Zebrafish as a Model for Studying Estrogen Signaling and Estrogenic Endocrine Disruption



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Estrogen signaling, mediated by estrogen receptors (ERs) and G-protein-coupled estrogen receptor (GPER), plays important roles in humans and wildlife. Perturbing estrogen signaling may lead to deleterious health problems such as infertility, developmental defects, metabolic disorders, and certain types of cancers. A broad range of chemicals, which are classified as estrogenic endocrine disruptors (EEDs), can bind to ERs or GPERs, thereby interfering with estrogen signaling and causing adverse effects. Zebrafish embryos/larvae are an emerging model used in xenoestrogen studies. However, endogenous estrogen signaling in developing zebrafish embryos/larvae is still poorly understood. In the studies of this dissertation, first a whole-genome analysis of estrogen regulated genes in zebrafish embryos/larvae at different developmental stages was performed. The identified estrogen-responsive genes were distinct between the four time points, however GO biological process enrichment of the estrogen-responsive genes revealed similar functional groups between the four time points. Tissue-specific effects of estrogen were also analyzed using tissue enrichment of estrogen-responsive genes and Tg(5xERE:GFP) transgenic fish. Brain, liver, heart, and pancreas were major estrogen-responsive organs in developing embryos and adults. Several candidate biomarkers were suggested from our studies, including f13a1a, cpn1, and zp3. Second, estrogenic and anti-estrogenic effects of ten bisphenols were assessed using zebrafish larvae. Selected estrogen-responsive genes identified from transcriptome analysis were tested in wild-type zebrafish larvae exposed to these bisphenols. Transgenic reporter fish Tg(5xERE:GFP) were used to study the tissue specific effects of the selected bisphenols and GFP quantification was used to compare the estrogenic effects of the bisphenols. Anti-estrogenic effects of the bisphenols were also studied in zebrafish larvae. Eight bisphenols showed tissue-specific and dose-dependent estrogenic effects in zebrafish larvae. One bisphenol showed only anti-estrogenic effects, and one bisphenol showed neither estrogenic nor anti-estrogenic effects. f13a1a was shown to be an effective new biomarker for assessing estrogenicity of chemicals. Third, high throughput screening of ToxCastTM Phase I pesticides library and NIH Clinical Collection drug library was performed using Tg(5xERE:GFP) reporter fish. A total of 26 environmental estrogens and 62 clinical drugs with estrogenic activities were identified from these two libraries. Then environmental estrogens identified from reporter zebrafish assays were compared to xenoestrogen datasets from in vitro assays, which are maintained in ToxCast Database (ToxCastDB). Seven chemicals were overlapping between our fish assays and all of the in vitro assays in ToxCastDB. In conclusion, zebrafish embryos/larvae are shown to be a good model for studying estrogen signaling and estrogenic endocrine disruption. These studies are important for understanding estrogen signaling in developing zebrafish and promoting the use of zebrafish larvae as a high-throughput screening model.



Zebrafish, Estrogen signaling, Endocrine disruption