N-Acetylbenzidine-N'-glucuronidation by human, dog and rat liver

Abstract

While N-glucuronidation is an important pathway for metabolism of aromatic amines, it has not been demonstrated for N-acetylbenzidine. A glucuronide of N-acetylbenzidine was synthesized and identified by mass spectrometry as N-acetylbenzidine-N'-glucuronide. This N'-glucuronide is acid labile with a t1/2 of 4 min at pH 5.3. A similar acid lability was also observed with benzidine-N-glucuronide. The formation of N-acetylbenzidine-N'-glucuronide was assessed with liver slices and microsomes prepared from human, dog and rat. When 0.014 mM [3H]N-acetylbenzidine was incubated with human liver slices a significant amount of N-acetylbendizine-N'-glucuronide was produced (8-26% of the total radioactivity recovered). With higher concentrations of [3H]N-acetylbenzidine (1 mM) rat slices also produced N-acetylbenzidine-N'-glucuronide. However, N'-glucuronide formation was not detected with dog liver slices incubated with either 0.014 or 1 mM [3H]N-acetylbenzidine. N-Acetylbenzidine-N'-glucuronide formation was observed with microsomes prepared from human, dog and rat. To assess maximum activity four detergents were used at two concentrations. With or without detergent activation the relative amount of glucuronidation was human > > dog > rat. The rate of benzidine N-glucuronide formation was 4.3- and 1.6-fold greater than N-acetylbenzidine-N'-glucuronide in dog and rat respectively, while in human both rates were similar (1.1-fold). With or without detergent activation the relative amount of benzidine-N-glucuronide formation was human> dog > > rat. N-Glucuronidation of [3H]N,N'-diacetylbenzidine was not observed. Thus N-actylbenzidine-N'-glucuronide formation appears to be an important pathway for metabolism of N-acetylbenzidine, especially in humans. Due to their acid lability, formation of the N-glucuronides of N-acetylbenzidine and benzidine provides a mechanism for hepatic detoxification and accumulation of these carcinogens in the bladder. A new model is described illustrating the effect of N-glucuronidation and the influence of N-acetylation on arylmono- and aryldiamine-induced bladder carcinogenesis.

NAT2 slow acetylation and bladder cancer in workers exposed to benzidine

“…Studies using human, dog and rat liver slices have demonstrated that both N-acetylbenzidine and benzidine are released from their glucuronides by acidic urine. 60 It has been hypothesized that the released primary aromatic amines could be further activated within the bladder to initiate carcinogenesis. 57 In vitro experiments have demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1, but not NAT2.…”

Evaluation of the metabolism of propranolol by linear ion trap technology in mouse, rat, dog, monkey, and human cryopreserved hepatocytes

“…showing that the N ‐oxidation of propranolol is mediated by FMO 35. To support our conclusion that the proposed N ‐hydroxylamine glucuronide formation is specific to dog and monkey hepatocytes, samples were prepared under neutral as well as acidic conditions to test glucuronide stability 36–39. No difference was observed in N‐ hydroxlamine peak intensity under either condition and the metabolite was not observed in any other species.…”

Aromatic Nitro and Amino Compounds

“…However, the Nglucuronide of N-OH-BZ was stable at an acidic pH (257). Although dog liver slices made an N-glucuronide from BZ (258), dog liver did not form such a conjugate from N-acetyl-BZ, but human and rat liver did (259). It was noted that liver microsomes from aged rats were more susceptible to the effects of BZ than those from younger animals (260).…”