Analysis of damage to human genes for estimating the carcinogenicity of environmental chemicals

脉冲场凝胶电泳（Pulsed-field gel electrophoresis，PFGE）已成为研究高分子量DNA的有力工具，常用于检测细胞DNA双链断裂。此外，我们还设计了一种PFGE检测DNA单链断裂和碱不稳定位点的实验方案，在本研究中，我们利用PFGE和DNA测序技术研究了金属离子存在下Ames试验阴性致癌物对细胞和分离DNA的损伤。用ESR自旋捕集法研究了引起DNA损伤的活性物种。我们已经表明，在Cu（II）的存在下，Ames试验阴性致癌物（苯代谢物，邻苯基苯酚代谢物，咖啡酸，五氯苯酚代谢物，色氨酸代谢物）通过H2 O2的形成对分离的DNA造成损伤。脉冲场凝胶电泳显示，Ames试验阴性致癌物诱导培养的人细胞中的Mn（II）的存在下的DNA链断裂。用碱处理，DNA单链断裂观察。链断裂增加3-氨基三唑（过氧化氢酶抑制剂）和减少过氧化氢酶，表明H2 O2的参与。邻菲咯啉可减轻DNA损伤，表明过渡金属离子参与了DNA损伤。这些结果表明，在Mn（II）或Cu（II）的存在下，Ames试验阴性致癌物产生过氧化氢，这是激活的过渡金属在体外引起DNA损伤，并可能在培养的细胞。

Pulsed-field gel electrophoresis (PFGE) has emerged as a powerful tool for the study of high-molecular weight DNA.PFGE is generally used for detection of cellular DNA double-strand breaks. In addition, we have designed an experimental protocol which allows the detection of DNA single-strand breaks plus alkali-labile sites by PFGE.In this study, we have investigated damage to cellular and isolated DNA by Ames-test negative carcinogens in the presence of metal ions using both PFGE and DNA sequencing techNique. Active species causing DNA damage were investigated by the ESR-spin trapping method. We have shown that in the presence of Cu (II), Ames-test negative carcinogens (benzene metabolites, o-phenylphenol metabolites, caffeic acid, pentachlorophenol metabolites, tryptophan metabolites) caused damage to isolated DNA through H2O2 formation. PFGE showed that Ames-test negative carcinogens induced DNA strand breaks in cultured human cells in the presence of Mn (II). With alkali treatment, DNA single-strand breaks were observed. The strand breakage was increased by 3-aminotriazol (a catalase inhibitor) and decreased by catalase, indicating the involvement of H2O2. The DNA damage was decreased by o-phenanthroline, indicating the involvement of transition metal ion. These results suggest that in the presence of Mn (II) or Cu (II), Ames-test negative carcinogens produce H2O2, which is activated by transition metals to cause damage to DNA in vitro and probably in cultured cells.