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Glucuronidation of N-hydroxy arylamines is thought to be a necessary step in their initiation of bladder cancer. This was evaluated for the N-hydroxy metabolites of N-acetylbenzidine (ABZ). N'-Hydroxy-N-acetylbenzidine (N'-HA), N-hydroxy-N-acetylbenzidine (N-HA) and N-hydroxy- N,N'-diacetylbenzidine (N-HDA) were synthesized. Except for N'-HA, these compounds were quite stable. Ascorbic acid and/or acidic pH increased the stability of N'-HA. When each N-hydroxy compound was added to reaction mixtures containing [14C]UDP-glucuronic acid, 3 mM ascorbic acid and human liver microsomes a new product was detected by HPLC. Emulgen 911 was a better detergent than Triton X-100 for expressing microsomal activity, with maximal glucuronidation observed with 0.3% Emulgen 911. At 0.125 mM amine the rate of glucuronidation was N-HDA >> N'-HA = benzidine > ABZ > N-HA. In contrast, at 0.5 mM amine the rate of glucuronidation of N-HA was only exceeded by N-HDA. At pH 5.5 and 37 degrees C the t1/2 for the enzymatically prepared glucuronide conjugates of ABZ, N'-HA and N-HA were 7.5 min and 3.5 and 1.8 h respectively. For N-HDA > 90% of this glucuronide remained after 24 h. At pH 7.4 and 37 degrees C the t1/2 for the glucuronide conjugates of ABZ and N-HA were 2.3 and 2 h respectively, with the amounts remaining after 24 h for N'-HA and N-HDA being 75 and 90% respectively. At pH 6.5 the t1/2 for N'-HA was 14 h. Thus only glucuronides of ABZ and N'-HA exhibit pH-dependent changes in t1/2. Compared with ABZ, glucuronides the N-hydroxy metabolites are more stable at acidic pH. Acidic urine would be more likely to hydrolyze the glucuronide conjugate of ABZ than those of its N-hydroxy metabolites. Because these results are different from that hypothesized for arylmonoamines, a new model was developed to explain the role of N-oxidation, N-glucuronidation and N-acetylation in the carcinogenesis of benzidine, an aryldiamine.

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.

The mechanism by which benzidine induces bladder cancer in dog was evaluated by assessing metabolism of [3H]benzidine by dog liver slices and microsomes. Slices incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min peak, which was also produced when microsomal incubations were supplemented with UDP-glucuronic acid. In contrast to microsomes, very little of the 32.5 min peak was produced with the 100,000 g supernatant fraction. Microsomal metabolism was increased 5-fold by pretreatment with Triton X-100. Very little activity was observed with rat microsomes in either the presence or absence of Triton X-100. This metabolite was also generated by incubating benzidine with glucuronic acid at 4 degrees C for 3 days. Thermospray MS identified this metabolite as benzidine N-glucuronide. At 37 degrees C, the t1/2 stability of purified N-glucuronide was 99, 25 and 3 min in dog urine adjusted to pH 7.3, 6.3 and 5.3 respectively. The N-glucuronide was quite stable at pH 9.3, in dog plasma, and in aprotic solvents for 4 h at 37 degrees C. Relative to benzidine, its N-glucuronide is weakly bound to plasma proteins but not more reactive with DNA. Thus, detoxification by liver provides a mechanism for accumulation of benzidine in acidic urine, uptake of benzidine into bladder epithelium, and activation of benzidine in bladder. The liver and N-glucuronidation play a potentially important role in the species specificity of benzidine carcinogenesis.