Metabolism of 3,3′ -dichlorobenzidine by horseradish peroxidase

Abstract

1. The peroxidatic oxidation of 3,3'-dichlorobenzidine by horseradish peroxidase in the presence of H2O2 was examined spectrophotometrically and the reactivity of the spectral species were compared to those formed from the peroxidative oxidation of benzidine. 2. The horseradish peroxidase-catalyzed oxidation of 3,3'-dichlorobenzidine yielded two transient and one stable spectral species with absorption maxima at 630 nm, 370 nm and 410 nm, respectively, whereas that of benzidine yielded three stable spectral species with absorption maxima at 610 nm, 425 nm and 370 nm, respectively. 3. The 425 nm species from benzidine, but not the 410 nm species from 3,3'-dichlorobenzidine was scavenged by butylated hydroxyanisole, glutathione, N-acetylcysteine or 2-deoxyguanosine. 4. H.p.l.c. mass spectrometric analysis, and comparative studies with potassium dichromate oxidation of 3,3'-dichlorobenzidine, indicated that the major product from the horseradish peroxidase-catalyzed oxidation of 3,3'-dichlorobenzidine is azo-3,3'-dichlorobenzidine. 5. None of the products from enzymic or chemical oxidation of either 3,3'-dichlorobenzidine or benzidine was directly mutagenic to S. typhimurium TA98 in the Ames test; however the chemically oxidized and enzymic products from 3,3'-dichlorobenzidine were mutagenic in the presence of H2O2. 6. The data indicate that despite apparent structural similarities between the intermediates formed during the peroxidatic oxidation of all benzidines, the intermediates and products of peroxidatic oxidation of dichlorobenzidine have reactivities and stabilities different from those of other benzidines.

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

“…In subsequent studies with sensitive tester strains which produce high levels of NAT/OAT, we have shown that this activation requires PHS peroxidase activity, but does not require exogenous PHS substrate (arachidonic acid), since S. typhimurium bacteria produce low levels of hydrogen peroxide (8). Both PHS peroxide activity and HRP readily metabolize benzidine by one-electron oxidation to reactive free radical and diimine products (27). Other aromatic amines are also oxidized to radical intermediates by peroxidases (26).…”

“…Indeed, since N-acetylbenzidine is a much poorer peroxidase reducing substrate than is benzidine, acetylation should simply divert substrate away from the activation pathway. Synthetic benzidine diimine, the product of peroxidative metabolism of benzidine, is not mutagenic in strains TA98 (27) or YG1012.2 Furthermore, HRP cannot activate benzidine to a mutagenic species, despite facile substrate oxidation (28).…”