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

The thyroid hormone system regulates physiological processes that are crucial for growth and metabolism. The homeostasis of thyroid hormones can be affected by test substances via multiple molecular interactions, one of them being the displacement of thyroid hormones from their binding proteins. The main thyroid hormone in the human bloodstream is L-thyroxine (T4), ≥ 99.5% of which is transported bound to three proteins: thyroxine binding globulin (TBG), transthyretin (TTR) and human serum albumin (HSA). To investigate the interactions of xenobiotics with the binding of T4 to these proteins, an in vitro thyroid hormone protein binding assay (THPB-assay) was developed. Samples containing 100 nM T4 were incubated with a T4-binding protein and increasing concentrations of a test compound, followed by removal of the unbound T4 via size exclusion chromatography and analysis of protein-bound T4. This assay allows for multiple adjustable parameters, including the choice of binding protein and the detection method for protein-bound T4, which can be extracted and measured via LC-MS or, when using ¹²⁵I-T4, by a gamma counting. Eight test substances were tested for the displacement of T4 from TTR and TBG respectively. For interference with T4-TTR binding, Tetrabromobisphenol A was established as a positive control with an average measured IC₅₀ of 287 nM. Displacement of T4 was observed for five more test substances, which displaced T4 from TTR with IC₅₀ ranging from 101 nM for Tetrac and 121 nM for 2,4,6-tribromophenol (TBP) to 1056 nM for Genistein. 6-propylthiouracil and L-DOPA did not displace T4 from TTR. LC-MS analysis and gamma counting of ¹²⁵I-T4 yielded similar results for each test substance. The five test substances which displaced T4 from TTR were additionally tested for displacement of T4 from TBG. Tetrac and Benziodarone displaced T4 with IC₅₀ values of 3 µM and 22 µM, respectively, whereas the remaining compounds did not. Finally, a mix of TTR, TBG and HSA, in concentrations of 536 nM, 26.5 nM and 65.35 µM respectively, was tested with the potent T4 displacer Tetrac. Tetrac displaced T4 from this protein mix with an IC₅₀ of 154 µM. Using the radioligand detection method, the T4 displacement of compounds from human and rat serum was tested to elucidate their effect in a physiological environment and identify potential species differences. Tetrac displaced T4 from human and rat serum at 10% serum concentration in samples, with IC₅₀ values of 38 µM and 242 µM respectively. TBP displaced T4 from 10% rat serum with an IC₅₀ of 1.3 mM. No displacement was observed in 10% human serum. At higher serum concentrations, no test substance was measured to displace T4 regardless of the species. While further research is essential, the insights regarding the absence of displacement at physiological serum concentrations challenge current risk assessment frameworks, which define changes in serum T4 levels as a key event in adverse outcome pathways related to the displacement of T4 from TTR.