Cell-based reporter assay can quantify epigenetic changes that have been chemical induced

Researchers developed a cost-effective assay to be used with chemicals and potential carcinogens associated with reversible changes to DNA

Cleaning agents, cosmetics, dyes, flavourings, preservatives, pesticides and other industrial chemicals are now more often being recognised as hazardous to human health. Widespread use has seen a rise in the prevalence of a variety of diseases which are chemical toxicity induced. These include cancers, disruption to hormones, neurological disorders and skin conditions.

Many chemicals have a carcinogenic effect where cancers can develop because of genotoxic effects. This means the direct or indirect interference of DNA replication and/or damage repair processes which can result in mutations and chromosomal change. Various in vitro genotoxicity assays help assess the interactions of potential carcinogens with DNA and elucidate their role in health and disease.

Also, epigenetic alterations, which are reversible changes to DNA and chromatin – packaged DNA-protein complexes – have been implicated in chemically induced carcinogenesis.

For example, DNA methylation where the addition of a methyl group to DNA will silence gene expression. Conversely, acetylation of histones – the addition of acetyl group to the proteins that bind DNA – opens up the chromatin structure, making DNA accessible for transcription.

Such dynamic epigenetic alterations in DNA and histones regulate gene expression in a cell- and tissue-specific manner. Environmental chemicals such as arsenic, benzene, bisphenol A, cadmium, pesticides and other carcinogens have been reported to induce disease-related epigenetic changes.

Understanding these chemical-induced epigenetic alterations can help with safety assessments of environmental chemical compounds.

Cell-based assays previously have been developed to detect epigenetic changes in a unidirectional manner – inactivation/reactivation of gene expression – based on the baseline status of the reporter gene. These do not however fully capture chemical-induced epigenetic alterations. A bidirectional assay needed to be developed to effectively detect a wide range of chemical exposures that could cause epigenetic change in human cells.

A team drawn jointly from the Graduate School of Science, Chiba University and the National Institute of Health Sciences both in Japan have developed a novel epi-genotoxicity assay to evaluate carcinogen-induced epigenetic changes.

“In the field of genome biology, mastering epigenetic analysis techniques is both challenging and costly, making it difficult to use in the safety assessment of chemicals,” said Associate Professor Akira Sassa at Chiba University.

“This led us to consider developing a universally accessible method through collaborative research across academia, industry, and government. Elucidation of previously unknown epigenetic mechanisms of chemical carcinogenesis can aid safer chemical use worldwide, including in developing countries,” he said.

Thymidine kinase (TK) gene mutation assay (TK assay) is a conventional in vitro genotoxicity test that detects mutations in the TK gene locus, an essential ‘housekeeping’ gene expressed by all cell types.

The research team had previously enhanced the TK assay and improved its detection sensitivity for the safety assessment of pharmaceutical, industrial, agricultural and environmental chemicals with potential genotoxic and cytotoxic effects. In the current study, they have further developed an epi-TK reporter assay by site-specifically methylating the TK promoter region.

They assessed the capability of the epi-TK assay to reflect global epigenetic changes by quantifying ‘TK reversion’ or the expansion of cells with a silenced-TK promoter following treatment with inhibitors of DNA methyl transferases (DNMT) with well-characterised mechanisms of action to elucidate the potential of the system to reflect epigenetic changes.

Notably, treatment with DNMT inhibitors resulted in unmethylated sites within the TK promoter region and expansion of TK revertant colonies. Additionally, treatment with 12-O-tetradecanoylphorbol-13-acetate, a widely studied non-genotoxic seed oil-derived carcinogen, led to a significant decrease in TK revertant frequency and histone acetylation levels. The epi-TK assay could thus reflect epigenetic changes associated with both gene silencing and activation.

Contrary to advanced sequencing analyses that require expensive reagents and instrumentation, the epi-TK reporter assay developed in this study offers a simpler, cost-effective, and quantitative approach to evaluate chemical epi-genomic toxicity.

 “Our research can enhance the understanding of the impact of chemicals on public health and disease prevention, thereby promoting safer management and use of chemicals.

“Understanding the relationship between environmental chemicals and diseases, and improving chemical safety evaluations, can aid the implementation of measures to reduce the exposure to harmful chemicals in both work and living environments,” concluded Associate Professor Sassa.

For further information please visit: 10.1038/s41598-025-92121-6