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The synthetic estrogen 17\u03b1-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20\u2009ng\/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n\u2009\u2265\u20094\/group\/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6\u2009h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post\u2013EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547\u20131556. \u00a9 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.<\/jats:p>","DOI":"10.1002\/etc.5879","type":"journal-article","created":{"date-parts":[[2024,5,24]],"date-time":"2024-05-24T11:15:27Z","timestamp":1716549327000},"page":"1547-1556","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17\u03b1-Ethynylestradiol"],"prefix":"10.1093","volume":"43","author":[{"given":"Ramya T.","family":"Kolli","sequence":"first","affiliation":[{"name":"Interdisciplinary Toxicology Program University of Georgia Athens Georgia USA"},{"name":"Pharmaceutical and Biomedical Sciences University of Georgia Athens Georgia USA"},{"name":"Student Services Authority Athens Georgia USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Travis 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