Bisphenol A analogues induce a feed-forward estrogenic response in zebrafish.

Because exposure to bisphenol A (BPA) has been linked to health problems in humans and wildlife, BPA analogues have been synthesized to be considered as replacement molecules. We here have examined estrogenic activity of BPA and five of its analogues, BPAF, BPE, BPC, BPC-Cl, and BPS by a combination of zebrafish-based in vivo and in vitro assays. We used transgenic estrogen reporter (5xERE:GFP) fish to study agonistic effects of bisphenols. Exposures to BPA, BPAF, BPE, and BPC, induced GFP expression in estrogen reporter fish at low exposure concentrations in the heart valves and at higher concentrations in the liver, whereas BPC-Cl activated GFP expression mainly in the liver, and BPS faintly in the heart only. The in vivo response was compared to in vitro estrogenicity of bisphenol exposure using reporter cells that express the zebrafish estrogen receptors driving expression of an estrogen response element (ERE)-luciferase reporter. In these cells, BPA, BPAF, BPC, BPE and BPS preferentially activated Esr1, whereas BPC-Cl preferentially activated Esr2a. By quantitative PCR we found that exposure to BPAF induced expression of the classical estrogen target genes vtg1, esr1, and cyp19a1b in a concentration response manner, but the most responsive target gene was f13a1a. Exposure to BPC-Cl resulted in a different expression pattern of vtg1 and f13a1a with an activation at low concentrations, followed by a declining expression at higher concentrations. Because expression of f13a1a was strongly activated by all compounds tested, we suggest including this mRNA as a biomarker for estrogenicity in larval fish. We further showed that exposure to BPAF and BPC-Cl increased E2 levels in zebrafish larvae, indicating that bisphenol exposures result in a feed-forward response that can further augment the estrogenic activity of these compounds.

Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model.

In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for the identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative concentration-response information on estrogenic activity of chemical compounds.