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Identifying Thyroid-Active Chemicals Using High-Throughput Screening
Citation:
Eytcheson, Stephanie A., A. Zosel, M. Hornung, AND S. Degitz. Identifying Thyroid-Active Chemicals Using High-Throughput Screening. SOT, Nashville, TN, March 19 - 23, 2023. https://doi.org/10.23645/epacomptox.24299056
Impact/Purpose:
High-throughput screening is an approach to rapidly test many chemicals for biological impacts. This abstract describes high-throughput screening assays used to assess chemical impact on thyroid-relevant targets. Specifically, chemicals from the ToxCast library were screened for inhibition of thyroid-hormone carrier proteins TBG and TTR. Screening results will allow for ranking and prioritization of chemicals to be tested in vivo and can be used to develop approaches for predicting toxicity while reducing the use of animals. This work is applicable to those interested in rapidly identifying chemicals with the potential to disrupt the thyroid system or in alternatives to animal testing.
Description:
High-throughput screening (HTS) assays allow for rapidly testing many chemicals for bioactivity at specific molecular targets. One goal of HTS is to reduce the time and cost of generating data to support evaluation of chemicals for potential endocrine disruption. Data from these assays can be used to develop a framework to predict in vivo effects with the goal of reducing animal testing. The US EPA’s Toxicity Forecaster (ToxCast) has a library of HTS data which can be used to prioritize chemicals of concern. Further, HTS can be used to identify potential molecular initiating events (MIEs) within adverse outcome pathways (AOPs). One gap in thyroid-related HTS includes the thyroid hormone carrier proteins transthyretin (TTR) and thyroxine-binding globulin (TBG) as potential MIEs of thyroid system disruption. TTR and TBG maintain the levels of free versus bound thyroid hormone and serve as circulating hormone transport proteins to deliver thyroid hormone to target tissue. To address this gap, a fluorescent high-throughput assay has been developed to assess inhibition of TTR or TBG. 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANSA) is a probe which fluoresces when bound to TTR or TBG. In the assay, displacement of ANSA from the protein by inhibitory chemicals results in a loss of fluorescence. A two-tiered approach was utilized to rank and prioritize chemicals for further testing. The first tier consisted of screening approximately 1800 chemicals from the ToxCast phase 1, phase 2, and e1k libraries for activity at a single high concentration. The total number of active chemicals in each assay (i.e. greater than 20% activity) were 777 in the TBG assay and 969 in the TTR assay. Chemicals with activity above the 80% threshold in the TBG assay (154 chemicals) and above the 95% threshold in the TTR assay (184 chemicals) were moved on to concentration response screening to define the IC50 values. Results from the assays will support ranking and prioritization of chemicals to be tested in vivo and will aid in the development of a framework to predict in vivo effects from in vitro HTS data. The contents of this abstract neither constitute, nor necessarily reflect, US EPA policy.
URLs/Downloads:
DOI: Identifying Thyroid-Active Chemicals Using High-Throughput Screening
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