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. 1H-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 N3 imidazole ring nitrogen. Km values for PhIP were 0.2-0.3 mM in both species; however, rabbit microsomes exhibited a 22-fold higher Vmax. 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.
Styczynski
P B
PB
Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205.
Blackmon
R C
RC
Groopman
J D
JD
Kensler
T W
TW
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004285:Dogs; D005965:Glucuronates / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D007093:Imidazoles / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D009153:Mutagens / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet
D005965:Glucuronates; D007093:Imidazoles; D009153:Mutagens; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D005966:Glucuronidase