Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase.
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 32P-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 microM and a Vmax 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.
Degen
Gisela H
GH
Institut für Arbeitsphysiologie an der Universität Dortmund, Ardeystr. 67, D-44139 Dortmund, Germany. degen@ifado.de
Schlattjan
Jan H
JH
Mähler
Solveigh
S
Föllmann
Wolfram
W
Golka
Klaus
K
D000818:Animals; D001560:Benzidines / Q000378:metabolism* / Q000493:pharmacokinetics* / Q000633:toxicity; D001711:Biotransformation; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology / Q000378:metabolism; D012756:Sheep; D013552:Swine; D019459:Urothelium / Q000201:enzymology / Q000378:metabolism
D001560:Benzidines; D018736:DNA Adducts; C007291:N,N'-diacetylbenzidine; C029876:benzidine; D011451:Prostaglandin-Endoperoxide Synthases; C034938:N-acetylbenzidine