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Epigenetic Mechanism Mediating the Regulation of the Estrogen Exposure Biomarker Vitellogenin in Zebrafish
Citation:
Kolli, R., B. Cummings, T. Glenn, R. Bringolf, AND J. Kenneke. Epigenetic Mechanism Mediating the Regulation of the Estrogen Exposure Biomarker Vitellogenin in Zebrafish. Gordon Research Conference on Cellular and Molecular Mechanisms of Toxicity, NH, Andover, August 13 - 18, 2017.
Impact/Purpose:
Presented at the Gordon Research Conference on Cellular and Molecular Mechanisms of Toxicity, August 13-18, Andover, NH
Description:
An increasing number of pharmaceuticals 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), a pharmaceutical estrogen used in oral contraceptives that can inadvertently be introduced into aquatic environments through waste water treatment plant effluent. 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 EDCs and resulting feminization. However, the mechanisms behind VTG induction are not fully known. We hypothesized that the expression of vtg1 is regulated via DNA methylation. To test this hypothesis, we used targeted genome bisulfite sequencing (TGBS) to determine the percent methylation at the 5’-flanking region of the vtg1 promoter. DNA methylation was assessed in the livers of adult male zebrafish exposed to 20 ng/L EE2 for 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 7 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. We also showed that EE2 exposure decreased the percent DNA methylation of vtg1 promoter, which remained decreased even after removal of EE2. Collectively, these data suggest a role for DNA methylation in VTG induction and subsequent feminization of male Zebrafish after exposure to EE2, and identifies a novel epigenetic mark of feminization that may serve as an indicator of early life exposure to EE2, which may aid in ecological risk assessment. Disclaimer: The opinions presented here are those of the authors and do not represent official policy of the US EPA.
