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

Abstract

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)

Genetic polymorphisms of N-acetyltransferase 1 and 2 and risk of cigarette smoking-related bladder cancer

“…In addition to NAT1 and NAT2, the metabolism of arylamines also involves a number of other enzymes including sulphotransferases (Kato and Yamazoe, 1994), prostaglandin H synthase (Smith et al, 1992), cytochrome P450 1A2 (Butler et al, 1989;Fleming et al, 1994) and glucuronyltransferases. These enzymes may also contribute to the metabolic steps of arylamines relevant to the development of bladder cancer.…”

T Cell Responses to Paraphenylenediamine and to Its Metabolites Mono- and Diacetyl-Paraphenylenediamine

“…This reaction was catalyzed by N -acetyltransferase 1 (NAT1). N -Acetylation reactions are considered as a detoxification step but recently Smith et al [4]and others [5]reported that the monoacetylated metabolite of benzidine is more toxic than benzidine itself. Therefore, we focussed in this study on the immunogenic capacities of the acetylated metabolites of PPD by measuring the proliferative responses of T cells.…”

Peroxygenase Metabolism of N-Acetylbenzidine by Prostaglandin H Synthase

“…NЈHA generates nitrenium ions that react with DNA to produce dGp-ABZ (46). It was proposed that PHS-derived 4Ј-nitro-4-acetylaminobiphenyl had to be reduced to NЈHA before DNA adduct formation would occur (15). Whereas the latter may happen, this study demonstrates that NЈHA is an intermediate available for immediate reaction with DNA.…”

“…Horseradish peroxidase does not form this metabolite. Metabolism of ABZ by PHS, but not by horseradish peroxidase, causes mutations that are attributed to 4Ј-nitro-4-acetylaminobiphenyl (15). The NЈ-hydroxy product of ABZ is not detected and is not thought to be an intermediate in 4Ј-nitro-4-acetylaminobiphenyl formation.…”

“…Benzidine, an aromatic amine reducing cofactor, is converted to a radical cation and subsequently converted to benzidinediimine, a two-electron oxidation product (12)(13)(14). In contrast, monoacetylated benzidine, N-acetylbenzidine (ABZ), yields an oxygenated metabolite, 4Ј-nitro-4-acetylaminobiphenyl (15). Horseradish peroxidase does not form this metabolite.…”