Screening the ToxCast Chemical Libraries for Binding to Transthyretin

23 May 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Transthyretin (TTR) is one of the serum binding proteins responsible for transport of thyroid hormones (TH) to target tissue and for maintaining the balance of available TH. Chemical binding to TTR and subsequent displacement of TH has been identified as an endpoint in screening chemicals for potential disruption of the thyroid system. To address the lack of data regarding chemicals binding to TTR, we optimized an in vitro assay utilizing the fluorescent probe 8-anilino-1-napthalenesulfonic acid (ANSA) and the human protein TTR to screen over 1,500 chemicals from the U.S. EPA’s ToxCast ph1_v2, ph2, and e1k libraries utilizing a tiered approach. Testing of a single high concentration (target 100 µM) resulted in 888 chemicals with 20% or greater activity based on displacement of ANSA from TTR. Of these, 282 chemicals had activity of 85% or greater and were further tested in 12-point concentration-response with target concentrations ranging from 0.015-100µM. An EC50 was obtained for 276 of these 282 chemicals. To date, this is the largest set of chemicals screened for binding to TTR. Utilization of this assay is a significant contribution towards expanding the suite of in vitro assays used to identify chemicals with the potential to disrupt thyroid hormone homeostasis.

Keywords

thyroid
screening
transthyretin
endocrine disruption
NAMs
in vitro

Supplementary materials

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Description
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TTR Supplemental Figures
Description
Plate layouts for single concentration and concentration response testing.
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TTR Supplemental Tables
Description
Tables outlining components of the assay reactions and lists of autofluorescent chemicals, chemicals that were excluded from analysis due to interference, and chemicals screened in single concentration and concentration response testing.
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