2005 Progress Report: 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 Period Covered by this Report: October 1, 2004 through September 30, 2005
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
The overall goal of this research project is to 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 endpoints in a screening assay to detect endocrine disrupting chemicals (EDCs). Our approach is to demonstrate that real-time quantitative-polymerase chain reaction (q-PCR) analysis of expressed genes in living zebrafish embryos can detect and characterize: (1) multiple subclasses of EDCs; (2) multiple gene- and tissue-targets of a given EDC subclass; and (3) EDCs that target genes both upstream and downstream of receptor binding.
The specific objectives of this research project are to: (1) address an urgent need for regulators to better predict which of the estimated 87,000 chemicals in the environment have the potential to disrupt hormone-dependent processes of physiology, reproduction, and development; (2) provide biologically relevant criteria for prioritizing chemicals for further testing; and (3) help interpret reports of reproductive and developmental abnormalities in wildlife and humans. Two major classes of persistent environmental pollutants with potential endocrine disrupting effects are estrogen-like chemicals (xenoestrogens) that interact with estrogen receptors (ER), estrogen signaling pathways, and dioxin-like chemicals (DLC) that interact with arylhydrocarbon receptors (AhR) to perturb AhR- and ER-signaling pathways. Thus, the current project focuses on specific molecular markers of ER- and AhR-mediated actions and effects. In addition, the project is viewed as a proof-of-principle study to develop approaches and protocols with universal applicability for screening chemicals with effects on any other nuclear receptor signaling pathway of interest, and to establish the feasibility of procedures and endpoints that are amenable to high throughput modifications.
Reproduction and development in man and in animals are essential for survival of species, species diversity, maintenance of ecosystems, and commercial activities. Thus, 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). We proposed the development of an assay using living zebrafish (Danio rerio) embryos as a whole animal in vitro screening system for simultaneous detection of multiple subsets of EDCs: (1) EDCs that act via ERs to induce brain P450 aromatase (P450aromB) and hepatic vitellogenin expression; (2) EDCs that act via AhRs to reduce gonadal aromatase (P450aromA) and increase P4501A1 expression; (3) EDCs that interact directly with pre-formed aromatase enzyme to block aromatization; and (4) EDCs that perturb ER, AhR, and P450 aromatase expression per se. An automated real-time reverse transcription (RT)-PCR approach will be used to measure targeted mRNAs in single and multiplex assays. The proposed zebrafish embryo system is a novel alternative to, and extension of, the current EDSP Tier 1 Screening Battery, which includes a mandate for ER binding, reporter assays, and an alternative placental aromatase (enzyme) assay, but does not presently include an assay for chemicals that disrupt endogenous estrogen signaling by altering aromatase or ER expression, nor does it include an assay that can detect possible AhR-mediated effects on reproductively relevant gene targets, or a screening assay that can simultaneously compare sensitivity and responsiveness of multiple genes to a given chemical. Although the proposed in vitro assay minimizes animal and chemical use, it has the advantages of an in vivo system for predicting agonist versus 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 coregulators. Resultant data will provide biologically relevant criteria for prioritizing chemicals for further testing and will help to interpret reports of reproductive and developmental effects in wildlife and humans. Validation of a zebrafish embryo gene expression assay for detecting known and suspected ER- and AhR-acting EDCs will have immediate applicability for routine chemical screening, and will demonstrate the feasibility of the same approach to detect chemicals that interact with other members of the nuclear receptor superfamily.
Significance and Relevance
Progress to date demonstrates that real-time q-PCR analysis of expressed genes in living zebrafish embryos can be used to detect and characterize: (1) multiple subclasses of EDCs (e.g., estrogen- and dioxin-like); (2) multiple gene- and tissue-targets of a given receptor class (e.g., ER- and AhR-responsive); (3) EDCs that target genes upstream of receptor binding (e.g., P450arom); and (4) the presence and nature of EDCs in samples from polluted environments. The assay has the power to detect and quantify both agonists and antagonists of a given receptor class, and to discriminate differentially spliced mRNA variants of a given gene.
We will: (1) complete characterization of q-PCR assays for all genes presently used as biomarkers; (2) accelerate routine screening of diverse classes of EDCs and agonist-antagonist combinations; (3) collect, process, and assay environmental samples from polluted sites (e.g., New Bedford Harbor and Cape Cod Superfund sites) and reference environments to link laboratory testing to actual problems in the natural environment; (4) expand the panel of genes used for screening EDCs, based on results of microarrays and subtraction libraries; and (5) complete and submit manuscripts for publication.