4-Aminobiphenyl N-glucuronidation by liver microsomes: optimization of the reaction conditions and characterization of the UDP-glucuronosyltransferase isoforms.
4-Aminobiphenyl (4-ABP) is an arylamine that has long been associated with human and animal urinary bladder cancer. N-glucuronidation is an important metabolic pathway that contributes significantly to 4-ABP-bladder carcinogenesis by facilitating transport of the active metabolites from the liver to the bladder. This pathway is carried out by UDP-glucuronosyltransferase (UGTs). These enzymes are located in the inner membrane of the endoplasmic reticulum. Full UGT activity is not achieved until membrane constraints are removed. This study was conducted to optimize the incubation conditions of 4-ABP N-glucuronidation. The kinetic parameters of the isozymes most commonly involved in arylamine glucuronidation, namely UGT1A4 and UGT1A9, were also determined. The UGT reaction was linear in the incubation time (0-90 min) and in the microsomal protein range of 0-0.5 mg. Alamethicin, a pore-forming agent, was found to be the best reagent to activate UGTs. It increased the enzyme activity by nearly 8-fold and this activation was at concentration of 50 microg mg(-1) protein. Interestingly, UGT1A4 glucuronidated 4-ABP with more affinity and efficiency than did UGT1A9. The K(m) and V(max) of UGT1A4 for 4-ABP were 58.8 microm and 234.9 pmol min(-1) mg(-1) protein, respectively, and 227.5 microm and 31.2 pmol min(-1) mg(-1) protein for UGT1A9. Furthermore, hecogenin was found to be a competitive inhibitor for UGT1A4. It increased the K(m) of UGT1A4 for 4-ABP by nearly 10 fold at a concentration of 50 microm. This is the first report that tried to optimize the incubation conditions for 4-ABP N-glucuronidation and characterized the enzyme kinetic parameters of UGT isoforms catalysing 4-ABP N-glucuronidation.
Al-Zoughool
Mustafa
M
McLaughlin Centre for Population Health Risk Assessment, University of Ottawa, Ottawa, Canada. malzough@uottawa.ca
Talaska
Glenn
G
D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D001713:Biphenyl Compounds / Q000378:metabolism*; D005260:Female; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans
C503507:4-aminobiphenyl-N-glucuronide; D001713:Biphenyl Compounds; D020719:Glucuronides; D007527:Isoenzymes; D000408:Alamethicin; D014453:Glucuronosyltransferase