THE DIRECT GLUCURONIDATION OF 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE (PHIP) BY HUMAN AND RABBIT LIVER-MICROSOMES

The glucuronidation of the food-borne heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) was investigated using hepatic microsomes from several species. PhIP-glucuronic acid conjugates were formed in an NADPH-free system using microsomes from rabbit, dog, guinea pig, and human. Rat, hamster, and mouse microsomes were incapable of directly producing PhIP-glucuronides. The PhIP-glucuronide generated with human microsomes could be resolved by reverse-phase HPLC from that produced with rabbit microsomes. In addition, the human PhIP-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase, whereas the rabbit PhIP-glucuronide did not undergo beta-glucuronidase catalyzed hydrolysis. Fast atom bombardment mass spectrometry of both glucuronides revealed the presence of ions with m/z 401 (M+ H+). Rabbit PhIP-glucuronide had a lambda(max) of 316 nm, similar to that of the parent PhIP. By contrast, a spectral shift in UV absorbance was observed for the human PhIP-glucuronide, which had a lambda max of 305 nm. H-1-NMR spectroscopy and nuclear Overhauser enhancements established that rabbit PhIP-glucuronide was conjugated at the exocyclic amine nitrogen, whereas human PhIP-glucuronide was conjugated at the N-3 imidazole ring nitrogen. K-m values for PhIP were 0.2-0.3 mM in both species; however, rabbit microsomes exhibited a 22-fold higher V-max. Collectively, these studies indicate that human and rabbit liver microsomes form structurally different glucuronides of PhIP and suggest the involvement of multiple isoforms of UDP-glucuronosyltransferase. Further, these data suggest that in certain species, including humans, the direct conjugation of PhIP with glucuronic acid may represent a primary route of PhIP metabolism and detoxication.