N-glucuronidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N_hydroxy-PhIP by specific human UDP-glucuronosyltransferases.
Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics. Recent studies have shown that in humans, UDP-glucuronosyltransferase (UGT)-mediated glucuronidation plays a critical role in the detoxification of food-borne carcinogenic heterocyclic amines. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant carcinogenic heterocyclic amine found in well-cooked meats, has been shown to be extensively glucuronidated in humans. To determine which UGT isozymes are involved in the biotransformation of PhIP and the cytochrome P4501A2-mediated reactive intermediate N-hydroxy-PhIP, microsomes expressing human UGT1A1, -1A4, -1A6 or -1A9 were incubated with PhIP and N-hydroxy-PhIP and the reaction products analyzed by HPLC and ESI-MS. Incubations containing N-hydroxy-PhIP and UGT1A1 expressing microsomes, with an apparent Km of 4.58 microM and a Vmax of 4.18 pmol/min/mg protein, had the highest capacity to convert N-hydroxy-PhIP to N-hydroxy-PhIP-N2-glucuronide. Microsomes expressing UGT1A9 produced N-hydroxy-PhIP-N3-glucuronide at the highest rate with an apparent Km and Vmax of 3.73 microM and 4.07 pmol/min/mg, respectively. A third previously undefined glucuronide accounted for 31% of the total glucuronides formed from the UGT1A4 expressing microsomes. No glucuronide conjugates were detected from microsomes expressing UGT1A6. Incubations containing PhIP as substrate formed direct PhIP-glucuronides in microsomes expressing UGT1A1, UGT1A4 and UGT1A9 but at levels averaging 53-fold lower than when N-hydroxy-PhIP was used as the substrate. Knowing the glucuronidation capacity of the specific UGT isozymes involved in PhIP and N-hydroxy-PhIP glucuronidation should help in determining the individual susceptibility to the potential cancer risk from exposure to PhIP.
Malfatti
M A
MA
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, PO Box 808, L-452, Livermore, CA 94551, USA.
Felton
J S
JS
D001711:Biotransformation; D002273:Carcinogens / Q000493:pharmacokinetics*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000493:pharmacokinetics*; D008861:Microsomes / Q000201:enzymology; D011725:Pyridines / Q000493:pharmacokinetics*; D021241:Spectrometry, Mass, Electrospray Ionization
D002273:Carcinogens; D007093:Imidazoles; D011725:Pyridines; C060533:2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D014453:Glucuronosyltransferase