In this proposal, support is requested to carry out studies in five areas related to the enzymatic basis of how chemicals are bioactivated and detoxicated: (I) The aggregation states of eight cytochromes P-450 (P-450), NADPH-P-450 reductase, epoxide hydrolase, cytochrome b5, NADH-Fe(CN)3-6 reductase, and microsomal flavin-containing monooxygenase will be examined in microsomal membranes using target inactivation analysis. (II) Methods will be developed in which P-450s, NADPH-P-450 reductase, epoxide hydrolase, glutathione (GSH) S-transferase, specific antibodies to some of these proteins can be inserted into cells which do not express them in order to establish causal relationships between metabolism and toxicity with several chemicals of environmental interest. (III) Several of the chemical and biological details of the mechanism of formation of 1,2-dibromoethane (DBE, ethylene dibromide) adducts with DNA and the fate of these adducts, including any potential repair, will be examined. (IV) A variety of substrate probes and electrochemical and spectroscopic approaches will be used to examine the validity of radicaloid pathways in P-450 and model metalloporphyrin oxidation mechanisms and, if the radicals exits, how they can be described in certain physical parameters. (V) Collaborative studies will be continued in the area of the rat liver P-450s in terms of development of monoclonal antibodies, immunohistochemical localization in extrahepatic tissues, and assignment of roles of individual P-450s in the metabolism of acetaminophen, 17 Beta-estradiol, 2-aminofluorene, 1-naphthylamine, 2-naphthylamine, styrene, and butylated hydroxyanisole. The results of these studies should enhance our knowledge of basic mechanisms about how the detrimental effects of chemicals are influenced by enzymes.
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