To safeguard human and environmental health in a cost effective manner, we must develop a mechanistic basis for predicting toxic risk and assessing environmental exposure. We will address aspects of these goals by developing an understanding of the biochemistry, regulation, and toxicological significance of hydrolytic pathways of metabolism. We have targeted two groups of enzymes both of which are induced by peroxisome proliferators and which add water to xenobiotics. Since the epoxide functionality is the reactive center of some of the most dangerous mutagens, carcinogens and toxins known, the cytosolic epoxide hydrolase (cEH) is targeted in objective I. Based on our recent isolation of the message and gene of the cEH, we will examine its regulation and subcellular targeting. Parallel approaches in photoaffinity labeling, amino acid modification, enzyme kinetics and site directed mutagenicity are being used to determine catalytic mechanism. This information will be used to design more effective substrates and inhibitors. All of the above technology will be used to test the hypothesis that the cEH has an endogenous role in the biosynthesis of diols and tetrahydrofuran diols of a variety of lipids including arachidonic acid. Similar approaches will be used to assess the in vitro and in vivo roles of cEH in ameliorating toxicity. Our second objective emphasizes the carboxylesterases which metabolize esters such as malathion, permethrin and a variety of pharmaceuticals. Similar techniques will be used. We will purify hepatic esterases using esterase specific affinity columns that we have designed based upon potent transition state mimics of the enzymes. Based on this work we will isolate cDNA and genomic clones as in objective I and use these to study the regulation of the enzymes. We are investigating the catalytic mechanism of esterases relying heavily on production of mutants in the baculovirus expression system. We are using a new class of spectral substrates yielding highly sensitive assays to monitor serum and tissue esterases following xenobiotic exposure. The above information will be used to extend our appreciation of the role of esterases in the metabolism on natural and man made toxins.
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