This application requests aggregate funding for continuation of current programs in enzymology and chemistry of bioactivation. Human cytochrome P-450 enzymes will be purified, particularly those involved in known polymorphisms of drug oxidation. The proteins will be characterized and the related genes cloned and identified; nucleic acid probes will be used to identify the molecular basis of catalytic polymorphism in each case. Enzyme reconstitution and immunoinhibition techniques will be used to determine the specificity of individual enzymes for specific drugs and pro-carcinogens. Another aspect of the proposal involves elucidation of the chemistry involved in the activation of the carcinogens ethylene dibromide and trichloroethylene and the parasympathomimetic alkaloid slaframine. The interactions of the ethylene dibromide/glutathione half-mustard/thiiranium ion with any specific sites on DNA will be probed and the biological roles of individual adducts will be examined. The roles of hepatic cytochrome P-450 forms in acute toxicity of chemicals to hepatocyte-derived cultured cells will be probed by microinjection of proteins and possibly gene tranfection. Finally, we propose to continue our studies on the chemistry involved in catalysis of oxygenation by cytochrome P-450, utilizing mechanism-based inactivators and diagnostic substrate probes (particularly cyclopropylamines and dihydropyridines), model metalloporphyrins, and physical descriptions of rate processes. These studies should enhance our understanding of how chemicals are activated to dangerous species and the inter-individual variations in susceptibility to these agents.
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