We plan to pursue our studies of factors that affect activity and inducibility of the microsomal ethanol oxidizing systems (MEOS) and associated functions, and hence their pathologic role in alcohol-induced liver injury. We will take advantage of the availability of liver specimens reflecting a large spectrum of human disease, and subject them to Western blot and PCR analysis for P4502E1 (2E1) protein and mRNA, respectively. We will first determine how the induction of microsomal 2E1 is regulated in man, with focus on possible roles of dose and type of alcoholic beverage, age and associated medications and/or deficiencies. We will also asses the role of dietary lipids, especially the species of phosphatidylcholine, in the activity of MEOS in vitro and its inducibility in vivo after chronic ethanol consumption. Specifically, we will evaluate in vitro how the fatty acid composition of the phospholipids may affect 2E1 catalytic activity. In vivo, we will determine how changes in dietary lipids alter the activity of enzymes (including 2E1) in liver microsomes and how the latter correlates with changes in phospholipid composition. The studies will be conducted in our experimental models of alcoholic liver injury in rodents and nonhuman primates and corroborated in human liver tissue, which we will obtain from a parallel study evaluating the effects of supplementation of dietary phospholipids on the outcome of alcoholic liver disease. Associated changes in microsomal phosphatidylethanolamine methyltransferase activity and membrane fluidity will be determined. At present, measurement of 2E1 activity requires liver tissue. However, since 2E1 metabolizes not only ethanol but also chlorozoxazone (CZX), a widely used drug, the suitability of CZX clearance in vivo as a marker of 2E1 induction will be evaluated. These studies will be extended to P4501A2(1A2), found to be able to sustain ethanol metabolism, inducible by factors commonly present in the heavy drinker (smoking, omeprazole treatment) and capable of activating carcinogens. A potentially important factor that may affect 2E1 and 1A2 activity is the extent of adduct formation between the proteins and acetaldehyde. The potential impact of this adduction on the catalytic conversion of ethanol to acetaldehyde, and other 2E1 and 1A2 mediated activities, will be investigated. The planned studies, by revealing some of the determining factors of enzyme activity, may provide the information needed to minimize ethanol's toxicity, or that resulting from the activation of other hepatotoxins. Finally, we will also evaluate the interaction of ethanol with microsomal P450s (especially P4502C8, 1A2 and also 3A3/4) in retinoid metabolism and hepatic retinol depletion, which may contribute to the increased incidence of various cancers in the alcoholic. The broad aim of our proposed studies is to define biochemical differences between heavy drinkers and more moderate consumers. We will focus on the microsomal cytochrome P450 system and its changes secondary to drinking, which have been shown to alter the response of the alcoholic to ethanol and other hepatotoxic agents and even nutrients. Such information is aimed at elucidating key aspects of alcohol mediated pathology in a way which may eventually affect alcohol related medical complications, their prevention and treatment.
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