Vol. 16, No. 5 September/October 1992

0 I45-6008/92/ 1605-0870$3.00/0 ALCOHOLISM: C L I N I C AAIN D EXPERIMFNTAL RESEARCH

Highlights of the RSA Symposium on Enzymes of Alcohol Metabolism Assembled by: David W. Crabb and William Lands

INTRODUCTION AND OVERVIEW*

Metabolism of alcohol represents the fortuitous removal of an exogenous substance that does not appear in appreciable amounts during normal vertebrate metabolism, but which is provided in small amounts by microorganisms in the gastrointestinal tract and some foods. Thus, metabolic removal of large amounts of ingested alcohol depend on the action of enzymes that probably have been preserved through evolutionary selection because of their ability to handle other metabolites as well as small amounts of ethanol. As a result, factors that regulate the expression and action of the alcohol-metabolizing enzymes may not relate clearly to the intake, metabolism, or blood levels of alcohol. Biomedical researchers need to evaluate carefully the mechanisms by which alcohol and its metabolites (acetaldehyde and acetate) might provide useful energy or affect physiological responses. A symposium held at the 199 1 annual meeting of the Research Society on Alcoholism reviewed recent research on three types of enzymes for alcohol metabolism: alcohol dehydrogenases (ADH), cytochromes P450 (CUP), and fatty acid ethyl ester synthases (FAEES). New information on these three types of enzymes presented at the annual meeting is summarized below. ALCOHOL DEHYDROGENASES (ADH)

Gastric ADH has potential importance in the prehepatic (first pass) metabolism of ethanol' and is lower in females and alcoholics.' Baraona et al. reported that about 80% of Japanese lack gastric ADH (seen as a band with slow cathodal mobility in starch gels). Gastric endoscopic biopsies and surgically resected stomach samples showed a lower rate at which this ADH reduced nitrobenzaldehyde (an excellent substrate for CT ADH,2 one of the two forms of ADH purified from s t o r n a ~ h ~ ?whereas ~), activities of class I and 111ADH were not lower. In addition to low K, From the Department ofktedicine, Indiana University Medical Centfr, Indianapolis, Indiana. Receivedfor publication November 6, 1991; accepted April 7, 1992 Reprint requests: David W. Crabb, M.D., Department of Medicine, Division of Gastroenterology/Hepatology,Medical Research and Library Building 424, Indiana University Medical Center, 975 W. Walnut Street, Indianapolis, IN 46202-5121. * This summary includes concepts and comments obtained from the symposium participants. Copyright 0 1992 by The Research Society on Alcoholism. 870

class I isozymes and high K, class 111 isozymes (similar to those present in human liver), there was another isozyme class with high K, for ethanol and slower cathodic mobility than class I isoenzymes on starch gel electrophoresis. By DEAE-Sephadex chromatography, it was eluted with the equilibration buffer after class I isozymes. It was not present in the human liver and seemed to correspond to a gastric isozyme. Nine of the 13 Japanese stomachs lacked this isozyme and had only those of class I and 111. The implications of this finding are that there is a genetic determinant of this deficiency and that individuals lacking gastric ADH may experience higher blood alcohol levels after drinking than those with the enzyme. In Orientals, deficient aldehyde dehydrogenase activity4 and the polymorphism of liver alcohol dehydrogenase (ADH) at the ADH2 gene locus of class I ADH isoenzymes5 are well recognized. Of especial relevance to alcoholism is the possibility that this enzyme deficiency may contribute to ethnic differences in the bioavailability of ingested ethanol6 or to the well known differences between Orientals and non-Orientals in their response to alcohol consumption' and in the intensity of the alcohol flush reaction in Japanese. Qulali et a1.' reported that the activity of kidney ADH and the abundance of its mRNA are increased in male or female rats by treatment with estradiol. ADH activity and mRNA in the kidney but not liver are responsive to estrogen. Northern blot analysis showed an 8- to 10-fold increase in ADH mRNA after treatment of male rats for 10 days with 1 rng/kg/day estradiol. In situ hybridization of kidney sections from control and treated male rats indicated an approximate 1 0-fold increase in ADH mRNA and showed that the mRNA was localized to the medulla. In females, ADH mRNA was detected in the inner cortex and the medulla. Northern blots showed that the increase occurs within 24 hr of injecting estradiol; nuclear run-on assays indicated that the induction was not associated with an increase in the rate of ADH transcription. This suggests that the effect of the estradiol may be to stabilize the mRNA or to increase the efficiency of processing of the nascent transcript. These are the most dramatic effects of hormones on ADH activity and mRNA yet observed in vivo. A metabolic role for kidney ADH was sought by examining the effect of ethanol on rates of gluconeogenesis in isolated kidney tubule cells. By analogy with the liver, it was hypothesized that the metabolism of ethanol would Alcohol Clin Exp Res, Vol 16, No 5, 1992: pp 870-874

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increase the NADH/NAD’ ratio in the cells and inhibit gluconeogenesis. Kidney tubules prepared by collagenase digestion of minced kidney from male and female rats had rates of gluconeogenesis from various substrates similar to published reports.’ Addition of 10 mM ethanol alone had little effect on glucose formation rates in cells from male or female kidneys, but with oleate (2 mM) in the incubation medium the rate of gluconeogenesis was reduced. Other fatty acids (palmitate or decanoate) did not exhibit this effect. The effect of added oleate was blocked by increasing the concentration of serum albumin in the medium, suggesting that the effect was due to increased concentrations of unbound oleate. The observed inhibition of gluconeogenesis appeared to be the result of interactions between ethanol and high concentration of free fatty acid. However, free fatty acids are unlikely to reach this level in vivo, and the phenomenon observed may not occur in intact animals. The medullary location of the enzyme is interesting in light of the association between chronic alcoholism and renal papillary necrosis. Hurley et al.’ reported on crystallized recombinant human ADH Dl ADH as well as a site-specific mutant of the enzyme. The structures of human plpl alcohol dehydrogenase (ADH) and a site-directed mutant with a Gly for Arg substitution at position 47 have been determined by x-ray crystallographyof the binary enzyme-NAD+complexes to 3.0 A and 2.4 A resolution, respectively. The overall structure of the recombinant plpl enzyme complexed with its coenzyme nicotinamide adenine nucleotide (NAD+) is similar to that of horse liver alcohol dehydrogenase.’ However, the approximately 50-fold difference between the activity of the horse and human enzymes results from subtle rearrangements of the coenzyme binding site due to the multiple sequence differences in other regions of the enzyme.’ Site-directed mutagenesis, replacing specific amino acids known to be substituted in naturally occurring human enzyme variant~,’~ permitted asking the question “How do these amino acid replacements affect the functional and structural properties of the enzyme?” The coenzyme binding site of alcohol dehydrogenase is remarkably adaptive. An example of this is seen when arginine 47-an important amino acid involved in cofactor (NAD’) binding-is replaced with a glycine. This substitution occurs naturally in the human (Y(Y alcohol dehydrogenase. When this amino acid substitution is created in the human p 1p 1 enzyme, it results in a dramatic structural rearrangement of the enzyme structure, which allows the enzyme to retain high affinity for NAD’ and alcohol oxidizing activity by compensating for the loss of this critical basic amino acid. Another basic amino acid, lysine 228, is recruited from a different region of the coenzyme binding site to replace the function of the mutated arginine. This enzyme has a substantial rearrangement of atoms near the active site and it has a different kinetic mechanism (Theorell-Chance rather than ordered Bi Bi).

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CYTOCHROME P450

Chen et al. reported on the distribution of several enzymes of alcohol metabolism, as detected by in situ hybridization. The distribution of ADH is controversial, with reports that ADH activity or immunoreactivity is predominantly pericentral, l4 periportal, l 5 or evenly16 distributed. This contrasts with the predominantly pericentral distribution of P450IIE1, which has been demonstrated by several laboratories. Lumeng’s group had reported that alcohol dehydrogenase (ADH) is found at similar levels in pericentral and perivenous hepatocytes”,’*; in contrast, P450IIE 1 is found predominantly in the perivenous one.''^^^ The technique of in situ hybridization, which detects specific mRNAs in tissue slices, was validated using probes for glutamine synthetase, which is found in one to two cell layers immediately adjacent to the central vein. mRNA for ADH was found nearly equally distributed across the lobule, whereas there was a gradient of both P450 IIEl mRNA and enzyme activity, with maximal activity in the perivenous cells. Adminstration of the drug 4-methylpyrazole (4MP) or chronic feeding of ethanol increased the activity of P450 IIEl, but did not alter the abundance of P450IIE1 mRNA, as can be semi-quantitatively estimated by image analysis from the in situ hybridization data. Liver sections from control and 4MP- or CCLtreated rats were processed on the same slides. Whereas 4MP injection was known to increase P450IIE 1 enzyme activities and protein contents, it failed to increase P450IIE 1 mRNA in the liver lobule. By comparison, CC4 treatment effectively eliminated all P450IIE 1-mRNA signals, The preferential vulnerability of the perivenous zone to ethanol is at least partly linked to the heterogeneity in expression of the P4501IE1 gene in the liver. This is consistent with other studies that indicate that in rats, alcohol and pyrazoles induce P450IIEl mainly by stabilizing the protein, and not by altering the mRNA Since P450IIE 1 catalyzes the oxidation of xenobiotics including ethanol (the latter to produce acetaldehyde, which is a potential hepatotoxin) and it generates free radicals, the induction by alcohol of the perivenous expression of P450IIEl may explain the preferential vulnerability of this zone to alcohol- or drug-induced liver injury in the rat. Human P450IIE1 protein has been purified and its cDNA has been cloned recently. However, there are no reports about the zonal distribution of the mRNA for alcohol dehydrogenase and P450IIE 1 in human liver. Peng et al.23reported on the developmental pattern of the rabbit cytochrome P540s that are alcohol-inducible, P450IIEl and P450IIE2; the second form does not occur in rats or humans. The P450IIE2 is 97% identical with the P450IIE 1 amino acid composition. However, P450IIE2 is expressed in neonatal rabbits, whereas P450IIEl is expressed in adult rabbits. Only the P450IIE1 mRNA is detected in the adult kidney, at about 50% the activity seen in the liver.24It is noteworthy that kidney expresses relatively high levels of three enzymes of alcohol

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metabolism (ADH, ALDH2, and P4501IEl), yet it does not seem to be susceptible to alcohol-induced tissue injury. Examination of the specific cellular distribution of these three enzymes by immunochemical methods or by in situ hybridization might provide useful clues to how the kidney avoids injury. In liver, P450IIE2 mRNA was detected immediately after birth, and reached slightly greater than the adult level in 2 weeks, while IIEl mRNA was not detected until the 2nd week, and then increased rapidly to a level approximately 2-fold greater than that of adults. P450IIE protein was detected in liver immediately after birth, coincident with the appearance of P450IIE2 mRNA, peaked at 2 weeks, and then, despite the continued elevation in P450IIE mRNA, decreased to the adult level at 5 weeks. In kidney, P450IIE2 mRNA was not detected at any age; P450IIE1 mRNA, however, was detected at 1 week, and the level increased to about 50% of the adult level at 5 weeks. P450IIE protein in kidney was elevated at 2 weeks, relative to mRNA levels, and reached the adult level, despite low RNA levels, at 5 weeks. This lack of close correlation between RNA and protein levels indicates that the post-transcriptional control of P450IIE enzyme levels that is dominant in adult animals is also active in neonates. The importance of this differential expression is not yet known, but may be related to the changes in diet that accompany weaning. The shift from high fat breast milk to a vegetable matter diet is likely to produce changes in a variety of enzymes, including the P450s. Moreover, P450IIE1 levels are altered by regimens that elevate blood ketone levels, and P450IIEl is active toward a variety of lipid and ketone substrates. It is possible that P450IIE2 has a role distinct from that of P450IIE 1 in the disposition of lipids in breast milk; another possibility is that P450IIE2 is involved in the metabolism of maternal steroids present in milk. As humans have only a single P450IIE enzyme, the rabbit may provide a paradigm to delineate the role that neonatal expression of an alcohol-inducible cytochrome P450 plays in both species. Menez et al. reported that P450IIE1 is phosphorylated in vitro by protein kinase A, calmodulin-dependent protein kinase I1 (CaM kinase 11), and protein kinase C. CaM kinase I1 was the most efficient to catalyze this phosphorylation: the maximum incorporation of 32Pwas 0.94 mol/ mol of cytochrome. Protein kinase A phosphorylated a maximum of 0.70 mol of P/mol cytochrome. The phosphorylation by protein kinase C was at a maximum of 0.19 mol of P/mol of cytochrome. In partial proteolytic digestion experiments, protein kinases A and C appeared to phosphorylate the same peptide, whereas CaM kinase I1 phosphorylated two peptides of different size. These results suggest the presence of one site of phosphorylation when the phosphorylation of this cytochrome is achieved by protein kinase A or protein kinase C and of two sites when phosphorylation is achieved by CaM kinase 11. This phenomenon is of interest to alcohol researchers because

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the stability of the enzyme seems to be the major means of regulating the activity of this cytochrome, and phosphorylation may increase the rate at which this enzyme is degraded.” It will be interesting to extend this work to phosphorylation of the protein in isolated hepatocytes (or renal tubule cells) and to examine the effects of hormones on the phosphorylation state of the protein. These results confirm previous work that shows that protein kinase A phosphorylates P450IIE 125 and provide additional evidence that protein kinase C and CaM kinase I1 phosphorylate this ethanol-metabolizing enzyme. However, these data were obtained with purified P4501IE l , and therefore do not prove that protein kinases A or C and CaM kinase TI phosphorylate this isozyme in intact hepatocytes. Protein kinase A-stimulated phosphorylation has been shown to cause an enhanced susceptibility of P4501IE1 to inactivation and d e g r a d a t i ~ n .It~ ~will be important to determine if the residue phosphorylated by protein kinase C or CaM kinase I1 is the same as that phosphorylated by protein kinase A. A comparison may then be made of phosphorylation sites and regulation of P450IIE 1 activity and inducibility by all three kinases. P450IIE 1 is induced by chronic alcohol consumption26 and contributes significantly to the metabolism of alcohol ingested by alcoholic individuals. The induction of this isozyme may be a factor in producing liver damage and cirrhosis observed in a subset of this population. Phosphorylation mechanisms and other factors which control the activity of P450IIE1 may be important in the prevention and/or treatment of liver damage and cirrhosis. Of great interest was the report of a new alcohol-inducible P450 by Martin Ronis. This work was prompted by the knowledge that chronic alcohol use has effects on drug metabolism that cannot be entirely explained by the induction of CYP 2EI. The proposed role of CYP 2E1 in liver damage caused by a variety of substances includes: ethanol, organic solvents such as carbon tetrachloride, anesthetics such as enflurane, and analgesics such as aceta m i n ~ p h e n . ~CYP ~ , ~ 2E1 ’ catalyses the activation of these compounds to radical intermediates with the co-production of active oxygen specie^.^^,^' However, the oxidative metabolism of many compounds that are not substrates for CYP 2E1 is also affected by alcohol c o n s ~ m p t i o n . ~ ~ These include rifamycin, methadone, gonadal steroids and the liposoluble vitamins A, D, and E. To date, the only other P450 isozymes affected by ethanol treatment have been reported to be CYP 2B1, the major phenobarbitalinducible f ~ r r n ~and ’ , ~ the ~ CYP 3A i s o z y m e ~ . These ~~?~~ isozymes are involved in the 6P-hydroxylation of gonadal steroids and bile acids, the metabolism of macrolide antibiotics and cyclosporins and the metabolism of opioids. In the current study, a member of P450 gene family CYP 2C, P450EtOH2, has been partially characterized. This protein co-purified with CYP 2E1 during its isolation from isoniazid-induced rat liver. It is induced 2- to 3-fold by short-term and chronic ethanol treatment in a diet-

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independent fashion and is regulated differently from CYP neuropathy, or pancreatitis. Thus, FAEES-111joins ADH, 2El by xenobiotic treatment and during d e ~ e l o p m e n t . ~ALDH2, ~ and CYP2El as candidate genes with known PreliminaryN-terminal sequence data and cross-reactivity polymorphisms that can be tested in the pursuit of the of an antibody specific in Western blot analysis for gene loci, which contribute to susceptibility to alcoholism P450EtOH2, suggests a relationship to the previously char- and/or its medical complications. acterized rat 2C family members CYP 2C6 and CYP 2C7. Genetic studies of both alcoholism and consequent endHowever, its physical properties and regulation differen- organ damage have been hampered by the absence of tiate this isozyme from both previously described forms. relevant genetic/molecular r n a r k e r ~ . In ~ ~recent , ~ ~ years These rat isozymes have orthologues in human liverthis search has included both the primary ethanol metabCYP 2C8, 2C9, and 2C10, which show polymorphisms olizing pathways, the alcohol and aldehyde dehydrogentowards the drugs mephenytoin and t ~ l b u t a m i d e . ~ ~ , ~ ases, ~ and nonoxidative and secondary oxidative pathways. Both CYP 2C7 and 2C8 catalyze the 4-hydroxylation In the case of the well-documented secondary organ effects of retinoic acid and retin01.~~-~' The new isozyme, P450- such as pancreatitis, cardiomyopathy, and brain damage, EtOH2, is under different xenobiotic and developmental the ethanol recognizing enzyme is FAEES-III.46-49 FAEESregulation than CYP 2E 1. Chronic alcohol consumption 111 represents a true candidate gene. To develop FAEESmay cause vitamin A deficiency in rats, baboons, and I11 into a useful genetic/molecular marker for studies of human alcoholic^.^^^^^ Retinoids are hepatic paracrine fac- secondary alcoholic end-organ damage, Devor et al. tors and have been implicated in hepatotoxicity and he- mapped the FAEES-111 locus to provide a physical position patofibr~sis.~',~~ Retinoic acid is a hormone that is known and also a DNA marker c o n t e ~ t ~for ~ , use ~ ' in future to modify the behavior of the Ito cells, the mesenchymal genetic studies of the role of this enzyme in the abovecells of the liver that are probably the major sources of mentioned secondary end-organ consequences of alcohol collagen and matrix proteins in hepatic fibrosis. Hence, abuse. the induction of CYP2C by ethanol may influence the REFERENCES fibrosis that can occur in alcoholic liver disease. Ethanol 1. 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