Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material.

Ram seminal vesicle (RSV) microsomal preparations activate benzidine and other arylamines to mutagenic species in a modified Ames assay. We have examined the mechanism of this activation process in more detail. The mutagenic effect was neither arachidonic acid-dependent nor indomethacin inhibitable. The mutagenic species was stable for at least 30 min in experiments in which addition of bacteria was delayed. Acetylbenzidine was a much more potent mutagen than benzidine in this system. Substitution of the acetylase-deficient tester strain TA98/1,8-DNP6 for strain TA98 markedly reduced the mutagenicity of acetylbenzidine and completely eliminated the mutagenicity of benzidine. Benzidine analogues 3,3'-dimethoxybenzidine (o-dianisidine), o-tolidine and 3,3',-5,5'-tetramethylbenzidine were not mutagenic in the RSV activation system. RSV-dependent activation of all radiolabeled congeners examined resulted in covalent binding to calfthymus DNA. The rank order of binding was: 3,3'-dichlorobenzidine greater than benzidine greater than o-dianisidine greater than acetylbenzidine greater than tetramethylbenzidine. This binding required active enzyme and arachidonic acid or hydrogen peroxide. The reactive species was short-lived: delayed addition of DNA reduced the level of binding nearly to zero. Binding was inhibitable by indomethacin, but this inhibition was incomplete in the cases of dichlorobenzidine and acetylbenizidine. We conclude that the extracellular generation of peroxidase-catalyzed oxidation products does not explain the RSV microsome-dependent mutagenicity observed with these compounds.