2014 Progress Report: In Vitro to In Vivo Screening of Thyroid Hormone Receptor Disrupting Chemicals

EPA 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, 2014 through February 28,2015
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

Objective:

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.

Progress Summary:

The major accomplishments during this funding period were:

  1. We validated the predominant class of active compounds from HTS screening as RXR active compounds interacting with TRs in a heterodimeric complex (manuscript in preparation).
  2. We developed and validated an induced metamorphosis assay in one week old tadpoles that is adaptable to medium throughput screening of potential positive compounds from HTS assays.

 Summary of Accomplishments:

  1. Validation of putative agonists from HTS screen: focus on rexinoids.

In order to identify man-made chemical compounds that disrupt TH signaling, we previously reported on a cell line, GH3.TRE-Luc, a stably transfected clonal line of rat pituitary GH3 cells, in which a canonical thyroid hormone response element for binding thyroid hormone receptors (TRs) drives expression of a destabilized luciferase (Luc) gene. In high-throughput screening experiments using these cells, several retinoid/rexinoid compounds induced Luc activity, although the retinoic acid receptor (RAR) specific synthetic agonist TTNPB did not. Here we characterize the retinoid response and show that even in the case of all trans retinoic acid (ATRA) and 13-cis retinoic acid (13cRA), the retinoids appear to be functioning through retinoid –X receptors (RXRs), which are heterodimer partners with TRs, as RXR antagonists abrogated the retinoid-induced Luc activation. We investigated whether this retinoid response also induced endogenous TR target genes in the GH3.TRE-Luc cells, and we show that Luc activity correlated well with TR target gene activation. In addition, synthetic RXR-specific agonists significantly activated all the tested TR target genes, but interestingly, the extent compared to the maximal T3-induction the retinoids/rexinoids could reach varied on a gene by gene basis, mostly through differences in the fold activation by T3. In contrast, the same retinoid compounds were unable to activate the identical Luc reporter construct in transient transfection assays in the human hepatocarcinoma cell line HuH7, nor did they activate two of the same genes, which were activated by T3 and the endogenous TRs expressed in HuH7 cells. These data demonstrate the suitability and sensitivity of the GH3.TRE-Luc cells for screening chemical compound libraries for TH disruption and suggest that the extent of disruption can vary both on a cell type and gene-specific basis, including an under appreciated contribution by RXRs.

  1. Development of one week induced metamorphosis assay for a convenient in vivo screen for HTS active compounds.

One of the most dramatic effects of any hormone in nature can be observed in amphibian metamorphosis, a process completely dependent on thyroid hormones (THs) that are structurally identical from frog to man. Most work on the molecular underpinnings of metamorphosis has been conducted using the frog Xenopus laevis, a system that has proven to be a particularly useful biological assay for identifying and characterizing novel thyroid hormone receptor agonists and antagonists, and more recently for screening environmental thyroid hormone disrupting chemicals. During metamorphosis, de novo adult tissue growth and larval tissue death during metamorphosis is coordinated by rising levels of TH.  TH also induces remodeling in tissues that undergo both larval cell death and adult cell proliferation and differentiation.  These morphological changes are accompanied by extensive tissue-specific gene expression changes that have been extensively characterized. Like their mammalian and avian counterparts, Xenopus laevis has two highly conserved receptor isoforms, xTRa and xTRb, encoded by two separate genes, and together with their heterodimer partner retinoid-x-receptor (RXR) modulate target gene transcription.  Both genes are also autoinduced by TH, particularly TRb.

There are multiple advantages of the Xenopus system for studying the basic biology of developmental endocrinology, as well as its application in environmental toxicology. For example, large numbers of embryos can be obtained year round, and they develop competence to respond to exogenously added TH and synthetic analogs shortly after hatching [4].  Given the conservation of the TH signaling pathway from frog to man, and the exquisite specificity of metamorphosis on TH, the EPA and OECD proposed the development of the Amphibian Metamorphosis Assay as a Tier 1 battery component for endocrine disrupting chemicals (https://www.epa.gov/scipoly/oscpendo/pubs/assayvalidation/tier1battery.htm; http://www.oecd-ilibrary.org/environment/test-no-231-amphibian-metamorphosis-assay_9789264076242-en). However, the assay is time consuming, uses a large number of animals and large water volumes (and compounds) with a specialized flow through water supply not routinely available in most laboratories, relies heavily on limb development as the primary endpoint for developmental staging along with thyroid histology, and does not provide direct mode of action information. Therefore, we have developed a simpler and more direct assay for TH action in development using young tadpoles only one week after fertilization. TH induced morphological and gene expression changes are accelerated and largely mirror those observed in natural metamorphosis. Key advantages are that the animals can be exposed in much smaller volumes than in the standard AMA, the variation between the animals is much smaller, and the bioassay only takes twelve days after fertilization to complete. In addition, augmenting the bioassay with transgenic reporter animals provides increased information about the mode of action of the compounds in question. This assay is highly suitable as an important second tier in screening for THD to confirm the results of the in vitro assays, but the speed of the assay and size of the animals may also provide an opportunity for some level of automating the assay for higher throughput.  We further increased the utility of the one week induced metamorphosis assay to include morphological endpoints directly associated with the expression of specific genes in growth versus resorption pathways. In this way, changes in specific gene expression (and reporter gene activity) caused by compound exposure can be linked to adverse morphological outcomes. To develop the assay, we exposed animals to endogenous hormones (T3 and T4), TR isotype selective agonists (CO23 and GC-1) and polybrominated environmental chemicals of concern with less certain modes of action (BDE-47, TBBPA, and HBCD). Nevertheless, the array of compounds used helped us establish the utility of this assay to detect TH disrupting compounds with increasing levels of specificity and sensitivity.

Journal Articles:

No journal articles submitted with this report: View all 10 publications for this project

Supplemental Keywords:

Thyroid hormone receptors, Rexinoids, Retinoids,Reporter gene assay, Endocrine disruption

Progress and Final Reports:

Original Abstract
  • 2012 Progress Report
  • 2013 Progress Report
  • Final Report