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DNA 甲基化焦磷酸测序检测法适用于评估具有表观遗传活性的环境或临床相关化学物质。

DNA methylation pyrosequencing assay is applicable for the assessment of epigenetic active environmental or clinical relevant chemicals.

机构信息

Environmental Toxicology, University of Trier, FBVI, Universitätsring 15, 54296 Trier, Germany.

出版信息

Biomed Res Int. 2013;2013:486072. doi: 10.1155/2013/486072. Epub 2013 Sep 4.

DOI:10.1155/2013/486072
PMID:24093099
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3777179/
Abstract

Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects induced by chemicals and drugs. An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels. In vitro cell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. The in vitro testing of epigenetically active chemicals (5-methyl-2'-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation). In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants.

摘要

细胞和生物体暴露于应激源可能导致表观遗传变化。本研究表明，使用焦磷酸测序法研究 DNA 甲基化是一种替代体外和体内化学物质和药物诱导的表观遗传毒性测试的方法。本文展示了用化学物质/药物处理细胞后，对整体和 CpG 位点特异性 DNA 甲基化的体外评估。亚硫酸氢盐基因组测序的甲基化对照显示，LINE1 中甲基化的阳性对照具有高甲基化，阴性对照具有低甲基化。LINE1 元件内的 CpG 位点被甲基化到不同的水平。体外细胞培养显示甲基化水平在 56%至 49%之间。与原始非耐药肿瘤细胞相比，耐药肿瘤细胞的培养物表现出明显的低甲基化。对具有表观遗传活性的化学物质（5-甲基-2'-脱氧胞苷和曲古抑菌素 A）的体外测试表明，在处理后 LINE1 甲基化状态发生显著变化，而特定的 CpG 位点比其他位点更容易去甲基化（局部甲基化）。总之，使用焦磷酸测序法进行 DNA 甲基化不仅可用于测试表观遗传毒素/药物，还可用于药物、食品和环境相关污染物的风险评估。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/302c/3777179/8d0c3e822680/BMRI2013-486072.008.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/302c/3777179/8d0c3e822680/BMRI2013-486072.008.jpg

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