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High-Throughput Screening and Chemotype-Enrichment Analysis of ToxCast Phase II Chemicals Evaluated for Human Sodium-Iodide Symporter (NIS) Inhibition
Citation:
Wang, J., D. Hallinger, A. Murr, A. Buckalew, R. Lougee, A. Richard, S. Laws, AND T. Stoker. High-Throughput Screening and Chemotype-Enrichment Analysis of ToxCast Phase II Chemicals Evaluated for Human Sodium-Iodide Symporter (NIS) Inhibition. ENVIRONMENT INTERNATIONAL. Elsevier B.V., Amsterdam, Netherlands, 126:377-386, (2019). https://doi.org/10.1016/j.envint.2019.02.024
Impact/Purpose:
The use of hNIS-HEK293T-EPA cells in the NIS-mediated iodide uptake and cell viability assays provide the U.S.EPA with a rapid approach for identifying chemicals that have the potential to disrupt a key molecular step essential for the synthesis of thyroid hormones. This manuscript demonstrates that the NIS HTS approach can identify chemicals that have the potential to inhibit the NIS and the newly developed ranking system can be used to prioritize the chemicals for further evaluation in both orthogonal assays and short-term in vivo studies. Here, we report the NIS inhibitor screening for 768 ToxCast Phase II (ph2) chemicals. The 172 chemicals that were active in the RAIU assay were ranked based on potency, effects on cell viability, and normalized to perchlorate. Some of the highest ranked chemicals included PFOS, tributyltin chloride, and triclocarban, which have been shown to disrupt the thyroid axis. The present results were then combined with the previous ph1v2 library screening results to produce two sets of binary hit-calls for 1028 unique chemicals, consisting of 273 positives exhibiting significant RAIU inhibition, and 63 positives following application of a cell viability filter. A ToxPrint chemotype-enrichment analysis identified more than 20 distinct chemical substructure features associated with each version of the NIS inhibition hit-call space. A shared set of 9 unique ToxPrint chemotypes enriched in both hit-call sets indicates strong chemotype signals that are insensitive to cell viability filters, that can help to guide SAR investigations and inform future experiments. This chemotype information and SAR for both active and inactive chemicals will be used to test predictions in the NIS screening of the E1K ToxCast library of over 800 chemicals and guide follow-up studies.
Description:
In support of the Endocrine Disruptor Screening Program (EDSP), the U.S. EPA’s Office of Research and Development (ORD) is developing high-throughput screening (HTS) approaches to identify chemicals that may alter target sites in the thyroid hormone (TH) pathway. The sodium iodide symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into the thyroid gland as the initial step of TH biosynthesis. Previously, we screened 293 ToxCast Phase I chemicals using a HEK293T cell line expressing human NIS in parallel radioactive iodide uptake (RAIU) and cell viability assays to identify potential environmental NIS inhibitors. Here, we expanded NIS inhibitor screening for 768 ToxCast Phase II chemicals. Following single-concentration screening (at 1×10-4 M with a 20% inhibition cutoff), 235 samples were further tested in multiple-concentration (1×10-9 - 1×10-4 M) format in both RAIU and cell viability assays. The 172 samples that exhibited significant RAIU inhibition were then prioritized using combined RAIU and cell viability responses, and normalized relative to the known NIS inhibitor sodium perchlorate. Some of the highest ranked chemicals, such as PFOS, tributyltin chloride, and triclocarban, have been previously reported to be thyroid disruptors. In addition, several novel chemicals were identified as potent NIS inhibitors. The present results were combined with the previous Phase I screening results to produce two sets of binary hit-calls for 1028 unique chemicals, consisting of 273 positives exhibiting significant RAIU inhibition, and 63 positives following application of a cell viability filter. A ToxPrint chemotype-enrichment analysis identified more than 20 distinct chemical substructural features, represented in more than 60% of the active chemicals, as significantly enriched in each NIS inhibition hit-call space. A shared set of 10 chemotypes enriched in both hit-call sets indicates highly stable chemotype signals that can help to guide structure-activity relationship (SAR) investigations and inform future research.
URLs/Downloads:
DOI: High-Throughput Screening and Chemotype-Enrichment Analysis of ToxCast Phase II Chemicals Evaluated for Human Sodium-Iodide Symporter (NIS) Inhibition