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

我们设计了一种实验方案，允许通过脉冲场凝胶电泳（PFGE）检测DNA单链断裂和碱不稳定位点。在碱处理下，异烟肼、肼和苯肼在Mn（II）预处理细胞的DNA中产生单链断裂和碱不稳定位点。在实验条件下，碱处理对对照DNA和限制性内切酶Not - i处理的DNA没有影响。因此，我们似乎有理由认为，碱处理后的DNA片段化水平比相应的碱未处理样品的DNA片段化水平增加是由于DNA损伤剂引入的单链断裂和碱不稳定位点。在Cu（II）存在下，1,2,4-苯三醇（苯代谢物）、2,5-二羟基联苯（邻苯酚代谢物）、四氯对苯二酚（PCP代谢物）、3-羟基苯甲酸、3-羟基尿氨酸（色氨酸代谢物）和咖啡酸通过过氧化氢（H2O2）形成对分离的DNA造成损伤。这些致癌物尚未被证明在细菌系统中具有诱变作用。PFGE显示，在Mn（II）、色氨酸代谢物和咖啡酸存在的情况下，细胞DNA链断裂。3-氨基三唑（一种过氧化氢酶抑制剂）对链断裂有增强作用，邻菲罗啉对链断裂有抑制作用。因此，认为Mn（II）催化某些色氨酸代谢物和咖啡酸的自氧化产生H2O2， H2O2被内源性过渡金属离子激活，对细胞DNA造成损伤。在过渡金属存在的情况下，非诱变致癌物或其代谢物引起氧化性DNA损伤是一个有趣的问题。

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.