GLUCURONIDATION OF N-HYDROXY METABOLITES OF N-ACETYLBENZIDINE

Glucuronidation of N-hydroxy arylamines is thought to be a necessary s tep in their initiation of bladder cancer, This was evaluated for the N-hydroxy metabolites of N-acetylbenzidine (ABZ), N'-Hydroxy-N-acetylb enzidine (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 reac tion mixtures containing [C-14]UDP-glucuronic acid, 3 mM ascorbic acid and human liver microsomes a new product was detected by HPLC, Emulge n 911 was a better detergent than Triton X-100 for expressing microsom al activity, with maximal glucuronidation observed with 0.3% Emulgen 9 11, 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 gluc uronidation of N-HA was only exceeded by N-HDA, At pH 5.5 and 37 degre es C the t(1/2) 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, Fo r N-HDA >90% of this glucuronide remained after 24 h, At pH 7.4 and 37 degrees C the t(1/2) for the glucuronide conjugates of ABZ and N-HA w ere 2.3 and 2 h respectively, with the amounts remaining after 24 h fo r N'-HA and N-HDA being 75 and 90% respectively, At pH 6.5 the t(1/2) for N'-HA was 14 h, Thus only glucuronides of ABZ and N'-HA exhibit pH -dependent changes in t(1/2). Compared with ABZ, glucuronides the N-hy droxy metabolites are more stable at acidic pH, Acidic urine would be more likely to hydrolyze the glucuronide conjugate of ABZ than those o f its N-hydroxy metabolites, Because these results are different from that hypothesized for arylmonoamines, a new model was developed to exp lain the role of N-oxidation, N-glucuronidation and N-acetylation in t he carcinogenesis of benzidine, an aryldiamine.