REACTIVE INTERMEDIATES IN METABOLISM AND DRUG TOXICITY

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Many drugs and chemicals which injure cells require biotransformation to chemically reactive metabolites to express their toxicity. One simple compound which models this behavior is bromobenzene, whose reactive metabolites become covalently bound to cellular proteins, eliciting tocixity in direct proportion to their binding. A key question which remains unanswered is "what are the structures of these reactive metabolites and the adducts they form with proteins? Both arene oxide and quinone metabolites are known to form, but neither has been shown conclusively to bind. One objective of the proposed research is to answer this question. Proteins labelled with [C-14]-bromobenzene metabolites will be hydrolyzed, modified amino acids isolated and their structures elucidated. Adduct profiles will be used to compare the effects of treatments such as enzyme induction which alter the metabolism and toxicity of bromobenzene, and to compare adducts arising directly from bromobenzene to those arising from secondary metabolism of its phenolic metabolites. This will help identify those adducts which are most significant toxicologically. A second objective of this research is to probe the mechanism of microsomal epoxide hydrolase, which detoxifies bromobenzene- and other epoxides. Kinetic isotope effects for enzymic hydration of deuterium-, 0-18 and C-13 labelled S-(+)1 and R-(-)-p- nitrostyrene oxide, and for related model studies in progress, will be determined and used to deduce the geometry of the transition state for the enzymic reaction. The corresponding imine (aziridine) analog will be investigated as a substrate and inhibitor to probe for involvement of an acid-catalysis component in the enzyme mechanism. Together with existing information about general base activation of water as a specific cosubstrate, the proposed studies should furnish a comprehensive picture of the mechanism of this important enzyme.