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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES002205-11
Application #
3249687
Study Section
Toxicology Study Section (TOX)
Project Start
1979-06-25
Project End
1992-05-31
Budget Start
1989-07-01
Budget End
1990-05-31
Support Year
11
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37203
Guengerich, F Peter (2004) Cytochrome P450: what have we learned and what are the future issues? Drug Metab Rev 36:159-97
Guengerich, F P; Kim, D H; Iwasaki, M (1991) Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 4:168-79
Guengerich, F P; Kim, D H (1991) Enzymatic oxidation of ethyl carbamate to vinyl carbamate and its role as an intermediate in the formation of 1,N6-ethenoadenosine. Chem Res Toxicol 4:413-21
Baron, J; Burke, J P; Guengerich, F P et al. (1988) Sites for xenobiotic activation and detoxication within the respiratory tract: implications for chemically induced toxicity. Toxicol Appl Pharmacol 93:493-505
Guengerich, F P (1988) Roles of cytochrome P-450 enzymes in chemical carcinogenesis and cancer chemotherapy. Cancer Res 48:2946-54
Guengerich, F P (1988) Cytochromes P-450. Comp Biochem Physiol C 89:1-4
Veltman, J C; Dekant, W; Guengerich, F P et al. (1988) Cytotoxicity and bioactivation mechanism of benzyl 2-chloro-1,1,2-trifluoroethyl sulfide and benzyl 1,2,3,4,4-pentachlorobuta-1,3-dienyl sulfide. Chem Res Toxicol 1:35-40
Schaefer, W H; Harris, T M; Guengerich, F P (1987) Reaction of the model thiol 2-mercaptoethanol and glutathione with methylvinylmaleimide, a Michael acceptor with extended conjugation. Arch Biochem Biophys 257:186-93
Guengerich, F P (1986) Covalent binding to apoprotein is a major fate of heme in a variety of reactions in which cytochrome P-450 is destroyed. Biochem Biophys Res Commun 138:193-8
Hogy, L L; Guengerich, F P (1986) In vivo interaction of acrylonitrile and 2-cyanoethylene oxide with DNA in rats. Cancer Res 46:3932-8

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