[14C]Benzidine is rapidly oxidized by a peroxidase/H2O2 system to products which bind irreversibly to DNA. The presence of exogenous DNA also prevented benzidine polymerization to 'benzidine brown' and azobenzidine. Two molar equivalents of H2O2 were required to oxidize the benzidine and achieve maximal DNA binding. Furthermore, 95% of the benzidine was trapped and 36 nmol benzidine was bound per mg DNA. Polyriboguanylic acid was as effective as DNA in binding benzidine, but polyriboadenylic acid, polyribouridylic acid and polyribocytidylic acid were much less effective. Binding of [14C]benzidine correlated well with the absorbance at 295 nm and 390 nm of the modified DNA or various synthetic homopolymers of ribonucleotides isolated from the reaction mixture. The peroxidase/H2O2 system also catalyzed the binding of dichlorobenzidine, o-tolidine and o-dianisidine to DNA but 3,5,3',5'-tetramethylbenzidine, a non-carcinogen, did not bind. The binding could be prevented by various biological hydrogen donors, thiols, or phenolic antioxidants. The mechanisms for DNA protection were investigated; the oxidized benzidine species involved in binding can be reduced with ascorbate, NADPH, or thiols, and trapped by thiols or phenolic antioxidants to form conjugates or adducts.

Addition of the tumor promoter phorbol myristate acetate to polymorphonuclear leukocytes results in the oxidation of the arylamine carcinogens; [14C]benzidine, N-[14C]methylaminoazobenzene and [14C]aminofluorene to reactive intermediate(s) that bind irreversibly to the leukocyte DNA. The binding was dependent on oxygen and was decreased by sulfhydryl inhibitors and phenolic antioxidants that inhibit the respiratory burst triggered by the phorbol myristate. Both the binding and the respiratory burst were increased by azide, presumably as a result of intracellular catalase inhibition. However higher concentrations of azide and cyanide prevented binding without affecting the respiratory burst indicating that myeloperoxidase is a catalyst for the binding. Granules isolated from the activated leukocytes and H2O2 catalyzed a cyanide sensitive benzidine binding to calf thymus DNA. Myeloperoxidase and H2O2 also catalysed extensive binding of these arylamines to calf thymus DNA. The leukocytes appear to be a useful model cell for studying one electron oxidation-catalyzed carcinogen activation.

Whilst much emphasis has been placed in chemical carcinogenesis on the 2 e oxidation catalytic activity of monooxygenase, it is clear that 1 e oxidation pathways mediated by prostaglandin synthetase activity, lipid peroxidation or monooxygenase activity can also form metabolites which readily bind to the informational macromolecules and could be a critical step in the initiation of neoplasia. The 1 e oxidation pathway is more active in catalysing this binding with phenols, amines and hydrazines and could explain the necrosis or carcinogenesis induced by acetamlnophen, diethylstilbestrol, methylhydrazine, benzidine, benzene, hair dyes, cyclophosphamide (1). It is widely held that the ultimate carcinogen is an electrophile however the 1 e oxidation pathway forms free radicals and chemical carcinogenesis mechanisms need to be re-examined.

Addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to polymorphonuclear leukocytes is known to trigger the respiratory burst leading to the formation of H2O2. We now show that this H2O2 can react with some peroxidase within the cell to peroxidatically oxidize the carcinogen N-methylaminoazobenzene to reactive intermediate(s) that bind irreversibly to cellular DNA. The antioxidant butylated hydroxyanisole inhibits this binding suggesting that free radicals may be involved in the activation process.