Screening Estrogenic Activities of Chemicals or Mixtures In Vivo Using Transgenic (cyp19a1b-GFP) Zebrafish Embryos

The tg(cyp19a1b-GFP) transgenic zebrafish expresses GFP (green fluorescent protein) under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i) it is only expressed in radial glial progenitors in the brain of fish and (ii) it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture), including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective

Early life exposure to ethinylestradiol enhances subsequent responses to environmental estrogens measured in a novel transgenic zebrafish

This is the author accepted manuscript. The final version is available from Nature Publishing Group via the DOI in this record.Estrogen plays fundamental roles in a range of developmental processes and exposure to estrogen mimicking chemicals has been associated with various adverse health effects in both wildlife and human populations. Estrogenic chemicals are found commonly as mixtures in the environment and can have additive effects, however risk analysis is typically conducted for single-chemicals with little, or no, consideration given for an animal's exposure history. Here we developed a transgenic zebrafish with a photoconvertable fluorophore (Kaede, green to red on UV light exposure) in a skin pigment-free mutant element (ERE)-Kaede-Casper model and applied it to quantify tissue-specific fluorescence biosensor responses for combinations of estrogen exposures during early life using fluorescence microscopy and image analysis. We identify windows of tissue-specific sensitivity to ethinylestradiol (EE2) for exposure during early-life (0-5 dpf) and illustrate that exposure to estrogen (EE2) during 0-48 hpf enhances responsiveness (sensitivity) to different environmental estrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. Our findings illustrate the importance of an organism's stage of development and estrogen exposure history for assessments on, and possible health risks associated with, estrogen exposure.This work was co-funded by a BBSRC CASE studentship with AstraZeneca (reference 620033640) supporting J.M.G, a BBSRC small grant (reference BB/L01548X/1) to C.R.T., and by the AstraZeneca Global Safety, Health and Environment research programme. S.F.O. is an employee of AstraZeneca, a biopharmaceutical company specialized in the discovery, development, manufacturing and marketing of prescription medicines. AstraZeneca provided support in the form of studentship and salary for author S.F.O. but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Special thanks to Dr. Nicola Rogers for editing this manuscript