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Changes in CpG-methylation of Vitellogenin 1 in Adult Male Zebrafish After Exposure to 17α-ethynylestradiol (EE2)
Citation:
Kolli, R., B. Cummings, T. Glenn, R. Bringolf, AND J. Kenneke. Changes in CpG-methylation of Vitellogenin 1 in Adult Male Zebrafish After Exposure to 17α-ethynylestradiol (EE2). 2018 SOT Annual Meeting and ToxExpo, San Antonio, TX, March 11 - 15, 2018.
Impact/Purpose:
Presented at the Society of Toxicology - Annual Meeting 2018.
Description:
An increasing number of pharmaceutical and industrial chemicals are being classified as endocrine disrupting compounds (EDCs). These chemicals can interfere with hormonal homeostasis and lead to developmental disorders, cancer and other pathologies. One such EDC is 17α-ethynylestradiol (EE2) used in oral contraceptives that can inadvertently be introduced into aquatic environments through municipal waste water treatment facilities. Exposure of male fish to EE2 is known to increase the expression of the egg yolk precursor protein vitellogenin (VTG). This induction has been predominantly used as a molecular marker of exposure to estrogenic EDCs and resulting feminization. However, the mechanisms behind VTG induction are not fully known and we hypothesized that it is regulated via DNA methylation. To test this hypothesis, we used a modified sequencing approach called targeted genome bisulfite sequencing (TGBS) to determine the percent methylation at the vtg1 promoter. DNA methylation was assessed in the livers of adult male zebrafish exposed to 20 ng/L EE2 for 0.25, 0.5, 4, 7 and 14 days. A significant increase in mRNA was observed in the EE2-exposed fish as early as 6 hrs. A decrease (≥35%) in DNA methylation at the CpG sites, however, was not observed until after 4 days of EE2 exposure. The decrease in DNA methylation brought the overall methylation of vtg1 promoter in male zebrafish to the same level as that of female controls, suggesting that it may lead to feminization. The persistence of these changes was assessed by discontinuing EE2 exposure. We observed that mRNA levels returned to basal levels after 7 days post EE2 removal. In contrast, DNA methylation levels remained significantly decreased. These data confirm the ability of EE2 to induce a genotypic change in VTG mRNA expression that can lead to feminization. Collectively, these data suggest a role for DNA methylation in VTG induction and subsequent feminization of male Zebrafish after exposure to EE2. Our work identifies a novel epigenetic mark of feminization that may serve as an indicator of previous exposure to EE2 which will aid in ecological risk assessment of EDCs.
