Glucuronidation of benzidine and its metabolites by cDNA-expressed human UDP-glucuronosyltransferases and pH stability of glucuronides

Although glucuronidation is considered a necessary step in aromatic amine-i nduced bladder cancer, the specific enzymes involved are not known. This st udy assessed the capacity of five different human recombinant UDP-glucurono syltransferases expressed in COS-1 cells to glucuronidate benzidine, its me tabolites and 4-aminobiphenyl. [C-14]UDP-glucuronic acid was used as co-sub strate, UGT1A1, UGT1A4 and UGT1A9 each metabolized all of the aromatic amin es, UGT1A9 exhibited the highest relative rates of metabolism with preferen ce for the two hydroxamic acids, N-hydroxy-N-acetylbenzidine and N-hydroxy- N,N'-diacetylbenzidine, UGT1A9 metabolized 4-aminobiphenyl similar to 50% f aster than benzidine or N-acetylbenzidine. UGT1A4 N-glucuronidated N'-hydro xy-N-acetylbenzidine at the highest relative rate compared with the other t ransferases, UGT1A6 was effective in metabolizing only four of the eight ar omatic amines tested. UGT1A1 demonstrated more extensive metabolism of the hydroxamic acid, N-hydroxy-N,N'-diacetylbenzidine, and the ring oxidation p roduct, 3-OH-N,N'-diacetylbenzidine, than it did for the other six amines, UGT2B7 was the only product of the UGT2 gene family examined and it metabol ized all the aromatic amines at similar low relative levels compared with a preferred substrate, 4-OH-estrone, The K-m values for N-acetylbenzidine me tabolism by UGT1A1 and UGT1A4 were 0.37 +/- 0.14 and 1.8 +/- 0.4 mM, respec tively, The O-glucuronide of 3-OH-N,N'-diacetylbenzidine was not hydrolyzed during a 24 h 37 degrees C incubation at either pH 5.5 or 7.4, Likewise, t he O-glucuronide of 3-OH-benzidine was stable at pH 7.4, with 52% remaining at pH 5.5 after 24 h, These results suggest the following relative ranking of transferase metabolism: UGT1A9 > UGT1A4 much greater than UGT2B7 > UGT1 A6 approximate to UGT1A1, The relative pH stability of O-glucuronides is co nsistent with a role in detoxification and excretion of aromatic amines, wh ile the acid lability of N-glucuronides is consistent with delivery of thes e amines to the bladder epithelium for activation, resulting in DNA adducts which may lead to mutations.