Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor alpha-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 microM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate), 2-amino-1-methyl-4'-hydroxy-6- phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4'-hydroxy-PhIP to 4'-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP.(ABSTRACT TRUNCATED AT 250 WORDS)