2013 Progress Report: In Vitro to In Vivo Screening of Thyroid Hormone Receptor Disrupting ChemicalsEPA Grant Number: R835164
Title: In Vitro to In Vivo Screening of Thyroid Hormone Receptor Disrupting Chemicals
Investigators: Furlow, David
Current Investigators: Furlow, David , Murk, Albertinka J.
Institution: University of California - Davis
Current Institution: University of California - Davis , Wageningen University & Research Centre
EPA Project Officer: Klieforth, Barbara I
Project Period: March 1, 2012 through February 29, 2016
Project Period Covered by this Report: March 1, 2013 through February 28,2014
Project Amount: $649,345
RFA: Developing High-Throughput Assays for Predictive Modeling of Reproductive and Developmental Toxicity Modulated Through the Endocrine System or Pertinent Pathways in Humans and Species Relevant to Ecological Risk Assessment (2011) RFA Text | Recipients Lists
Research Category: Computational Toxicology , Endocrine Disruptors , Health , Ecosystems , Safer Chemicals
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.
The major accomplishments during this funding period were:
- We completed our analysis of high throughput screening of GH3.TRE.LUC cells for thyroid hormone active compounds; those results were published in Freitas, et al. Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform. Curr Chem Genomics Transl Med. 2014;8:36-46.
- We completed our analysis of endogenous gene responses to thyroid hormone, synthetic analogs, and selected endocrine disruptors to validate the GH3 cells for further downstream analysis of the high throughput screening assays. A manuscript describing these results was submitted for publication.
Summary of Accomplishments:
1. High throughput screening of GH3.TRE.LUC cells: Initial characterization.
To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. Five potential TR antagonists were identified. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (> 100 µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 μM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening.
2. Characterization of the GH3 cell transcriptional response to thyroid hormone and endocrine disrupting chemicals.
While the GH3 cell system, and the GH3.TRE-Luc line in particular, has great potential for thyroid hormone (TH) endocrine disruption studies, the cells have not been fully characterized with regards to their TH signaling components, endogenous TH responsive genes, and the suitability of the integrated reporter gene as a surrogate for endogenous gene responses. We obtained microarray data that identified the T3 signaling components in these cells as well as a cadre of TH responsive genes, including known direct target genes like the corepressor hairless (Hr) and other genes related to angiogenesis such as HIF2. We then demonstrated that responses to 3,3’,5-triiodothyronine (T3) and two synthetic TR isotype selective thyromimetics in the reporter gene assay showed good concordance with cell proliferation and endogenous TH responsive gene expression assays. Lastly, we demonstrated that bisphenol A inhibited T3 mediated induction of the reporter gene as well as endogenous gene expression. Therefore, the reporter gene assay provided by these highly TH responsive cells provides an excellent tool for rapidly and sensitively screening for TR active compounds. Further, identification of TH response genes in these cells is important for secondary screening of high throughput assays for new synthetic TR modulators and TR disrupting compounds, and to determine their potential genomic versus nongenomic modes of action.
- We will validate the predominant class of active compounds from HTS screening as RXR active compounds interacting with TRs in a heterodimeric complex and prepare a manuscript for publication.
- We will develop and validate an induced metamorphosis assay in 1-week old tadpoles that is adaptable to medium throughput screening of potential positive compounds from HTS assays .