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

我们设计了一种实验方法，可以用脉冲场凝胶电泳法(PFGE)检测DNA单链断裂和碱不稳定位点。碱处理后，异烟肼、肼和苯肼可导致细胞DNA出现单链断裂和碱不稳定位点。在所用的实验条件下，对照DNA和限制性内切酶未处理的DNA没有观察到碱处理的效果。因此，似乎可以合理地假设，与相应的碱处理样品相比，碱处理的DNA断裂水平的增加是由于DNA损伤剂引入的单链断裂和碱不稳定部位。在铜(II)、1，2，4-苯三酚(苯代谢物)、2，5-二羟基联苯(邻苯基苯酚代谢物)、四氯对苯二酚(PCP代谢物)、3-羟基邻氨基苯甲酸、3-羟基犬尿氨酸(色氨酸代谢物)和咖啡酸通过过氧化氢(H_2O_2)的形成对分离的DNA造成损伤。这些致癌物在细菌系统中尚未被证明具有致突变性。PFGE结果表明，在Mn(II)存在下，色氨酸代谢产物和咖啡酸可引起细胞DNA的断裂，过氧化氢酶抑制剂3-氨基三唑对DNA断裂有促进作用，邻菲咯啉对DNA断裂有抑制作用。因此，人们认为，某些色氨酸代谢物和咖啡酸在Mn(II)催化下自氧化生成H_2O_2，而H_2O_2被内源过渡金属离子激活，从而对细胞DNA造成损伤。

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.