Citation:

Laws, S., A. Buckalew, J. Wang, D. Hallinger, A. Murr, AND T. Stoker. Use of FRTL-5 Cell Line as a Complementary Assay for Chemicals Identified During High-Throughput Screening as Sodium/Iodide Symporter (NIS) Inhibitors. Society of Toxicology, San Antonio, TX, March 11 - 15, 2018.

Impact/Purpose:

This research supports the Office of Research & Development's initiative to develop and implement high throughput screening approaches for the Endocrine Disruptor Screening Program that expand the current coverage of molecular targets of thyroid hormone pathway. The work is a extension of the screening of 2 large ToxCast chemical libraries to identify chemicals that have the potential to disrupt thyroid hormone synthesis by inhibiting the uptake of iodide via the sodium iodide symporter (NIS). Here, we are evaluating the use of a rat thyroid follicular cell line as a secondary assay to test chemicals originally identified during high throughput screening (HTPS) as potential thyroid disruptors. Results will validate the HTPS assay as well as provide additional information on the mechanism of NIS inhibition and potential impact on thyroid hormone synthesis.

Description:

Confirmation of Test Chemicals Identified by a High-Throughput Screen (HTPS) as Sodium Iodide Symporter (NIS) Inhibitors in FRTL-5 Model S. Laws1, A. Buckalew1, J. Wang2, D. Hallinger1, A. Murr1, and T. Stoker1. 1Endocrine Toxicology Branch, TAD, NHEERL, ORD, U.S. Environmental Protection Agency, RTP, NC; and 2Oak Ridge Institute for Science and Education, U.S. Dept. of Energy, Oak Ridge, TN. The Fischer rat thyroid follicular cell line (FRTL-5) has been widely used to study thyroid function. This cell line was evaluated for its use in a secondary test for ToxCast chemicals previously identified as potential NIS inhibitors in a HTPS radioactive iodide uptake (RAIU) assay utilizing a HEK293T cell line expressing human NIS (hNIS). The FRTL-5 RAIU assay was optimized in a 96-well format over a 2-hr exposure period, and validated with a set of known NIS inhibitors and inactive compounds. The 12 top-ranked chemicals (ranking scores 58.5 - 150.4, relative to a score of 200 for sodium perchlorate) of the HTPS hNIS RAIU assay were then tested in the FRTL-5 RAIU assay. Chemicals (0.001-100µM) were tested in parallel RAIU and cell viability assays across 3 independent biological replicates. Dose responses of test chemicals from RAIU and cell viability assays were analyzed and sorted according to relative NIS inhibition potencies using a unique ranking system for RAIU responses with adjustment for cytotoxicity. Results from assay controls, as well as reference and test chemicals, demonstrated robust, highly reproducible performance of FRTL-5 RAIU and cell viability assays. Eleven of the 12 chemicals confirmed significant RAIU inhibition in the FRTL-5 assay, of which 7 obtained a ranking score >50. These data demonstrate the utility of a complementary RAIU assay with a physiologically relevant thyroid follicular cell with endogenous NIS expression to further investigate chemicals identified in HTPS for NIS inhibition. Chemicals displaying strong NIS inhibition in both the FRTL-5 and hNIS RAIU assays will be prioritized for additional testing using in vitro/in vivo assays to characterize the mechanism of inhibition and potential impact on thyroid hormone synthesis. This abstract does not necessarily reflect EPA policy.

Record Details: