Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase

Abstract

Benzidine and N-acetylbenzidine are activated to genotoxic metabolite(s) within the urothelial target tissue, with phase-I and phase-II enzymes being relevant. In principle, both benzidine and N-acetylbenzidine are activated by prostaglandin H synthase (PHS) to reactive intermediates. However, the relative impacts of benzidine and N-acetylbenzidine in this process remain unclear. Two experimental in vitro systems were used in the present comparative investigation: ram seminal vesicle microsomes rich in PHS and porcine urinary bladder epithelial cells (PUBEC) as a model system mimicking the general metabolic situation within the human urothelium. Benzidine, N-acetylbenzidine and N, N′-diacetylbenzidine were incubated with ram seminal vesicle microsomes and arachidonic acid and control incubations were performed with heat-inactivated microsomes. The metabolic disappearance of benzidine, N-acetylbenzidine or N, N′-diacetylbenzidine indicated a rapid turnover by PHS of benzidine and a slower turnover of N-acetylbenzidine. There was almost no PHS-associated metabolism of N, N′-diacetylbenzidine, suggesting that diacetylation of benzidine could represent a pathway of biological inactivation. Under similar conditions, incubations were performed with ram seminal vesicles and benzidine or N-acetylbenzidine upon addition of calf thymus DNA. After re-isolation of the DNA and 32 P -postlabeling, with benzidine 2 distinct adducts were found of unknown nature, and with N-acetylbenzidine a single adduct appeared with co-migrated with the N′-(3′-monophosphodeoxyguanosin-8-yl)- N-acetylbenzidine. PUBEC cells were also incubated with benzidine or N-acetylbenzidine. No DNA adduct was found with benzidine, but a total of five adducts was produced from N-acetylbenzidine. The major adduct again co-migrated with N′-(3′-monophosphodeoxyguanosin-8-yl)- N-acetylbenzidine. When benzidine was incubated with PUBEC cells N-acetylbenzidine and, with some delay, N, N′-diacetylbenzidine were formed. Application of Lineweaver–Burk plots for the formation of N-acetylbenzidine from benzidine revealed a K m of 56.4 μM and a V max of 7.05 nmol/h per 10 6 PUBEC cells. The investigations generally support a key role of N-acetylbenzidine at the target site of the urothelium.