Two novel in vitro assays to screen chemicals for their capacity to inhibit thyroid hormone transmembrane transporter proteins OATP1C1 and OAT4

Abstract

Early brain development depends on adequate transport of thyroid hormones (THs) from the maternal circulation to the fetus. To reach the fetal brain, THs have to cross several physiological barriers, including the placenta, blood-brain-barrier and blood-cerebrospinal fluid-barrier. Transport across these barriers is facilitated by thyroid hormone transmembrane transporters (THTMTs). Some endocrine disrupting chemicals (EDCs) can interfere with the transport of THs by THTMTs. To screen chemicals for their capacity to disrupt THTMT facilitated TH transport, in vitro screening assays are required. In this study, we developed assays for two THTMTs, organic anion transporter polypeptide 1C1 (OATP1C1) and organic anion transporter 4 (OAT4), both known to play a role in the transport of THs across barriers. We used overexpressing cell models for both OATP1C1 and OAT4, which showed an increased uptake of radiolabeled T4 compared to control cell lines. Using these models, we screened various reference and environmental chemicals for their ability to inhibit T4 uptake by OATP1C1 and OAT4. Tetrabromobisphenol A (TBBPA) was identified as an OATP1C1 inhibitor, more potent than any of the reference chemicals tested. Additionally perfluorooctanesulfonic acid (PFOS), perfluoroctanic acid (PFOA), pentachlorophenol and quercetin were identified as OATP1C1 inhibitors in a similar range of potency to the reference chemicals tested. Bromosulfophthalein, TBBPA, PFOA and PFOS were identified as potent OAT4 inhibitors. These results demonstrate that EDCs commonly found in our environment can disrupt TH transport by THTMTs, and contribute to the identification of molecular mechanisms underlying TH system disruption chemicals.

Keywords: Endocrine disrupting chemicals; In vitro bioassays; Organic anion transporter 4; Organic anion transporter polypeptide 1C1; Thyroid hormones.

Fig. 1

Kinetics of the CHO-K1 OATP1C1 and MDCK-OAT4 T4 uptake assay. A The uptake of ¹²⁵I-T4 in time, expressed as a percentage of total T4, in CHO-K1 OATP1C1 or CHO-K1 wildtype cells. B Comparison between the percentage of ¹²⁵I-T4 uptake in CHO-K1 OATP1C1 and CHO-K1 WT cells after 40 min incubation, in the absence and presence of 100 µM ICG. C Uptake of ¹²⁵I-T4 uptake in time expressed as a percentage of total T4 in MDCK-OAT4 and MDCK-DEST cells over time. D Comparison of the percentage of ¹²⁵I-T4 uptake in MDCK-OAT4 and MDCK-DEST cells after 40 min incubation, with and without 100 µM LESI. Data is shown as the mean ± SD, N = 3. Comparisons with a p values below 0.05 are indicated by a star

Fig. 2

Percentage of ¹²⁵I-T4 uptake compared to the vehicle control 0.1% DMSO in A CHO-K1 OATP1C1 and B MDCK-OAT4 cells in the presence of selected chemicals. CHO-K1 OATP1C1 and MDCK-OAT4 cells were exposed to 10 nM T4 and 10–100 µM of selected chemicals for 30 min and 20 min respectively. Chemicals that decreased T4 uptake below 80% (i.e. 20% inhibition) after exposure were considered positive hits. 0.1% DMSO was used as vehicle control, 100 µM ICG was used as a positive control for OATP1C1 and 100 µM LESI for OAT4 (in black). Results are shown as mean ± SD, N = 3. Comparisons with a p values below 0.05 are indicated by a star

Fig. 3

Concentration–response curves for test chemicals that showed inhibition towards OATP1C1 mediated uptake of T4 (10 nM) in the CHO-K1 OATP1C1 TH uptake assay. The fraction of ¹²⁵I-T4 uptake is normalized to the positive control, 100 µM ICG, (lower limit = 0.0) and to the DMSO control (upper limit = 1.0). Blue dots indicate the plate medians from three independent experiments, all control and chemical concentrations were tested in triplicates per plate. The black line indicates the best-fit concentrations response curve, the dotted line the corresponding 95% confidence interval. The blue dotted line indicates the BMC20, the concentration that inhibits the ¹²⁵I-T4 uptake by 20%. The concentration range at which cytotoxicity was observed is indicated with a blue box. Compounds for which a control renormalization step was performed are marked with an¹ (color figure online)

Fig. 4

Concentration–response curves for test chemicals that showed OAT4 mediated uptake of T4 (10 nM) inhibition in the MDCK-OAT4 TH uptake assay. The fraction of ¹²⁵I-T4 uptake is normalized to the positive control, 100 µM LESI (lower limit = 0.0) and to the DMSO control (upper limit = 1.0). Blue dots indicate the plate medians from three independent experiments, all control and chemical concentrations were tested in triplicates per plate. The black line indicates the best-fit concentrations response curve, the dotted line the corresponding 95% confidence interval. The blue dotted line indicates the BMC20, the concentration that inhibits the ¹²⁵I-T4 uptake by 20%. The concentration range at which cytotoxicity was observed is indicated with a blue box. Compounds for which a control renormalization step was performed are marked with an¹ (color figure online)

Fig. 5

Overview of the potency of THTMT inhibitors on different THTMT TH uptake assays. Data for the MCT8 column was taken from Wagenaars et al. (2023). The calculated BMC20 value is given for each THTMT TH uptake assay, as well as a color graded range of BMC20 as shown in the figure legend. Compounds for which no inhibition below 20% was observed are marked in red. ND = no BMC20 value was determined, NR = no significant response observed, NT = not tested, CT = cytotoxic at BMC20 (color figure online)