MITOCHONDRIAL OXIDATION OF 3, 3'-DIAMINOBENZIDINE AND RELATED COMPOUNDS, AND THEIR POSSIBLE RELATION TO CARCINOGENESIS

1. The enzymic oxidation of benzidine compounds by rat liver and beef heart cytochrome a was studied spectrophotometrically. 2. The mitochondrial oxidation of 3, 3'-diaminobenzidine was inhibited by cyanide, azide, and heat-treatment for 10min at 60°, while it was activated by the addition of cytochrome c. Although approximately 70% inhibition was observed when 10mM succinate was added, activity was restored after the further addition of malonate, which suppressed succinate dehydrogenase activity. Oxidation was confirmed to be due to cytochrome oxidase activity. 3. 3, 3'-Diaminobenzidine was successfully oxidized by beef heart cytochrome a, and stimulated by the addition of cytochrome c. The molecular activity of cytochrome a alone was estimated as 0.72 (mot of diaminobenzidine/mol of cytochrome a/min). The rate of oxidation by cytochrome a and c decreased in the order of 3, 3'-diaminobenzidine, 3, 3'-dimethoxybenzidine, 3, 3'-dimethylbenzidine, and benzidine. 4. A possible relationship between the cytochrome c-reducing ability of benzidine derivatives and their carcinogenicity is discussed. Potent carcinogens, 3, 3'-dichlorobenzidine and p-aminobiphenyl could not reduce cytochrome c; and benzidine, 3, 3'-dimethylbenzidine, and 3, 3'-dimethoxybenzidine were shown to be moderate reductants. 3, 3'-Diaminobenzidine, a mild carcinogen, markedly reduced cytochrome c. Carcinogenic diphenylamine did not react with cytochrome c, but p-aminodiphenylamine quickly reduced it, suggesting that it has little or no carcinogenicity.