Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2022.915096

Figure Lengend Snippet: (A) Evaluation of cell iodide uptake, at basal condition (Basal)and upon stimulation with 1 mU/ml thyroid stimulating hormone (TSH), in FRTL-5 normal rat thyrocyte cell line exposed for 24 hours to acetic acid, 2,2-difluoro-2-[(2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy]-ammonium salt (1:1) (C6O4), perfluoro-octanoic acid (PFOA) perfluoro-octane-sulfonic acid (PFOS) at concentration ranging from 0.1 to 100 ng/mL. Reference inhibition of iodide uptake was obtained by incubation with 50 μM sodium thiocyanate (NaSCN). Experiments were performed in triplicate and normalized as percentage fold increase on basal control laking TSH stimulus and exposure to perfluoroalkyl substances (CTRL). Data are reported as mean values ± standard deviation. Significance: *P < 0.05, **P < 0.01 vs corresponding CTRL; a=p < 0.05, b=p < 0.01 and c=p < 0.001 vs corresponding basal). Evaluation of FRTL-5 cell viability (B) and cell mebrane fluidity (C) upon exposure to C6O4, PFOA or PFOS exposed for 24 hours at concentration ranging 0.1 to 100 ng/mL. In CTRL, exposure was omitted. Cell viability was evaluated by MTT assay and membrane fluidity by merocyanine-540 staining as described in methods. Experiments were performed in triplicate and normalized as percentage fold increase on CTRL. Data are reported as mean values ± standard deviation.

Article Snippet: Perfluoro-octanoic acid and perfluoro-octane-sulfonic acid were purchased by Wellington Laboratories (Ontario, Canada).

Techniques: Concentration Assay, Inhibition, Incubation, Control, Standard Deviation, MTT Assay, Membrane, Staining

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2022.915096

Figure Lengend Snippet: Computer-based Molecular Docking and Molecular Dynamics Analysis of the possible docking of 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1) (C6O4), perfluoro-octanoic acid (PFOA) or perfluoro-octane sulphonic acid (PFOS), to thyroid stimulating hormone-receptor (TSH-R), whose extracellular domain is shown in (A) . Representative model of thyroid stimulating hormone (TSH) eterodimeric chain (TSH-α and TSH-β) bound to TSH-R is reported in (B) . Possible binding sites of C6O4, PFOA or PFOS to TSH-R are reported in (C) , with the the corresponding free Gibbs energy variations (ΔG) of the complex formation and aminoacids involved in the binding. Representative images of the best conformers of unliganded receptor (blue) overimposed with the receptor bound to C6O4 (ocra), PFOA (purple) or PFOS (orange), are shown in (D) . The interaction interface between TSH and the extracellular domain of TSH-R and the the root-mean-square deviation (RMSD) profile of the LEU100-GLN170 domain of the receptor, mainly involved in the binding to TSH, of unliganded receptor and the receptor bound to C6O4, PFOA, or PFOS, are shown in (E) .

Article Snippet: Perfluoro-octanoic acid and perfluoro-octane-sulfonic acid were purchased by Wellington Laboratories (Ontario, Canada).

Techniques: Binding Assay

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2022.915096

Figure Lengend Snippet: (A) Evaluation of intracellular cAMP levels in FRTL-5 normal rat thyrocyte cell line at basal condition, upon stimulation with 1 mU/ml thyroid stimulating hormone (TSH) or after the exposure for 24 hours to acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1) (C6O4), perfluoro-octanoic acid (PFOA) perfluoro-octane-sulfonic acid (PFOS), at concentration ranging from 0.1 to 100 ng/mL, followed by stimulation with TSH. Experiments were performed in triplicate and normalized as percentage fold increase on basal control lacking TSH stimulus and exposure to perfluoroalkyl substances (CTRL). Data are reported as mean values ± standard deviation Significance: *P < 0.05, **P < 0.01, ***P < 0.001 vs unexposed cells stimulated with TSH. (B) Evaluation of TSH-receptor (TSH-R), sodium-iodide symporter (NIS) and thyroperoxidase (TPO) gene expression FRTL-5 cell at different combinations of exposure to C6O4, PFOA and PFOS, at concentration ranging from 0.1 to 100 ng/mL, and upon stimulation with TSH. Experiments were performed in triplicate and normalized on cell stimulated with TSH. Significance: *P < 0.05, **P < 0.01, ***P < 0.001 vs TSH.

Article Snippet: Perfluoro-octanoic acid and perfluoro-octane-sulfonic acid were purchased by Wellington Laboratories (Ontario, Canada).

Techniques: Concentration Assay, Control, Standard Deviation, Expressing