Development of a new in vitro assay system for evaluating the effects of chemicals on dna methylation

Maky Ideta-Otsuka, Misato Miyai, Naoki Yamamoto, Ayaka Tsuchimoto, Hideki Tamura, Kentaro Tanemura, Makoto Shibutani, Katsuhide Igarashi

Research output: Contribution to journalArticlepeer-review


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 proto-type 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 cyto-sine 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.

Original languageEnglish
Pages (from-to)83-90
Number of pages8
JournalJournal of Toxicological Sciences
Issue number2
Publication statusPublished - 2021
Externally publishedYes


  • 5-azacytidine
  • Agouti IAP promoter
  • DNA methylation
  • Daz1 promoter
  • Reporter assay

ASJC Scopus subject areas

  • Toxicology


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