Evidence for activation of carcinogenic o-anisidine by prostaglandin H synthase: P-32-postlabelling analysis of DNA adduct formation

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activi ty, we examined the ability of prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, to activate this carcinogen to metabolites b inding to macromolecules. Using [C-14]-labeled o-anisidine, we observed sub stantial PHS-dependent binding of o-anisidine to protein, DNA and polydeoxy ribonucleotides (poly(dX)]. This binding is inhibited by radical scavengers glutathione, ascorbate and NADH. The nuclease PI and 1-butanol extraction enrichment procedure of the P-32-postlabeling analysis of DNA modified by a ctivated o-anisidine provide evidence that covalent binding to DNA is the p rincipal type of DNA modification. Deoxyguanosine is determined to be the m ajor target for binding of o-anisidine in DNA. The possibility that o-anisi dine is carcinogenic to the rodent urinary bladder via its activation by bl adder PHS is suggested. The results presented here are the first report dem onstrating a PHS-mediated activation of o-anisidine to reactive species for ming covalent DNA adducts.