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DEVELOPMENT OF AN IN VITRO RADIOACTIVE IODIDE UPTAKE ASSAY (RAIU) WITH HUMAN NIS-EXPRESSING HEK293T-EPA CELL LINE
Citation:
Murr, A., D. Hallinger, A. Buckalew, Steve Simmons, T. Stoker, AND S. Laws. DEVELOPMENT OF AN IN VITRO RADIOACTIVE IODIDE UPTAKE ASSAY (RAIU) WITH HUMAN NIS-EXPRESSING HEK293T-EPA CELL LINE. To be Presented at Society of Toxicology, New Orleans, LA, March 13 - 17, 2016.
Impact/Purpose:
This poster presents the validation of a new cell based assay for evaluating potential thyroid toxicants in a radioactive iodide uptake assay.
Description:
Many high-throughput screening (HTPS) assays are available in the US EPA ToxCast program for estrogen and androgen pathways; only a limited number of assays exist for thyroid pathways. One potential target of thyroid-disrupting chemicals is the active uptake of iodide into the thyroid follicular cell via the sodium/iodide symporter (NIS), which is essential for the synthesis of thyroid hormones. Our laboratory evaluated a RAIU assay to screen for environmental chemicals that inhibit iodide uptake via NIS. We initially evaluated this assay with stably transfected hNIS-HEK293 cells (CEA, France) and then adapted it for use with a second-generation cell line, hNIS-HEK293T-EPA. These new cells stably express human NIS and efficiently uptake 125I-. In both cell lines, we optimized parameters such as cell density, incubation time, and 125I- exposure to ensure reliability (current Z’>0.8 ±0.08 for both cell lines) and sensitivity (dynamic range 63 fold and 220 fold, respectively) for detecting inhibitors of iodide uptake. IC₅₀ values were determined using nonlinear regression analyses of RAIU assay activity from cells exposed to multiple concentrations of each test compound. All response values were normalized to percent vehicle (HBSS, 10mM HEPES). The relative inhibition ranked by potency of the tested compounds (in both cell lines) are as follows: KPF₆>KClO₄>NaClO₄>NaBF₄>>NaSCN>>NaNO₃. NaF, a negative control, had no impact on iodide uptake. After test compound exposure, cells were evaluated for cytotoxicity to differentiate iodide uptake inhibition in viable cells from cell death. Based on their reliable performance in the RAIU assay, the hNIS-HEK293T-EPA will soon be used to screen ToxCast chemicals for inhibitors of iodide uptake via NIS. This abstract does not necessarily reflect EPA policy.