Laboratory of Instrumental Analysis, School of Pharmacy and Pharmaceutical Sciences, Hoshi University.
Laboratory of Biofunctional Science, School of Pharmacy and Pharmaceutical Sciences, Hoshi University.
J Toxicol Sci. 2021;46(2):83-90. doi: 10.2131/jts.46.83.
Epigenetic toxicity, a phenomenon in which chemicals exert epigenetic effects and produce toxicity, has been attracting attention in recent years due to advances in toxicology accompanying the development of life sciences. However, it has been difficult to identify epigenetic toxicants due to the lack of a simple experimental system to evaluate epigenetic toxicity. In this study, we developed a prototype of an in vitro reporter assay system for assessing the effects of chemicals on DNA methylation using two promoters showing different degrees of DNA methylation, Agouti IAP and Daz1 promoters, and a luciferase reporter. The system successfully detected DNA demethylating activity using 5-azacytidine, a chemical having DNA demethylation activity, as a positive control chemical, and demethylation of cytosine of CpG in the promoter was confirmed by pyrosequencing analysis. Next, in order to improve the detection sensitivity of the DNA demethylating activity of this system, we tried to increase the basal level of methylation of the Daz1 promoter by pre-methylase treatment of the reporter vectors. As a result, the detection sensitivity of the system was successfully improved in cells where the basal level of methylation was indeed increased by methylase treatment. Thus, the developed assay system here is effective for the simple evaluation of chemicals that affect DNA methylation.
表观遗传毒性是指化学物质产生表观遗传效应并产生毒性的现象，近年来随着生命科学的发展，毒理学的进步，引起了人们的关注。然而，由于缺乏评估表观遗传毒性的简单实验系统，因此很难识别表观遗传毒性剂。在这项研究中，我们使用两种显示不同程度 DNA 甲基化的启动子（Agouti IAP 和 Daz1 启动子）和一个荧光素酶报告基因，开发了一种用于评估化学物质对 DNA 甲基化影响的体外报告基因检测系统原型。该系统成功地使用具有 DNA 去甲基化活性的 5-氮杂胞苷作为阳性对照化学物质检测到 DNA 去甲基化活性，并通过焦磷酸测序分析证实了启动子中 CpG 胞嘧啶的去甲基化。接下来，为了提高该系统检测 DNA 去甲基化活性的灵敏度，我们尝试通过预甲基化处理报告载体来增加 Daz1 启动子的基础甲基化水平。结果，通过甲基化酶处理确实增加了基础甲基化水平的细胞中，该系统的检测灵敏度得到了成功提高。因此，这里开发的测定系统可有效地用于简单评估影响 DNA 甲基化的化学物质。
