The long-term objective is to provide the tools which will allow the assessment of risks to man upon exposure to epoxides. To do so we must understand the relative contribution of various epoxide metabolizing enzymes (EME's) in the degradation/activation of epoxides, some of which are cytotoxic, mutagenic and/or carcinogenic, in vivo. Thus cell free systems will initially be used, both to develop methodology and to better understand the various EME's, in particular the cytosolic and mitochondrial epoxide hydrolases (EH's) in the cell. Knowledge gained from these studies will then be used for hepatocyte culture and isolated liver systems, and finally to in vivo systems.
The specific aims of this proposal are: I. Evaluate the relative role of EME's in cell free systems.
This aim will develop assay methodology, monitor metabolism of endogenous epoxides, localize the mitochondrial EH, monitor the relative distribution of EME's in different species, and evaluate various compounds as inhibitors of EME's. The cytosolic EH will be purified, antibodies developed, and immunoassays (ELISA) developed. II. Evaluate EME's in hepatocytes. Levels of EME's will be determined and epoxide metabolism monitored. The effect of various inducers and inhibitors on epoxide metabolism in hepatocytes will be assessed. The cytotoxicity and genotoxicity of selected epoxides will be evaluated. III. Determine epoxide metabolism in isolated liver, and finally, IV. Perform initial experiments to determine epoxide metabolism in vivo. Various methodologies will be used. Epoxide hydration and/or conjugation will be monitored by partition, tlc, glc, and/or hplc assays. Cytosolic EH purification will be by ion exchange, hydrophobic, affinity and gel filtration chromatography, while characterization will include pI, substrate selectivity, kinetics, and active site analysis. Hepatocytes will be isolated following procedures by Seglen and monolayer cultures used. Pharmacokinetics and epoxide metabolism in isolated liver and in vivo will be determined by monitoring and identifying metabolites in perfusion media, blood, urine, feces and tissues. The practicality of using selective epoxide substrates, inducers, inhibitors and different species to unravel the complexity and relative role of the various EME's in the vivo metabolism of epoxides will be assessed.
| Gill, S S; Kaur, S (1987) Hepatic epoxide hydrolase activities and their induction by clofibrate and diethylhexylphthalate in various strains of mice. Biochem Pharmacol 36:4221-7 |
| Grayson, M H; Gill, S S (1986) Effect of in vitro exposure to styrene, styrene oxide, and other structurally related compounds on murine cell-mediated immunity. Immunopharmacology 11:165-73 |
| Kaur, S; Gill, S S (1986) Distribution and nature of epoxide hydrolase activity in subcellular organelles of mouse liver. Biochem Pharmacol 35:1299-308 |
| Kaur, S; Gill, S S (1986) Effect of aging on testicular epoxide hydrolase and glutathione-S-transferase activities in mice. Toxicol Lett 30:113-20 |
| Kaur, S; Gill, S S (1985) Age-related changes in the activities of epoxide hydrolases in different tissues of mice. Drug Metab Dispos 13:711-5 |
| Pichare, M M; Gill, S S (1985) The regulation of cytosolic epoxide hydrolase in mice. Biochem Biophys Res Commun 133:233-8 |
