In the requested period of support, we propose to continue work on the bioactivation of haloalkenes and acrylonitrile, extend our efforts on the immunohistochemical localization of epoxide hydrolase, and re-initiate studies on the mechanism of oxidative bioactivation of the parasympathomimetic alkaloid slaframine. (I) Several aspects of the oxidation of the model haloalkene trichloroethylene (TCE) will be addressed, including: (A) the mechanism of chemical degradation of TCE-oxide, (B) the nature of the covalent adducts formed by TCE metabolites with proteins, (C) establishment of the stereochemistry of oxidative migration of halide groups in TCE analogs using 19F NMR methods, (D) characterization of any DNA adducts formed in vitro and in vivo using neutral thermal hydrolysis and 32P postlabeling techniques, and (E) the nature of heme adducts formed during mechanism- based inactivation of cytochrome P-450. (II) DNA adducts formed in rat liver, forestomach, and brain during chronic administration of the carcinogen acrylonitrile will be characterized using several chemical methods in order to evaluate their biological roles. Chemical (NaB3H4) and enzymatic (32P) postlabeling methods will be emphasized. (III) Immunohistochemical localization studies involving epoxide hydrolase, a major enzyme involved in the biotransformation of epoxide, will be continued in various rat tissues, including liver, lung, skin, pancreas, gut, and testis. (IV) The bioactivation of the parasympathomimetic natural product slaframine (1S, 6S, 8aS-1-1-acetoxy-6-aminooctahydro-indolizine) will be investigated. (A) NMR and adduct formation methods will be used to define the chemistry of oxidation in model systems. (B) A radioligand competition assay will be used to measure binding of activated slaframine to rat hog heart muscarinic acetylcholine receptors, and the tissue and subcellular localization of the activating enzyme and its cofactor requirements will be determined; purified enzymes will be examined for their roles. (C) Chromatographic and spectral methods will be used to relate to the chemistry occurring in enzymatic systems to bio-organic models. (D) Efforts will be made to directly prepare active metabolites by chemical methods to test their receptor-binding activity. These studies should provide insights into interesting questions related to the chemistry and biochemistry of activation of potentially toxic chemicals which are present in the environment.
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