Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene

Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene Akira Sassa, Haruto Yamada, Mizuki Odagiri, Keigo Yamakita, Aoi Chiba, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5623363/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase ( TK ) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure. Biological sciences/Genetics/Epigenetics Biological sciences/Genetics/Eukaryote Biological sciences/Genetics/Genomic instability Biological sciences/Biological techniques/Genetic techniques toxicology epi-genotoxicity reporter assay 12-O-tetradecanoylphorbol-13-acetate DNMT inhibitor TK gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Numerous chemical agents have been implicated in carcinogenesis through diverse mechanisms, including genetic, epigenetic, and metabolic alterations. Among these processes, the ability of chemicals to induce genetic changes is defined as genotoxicity. Genotoxic agents can bind directly to DNA and act indirectly by interfering with enzymatic pathways such as DNA replication, DNA repair, and DNA damage signaling, ultimately leading to gene mutations and chromosomal aberrations. The potential risk of chemicals interacting with DNA is evaluated through genotoxicity testing. The Organization for Economic Co-operation and Development (OECD) has established guidelines for assessing chemicals using in vitro genotoxicity tests, including the bacterial Ames test, the mammalian cell chromosomal aberration test, and the mammalian cell gene mutation test 1 . In addition to genotoxic potential, epigenetic alterations are believed to contribute to chemically induced carcinogenesis 2 . Among these, cytosine C5 methylation in CpG dinucleotides represents a key epigenetic modification, playing crucial roles in processes such as X chromosome inactivation, genomic imprinting, and repression of retrotransposons through gene silencing. In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs). Of the active DNMTs, DNMT1 is responsible for maintaining DNA methylation during replication, while DNMT3A/3B are involved in de novo DNA methylation under specific cellular conditions. Beyond DNA methylation, gene expression is further regulated by post-translational histone modifications, which serve as structural elements for packaging genomic DNA within the nucleus. Histone H3K4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27Ac) are transcriptionally active marks that exhibit an inverse correlation with DNA methylation at gene promoter regions. These cellular epigenetic patterns can be influenced by exposure to certain chemical reagents that directly or indirectly modify DNA methylation and/or histone modifications 3 . Several therapeutic drugs have been developed as epigenetic modifiers with anticancer potential. Aberrant DNA methylation and the hyperactivation of histone deacetylases (HDACs) are closely linked to cell proliferation and metastasis in various cancers, making DNMTs and HDACs promising therapeutic targets 4 . 5-Aza-2′-deoxycytidine (5-azadC) is a widely used demethylating agent that inhibits DNMTs through its incorporation into genomic DNA 5 . Similarly, HDAC inhibitors promote hyperacetylation of core histones, thereby modulating chromatin structure and altering gene expression patterns 6 . Beyond these designed therapeutic agents, environmental chemicals such as bisphenol A, arsenic, cadmium, benzene, and pesticides have been reported to induce aberrant epigenetic changes, contributing to the onset of various diseases 7 . Notably, many carcinogens are believed to exert their effects through non-genotoxic mechanisms 8 . Therefore, evaluating the potential epigenetic effects and underlying mechanisms of action is a critical component in the safety assessment of environmental chemical compounds. To evaluate epigenetic alterations induced by chemical reagents, researchers have developed various cell-based reporter assays. Johnson et al. introduced a system using an epigenetically silenced green fluorescent protein ( GFP ) reporter gene controlled by an exogenous cytomegalovirus promoter 9 . More recently, endogenous tumor suppressor genes such as SFRP1 and BRCA1 have been employed as GFP -based reporters to study epigenetic changes 10,11 . Similarly, Okochi-Takada et al. developed a high-throughput screening platform using luciferase and GFP reporter genes driven by the inactivated UCHL1 locus in HCT116 cells 12 . These systems generally detect epigenetic modifications in unidirectional manner, either inactivation or reactivation of gene expression, based on the initial epigenetic state of the reporter gene locus. A notable approach is an in vitro reporter system utilizing DNMT-transformed yeast cells, enabling rapid and efficient detection of epigenetic effects caused by chemical reagents in bidirectional 13-15 . However, to ensure the extrapolation of experimental results to humans, a flexible bidirectional reporter assay needs to be designed in mammalian cells to effectively detect diverse modifications resulting from chemical exposure. Currently, the epigenetic impacts of chemicals on housekeeping genes remain poorly understood, emphasizing the need to evaluate these genes as potential reporter loci to broaden the scope and utility of such assays. In this study, we aimed to develop an epi-genotoxicity assay to evaluate epigenetic modifications induced by carcinogens, based on the thymidine kinase ( TK ) gene mutation assay (TK assay) technique. The TK assay is a conventional mammalian in vitro genotoxicity test that detects various mutations at the housekeeping TK gene locus, utilizing the human lymphoblastoid TK6 cell line 16 . The OECD Test Guideline (TG490) adopted the conventional TK assay for the safety assessment of pharmaceutical, industrial, agricultural, and environmental chemicals 17 . We recently enhanced the TK assay by developing genome-edited TK6 cell lines, improving its sensitivity for detecting genotoxic and cytotoxic effects of chemicals 18-20 . In the current study, we further advanced this approach by establishing epigenetically modified TK6 derivative (mTK6) cells as the epi-genotoxicity test system. Using the CRISPR/dCas9-SunTag-DNMT3A system, CpG sites within the endogenous TK promoter region were selectively methylated 21 , resulting in mTK6 cells with a stably methylated TK gene. These cells can be propagated in the presence of trifluorothymidine (TFT), which selectively exerts cytotoxicity against TK -proficient cells. The developed method, termed the “epi-TK assay,” enables accurate quantification of epigenetic changes induced by chemical exposure. This is achieved by measuring the frequency of TK revertant colonies in hypoxanthine, aminopterin , and thymidine (HAT) selection medium (Fig. 1A). To assess the ability of the epi-TK assay to quantify global epigenetic effects, we tested DNMT inhibitors (5-Aza-2'-deoxycytidine (5-azadC), GSK-3484865) and HDAC inhibitors (vorinostat and trichostatin A) as model substances with well-characterized mechanisms of action (Fig. 1B). Additionally, we examined the epigenetic effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a widely studied non-genotoxic tumor promoter/inflammation inducer derived from the seed oil of Jatropha curcas L. Using the established epi-TK assay, we observed not only an increase in TK reversion frequency induced by 5-azadC and GSK-3484865 but also a significant decrease in TK revertant frequency following TPA exposure. Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment caused a global reduction in H3K27Ac levels, likely linked to TPA-mediated chronic inflammation. These findings highlight the utility of the epi-genotoxicity assay as a tool for evaluating epigenetic alterations induced by chemical exposure in both directions. 2. Results Establishment of human TK6 derivative cells assessing chemical-induced epigenetic alterations We developed a reporter system to assess chemical-induced epigenetic alterations using the mammalian TK gene mutation assay as a platform. To create a cell line capable of quantifying epigenetic effects, we employed CRISPR/dCas9-SunTag-DNMT3A along with sgRNA expression plasmids specifically designed to methylate CpG loci within the promoter region of the endogenous TK gene. As illustrated in Figure 2A, transient expression of the sgRNAs resulted in a significant increase in TFT-resistant colonies (1.0 × 10 −3 ) compared to cells transfected with empty sgRNA vectors (3.2 × 10 −5 ). Among 12 clones isolated, we selected a representative clone that exhibited stable proliferation in TFT-containing medium. This isolated clone, designated as the “mTK6” cell line, had a population doubling time of 14 ± 1.0 h in the absence of TFT and 13 ± 0.52 h in its presence, similar to the original TK6 cell line’s doubling time of 14 ± 0.26 h (Fig. 2B). RT-qPCR analysis revealed abolished TK gene expression in mTK6 cells compared to TK6 cells (Fig. 2C), indicating that TK gene expression was repressed due to DNA methylation in its promoter region. We also evaluated the TK revertant frequency during cell proliferation up to 3 days following TFT removal from the medium (Fig. 2D). Spontaneous TK reversion was observed in a time-dependent manner: 0 day (0.77 ± 0.27 × 10 -4 ), 1 day (3.6 ± 1.3 × 10 -4 ), 2 days (6.4 ± 2.7 × 10 -4 ), and 3 days (8.8 ± 1.3 × 10 -4 ). Based on this background TK revertant frequency, we hypothesize that epigenetic alterations induced by chemical exposure can be quantified by measuring changes in the number of TK revertant colonies. Quantification of epigenetic effects of covalent and non-covalent DNA methyltransferase inhibitors We first evaluated the capability of the epi-TK assay to quantify the effects of typical DNA demethylating agents. 5-Aza-2'-deoxycytidine (5-azadC), a widely used potent DNMT inhibitor, was tested on mTK6 cells across a range of concentrations (0.02–0.1 µM), determined based on cytotoxicity assays (Fig. 3A). As shown in Figure 3B, treatment with 5-azadC led to a 230-fold increase in TK revertant frequency (7.7 ± 1.0 × 10 -2 ) at the highest concentration (0.1 µM) compared to the solvent control (3.3 ± 0.67 × 10 -4 ). Notably, 5-azadC is known for its high cytotoxicity and genotoxicity due to the formation of covalent DNA-DNMT adducts. To evaluate the assay’s versatility, we tested the epigenetic effects of non-covalent DNMT inhibitor GSK-3484862. As shown in Figure 3C and 3D, GSK-3484862 treatment significantly increased TK revertant frequency in a dose-dependent manner (0.12 ± 0.033 at 0.5 µM, 0.19 ± 0.052 at 2.0 µM, and 0.29 ± 0.044 at 5.0 µM) compared to the DMSO control (1.6 ± 0.46 × 10 -4 ). We next investigated the DNA methylation pattern of the TK gene promoter region in mTK6 cells. Bisulfite sequencing revealed methylation of cytosine at 26 CpG dinucleotides within a 230 bp region upstream of the TK gene’s start codon (Fig. 3E). To further examine the methylation status of the TK promoter after exposure to DNMT inhibitors, we analyzed TK revertant colonies. Notably, all CpG sites within the TK promoter were unmethylated in both spontaneous and DNMT inhibitor-mediated (5-azadC and GSK-3484862) TK revertant colonies. These findings suggest that the DNA methylation pattern of the TK promoter is closely associated with its gene expression status. Determination of histone modification status in the methylated TK gene promoter Transcriptionally active chromatin is characterized by the presence of H3K27ac and H3K4me3 near the transcription start site of the target gene. Thus, we investigated whether the status of H3K27ac and H3K4me3 was altered following DNA methylation in the TK promoter region. Interestingly, ChIP-qPCR analyses revealed that enrichment levels of K3K27Ac and H3K4me3 were comparable to those observed in TK6 cells (Fig. 4A and 5B). Consistent with these findings, exposure to HDAC inhibitors vorinostat (0.2 µM) and trichostatin A (0.01 µM) for 24 and 48 hours did not affect the frequency of TK revertants (Fig. 4C). This lack of effect may be due to the sustained levels of H3K27Ac and H3K4me3 in the presence of repressive DNA methylation at the TK promoter region in mTK6 cells. Detection of the epigenetic consequence of a non-genotoxic carcinogen/inflammation inducer 12-O-Tetradecanoylphorbol-13-acetate Based on the unique epigenetic pattern of the TK gene in mTK6 cells described above, we hypothesized that chemically induced global histone modifications could be detected through changes in the frequency of TK reversion. To test this hypothesis, we selected TPA, a potent non-genotoxic carcinogen with potential effects on histone modifications, though the persistent epigenetic consequences remain poorly understood. As shown in Figure 5A, treatment with TPA at concentrations ranging from 0.02–1.0 µg/ml resulting in approximately 40–50% cell survival. Under these conditions, the frequency of TK revertants decreased significantly, with a maximum of 28-fold reduction (0.090 ± 0.0058 × 10 -4 ) compared to the solvent control (2.5 ± 0.42 × 10 -4 ) (Fig. 5B). To further investigate histone modification status following TPA treatment, cells were harvested three days of cultivation following the exposure to TPA and subjected to ChIP-qPCR and western blotting analysis. ChIP-qPCR analysis revealed a significant reduction in H3K27Ac levels at the GAPDH and TK loci after TPA exposure (Fig. 5C). In contrast, the enrichment of H3K4me3 did not significantly change between ethanol- and TPA-treated cells (Fig. 5D). Consistently, western blotting analysis confirmed a significant reduction in H3K27Ac levels following TPA treatment (Fig. 5E and F). According to a recent study, differential gene expression patterns of epigenetic regulators, including DNMTs and chromatin remodelers, were observed depending on the duration of TPA exposure 22 . To investigate whether the reduction in H3K27Ac levels observed in our results is due to altered expression of epigenetic modulators, we performed RNA-seq analysis to identify DEGs resulting from TPA treatment. A 24-h treatment with TPA led to the upregulation of genes associated with the “ERK1 and ERK2 cascade” and “MAPK cascade,” followed by the “inflammatory response” (Fig. S1A-D). After washing out TPA and culturing the cells for 3 days, Gene Ontology analysis revealed that the biological pathways of upregulated genes were enriched in “leukocyte activation,” “innate immune response,” and “lymphocyte activation.” For the downregulated genes associated with TPA treatment, fewer pathways were enriched: “leukocyte tethering or rolling” for the 24-h TPA treatment, and “regulation of cell migration,” “cellular response to chemokine,” and “leukocyte activation” for the 3-day culture following TPA treatment. No significant differential expression of genes involved in epigenetic regulation, such as DNMT1, DNMT3A/B, histone acetyltransferases, and HDACs, was observed. 3. Discussion Over the past two decades, next-generation sequencing-based technologies have been developed to explore the epigenetic regulatory landscape of cells and tissues. Pioneered by Bisulfite-seq and ChIP-seq detecting DNA methylation and histone modifications, a wide range of epigenome profiling techniques has developed. These include Micrococcal Nuclease sequencing (MNase-seq), DNase I hypersensitive site sequencing (DNase-seq), Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-Seq), and Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), all of which are commonly used to assess chromatin accessibility 23-26 . An emerging technology, Hi-C (high-resolution chromosome conformation capture), provides a high-throughput method for mapping the 3D structure of chromosomes within the nucleus 27 . While these techniques have advanced epigenetic research, their implementation requires considerable experimental and analytical expertise, as well as expensive instruments and reagents. In this context, the development of reporter assays to detect specific patterns of epigenetic alteration offers a simpler, more cost-effective, and quantitative approach for evaluating chemical toxicity, providing framework for such assays. Thus, the experimental procedure of the epi-TK assay (Fig. 1), based on the standard genotoxicity testing outlined in OECD TG490, meets the criteria for an effective reporter system, contributing to safety assessment. For the development of the epi-TK assay, CRISPR/dCas9-SunTag-DNMT3A was transiently expressed in TK6 cells, leading to increased DNA methylation at CpG dinucleotides within the TK promoter region (Fig. 3E). Given that the expression of the TK gene is regulated by transcription factors such as E2F1 and SP1 28,29 , it is likely that DNA methylation at these CpG loci inhibits the binding of transcription factors, thereby preventing the transcription of the TK gene. Interestingly, the spontaneous frequency of TK revertant increased with the duration of cell culture after the removal of TFT (Fig. 2D), suggesting that the CpG sites in the promoter region were spontaneously demethylated during cell division. This reversible state of the epigenetically edited TK gene may be attributed to the persistent presence of active histone marks, such as H3K27Ac and H3K4me3 (Fig. 4A and B). Moreover, a recent study demonstrated that artificially introduced histone modifications, including H3K4me3, H3K27Ac, H3K27me3, H3K9me2/3, and H2AK119Ub, are progressively diluted after cell division 30 . Although the mechanistic basis of such epigenetic restoration remains unclear, a potential regulatory mechanism could involve the three-dimensional chromatin architecture within the nucleus 31 . Nevertheless, the epigenetic state of the TK gene is stably maintained when cells are cultured in the presence of TFT (Fig. 2B), ensuring the reproducibility of the assay. Using the housekeeping TK gene as a reporter locus, the effects of DNMT inhibitors were quantified (Fig. 3). The frequency of TK reversion following exposure to GSK-3484862 was substantially higher than that observed with 5-azadC. These results align with the observation that GSK-3484862 induces demethylation more efficiently than 5-azadC in murine cells 32 . This difference in demethylating efficacy is attributed to the cytotoxicity of 5-azadC, which forms covalent protein-DNA adducts 33 , whereas GSK-3484862 inhibits DNMT1 in a non-covalent manner. Regarding HDAC inhibitors, treatment of mTK6 cells with trichostatin A and vorinostat did not significantly increase the TK revertant frequency (Fig. 4C), likely due to the persistent levels of H3K27Ac and H3K4me3 at the TK gene locus. TPA stimulates cellular responses, including cell proliferation, migration, and differentiation through the activation of specific types of protein kinase C (PKC) 34-36 . Previous reports have shown that TPA treatment rapidly increases H3S10/H3S28 phosphorylation and H3 acetylation levels via mitogen- and stress-activated protein kinases 37-39 . These responses are associated with the transcriptional activation of immediate early response genes, with phosphorylation of both serine residues peaking around 1 hour following TPA treatment and then declining 37,38 . Interestingly, in our study, treatment of cells with TPA resulted in a significant decrease in TK reversion frequency (Fig. 5B), accompanied by a global reduction in H3K27Ac levels (Fig. 5C-F). The differences observed in our study may be due to the varying time scales used to assess histone modifications following TPA exposure. Specifically, H3K27Ac and H3K4me3 levels were measured after a 3-day recovery period following TPA treatment, allowing to distinguish immediate-early responses from persistent epigenetic changes. In agreement with our findings, prolonged TPA exposure leads to a global reduction of H3S10 phosphorylation in HepG2 cells 40 . Therefore, the TPA-induced reduction in H3K27Ac may reflect changes distinct from the histone modifications involved in the immediate-early responses to cellular stimuli. Initially, we hypothesized that TPA exposure might induce changes in the expression of epigenetic regulators as a potential mechanism behind the observed epigenetic alterations. Supporting this idea, a recent study reported that transcriptome analysis of Bhas 42 cells exposed to TPA revealed altered expression patterns of key epigenetic factors, including DNMT1, DNMT3A, MBD3 , and Mi2 22 . However, in our study, TPA treatment did not induce DEGs in epigenetic-related pathways, such as those involving DNMTs, HDACs, and histone acetyltransferases (Fig. S1 D and E). Thus, the decreased levels of H3K27Ac are not due to changes in the expression of epigenetic factors that directly regulate chromatin status. These differing results may reflect the malignant status of the cells, including mutations or inactivation of tumor suppressor genes and proto-oncogenes. While Bhas 42 cells are characterized by Tp53 deficiency, TK6 cells retain the native TP53 gene and are capable of undergoing the normal apoptotic process, which could explain the differential expression pattern observed in response to the tumor promoter TPA. TPA is known to induce inflammation through activation of the NFκ-B signaling pathway 41,42 . Notably, inflammatory stresses is closely linked to epigenetic modifications, including DNA methylation and histone acetylation 43,44 . Chronic inflammatory stress was observed in TPA-treated mTK6 cells even after three days of cultivation following TPA treatment (Fig. S1E), suggesting that TPA-mediated inflammation may contribute to global epigenetic modifications. A potential relationship between the progression of inflammation and histone deacetylation has been reported in both in vitro and in vivo studies 45-47 . While the precise mechanism behind TPA-mediated H3K27Ac reduction remains unclear, this epigenetic alteration could serve as a biological marker for specific inflammatory responses. Further research is needed to elucidate the mechanistic basis of the epigenetic toxicity associated with this chemical reagent. 4. Method Cell culture. The human lymphoblastoid TK6 cell line and its derivative were cultured in RPMI-1640 medium (Nacalai Tesque) supplemented with 200 μg/mL sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Nichirei Biosciences, Inc.). The cultures were maintained at 37°C in a 5% CO 2 atmosphere with 100% humidity. Generation of human mTK6 cell line. To generate TK6 derivative cells with an epigenetically modified TK gene, the dCas9-SunTag DNMT3A system was employed to induce DNA methylation at the CpG loci of the TK gene promoter. The gRNA cloning vector was generously provided by Dr. George Church (Addgene plasmid #41824; http://n2t.net/addgene:41824; RRID:Addgene_41824) 48 and LLP252 pEF1a-NLS-scFvGCN4-DNMT3a (Addgene plasmid #100941; http://n2t.net/addgene:100941; RRID:Addgene_100941) and LLP457 pGK-dCas9-Suntag-BFP (Addgene plasmid# 100957; http://n2t.net/addgene:100957; RRID:Addgene_100957) were gift from Dr. Ryan Lister 21 . Four single-guide RNA (sgRNA) targets were designed for the TK promoter region, as detailed in Supplementary Table S1. A plasmid pool consisting of the four sgRNA (21 µg), pGK-dCas9-Suntag-BFP (21 µg), and pEF1a-NLS-scFvGCN4-DNMT3a (8 µg) was transfected into TK6 cells (5 × 10 6 ) using 0.1 ml Nucleofector solution V (Lonza) and a Nucleofector 2b device, following the manufacturer’s instructions. After 48 h of incubation, the cells were seeded into 96-microwell plates at concentrations of either 20,000 or 200,000 cells/mL in the presence of 3.0 μg/ml trifluorothymidine (TFT). To determine the plating efficiency, cells were also seeded at a concentration of 8 cells/mL in the absence of TFT. After 10 days of incubation, TFT-resistant clones were isolated and maintained in RPMI-1640 medium containing 3.0 μg/ml TFT for at least 10 passages before preparing frozen stocks. The integrity of the TK promoter region sequences in the clones was validated using PCR primers listed in Supplementary Table S2. Real-time RT-PCR. Total RNA was extracted from cell using the NucleoSpin RNA Plus kit (Macherey-Nagel). cDNAs were synthesized from the extracted RNA with ReverTra Ace (Toyobo Co., Ltd.). Quantitative PCR was performed using Thunderbird ® Next SYBR qPCR Mix (Toyobo Co., Ltd.) and specific primers listed in Supplementary Table S2. The expression levels of the TK gene were normalized to the internal GAPDH expression levels Chemical substances. 5-Aza-2'-deoxycytidine and vorinostat were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), while trichostatin A was procured from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). GSK-3484862 was purchased from ChemieTek (IN, USA), and 12- O -tetradecanoylphorbol-13-acetate (TPA) were sourced from Merck (Darmstadt, Germany). 5-Aza-2'-deoxycytidine was dissolved and diluted in H 2 O. Trichostatin A, vorinostat, and TPA were dissolved in ethanol, while GSK-3484862 were prepared in dimethyl sulfoxide. Epi-TK assay. mTK6 cells were cultured to the exponential phase in the presence of 3.0 μg/ml TFT. Afterward, cells were washed to remove TFT and resuspended in RPMI-1640 medium supplemented with 200 μg/mL sodium pyruvate and 10% FBS. For chemical exposure, 10 ml aliquots of cell suspension were prepared in 100 mm Petri dishes at a concentration of 250,000 cells/ml. Test chemicals at various concentrations were added to the dishes, which were then incubated at 37℃ for 24 h. Following chemical treatment, cells were washed and resuspended in fresh RPMI-1640 medium. To measure the TK revertant frequency, cells were seeded into 96-microwell plates containing 200 μM hypoxanthine, 0.1 μM aminopterin, and 17.5 μM thymidine (HAT). Seeding densities ranged from 100 to 100,000 cells/ml (20 to 20,000 cells/well), depending on the test chemicals evaluated. Concurrently, cells were plated in 96-microwell plates at a density of 8 cells/ml (1.6 cells/well) without HAT to assess the cloning efficiency (CE). After 21 days of incubation, the number of colonies in both the CE and HAT plates was recorded. CE was calculated using Eq. 1 based on the Poisson distribution 49 , where EW represents the number of wells without colonies, TW represents the total number of wells, and N is the average number of cells per well (N = 1.6) in the CE plates. CE = − ln (EW / TW) / N (1) Relative survival (%) was determined by comparing the CE values of chemical-treated cells to those of the solvent control. The TK revertant frequency was calculated using eq. 2 also based on the Poisson distribution. Here, N corresponds to the number of cells per well (N = 20 to 20,000) in the HAT plates. The results were statistically analyzed using Dunnett’s test and compared with solvent control values. TK revertant frequency = [− ln (EW / TW) / N] / treated CE (2) Bisulfite sequencing. Genomic DNA was extracted from cells (1 × 10 6 ) using the Nucleospin Tissue kit (Macherey-Nagel). The extracted DNA, dissolved in 50 µL H 2 O (2 µg), was treated with 5.5 µL of 2 M NaOH and incubated at 37℃ for 15 min. Subsequently, 30 µL of 10 mM hydroquinone and 520 µL of 2 M sodium metabisulfite were added to the mixture, followed by incubation at 50℃ for 16 h. The bisulfite-converted DNA was purified using the Wizard DNA Clean-Up System (Promega) and subjected to PCR amplification using EpiTaq HS (TAKARA). A 451 bp DNA fragment containing the TK promoter region was amplified using primers listed in Supplementary Table S2. The resulting PCR products was ligated into the pTA2 Vector using the Target Clone TA cloning system (Toyobo Co., Ltd.). The ligation mixture was transformed into Escherichia coli TOP10 cells , and recombinant clones were verified by Sanger sequencing. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). To assess H3K27Ac and H3K4me3 levels following TPA exposure, cells were treated with 20 ng/mL TPA at 37℃ for 24 h. After treatment, the cells were washed twice with RMPI-1640 medium and cultured exponentially for 3 days to allow phenotypic expression. A total of 3 × 10 6 cells were then cross-linked with 1% formaldehyde at room temperature for 10 min. The cross-linking reaction was terminated by adding 125 mM glycine. Following this, cells were washed with phosphate buffered saline and resuspended in 300 µl sonication buffer (10 mM Tris-HCl (pH8.0), 2 mM EDTA, 0.25% SDS). DNA was fragmented by sonication, and the resulting chromatin was incubated at 4℃ for 16 h with specific antibodies (anti-H3K27Ac monoclonal antibody (AB_2793797, ACTIVE MOTIF) and anti-H3K4me3 monoclonal antibody (ab8580, abcam)) and Protein A Sepharose 4 beads (Cytiva). The Sepharose beads were washed, and the immunoprecipitated DNA was eluted using elution buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS, 0.3 µg/ml Proteinase K) at 65℃ for 6 h. Purification of DNA was performed using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). The abundance of immunoprecipitated DNA was quantified using the Thunderbird ® Next SYBR qPCR Mix (Toyobo Co., Ltd.) with specific primers listed in Supplementary Table S2 . Western blotting Total cell extracts were fractioned on gradient 5–20% SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with 3% skim milk before incubation with primary antibodies. To detect H3K27Ac, Histone H3, and GAPDH, membranes were incubated overnight at 4°C in Hikari A solution (Nacalai Tesque) with the following antibodies: 1:1000 dilution of anti-H3K27Ac monoclonal antibody (AB_2793797, ACTIVE MOTIF), 1:1000 dilution of anti-Histone H3 monoclonal antibody (gifted from Dr. Kimura), or 1:1000 dilution of anti-GAPDH monoclonal antibody (sc-32233, Santa Cruz). After washing with tris-buffered saline containing 0.05% Tween 20, the membranes were incubated with a 1:4000 dilution of anti-mouse IgG or anti-rat IgG conjugated to horseradish peroxidase (Cytiva) in Hikari B solution (Nacalai Tesque). The chemiluminescent signal were detected using Chemi-Lumi One Super (Nacalai Tesque). Declarations Acknowledgments We are grateful to Dr. Hiroshi Honda (Kao Corporation) and Dr. Takayuki Fukuda (Bozo Research Center) for their helpful comments and discussions. We also thank Dr. Hiroshi Kimura (Tokyo Institute of Technology, Japan) for generously providing the antibody used to identify histone H3. This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare (21KA1001), the Consumer Affairs Agency Program Grant (24KA1008), and JSPS KAKENHI (22H03748 and 19K12339). Additional funding was provided by grants from the Takeda Science Foundation [to A.S. and K.U.]. Author contributions H.Y., M.O., M.Y., and A.S. conceptualized the research, H.Y., M.O., K.Y., M.Y., M.H., K.S., K.U., and A.S. participated in study discussions and experimental design. H.Y., M.O., K.Y., A.C., A.U., M.Y., and A.S. carried out the experiments and analyzed the data. A.S. authored the manuscript and all authors reviewed and approved the final manuscript. Data availability statement The transcriptome data raw files have been deposited to the DDBJ with accession number PRJDB19707: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJDB19707 . Additional Information Competing Interests The authors declare that there are no competing interests. References Organisation for Economic Co-operation and Development. Environment Directorate., Organisation for Economic Co-operation and Development. Joint Meeting of the Chemicals Committee and the Working Party on Chemicals Pesticides and Biotechnology. & Inter-Organization Programme for the Sound Management of Chemicals. Guidance for conducting retrospective studies on socio-economic analysis. OECD, (1999). Sharma, S., Kelly, T. K. & Jones, P. A. Epigenetics in cancer. Carcinogenesis 31 , 27–36. 10.1093/carcin/bgp220 (2010). Baccarelli, A. & Bollati, V. Epigenetics and environmental chemicals. Curr. Opin. Pediatr. 21 , 243–251. 10.1097/mop.0b013e32832925cc (2009). Eglen, R. M. & Reisine, T. 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Biochem. 110 , 1–8. 10.1016/0003-2697(81)90103-2 (1981). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 15 Jan, 2025 Reviews received at journal 14 Jan, 2025 Reviewers agreed at journal 04 Jan, 2025 Reviews received at journal 04 Jan, 2025 Reviewers agreed at journal 03 Jan, 2025 Reviewers agreed at journal 02 Jan, 2025 Reviewers invited by journal 02 Jan, 2025 Editor assigned by journal 02 Jan, 2025 Editor invited by journal 24 Dec, 2024 Submission checks completed at journal 23 Dec, 2024 First submitted to journal 11 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5623363","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":402191254,"identity":"17fe0dd4-421a-4efb-8da8-f19dc7d7a75f","order_by":0,"name":"Akira Sassa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBADOQYJxjYwi7GBSC3GpGtJbJBgYCNOKX8D+8OPP/7YpffPbm578IPhMAPzbALWSBzgMZbmbUvOnXHnYLthD1AL45wDBKy5/4ZBmrGBOXeDRGKbBO8/oJYZCfh1yB9gf/zzx5/6dAOgFsk/DERoMTjAYCbBw3Y4AaRFmocYLYYHeMyseduOG864kdhuLMOQzkPQL3JAh9388adann9G+rOHbxis5QwJhRgG4DGcQaIOYIBIkKxlFIyCUTAKhjkAAImnQENV0nNFAAAAAElFTkSuQmCC","orcid":"","institution":"Chiba University","correspondingAuthor":true,"prefix":"","firstName":"Akira","middleName":"","lastName":"Sassa","suffix":""},{"id":402191255,"identity":"89b1f0d6-5938-4af7-bf74-4fb25b4f211c","order_by":1,"name":"Haruto Yamada","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Haruto","middleName":"","lastName":"Yamada","suffix":""},{"id":402191256,"identity":"71a1f757-f298-4af7-9a6f-8349a3a5a849","order_by":2,"name":"Mizuki Odagiri","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Mizuki","middleName":"","lastName":"Odagiri","suffix":""},{"id":402191257,"identity":"ea39b95b-c061-4f72-ab62-54eebca133ef","order_by":3,"name":"Keigo Yamakita","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Keigo","middleName":"","lastName":"Yamakita","suffix":""},{"id":402191259,"identity":"05b3f614-fd04-4022-96b1-ccb5ece12701","order_by":4,"name":"Aoi Chiba","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Aoi","middleName":"","lastName":"Chiba","suffix":""},{"id":402191261,"identity":"1cbecd12-b451-4190-bc12-5d4bc9602ba8","order_by":5,"name":"Akiko Ukai","email":"","orcid":"","institution":"National Institute of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Ukai","suffix":""},{"id":402191262,"identity":"c5b47440-20bb-4c3f-9ad2-a8bb00ce7f2d","order_by":6,"name":"Manabu Yasui","email":"","orcid":"","institution":"National Institute of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Manabu","middleName":"","lastName":"Yasui","suffix":""},{"id":402191263,"identity":"291759f2-d715-4d9d-ab7a-a1c49bf1f802","order_by":7,"name":"Masamitsu Honma","email":"","orcid":"","institution":"National Institute of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masamitsu","middleName":"","lastName":"Honma","suffix":""},{"id":402191264,"identity":"8d71e9ce-8e07-4dee-bb9e-a290595e4cab","order_by":8,"name":"Kei-ichi Sugiyama","email":"","orcid":"","institution":"National Institute of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kei-ichi","middleName":"","lastName":"Sugiyama","suffix":""},{"id":402191265,"identity":"65e729c3-755c-487c-a56d-d694361d36c5","order_by":9,"name":"Kiyoe Ura","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Kiyoe","middleName":"","lastName":"Ura","suffix":""}],"badges":[],"createdAt":"2024-12-11 10:23:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5623363/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5623363/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-92121-6","type":"published","date":"2025-03-05T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73886440,"identity":"34bde52a-0689-4e81-adbf-4bc7e5de1a3e","added_by":"auto","created_at":"2025-01-15 14:41:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2312803,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the epi-TK assay. Based on human TK6 cells, site-specific methylation of CpG sites in the promoter region of the TK gene achieved via the CRISPR/dCas9-SunTag-DNMT3A system. The resulting mTK6 cell line was cultured in medium containing TFT. After exposure to test compound, the cells were seeded into 96-microwell plates with HAT to assess TK revertant frequency. The cells were plated into 96-microwell plates without HAT to determine CE. Colony numbers from CE and HAT plates were recorded, and the TK revertant frequency was calculated as described in the Methods section.\u003c/p\u003e","description":"","filename":"Figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/8d7fd616aa3f427a9cfa886f.jpg"},{"id":73886441,"identity":"64dd5641-78a6-4d5c-a816-829dde3e91eb","added_by":"auto","created_at":"2025-01-15 14:41:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":803608,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of human mTK6 cells for assessing chemical-induced epigenetic effects. (A) Representative data showing the TFT resistant frequency after transfection with plasmids encoding CRISPR/dCas9-SunTag-DNMT3A and sgRNAs. The frequency of TFT-resistant colonies was assessed following transient expression of CRISPR/dCas9-SunTag-DNMT3A and sgRNAs targeting the \u003cem\u003eTK\u003c/em\u003e gene (+). Transfection with the CRISPR/dCas9-SunTag-DNMT3A plasmid and an empty sgRNA vector served as negative control (-). (B) Population doubling time of TK6 and mTK6 cells. Doubling time were measured in both the absence or presence of TFT. (C) Expression levels of the \u003cem\u003eTK\u003c/em\u003egene in TK6 and mTK6 cells. Relative mRNA abundance was normalized to \u003cem\u003eGAPDH\u003c/em\u003e expression. (D) Spontaneous TK revertant frequency of mTK6 cells. Data are presented as the mean ± S.E. of three independent experiments. * A Significant difference between the assessed cells; P \u0026lt; 0.05 (Dunnett’s Multiple Comparison Test).\u003c/p\u003e","description":"","filename":"Figure21.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/fcb3c77ac69c577617577f7f.jpg"},{"id":73886734,"identity":"8195a103-104e-4f65-b086-29085aa52ddc","added_by":"auto","created_at":"2025-01-15 14:49:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2141917,"visible":true,"origin":"","legend":"\u003cp\u003eRelative survival and TK revertant frequency in the epi-TK assay for 5-aza-2'-deoxycytidine and GSK-3484862. Cytotoxic and epigenetic effects were measured as relative survival (%) and TK revertant frequency (×10\u003csup\u003e-2\u003c/sup\u003e), respectively, following treatment with 5-azadG (A and B) and GSK-3484862 (C and D). The data on TK revertant frequencies were statistically analyzed using Dunnett’s test and compared to the solvent control. Results from three independent experiments are presented. (E) Bisulfite sequencing analysis of the TK gene in mTK6 cells. Each circle represents a CpG site located within or near the promoter region of the TK gene. Black circles indicate methylated CpG site, while white circles represent unmethylated CpG site. Each row corresponds to the CpG sites of a single clone.\u003c/p\u003e","description":"","filename":"Figure31.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/4164c3991a0fb5d2824fbfb5.jpg"},{"id":73886461,"identity":"cfd780c1-94af-4533-b1e3-4ee0ebc08c18","added_by":"auto","created_at":"2025-01-15 14:41:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":700210,"visible":true,"origin":"","legend":"\u003cp\u003eHistone modification status of the \u003cem\u003eTK\u003c/em\u003e gene. Active histone marks H3K27Ac (A) and H3K4me3 (B) at the transcription start site of the \u003cem\u003eTK\u003c/em\u003e gene were analyzed by chromatin immunoprecipitation and qPCR analysis. The\u003cem\u003e HBB\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003egenes were analyzed for comparison. (C) TK revertant frequencies following treatment with HDAC inhibitors vorinostat and trichostatin A. Ethanol (EtOH) served as a negative control. Data are presented as the mean ± S.E. of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure41.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/654e3afa02876cb470bc5da8.jpg"},{"id":73886442,"identity":"e42d630d-073e-49fb-83da-b811021d4cb1","added_by":"auto","created_at":"2025-01-15 14:41:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1420775,"visible":true,"origin":"","legend":"\u003cp\u003eEpigenetic effects of 12-\u003cem\u003eO\u003c/em\u003e-tetradecanoylphorbol-13-acetate assessed by the epi-TK assay. Cytotoxicity and epigenetic effects were assessed as relative survival (A) and TK revertant frequency (B) after cell treatment with TPA. The data on TK revertant frequencies were statistically analyzed using Dunnett’s test and compared to the solvent control. Results from three independent experiments are shown. (C) Enrichment of H3K27Ac in response to TPA treatment. H3K27Ac levels were determined by chromatin immunoprecipitation and qPCR analysis. The \u003cem\u003eHBB\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e genes were analyzed for comparison. Dataare presented as the mean ± S.E. of three independent experiments. * A significant difference between the assessed samples; P \u0026lt; 0.05 (student’s t-test). (D) Enrichment of H3K4me3 in response to TPA treatment were determined by chromatin immunoprecipitation and qPCR analysis. Cells were harvested three days of cultivation following the exposure to ethanol or TPA. The \u003cem\u003eHBB\u003c/em\u003eand \u003cem\u003eGAPDH\u003c/em\u003e genes were analyzed for comparison. Data are presented as the mean ± S.E. of three independent experiments. (E) Western blotting analysis for H3K27Ac following TPA treatment. Whole cell extracts were prepared from cells harvested after three days of cultivation following the exposure to ethanol or TPA. Extracted samples were analyzed by immunoblotting and the blots were probed with the indicated antibodies. (F) Line graph showing quantification of H3K27Ac levels after normalization with total H3 levels from western blotting analysis. Data are presented as the relative intensity compared to ethanol-treated cells and expressed as the mean ± S.E. from three independent biological replicates. ** A significant difference between the assessed samples; P \u0026lt; 0.01 (student’s t-test). Uncropped blots are shown in supplementary Figure S2.\u003c/p\u003e","description":"","filename":"Figure51.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/7f7d7145632171fe9012acb6.jpg"},{"id":78181477,"identity":"cdb79375-bd79-4eff-bd80-2d168ccd6efe","added_by":"auto","created_at":"2025-03-10 17:46:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8251722,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/81c04052-79b4-445d-8b06-6e5e63d6799a.pdf"},{"id":73886458,"identity":"5a5b1ead-97f2-40f0-aca9-a604ac670f09","added_by":"auto","created_at":"2025-01-15 14:41:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":281569,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5623363/v1/6ff05651117c733402c2b505.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNumerous chemical agents have been implicated in carcinogenesis through diverse mechanisms, including genetic, epigenetic, and metabolic alterations. Among these processes, the ability of chemicals to induce genetic changes is defined as genotoxicity. Genotoxic agents can bind directly to DNA and act indirectly by interfering with enzymatic pathways such as DNA replication, DNA repair, and DNA damage signaling, ultimately leading to gene mutations and chromosomal aberrations. The potential risk of chemicals interacting with DNA is evaluated through genotoxicity testing. The Organization for Economic Co-operation and Development (OECD) has established guidelines for assessing chemicals using \u003cem\u003ein vitro\u003c/em\u003e genotoxicity tests, including the bacterial Ames test, the mammalian cell chromosomal aberration test, and the mammalian cell gene mutation test\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to genotoxic potential, epigenetic alterations are believed to contribute to chemically induced carcinogenesis\u003csup\u003e2\u003c/sup\u003e. Among these, cytosine C5 methylation in CpG dinucleotides represents a key epigenetic modification, playing crucial roles in processes such as X chromosome inactivation, genomic imprinting, and repression of retrotransposons through gene silencing. In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs). Of the active DNMTs, DNMT1 is responsible for maintaining DNA methylation during replication, while DNMT3A/3B are involved in de novo DNA methylation under specific cellular conditions. Beyond DNA methylation, gene expression is further regulated by post-translational histone modifications, which serve as structural elements for packaging genomic DNA within the nucleus. Histone H3K4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27Ac) are transcriptionally active marks that exhibit an inverse correlation with DNA methylation at gene promoter regions. These cellular epigenetic patterns can be influenced by exposure to certain chemical reagents that directly or indirectly modify DNA methylation and/or histone modifications\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSeveral therapeutic drugs have been developed as epigenetic modifiers with anticancer potential. Aberrant DNA methylation and the hyperactivation of histone deacetylases (HDACs) are closely linked to cell proliferation and metastasis in various cancers, making DNMTs and HDACs promising therapeutic targets\u003csup\u003e4\u003c/sup\u003e. 5-Aza-2\u0026prime;-deoxycytidine (5-azadC) is a widely used demethylating agent that inhibits DNMTs through its incorporation into genomic DNA\u003csup\u003e5\u003c/sup\u003e. Similarly, HDAC inhibitors promote hyperacetylation of core histones, thereby modulating chromatin structure and altering gene expression patterns\u003csup\u003e6\u003c/sup\u003e. Beyond these designed therapeutic agents, environmental chemicals such as bisphenol A, arsenic, cadmium, benzene, and pesticides have been reported to induce aberrant epigenetic changes, contributing to the onset of various diseases\u003csup\u003e7\u003c/sup\u003e. Notably, many carcinogens are believed to exert their effects through non-genotoxic mechanisms\u003csup\u003e8\u003c/sup\u003e. Therefore, evaluating the potential epigenetic effects and underlying mechanisms of action is a critical component in the safety assessment of environmental chemical compounds.\u003c/p\u003e\n\u003cp\u003eTo evaluate epigenetic alterations induced by chemical reagents, researchers have developed various cell-based reporter assays. Johnson et al. introduced a system using an epigenetically silenced green fluorescent protein (\u003cem\u003eGFP\u003c/em\u003e) reporter gene controlled by an exogenous cytomegalovirus promoter\u003csup\u003e9\u003c/sup\u003e. More recently, endogenous tumor suppressor genes such as \u003cem\u003eSFRP1\u003c/em\u003e and \u003cem\u003eBRCA1\u003c/em\u003e have been employed as \u003cem\u003eGFP\u003c/em\u003e-based reporters to study epigenetic changes\u003csup\u003e10,11\u003c/sup\u003e. Similarly, Okochi-Takada et al. developed a high-throughput screening platform using luciferase and \u003cem\u003eGFP\u003c/em\u003e reporter genes driven by the inactivated \u003cem\u003eUCHL1\u003c/em\u003e locus in HCT116 cells\u003csup\u003e12\u003c/sup\u003e. These systems generally detect epigenetic modifications in unidirectional manner, either inactivation or reactivation of gene expression, based on the initial epigenetic state of the reporter gene locus. A notable approach is an \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003ereporter system utilizing DNMT-transformed yeast cells, enabling rapid and efficient detection of epigenetic effects caused by chemical reagents in bidirectional\u003csup\u003e13-15\u003c/sup\u003e. However, to ensure the extrapolation of experimental results to humans, a flexible bidirectional reporter assay needs to be designed in mammalian cells to effectively detect diverse modifications resulting from chemical exposure. Currently, the epigenetic impacts of chemicals on housekeeping genes remain poorly understood, emphasizing the need to evaluate these genes as potential reporter loci to broaden the scope and utility of such assays.\u003c/p\u003e\n\u003cp\u003eIn this study, we aimed to develop an epi-genotoxicity assay to evaluate epigenetic modifications induced by carcinogens, based on the thymidine kinase (\u003cem\u003eTK\u003c/em\u003e) gene mutation assay (TK assay) technique. The TK assay is a conventional mammalian \u003cem\u003ein vitro\u003c/em\u003e genotoxicity test that detects various mutations at the housekeeping \u003cem\u003eTK\u003c/em\u003e gene locus, utilizing the human lymphoblastoid TK6 cell line\u003csup\u003e16\u003c/sup\u003e. The OECD Test Guideline (TG490) adopted the conventional TK assay for the safety assessment of pharmaceutical, industrial, agricultural, and environmental chemicals\u003csup\u003e17\u003c/sup\u003e. We recently enhanced the TK assay by developing genome-edited TK6 cell lines, improving its sensitivity for detecting genotoxic and cytotoxic effects of chemicals\u003csup\u003e18-20\u003c/sup\u003e. In the current study, we further advanced this approach by establishing epigenetically modified TK6 derivative (mTK6) cells as the epi-genotoxicity test system. Using the CRISPR/dCas9-SunTag-DNMT3A system, CpG sites within the endogenous \u003cem\u003eTK\u003c/em\u003e promoter region were selectively methylated\u003csup\u003e21\u003c/sup\u003e, resulting in mTK6 cells with a stably methylated \u003cem\u003eTK\u003c/em\u003e gene. These cells can be propagated in the presence of trifluorothymidine (TFT), which selectively exerts cytotoxicity against \u003cem\u003eTK\u003c/em\u003e-proficient cells. The developed method, termed the \u0026ldquo;epi-TK assay,\u0026rdquo; enables accurate quantification of epigenetic changes induced by chemical exposure. This is achieved by measuring the frequency of TK revertant colonies in hypoxanthine, \u003cem\u003eaminopterin\u003c/em\u003e, and thymidine (HAT) selection medium (Fig. 1A).\u003c/p\u003e\n\u003cp\u003eTo assess the ability of the epi-TK assay to quantify global epigenetic effects, we tested DNMT inhibitors (5-Aza-2\u0026apos;-deoxycytidine (5-azadC), GSK-3484865) and HDAC inhibitors (vorinostat and trichostatin A) as model substances with well-characterized mechanisms of action (Fig. 1B). Additionally, we examined the epigenetic effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a widely studied non-genotoxic tumor promoter/inflammation inducer derived from the seed oil of \u003cem\u003eJatropha curcas\u003c/em\u003e L. Using the established epi-TK assay, we observed not only an increase in TK reversion frequency induced by 5-azadC and GSK-3484865 but also a significant decrease in TK revertant frequency following TPA exposure. Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment caused a global reduction in H3K27Ac levels, likely linked to TPA-mediated chronic inflammation. These findings highlight the utility of the epi-genotoxicity assay as a tool for evaluating epigenetic alterations induced by chemical exposure in both directions.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003eEstablishment of human TK6 derivative cells assessing chemical-induced epigenetic alterations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe developed a reporter system to assess chemical-induced epigenetic alterations using the mammalian \u003cem\u003eTK\u003c/em\u003e gene mutation assay as a platform. To create a cell line capable of quantifying epigenetic effects, we employed CRISPR/dCas9-SunTag-DNMT3A along with sgRNA expression plasmids specifically designed to methylate CpG loci within the promoter region of the endogenous \u003cem\u003eTK\u003c/em\u003e gene. As illustrated in Figure 2A, transient expression of the sgRNAs resulted in a significant increase in TFT-resistant colonies (1.0 \u0026times; 10\u003csup\u003e\u0026minus;3\u003c/sup\u003e) compared to cells transfected with empty sgRNA vectors (3.2 \u0026times; 10\u003csup\u003e\u0026minus;5\u003c/sup\u003e). Among 12 clones isolated, we selected a representative clone that exhibited stable proliferation in TFT-containing medium. This isolated clone, designated as the \u0026ldquo;mTK6\u0026rdquo; cell line, had a population doubling time of 14 \u0026plusmn; 1.0 h in the absence of TFT and 13 \u0026plusmn; 0.52 h in its presence, similar to the original TK6 cell line\u0026rsquo;s doubling time of 14 \u0026plusmn; 0.26 h (Fig. 2B). RT-qPCR analysis revealed abolished \u003cem\u003eTK\u003c/em\u003e gene expression in mTK6 cells compared to TK6 cells (Fig. 2C), indicating that \u003cem\u003eTK\u003c/em\u003e gene expression was repressed due to DNA methylation in its promoter region. We also evaluated the TK revertant frequency during cell proliferation up to 3 days following TFT removal from the medium (Fig. 2D). Spontaneous TK reversion was observed in a time-dependent manner: 0 day (0.77 \u0026plusmn; 0.27 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e), 1 day (3.6 \u0026plusmn; 1.3 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e), 2 days (6.4 \u0026plusmn; 2.7 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e), and 3 days (8.8 \u0026plusmn; 1.3 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e). Based on this background TK revertant frequency, we hypothesize that epigenetic alterations induced by chemical exposure can be quantified by measuring changes in the number of TK revertant colonies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of epigenetic effects of covalent and non-covalent DNA methyltransferase inhibitors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first evaluated the capability of the epi-TK assay to quantify the effects of typical DNA demethylating agents. 5-Aza-2\u0026apos;-deoxycytidine (5-azadC), a widely used potent DNMT inhibitor, was tested on mTK6 cells across a range of concentrations (0.02\u0026ndash;0.1 \u0026micro;M), determined based on cytotoxicity assays (Fig. 3A). As shown in Figure 3B, treatment with 5-azadC led to a 230-fold increase in TK revertant frequency (7.7 \u0026plusmn; 1.0 \u0026times; 10\u003csup\u003e-2\u003c/sup\u003e) at the highest concentration (0.1 \u0026micro;M) compared to the solvent control (3.3 \u0026plusmn; 0.67 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e). Notably, 5-azadC is known for its high cytotoxicity and genotoxicity due to the formation of covalent DNA-DNMT adducts. To evaluate the assay\u0026rsquo;s versatility, we tested the epigenetic effects of non-covalent DNMT inhibitor GSK-3484862. As shown in Figure 3C and 3D, GSK-3484862 treatment significantly increased TK revertant frequency in a dose-dependent manner (0.12 \u0026plusmn; 0.033 at 0.5 \u0026micro;M, 0.19 \u0026plusmn; 0.052 at 2.0 \u0026micro;M, and 0.29 \u0026plusmn; 0.044 at 5.0 \u0026micro;M) compared to the DMSO control (1.6 \u0026plusmn; 0.46 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next investigated the DNA methylation pattern of the \u003cem\u003eTK\u003c/em\u003e gene promoter region in mTK6 cells. Bisulfite sequencing revealed methylation of cytosine at 26 CpG dinucleotides within a 230 bp region upstream of the \u003cem\u003eTK\u003c/em\u003e gene\u0026rsquo;s start codon (Fig. 3E). To further examine the methylation status of the TK promoter after exposure to DNMT inhibitors, we analyzed TK revertant colonies. Notably, all CpG sites within the \u003cem\u003eTK\u003c/em\u003e promoter were unmethylated in both spontaneous and DNMT inhibitor-mediated (5-azadC and GSK-3484862) TK revertant colonies. These findings suggest that the DNA methylation pattern of the \u003cem\u003eTK\u003c/em\u003e promoter is closely associated with its gene expression status.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of histone modification status in the methylated\u003cem\u003e\u0026nbsp;TK\u003c/em\u003e gene promoter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscriptionally active chromatin is characterized by the presence of H3K27ac and H3K4me3 near the transcription start site of the target gene. Thus, we investigated whether the status of H3K27ac and H3K4me3 was altered following DNA methylation in the \u003cem\u003eTK\u003c/em\u003e promoter region. Interestingly, ChIP-qPCR analyses revealed that enrichment levels of K3K27Ac and H3K4me3 were comparable to those observed in TK6 cells (Fig. 4A and 5B). Consistent with these findings, exposure to HDAC inhibitors vorinostat (0.2 \u0026micro;M) and trichostatin A (0.01 \u0026micro;M) for 24 and 48 hours did not affect the frequency of TK revertants (Fig. 4C). This lack of effect may be due to the sustained levels of H3K27Ac and H3K4me3 in the presence of repressive DNA methylation at the \u003cem\u003eTK\u003c/em\u003e promoter region in mTK6 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of the epigenetic consequence of a non-genotoxic carcinogen/inflammation inducer 12-O-Tetradecanoylphorbol-13-acetate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the unique epigenetic pattern of the \u003cem\u003eTK\u003c/em\u003e gene in mTK6 cells described above, we hypothesized that chemically induced global histone modifications could be detected through changes in the frequency of TK reversion. To test this hypothesis, we selected TPA, a potent non-genotoxic carcinogen with potential effects on histone modifications, though the persistent epigenetic consequences remain poorly understood. As shown in Figure 5A, treatment with TPA at concentrations ranging from 0.02\u0026ndash;1.0 \u0026micro;g/ml resulting in approximately 40\u0026ndash;50% cell survival. Under these conditions, the frequency of TK revertants decreased significantly, with a maximum of 28-fold reduction (0.090 \u0026plusmn; 0.0058 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e) compared to the solvent control (2.5 \u0026plusmn; 0.42 \u0026times; 10\u003csup\u003e-4\u003c/sup\u003e) (Fig. 5B). To further investigate histone modification status following TPA treatment, cells were harvested three days of cultivation following the exposure to TPA and subjected to ChIP-qPCR and western blotting analysis. ChIP-qPCR analysis revealed a significant reduction in H3K27Ac levels at the \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eTK\u003c/em\u003e loci after TPA exposure (Fig. 5C). In contrast, the enrichment of H3K4me3 did not significantly change between ethanol- and TPA-treated cells (Fig. 5D). Consistently, western blotting analysis confirmed a significant reduction in H3K27Ac levels following TPA treatment (Fig. 5E and F).\u003c/p\u003e\n\u003cp\u003eAccording to a recent study, differential gene expression patterns of epigenetic regulators, including DNMTs and chromatin remodelers, were observed depending on the duration of TPA exposure\u003csup\u003e22\u003c/sup\u003e. To investigate whether the reduction in H3K27Ac levels observed in our results is due to altered expression of epigenetic modulators, we performed RNA-seq analysis to identify DEGs resulting from TPA treatment. A 24-h treatment with TPA led to the upregulation of genes associated with the \u0026ldquo;ERK1 and ERK2 cascade\u0026rdquo; and \u0026ldquo;MAPK cascade,\u0026rdquo; followed by the \u0026ldquo;inflammatory response\u0026rdquo; (Fig. S1A-D). After washing out TPA and culturing the cells for 3 days, Gene Ontology analysis revealed that the biological pathways of upregulated genes were enriched in \u0026ldquo;leukocyte activation,\u0026rdquo; \u0026ldquo;innate immune response,\u0026rdquo; and \u0026ldquo;lymphocyte activation.\u0026rdquo; For the downregulated genes associated with TPA treatment, fewer pathways were enriched: \u0026ldquo;leukocyte tethering or rolling\u0026rdquo; for the 24-h TPA treatment, and \u0026ldquo;regulation of cell migration,\u0026rdquo; \u0026ldquo;cellular response to chemokine,\u0026rdquo; and \u0026ldquo;leukocyte activation\u0026rdquo; for the 3-day culture following TPA treatment. No significant differential expression of genes involved in epigenetic regulation, such as DNMT1, DNMT3A/B, histone acetyltransferases, and HDACs, was observed.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eOver the past two decades, next-generation sequencing-based technologies have been developed to explore the epigenetic regulatory landscape of cells and tissues. Pioneered by Bisulfite-seq and ChIP-seq detecting DNA methylation and histone modifications, a wide range of epigenome profiling techniques has developed. These include Micrococcal Nuclease sequencing (MNase-seq), DNase I hypersensitive site sequencing (DNase-seq), Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-Seq), and Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), all of which are commonly used to assess chromatin accessibility\u003csup\u003e23-26\u003c/sup\u003e. An emerging technology, Hi-C (high-resolution chromosome conformation capture), provides a high-throughput method for mapping the 3D structure of chromosomes within the nucleus\u003csup\u003e27\u003c/sup\u003e. While these techniques have advanced epigenetic research, their implementation requires considerable experimental and analytical expertise, as well as expensive instruments and reagents. In this context, the development of reporter assays to detect specific patterns of epigenetic alteration offers a simpler, more cost-effective, and quantitative approach for evaluating chemical toxicity, providing framework for such assays. Thus, the experimental procedure of the epi-TK assay (Fig. 1), based on the standard genotoxicity testing outlined in OECD TG490, meets the criteria for an effective reporter system, contributing to safety assessment.\u003c/p\u003e\n\u003cp\u003eFor the development of the epi-TK assay, CRISPR/dCas9-SunTag-DNMT3A was transiently expressed in TK6 cells, leading to increased DNA methylation at CpG dinucleotides within the \u003cem\u003eTK\u003c/em\u003e promoter region (Fig. 3E). Given that the expression of the \u003cem\u003eTK\u003c/em\u003e gene is regulated by transcription factors such as E2F1 and SP1\u003csup\u003e28,29\u003c/sup\u003e, it is likely that DNA methylation at these CpG loci inhibits the binding of transcription factors, thereby preventing the transcription of the \u003cem\u003eTK\u003c/em\u003e gene. Interestingly, the spontaneous frequency of TK revertant increased with the duration of cell culture after the removal of TFT (Fig. 2D), suggesting that the CpG sites in the promoter region were spontaneously demethylated during cell division. This reversible state of the epigenetically edited \u003cem\u003eTK\u003c/em\u003e gene may be attributed to the persistent presence of active histone marks, such as H3K27Ac and H3K4me3 (Fig. 4A and B). Moreover, a recent study demonstrated that artificially introduced histone modifications, including H3K4me3, H3K27Ac, H3K27me3, H3K9me2/3, and H2AK119Ub, are progressively diluted after cell division\u003csup\u003e30\u003c/sup\u003e. Although the mechanistic basis of such epigenetic restoration remains unclear, a potential regulatory mechanism could involve the three-dimensional chromatin architecture within the nucleus\u003csup\u003e31\u003c/sup\u003e. Nevertheless, the epigenetic state of the \u003cem\u003eTK\u003c/em\u003e gene is stably maintained when cells are cultured in the presence of TFT (Fig. 2B), ensuring the reproducibility of the assay.\u003c/p\u003e\n\u003cp\u003eUsing the housekeeping\u003cem\u003e\u0026nbsp;TK\u003c/em\u003e gene as a reporter locus, the effects of DNMT inhibitors were quantified (Fig. 3). The frequency of TK reversion following exposure to GSK-3484862 was substantially higher than that observed with 5-azadC. These results align with the observation that GSK-3484862 induces demethylation more efficiently than 5-azadC in murine cells\u003csup\u003e32\u003c/sup\u003e. This difference in demethylating efficacy is attributed to the cytotoxicity of 5-azadC, which forms covalent protein-DNA adducts\u003csup\u003e33\u003c/sup\u003e, whereas GSK-3484862 inhibits DNMT1 in a non-covalent manner. Regarding HDAC inhibitors, treatment of mTK6 cells with trichostatin A and vorinostat did not significantly increase the TK revertant frequency (Fig. 4C), likely due to the persistent levels of H3K27Ac and H3K4me3 at the \u003cem\u003eTK\u003c/em\u003e gene locus.\u003c/p\u003e\n\u003cp\u003eTPA stimulates cellular responses, including cell proliferation, migration, and differentiation through the activation of specific types of protein kinase C (PKC)\u003csup\u003e34-36\u003c/sup\u003e. Previous reports have shown that TPA treatment rapidly increases H3S10/H3S28 phosphorylation and H3 acetylation levels via mitogen- and stress-activated protein kinases\u003csup\u003e37-39\u003c/sup\u003e. These responses are associated with the transcriptional activation of immediate early response genes, with phosphorylation of both serine residues peaking around 1 hour following TPA treatment and then declining\u003csup\u003e37,38\u003c/sup\u003e. Interestingly, in our study, treatment of cells with TPA resulted in a significant decrease in TK reversion frequency (Fig. 5B), accompanied by a global reduction in H3K27Ac levels (Fig. 5C-F). The differences observed in our study may be due to the varying time scales used to assess histone modifications following TPA exposure. Specifically, H3K27Ac and H3K4me3 levels were measured after a 3-day recovery period following TPA treatment, allowing to distinguish immediate-early responses from persistent epigenetic changes. In agreement with our findings, prolonged TPA exposure leads to a global reduction of H3S10 phosphorylation in HepG2 cells\u003csup\u003e40\u003c/sup\u003e. Therefore, the TPA-induced reduction in H3K27Ac may reflect changes distinct from the histone modifications involved in the immediate-early responses to cellular stimuli.\u003c/p\u003e\n\u003cp\u003eInitially, we hypothesized that TPA exposure might induce changes in the expression of epigenetic regulators as a potential mechanism behind the observed epigenetic alterations. Supporting this idea, a recent study reported that transcriptome analysis of Bhas 42 cells exposed to TPA revealed altered expression patterns of key epigenetic factors, including \u003cem\u003eDNMT1, DNMT3A, MBD3\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Mi2\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e. However, in our study, TPA treatment did not induce DEGs in epigenetic-related pathways, such as those involving DNMTs, HDACs, and histone acetyltransferases (Fig. S1 D and E). Thus, the decreased levels of H3K27Ac are not due to changes in the expression of epigenetic factors that directly regulate chromatin status. These differing results may reflect the malignant status of the cells, including mutations or inactivation of tumor suppressor genes and proto-oncogenes. While Bhas 42 cells are characterized by \u003cem\u003eTp53\u003c/em\u003e deficiency, TK6 cells retain the native \u003cem\u003eTP53\u003c/em\u003e gene and are capable of undergoing the normal apoptotic process, which could explain the differential expression pattern observed in response to the tumor promoter TPA.\u003c/p\u003e\n\u003cp\u003eTPA is known to induce inflammation through activation of the NF\u0026kappa;-B signaling pathway\u003csup\u003e41,42\u003c/sup\u003e. Notably, inflammatory stresses is closely linked to epigenetic modifications, including DNA methylation and histone acetylation\u003csup\u003e43,44\u003c/sup\u003e. Chronic inflammatory stress was observed in TPA-treated mTK6 cells even after three days of cultivation following TPA treatment (Fig. S1E), suggesting that TPA-mediated inflammation may contribute to global epigenetic modifications. A potential relationship between the progression of inflammation and histone deacetylation has been reported in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003estudies\u003csup\u003e45-47\u003c/sup\u003e. While the precise mechanism behind TPA-mediated H3K27Ac reduction remains unclear, this epigenetic alteration could serve as a biological marker for specific inflammatory responses. Further research is needed to elucidate the mechanistic basis of the epigenetic toxicity associated with this chemical reagent.\u003c/p\u003e"},{"header":"4. Method","content":"\u003cp\u003e\u003cstrong\u003eCell culture.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human lymphoblastoid TK6 cell line and its derivative were cultured in RPMI-1640 medium (Nacalai Tesque) supplemented with 200 \u0026mu;g/mL sodium pyruvate, 100 U/mL penicillin, and 100 \u0026mu;g/mL streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Nichirei Biosciences, Inc.). The cultures were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere with 100% humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of human mTK6 cell line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate TK6 derivative cells with an epigenetically modified\u003cem\u003e\u0026nbsp;TK\u003c/em\u003e gene, the dCas9-SunTag DNMT3A system was employed to induce DNA methylation at the CpG loci of the \u003cem\u003eTK\u0026nbsp;\u003c/em\u003egene promoter. The gRNA cloning vector was generously provided by Dr. George Church (Addgene plasmid #41824; http://n2t.net/addgene:41824; RRID:Addgene_41824)\u003csup\u003e48\u003c/sup\u003e and LLP252 pEF1a-NLS-scFvGCN4-DNMT3a (Addgene plasmid #100941; http://n2t.net/addgene:100941; RRID:Addgene_100941) and LLP457 pGK-dCas9-Suntag-BFP (Addgene plasmid# 100957; http://n2t.net/addgene:100957; RRID:Addgene_100957) were gift from Dr. Ryan Lister\u003csup\u003e21\u003c/sup\u003e. Four single-guide RNA (sgRNA) targets were designed for the \u003cem\u003eTK\u003c/em\u003e promoter region, as detailed in Supplementary Table S1. A plasmid pool consisting of the four sgRNA (21 \u0026micro;g), pGK-dCas9-Suntag-BFP (21 \u0026micro;g), and pEF1a-NLS-scFvGCN4-DNMT3a (8 \u0026micro;g) was transfected into TK6 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) using 0.1 ml Nucleofector solution V (Lonza) and a Nucleofector 2b device, following the manufacturer\u0026rsquo;s instructions. After 48 h of incubation, the cells were seeded into 96-microwell plates at concentrations of either 20,000 or 200,000 cells/mL in the presence of 3.0 \u0026mu;g/ml trifluorothymidine (TFT). To determine the plating efficiency, cells were also seeded at a concentration of 8 cells/mL in the absence of TFT. After 10 days of incubation, TFT-resistant clones were isolated and maintained in RPMI-1640 medium containing 3.0 \u0026mu;g/ml TFT for at least 10 passages before preparing frozen stocks. The integrity of the TK promoter region sequences in the clones was validated using PCR primers listed in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time RT-PCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cell using the NucleoSpin RNA Plus kit (Macherey-Nagel). cDNAs were synthesized from the extracted RNA with ReverTra Ace (Toyobo Co., Ltd.). Quantitative PCR was performed using Thunderbird\u003csup\u003e\u0026reg;\u003c/sup\u003e Next SYBR qPCR Mix (Toyobo Co., Ltd.) and specific primers listed in Supplementary Table S2. The expression levels of the \u003cem\u003eTK\u003c/em\u003e gene were normalized to the internal\u003cem\u003e\u0026nbsp;GAPDH\u003c/em\u003e expression levels\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical substances.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5-Aza-2\u0026apos;-deoxycytidine and vorinostat were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), while trichostatin A was procured from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). GSK-3484862 was purchased from ChemieTek (IN, USA), and 12-\u003cem\u003eO\u003c/em\u003e-tetradecanoylphorbol-13-acetate (TPA) were sourced from Merck (Darmstadt, Germany). 5-Aza-2\u0026apos;-deoxycytidine was dissolved and diluted in H\u003csub\u003e2\u003c/sub\u003eO. Trichostatin A, vorinostat, and TPA were dissolved in ethanol, while GSK-3484862 were prepared in dimethyl sulfoxide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpi-TK assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emTK6 cells were cultured to the exponential phase in the presence of 3.0 \u0026mu;g/ml TFT. Afterward, cells were washed to remove TFT and resuspended in RPMI-1640 medium supplemented with 200 \u0026mu;g/mL sodium pyruvate and 10% FBS. For chemical exposure, 10 ml aliquots of cell suspension were prepared in 100 mm Petri dishes at a concentration of 250,000 cells/ml. Test chemicals at various concentrations were added to the dishes, which were then incubated at 37℃ for 24 h. Following chemical treatment, cells were washed and resuspended in fresh RPMI-1640 medium. To measure the TK revertant frequency, cells were seeded into 96-microwell plates containing 200 \u0026mu;M hypoxanthine, 0.1 \u0026mu;M aminopterin, and 17.5 \u0026mu;M thymidine (HAT). Seeding densities ranged from 100 to 100,000 cells/ml (20 to 20,000 cells/well), depending on the test chemicals evaluated. Concurrently, cells were plated in 96-microwell plates at a density of 8 cells/ml (1.6 cells/well) without HAT to assess the cloning efficiency (CE). After 21 days of incubation, the number of colonies in both the CE and HAT plates was recorded. CE was calculated using Eq. 1 based on the Poisson distribution\u003csup\u003e49\u003c/sup\u003e, where EW represents the number of wells without colonies, TW represents the total number of wells, and N is the average number of cells per well (N = 1.6) in the CE plates.\u003c/p\u003e\n\u003cp\u003eCE = \u0026minus; ln (EW / TW) / N \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (1)\u003c/p\u003e\n\u003cp\u003eRelative survival (%) was determined by comparing the CE values of chemical-treated cells to those of the solvent control. The TK revertant frequency was calculated using eq. 2 also based on the Poisson distribution. Here, N corresponds to the number of cells per well (N = 20 to 20,000) in the HAT plates. The results were statistically analyzed using Dunnett\u0026rsquo;s test and compared with solvent control values.\u003c/p\u003e\n\u003cp\u003eTK revertant frequency = [\u0026minus; ln (EW / TW) / N] / treated CE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(2)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBisulfite sequencing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from cells (1\u0026nbsp;\u0026times; 10\u003csup\u003e6\u003c/sup\u003e) using the Nucleospin Tissue kit\u0026nbsp;(Macherey-Nagel). The extracted DNA, dissolved in 50 \u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO (2 \u0026micro;g), was treated with 5.5 \u0026micro;L of 2 M NaOH and incubated at 37℃ for 15 min. Subsequently, 30 \u0026micro;L of 10 mM hydroquinone and 520 \u0026micro;L of 2 M sodium metabisulfite were added to the mixture, followed by incubation at 50℃ for 16 h. The bisulfite-converted DNA was purified using the Wizard DNA Clean-Up System (Promega) and subjected to PCR amplification using EpiTaq HS (TAKARA). A 451 bp DNA fragment containing the \u003cem\u003eTK\u0026nbsp;\u003c/em\u003epromoter region was amplified using primers\u0026nbsp;listed in Supplementary Table S2. The resulting PCR products was ligated into the pTA2 Vector using the Target Clone TA cloning system (Toyobo Co., Ltd.). The ligation mixture was transformed into\u003cem\u003e\u0026nbsp;Escherichia coli\u003c/em\u003e\u003cem\u003e\u0026nbsp;TOP10 cells\u003c/em\u003e, and recombinant clones were verified by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation-quantitative PCR (ChIP-qPCR).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess H3K27Ac and H3K4me3 levels following TPA exposure, cells were treated with 20 ng/mL TPA at 37℃ for 24 h. After treatment, the cells were washed twice with RMPI-1640 medium and cultured exponentially for 3 days to allow phenotypic expression. A total of 3\u0026nbsp;\u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were then cross-linked with 1% formaldehyde at room temperature for 10 min. The cross-linking reaction was terminated by adding 125 mM glycine. Following this, cells were washed with phosphate buffered saline and resuspended in 300 \u0026micro;l sonication buffer (10 mM Tris-HCl (pH8.0), 2 mM EDTA, 0.25% SDS). DNA was fragmented by sonication, and the resulting chromatin was incubated at 4℃ for 16 h with specific antibodies (anti-H3K27Ac monoclonal antibody (AB_2793797, ACTIVE MOTIF) and anti-H3K4me3 monoclonal antibody (ab8580, abcam)) and Protein A Sepharose 4 beads (Cytiva). The Sepharose beads were washed, and the immunoprecipitated DNA was eluted using elution buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS, 0.3 \u0026micro;g/ml Proteinase K) at 65℃ for 6 h. Purification of DNA was performed using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). The abundance of immunoprecipitated DNA was quantified using the Thunderbird\u003csup\u003e\u0026reg;\u003c/sup\u003e Next SYBR qPCR Mix (Toyobo Co., Ltd.) with specific primers listed in Supplementary Table S2\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal cell extracts were fractioned on gradient 5\u0026ndash;20% SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with 3% skim milk before incubation with primary antibodies. To detect H3K27Ac, Histone H3, and GAPDH, membranes were incubated overnight at 4\u0026deg;C in Hikari A solution (Nacalai Tesque) with the following antibodies: 1:1000 dilution of anti-H3K27Ac monoclonal antibody (AB_2793797, ACTIVE MOTIF), 1:1000 dilution of anti-Histone H3 monoclonal antibody (gifted from Dr. Kimura), or 1:1000 dilution of anti-GAPDH monoclonal antibody (sc-32233, Santa Cruz). After washing with tris-buffered saline containing 0.05% Tween 20, the membranes were incubated with a 1:4000 dilution of anti-mouse IgG or anti-rat IgG conjugated to horseradish peroxidase (Cytiva) in Hikari B solution (Nacalai Tesque). The chemiluminescent signal were detected using Chemi-Lumi One Super (Nacalai Tesque).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr. Hiroshi Honda (Kao Corporation) and Dr. Takayuki Fukuda (Bozo Research Center) for their helpful comments and discussions. We also thank Dr. Hiroshi Kimura (Tokyo Institute of Technology, Japan) for generously providing the antibody used to identify histone H3. This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare (21KA1001), the Consumer Affairs Agency Program Grant (24KA1008), and JSPS KAKENHI (22H03748 and 19K12339). Additional funding was provided by grants from the Takeda Science Foundation [to A.S. and K.U.].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Y., M.O., M.Y., and A.S. conceptualized the research, H.Y., M.O., K.Y., M.Y., M.H., K.S., K.U., and A.S. participated in study discussions and experimental design. H.Y., M.O., K.Y., A.C., A.U., M.Y., and A.S. carried out the experiments and analyzed the data. A.S. authored the manuscript and all authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome data raw files have been deposited to the DDBJ\u003cem\u003e\u0026nbsp;with accession number\u0026nbsp;\u003c/em\u003ePRJDB19707: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJDB19707 .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOrganisation for Economic Co-operation and Development. Environment Directorate., Organisation for Economic Co-operation and Development. 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Biochem.\u003c/em\u003e \u003cb\u003e110\u003c/b\u003e, 1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0003-2697(81)90103-2\u003c/span\u003e\u003cspan address=\"10.1016/0003-2697(81)90103-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1981).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"toxicology, epi-genotoxicity, reporter assay, 12-O-tetradecanoylphorbol-13-acetate, DNMT inhibitor, TK gene","lastPublishedDoi":"10.21203/rs.3.rs-5623363/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5623363/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNumerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase (\u003cem\u003eTK\u003c/em\u003e) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous \u003cem\u003eTK\u003c/em\u003e gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.\u003c/p\u003e","manuscriptTitle":"Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-15 14:41:40","doi":"10.21203/rs.3.rs-5623363/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-15T06:52:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-14T21:17:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16755494520126501484411372012646441318","date":"2025-01-04T09:42:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-04T06:26:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36695873046771311366380025965162335974","date":"2025-01-04T04:05:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286983345649108904026607718230109674916","date":"2025-01-02T15:24:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-02T08:20:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-02T06:04:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-12-24T18:29:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-23T08:30:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-12-11T10:07:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d66d5955-4177-4a2d-b808-e01182348ff3","owner":[],"postedDate":"January 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42867970,"name":"Biological sciences/Genetics/Epigenetics"},{"id":42867971,"name":"Biological sciences/Genetics/Eukaryote"},{"id":42867972,"name":"Biological sciences/Genetics/Genomic instability"},{"id":42867973,"name":"Biological sciences/Biological techniques/Genetic techniques"}],"tags":[],"updatedAt":"2025-03-10T17:09:39+00:00","versionOfRecord":{"articleIdentity":"rs-5623363","link":"https://doi.org/10.1038/s41598-025-92121-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-05 15:57:27","publishedOnDateReadable":"March 5th, 2025"},"versionCreatedAt":"2025-01-15 14:41:40","video":"","vorDoi":"10.1038/s41598-025-92121-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-92121-6","workflowStages":[]},"version":"v1","identity":"rs-5623363","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5623363","identity":"rs-5623363","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}