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P5 (catalog #P9129), P4 (P0130), T (T-1500), E2 (E-8875), dimethyl sulfoxide (DMSO, D8418), NADPH (N7505), and NAD+ (N7004) were purchased from Sigma–Aldrich (St. Louis, MO, USA). PFBS (C4, CAS#375-73-5, Cat#387933, purity 98%) was purchased from J&K Scientific (Beijing, China), PFHpS (C7, CAS#375-92-8, Cat#C15986880, purity 95.3%) and 8:2FTS (CAS#39108-34-4, Cat#C15986585, purity 95.3%) were purchased from Dr. Ehenstorfer (Augsburg, Germany), PFPS (C5, CAS#2706-91-4, Cat#ACM2706914, purity 96%) was purchased from Alfa Chemistry (Ronkonkoma, NY, USA), PFOS (C8, CAS#1763-23-1, Cat#H0781, purity 98%) was purchased from TCI (Tokyo, Japan), PFHxS (C6, CAS#355-46-4, Cat#P999738, purity 98%) and PFDS (C10, CAS#335-77-3, Cat#P286540, purity 95%) were purchased from TRC (Toronto, Canada), 6:2FTS (C10, CAS#27619-97-2, Cat#T923214, purity 98%) was purchased from Macklin (Shanghai, China), PFDoS (C12, CAS#1260224-54-1, Cat#1ST14848, purity 98%) was purchased from Alta Scientific (Tianjin, China). PFSA compounds were dissolved in DMSO. Pregnant Sprague-Dawley rats (280–320 g) were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). Rat placentas were obtained from dams on gestational day 20 after the rats were euthanized by CO2 and cervical dislocation. The Animal Care and Use Committee of Wenzhou Medical University approved all animal protocols. The Second Affiliated Hospital of Wenzhou Medical University provided full-term human placental samples under the endorsement of the Hospital Ethics Committee and the subjects’ agreement (Protocol no. 2022-K-81-01). The human choriocarcinoma cell line JEG-3 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).
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Microsomes were prepared from the placenta at 4 °C as described previously[16]. Briefly, homogenized placental suspension in 0.01 mol/L phosphate-buffered saline (PBS, pH 7.2) supplemented with 0.25 mol/L sucrose was sequentially centrifuged at 700 ×g for 30 min, 14,500 ×g for 30 min, and then 105,000 ×g twice for 1.5 h for obtaining the microsomal pellet, which was resuspended in ice-cold PBS, and its protein content was measured by an enhanced BCA protein assay kit (Cat#P0010, Beyotime Biotech; Shanghai, China) according to the manufacturer's instruction.
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Both human h3β-HSD1 and rat r3β-HSD4 catalyze P5 conversion to P4, which can be measured using the HPLC/MS–MS method[16]. The following operations were performed: 1) a linear reaction condition for 3β-HSD was established after incubating the assay system (a 1.5 mL tube containing 200 nmol/L P5, 200 μmol/L NAD+, and 5 μg placental microsome in 100 μL pH 7.2 0.01 mol/L PBS) for 0–90 min in a shaking water bath (75 rpm) at 37 °C; 2) Michaelis–Menten kinetics of each enzyme was established after incubating the assay system (a 1.5 mL tube containing 0–2 μmol/L P5, 200 μmol/L NAD+, and 5 μg placental microsome in 100 μL 0.01 mol/L PBS) for 60 min; 3) a screening test for PFSA-mediated 3β-HSD inhibition was established after incubating the assay system (a 1.5 mL tube containing 200 nmol/L P5, 200 μmol/L NAD+, 5 μg placental microsome, and 100 μmol/L PFSA in 100 μL 0.01 mol/L PBS) for 60 min; 4) the dose response of each PFSA was measured after incubating the assay system (a 1.5 mL tube containing 200 nmol/L P5, 200 μmol/L NAD+, 5 μg placental microsome, and 0–200 μmol/L PFSA 100 μL 0.01 mol/L PBS) for 60 min; 5) the enzyme kinetics inhibition assay was established after incubating the assay system (a 1.5 mL tube containing 0–5 μmol/L P5, 200 μmol/L NAD+, 5 μg placental microsome, and 0–200 μmol/L PFSA in 100 μL 0.01 mol/L PBS) for 60 min. At the end of the reaction, 10 μL internal standard (IS, T-d5, Shanghai Zzbio Co., China) together with 200 μL acetonitrile (Merck Supelco, PA) was added to the tube in an ice bath, and the tube was centrifuged at 20,000 ×g for 10 min. Then, 10 μL extract was injected into HPLC–MS/MS system for measuring P4 amount.
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HPLC–MS/MS (Waters, USA) equipped with an Acquity BEH C18 column (2.1 mm × 50.0 mm, 1.7 μm particle size) was used to determine P4[16]. A gradient procedure in mixed solvent A (0.1% aqueous formic acid solution) and solvent D (acetonitrile) was programmed as follows: 70%–30% D (0–0.3 min), 10%–90% D (0.3–1.9 min), 70%–30% A (1.9–2.0 min). The flow rate was 0.40 mL/min and the injection volume was 10 μL. The column and sample temperatures were 30 and 4 °C, respectively. The XEVO TQD triple quadrupole mass spectrometer equipped with ESI source (Waters) was used for mass determination in a multiple-reaction monitoring mode and the MRM mode transitions for P4 and IS were m/z 315.16→96.92 and 289.07→96.92, respectively. Masslynx 4.1 (Waters) software was used for data acquisition and control. The P4 amount was calculated using the standard curve method with IS.
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The JEG-3 choriocarcinoma cell line is widely used as a placental steroid production model[19]. This cell line is derived from a human choriocarcinoma, produces a substantial P4 amount, and contains h3β-HSD1 and CYP19A1[19]. Therefore, it was used as a placental syncytiotrophoblast model in this study. Briefly, 105 cells were cultivated per well in 24-well plates using Minimum Essential Medium (MEM) supplemented with phenol red, 10% fetal calf serum (FCS), and various PFOS and PFDS concentrations at 37 °C and 5% CO2 for P4 production for 24 h. The media were collected for measuring P4 using HPLC–MS/MS method. For cytotoxicity analysis, the cells were treated with 100 μmol/L PFOS and PFDS and their viability was tested. For viability assay, 5,000 cells were seeded per well in 100 μL medium in 96-well plates and treated with 100 μmol/L PFOS and PFDS for 24 h. The optical density (OD) at 450 nm was measured using the Cell Counting Kit-8 (CCK-8, Sigma–Aldrich). Five samples were measured at each point and the results were averaged.
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Human CYP19A1 catalyzes T to E2 conversion, which can be measured using a radioimmunoassay[21]. Briefly, the following operations were performed: 1) a linear reaction condition for CYP19A1 was established after incubating the assay system (a 1.5 mL tube containing 100 nmol/L T, 200 μmol/L NADPH, and 5 μg human placental microsome in 200 μL pH 7.2 0.01 mol/L PBS) for 0–120 min in a shaking water bath (75 rpm) at 37 °C; 2) Michaelis–Menten kinetics of CYP19A1 was established after incubating the assay system (a 1.5 mL tube containing 0–1 μmol/L T, 200 μmol/L NADPH, and 5 μg placental microsome in 200 μL 0.01 mol/L PBS) for 60 min; 3) a screening test for PFSA-mediated CYP19A1 inhibition was established after incubating the assay system (a 1.5 mL tube containing 100 nmol/L T, 200 μmol/L NADPH, 5 μg placental microsome, and 100 μmol/L PFSA in 200 μL 0.01 mol/L PBS) for 60 min. The reactions were terminated by adding 10 μL 1 N HCl. E2 amount in the reaction medium was measured using radioimmunoassay[21]. A standard curve was prepared using 10–2,000 pg/mL E2, the bound and free steroids were separated with a charcoal–dextran suspension, and radioactivity was determined using a β-counter (PE). The minimum detectable E2 amount was 5 pg/mL.
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Dose response and mode of action (MOA) were analyzed as previously described[16]. Control (DMSO) activity was set at 100%. Residual activity after PFSA treatment was normalized to that of the control. When the residual activity after PFSA treatment was less than or equal to 50%, the half-maximum inhibitory concentration (IC50), which was calculated from the nonlinear regression (curve fit) of the dose response inhibition model, in which the inhibitor and response (three parameters) were calculated using GraphPad (GraphPad Inc., CA, USA). For MOA, enzyme kinetics inhibition (mixed model) with the following equations were used:
$$ {V}_{maxApp}=\frac{{V}_{max}}{1+\dfrac{I}{\alpha \times {K}_{i}}} $$ (1) $$ {K}_{mApp}={K}_{m}\times \frac{1+\dfrac{I}{{K}_{i}}}{1+\dfrac{I}{\alpha \times {K}_{i}}} $$ (2) $$ Y=\frac{{V}_{maxApp}\times X}{{K}_{mApp}+X} $$ (3) where Vmax is the maximum velocity (pmol∙mg−1∙min−1), I is the inhibitor concentration, Ki is the inhibition constant, Km is Michaelis–Menten kinetics constant, and α is a factor to judge the MOA. “α = 1” indicates noncompetitive inhibition, “α > 1 or < 1” indicates mixed inhibition, “α near 0 but not equal to 0” indicates uncompetitive inhibition; “α > ∞” indicates competitive inhibition. A Lineweaver–Burk plot (LBP) was drawn after comparing 1/V (velocity) with 1/substrate concentration (1/[P5]) to further evaluate the MOA of PFSA.
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AlphaFold human h3β-HSD1 model (AF-P14060-F1-model) [https://alphafold.ebi.ac.uk/entry/P14060][22,23], with 94.16 average model confidence (pLDDT), was selected as the human h3β-HSD1 target for in silico docking analysis. PFSA was sketched in ChemBioDraw Ultra 12.0 (Cambridge, UK) in the mol2 format. Docking analysis was performed with SwissDock web Server[24], and the lowest binding energy was calculated and visualized by Chimera 1.1.1 software (San Francisco, CA, USA). Hydrogen bonds and amino acid residues were labeled using a chimera. PyMOL software was used for the 3D structure and measurement of the chemical length. 2D graphics of the superimposed structures of PFSA with P5 were analyzed using LigPlot[25].
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The assays were repeated 4–8 times. Enzymatic results were analyzed using one-way ANOVA followed by post hoc Tukey’s multiple comparisons to identify significant differences between the groups for the screening test. Significant differences are denoted as *P < 0.05, **P < 0.01, ***P < 0.001, $P < 0.05, and $$$P < 0.001. Data are presented as means ± standard deviation (SD).
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Time-dependent h3β-HSD1 activity was measured after mixing 200 nmol/L P5 and 0.2 mmol/L NAD+ with human placental microsome for 0–90 min. P5 to P4 conversion rate was linear within 90 min (Figure 2A). When 0–2 μmol/L P5 was incubated with 0.2 mmol/L NAD+ and 5 μg human placental microsome for 60 min, the Km and Vmax for h3β-HSD1 were 0.228 ± 0.053 μmol/L and 1.152 ± 0.044 nmol∙mg−1min−1, respectively (Figure 2B), which is within the previously reported range[26]. The screening results showed that PFHpS, PFOS, and PFDS significantly inhibited h3β-HSD1 activity, resulting in residual activity close to or lower than 50% of the control activity (Figure 2C). Although PFOS and 6:2FTS have the same number of carbon atoms, 100 μmol/L 6:2FTS did not inhibit h3β-HSD1 activity. This was also true for 8:2FTS and PFDS (Figure 2C). These results indicate that PFSA structure-dependently inhibits h3β-HSD1 activity. The dose response of PFSA molecules with approximately 50% or lower residual activity than control was determines using a concentration series. The IC50 of PFOS, PFDS, and PFHpS were 9.03 ± 4.82, 42.52 ± 8.99, and 112.6 ± 29.39 μmol/L, respectively (Table 1 and Figure 2D–F). The enzyme kinetics inhibition analysis revealed that the Ki of PFOS, PFDS, and PFHpS were 8.32, 43.41, and 114.7 μmol/L, respectively (Table 1 and Figure 3). These results indicate that the inhibitory potency is PFOS > PFDS > PFHpS > PFBS = PFPS = PFHxS = PFDoS for h3β-HSD1, showing a V-shaped turn at PFOS (C8). Enzyme kinetics inhibition (mixed model) and Lineweaver–Burk plot analysis showed that PFOS and PFHpS were mixed inhibitors, whereas PFDS was a competitive P5 inhibitor (Figure 3 and Table 1).
Figure 2. Time-curve reaction, Michaelis–Menten kinetics, screening assay, and dose response of PFSA to inhibit placental h3β-HSD1 activity. (A) Time-curve reaction; (B) Michaelis–Menten kinetics; (C) Screening for PFSA to inhibit human placental h3β-HSD1 activity: means ± SD (n = 4–8); ***Indicates significant difference compared to CON at P < 0.001, $$$PFOS vs. 6:2FTS at P < 0.001, $PFDS vs. 8:2FTS at P < 0.05; (D–F) IC50 of PFHpS, PFOS, and PFDS, respectively. 6:2FTS, 1H, 1H, 2H, and 2H-perfluorooctanesulfonic acid; 8:2FTS, 1H, 1H, 2H, and 2H-perfluorodecanesulfonic acid; CON, control; h3β-HSD1, human 3β-hydroxysteroid dehydrogenase 1; IC50, half-maximum inhibitory concentration; PFDS, perfluorodecanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFOS, perfluorooctanesulfonic acid; PFSA, polyfluoroalkyl sulfonic acid; SD, standard deviation.
Name Length (Å) IC50 (μmol/L) Ki/Km (μmol/L) LBE, (kcal/mol) Mode action Binding site P5 12.0 ND 0.308 −8.754 competitive Steroid PFBS (C4) 7.2 NI ND −6.620 ND Steroid PFPS (C5) 8.9 NI ND −6.320 ND Steroid PFHxS (C6) 9.9 NI ND −6.555 ND Steroid PFHpS (C7) 10.2 112.6 ± 29.39 114.7 −6.800 ND Steroid PFOS (C8) 11.6 9.03 ± 4.83 8.32 −7.272 mixed Steroid 6:2FTS (C8) 12.0 NI ND −6.661 ND Steroid PFDS (C10) 14.4 42.52 ± 8.99 43.41 −7.153 competitive Steroid 8:2FTS (C10) 14.4 NI ND −6.709 ND Steroid PFDoS (C12) 16.7 NI ND −6.531 ND Steroid Note. IC50 = half-maximal inhibitory concentration, Ki = measured inhibition constant, LBE = lowest binding energy; NI = No inhibition at 100 μmol/L; ND = Not determined. IC50, half-maximum inhibitory concentration; PFBS, perfluorobutanesulfonic acid; PFDS, perfluorodecanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; h3β-HSD1, human 3β-hydroxysteroid dehydrogenase 1; PFHpS, perfluoroheptanesulfonic acid; PFHxS, perfluorohexanesulfonic acid; PFOS, perfluorooctanesulfonic acid; PFDoS, perfluorododecanesulfonic acid; 6:2FTS, 1H, 1H, 2H, and 2H-perfluorooctanesulfonic acid; 8:2FTS, 1H, 1H, 2H, and 2H-perfluorodecanesulfonic acid; SD, standard deviation. Table 1. Information of PFSA to inhibit human 3β-HSD1
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3D h3β-HSD1 structure (AF-P14060-F1) constructed using AlphaFold2 was used. P5 docked to h3β-HSD1. Docking revealed that P5 forms two hydrogen bonds with h3β-HSD1 catalytic residues, Ser125 and Tyr155 (Figure 4A), and contacts 13 residues (Val88, Ser125, Ile126, Glu127, Tyr155, Pro187, Met188, Tyr189, Ile190, Phe197, Tyr265, Ile319, and Leu322) (Figure 4B). This is consistent with the results of a previous docking model by Autodock for h3β-HSD1 with catalytic residues Ser124 and Tyr154, because it lacks one amino acid[27]. All PFSA were docked to the steroid-binding active site of h3β-HSD1. PFOS contacts five residues (Table 2), overlaps with four substrate-binding residues, and forms two hydrogen bonds (Figure 4C and 4D), and the lowest binding energy is −7.272 kcal/mol (Table 1). PFDS contacts eight residues (Table 2), overlaps with six substrate-binding residues, and forms one hydrogen bond with residue Tyr155 (Figure 4E and 4F), and the lowest binding energy is −7.153 kcal/mol (Table 1). PFHpS contacts 11 residues (Table 2), overlaps with six substrate-binding residues, and forms three hydrogen bonds (Figure 4G and 4H); the lowest binding energy was −6.800 kcal/mol (Table 1). Interestingly, the lowest binding energy values of other PFSA molecules were greater than −6.800 kcal/mol (Table 1), although they overlap with more than three substrate-binding residues (Supplementary Figure S1, available in www.besjournal.com). FTS connect with several substrate-binding residues and form hydrogen bonds with the catalytic residue Tyr155. However, the hydrogen atoms near the sulfonic acid moiety, instead of those near the fluorine atoms, may cause less negative attraction with h3β-HSD1, which may decrease the binding affinity (Supplementary Figure S1). The short-chain PFSA molecules, (such as PFBS), are extremely small, possibly decreasing the binding affinity (Supplementary Figure S2, available in www.besjournal.com). We further measured the molecular length of P5 and each PFSA, and found that the appropriate molecular length of P5, PFOS, PFBS, and PFDoS are 12, 11.6, 7.1, and 16.7 Å, respectively. We calculated the correlation between the Km of P5, Ki of PFHpS and PFOS, the molecular size, and the correlation between Ki and the lowest binding energy of PFHpS, PFOS, and PFDS. Ki/Km were inversely correlated with P5, PFHpS, and PFOS size (Figure 5A, R2 = 0.978), indicating that PFHpS and PFOS inhibit human 3β-HSD1, and Ki positively correlates with the lowest binding energy (Figure 5B, R2=0.993). This indicates that PFSA size should match the binding cavity like substrate P5 (12 Å), and lowest binding energy can well predict the inhibitory potency of PFSA.
Figure 4. In silico analysis of perfluoroalkyl sulfonic acid with h3β-HSD1. The 3D model of h3β-HSD1 contains the catalytic residues Ser125 and Tyr155 (pink in A). (A, C, E, G) 3D structure of P5 (cyan), PFOS (red), PFDS (red), PFHpS (red), respectively; (B) 2D for P5; (D, F, H) superimposed images for PFOS (purple), PFDS (purple), PFHpS (purple): hydrogen bonds (green line), overlapping residues (red circled), P5 (cyan) as background. h3β-HSD1, human 3β-hydroxysteroid dehydrogenase 1; P5, pregnenolone; PFDS, perfluorodecanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFOS, perfluorooctanesulfonic acid.
Compd. Contacting residues H bonds Overlapping residues PFBS Ile86, Glu127, Tyr155, Val88, Tyr189, Ile190, Phe197, Leu322 Glu127(3.27Å), Tyr155(2.76Å) Val88, Glu127, Tyr155, Tyr189, Ile190, Phe197, Leu322 PFPS Ile86, Val88, Glu127, Tyr189, Phe197, Leu322 Glu127(2.84Å) Val88, Glu127, Tyr189, Phe197, Leu322 PFHxS Ile86, Val88, Glu127, Phe197 Glu127(2.71Å) Val88, Glu127, Phe197 PFHpS Thr82, Cys84, Ile86, Thr 123, Ser124, Glu127, Tyr155, Met188, Tyr189, Ile190, Phe197 Cys84(3.01Å), Tyr155(2.88Å), Glu127(2.88Å), Ile190(2.92Å) Glu127, Tyr155, Met188, Tyr189, Ile190, Phe197 PFOS Glu127, Tyr189, Phe197, Asn269, Leu322 Glu127(2.78Å), Asn269(3.32Å) Glu127, Tyr189, Phe197, Leu322 6:2FTS Glu127, Ser125, Tyr155, Met188, Tyr189, Ile190, Phe197 Tyr155(2.80Å) Glu127, Ser125, Tyr155, Met188, Tyr189, Ile190, Phe197 PFDS Ser125, Tyr155, Phe197, Val215, Tyr265, Ile319, Ser323, Leu322 Tyr155(2.78Å), Ser125, Tyr155, Phe197, Tyr265, Ile319, Leu322 8:2FTS Ser124, Ser125, Tyr155, Tyr189, Ile190, Phe197, Tyr265, Leu322 Ser155(2.89Å), Tyr155(3.00Å), Ser125, Tyr155, Tyr189, Ile190, Phe197, Tyr265, Leu322 PFDoS Ile86, Ser125, Glu127, Tyr155, Tyr189, Ile190, Phe197, Ans269, Leu322 Asn269(3.00Å) Ser125, Glu127, Tyr155, Tyr189, Ile190, Phe197, Ans269, Leu322 Note. PFBS, perfluorobutanesulfonic acid; PFDS, perfluorodecanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; h3β-HSD1, human 3β-hydroxysteroid dehydrogenase 1; PFHpS, perfluoroheptanesulfonic acid; PFHxS, perfluorohexanesulfonic acid; PFOS, perfluorooctanesulfonic a. Table 2. Contacting residues, hydrogen (H) bonds, and overlapping residues of PFSA with pregnenolone to human 3β-HSD1
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To test whether PFOS and PFDS can inhibit P4 synthesis, JEG-3 cells were treated with 1–100 μmol/L PFOS and PFDS, where the highest concentration (100 µmol/L) was based on the highest serum PFSA levels (114.1 mg/L, 100–250 μmol/L PFOS) in occupational workers[28]. We found that ≥ 1 μmol/L PFOS significantly decreased P4 output and 10–100 μmol/L PFDS significantly reduced P4 levels (Figure 6). These results indicated that PFOS and PFDS can inhibit P4 biosynthesis.
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Rat placental r3β-HSD4 is a human placental h3β-HSD1 homologue that is highly similar to human h3β-HSD1[15]. Previous studies have shown that some PFAS molecules, such as perfluoroundecanoic acid (PFUnA), inhibit rat r3β-HSD4 activity[16]. Michaelis–Menten kinetics analysis showed that the Km and Vmax of r3β-HSD4 were 0.228 ± 0.078 μmol/L and 37.38 ± 4.06 pmol/mg/min, respectively, (Figure 7A), and the Km is similar to the previously reported value[15]. We screened the inhibitory effect of 100 μmol/L PFSA on r3β-HSD4. None of the PFSA tested inhibited rat 3β-HSD4 (Figure 7B), indicating species-dependent difference in placental 3β-HSD inhibition.
Figure 7. (A) Michaelis–Menten kinetics and (B) screening assay of perfluoroalkyl sulfonic acid to inhibit rat placental r3β-HSD4 activity. means ± SD (n = 4); No significant (“ns”) difference compared to CON was observed. CON, control; r3β-HSD4, rat 3β-hydroxysteroid dehydrogenase 4; SD, standard deviation.
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Human placental CYP19A1 catalyzes E2 formation from T. Michaelis–Menten kinetics analysis showed that the Km and Vmax of human placental CYP19A1 were 0.183 ± 0.053 μmol/L and 66.81 ± 6.63 pmol∙mg−1∙min−1, respectively (Figure 8A), and the Km is similar to the previously reported value [21]. We screened the inhibitory effect of 100 μmol/L PFSA on human placental CYP19A1. None of the PFSA tested inhibited human CYP19A1 (Figure 8B), indicating that PFSA specifically inhibits human placental 3β-HSD1 activity.
Figure 8. (A) Michaelis–Menten kinetics and (B) screening assay of PFSA to inhibit human placental CYP19A1 activity. means ± SEM (n = 4); No significant (“ns”) difference compared to CON was observed. CON, control; CYP19A1, aromatase; PFSA, polyfluoroalkyl sulfonic acid; SEM, standard error of mean.
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Chemicals and Animals
Microsomal Preparation
3β-HSD Assay
P4 Amount Determination Using HPLC–MS/MS
JEG-3 Cell Culture and Treatment
CYP19A1 Activity Assay
Dose Response and Enzyme Kinetics Inhibition Analysis
Molecular Simulation Analysis for PFSA with h3β-HSD1
Statistics
PFSA Structure-Dependently Inhibit h3β-HSD1 Activity
Docking Analysis of PFSA with h3β-HSD1
PFOS and PFDS Inhibit P4 Production in JEG-3 Cells
PFSA Do not Affect Rat Placental r3β-HSD4 Activity
PFSA Do not Affect Human Placental CYP19A1 Activity
22411+Supplementary Materials.pdf |