Orthogonal screening for thyroid stimulating hormone receptor modulators in human thyroid assays.

Area of Science:

• Environmental Toxicology
• Endocrinology
• Chemical Safety Assessment

Background:

• The U.S. Environmental Protection Agency (EPA) utilizes high-throughput screening (HTS) assays within the ToxCast and Tox21 programs to evaluate environmental chemicals.
• The hypothalamic-pituitary-thyroid axis, particularly the thyroid stimulating hormone receptor (TSHR), is crucial for thyroid development and hormone synthesis.
• Previous HTS identified numerous potential TSHR modulators, but their human relevance and mechanisms remain unclear.

Purpose of the Study:

• To select and screen chemicals active in the Tox21 TSHR assay for agonist/antagonist activity in human thyrocytes.
• To evaluate mechanistic effects on the native TSHR and functional impacts on thyroid hormone synthesis in human thyroid microtissues.
• To establish a tiered testing strategy for assessing thyroid-disrupting chemicals.

Main Methods:

• Selected subsets of chemicals from the Tox21 TSHR HTS assay for further screening.
• Employed a two-dimensional primary screening assay using thyroglobulin production in primary thyrocytes as a biomarker.
• Utilized three-dimensional thyroid microtissue assays for secondary screening to assess thyroid hormone synthesis.

Main Results:

• Screened 72 chemicals in agonist mode and 64 in antagonist mode.
• Identified two agonist and 13 antagonist chemicals with consistent activity across both assay formats.
• Demonstrated concordant results between primary thyrocyte and thyroid microtissue assays.

Conclusions:

• The study successfully validated TSHR modulators using a tiered testing approach.
• Results support the biological relevance of HTS-identified chemicals to human thyroid function.
• This tiered strategy enhances hazard characterization for potential thyroid-disrupting chemicals.