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Metabolic activation of benzidine

Dear Sir, In reviewing the response by Carreon et al., the framework of our conclusions regarding studies concerning benzidine can be summarized as follows. The mechanisms by which benzidine causes urinary bladder cancer is of scientific interest and, more importantly, as a public health issue. As stated in our original communication, we agree with Carreon et al. that rapid acetylators appear to be at greater risk. Our primary concern with their original paper and their response is that they give unwarranted strength to the idea that prostanglandin H synthase (PHS) is involved in the activation of N-acetylbenzidine to N0-hydroxy-N-acetylbenzidine in the urothelium, a target of benzidine in humans. We agree with their newly submitted proposed metabolic scheme that PHS is unlikely to produce a reactive metabolite in the urothelium. This is consistent with previous studies comparing the activation of benzidine and N-acetylbenzidine by PHS. The experimental evidence provided in Table I is from Morton et al. As can be seen, PHS activation of N-acetylbenzidine is less than 1% that of benzidine, about one half that of 2-aminofluorene or 2-aminonaphthalene and slightly greater than 4-aminobiphenyl. Recognition of Zenser’s ability to detect N0-hydroxy-N-acetylbenzidine on incubation with N-acetylbenzidine and PHS is not convincing that it would actually influence DNA modification in the urothelium. If this were to be the case, one could argue that activation by PHS would be important for simple aromatic monoamines such as 2-aminofluorene and 2-aminonaphthalene. It is likely that most investigators in this area would agree that the presentation of N-oxidized aromatic amines to the urothelium is probably a crucial requirement for a carcinogenic outcome. A final metabolic transformation, such as the production of a reactive species by O-acetylation, would provide for chemical modification of DNA within the urothelial cell. The oxidized derivatives are generally believed to be derived from the urine; urinary diversion in the dog abolished the bladder carcinogenicity of 2-aminonaphthalene. Much attention has been given to the possibility that urinary pH is involved in the stabilization, or activation, of various N-oxidized metabolites. The idea that the lower pH of urine might stabilize some N-oxidized metabolites would seem to be more important than the enhancement of modification of DNA by these metabolites. This comes from recognition that decreases in tightly regulated intracellular pH would be expected to compromise cellular viability and not provide for survival of modified cells capable of the development of tumors. Previous studies that have isolated benzidine-DNA adducts from exfoliated cells in the urine cannot exclude the possibility that these adducts came from acid-catalyzed reactions of hydroxylamines with nonviable cells that no longer had intact external cellular membranes. We agree that the question of the identification of metabolites that are unstable, as with many of the postulated benzidine derivatives, presents a dilemma when trying to evaluate biological mechanisms. These qualitative and quantitative limitations make it important to incorporate observational experiments with those that can be adequately controlled. This is why the heterotopic bladder system (HTB), as developed by Oyasu, is useful in attempting to better relate metabolism to biological consequences. In this system, the carcinogenicity of the N-OH-N-glucuronide is not correlated to the rate of its hydrolysis. As shown in our article, 50% of the N-hydroxy-Nacetyl-2-aminofluorene (N-OH-AAF) applied to the HTB was absorbed within 18 min whereas only 30% of the N-glucuronide of N-OH-2-aminofluorene disappeared from the bladder fluid within 4 hr in a pH range of 7.1–7.4. Yet, the N-glucuronide produced urothelial tumors in 95% of the rats and N-OHAAF produced tumors in only 43% of the rats. Similarly, N0OH-N0-glucuronide-N-acetylbenzidine and N0-OH-N-acetylbenzidine produced bladder tumors in 100 and 60% of the rats, respectively. Zenser’s group has reported that N0-OH-N0-glucuronide-N-acetylbenzidine can not be hydrolyzed using a metabolite prepared from N0-OH-N-acetylbenzidine in the presence of a glucuronosyltransferase system. From a chemical structure viewpoint, the stability of this conjugate is not expected to be different from the N-OH-N-glucuronide of 2aminofluorene, 4-aminobiphenyl and 2-aminonapthalene. The TABLE I – PHS-CATALYZED REACTION OF VARIOUS SUBSTRATES WITH tRNA