Some thyroid-disrupting chemicals (TDCs) affect thyroid function by activating the pathways mediated by a typical thyroid hormone (TH) membrane receptor, integrin αvβ3. The present study introduces improved competitive binding assays for the rapid and sensitive evaluation of the binding affinities of TDCs for integrin αvβ3. Based on different probes, two assays were modified: a fluorescence competitive binding assay and a radioligand competitive binding assay. The chemicals tested included the known TH, 3,3',5,5'-tetraiodo-l-thyronine (T4); a deaminated analog of T4, tetraiodothyroacetic acid (tetrac); and phthalate esters (PAEs). The relative binding potency of T4 was studied, and the concentration required to displace 50% of the ligands from their receptors (RIC50) of T4 was 4.9 × 105 and 9.7 × 104 nM for the fluorescence and radioligand competitive binding assays, respectively, suggesting that the radioligand competitive binding assay might be more sensitive for the evaluation of the binding affinity for integrin αvβ3. The three PAEs, including diethyl hexyl phthalate (DEHP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DnBP), demonstrated binding affinities for integrin αvβ3 in the following order of potency: DnBP > DEHP > BBP tested by the radioligand competitive binding assay. A docking simulation of each of the three PAEs with integrin αvβ3 confirmed the calculated binding energies, which had a strong positive relationship with the log RIC20 values of the 3 PAEs (R = 0.99, p < 0.001). The present study shows that the established radioligand competitive binding assay could be used as a valuable tool for quantifying the affinity of TDCs for integrin αvβ3.
Keywords: (99m)Tc-3PRGD(2); Competitive binding assay; Integrin α(v)β(3); Phthalate esters; T4-BSA-F; Thyroid-disrupting chemicals.
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