Distinct Mechanisms of Oxidative DNA Damage by Two Metabolites of Carcinogenic o-Toluidine

Mechanisms of DNA damage by metabolites of carcinogenic o-toluidine in the presence of metals were investigated by the DNA sequencing technique using 32P-labeled human DNA fragments. 4-Amino-3-methylphenol, a major metabolite, caused DNA damage in the presence of Cu(II). Predominant cleavage sites were thymine and cytosine residues. o-Nitrosotoluene, a minor metabolite, did not induce DNA damage even in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. The DNA cleavage pattern was similar to that in the case of 4-amino-3-methylphenol. Bathocuproine and catalase inhibited DNA damage by these o-toluidine metabolites, indicating the participation of Cu(I) and H2O2 in the DNA damage. Typical free hydroxyl radical scavengers showed no inhibitory effects on the DNA damage. o-Toluidine metabolites increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine in calf thymus DNA in the presence of Cu(II). UV–visible and ESR spectroscopic studies have demonstrated that 4-amino-3-methylphenol is autoxidized to form the aminomethylphenoxyl radical and o-nitrosotoluene is reduced by NADH to the o-toluolhydronitroxide radical in the presence and absence of Cu(II). Consequently, it is considered that these radicals react with O2 to form O−2 and subsequently H2O2, and that the reactive species generated by the reaction of H2O2 with Cu(I) participate in the DNA damage. Metal-mediated DNA damage by o-toluidine metabolites through H2O2 seems to be relevant for the expression of the carcinogenicity of o-toluidine.