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Towards an Integrated Endocrine Disrupter Screen: Identification of Biomarkers of Estrogen- and Arylhydrocarbon-Receptor Signaling Networks in Zebrafish EmbryosEPA Grant Number: F5D10654
Title: Towards an Integrated Endocrine Disrupter Screen: Identification of Biomarkers of Estrogen- and Arylhydrocarbon-Receptor Signaling Networks in Zebrafish Embryos
Investigators: Burnam, Lucinda
Institution: Boston University
EPA Project Officer: Jones, Brandon
Project Period: September 6, 2005 through August 31, 2008
Project Amount: $103,496
RFA: STAR Graduate Fellowships (2005) RFA Text | Recipients Lists
Research Category: Academic Fellowships
Many small molecular weight environmental chemicals have the potential to perturb reproduction, development and normal physiology by interfering with nuclear receptor-mediated hormone signaling pathways (termed endocrine disrupting chemicals, EDC); however, few of the estimated 87,000 chemicals added to the environment have actually been tested for endocrine disrupting activity, nor do available assays address the diversity and complexity of the endocrine system in the whole organism. To obtain information necessary to assess the extent of the EDC problem in the natural environment, and better predict effects of EDC exposure in humans and animals, the objectives of proposed studies are to:
- develop an in vitro assay for routine screening of multiple classes of EDC and mixtures using zebrafish embryos as a whole animal model of the vertebrate endocrine system;
- characterize the genome wide estrogen receptor (ER) mediated signaling network, and identify points of interface with arylhydrocarbon receptor (AhR) activated pathways;
- and obtain profiles of representative estrogen- and dioxin-like EDC based on their ability to differentially or selectively perturb elements of the ER network and AhR interface.
Proposed research is predicated on the hypothesis 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 read-out to detect and predict effects of multiple classes of individual and interacting EDC. This proposal is intended as a proof-of-principle study that can, in future, be applied to chemicals that perturb any nuclear receptor-mediated hormone-signaling pathway.
Zebrafish have a typical vertebrate endocrine system and developmental program. Rapid ex utero development facilitates in vitro testing, in which it is possible to simultaneously assess multiple chemicals, doses, and exposure times, but has the added value of a whole organism in which all tissue types are represented and in their normal physiological interrelationships. To devise and validate an assay for routine chemical screening and identification of previously unrecognized EDC, we are using zebrafish embryos as a whole animal model of the vertebrate endocrine system. To quantify EDC-induced perturbations in the amount or timing of gene expression, real time quantitative polymerase chain reaction (qPCR) analysis will be applied. For optimization of test protocols, and determination of dose-response relations, initial focus will be on EDC that affect established estrogen- and dioxin-responsive genes. To develop a comprehensive (genome wide) approach to estrogen signaling, additional estrogen and estrogen/dioxin responsive genes will be identified through DNA microarray analysis, and verified by qPCR. To assemble these genes in a whole embryo, global estrogen signaling network, and identify points of convergence with AhR-mediated pathways, experimental studies which characterize estrogen-/dioxin- dose, time course and tissue specificity will be supplemented by genome database mining and computational approaches to determine gene ontology, upstream regulatory regions, and putative transcription factor elements. Information obtained can then be combined with experimental results to devise perturbation profiles of representative estrogen- and dioxin-like EDC. Such information will help in determining the nature and direction of their effects, cellular sites of action, and mechanism of action (e.g., agonist in brain, but not in liver). Although characterization of signaling networks has been applied to simple organisms like yeast, and to hormone regulated signaling in individual tissue- and cell-types in mammals, proposed studies, if successful, will provide a wealth of novel information with general relevance to vertebrates.
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. Although proposed studies are not intended to bring the screen to high-throughput capacity, it is feasible that an optimized system can be modified for large scale screening of compounds in the near future. Additionally, results of this study of ER-/AhR-mediated signaling will serve as a prototype for developing assays to detect chemicals that interact with other members of the nuclear receptor superfamily or otherwise alter gene expression.