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 P-32-labeled human DNA fragments. 4-Amino-3-methylphenol, a major metabolit e, caused DNA damage in the presence of Cu(II), Predominant cleavage sites were thymine and cytosine residues, o-Nitrosotoluene, a minor metabolite, d id not induce DNA damage even in the presence of Cu(II), but addition of NA DH induced DNA damage very efficiently. The DNA cleavage pattern was simila r to that in the case of 4-amino-3-methylphenol, Bathocuproine and catalase inhibited DNA damage by these o-toluidine metabolites, indicating the part icipation of Cu(I) and H2O2 in the DNA damage. Typical free hydroxyl radica l scavengers showed no inhibitory effects on the DNA damage. o-Toluidine me tabolites 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 spectroscopi c studies have demonstrated that 4-amino-3-methylphenol is autoxidized to f orm 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 O-2 to for m 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-mediat ed DNA damage by o-toluidine metabolites through H2O2 seems to be relevant for the expression of the carcinogenicity of o-toluidine. (C) Academic Pres s.