Grundy, 1984). In several studies, (with some exceptions) (Barnes et al., 1965), administration of alcohol to moderate drinkers resulted In increased oxygen consumption (Tremolleres and Carte, 1961; Perman, 1962) and thls effect was much greater in alcoholics (Tremolleres and Carre, 1961). Substitution of ethanol for carbohydrate increased the metabolic rate and thermogenesls of humans and rodents (Stock and Stuart, 1974). Although some of the energy wastage was attributable to brown fat thermogenesls in rats, most of it could not be so explained (Rothwell and Stock, 1984). One postulated mechanism of energy wastage when ethanol Is consumed Is via oxidation without phosphorylatlon by the mlcrosomal ethanol oxidizing system (MEOS) (Plrola and Lleher, 1976). Indeed, whereas

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Flg. 3. Oxidation of ethanol in the hepatocyte and link of the two products (acetaldehyde and H) to disturbances in intermediary metabolism. NAD, nlcotlnamide adenine dlnucleotlde; NADH, reduced NAD; GSH, reduced glutathlone; GSSG, oxldzed glutathlone. The broken lines indicate pathways that are depressed by ethanol. The symbol -[ denotes interference or binding; --~--~--, stimulation or activation.

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ethanol oxidation to acetaldehyde via the ADH pathway is associated with the generation of NADH, a high energy compound, this is not the case when ethanol is oxidized via MEOS (Fig. 3). In the latter pathway, a high energy compound (NADPH) is in fact utilized and no high energy compound is being formed. The reaction only generates heat. To the extent that this calorigenesis exceeds the needs for thermoregulation, this can be considered as energy wastage. Contrasting with the NADH pathway, which has a low Km (about ImM), the MEOS has a relatively high Km (about 10mM), and therefore its impact can be expected to be greater at high rather than at low blood ethanol levels. Furthermore, this pathway is induced by hronic ethanol consumption, which was noted to aggravate the energy wastage (Pirola and Lieber, 1972, 1976). Another mechanism to explain energy wastage is the uncoupling of mitochondrial NADH re-oxidatlon, perhaps promoted by catecholamine release or a hyperthyroid state (Israel et al., 1975), although the hyperthyroid aspect of the mechanism is controversial (Teschke et al., 1983). Hepatic ATP content is reduced after chronic ethanol feeding (French, 1966; Bernstein et al., 1973; Spach et al., 1982). This decrease in hepatic ATP content is consistent with decreased energy available from ethanol and also decreased production of ATP secondary to mitochondrial damage. The latter has been demonstrated in a variety of species, including man (Lane and Lieber, 1966) and subhuman primates (Arai et al., 1984a,b), with damage to the electron transport chain and altered oxidative phosphorylation. Increased ATPase activity may also be involved (Videla et al., 1973)$ However, some reports indicate that the magnitude of the increase in Na+,K -ATPase activity is only 15% (Gordon, 1977), an amount unlikely to account for changes in ATP levels. Inhibition of glycolysls by ethanol is another possible mechanism for decreased ATP, at least in vitro and under hypoxic conditions (Younes and Strubelt, 1987). However, whether hypoxia plays a significant role in alcoholic liver injury is the subject of debate (vide Infra). Whereas those hospitalized for medical complications (particularly liver disease) may be severely malnourished with antecedents of inadequate dietary protein (Patek et al., 1975) and signs of protein malnutrition (Iber, 1971; Mendenhall et al., 1985), the vast majority of the alcohol consuming public has slight (if any) detectable nutritional impairment and many ambulatory subjects who drink to excess are not malnourished. However, as alcohol intake increases, there is a decrease in percent of energy derived from protein, fat and carbohydrate and the nutritional quality of the diet declines (Hillers and Massey, 1985; Sherlock, 1984). In addition, ethanol ingestion enhances nitrogen loss in urine of both rats (Klatskln, 1961) and man (McDonald and Margen, 1976; Bunout et al., 1987) thereby possibly increasing protien requirements. Protein, methlonine and choline deficiency has been incriminated in the pathogenesis of liver injury for several decades because in growing rats, deficiencies in dietary protein and lipotroplc factors (choline and methlonine) can produce a fatty liver (Best et al., 1949) and it has been reported that ethanol increases choline requirements in the rat (Klatskin et al., 1954), possibly by enhancing choline oxidation (Thompson and Reltz, 1976). Primates, however, are far less susceptible to protein and lipotropic deficiency than rodents (Hoffbauer and Zakl, 1965). Clinically, choline treatment of patients suffering from alcoholic liver injury has been found to be ineffective in the face of continued alcohol abuse (Olson, 1964; Phillips and Davldson, 1954; Post et al., 1952; Volwiler et al., 1948). Furthermore, massive supplementation with choline failed to prevent the fatty liver produced by alcohol in volunteer subjects (Rubln and Lieber, 1968). This is not surprising since, unlike rat liver, human liver contains very little choline oxldase activity, which may explain the species differences with regard to choline deficiency. Moreover, fatty liver as well as fibrosis (including cirrhosis) developed in baboons despite liberal amounts of methionine (Lieber and DeCarll, 1974) and massive supplementation with choline, even to the point of

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toxicity (Lieber et el., 1985). However, in addition to its possible role as lipotrope, methionine may also exert a more specific effect as a selective precursor of cysteine (vide infra). Alterations of other nutritent activation, utilization and degradation, resulting in altered bioavailability, and their possible impact on liver function is discussed in detail elsewhere (Lieber, 1988). In summary, at the subcellular biochemical level, the original dichotomy between nutritional and toxic effects of ethanol has been bridged, and, in that regard, the classic separation between nutritional and toxic effects of ethanol is now one of semantics rather than of substance. ADH Mediated Ethanol Metabolism and Associated Metabolic and Pathologic Effects. Liver ADH exists in multiple molecular forms which arise from the association, in various permutations, of different types of subunits (Li, 1988). Whatever the form, in ADH-mediated oxidation of ethanol, hydrogen is transferred from the substrate to the cofactor nicotinamide adenine dinucleotide (NAD), converting it to reduced form (NADH) (Fig. 3). As a net result, ethanol oxidation by ADH generates an excess of reducing equivalents as free NADH in hepatic cytosol, primarily because the metabolic systems involved in NADH removal are not able to fully offset the accumulation of NADH. The acetaldehyde produced in this reaction is converted to acetate by aldehyde dehydrogenase, which is also associated with conversion of HAD to NADH. The increased NADH/NAD ratio is a sign of a major change in liver metabolism during ethanol oxidation to which several hepatic and metabolic disorders associated with alcohol abuse have been attributed (Lieber et al., 1959; Lieber and Schmid, 1961; Lieber and Davidson, 1962). This notion was useful in furthering our understanding of ethanol-induced changes in lipid, protein, carbohydrate (hypoglycemia) and uric acid metabolism, as reviewed elsewhere (Lieber, 1982). This concept was more recently revived following the demonstration that ethanol-induced redox changes are exacerbated in the perivenular areas of the liver (Jauhonen et al., 1982). Perivenular Injury A characteristic feature of liver injury in the alcoholic is the predominance of lesions in the perivenular (also called centrilobular) zone or zone 3 of the hepatic acinus. The mechanism for this zonal selectivity of the toxic effects of ethanol remains unknown. Two distinct but not mutually exclusive hypotheses have been raised: one claims that ethanol can produce hypoxic damage of perivenular hepatocytes, whereas the other postulates that conditions normally prevailing in the perivenular zone enhance the metabolic toxicity of ethanol. Hypoxia The hypoxia hypothesis originated from the observation that liver slices from rats fed alcohol chronically consume more oxygen than those of controls (Videla and Israel, 1970). It was then postulated that the increased consumption of oxygen would increase the gradient of oxygen tensions along the sinusoids to the extent of producing anoxic ~njury of ~erivenular hepatocytes (Israel et al., 1975). Such a mechanism was illustrated experimentally when centrilobular liver cell necrosis was induced by hypoxia in chronic ethanol-fed rats (French et al., 1984). Furthermore, both in human alcoholics (Kessler et al., 1954) and in animals fed alcohol chronically (Jauhonen et al., 1982; Sato et al., 1983), decreases in either hepatic venous oxygen saturation (Kessler et al., 1954) or PO 2 (Jauhonen et al., 1982) and in tissue oxygen tensions (Sato et el., 1983) have been found during the withdrawal state. However, this decrease is within

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the range of values found in normal subjects and hepatic venous oxygen content, determined as either oxygen saturation or oxygen tension, was found to be normal in patients with chronic alcoholic liver disease (Bendtsen et al., 1987). Moreover, the differences in hepatic oxygenation found during the withdrawal state disappeared (Jauhonen et al., 1982; Shaw et al., 1977) or decreased (Sato et al., 1983) when alcohol was present in the blood. Acute ethanol administration increased splanchnic oxygen consumption in naive baboons, but the consequences of this effect on oxygenation in the perivenular zone were offset by increased blood flow resulting in unchanged hepatic venous oxygen tension (Jauhonen et al., 1982). Indeed, ethanol in fact induces an increase in portal hepatic blood flow (Stein et al., 1963; Shaw et al., 1977; Jauhonen et al., 1982; Carmichael et al., 1987). In baboons, defective O_ utilization rather than lack z of 02 blood supply characterized liver injury produced by high concentrations of ethanol (Lieber et al., 1988). Indeed, in 5 baboons fed control diets, hepatic vein catheterization revealed that a moderate ethanol dose, resulted in increased splanchnlc O^ consumption, offset by an increase in flow sufficient to prevent z lack of Op, as judged from normal hepatic venous PO2, tissue hemoglobin concentration (Hb) and 09 saturation of Hb (SO9). Flow independent tissue O 9 consumption (VO2) also in£reased. Hb, SO 2 and VOp were measured by hepatic surface reflectance spectrophotometry through perltoneoscopy. After high ethanol, VO? failed to increase (or even decreased), without any decrease in SO^ or in hepatic venous PO2, indicating impaired capacity of the hepatocytes to tak~ up 02 , even in the presence of an ample O9 supply. Thus, defective O? utilization rather than lack of blood 02 supply characterizes ethanol-inducea liver injury. The latter, however, may sensitize the liver to extraneous hypoxia (Miyamoto and French, 1988) and conversely, hypoxia may potentiate the hepatotoxlcity of ethanol (Younes and Strubelt, 1987; Desmoulln et al., 1987). Although the theory of alcohol induced liver necrosis secondary to a "hypermetabolic state" is still controversial, it has led to therapeutic trials such as the treatment of alcoholic liver disease with propylthlouracil. Previous short-term treatment efforts have yielded conflicting results, but a recent long-term study reported a lowered mortality in the treated group (Orrego et al., 1987). Curiously, the beneficial effect was not observed in patients with high alcohol consumption: it was restricted to those in whom alcohol intake was moderate, a group known to have a good outcome even without specific treatment. The mechanism of the beneficial effect is not clear. Propylthiouracil has been shown experimentally to protect against alcohol-lnduced hepatocellular necrosis in hypoxlc conditions but such hypoxic conditions were not documented in the type of patients in whom the beneficial effects were obtained. One wonders to what extent a change in alcohol consumption might have been responsible for the better outcome. Redox shift. An alternative hypothesis to explain the selective perivenular hepatotoxicity of ethanol postulates that the low oxygen tensions normally prevailing in perlvenular zones could exaggerate the redox shift produced by ethanol (Jauhonen et al., 1982). To study the magnitude of such a shift in the baboon, the effects of ethanol on the lactate/pyruvate ratio in hepatic venous blood (an approximation of that in perlvenular hepatocytes) were compared with the ratio in total liver. Ethanol increased the lactate/pyruvate ratio and decreased pyruvate more in hepatic venous blood than in total liver. In isolated rat hepatocytes, the ethanol-lnduced redox shift was markedly exaggerated by lowering the oxygen to a tension similar to those found in centrilobular zones. The process was also assessed in the isolated perfused rat liver, by varying the oxygen supply, to reproduce the oxygen tensions prevailing in vlvo along the slnusold (Jauhonen et al., 1985). Varying the oxygen tensions within the physiological range produced

P a t h o p h y s i o l o g y of A l c o h o l i c Liver Disease

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+ a r e d o x g r a d i e n t o f b o t h cytochrome o x i d a s e and NAD . The d e g r e e o f r e d u c t i o n o f cytochrome o x i d a s e a t t h e s e p h y s i o l o g i c a l oxygen t e n s i o n s was n o t a s s o c i a t e d w i t h impairment in the ability of the liver t o consume oxygen and t o produce ATP, suggesting a lack of cellular anoxia. ~enty-five ~LH e t h a n o l i n c r e a s e d h e p a t i c oxygen c o n s u m p t i o n , b u t had no d i r e c t effect on t h e s t a t e o f r e d u c t i o n o f cytochrome o x i d a s e . The e f f e c t s o f e t h a n o l and oxygen t e n s i o n s on NADH change were additive, indicating that a greater redox shift should occur when ethanol is oxidized at oxygen tensions similar to those normally prevailing in perivenular zones than at those in periportal zones. This dependence of the ethanol-induced redox shift on oxygen tensions may contribute to the selective perfvenular hepatotoxlcity of alcohol (Jauhonen et al., 1982). Indeed, many toxic effects of ethanol are linked to the ability of ethanol to shift the redox equilibrium toward a reduced state. Interaction of the redox changes with lipid metabolism have been discussed before (Lieber, 1982; Lieber and Pignon, 1987), including inhibition of fatty acid oxidation (Lieber and Schmid, 1961), which may be of relevance for the perivenular exacerbation of fat accumulation. Inhibition of protein synthesis has also been observed after addition of ethanol to various preparations in vitro (Jeejeebhoy et al., 1975; Rothschild et al., 1971). In vivo, the acute effects of ethanol on protein synthesis have been less consistent than those described in vitro. No changes in the synthesis of total liver protein were found after administration of ethanol to well fed naive rats (Baraona et al., 1980). It is noteworthy that with progression of fibrosis, estimated regional hepatic tissue hemoglobin concentration may decrease; this decreased oxygen supply to the liver may have an important role in the progression of alcoholic live~ disease (Hayashi et al., 1985). Indeed, NADH inhibits the activity of NAD -dependent xanthine dehydrogenase (XD), thereby favoring that of oxygen-dependent xanthine oxidase (XO) (Kato et al., 1988). It has been postulated that due to hypoxia and ethanol, purlne metabolltes and acetaldehyde accumulate and could be metabolized via XO. This process may lead to the production of oxygen radicals which most probably mediate both inhibition of glycolysis and the direct toxic effects towards liver cells, including peroxldation (Younes and Strubelt, 1987). However, cyanamide (a potent aldehyde dehydrogenase inhibitor) increased acetaldehyde levels 5 fold after ethanol, while lipid peroxidation was significantly decreased by this treatment, indicating that acetaldehyde was not the substrate involved (Kato et al., 1988). Physiological substrates for XO, hypoxanthine and xanthine, as well as AMP, significantly increased in the liver after ethanol, together with an enhanced urinary output of allantoin (a final product of xanthine metabolism). Allopurinol pretreatment resulted in 90% inhibition of XO activity, and also significantly decreased ethanol induced lipid peroxldatlon (Kato et al., 1988). Zonal distribution of enzyme activities. Zonal distribution of some enzymes can influence the selective perivenular toxicity. Proliferation of the smooth endoplasmic reticulum after chronic ethanol consumption is maximal in the perivenular zone with associated enzyme induction and related effects (discussed subsequently). There also might be more ADH activity in the perivenular zone, although this has been the subject of a longstanding debate. Early hlstochemical studies carried out on rat liver either showed periportal maxima of ADH activity (Greenberger et al., 1965) or were unable to demonstrate any uneven distribution pattern (Berres et al., 1970), contrasting with th~ microquantltatlve measurements of Morrison and Brock (1967), which had originally revealed that the activity of ADH in the perlcentral area of the liver lobule in man and in female rats was about 1.7 times higher than in the perlportal area. By means of modern immunohlstochemical techniques, human ADH has now been demonstrated malnly'In hepatocytes around the terminal hepatic venule (Buehler et al., 1982). Thus, the differential distribution of ADH, with a presumably higher level in the perlvenular zone, could contribute to the

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increased hepatotoxicity of ethanol by providing (together with the "induced" microsomal pathway) an increased amount of the toxic metabolite acetaldehyde (Lieber, 1985). One must, however, also take into account that after chronic ethanol consumption, unlike the activity of MEOS, which is induced, that of ADH may not change or even may decrease. Indeed, although some discrepant results had been published before (Hawkins et al., 1966; Hawkins and Kalant, 1972), the same group has subsequently reported that ADH activity does not increase after chronic ethanol feeding (Videla et al., 1973; Kalant et al., 1975), a finding consistent with the observation of others (Singlevich and Barboriak, 1971; Raskin and Sokoloff, 1972; de Saint-Blanquat et al., 1972). In some studies, there was actually a decrease of ADH activity in the liver after chronic ethanol consumption (Lieber and DeCarli, 1970a; Brighenti and Pancaldi, 1970; Salaspuro et al., 1981), a finding which is consistent with the observation that alcoholics may display decreased hepatic ADH activity even in the absence of liver damage (Ugarte et al., 1967). In view of the observation of Crow et al. (1977) that, under certain circumstances, the level of ADH activity may become a major rate-limitlng factor for the metabolism of ethanol, the decrease of ADH after chronic ethanol consumption may, on occasion, acquire functional significance. Fibrosis. The zonal nature of alcohol induced liver injury is particularly important concerning fibrosis. Indeed, regardless of mechanism, cellular injury triggers a fibrotic response with, as its most striking early manifestation, a ring of perivenular fibrosi& (PVF). The role of the redox-mediated increase of lactate in collagen production has been reviewed (Lieber, 1981, 1982) and the possible effects of acetaldehyde are discussed subsequently. Of practical importance is the fact that once PVF appears, it reflects initiation of the fibrotic process. Individual susceptibility to the development of cirrhosis varies and therefore, it is important to recognize, already at an early and still reversible stage, those individuals prone to progress to cirrhosis. Sequential studies, both in baboon and man, have shown that PVF has prognostic value for the ultimate development of cirrhosis. PVF is commonly associated with some interstitial pericellular fibrosis (Van Waes and Lieber, 1977); the latter has been confirmed and emphasized (Nasrallah et al., 1980). From a prognostic point of view, however, it is the recognition of PVF in needle biopsies of liver tissue which is most practical and valuable. This lesion had been described before in association with full-blown alcoholic hepatitis (Edmondson et al., 1967), but it can already occur at the fatty liver stage in the absence of hepatitis (Van Waes and Lieber, 1977). Experimental studies in alcohol-fed baboons have shown that in those animals which progressed to cirrhosis, PVF invariably occurred already at the fatty liver stage; in contrast, animals that did not show the lesion did not progress beyond the stage of fatty liver. These studies have now been expanded by Worner and Lieber in man (1985): they showed that patients with PVF at the fatty liver stage were much more likely to progress to more severe stages of alcoholic liver disease, if they continued to consume alcohol. Thus, PVF can now be considered as an early warning sign of impending cirrhosis, if drinking continues. The concept of PVF as a precirrhotic lesion in alcoholics is further strengthened by similar observations in patients with fatty livers after bypass operation for morbid obesity (Marrubbio et al., 1976) and in diabetic individuals (Falchuck et al., 1980). To detect this preclrrhotic lesion, liver biopsy is the only reliable method at present, but it has obvious limitations in its large scale applicability and does not necessarily reflect ongoing fibrogenic activity. As a non-invasive test to assess liver fibrosis, blood measurements of several substances or enzymes involved in collagen metabolism such as prollne, hydroxyproline, procollagen peptides, lactic acid and prolyl hydroxylase activity have been carried out. Among these tests, radloimmunoassay for the aminoterminal peptldes of type III procollagen (P-Ill-P), either using the original technique

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of Rohde et al. (1983) or some modification thereof (Pierard et al., 1984; NiemelH, 1985), appears to be the most widely used. However, the assay for P-III-P failed to differentiate patients with fatty liver from those with fatty liver and early fibrosis (Savolalnen et al., 1984). More recently, a modified P-III-P assay using Fab fragments of the antibody (Fab-P-III-P) has been developed; it detects degraded fragments of propeptides in addition to the intact propeptide for which the original P-III-P assay has a high selectivity (Rohde et al,, 1983). Values of Fab-P-III-P above 45 mg/ml detected the majority of patients with fibrosis, including 55% of the subjects with perivenular fibrosis, 62% with septal fibrosis and more than 90% of the patients with cirrhosis (Nouchi et al., 1987). Although several studies have dealt with the relationship between propeptides and the biosynthesis and degradation of collagen, the mechanism controlling this process is still obscure. Interstitial collagens are synthesized intracellularly as procollagens that contain extension propeptldes at the amino and carboxyl ends of their three polypeptide chains. After secretion, conversion of procollagen to collagen and assembly into fibrils occurs. While the aminopropeptides of Type I procollagen are released at an early phase of flbrillogenesis, those of Type III procollagen are retained in epidermal collagen fibrils even at a mature stage in the dermis (FleischmaJer et al., 1983) and liver (Sato et al., 1986). The presence of propeptldes in hepatic fibrils is relevant for measurements of their fragments in the serum. As a reflection of the concept that the extension propeptides are cleaved from procollagen upon extrusion from the cell, it had been proposed that measurement of these circulating peptides may reflect the process of collagen synthesis. The documentation that such peptides are part of the collagen fibers Indicates that their appearance in blood reflects not only synthesis but also breakdown. Such a process might explain why the elevated serum levels observed in patients with liver diseases correlate not only with the degree of hepatic fibrosis but also with the severity of inflammation (Savolainen et al., 1984; Nouchi et al., 1987). Role of Gastric ADH in the Bioavailability of Ethanol It is generally recognized that the liver is the main site of ethanol metabolism (Lieber, 1982), and the magnitude of gastrointestinal ethanol metabolism was assumed to be small (Lamboeuf et al., 1981, 1983). Some authors (Lin and Kessler 1981) could not demonstrate any significant gastrointestinal ethanol oxidation when they gave an acute high dose to rats and concluded that this process was of negligible quantitative significance. The issue was reopened more recently by showing that a significant fraction of alcohol ingested in doses in keeping with usual "social drinking" does not enter the systemic circulation in the rat and is oxidized mainly in the stomach (Julkunen et al., 1985a,b). This process was also shown to occur in man (Fig. 4) (DiPadova et al., 1987a). The magnitude of this process in the rat was found to amount to about 20% of the ethanol administered when given at a low dose (Caballeria et al., 1987). In alcoholics, first pass metabolism was much smaller and after fasting, the first pass metabolism had virtually disappeared (DiPadova et al., 1987a) (Fig. 4). Reduced first pass metabolism in alcoholics may be due, in part, to diminished ADH activity, since a significant decrease of gastric ADH activity has been documented in rats fed alcohol-contalnlng diets chronically (Julkunen et al., 1985a,b) and since alterations in the rate of gastric emptying do not appear to be contributory (Keshavarzian et al., 1986). Thus, it is appears that gastric ADH and the associated ethanol metabolism decrease the bioavailabil~ty of ethanol and may represent a "barrier" to penetration of ethanol into the body and thereby could modulate its potential toxicitF. After chronic ethanol consumption, much of that "barrier" appears to be lost and, hence, systemic effects of ethanol will be exacerbated. This barrier may also be affected by common drugs, such as

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cimetidine, which decrease the activity of gastric ADH, peripheral blood levels of ethanol (Caballeria et al., 1987).

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Fig. 4. First-pass metabolism of ethanol in 5 nonalcohollcs and 7 alcoholics after ethanol (0.15 g/kg body wt). Ethanol was administered in a 5% dextrose solution (5 g/100 ml) orally or intravenously 1 h after a standard breakfast. Drinking time was 10 mln and that of the intravenous infusion was 20 min. The black area in the figure represents the amount of ingested alcohol that did not enter the systemic circulation and expresses the magnitude of the flrst-pass metabolism of ethanol. It was much lower in alcoholics than in nonalcoholics (DiPadova et al., 1987a). Metabolism of Alcohol via the Microsomal Ethanol Oxidizing System (MEOS): Associated Hepatotoxicity and Interaction with other Drugs and Hepatotoxic Agents Role of ME0S in Ethanol Metabolism It was formerly assumed that the primary pathway for hepatic ethanol metabolism involves cytosolic ADH. Non-ADH mediated ethanol oxidation occurring in liver mlcrosomes was attributed to a hydrogen peroxide-dependent reaction promoted by contaminating catalase, because this oxidative reaction exhibited a substrate specificity for methanol rather than long-chaln aliphatic alcohols and was exquisitely sensitive to inhibitors of catalase activity such as azide and cyanide (Orme-Johnson and Ziegler, 1965; Ziegler, 1972). The observation in rats (Iseri et al., 1964, 1966) as well as in man (Lane and Lieber, 1966) that chronic ethanol consumption was associated with proliferation of microsomal membranes prompted the suggestion that liver microsomes could be a site for a distinct and adaptive system of ethanol oxidation. Indeed, such a system was demonstrated in vitro and named the mlcrosomal ethanol oxidizing system (MEOS) (Lieber and DeCarll, 1968, 1970a). Rates of MEOS-catalyzed ethanol oxidation were ten-fold higher than those originally reported by Orme-Johnson and Ziegler and, based on various studies, it was concluded that the MEOS was distinct from ADH and

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catalase and dependent on cytochrome P450. This proposal initiated a decade of research and a lively debate that was finally resolved after: a) isolation of a P450-containing fraction from liver mlcrosomes which, although devoid of any ADH or catalase activity, could still oxidize ethanol as well as higher aliphatic alcohols (e.g., butanol which is not a substrate for catalase) (Teschke et al., 1972, 1974; Mezey et al., 1973) and b) reconstitution of ethanol-oxldizlng activity using NADPH-cytochrome P450 reductase, phospholipid, and either partially-purified or highly-purifled microsomal P450 from untreated (Ohnlshi and Lieber, 1977) or phenobarbltal-treated (Miwa et al., 1978) rats. That chronic ethanol consumption results in the induction of a unique P450 was shown by Ohnishl and Lieber (1977) using a liver mlcrosomal P450 fraction isolated from ethanol-treated rats. An ethanol-lnducible form of P450 (LM3a), purified from rabbit liver microsomes (Koop et al., 1982; Ingelman-Sundberg and Johansson, 1984), catalyzed ethanol oxidation at rates much higher than other P450 isozymes, and also had an enhanced capacity to oxidize l-butanol, l-pentanol, and aniline (Morgan et al., 1982), acetaminophen (Morgan et al., 1983), CC14 (Ingelman-Sundberg and Johansson, 1984), acetone (Koop and Cassaza, 1985), and N-nitrosodimethylamine (NDMA) (Yang et al., 1985). Similar results have been obtained with cytochrome P450j, a major hepatic P450 isozyme purified from ethanol- or isoniazid-treated rats (Ryan et al., 1985, 1986). Others have also provided evidence for the existence of a P450j-like Isozyme in humans (Wrighton et al., 1986, 1987; Song et al., 1986) however, its catalytic activity toward

]

2:5

4

5

6

7

8

Fig. 5. SDS-PAGE of human mlcrosomes and purified cytochromes P-450. Samples were analyzed on a slab gel 0.75 mm thick containing 7.5% acrylamide using a discontinuous buffer system. Migration proceeds from top to bottom. Lanes 2 and 7, mlcrosomes (I0 ~g); lanes 3, 4 and 5, cytochrome P-450-B, P450-ALC (P45011EI) and P450-C, respectively, (0.5 ~g); lane 6, mix of all three P450s (0.25 pg each); lanes 1 and 8, protein standards with molecular weights of 98,000, 68,000, 58,000, 53,000, 43,000 and 29,000 (0.5 ug each) (From Lasker et al., 1987a).

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ethanol was not described. We now have succeeded in obtaining the purified human protein (Fig. 5) in a catalytically active form, with a high turnover rate for ethanol and other specific substrates (Lasker et al., 1987a). In a new nomenclature of cytochromes P450, it was proposed that the ethanol-inducible form be designated as P45011EI (Nebert et al., 1987). However, other microsomal cytochrome P450 isozymes can also contribute to ethanol oxidation (Lasker et al., 1987b). Thus, the designation P45011EI should be reserved for this specific P450 alcohol oxygenase, whereas the term microsomal ethanol oxidizing system (MEOS) should be maintained when one refers to the overall capacity of the microsomes to oxidize ethanol rather than to that fraction of the activity which is specifically catalyzed by P45011EI. MEOS has a relatively high K for ethanol (8-10 mM compared with 0.2-2 mM for m ADH) and thus normally ADH accounts for the bulk of ethanol oxidation at low blood ethanol levels; however, this may not be true at high ethanol levels and/or after chronic use of alcohol. MEOS may play a highly significant role in ethanol oxidation under these circumstances, in part because of its inducibility (Lieber and DeCarli, 1968, 1970a). Indeed, most inhibitor studies have supported the idea of a significant role for non-ADH pathways, especially after chronic ethanol treatment or at high ethanol concentrations (Lieber and DeCarli, 1970a, 1972; Papenberg et al., 1970; Grunnet et al., 1973; Matsuzaki et al., 1981). While data obtained with inhibitors are suggestive of MEOS involvement, they cannot be considered conclusive since inhibitors are not sufficiently specific. A mutant deermouse strain that lacks ADH nevertheless actively oxidizes ethanol (Burnett and Felder, 1978, 1980; Shigeta et al., 1984), providing clear evidence that non-ADH pathways can be important in vivo. Availability of this strain has also stimulated research on the respective roles of MEOS and catalase in non-ADH ethanol metabolism (Shigeta et al., 1984; Glassman et al., 1985; Takagi et al., 1986; Handler et al., 1986; Kato et al., 1987). Catalase has generally been assumed to play a minor role, due principally to the limited intracellular production of H20 p (Boveris et al., 1972), Recently, however, the debate over the respective roles of MEOS and catalase in non-ADH ethanol oxidation has intensified with much of it centering on the true effects of the inhibitors employed. The catalase inhibitor, azide, does not significantly affect other ethanol pathways in vitro at low concentrations (Teschke et al., 1974, 1976). In hepatocytes from ADH- deermice, azide has little non-specific effects (Kato et al., 1987) but it is a potent metabolic poison and cannot be used in vivo. The other commonly used catalase inhibitor, 3-amino-l,2,4-triazole (AT), is used in vivo but it also inhibits microsomal enzymes (Kato, 1967) and ADH (Feytmans and Leighton, 1973). The current controversy over whether catalase plays a role in non-ADH metabolism originated, to some degree, from the failure to take into account the multiple effects of AT. Thus, the small depression in ethanol metabolism sometimes reported after AT administration was found to quantitatively match a moderate inhibition in MEOS activity (Kato et al., 1987). Another study (Handler et al., 1986) reported that 1.5 hrs after AT administration to deermice, ethanol elimination is inhibited by 85%. While this contradicts virtually every other report on the effects of AT in either deermlce (Shigeta et al., 1984; Takagi et al., 1986; Kato et al., 1987) or rats (Kinard et al., 1956; Tephly et al., 1964; Roach et al., 1972), it does illustrate the kind of problem associated with inhibitors: secondary effects of unknown magnitude are likely. The ADHdeermouse offered the possibility of studying non-ADH pathways without the use of inhibitors. Even in this animal, however, there remain two non-ADH pathways to be distinguished. This can also be done without inhibitors, by using the fact that each pathway of ethanol metabolism displays a characteristic intrinsic isotope effect. The bond of iR-hydrogen in ethanol is enzymatically broken in the first step of ethanol metabolism. If the IR-hydrogen is replaced by heavier deuterium, the bond breaking step will be slowed. This is termed as the intrinsic isotope effect which can alter the kinetic parameters of the enzymatic reaction.

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Intrinsic isotope effects were used to calculate flux through the ADH, MEOS, and catalase pathways (Takagi et al., 1985; Alderman et al., 1987). In hepatocytes of ADH- deermice, the results were consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor AT did not produce a significant change. Even when ADH is present (in the ADH + strain), non-ADH pathways (mostly MEOS) participated significantly in ethanol metabolism at all concentrations tested and played a major role at high levels. These studies support the conclusion reached before with kinetic studies in subhuman primates and humans (LieSer, 1982). In addition to its role for ethanol metabolism per se, ethanol oxidation via MEOS may have significant consequences for the pathogenesis of liver injury, either directly (through production of acetaldehyde) or indirectly through the microsomal activation of other xenobiotics (vide infra). Hepatotoxicity Through Activation of Xenohiotic Agents. In view of its inducibility and broad substrate specificity, the ethanol-speciflc form of cytochrome P450 has provided a major clue to the understanding of multiple ethanol-drug interactions, particularly with respect to the enhanced susceptibility of alcoholics to the hepatotoxic effects of various xenobiotic agents. Interaction of alcohol and drugs occurs at many sites, as reviewed elsewhere (Lieber, 1982, 1985). In this paper, the focus will be on the interaction of ethanol with cytochrome P450-dependent microsomal drug metabolism (Fig. 6).

O

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.ETABOU~

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Fig. 6. Schematic representation of hepatic alcohol-drug interactions involving the alcohol dehydrogenase patway and the mlcrosomes: (A) Metabolism of alcohol by ADH and drugs by microsomes; (B) Contribution of the microsomal pathway to alcohol metabolism at high blood ethanol levels; (C) Inhibition of mlcrosomal drug metabolism in the presence of alcohol, in part through competition for a common microsomal detoxification process; (D) Induction of microsomal activity after chronic alcohol consumption; (E) Acdelerated drug metabolism because of microsomal induction after chronic alcohol consumption (From Lieber, 1986).

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C.S. Lieber

Acutelnteractions.

The main effect of the presence of ethanol is the inhibition of hepatic drug metabolism (Fig. 6C) as reviewed elsewhere (Lieber, 1982). One mechanism involved appears to be direct competition between ethanol and other drugs for a common metabolic process involving cytochrome P450 (Lieber, 1982). Ethanol may also interfere with microsomal drug metabolism indirectly by decreasing the supply of NADPH; in addition, it inhibits glucuronidation (Moldeus et al., 1978), including that of morphine, due to diminished levels of UDP-glucuronic acid, probably through decreased UDP-glucose dehydrogenase activity secondary to the altered intracellular NADH/NAD ratio accompanying ethanol oxidation (Bodd et al., 1986). In contrast, other drug detoxification processes such as acetylatlon (Hutchings et al., 1984) and sulfation (Sundheimer and Brendel, 1984) are unaltered by ethanol. Chronic interactions. It is well known that chronic alcohol abusers develop tolerance to ethanol. Such tolerance is due, in part, to central nervous system adaptation (pharmacodynamic tolerance). In addition, there is metabolic tolerance that results from an increased capacity to oxidize ethanol. This occurs because of the adaptive increase in activity of non-ADH pathways involved in ethanol metabolism, most likely MEOS, as discussed before (Fig. 6D). In addition to tolerance to ethanol, alcoholics tend to display tolerance to various other drugs; this has generally been attributed to central nervous system adaptation. However, metabolic adaptation must also be considered. Indeed, the induction of MEOS activity after chronic alcohol consumption "spills over" to various other drug-metabolizing systems in liver mlcrosomes, thereby accelerating drug metabolism in general (Fig. 6E). As a consequence, the administration of ethanol to volunteers under metabolic ward conditions resulted in a striking increase in the rate of blood clearance of meprobamate and pentobarbital (Misra et al., 1971). Similarly, increases in the metabolism of aminopyrine, tolbutamlde, propranolol, and rifampicln due to ethanol administration have been described. The general increase in microsomal enzyme activities also applies to those that convert exogenous substrates to toxic compounds and it particularly affects those xenobiotics for which the ethanol-inducible form of cytochrome P450 (P450IIEI) has a high affinity. For instance, CCI 4 exerts its toxicity after conversion to an active metabolite in the mlcrosomes. As discussed before, P4501IEI is particularly active in that regard and indeed alcohol pretreatment remarkably enhances CCI 4 hepatotoxicity (Hasumura et al., 1974). Thus, the clinical observation of the enhanced susceptibility of alcoholics to the hepatotoxic effect of CCI. may be due, at least in part, to increased metabolic activation of this compound. Liver toxicity of bromobenzene was also found to increase following chronic alcohol consumption (Hetu et al., 1983). It is likely that a large number of other toxic agents will be found to display a selective injurious action in the alcoholic patient. This pertains not only to industrial solvents, hut also to a variety of prescribed drugs. For instance, the increased hepatotoxicity of isoniazid observed in alcoholics may well be due to increased microsomal production of an active metabollte of the acetyl derivative of the drug (Timbrell et al., 1980). The capacity of isoniazid to induce the ethanol-specific form of cytochrome P450 has been noted before. Similarly, chronic ethanol administration enhances phenylbutazone hepatotoxicity, possibly because of increased biotransformation (Beskid et al., 1980). The

above

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Pathophysiology of Alcoholic Liver Disease

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over-the-counter medications. Acetaminophen (paracetamol, N-acetyl-paminophenol), widely used as an analgesic and an antipyretic, is generally safe when taken in recommended doses. However, large doses of acetaminophen, mostly taken in suicide attempts, have been shown to produce fulminant hepatic failure. It is now clear that in addition to glucuronldation and sulfation, acetaminophen is also metabolized in the liver by the microsomal cytochrome P450 system; the latter biotransformation yields an active metabolite highly toxic to the liver. Furthermore, in a reconstituted system, P45011EI was found to have a high capacity to oxidize acetaminophen to a reactive intermediate which readily formed a conjugate with reduced glutathione (Morgan et al., 1983). Moreover, experimentally, enhanced covalent binding of reactive metabolite(s) of acetaminophen to liver microsomes from ethanol-fed rats was observed to be associated with increased hepatotoxlcity (Sato et al., 1981). Therefore, it was to be expected that a history of alcohol consumption might favor the hepatotoxicity of acetaminophen, as indeed suggested by various case reports (Wright and Prescott, 1973; Seef et al., 1986). However, unlike pretreatment with alcohol, which (as discussed above) accentuates toxicity, the presence of ethanol in part prevents the acetaminophen-induced hepatoxlcity, most likely because of inhibition of the biotransformation of acetaminophen to reactive metabolltes (Sato and Lieber, 1981; Altomare et al., 1984a,b). Thus, the greatest vulnerability of the alcoholic to acetaminophen is not necessarily during drinking, when ethanol may compete with acetaminophen for its microsomal metabolism, but rather after alcohol withdrawal, at which time there is no inhibitory effect of alcohol any more but the induction of the microsomal metabolism still persists. This is also the period when the need for analgesics may be high because of the well known withdrawal symptomatology. At that time, amounts of acetaminophen, large but usually still considered safe (2.5 to 5 g per day), may cause severe toxic side effects in the alcoholic. Alcohol abuse is also associated with an increased incidence of upper alimentary and respiratory tract cancers; many factors have been incriminated in the cocarcinogenic effect of ethanol (Lieber et al., 1986). One of the mechanisms is the effect of ethanol on enzyme systems involved in cytochrome P450 dependent carcinogen activation. This has been demonstrated by using microsomes derived from a variety of tissues including the liver (the principal site of xenobiotlc metabolism), the lungs and intestines (the major portals of entry for tobacco smoke and dietary carcinogens, respectively) and the esophagus (where ethanol consumption is a major risk factor in cancer development). Ethanol has a unique effect on the chemical carcinogen N-nitrosodimethylamine (NDMA): it induces a microsomal NDMA demethylase which functions at low NDMA concentrations (Garro et al., 1981). This is in contrast to other microsomal enzyme inducers such as phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls. These compounds increase the activity of other NDMA demethylases -- the activity of which is detectable only at relatively high NDMA concentrations -- while repressing the activity of low Km NDMA demethylases (Guttenplan and Garro, 1977). Furthermore, selective affinity for NDMA has been demonstrated with the ethanol-lnducible form of cytochrome P450 (Yang et al., 1985). Alcoholics are also commonly heavy smokers and epidemiologically, a synergistic effect of alcohol consumption and smoking has been described, as reviewed elsewhere (Lieber et al., 1986). Interactions at various levels are possible: chronic ethanol consumption was found to enhance the mutagenicity of tobacco pyrrolysates (Garro et al., 1981). More recently benzoflavone, a tobacco llke inducer, was also found to induce a liver cytochrome P450 which is structurally different but catalytically similar to P450EIII. This new P450 could be involved in some of the pathological effects associated with combined habitual alcohol and tobacco use (Lasker et al., 1987b). Ethanol also affects mlcrosomal metabolism of exogenous and endogenous steroids,

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as discussed in detail elsewhere (Lieber, 1982); the effects include enhanced testosterone degradation and conversion to estrogens, as well as decreased testicular steroid synthesis. Furthermore, ethanol alters the metabolism of a structurally related vitamin, such as vitamin D (Gascon-Barre, 1982). These and other micronutrients may serve as substrates for the mlcrosomal enzymes. The induction of the microsomal oxidative activities may alter vitamin requirements and even affect the integrity of liver and other tissues, as exemplified by vitamin A. It had been known that plasma vitamin A as well as retinol binding protein (RBP) levels (McClain et al., 1979) are decreased in patients with alcoholic cirrhosis. In addition, it has been found that already at the early fatty liver stage, alcoholics commonly have very low hepatic vitamin A concentration despite normal circulating vitamin A levels and the absence of obvious dietary vitamin A deficiency (Leo and Lieber, 1982). In experimental animals, ethanol administration was shown to depress hepatic vitamin A levels, even when administered with diets containing adequate amounts of vitamin A (Sato and Lieber, 1981). When dietary vitamin A was virtually eliminated, the depletion rate of hepatic vitamin A stores was two to three times faster in ethanol-fed rats than in controls, possibly because of accelerated mlcrosomal degradation of vitamin A (Leo et al., 1984). Furthermore, it has been shown, using reconstituted systems with purified forms of cytochrome P450, that retinoic acid can serve as a substrate for microsomal oxidation (Leo et al., 1984). An even greater microsomal metabolism was found for retinol. Indeed, a new pathway of microsomal retinol metabolism, inducible by either ethanol or drug administration and capable of degrading an amount of retinol comparable to the daily intake, has been discovered in the liver (Leo and Lieber, 1985). The hepatic depletion was s~rikingly exacerbated when ethanol and drugs were combined (Le~ et al., 1987a), which mimicks a common clinical occurence. A new microsomal NAD--dependent retlnol dehydrogenase has also been described (Leo et al., 1987b). Furthermore, ethanol contributes to hepatic retinold depletion by enhanced mobilization of the vitamin A (Leo et al., 1986a). Hepatic vitamin A depletion is associated with lysosomal lesions (Leo et al., 1983), decreased NDMA detoxification (Leo et al., 1986) and probably a score of other adverse effects. For all these reasons, vitamin A supplementation should be given to the alcoholic not only to correct his problems of night blindness and sexual dysfunction, but also to alleviate potential liver dysfunction. The therapeutic administration of vitamin A, however, is complicated by the fact that excessive amounts of this vitamin are known to be hepatotoxic (Leo and Lieber, 1988) and that the alcoholic has an enhanced susceptibility to this effect (Leo et al., 1982; Leo and Lieber, 1983). In control rats, amounts of vitamin A equivalent to those commonly used for the treatment of the alcoholic did not have significant toxic effects on the liver. However, in animals chronically fed alcohol, for the same dose of vitamin A, signs of toxicity developed, such as striking morphologlc and functional alterations of the mitochondria (Leo et al., 1982), along with hepatic necrosis and fibrosis (Leo and Lieber, 1983). Enhanced toxicity was not necessarily associated with an increased vitamin A level in the liver. One possible explanation, still to be proven, is that vitamin A toxicity may be mediated, at least in part, by the enhanced production of a toxic metabolite. In any event, there clearly is a narrowed "therapeutic window" for vitamin A in the alcoholic patient who has increased requirements of vitamin A, with, at the same time, enhanced susceptibility to its toxicity. Adverse Effects of Acetaldehyde All known pathways of ethanol metabolism result in the production of acetaldehyde. Its metabolism and general effects have been reviewed elsewhere (Lieber, 1982). We shall focus here on aspects of the toxicity of acetaldehyde most conspicuously related to liver injury.

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Hepatotoxicity Directly Associated with the Induction of MEOS Activity. Nomura and Lieber (1981) found covalent binding of exogenously added acetaldehyde to proteins of liver microsomes and an even greater effect was found with "endogenous acetaldehyde" (when ethanol was used). These results suggested a special capacity of "native" acetaldehyde (generated in sltu in the membranes) to form covalent links with the associated proteins. Furthermore, this effect was significantly increased after chronic ethanol consumption, in parallel with the induction of MEOS activity. We have now found that this process occurs in vivo and that acetaldehyde selectively forms a stable adduct in rat liver microsomes with the ethanol-induclble cytochrome P-45011EI (Hoerner et al., 1988). It is tempting to postulate that this increased acetaldehyde-proteln adduct formation may be responsible, at least in part, for the appearance of antibodies against such adducts. Indeed, using an animal model, Israel et al. (1986) demonstrated that acetaldehyde adducts may serve as antigens generating an immune response in mice. Our own studies (Hoerner et al., 1986) have shown that antibodies against acetaldehyde adducts (produced in vitro) are present in the serum of most alcoholics (Fig. 7). In addition to the acetaldehyde-proteln adducts formed in liver microsomes referred to above, acetaldehyde-protein adducts could be found in a variety of other tissues and serve as neoantigens. Indeed acetaldehyde binds covalently to other hepatic macromolecules (Mauch et al., 1985), hepatic cell membranes (Barry and McGiven, 1985) and other proteins such as human serum albumin (Donohue et al., 1983), hemoglobin (Stevens et al., 1981), red blood cell membrane proteins (Gaines et al., 1977) and tubulin (Baraona and Lieber, 1983). Adduct formation in the endoplasmic reticulum, however, may play a significant role in view of the high level of acetaldehyde generated at that site. Im

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Microtubules,

Swelling

of the Hepatocyte and their

Relation to

Two of the earliest and most conspicuous features of the hepatic damage produced

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Lieber

by alcohol are the deposition of fat and the enlargement of the liver. This hepatomegaly was traditionally attributed to the accumulation of liplds. However, in animals fed alcohol-contalnlng diets, it was shown that lipids account for only half of the increase in liver dry weight (Lieber et al., 1965), while the other half is almost totally accounted for by an increase in proteins (Baraona et al., 1975). The latter is secondary, in part, to acetaldehyde induced impairment of microtubular mediated protein secretion. Indeed, hepatic mlcrotubules are significantly decreased in alcoholic liver disease (Matsuda et al., 1979, 1985). Experimentally, feeding rats with diets providing 36% of energy as ethanol resulted in enlargement of hepatocytes, associated with a decrease in the concentration of total tubulin (Baraona et al., 1975, 1977, 1984). Alcohol feeding decreased the difference between total hepatic tubulin and free tubulin by approximately 30% with a proportional increase in the free tubulin fraction. This suggested that alcohol consumption has a major effect on tubulin polymerization to form microtubules (Baraona et al., 1977; Matsuda et al., 1979). The decrease of polymerized tubulin by ethanol was documented in rats (Baraona et al., 1981), baboons (Matsuda et al., 1978) and in alcoholics with liver disease (Matsuda et al., 1983). Morphometrlc studies by Matsuda et al. (1979) and 0kanoue et al. (1984) revealed that the ethanol-lnduced decrease in polymerized tubulin was associated with a decrease in the hepatic microtubular mass. Incubation of hepatocytes isolated from normal rats (Matsuda et al., 1979) with 50 mM ethanol produced accumulation of acetaldehyde (up to 130 uM) and decreased polymerized tubulin and the volume of visible microtubules. In vlvo, the acute intravenous administration of ethanol to naive rats decreased hepatic polymerized ~ tubulln and microtubules. This effect was partially prevented with the ADH inhibitor 4-methylpyrazole. Inhibition of acetaldehyde oxidation by pretreatment of the rats with disulflram increased the accumulation of acetaldehyde in the liver and produced a further decrease in mlcrotubules (Baraona et al., 1981; Baraona and Lieber, 1983). The acute effects of ethanol were markedly enhanced by prior consumption of ethanol-contalnlng diets. By inhibiting ADH with pyrazole and maintaining the concentration of acetaldehyde by multiple additions, we observed inhibitory effect of acetaldehyde (approximately 200 ~M) on colchlcine binding by liver tubulln (Baraona et al., 1981; Baraona and Lieber, 1983). The sulfhydryl groups of cystelne, present in tubulln, are involved in polymerization and colchlcine binding and binds to brain tubulln (Baraona et al., 1981). Acetaldehyde also has high affinity for -SH groups. Acetaldehyde binding to tubulin may therefore contribute to the decreased colchiclne-binding capacity of tubulln in alcohol-fed animals, but more importantly it may alter the capacity of tubulin to polymerlze. The disruption of liver mlcrotubules in alcohol-fed rats and in isolated hepatocytes incubated with ethanol was associated with a prominent accumulation of secretory vesicles (Matsuda et al., 1979). In isolated hepatocytes, the increase in volume and surface densities of the secretory vesicles and Golgl apparatus was prevented by pyrazole and reproduced by acetaldehyde and acetate (Matsuda et al., 1979). The time-course of incorporation of labeled leucine into i~nunoreactive albumin and transferrln revealed a significant delay in the secretion of these newly labeled proteins into the plasma with a corresponding retention in the liver of alcohol-fed rats (Baraona et al., 1977). These effects were associated with a significant accumulation of both albumin and transferrin in the liver. Similar, though less severe, alterations of albumin (Baraona et al., 1980) and glycoprotelns (Volentlne et al., 1984) were found after acute ethanol administration in rats. Inhibition of secretion was also observed after the addition of ethanol to liver slices (Tuma and Sorrell, 1981; Sorrell et al., 1983), but not to isolated hepatocytes (M6rland et al., 1981); however, in both preparations there was interference from inhibitory effects of ethanol on protein synthesis which must be taken into consideration.

Pathophysiology of Alcoholic Liver Disease

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Transferrin, one of the retained export proteins, was clearly stained in the ballooned hepatocytes of alcoholic liver disease but not in nonalcoholic liver disease (Matsuda et al., 1985). The degree to which transferrin was stained was related to hepatic microtubular contents and also related to the appearance of the heterogeneity of serum transferrin in alcoholic liver disease. These findings indicate that ballooning of hepatocytes in alcoholic liver disease, but not in nonalcoholic liver disease, is caused by the accumulation of exportable proteins due to impairment of microtubular polymerization. This accumulation might be related to the inhibition of secretion. However, the measured increase in export proteins such as albumin and transferrin accounted for only a relatively small fraction of the total increase in cytosolic protein. This raised the possibility that other export or constituent proteins of the cytosol could also contribute to the increase. Indeed, Pignon et al. (1987) found that an increase in fatty acid-binding protein (FABP) accounts for I/6 to I/3 of the increase in cytosolic protein induced by chronic ethanol feeding, thus becoming the largest known contributor to the ethanol-induced increase in these proteins. The increase in hepatic protein observed after ethanol was not associated with changes in concentration (Baraona et al., 1977), indicating that water was retained in proportion to the increase in protein. The mechanism of the water retention is not fully elucidated, but the rise in both protein and amino acids, plus a likely increase in associated small ions, could retain osmotically a large fraction of the water. The increases in lipid, protein, amino acid, water and electrolytes result in increased size of the hepatocytes. In the rat, the swelling affects both perivenular and periportal hepatocytes. Similar changes have been observed in baboons fed 50% of dietary energy as ethanol, although in this species the accumulation of fat greatly exceeds that of protein (Savolainen et al., 1984) and the enlarged hepatocytes have, as in man, a clear centrilobular distribution (Miyakawa et al., 1985). The number of hepatocytes and the hepatic content of deoxyribonucleic acid (DNA) do not change after alcohol treatment and thus the hepatomegaly is entirely accounted for by the increased cell volume (Baraona et al., 1975, 1977). There is also an increase in the number of hepatic mesenchymal cells after ethanol feeding (Baraona et al., 1975), but this increase does not significantly contribute to the hepatomegaly. The swelling of the hepatocytes after chronic alcohol administration was found to be associated with a reduction in intercellular space and with portal hypertension (Orrego et al., 1981). One suspects that ballooning and associated increase of the hepatocyte volume may result in severe impairment of key cellular functions. In alcoholic liver disease, some hepatocytes not uncommonly have a diameter which is increased 2-3 times, and thereby the volume may be increased more than i0 fold. It is not difficult to imagine that this type of cellular disorganization, with protein retention and ballooning, may promote progression of the liver injury in the alcoholic. Indeed, other causes of protein retention in the liver, such as alpha, antitrypsin deficiency, are known to be associated with progression to 1 fibrosis and cirrhosis. By analogy, one can assume that protein retention in the alcoholic also may in some way favor progression of,liver disease. Structural and Functional Alterations of the Mitochondria and Plasma Membranes. In the liver mltochondria of alcoholics, electronmicroscoplc studies have revealed striking morphologic alteratlens, including swelling and abnormal crlstae. Controlled studies in animals and man (Iseri et al., 1966; Lane and Lieber, 1966; R u b i n and Lieber, 1967; Lieber and Rubin, 1968) have shown that these changes are caused by alcohol itself, rather than other factors, such as a poor diet. These structural abnormalities are associated with functional impairments, especially decreased oxidation of fatty acids and a variety of other substrates, including acetaldehyde (Hasumura et al., 1976), as well as a reduction in cytochrome a and b content, respiratory capacity and oxidative

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phosphorylatlon (Lieber, 1982). In human volunteers given ethanol, mitochondrial lesions developed even in the presence of a hlgh-protein low-fat diet (Lieber and Rubln, 1968). It is noteworthy that high concentrations of acetaldehyde, the product of ethanol metabolism, mimic the defects produced by chronic ethanol consumption on oxidative phosphorylation (Cederbaum et al., 1974). One may wonder to what extent chronic exposure to acetaldehyde is the cause for the defect observed after chronic ethanol consumption. Alcoholics may exhibit higher acetaldehyde levels than nonalcoholics for a given ethanol load and blood level (Korsten et al., 1975; DiPadova et al., 1987b). It is therefore reasonable to speculate that exposure to high acetaldehyde levels may in turn affect mltochondrial function. In normal liver, acetaldehyde concentrations higher than those usually achieved after alcohol ingestion are required to produce toxicity. However, after chronic alcohol consumption, the liver mitochondria are unusually susceptible to the toxic effects of acetaldehyde, and important mitochondrlal functions, such as fatty acid oxidation, are depressed, even in the presence of relatively low acetaldehyde concentrations (Matsuzaki and Lieber, 1977). Paradoxically, in patients with alcoholic hepatitis, the presence of megamitochondria seemed to be associated with a better outcome in terms of mortality (Chedid et al., 1986). Furthermore, no complications were present in 72% of patients with megamitochondrla vs 39% for those without. Infection, gastrointestinal bleeding, pancreatltis, hyperglycemia, azotemia, delirium tremens, seizures, and hepatic encephalopathy were all more common in patients without megamitochondria. Thus, the patients with megamitochondrla appeared to represent a subcategory of alcoholic hepatitis with a milder degree of clinical severity. Alternatively, the presence of megamitochondrla may represent an earlier step of the disease. Indeed, megamitochondria appear already at the initial fatty liver stage (Lane and Lieber, 1966). While they persist through early stages of fibrosis and cirrhosis (Floersheim and Bianchi, 1985), they appear to decrease and even disappear with more severe disease. This is not to imply that the megamitochondrial lesion in and of itself is benign or even represents a more favorable change. Megamitochondria have not been isolated in pure form and therefore their function has not been assessed. However, alterations of a variety of mitochondrial functions have been reported in the rat, as reviewed by Lieber (1985). Morphologically and functionally, mitochondrla isolated from baboons fed ethanol chronically were also altered; these changes were fully developed already at the fatty liver stage and appeared to reflect the damage of mitochondrial membranes rather than an adaptive hypertrophy (Arai et al., 1984). It has been proposed that the functional changes in mitochondria may also be related to altered membranes produced as a consequence of prolonged ethanol consumption (Warlng et al., 1981, 1982). There have been several reports concerning the effect of ethanol on the fluidity of membranes. A currently held view is that, in vitro, ethanol renders membranes more fluid and that chronic ethanol administration alters membrane lipid composition, resulting in decreased fluidity, which is considered as an adaptation to the acute fluidizlng effect. Such an adaptation has been reported to occur in brain synaptosomes and erthyrocytes (Chin and Goldstein, 1977; Rottenberg et al., 1981), as well as in liver mitochondria and microsomes (Waring et al., 1981; Ponnappa et al., 1982). Membrane penetration of ethanol, anesthetics and hydrophoblc molecules in brain synaptosomes and liver mitochondrla of rats is reduced after long-term consumption of ethanol. Membranes became resistant to structural disordering by ethanol and halothane. It has been suggested that this increased rigidity impairs normal membrane function, although such a correlation has not always been verified (Gordon et al., 1982). Ethanol also strikingly alters other liver membranes, as reviewed by Sun and Sun (1985). Plasma membrane glycoprotein assembly in the liver following acute

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ethanol administration was significantly impaired (Mailllard et al., 1984). Furthermore, hepatocytes isolated from ethanol-fed animals exhibited pronounced morphologic alterations of their plasma membranes by scanning electron mlcrosocopy. Incubation of these hepatocytes with 50 mM ethanol also resulted in release and reduced content of alkaline phosphatase despite a higher initial total liver alkaline phosphatase. Moreover, chronic ethanol feeding resulted in reduced recovery of alkaline phosphatase in hepatic plasma membranes. These findings suggested that the increased serum alkaline ph0sphatase levels observed in response to chronic ethanol feeding may be due, at least in part, to increased lability of this plasma membrane enzyme (Yamada et al., 1985). It is well known that ethanol also has striking effects on another plasma membrane enzyme, namely ga~a-glutamyltranspeptldase (GGT). GGT activity is frequently elevated in alcoholics (Wietholz and Colombo, 1976; Shaw and Lieber, 1978; Horner et al., 1979) and in rats given ethanol (Shaw and Lieber, 1978; M6rland et al., 1977; Ishii et al., 1978; Lahrichi et al., 1982; Nishimura and Teschke, 1982; Yamada et al., 1985). ATPase is the third plasma membrane enzyme reported to be affected by ethanol. However, when the effects of ethanol and acetaldehyde on the + + Z+ activities of two hepatic plasma membrane ATPases - Na ,K -ATPase and Mg -ATPase - were investigated with concentrations ranging from 8-90 mM, ethanol did not cause+significant Inhibition. Acetaldehyde produced non-competitive inhibition of Na ,K -ATPase and Mg - -ATPase at concentrations of 6 and 56 mM, respectively and 5'-nucleotidase activity was also inhibited at these concentrations. Because the inhibitory concentrations of both substances are higher than are ususally found in alcoholic subjects or in experimental animals after alcohol feeding, it seems unlikely that direct suppression of ATPase activity by ethanol or acetaldehyde is responsible for the morphological abnormalities of alcohol-induced liver disease (Gonzalez-Calvin et al., 1983). Various studies have demonstrated that the cell surface plays an important role in intracellular homeostasis through activities of membrane enzymes, hormone receptors, and transmembrane transport processes. Many of these dynamic features are influenced, in part, by the fluidity of the lipid bilayer because of its effect on the mobility and/or exposure of membrane proteins. When the effect of chronic ethanol consumption on liver plasma membranes (PM) was studied (Yamada and Lieber, 1984) it was found that, in contrast to other membranes, the fluidity of hepatic PM, as assessed by fluorescence anlsotropy, increased. Similar results were observed by Polokoff et al. (1985). This alteration was associated with an increase in the cholesterol esters (Kim et al., 1988). Thus chronic ethanol feeding does not appear to result in a 'homeoviscous adaptation' of liver PM as reported for other membranes. However, hepatocytes, isolated from rats fed ethanol for 5 to 7 weeks had a decreased ability to bind concanavalin A (Metcalf et al., 1987) and chronic ethanol administration impaired the binding and endocytosis of asialo-orosomucoid in isolated hepatocytes (Casey et al., 1987). The major cause of this impairment appears to be a decreased number of cell surface asialoglycoprotein receptors, along with a decreased ability to internalize the surface-bound ligand. The process of receptor-medlated endocytosis is an important part of the overall means by which the biological effects of polypeptide hormones, growth factors, and other molecules are elicited. Alterations of this process by chronic ethanol ingestion could play a role in the pathogenesis of alcoholic liver injury and in the impairment of cellular repair processes. Promotion of Lipid Peroxidation; Alterations in Cysteine, Glutathlone and Iron. Aldehydes react quite readily with mercaptans (Schubert, 1936, 1937; Ratner and Clarke, 1937). L-cystelne can complex with acetaldehyde to form a hemiacetal, which could then transforms to L-2-methylthiazolidine-4-carboxylic acid (Schubert, 1937; Debey et al., 1958). Cysteine is one of the three amino acids

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which constitute glutathione (GSH). Binding of acetaldehyde with cysteine and/or GSH may contribute to a depression of liver GSH (Shaw et al., 1981), which may reduce the scavenging of toxic free radicals by this tripeptide. Although GSH depletion is not sufficient to cause lipid peroxidatfon, it is generally agreed upon that it may favor the peroxidation produced by other factors. GSH is important in the protection of cells against electrophilic drug injury in general, and against reactive oxygen species in particular. Rats fed ethanol chronically had significantly increased rates of GSH turnover associated with increased activity of hepatic GGT (Morton and Mitchell, 1985). The increase in turnover of GSH was not due to an increase in oxidation of GSH, consistent with the results of Vendemiale et al. (1984) who had found evidence for increased synthesis. In contrast to these effects of chronic ethanol consumption, acute ethanol administration inhibited GSH synthesis and produced an increased loss from the liver (Speisky et al., 1985). GSH transferase activity (Kocak-Toker et al., 1985) was decreased by acute ethanol administration, and GSH peroxidase after chronic treatment (Morton and Mitchell, 1985). A severe reduction in glutathione favors peroxldation (Wendel et al., 1979), and the damage may possibly be compounded by the increased generation of active radicals by the "induced" microsomes following chronic ethanol consumption. It is well known that the microsomal pathway, which requires Op and NADPH, is capable of generating lipid peroxides. Enhanced lipid peroxXdation, possibly mediated by acetaldehyde (DiLuzfo and Stege, 1977), has been proposed as a mechanism for ethanol induced fatty liver (DiLuzlo and Hartman, 1967). The capacity of acetaldehyde to cause lipid peroxidation in the liver has been demonstrated in isolated perfused livers (MUller and Sies, 1982). Acetaldehyde may promote lipid peroxidation indirectly, i.e. through depletion of GSH, or by a more direct mechanism. Indeed, peroxidation has been linked to acetaldehyde oxidation (MUller and Sies, 1983). However, to what extent alcohol administration in vivo results in lipid peroxidation and injury is still uncertain (Hashimoto and Recknagel, 1968; Scheig and Klatskin, 1969; Bunyan et al., 1969; Comportl et al., 1971). Theoretically, increased activity of microsomal NADPH oxidase following ethanol consumption (Lieber and DeCarli, 1970b; Reitz, 1975) could result in enhanced H 0 production, thereby also favoring lipid peroxidatfon. It has also been 2 ~laimed that the ethanolinducible form of rabbit liver microsomal cytochromes P450 is associated with increased hydroxyl radical formation (Ingelman-Sundberg and Johansson, 1984). However, when the effect of chronic alcohol feeding on lipid peroxidation was studied in rat liver mlcrosomes, no correlation with the generation of hydroxyl radicals was observed (Shaw et al., 1984). Studies of Thomas et al. (1985) showed that ferritin can provide the iron necessary for initiation of lipid peroxidation. OTo, as generated by xanthine oxidase, reductfvely releases ferritin-bound iron. Once released, this iron can promote the peroxidation of phospholipid liposomes. Catalase markedly stimulates malondialdehyde (MDA) formation in this system, suggesting that initiation is not dependent upon H_O 2. These results were further supported by the use of EPR spin trapping w~ich demonstrated a negative correlation between OH" formation and lipid peroxidation. It is noteworthy that in alcoholics, the serum ferritin level was elevated in both groups immediately after a drinking bout, significatnly more so in men with, than in those without, biochemical signs of liver injury (V~lim~ki et al., 1983). The serum iron concentration was equally increased, but returned to normal during the first week of ethanol withdrawal. Experimentally, iron and ethanol treatments enhanced liver lipid peroxidation (MDA formation) by 70 and 35%, respectively. Since the hepatic MDA formation increased by 92% after the joint iron-ethanol treatment, it was suggested that an additive effect on lipid peroxldation occurs under these conditions. In naive rats, very large amounts of ethanol (5-6g/kg)

are required to produce

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lipid peroxldation (DILuzlo and Hartman, 1967; MacDonald, 1973), whereas a smaller dose (3g/kg) had no effect (Shaw et al., 1981). By contrast, after chronic ethanol administration, even a smaller dose of ethanol administered acutely induced liver peroxldatlon and this effect could he prevented, at least partially, by the administration of methlonlne, a precursor of glutathlone (Shaw et al., 1981). In the baboon, the ethanol induced lipid peroxldatlon was even more striking: administration of relatively small doses of ethanol (l-2g/kg) produced lipid peroxldation and GSH depletion after 5-6 hours. In the baboon chronically fed alcohol (50% of total calories for I-4 years), alcoholic liver disease, including cirrhosis in some, developed and such animals showed evidence of enhanced hepatic lipid peroxldation and GSH depletion. These changes were observed following an overnight withdrawal from ethanol and were exacerbated by the readmlnistratlon of ethanol. Evidence for GSH depletion and lipid peroxldatlon (enhanced dlene conjugates) was found in liver biopsies of alcoholics who were withdrawn from alcohol (Shaw et al., 1983). Experimentally, as discussed before, acute ethanol intake diminished GSH content and enhanced that of GSSG, with a net decrease in the total GSH equivalents (GSH + 2GSSG). Depression of liver GSH has been found to predominate in the mltochondrial compartment (Fernandez-Chez et al., 1987). It could contribute to the striking functional and structlonal damage produced by chronic alcohol consumption in that organelle (vide supra). Billary release of total GSH was reduced under these conditions. The combined administration of iron and ethanol further influenced the decrease in hepatic GSH and the increase in GSSG levels elicited by the separate treatments. These data suggest that iron exposure accentuates those changes in lipid peroxldation and in the glutathlone status of the liver cell induced by acute ethanol intoxication (Valenzuela et al., 1983). It is tempting to speculate that the propensity of the baboon, after chronic ethanol consumption, to develop more severe lesions than the rat may in some way be related, at least in part, to its greater susceptibility to GSH depletion, resulting in the initiation of lipid peroxldatlon. It is apparent, however, that GSH depletion per se does not suffice to produce liver damage (Slegers et al., 1977). As mentioned before, concomitant enhanced production of active radicals may be required, possibly resulting from the mlcrosomal "induction" discussed in this review. Free radical generation by neutrophils may provide another potential mechanism of cellular injury in acute alcoholic liver disease (Williams and Barry, 1987). This hypothesis is based on the observation that acetaldehyde altered hepatocyte membranes stimulate neutrophils to produce superoxlde anion. However, the concentration of acetaldehyde to which the liver membrane vesicles prepaKed from liver biopsies were exposed was extremely high (I mM), several fold the maximum concentration ever reported in the liver. In another study, an elevation of chemotractic factor Inactlvator was shown in alcoholic liver disease (Robblns et al., 1987). This may explain impaired neutrophll chemotaxls in patients with alcoholic hepatitis and may be important in the pathogenesis of both localized infections and overwhelming sepsis (by altering neutrophll influx to sites of inflammation). Other modes of toxicity involve the interference with enzyme activity secondary to acetaldehyde binding with critical -SH groups, as discussed by Sorrel and Tuma (1987) and in another article of this series (Peters, 1988). Metabolism of ethanol by rat liver homogenates produced transaminase inhibition similar to that described in vivo and this effect required acetaldehyde generation (Solomon, 1987). One of the functlon~ most sensitive to acetaldehyde is the repair of alkylated nucleoproteins, which was found to be inactivated by minute concentrations of acetaldehyde (Esplna et al., 1988).

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A clinical situation in which acetaldehyde may be potentially involved as a source of liver injury is the occasional development of abnormal liver test in patients undergoing treatment with the drug disulfiram. Disulfiram, an inhibitor of acetaldehyde dehydrogenase, raises the acetaldehyde levels upon drinking and thereby causes flushing and other adverse effects. Abnormalities of liver tests are occasionally encountered in patients undergoing treatment with this drug. It is sometimes difficult to unravel whether alcohol, disulfiram, or combinations of the two is responsible for changes in liver status. Moderate changes in liver tests were found to he more related to drinking than to the taking of disulfiram (Iber et el., 1987). The practical recommendation was that if allergic symptoms, fever, and clinical picture are sufficently typical of drug reactions (and unlike alcoholic liver disease), particularly if the AST are above 300 units, the prompt discontinuation of dlsulfiram is necessary until the situation is clarified; on the other hand, when modest changes are encountered, extensive effort to determine whether the patient is drinking or not should be made. Acetaldehyde may also be involved in the promotion of hepatic flbrogenesis, in concert with the redox changes discussed before. Indeed, in myoflbroblast cultures, lactate was found to stimulate collagen synthesis and acetaldehyde had a similar effect (Savolalnen et el., 1984a) but ethanol did not. The stimulation of collagen formation by acetaldehyde but not ethanol was found to be a common property of fihroblasts from hepatic and nonhepatlc sources (Holt et al., 1984). The mechanisms underlying the effect of acetaldehyde upon myofibroblast and fibroblast synthetic activity await explanation. It is not known to what extent these in vitro observations pertain to in vivo conditions. Role of Endotoxin and Other Factors in Alcoholic Liver Injury, Including Vitamin A. A detailed discussion of the role of inflammation, immune-medlated mechanism and genetic factors are provided in detail in accompanied articles (Keppler, 1988; Eddleston, 1988). The possible contributory role of concommltant viral infection is discussed elsewhere (Lieber, 1986, 1987). In the pathogenesis of progressive liver disease, the role of endotoxin is of special interest but remains controversial. Rats fed a nutritionally adequate liquid alcohol-containlng diet according to the formula of Lieher and DeCarli developed fatty livers. Littermates fed an identical diet and challenged with small I.V. doses of E. coli llpopolysaccharlde endotoxin ( L P S ) developed focal necrotizing hepatitis. Control littermates fed an identical but alcohol free diet and challenged with identical doses of LPS did not develop any liver lesions. The hepatocyte necrosis with associated inflammatory changes had some of the features of early human alcoholic hepatitis and suggested a possible role for endotoxin in the latter (Bhagwandeen et el., 1987). In fact, heavy alcohol abuse was found to lead to transient endotoxemia even in patients with no signs of chronic liver disease (Bode et el., 1987a). There was also a significantly higher percentage of endotoxemia in patients with alcoholic cirrhosis as compared to non-alcohollc cirrhosis. These clinical findings, together with the corresponding experimental results, are consistent with the hypothesis that gut-derived endotoxlns may contribute to the pathogenesis of alcohol induced liver damage. One may wonder to what extent a similar mechanism is responsible for the observation that alcohol administration exerts a synergistic effect on jejunoileal bypass-induced liver dysfunction in rats (Bode et al., 1987b). One of the hidden factors which may aggravate ethanol induced fibrosis is alteration of vitamin A status, both hypo- and hyper-vitaminosls A (vide supra). Hypervitaminosis A per se can induce fibrosis and even cirrhosis (Leo and Lieber, 1988). Cirrhosis due to hypervltamlnosls A alone is an unusual occurance but potentiation of the fibrogenic effect of ethanol by hypervitaminosis A may be

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more common. It must also be pointed out that the relationship between vitamin A and fibrogenesis is complex: in addition to the evidence discussed before of vitamin A promoting fibrogenesis, under certain circumstances, the opposite effect has been described, for instance in isolated fibroblasts (Oikarlnen et al., 1985) or CCI 4 induced hepatic fibrosis (Senoo and Wake, 1985). Thus, it is conceivable that the striking depletion of hepatic vitamin A associated with alcoholic liver injury (Leo and Lieber, 1982) could also promote the fibrogenesis. The possible pathogenic role of a non-oxidative pathway of ethanol to form fatty acid ethyl esters was raised by Laposata and Lange (1986). The capacity of ethanol to form ethyl esters in vivo had already been demonstrated by Goodman and Deykin (1963). Laposata and Lange found that in acutely intoxicated subjects, concentrations of fatty acid ethyl esters were significantly higher than in controls in pancreas, liver, heart, and adipose tissue. Since this non-oxidative ethanol metabolism occurs in humans in the organs most commonly injured by alcohol abuse, and since some of these organs lack oxidative ethanol metabolism, Laposata and Lange (1986) postulated that fatty acid ethyl esters and their metabolism may have a role in the production of alcohol-lnduced injury. Further experiments are of course needed to verify this interesting hypothesis. CONCLUSIONS Two decades of productive research in ethanol metabolism have culminated in the elucidation of the various molecular forms of alcohol dehydrogenase and that of an ethanol-inducible cytochrome P450 (P450IIEI). Previous reviews have summarized metabolic alterations associated with ethanol metabolism via ADH and the associated redox changes, including alterations in carbohydrate, lipid and protein metabolism. The present article focuses on more recent information relating to the exacerbation of the redox change in the perivenular zone and associated pathology. Major emphasis is also placed on the role of P450IIEI, not only in terms of ethanol metabolism and ethanol tolerance, but also with regard to its unique potency to activate xenobiotlc agents; this now provides a better understanding for the increased susceptibility of the heavy drinker to the hepatotoxicity of industrial solvents, commonly used drugs, over-the-counter medications, and chemical carcinogens. All pathways of ethanol metabolism result in the production of acetaldehyde, the toxicity of which has been reviewed in detail before (Lieber, 1982). New aspects discussed here pertain to the formation of acetaldehyde-protein adducts. Also included is a discussion of the role of ethanol-induced alterations in microtubules, mitochondria and plasma membranes, as they relate, in part, to acetaldehyde induced toxicity, to free-radicals or to injury mediated by lipid peroxidation. Thus, elucidation of the pathways of ethanol metabolism has resulted in a better understanding of the pathophysiology of alcohol induced liver damage which may eventually provide a handle for better prevention and treatment. Acknowledgements Original studies reviewed in this paper were supported, in part, by DHHS Grants #AA03508, #DK23810, #AA05934 and the Veterans Administration. The author wishes the thank Ms. Patricia Walker for expert secretarial assistance. References Alderman, J., Takagi, T. and Lieber, C.S. (1987). Ethanol metabolizing pathways in deermice: estimation of flux calculated from isotope effects. J. Biol. Chem. 262, 7497-7503. Altomare, E., Leo, M.A. and Lieber, C.S. (1984a). Interaction of acute ethanol

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