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IN VITRO TO IN VIVO SCREENING OF THYROID HORMONE RECEPTOR DISRUPTING CHEMICALS
Impact/Purpose:
Thyroid hormones (TH) are critical regulators of vertebrate development and metabolism. Thus, exposure to environmental agents that affect TH synthesis, transport, metabolism, and/or receptor activity has profound consequences for the organism. The development of sensitive and reliable screening methods for TH disrupting chemicals should be an important component of a larger endocrine disruptor screening program. We recently developed a stable reporter cell line suitable for screening compounds that alter the transcriptional activity of the TH receptor (TR) in vitro (GH3.TRE-LUC). TH responsiveness of the cell line is highly sensitive, reliable, and rapid, and has been used in pilot high throughput screening assays at the NIH National Chemical Genomics Center. Our hypothesis is that chemicals that alter transcriptional control of the TRE luciferase reporter gene in GH3.TRE-LUC cells will also affect endogenous TH target gene expression and impact TH action in vivo, specifically Xenopus laevis metamorphosis. To test this hypothesis, we will validate chemicals acting as agonists or antagonists in the GH3.TRE-LUC cell line against endogenous TH target genes in GH3 cells, and determine the TR isotype dependence of the observed effects. Next, we will screen potential TR disrupting chemicals in wildtype Xenopus laevis tadpoles undergoing induced and spontaneous metamorphosis, and compare those findings to effects on reporter gene activity in newly developed TRE-Luciferase transgenic Xenopus laevis lines.
Description:
Upon completion of these studies, we will have established the predictive value of the GH3.TRE-LUC cell line to detect chemicals that can impact TH regulated gene expression and TH regulated developmental events in vivo. These studies have excellent potential to discover new chemicals that warrant further testing for significance to human development and overall health, and effects on sensitive life cycle stages in wildlife. These studies also lay the groundwork for future investigation into the underlying molecular mechanisms of action, and screening larger chemical libraries and complex mixtures for TR disrupting activity.