Na+/Taurocholate Cotransporting Polypeptide and Apical Sodium-Dependent Bile Acid Transporter Are Involved in the Disposition of Perfluoroalkyl Sulfonates in Humans and Rats

Abstract

Among the perfluoroalkyl sulfonates (PFASs), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) have half-lives of several years in humans, mainly due to slow renal clearance and potential hepatic accumulation. Both compounds undergo enterohepatic circulation. To determine whether transporters involved in the enterohepatic circulation of bile acids are also involved in the disposition of PFASs, uptake of perfluorobutane sulfonate (PFBS), PFHxS, and PFOS was measured using freshly isolated human and rat hepatocytes in the absence or presence of sodium. The results demonstrated sodium-dependent uptake for all 3 PFASs. Given that the Na(+)/taurocholate cotransporting polypeptide (NTCP) and the apical sodium-dependent bile salt transporter (ASBT) are essential for the enterohepatic circulation of bile acids, transport of PFASs was investigated in stable CHO Flp-In cells for human NTCP or HEK293 cells transiently expressing rat NTCP, human ASBT, and rat ASBT. The results demonstrated that both human and rat NTCP can transport PFBS, PFHxS, and PFOS. Kinetics with human NTCP revealed Km values of 39.6, 112, and 130 µM for PFBS, PFHxS, and PFOS, respectively. For rat NTCP Km values were 76.2 and 294 µM for PFBS and PFHxS, respectively. Only PFOS was transported by human ASBT whereas rat ASBT did not transport any of the tested PFASs. Human OSTα/β was also able to transport all 3 PFASs. In conclusion, these results suggest that the long half-live and the hepatic accumulation of PFOS in humans are at least, in part, due to transport by NTCP and ASBT.

Keywords: hepatocytes; perfluoroalkyl sulfonates; perfluorobutane sulfonate; perfluorohexane sulfonate; perfluorooctane sulfonate.

FIG. 1.

PFAS uptake into freshly isolated human (A–C) and rat (D–F) hepatocytes. Uptakes of 50 µM PFBS, PFHxS, and PFOS in the absence or presence of 100 µM bromosulfophthalein (BSP) was measured into freshly isolated human (HH) and rat hepatocytes (RH) for 2 min in the absence (white bars) or presence (gray bars) of sodium. Net sodium-dependent uptake (black bars) was calculated by subtracting the value of uptake in the absence of sodium from uptake in the presence of sodium. Each bar represents the mean ± SD from 3 independent experiments with triplicate determinations. The results were corrected for total protein concentration in each well. *p < .05.

FIG. 2.

Inhibition of ³H-taurocholate uptake mediated by human (A, hNTCP) and rat (B, rNTCP) NTCP by PFASs. Human NTCP-mediated 30 nM [³H]-taurocholate uptake was measured at 37°C for 1 min in the absence (DMSO) or presence of 10 µM PFBS, PFHxS, and PFOS using the CHO-hNTCP cell line. Rat NTCP-mediated 30 nM [³H]-taurocholate uptake was measured at 37°C for 1 min in the absence or presence of 10 µM PFBS, PFHxS, and PFOS using HEK293 cells transiently transfected with rNTCP. Each bar represents the mean ± SD of triplicate determinations. The results were corrected for total protein concentration in each well. *p < .05 compared to DMSO control.

FIG. 3.

Uptake of PFAS by human (A, hNTCP) and rat (B, rNTCP) NTCP. CHO-hNTCP cells or HEK293 cells transiently transfected with rNTCP were used to measure the uptake of 10 µM PFBS, PFHxS, and PFOS in the absence (white bars) and presence (gray bars) of sodium. Net sodium-dependent uptake (black bars) was calculated by subtracting the values of uptake in the absence of sodium from uptake in the presence of sodium. Each bar represents the mean ± SD from 2 independent experiments each performed with triplicate determinations. The results were corrected for total protein concentration in each well. *p < .05.

FIG. 4.

Time-dependent uptake of PFASs by human (A–C; hNTCP) and rat (D–F; rNTCP) NTCP. Uptake of 10 µM PFBS (A, D), PFHxS (B, E), and PFOS (C, F) was measured at 37°C at the indicated time points using CHO-hNTCP cells and HEK293 cells transiently transfected with rNTCP in the absence (circles) and presence (squares) of sodium. Net sodium-dependent uptake (triangles) was calculated by subtracting the value of uptake in the absence of sodium from uptake in the presence of sodium. The results were corrected for total protein concentration in each well. Each point represents the mean ± SD of triplicates.

FIG. 5.

Kinetics of human (A–C) and rat (D, E) NTCP-mediated transport of PFBS (A, D), PFHxS (B, E), and PFOS (C). Uptake of increasing concentrations of PFBS, PFHxS, and PFOS was measured within the initial linear range of transport using CHO-hNTCP cells (A–C) and HEK293 cells transiently transfected with rNTCP (D, E). Net uptake was calculated by subtracting the values of uptake in the absence of sodium from uptake in the presence of sodium and was corrected for total protein concentration. Resulting data were fitted to the Michaelis–Menten equation to obtain K_m and V_max values. Each point represents the mean ± SD from 3 to 4 independent experiments performed in triplicates.

FIG. 6.

Inhibition of human MRP2 (A), BCRP (B), and BSEP (C) transport by PFASs. Transport of 5 µM CDCF, 0.01 µM [³H]-estrone-3-sulfate or 0.5 µM [³H]-taurocholate by human MRP2, BCRP and BSEP vesicles were measured with or without 10 µM (Black bar) or 100 µM (white bar) PFBS, PFHxS, and PFOS. Each bar represents the mean ± SD of triplicate determinations. *p < .05 compared to DMSO control.

FIG. 7.

Uptake of PFASs by human (A, C, D) and rat (B) ASBT. HEK293 cells transiently transfected with hASBT (A) or rASBT (B) were used to measure uptake of 10 µM PFBS, PFHxS, and PFOS in the absence (white bars) or presence (gray bars) of sodium for 2 min. Net sodium-dependent uptake (black bars) was calculated by subtracting the values of uptake in the absence of sodium from uptake in the presence of sodium. Each bar represents the mean ± SD of triplicates from 2 independent experiments. (C, D) Uptake of 10 µM PFOS was measured at 37°C for the indicated time points by HEK293 cells transiently transfected with hASBT in the absence (circles) or presence (squares) of sodium. Net sodium-dependent uptake (triangles) was calculated by subtracting the values of uptake in the absence of sodium from uptake in the presence of sodium. The results were corrected for total protein concentration in each well. Each point represents the mean ± SD of triplicates. *p < .05.

FIG. 8.

Uptake of PFASs by human OSTα/β. HEK293 cells transiently transfected with hOSTα/β (gray bars) or empty vector pcDNA5/FRT (white bars) were used to measure uptake of 10 µM PFBS (A), PFHxS (B) or PFOS (C) at 2 and 10 min. Net uptake (black bars) was calculated by subtracting the values of uptake in empty vector transfected cells from uptake in the hOSTα/β transfected cells. The results were corrected for total protein concentration in each well. Each bar represents the mean ± SD of triplicates from 3 independent experiments.