Background

A broad range of chemical substances has been implicated in carcinogenesis through genetic, epigenetic, and metabolic mechanisms. Chemical-induced genetic alterations, collectively referred to as genotoxicity, arise from direct DNA damage or from disruption to DNA replication, repair, and damage response pathways. To evaluate these risks, standardized in vitro genotoxicity assays are widely applied in accordance with the recommendations of the Organization for Economic Co-operation and Development guidelines [1]. However, increasing evidence indicates that many chemical agents contribute to carcinogenesis not only through the induction of DNA mutations but also via perturbation of epigenetic regulation, including changes in DNA methylation and histone modifications. Current chemical safety testing frameworks rely primarily on in vitro mutation-based assays, such as the Ames test and mammalian cell gene mutation assays [2-4], which are unable to assess epigenetic alterations. Although high-resolution sequencing-based epigenome profiling technologies can generate detailed information, their high cost, technical complexity, and significant analytical burden limit their routine application in regulatory testing, highlighting the need for practical approaches to assess epigenetic toxicity. To address this need, we developed a practical approach for assessing epigenetic alterations at a housekeeping gene locus by adapting the TK gene mutation assay [2,5], which utilizes the human lymphoblastoid TK6 cell line widely employed in genotoxicity testing. The TK gene mutation assay quantifies mutant frequency by measuring colony formation according to the TK genotype. Trifluorothymidine (TFT) is used to select TK-deficient mutant colonies because cells expressing functional thymidine kinase convert TFT into a toxic nucleotide and undergo cell death, whereas TK-deficient cells survive in the presence of TFT. Conversely, cells carrying a functional or reverted TK gene can be selectively detected using hypoxanthine–aminopterin–thymidine medium, in which only cells with restored thymidine kinase activity survive through the salvage pathway under aminopterin-mediated inhibition of de novo nucleotide synthesis. Building upon standardized in vitro genotoxicity testing frameworks, we developed the epi-TK assay, a mammalian cell–based reporter system in which the promoter region of the housekeeping TK gene in human lymphoblast TK6 cells is site-specifically methylated using CRISPR/dCas9-based epigenome-editing technology [6]. The engineered reporter cell line enables quantitative and bidirectional detection of chemically induced epigenetic effects through changes in frequency of TK revertant colony formation. Following chemical exposure, epigenetic alterations at the TK locus are assessed using hypoxanthine–aminopterin–thymidine selection, with restoration of TK gene expression quantified as the frequency of TK revertant colonies. Because the methylated TK locus retains active histone acetylation marks, it exists in a transcriptionally suppressed but reversible state, allowing spontaneous TK reactivation to occur at a measurable baseline frequency during culture [6]. An increase in revertant frequency suggests inhibition of DNA methylation or promotion of DNA demethylation, whereas a decreased reversion may reflect changes in histone modifications associated with reduced chromatin accessibility. These trends can be further validated by locus-specific DNA methylation analysis, chromatin immunoprecipitation, or protein-level assessment, depending on the expected mode of action of the test compound. Using GSK3484862 as a model DNA methyltransferase 1 inhibitor [7], this protocol quantitatively evaluates its epigenetic effects using the epi-TK assay. As previously reported, the assay also enables quantitative assessment of inflammation-associated global reductions in histone acetylation [6], further highlighting its bidirectional capability to detect distinct modes of epigenetic alteration. Collectively, this assay provides a simple, cost-effective, and biologically relevant platform for evaluating epigenetic toxicity and offers a practical approach for routine chemical safety assessment.