A zebrafish (Danio rerio) embryo bioassay for endocrine disrupting chemicals: biomarkers of estrogen- and arylhydrocarbon- receptor actions and interactions
Griffin, Lucinda Burnam
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Accumulating evidence from epidemiological, wildlife, and laboratory studies indicates that abnormalities of reproduction, development and physiology can be ascribed to environmental contaminants that mimic or block essential hormone-regulated processes. Few of the >87,000 chemicals added to the environment have actually been tested for endocrine disrupting bioactivity, and the diversity and complexity of the vertebrate endocrine system presents an additional challenge. This thesis examines the proposition that perturbations in the normal amount, location or timing of a hormone-regulated gene product in living zebrafish embryos can be taken as evidence of endocrine disruption, and used as a convenient in vitro bioassay for detecting endocrine disrupting chemicals (EDC) without a priori knowledge of their chemical nature, concentration, uptake, metabolism, receptor binding, or mechanism of action. First, a panel of genes was identified in signaling pathways impacted by two of the major known classes of EDC: estrogen-like EDC targeting estrogen receptors (ER) and dioxin-like EDC targeting arylhydrocarbon receptors (AhR). Methods for quantitative (Q)PCR analysis of the targeted mRNAs were developed and validated. Exposure conditions (dose, duration, developmental stage) were optimized for detecting changes in mRNA expression in response to authentic ER and AhR ligands (estradiol, dioxin). Sensitivity, specificity and applicability of the zebrafish embryo bioassay was verified by testing known or suspected ER and AhR agonists and antagonists, chemicals that impact estrogen and dioxin signaling upstream or downstream of receptor binding, and chemical mixtures. To resolve the role of different ER subtypes in mediating observed gene-, tissue- and EDC-specific responses, oligonucleotide morpholinos were microinjected into fertilized eggs to knock down expression of each ER gene (esrl, esr2a, esr2b) prior to EDC exposure. Microarray analysis was then applied to identify on a global scale additional biomarkers in ER-, AhR- and ER-/AhR- overlapping signaling networks. Results described here demonstrate that the zebrafish embryo bioassay, in principle, is amenable to high throughput modifications and can be adapted to detect EDC that interact with any other nuclear receptor signaling pathway. Because the endocrine system is fundamentally conserved in vertebrates, research in zebrafish can facilitate assessment of risk to wildlife and human health from EDC in the natural environment.
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