Analysis of DNA damage in cultured cells induced by Ames test-negative carcinogens.

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We have designed an experimental protocol which allows the detection of DNA singlestrand breaks plus alkali-labile sites by pulsed field gel electrophoresis (PFGE). With alkali treatment, isoniazid, hydrazine and phenylhydrazine were shown to produce single-strand breaks plus alkali-labile sites in DNA of Mn(II)-pretreated cells. Under the experimental conditions employed, no effect of alkali treatment was observed with control DNA and restriction endonuclease Not I-treated DNA.Therefore, it seems reasonable to suppose that the increase of the level of DNA fragmentation by alkali treatment compared to that of the corresponding alkali-nontreated sample was due to single-strand breaks and alkali-labile sites introduced by DNA-damaging agents.In the presence of Cu(II), 1,2,4-benzenetriol (a benzene metabolite), 2,5-dihydroxybiphenyl (an o-phenylphenol metabolite ), tetrachlorohydroquinone ( PCP metabolite ), 3-hydroxyanthranilic acid, 3-hydroxykynurenine (tryptophan metabolites) and caffeic acid caused damage to isolated DNA through hydrogen peroxide (H2O2) formation. These carcinogens have not been proved to be mutagenic in bacterial systems. The PFGE showed that in the presence of Mn(II), tryptophan metabolite and caffeic acid produced strand breaks in DNA of the cells.3-Aminotriazol (a catalase inhibitor) showed enhancing effect on the strand breakage, whereas o-phenanthroline showed inhibitory effect on the strand breakage. Therefore, it is considered that Mn(II)-catalyzed autoxidation of certain tryptophan metabolite and caffeic acid produce H2O2, which is activated by endogenous transition metal ion to cause damage to cellular DNA.It is of interest that the nonmutagenic carcinogens or their metabolites cause oxidative DNA damage in the presence of transition metals.