Carcinogenesis, Vol. 20, No. 10, 1963-1969,
October 1999
© 1999 Oxford University Press
Carcinogenesis |
Glucuronidation of benzidine and its metabolites by cDNA-expressed human UDP-glucuronosyltransferases and pH stability of glucuronides
1 Heritable Disorders Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA and
2 VA Medical Center,
3 Department of Biochemistry and
4 Division of Geriatric Medicine, St Louis University School of Medicine, St Louis, MO 63125, USA
Although glucuronidation is considered a necessary step in aromatic amine-induced bladder cancer, the specific enzymes involved are not known. This study assessed the capacity of five different human recombinant UDP-glucuronosyltransferases expressed in COS-1 cells to glucuronidate benzidine, its metabolites and 4-aminobiphenyl. [14C]UDP-glucuronic acid was used as co-substrate. UGT1A1, UGT1A4 and UGT1A9 each metabolized all of the aromatic amines. UGT1A9 exhibited the highest relative rates of metabolism with preference for the two hydroxamic acids, N-hydroxy-N-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidine. UGT1A9 metabolized 4-aminobiphenyl ~50% faster than benzidine or N-acetylbenzidine. UGT1A4 N-glucuronidated N'-hydroxy- N-acetylbenzidine at the highest relative rate compared with the other transferases. UGT1A6 was effective in metabolizing only four of the eight aromatic amines tested. UGT1A1 demonstrated more extensive metabolism of the hydroxamic acid, N-hydroxy-N,N'-diacetylbenzidine, and the ring oxidation product, 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 metabolized all the aromatic amines at similar low relative levels compared with a preferred substrate, 4-OH-estrone. The Km values for N-acetylbenzidine metabolism by UGT1A1 and UGT1A4 were 0.37 ± 0.14 and 1.8 ± 0.4 mM, respectively. The O-glucuronide of 3-OH-N,N'-diacetylbenzidine was not hydrolyzed during a 24 h 37°C incubation at either pH 5.5 or 7.4. Likewise, the 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 > > UGT2B7 > UGT1A6 
UGT1A1. The relative pH stability of O-glucuronides is consistent with a role in detoxification and excretion of aromatic amines, while the acid lability of N-glucuronides is consistent with delivery of these amines to the bladder epithelium for activation, resulting in DNA adducts which may lead to mutations.
Abbreviations: PBS, phosphate-buffered saline; UCE, UGT1 common end.
5 To whom correspondence should be addressed at: VA Medical Center (11G-JB), St Louis, MO 63125-4199, USA Email: zensertv{at}slu.edu
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