A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting ChemicalsEPA Grant Number: R831301
Title: A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting Chemicals
Investigators: Callard, Gloria V.
Institution: Boston University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2003 through September 30, 2007
Project Amount: $400,000
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Health , Safer Chemicals , Children's Health , Endocrine Disruptors
Reproduction and development in man and animals are essential for survival of species, species diversity, maintenance of ecosystems, and commercial activities; therefore, there is an urgent need for regulators to develop methods to better predict which of the estimated 87,000 chemicals in the environment have the potential to disrupt hormone-dependent processes of development, physiology and reproduction (EDC, endocrine disrupting chemicals). Here we investigate the proposition that perturbations in the normal amount or timing of a hormone-regulated gene product can be taken as evidence of chemical exposure and used as an end-point in a screening assay to detect potential endocrine disrupting activity.
The objective of this project is development of an assay measuring expressed genes in living zebrafish (Danio rerio) embryos as a whole animal in vitro screening system for simultaneous detection of multiple subsets of EDC: (a) EDC that act via estrogen receptors (ER) to induce brain P450 aromatase (P450aromB) and hepatic vitellogenin (vtg) expression; (b) EDC that act via arylhydrocarbon receptors (AhR) to reduce gonadal aromatase (P450aromA) and increase hepatic P4501A1 expression; (c) EDC that interact directly with preformed aromatase enzyme to block aromatization; and (d) EDC that perturb ER and AhR expression per se. Following exposure of embryos to a range of concentrations of known and unknown chemicals, alone or in combination, in a multiwell plate format, an automated real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) approach will be applied to measure targeted mRNAs in single and multiplex assays. Although the proposed in vitro assay minimizes animal and chemical use, it has the advantages of an in vivo system for predicting agonist vs. antagonist properties of a chemical without a priori knowledge of uptake and accumulation, activating or metabolizing pathways, access to targets, receptor binding and activation, or required co-regulators. Proposed research is not intended to put into practice a high throughput fully automated assay but to show that procedures used and endpoints measured are amenable to high throughput modifications.
Resultant data will improve risk assessment by regulators by providing biologically relevant criteria for prioritizing chemicals for further testing and by helping to interpret reports of reproductive and developmental effects in wildlife and humans in the natural environment. Validation of a zebrafish embryo gene expression assay for detecting known and suspected ER- and AhR-acting EDC will have immediate applicability for routine screening of chemicals that act via these pathways, and will serve as a prototype for assays that can detect chemicals that interact with other members of the nuclear receptor superfamily.