Having-elucidated the role of cytochrome P450IIEl in the microsomal ethanol oxidizing system (MEOS) and having purified an active preparation from human liver, we now propose to determine the affinity of this isozyme for other substrates of biological interest such as retinoids, carcinogens and hepatotoxic agents using, in part, this human form. We plan to characterize the human cytochrome P450IIEl with respect to its ability to activate the structurally diverse chemical carcinogens dimethyl- and diethyl- nitrosamines, nitrosopyrolidine and vinylchloride. We also will determine whether a parallelism exists between the induction of the ethanol specific form of P450 and the potentiation of the hepatotoxicity of various solvents (e.g. carbon tetrachloride) and commonly used drugs (such as acetaminophen) and substances of abuse (e.g. cocaine). Toxicity will be determined by the leakage of liver enzymes into the bloodstream and by morphologic changes determined by light and electron microscopy. Microsomal induction will be evaluated by the determination of total cytochrome P450 as well as the visualization of the various isozymes by SDS-PAGE and the immunoquantitation of some of these forms using specific antibodies. The role of other factors (such as ketones) in the induction of liver microsomes and the potentiation of hepatotoxicity will also be assessed; these latter studies may provide new insight into the question of interactions between ethanol toxicity and malnutrition. The relative importance of ADH, MEOS and catalase for total hepatic ethanol metabolism will be quantitated in vivo and in isolated hepatocytes, focusing on changes resulting from chronic alcohol consumption. Three approaches will be employed: tritium as a radiotracer for the fate of reducing equivalents derived from ethanol, deuterated ethanol to determine pathway-selective isotope effects and metabolic studies with a deermouse strain lacking ADH. Our broad aim is to define biochemical differences between alcoholics and non-alcoholics and to determine to what extent the changes produced by ethanol consumption in the nature of the microsomal P450 system alter the response of the alcoholic to physiologic and pharmacologic substrates in a way which may eventually affect alcohol related pathology, its prevention and treatment. Our ultimate goal is to define in molecular terms some of the most far-reaching changes brought about by chronic ethanol consumption in the internal milieu of the body and the resulting response to the environment.
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