Qian Yan, Wang Xiaoli, Lv Zhanlu, Guo Chen, Han Mei, Wu Jiabing, Yang Yongjian, Yang Yishu, Jiang Yan, Wei Yongjie, Nie Jing, Liang Bao, Zhang Jinliang, Wang Xianliang
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China.
College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China.
BMC Biotechnol. 2015 Nov 26;15:107. doi: 10.1186/s12896-015-0224-y.
The demethylation potential of environmental pollutants is possibly an innate part of their comprehensive health risk. This paper develops a novel method called TDQ to quantify the demethylation epigenetic toxicity, termed the 5-AZA-CdR demethylation toxic equivalency, of aquatic samples from the heavily polluted Bohai Bay using Hep G2 cell lines transiently transfected with the pEGFP-C3 plasmid containing a methylated promoter of the EGFP reporter gene inserted artificially in vitro.
If the aquatic sample extract has strong total demethylation potential to the promoter, its methylation level will decrease, and increased green fluorescence will be observed under microscopy after TDQ co-incubation. The 5-AZA-CdR was selected as a representative demethylation agent to validate the principle of the TDQ method on three levels: significant dose-response relationships between the concentration of 5-AZA-CdR and the methylation level of promoters, mRNA expression level of the EGFP gene, and the fluorescence intensity of EGFP proteins. Twenty extracts from aquatic samples are successfully quantified with the TDQ test. Eight of them return meaningful results ranging from 0.00004 to 0.20053 μM 5-AZA-CdR toxicity equivalents.
The TDQ method is a reliable and rapid assay for the quantification of the DNA demethylation potential of aquatic sample extracts, which may shed light on the safety evaluation of food material.
环境污染物的去甲基化潜力可能是其综合健康风险的固有组成部分。本文开发了一种名为TDQ的新方法，用于量化来自重度污染的渤海湾水样的去甲基化表观遗传毒性，即5-氮杂胞苷（5-AZA-CdR）去甲基化毒性当量，该方法使用在体外人工插入含有增强绿色荧光蛋白（EGFP）报告基因甲基化启动子的pEGFP-C3质粒进行瞬时转染的Hep G2细胞系。
如果水样提取物对启动子具有较强的总去甲基化潜力，其甲基化水平将降低，并且在TDQ共同孵育后，在显微镜下会观察到绿色荧光增强。选择5-氮杂胞苷作为代表性去甲基化剂，在三个水平上验证TDQ方法的原理：5-氮杂胞苷浓度与启动子甲基化水平、EGFP基因的mRNA表达水平以及EGFP蛋白的荧光强度之间存在显著的剂量-反应关系。通过TDQ测试成功量化了20种水样提取物。其中8种得到了有意义的结果，范围为0.00004至0.20053 μM 5-氮杂胞苷毒性当量。
TDQ方法是一种可靠且快速的测定方法，用于量化水样提取物的DNA去甲基化潜力，这可能为食品原料的安全性评估提供线索。
