Optimized methods for measuring competitive binding of chemical substances to thyroid hormone distributor proteins transthyretin and thyroxine binding globulin

Abstract

Transthyretin (TTR) and thyroxine-binding globulin (TBG) are two major thyroid hormone (TH) distributor proteins in human plasma, playing important roles in stabilizing the TH levels in plasma, delivery of TH to target tissues, and trans-barrier transport. Binding of xenobiotics to these distributor proteins can potentially affect all these three important roles of distributor proteins. Therefore, fast and cost-effective experimental methods are required for both TTR and TBG to screen both existing and new chemicals for their potential binding. In the present study, the TTR-binding assay was therefore simplified, optimized and pre-validated, while a new TBG-binding assay was developed based on fluorescence polarization as a readout. Seven model compounds (including positive and negative controls) were tested in the pre-validation study of the optimized TTR-binding assay and in the newly developed TBG-binding assay. The dissociation constants of the natural ligand (thyroxine, T4) and potential competitors were determined and compared between two distributor proteins, showing striking differences for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA).

Keywords: Fluorescence polarization; Per- and polyfluoroalkyl substances; TBG binding; TTR binding; Thyroid hormone system disruptors.

Fig. 1

Reference compounds used for the TTR-binding assay and TBG-binding assay. D-mannitol served as a negative control

Fig. 2

TTR saturation binding curves obtained with different experimental conditions. Assays were performed with 30 nM TTR at a different incubation temperatures (RT = room temperature) analysed after 120 min and b different time points at room temperature (N = 3, n = 3). Data are presented as the average (± SD) of separate experiments

Fig. 3

TBG saturation binding curves assays obtained with 5 nM FITC-T4 at two different temperatures (RT and 37 °C) (N = 2, n = 2, data are presented as the average (± SD) of separate experiments). The inserted graph shows the same data plotted with a logarithmic scale on the x-axis

Fig. 4

Concentration–response curves of T4 in the TTR-binding assay (left y axis, N = 3, n = 3) and in TBG-binding assay (right y axis, N = 5, n = 2). Data are presented as the average (± SD) of separate experiments

Fig. 5

Concentration–response curves for seven model compounds a tested with 110 nM FITC-T4 and 30 nM TTR at RT (N = 3, n = 3); b tested with 5 nM FITC-T4 and 10 nM TBG at RT (N = 5, n = 2 for T4 and N = 3, n = 3 for other model compounds). Data are presented as the average (± SD) of data from separate experiments