Acute Toxicity, Teratogenic and Estrogenic Activity of Test Chemicals Using a Combination of Two Zebrafish Embryos-Based Assays Using the Cyp19A1B-Gfp Line
Abstract
The zebrafish embryo has become an essential alternative vertebrate model to animal testing for the environmental hazard and risk assessments of chemicals. Further interest of the zebrafish embryo model has gained through the development of transgenic models and their use to assess endocrine disrupting chemicals (EDCs). Among them, the EASZY assay was designed to detect and quantify the estrogenic activity of chemicals acting through estrogen receptors (ERs) to induce GFP driven by the ER-regulated brain aromatase cyp19a1b gene. This assay, for which a draft OECD TG is currently under adoption, is considered as a level 3 assay of the OECD conceptual framework for EDCs. However, its integration within fish testing strategy for the hazard and risk assessments of chemicals needs to be experienced. The objective of this study was therefore to set-up a functional testing strategy based on cyp19a1b-GFP embryos combining a refined FET assay and the EASZY assay to assess the toxicity, teratogenicity and estrogenic activity. This demonstration study was undertaken and applied to a panel of bisphenol A substitutes for which such toxicological information was lacking. Eleven bisphenols (BPs) were sequencely tested in the refined FET and EASZY assays. Mortality (LC50), teratogenic effetcs (EC50) were assessed for each compound demonstrating that BPs have variable toxicities and elicited various teratogen phenotypes, some BPs being classified as teratogens. The use of cyp19a1b-GFP embryos in the refined FET provided preliminary information on their potential estrogenic activity which was further investigated in EASZY. Chemical exposures to BPA and BPA substitutes led to concentration-dependant inductions of GFP except for TCBPA. BPs could be ranked, based on their EC50, from the most to the least active, highlighting that most of them elicit higher estrogenic activities than BPA. Altogether, we successfully combined two zebrafish-based embryo assays, FET and EASZY, through the use of transgenic cyp19a1b-GFP embryos within a functional testing strategy to efficiently acquire novel data on acute toxicity, teratogenic and estrogenic activity of BPA substitutes thereby contributing to their hazard assessment. This study also provides further evidence that chemicals modulate cyp19a1b expression during early brain development which potential adverse effects still need to be investigated to accuratly assess the risk posed by EDCs acting in brain.