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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM021784-15
Application #
3270669
Study Section
Toxicology Study Section (TOX)
Project Start
1978-08-01
Project End
1994-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
15
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Kansas Lawrence
Department
Type
Schools of Pharmacy
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Sukthankar, Pinakin; Avila, L Adriana; Whitaker, Susan K et al. (2014) Branched amphiphilic peptide capsules: cellular uptake and retention of encapsulated solutes. Biochim Biophys Acta 1838:2296-305
Koen, Yakov M; Sarma, Diganta; Hajovsky, Heather et al. (2013) Protein targets of thioacetamide metabolites in rat hepatocytes. Chem Res Toxicol 26:564-74
Hanzlik, Robert P; Koen, Yakov M; Fang, Jianwen (2013) Bioinformatic analysis of 302 reactive metabolite target proteins. Which ones are important for cell death? Toxicol Sci 135:390-401
Hajovsky, Heather; Hu, Gang; Koen, Yakov et al. (2012) Metabolism and toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes. Chem Res Toxicol 25:1955-63
Koen, Yakov M; Sarma, Diganta; Williams, Todd D et al. (2012) Identification of protein targets of reactive metabolites of tienilic acid in human hepatocytes. Chem Res Toxicol 25:1145-54
Koen, Yakov M; Hajovsky, Heather; Liu, Ke et al. (2012) Liver protein targets of hepatotoxic 4-bromophenol metabolites. Chem Res Toxicol 25:1777-86
Sarma, Diganta; Hajovsky, Heather; Koen, Yakov M et al. (2012) Covalent modification of lipids and proteins in rat hepatocytes and in vitro by thioacetamide metabolites. Chem Res Toxicol 25:1868-77
Sarma, Diganta; Hanzlik, Robert P (2011) Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide. J Labelled Comp Radiopharm 54:795-798
Hanzlik, Robert P; Fang, Jianwen; Koen, Yakov M (2009) Filling and mining the reactive metabolite target protein database. Chem Biol Interact 179:38-44
Ikehata, Keisuke; Duzhak, Tatyana G; Galeva, Nadezhda A et al. (2008) Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo. Chem Res Toxicol 21:1432-42

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