Quantifying Epigenetic Changes Induced by Chemical Exposure Using the epi-TK Assay

Epigenetic modifications play essential roles in regulating gene expression and maintaining cellular identity. Accumulating evidence suggests that chemical agents can contribute to carcinogenesis through epigenetic alterations, such as changes in DNA methylation and histone modifications, even in the absence of direct DNA damage. Here, we have developed a simple, cost-effective, and quantitative reporter assay, termed the epi-TK assay, to evaluate chemically induced epigenetic alterations. The assay is built upon the thymidine kinase (TK) gene mutation assay, a standardized and widely used in vitro genotoxicity assay for chemical safety evaluation. This system is based on an engineered human lymphoblastoid cell line (mTK6), in which the promoter region of the endogenous housekeeping TK gene is site-specifically methylated using epigenome-editing technology, resulting in stable transcriptional repression. Following chemical exposure, epigenetic perturbations at the TK locus are detected by culturing cells under hypoxanthine-aminopterin-thymidine selection and quantifying the frequency of TK revertant colonies, which reflects restoration of TK gene expression. Using the DNA methyltransferase 1 inhibitor GSK3484862 as a model compound, this protocol demonstrates that the epi-TK assay enables sensitive and quantitative detection of epigenetic state transitions. Importantly, this assay allows bi-directional detection of epigenetic changes, including DNA demethylation events and broader alterations in histone modification landscapes. Together, the epi-TK assay provides a practical and quantitative platform for evaluating epigenetic toxicity, with potential applications in chemical safety assessment frameworks. Key features • This protocol describes testing of the epigenetic effects of chemicals using the mTK6 cell line and a modified version of the TK gene-mutation assay. • By employing a DNA-methylated housekeeping TK gene and colony formation as the readout, the assay enables quantitative epigenetic changes without the need for specialized equipment. • The protocol offers a simple, quantitative, and cost-effective platform that is suitable for routine testing and comparative assessment of multiple compounds.