Mutagenic activation of benzidine requires prior bacterial acetylation and subsequent conversion by prostaglandin H synthase to 4-nitro-4'-(acetylamino)biphenyl.

We have used the Ames test in combination with prostaglandin H synthase (PHS) to study the bioactivation of benzidine as well as other aromatic amines. Previous investigations established that the formation of benzidine mutagens by PHS is dramatically enhanced in Salmonella typhimurium strains with high levels of acetyl CoA-dependent arylamine N-acetyltransferase/arylhydroxylamine O-acetyltransferase activity despite the fact that acetylation of aromatic amines decreases their susceptibility to oxidation by peroxidases. In this study, we used a new strain (YG1012) that has very high acetylation capability to investigate the metabolism and mutagenicity of benzidine and N-acetylbenzidine catalyzed by PHS (from ram seminal vesicle microsomes) and horseradish peroxidase (HRP). YG1012 bacteria rapidly acetylated benzidine to N-acetylbenzidine and N,N'-diacetylbenzidine. Preincubation of the bacteria with benzidine before addition of PHS increased the mutagenicity. Under conditions identical to those used to assess mutagenicity, PHS metabolized benzidine rapidly, but the substrate was not totally consumed, with about 40% of the original concentration remaining intact. These data suggest that conversion to N-acetylbenzidine may be the initial step in the bioactivation of benzidine in the PHS-mediated Ames assay. N-Acetylbenzidine is a cosubstrate for PHS peroxidase activity as measured by 5-phenyl-4-pentenyl hydroperoxide reduction, spectral changes, and formation of protein adducts. N-Acetylbenzidine was converted to mutagens by PHS but not HRP, with enhanced mutagenicity observed in bacteria with high acetylation activity. We used reverse- phase HPLC to characterize the metabolites of N-acetylbenzidine formed by PHS and HRP.(ABSTRACT TRUNCATED AT 250 WORDS)