基因组DNA氧化水平对甲基化的影响

The association of DNA methylation and DNA oxidation induced by H202

目的分析体外培养细胞在H20：染毒后8-oxo-dG的形成、细胞DNA5-mE含量的变化规律，探讨其时间一剂量一效应关系和DNA氧化与DNA甲基化的关联性。方法利用0、20、40、60、80和100μmol／LH202作用于人肺泡Ⅱ型上皮细胞（A549），每天染毒1次，每次2h，共持续4d，继续培养8d。应用高压液相色谱串联电化学检测技术分析细胞DNA8-oxo—dG水平、高效毛细管电泳（high－performance capillary electrophoresis，HPCE）法检测细胞全基因组DNA5-mE的含量。结果DNA甲基化与DNA氧化呈现不同特点，A549细胞染毒80μmol／LH2O2后DNA甲基化水平的改变需要10d以上的时间，而相同条件下DNA氧化水平的改变仅需12h，DNA甲基化的改变滞后于DNA氧化。H2O2可引起A549细胞甲基化程度降低，呈剂量依赖性关系。在一定的时间剂量范围内，DNA氧化与DNA甲基化作用间呈负相关关系（r=-0．72，P〈0．01）。结论DNA氧化能导致细胞DNA甲基化水平降低，DNA氧化可能是影响细胞甲基化的调控机制之一，在化学物质的毒作用效应中，DNA氧化可能是比DNA甲基化更早的一个重要分子事件。

Objective To study the potential association of DNA oxidation and DNA methylation, in vitro cultured cells were exposed to different doses of H202, 8-oxo-dG formation, cell DNA 5-mC contents were analyzed to explore the time- dose-response relationship of DNA oxidation and DNA methylation. Methods A549 cells were exposed to different doses of H202, 8-oxo-dG formation and cell genomic DNA 5-mC contents were analyzed by a high-performance liquid chromatography system and high performance capillary electrophoresis （HPCE）, respectively. Results H202 induced the formation of 8-oxo-dG and 5-mC in different characteristics, it need at least 10 days for significant changes in the level of DNA methylation, whereas under the same conditions, changes in the level of DNA oxidation cast only 12 hours. H202 induced decreased levels of DNA methylation in A549 cells in a dose-dependent manner. In a certain range of time and dose, it showed a negative correlation between DNA oxidation and DNA methylation. Conclusion The study suggests that oxidative DNA could lead to reduced levels of DNA methylation, DNA oxidation may affect the regulation of cellular methylation mechanisms, in the course of chemical mutagenesis, DNA oxidation may be an earlier important molecule event than DNA methylation.