Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene
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...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-03-01
|
| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-025-92121-6 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850029843309133824 |
|---|---|
| author | Haruto Yamada Mizuki Odagiri Keigo Yamakita Aoi Chiba Akiko Ukai Manabu Yasui Masamitsu Honma Kei-ichi Sugiyama Kiyoe Ura Akira Sassa |
| author_facet | Haruto Yamada Mizuki Odagiri Keigo Yamakita Aoi Chiba Akiko Ukai Manabu Yasui Masamitsu Honma Kei-ichi Sugiyama Kiyoe Ura Akira Sassa |
| author_sort | Haruto Yamada |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-12974871e88949fa97bc16fd187005dd |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-12974871e88949fa97bc16fd187005dd2025-08-20T02:59:24ZengNature PortfolioScientific Reports2045-23222025-03-0115111210.1038/s41598-025-92121-6Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK geneHaruto Yamada0Mizuki Odagiri1Keigo Yamakita2Aoi Chiba3Akiko Ukai4Manabu Yasui5Masamitsu Honma6Kei-ichi Sugiyama7Kiyoe Ura8Akira Sassa9Department of Biology, Graduate School of Science, Chiba UniversityDepartment of Biology, Graduate School of Science, Chiba UniversityDepartment of Biology, Graduate School of Science, Chiba UniversityDepartment of Biology, Graduate School of Science, Chiba UniversityDivision of Genome Safety Science, National Institute of Health SciencesDivision of Genome Safety Science, National Institute of Health SciencesDivision of Genome Safety Science, National Institute of Health SciencesDivision of Genome Safety Science, National Institute of Health SciencesDepartment of Biology, Graduate School of Science, Chiba UniversityDepartment of Biology, Graduate School of Science, Chiba UniversityAbstract 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.https://doi.org/10.1038/s41598-025-92121-6ToxicologyEpi-genotoxicityReporter assay12-O-tetradecanoylphorbol-13-acetateDNMT inhibitorTK gene |
| spellingShingle | Haruto Yamada Mizuki Odagiri Keigo Yamakita Aoi Chiba Akiko Ukai Manabu Yasui Masamitsu Honma Kei-ichi Sugiyama Kiyoe Ura Akira Sassa Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene Scientific Reports Toxicology Epi-genotoxicity Reporter assay 12-O-tetradecanoylphorbol-13-acetate DNMT inhibitor TK gene |
| title | Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene |
| title_full | Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene |
| title_fullStr | Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene |
| title_full_unstemmed | Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene |
| title_short | Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene |
| title_sort | dual directional epi genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping tk gene |
| topic | Toxicology Epi-genotoxicity Reporter assay 12-O-tetradecanoylphorbol-13-acetate DNMT inhibitor TK gene |
| url | https://doi.org/10.1038/s41598-025-92121-6 |
| work_keys_str_mv | AT harutoyamada dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT mizukiodagiri dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT keigoyamakita dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT aoichiba dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT akikoukai dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT manabuyasui dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT masamitsuhonma dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT keiichisugiyama dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT kiyoeura dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene AT akirasassa dualdirectionalepigenotoxicityassayforassessingchemicallyinducedepigeneticeffectsutilizingthehousekeepingtkgene |