基于重组发光酵母的遗传毒性检测技术及应用

Genotoxicity detection technology and application based on recombinant luminescent yeast

基于酵母DNA损伤响应网络,本研究选择4种生物标志物,以丝裂霉素C(MMC)为模型遗传毒物,双酚A为阴性对照,通过优化实验条件,建立了一种基于重组发光酵母的遗传毒性检测技术.结果表明:菌液培养体积为60μL、微板阅读器增益设置为100是最佳的检测条件;并在此条件下,将MMC暴露在酵母中,其毒性特征谱图在2 h分析长度内呈现出明显的浓度与时间依赖性,从剂量-响应曲线上得出其遗传毒性作用的最低浓度为0.04 mg·L^(-1);阴性对照双酚A的实时蛋白表达图谱则与MMC完全相反,并未呈现出浓度与时间的依赖关系.将上述方法应用于中国北方黄河中下游某村镇地表水及可疑污染源水样的遗传毒性测试,结果表明:当浓缩倍数为100倍时,14个测试点位中有6个点位处的水样被检出阳性;当浓缩倍数为10倍时,水样均呈阴性.

Based on the yeast DNA damage response network,four biomarkers were selected in this study,with mitomycin C(MMC)as the model genotoxicant and bisphenol A as the negative control.A recombinant luminescent yeast-based genotoxicity assay was established by optimizing the experimental conditions 60μL culture volume and 100 microplate reader gain setting.the toxicity profile of MMC exposed to yeast under these conditions showed a significant concentration and time dependence within 2 h analysis length,with 0.04 mg·L^(-1) being the lowest showing genotoxicity from doseresponse curve.In contrast,real-time protein expression profile of bisphenol A was completely opposite to that of MMC and did not show concentration or time dependence.This method was applied to test surface water samples taken from a village in the middle and lower reaches of the Yellow River in northern China,and 6 out of 14 sites tested positive when concentrated 100-fold,but all sites were negative when concentrated 10-fold.