High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish

Abstract

Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.

Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish

“…50 This translucent model extends the use of fluorescent reporters to life-stages >5 dpf, which would otherwise gain skin pigmentation that interferes with GFP detection. 48 This is an important feature of the model, as EDC effects may vary both within a tissue and between body tissues at different life-stages. 4,51,52 The life-stages selected for this study represent two key landmarks in CV development: 5 days post fertilization (dpf) marking formation of the endocardial rings (precursors to the heart valve leaflets); 15 dpf marking elongation of the valve leaflets.…”

“…39,71,72 Both lower and 10× higher (worst case) BPA concentrations were substantially below maximum tolerable concentrations. 48 MBP exposure concentrations were based on a relative potency of 250× compared to BPA, measured in vivo in juvenile medaka. 43 Stock solutions were prepared by dissolving pure test chemicals in analytical grade dimethyl sulfoxide (DMSO) and then diluting (200× ) in 400 mL of embryo culture water 73 to give the desired nominal exposure concentrations in 0.5% DMSO.…”

Multiplex Analysis Platform for Endocrine Disruption Prediction Using Zebrafish

“…( a ) Comparative graph showing EC50s from different endocrine-disrupting studies in small fish in early and late stages [9,16,17,18,19,23,27,29,34,35,40,42,59,60,61,62,63,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79] Compounds not previously tested by others are excluded. Details for each study are shown in Table S4.…”

“…All of them rely, among other parameters, on the quantification of plasma sex-specific proteins (vitellogenin and spiggin) after at least 21 days of exposure, belonging therefore to the category of animal testing. Within the early stages, most of the efforts so far have been focused on the validation and use of gene expression markers to assess estrogenicity [16] and the subsequent construction of specific transgenic lines combining estrogenic responsive promoters and fluorescent reporter genes [17,18]. Surprisingly, additional endocrine axes such as androgens and thyroid have been widely neglected.…”

Health Effects and Life Stage Sensitivities in Zebrafish Exposed to an Estrogenic Wastewater Treatment Works Effluent

“…Biosensor transgenic fish offer great potential for both mixture and temporal effects analyses for exposure to chemicals and their mixtures (34)(35)(36). Here we applied an Estrogen Responsive Element (ERE) -Green Fluorescent Protein (GFP) transgenic zebrafish (Danio rerio) model in a pigment free (Casper) strain of zebrafish [ERE-GFP-Casper; (37)] to investigate for health effects and life stage sensitivities following chronic and acute exposures to a WwTW effluent with known (measured) estrogenic content. The responses measured included assessments on survivorship, growth, sex and sexual development (via gonadal histopathology), fluorescence induction in both target tissues (via fluorescence microscopy) and in whole bodies via quantification of gfp mRNA (via qRT-PCR).…”