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

Abstract

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. [(14)C]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 approximately 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 K(m) 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 degrees 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 approximately 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.

CHARACTERIZATION OF AFLOQUALONE N-GLUCURONIDATION: SPECIES DIFFERENCES AND IDENTIFICATION OF HUMAN UDP-GLUCURONOSYLTRANSFERASE ISOFORM(S)

“…Unfortunately, we could not examine clearly the contribution of UGT1A3 isoform to the AFQ N-glucuronidation in human jejunum microsomes due to the absence of specific substrates or inhibitors for UGT1A3. It has been reported that primary amine glucuronidation was seen with UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, and UGT2B7 Ciotti et al, 1999;Hiller et al, 1999;Kaivosaari et al, 2002). The present results were almost consistent with these reports.…”

Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor

“…We were unable to detect desloratadine N-glucuronide or 3-hydroxydesloratadine N-glucuronide by LC-MS/MS in our previous study (Kazmi et al, 2015). Instability is a characteristic property of certain N-glucuronides (Ciotti et al, 1999).…”

A Long-Standing Mystery Solved: The Formation of 3-Hydroxydesloratadine Is Catalyzed by CYP2C8 But Prior Glucuronidation of Desloratadine by UDP-Glucuronosyltransferase 2B10 Is an Obligatory Requirement

“…These glucuronides appear to be very unstable, which is a characteristic of certain other N-glucuronides (Ciotti et al, 1999). Despite this limitation, the identification of CYP2C8 in combination with UGT2B10 in the formation of 3-hydroxydesloratadine contributes to our understanding of the long-standing mystery surrounding the enzymology of 3-hydroxydesloratadine formation in humans, providing a pathway for future investigation of the genetic basis for the desloratadine poor metabolizer phenotype.…”