Comparative Evaluation of the Effects of Legacy and New Generation Perfluoralkyl Substances (PFAS) on Thyroid Cells In Vitro

Abstract

BackgroundPer- and poly-fluorinated alkyl substances (PFAS) are environment-persitent emerging endocrine disrupting chemicals raising health concerns worldwide. Exposure to PFAS has been associated with the imbalance of thyroid hormones. However, available studies addressing the cell mechanism underlying thyroid disrupting feature of legacy PFAS, such as perfluoro-octanoic acid (PFOA), perfluoro-octane-sulfonic acid (PFOS), and the new generation substitutes, such as C6O4, are still lacking. In this study the potential disrupting effect of PFOA, PFOS, and C6O4 on a murine thyroid cell model was assessed.MethodsA rat FRTL-5 cell line was used as the normal thyroid follicular cell model. Cell iodide-uptake, induced by thyroid stimulating hormone (TSH), was used to assess the functional impact of PFAS exposure on cell function. Tetrazolium salt-based cell viability assay and merocyanine 540-based cell staining were used to address the possible involvement of cell toxicity and membrane biophysical properties on altered cell function. The possible direct interaction of PFAS with TSH-receptor (TSH-R) was investigated by computer-based molecular docking and analysis of molecular dynamics. Evaluation of intracellular cAMP levels and gene expression analysis were used to validate the direct impairment of TSH-R-mediated downstream events upon PFAS exposure.ResultsDifferent from PFOS or C6O4, exposure to PFOA at a concentration ≥ 10 ng/mL was associated with significant impairment of the iodide uptake upon TSH stimulation (respectively: basal 100.0 ± 19.0%, CTRL + TSH 188.9 ± 7.8%, PFOA 10 ng/mL + TSH 120.4 ± 20.9%, p= 0.030 vs CTRL + TSH; PFOA 100 ng/mL + TSH 115,6 ± 12,3% p= 0.017 vs CTRL + TSH). No impairment of cell viability or membrane stability was observed. Computational analysis showed a possible direct differential interaction of C6O4, PFOA, and PFOS on a same binding site of the extracellular domain of TSH-R. Finally, exposure to PFOA was associated with a significant reduction of downstream intracellular cAMP levels and both sodium-iodide transporter and thyroperoxidase gene expression upon TSH-R stimulation.ConclusionsOur data suggest that legacy and new generation PFAS can differentially influence TSH dependent signaling pathways through the direct interaction with TSH-R.

Investigation of the Interaction between Human Serum Albumin and Branched Short-Chain Perfluoroalkyl Compounds

“…17,31 Oppositely, less information is available regarding C6O4, an alternative to longchain PFASs, which was considered safe for use in food contact materials by the European Food Safety Agency, 32 and has shown less toxicity in various cell models in vitro. 33,34 Major health effects of long-chain PFASs are associated with their low elimination rates and high accumulation levels in the blood and in vital organs. 35 Indeed, the average half-life values for serum elimination of legacy PFASs, such as PFOA and PFOS, in environmentally exposed human populations are estimated to be in the order of 1−5 years, depending on the type of compound.…”

“…HPFO-DA was initially introduced as a safer alternative of PFOA and widely applied in manufacturing . Since its introduction, the environmental impact of HPFO-DA was carefully monitored and its adverse effects on human health found to be similar to those of PFOA and PFOS. , These findings led the U.S. Environmental Protection Agency and other competent authorities to develop large-scale monitoring plans and declare HPFO-DA a potential carcinogenic agent. , Oppositely, less information is available regarding C6O4, an alternative to long-chain PFASs, which was considered safe for use in food contact materials by the European Food Safety Agency, and has shown less toxicity in various cell models in vitro. , …”

“… 17 , 31 Oppositely, less information is available regarding C6O4, an alternative to long-chain PFASs, which was considered safe for use in food contact materials by the European Food Safety Agency, 32 and has shown less toxicity in various cell models in vitro. 33 , 34 …”