Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rat and human hepatocytes.

Metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been compared in human and rat hepatocytes. The identities of seven metabolites were confirmed by UV and mass spectroscopy and by co-elution with reference standards using HPLC. In human hepatocytes, the major biotransformation pathway of PhIP was cytochrome P4501A2 (CYP1A2)-mediated N-oxidation to form the genotoxic metabolite 2-(hydroxyamino)-1-methyl6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which underwent glucuronidation at the N 2 and N3 positions of PhIP to form stable conjugates. These products combined accounted for as much as 60% of the added PhIP. Direct glucuronidation of PhIP at the N 2 and N3 positions also occurred, accounting for up to 20% of the amount added. Glucuronide and sulfate conjugates of 2-amino-4-hydroxy1-methyl-6-phenylimidazo[4,5-b]pyridine (4-HO-PhIP) were also detected, comprising 5 and 12% of the products, respectively. The CYP1A2 inhibitor furafylline diminished the formation of both HONH-PhIP glucuronide conjugates in a concentration-dependent manner, however, levels of 4HO-PhIP were unchanged, indicating that CYP1A2 does not significantly contribute to 4-hydroxylation of PhIP. Hepatocytes of male rats, both untreated and pretreated with the CYP1A2 inducer 3-methylcholanthrene (3-MC) transformed PhIP into 4-HO-PhIP as the prominent product. Unconjugated and conjugated 4-HO-PhIP metabolites combined accounted for 18 and 46% of the PhIP products in untreated and in 3-MC-pretreated rat hepatocytes, respectAbbreviations: CID, collision-induced dissociation; CYP, cytochrome P450; EROD, ethoxyresorufin O-deethylation; FCS, fetal calf serum; HAA, heterocyclic aromatic amine; LC-ESI-MS, liquid chromatography electrospray ionization mass spectrometry; 3-MC, 3-methylcholanthrene; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NOE, nuclear Overhauser enhancement; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 4’-HO-PhIP, 2-amino-4’-hydroxyl-1-methyl-6-phenylimidazo[4,5-b]pyridine; HONH-PhIP, 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine; 5-HO-PhIP, 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine; PhIP-4’-OSO3H, 4-(2-amino-1-methylimidazo[4,5-b]pyridin-6-yl)phenyl sulfate; HON-PhIP-N 2 -Gl, N 2 -β-D-glucosiduronyl-2-(hydroxyamino)-1-methyl-6phenylimidazo[4,5-b]pyridine; PhIP-N 2 -Gl, N 2 -β-D-glucosiduronyl-2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine; HON-PhIP-N3-Gl, N3-β-D-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIPN3-Gl, N3-β-D-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine; PhIP-4’-O-Gl; 4’-β-D-glucosiduronyloxy-2-amino-4’-hydroxyl-1