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A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting Chemicals

EPA 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: Mustra, David
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)
Research Category: Endocrine Disruptors , Children's Health

Description:

Objective:

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.

Approach:

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.

Expected Results:

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.

Publications and Presentations:

Publications have been submitted on this project: View all 55 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 6 journal articles for this project

Supplemental Keywords:

POLLUTANTS/TOXICS, ENVIRONMENTAL MANAGEMENT, INTERNATIONAL COOPERATION, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Endocrine Disruptors - Environmental Exposure & Risk, Risk Assessment, Risk Assessments, Health Risk Assessment, Physical Processes, endocrine disruptors, Chemicals, Children's Health, Biochemistry, Environmental Policy, Environmental Chemistry, Endocrine Disruptors - Human Health, gene expression, endocrine disruptor screening program, animal models, endocrine disrupting chemicals, health effects, zebrafish embryo gene expression, childhood development, assessment of exposure, zebrafish embryo gene expression system, age-related differences, human health risk, susceptibility, screening assay, biogeochemistry, exposure, environmentally-caused disease, children's vulnerablity, endocrine disrupting chemcials, exposure studies, Human Health Risk Assessment

Progress and Final Reports:
2004 Progress Report
2005 Progress Report
2006 Progress Report
Final Report

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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