Thyroxin displacement from single and combined thyroid hormone binding proteins by various test substances

Area of Science:

• Endocrinology and Metabolism
• Toxicology and Environmental Health
• Biochemistry and Molecular Biology

Background:

• Thyroid hormones (TH), primarily L-thyroxine (T4), are essential for growth and metabolism.
• TH homeostasis is maintained by binding to plasma proteins like thyroxine binding globulin (TBG), transthyretin (TTR), and human serum albumin (HSA).
• Test substances can interfere with TH homeostasis by displacing T4 from these binding proteins.

Purpose of the Study:

• To develop and validate an in vitro thyroid hormone protein binding (THPB) assay to investigate xenobiotic interactions with T4 binding proteins.
• To assess the potential of eight test substances to displace T4 from TTR and TBG.
• To evaluate T4 displacement in human and rat serum to understand physiological relevance and potential species differences.

Main Methods:

• Development of an in vitro THPB assay using T4 and specific binding proteins (TTR, TBG, HSA).
• Quantification of protein-bound T4 after incubation with increasing concentrations of test compounds using size exclusion chromatography.
• Detection of T4 using liquid chromatography-mass spectrometry (LC-MS) or gamma counting with ¹²⁵I-T4.
• Testing displacement in human and rat serum using the radioligand detection method.

Main Results:

• The THPB assay successfully identified substances displacing T4 from TTR and TBG.
• Tetrabromobisphenol A (TBBPA), Tetrac, 2,4,6-tribromophenol (TBP), and Genistein displaced T4 from TTR.
• Tetrac and Benziodarone displaced T4 from TBG.
• Tetrac displaced T4 from a protein mix (TTR, TBG, HSA).
• Tetrac and TBP displaced T4 from rat serum, and Tetrac displaced T4 from human serum at 10% concentration, but not at physiological serum concentrations.
• No displacement was observed in 10% human serum for TBP, and no displacement occurred at higher serum concentrations for any tested substance.

Conclusions:

• The developed THPB assay is a versatile tool for assessing T4 displacement by xenobiotics.
• Several tested compounds demonstrate in vitro T4 displacement from TTR and TBG.
• The lack of significant T4 displacement at physiological serum concentrations challenges existing risk assessment frameworks that rely on T4 displacement as a key event.