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 and resulted in the formation of DNA adducts which can be used as in vivo markers for PHS-dependent oxidation. We have further studied the formation of amine mutagens by PHS using bacterial Test System. 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 studied the formation of tyrosyl radicals during the oxygenation of arachidonic acid by prostaglandin H synthase. Several tyrosyl radicals were observed by ESR that were characterized as radicals with two different conformations. These radicals appear to be formed as the result of enzyme catalysis and appear not be catalytic intermediates. Replacement of the Fe in PHS with Mn gives an enzyme that contains cyclooxygenase activity but no peroxidase activity. Tyrosyl radicals were not observed with this Mn-PHS. Other data suggest that tyrosyl radicals are formed as the result of enzyme inactivation. We are currently attempting to express PHS and generate forms of PHS to more fully characterize the enzymatic mechanism. Also, we have recently detected a mitogen inducible form of PHS that has a different substrate preference than the constitutive PHS.
