The long range goal of this project is to study the oxidation of chemicals to toxic or carcinogenic metabolites by prostaglandin H synthase (PHS) and to demonstrate the importance of this enzyme system in chemical- induced toxicity or carcinogenesis. We have shown that aromatic amine carcinogens, are metabolized to mutagens by PHS. PHS dependent oxidation occurred by a free radical mechanism. Amines are first acetylated to the acetylamine. PHS but not HRP then metabolizes the acetylamine to nitroacetylamine. The nitro metabolites are potent direct acting mutagens. This explains the role of bacterial acetylation and the difference between PHS and HRP. Our data suggest that PHS is a versatile enzyme system that can catalyze a variety of reactions which are important in the conversion of chemicals to carcinogenic metabolites in extra hepatic tissue. We have continued our studies on the formation of tyrosyl radicals during the oxygenation of arachidonic acid by prostaglandin H synthase. Two tyrosyl radicals were observed by ESR that were characterized as radicals with two different conformations. Data obtained from recent experiments further suggest that these radicals are not catalytic intermediates but are formed during the process of self-inactivation that occurs during enzyme catalysis. Studies also indicate that the tryosyl radicals formed by PHS have a different reactivity than the tyrosyl radicals formed by ribonucleotide reductase suggesting that the radicals have very different and unrelated roles in enzyme catalysis. We have also continued our studies on the mechanism by which aromatic amines are oxidized by PHS. We have obtained addition data that suggest that for some carcinogenic amines that are poor peroxidase substrates that direct oxygen transfer from the enzyme to the amine occurs with the purified enzyme. With crude microsomal preparations both direct transfer and peroxyl radical mediated oxygenation occurred. Further studies are in process. Future direction is to use molecular biology techniques to generate mutant forms of PHS.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Intramural Research (Z01)
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