Engineering Research Center of Groundwater Pollution Control and Remediation, Ministry of Education, College of Water Sciences, Beijing Normal University, Beijing, 100875, China.
Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China.
Chemosphere. 2020 Jun;249:126034. doi: 10.1016/j.chemosphere.2020.126034. Epub 2020 Feb 1.
Some thyroid-disrupting chemicals (TDCs) affect thyroid function by activating the pathways mediated by a typical thyroid hormone (TH) membrane receptor, integrin αβ. The present study introduces improved competitive binding assays for the rapid and sensitive evaluation of the binding affinities of TDCs for integrin αβ 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 × 10 and 9.7 × 10 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 αβ. The three PAEs, including diethyl hexyl phthalate (DEHP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DnBP), demonstrated binding affinities for integrin αβ 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 αβ 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 αβ.
一些扰乱甲状腺功能的化学物质（TDCs）通过激活典型的甲状腺激素（TH）膜受体整合素 αβ 介导的途径来影响甲状腺功能。本研究引入了改进的竞争性结合测定法，用于快速灵敏地评估 TDCs 与整合素 αβ 的结合亲和力。基于不同的探针，对两种测定法进行了修改：荧光竞争性结合测定法和放射性配体竞争性结合测定法。所测试的化学物质包括已知的 TH、3,3',5,5'-四碘-l-甲状腺素（T4）；T4 的脱氨酶类似物四碘甲状腺乙酸（tetrac）；和邻苯二甲酸酯（PAEs）。研究了 T4 的相对结合效力，并且 T4 从其受体上置换 50%配体所需的浓度（RIC50）分别为荧光和放射性配体竞争性结合测定法的 4.9×10 和 9.7×10 nM，表明放射性配体竞争性结合测定法可能更灵敏地评估整合素 αβ 的结合亲和力。三种 PAEs，包括邻苯二甲酸二乙酯（DEHP）、邻苯二甲酸丁基苄基酯（BBP）和邻苯二甲酸二丁酯（DnBP），通过放射性配体竞争性结合测定法，显示出对整合素 αβ 的结合亲和力，其效力顺序为 DnBP>DEHP>BBP。对三种 PAEs 中的每一种与整合素 αβ 的对接模拟证实了计算出的结合能，这与 3 种 PAEs 的 log RIC20 值具有很强的正相关关系（R=0.99，p<0.001）。本研究表明，建立的放射性配体竞争性结合测定法可作为定量 TDCs 与整合素 αβ 亲和力的有用工具。
