Disruption of Thyroid Hormone Sulfotransferase Activity by Brominated Flame Retardant Chemicals in the Human Choriocarcinoma Placenta Cell Line, BeWo
Brominated flame retardants (BFRs) have been shown to disrupt thyroid hormone (TH) homeostasis through multiple mechanisms, including inhibition of enzymes that regulate intracellular levels of THs, such as sulfotransferases (SULTs). The placenta plays a critical role in helping to maintain TH levels during fetal development and expresses SULTs. This is concerning given that disruption of TH regulation within the placenta could potentially harm the developing fetus. In this study, we investigated the effects of two polybrominated diphenyl ethers (PBDEs), two hydroxylated PBDEs, and 2,4,6-tribromophenol (2,4,6-TBP) on TH SULT activity in a choriocarcinoma placenta cell line (BeWo). BeWo cells were exposed to BFR concentrations up to 1 \xce\xbcM for 1 \xe2\x80\x93 24 h to investigate changes in basal SULT activity and in mRNA expression of several TH regulating genes. 2,4,6-TBP was the most potent inhibitor of basal 3,3\xe2\x80\xb2-T2 SULT activity at all exposure durations, decreasing activity by as much as 86% after 24 h of exposure. BDE-99, 3-OH BDE-47, and 6-OH BDE-47 also decreased 3,3\xe2\x80\xb2-T2 SULT activity by 23 \xe2\x80\x93 42% at concentrations of 0.5 \xce\xbcM and 1.0 \xce\xbcM following 24 h exposures. BDE-47 had no effect on SULT activity, and there was no observed effect of any BFR exposure on expression of SULT1A1, or thyroid nuclear receptors alpha or beta. This research demonstrates that total TH SULT activity in placental cells are sensitive to BFR exposure; however, the mechanisms and consequences have yet to be fully elucidated.
Introduction<!>Reagents and Materials<!>Cell Culture<!>Dosing<!>Cytotoxicity<!>Basal Sulfotransferase Activity Assays<!>Measurement of Thyroid Hormone Levels and Sulfated/Hydroxylated Metabolites in Cell Culture Growth Media<!>Enzyme Kinetics and Inhibition Assays<!>mRNA Expression Analysis<!>Statistics<!>SULT Inhibition in Cell Homogenates<!>Enzyme Inhibition Assay \xe2\x80\x93 Michaelis-Menten Curves<!>Exposures in Cell Culture<!>Growth Media TH Analysis<!>Growth Media Sulfated Metabolite Analysis<!>RT-qPCR<!>Discussion
<p>Brominated flame retardants (BFRs) such as polybrominated diphenyl ethers (PBDEs) have been historically used in various consumer products to meet flammability regulations. However, due to their demonstrated toxicity and environmental persistence, the PBDE commercial mixtures PentaBDE and OctaBDE were banned or phased-out, depending on the region, in the mid-2000s, while DecaBDE was phased-out at the end of 20131. Despite this phase-out, human exposure to PBDEs remains ubiquitous, and occurs primarily through inadvertent ingestion of contaminated house dust, with infants and toddlers experiencing higher rates of exposure due to their frequent hand-to-mouth contact2,3.</p><p>There is growing human epidemiological data suggesting that PBDE exposure may lead to poorer health outcomes, including associations with adverse birth outcomes, neurodevelopmental deficits, difficulties in becoming pregnant, and thyroid hormone levels4–6. The human placenta plays a critical role in the delivery of maternally-derived thyroid hormones (THs) to the fetal compartment during pregnancy. This is especially important during the first trimester of pregnancy when the fetus is undergoing many TH-mediated neurodevelopmental processes7. Therefore, gestation represents an extremely vulnerable window of fetal development that is susceptible to disruption by environmental contaminants.</p><p>Previous research from our laboratory demonstrated that some BFRs can inhibit thyroid deiodinase (DIO) and thyroid sulfotransferase (SULT) activity in human liver sub-cellular fractions8,9. In particular, human hepatic SULT activity was more sensitive to inhibition compared to DIO, especially for several halogenated BFRs (e.g. hydroxylated PBDEs, OH-BDEs, and 2,4,6-tribromophenol). Recently, we also measured the accumulation of BFRs in archived human placental tissues and examined associations between BFRs, THs, and DIO and SULT activity10. These studies suggest that exposure to BFRs may be altering local TH levels in the placenta through DIO and SULT activity. There are eight SULT isozymes that have been shown to perform TH sulfation (SULT1A1, 1A3, 1A5, 1B1, 1B2, 1C1, 1E1, and A1) and SULT1A1 and SULT1A3 are the dominant isoforms present in human placenta11, 12. However, given the complexity involved in using human tissues and the numerous confounding variables present in human studies, the use of a more controlled in vitro experimental system is needed to provide additional clarity into the underlying mechanisms of action. To this end, this project was undertaken to evaluate the effects of BFR exposure on TH SULT activity using the human placenta cell line, BeWo.</p><p>The BeWo cell line is a malignant human choriocarcinoma cell line derived from a methotrexate-resistant tumor isolated from a 13. Studies have confirmed the BeWo cell line to be ultrastructurally similar to in utero trophoblast cells, indicating that they are an appropriate model for placenta cell culture experiments14. Additionally, because their cell type composition is dominated by cytotrophoblasts, the BeWo cell line is more similar to an early-stage/first trimester placenta before subsequent differentiation and proliferation of syncytiotrophoblasts. This is important because the early-stage placenta represents the most vulnerable developmental window of exposure for the fetus. Thus, the use of the BeWo cell line can provide insight into this difficult-to-study developmental period.</p><p>The goal of this study was to investigate the effects of BFRs on TH SULT activity in the BeWo cell line, as a surrogate for placental tissues. We sought to determine if these specific BFRs will inhibit sulfation of thyroid hormones, and lead to increases in the intracellular concentrations of THs (e.g. triiodothyronine, T3). We also measured gene expression of several SULT isoforms, and thyroid hormone nuclear receptors.</p><!><p>2,4,6-TBP (99% pure), T4 (> 99%), T3 (> 99%), rT3 (> 99%), 3,3′-T2 (> 99%), and adenosine 3′-phosphate 5′-phosphosulfate (PAPS) lithium salt hydrate (> 60%) were purchased from Sigma-Aldrich (St. Louis, MO). BDE-47 (> 97%), BDE-99 (> 97%), 3-OH 2,2′,4,4′-BDE-47 (3-OH BDE-47; 99%), and 6-OH 2,2′,4,4′-BDE-47 (6-OH BDE-47; 100%) were purchased as neat standards from AccuStandard (New Haven, CT). [13C6]-3,3′-T2 was purchased from Isotec (Miamisburg, OH). 3,3′-T2 sulfate (3,3′-T2S; 98%) was custom synthesized by the Duke University Small Molecule Synthesis Facility (Durham, NC). T3 sulfate (T3S) was purchased from Toronto Research Chemicals (Toronto, ON; 98.6%).</p><p>Ham's F-12K (Kaighn's Modification) Medium cell culture media and other cell culture reagents were purchased from Life Technologies (Carlsbad, CA). All solvents and other reagents were purchased from VWR (Radnor, PA). BeWo cells (ATCC® CCL-98™) were obtained from the Duke University Cell Culture Facility (Durham, NC). All cells were grown in Tissue Culture treated flasks (75 and 182 cm2) and dishes (36.3 cm2) from Genesee Scientific (San Diego, CA).</p><!><p>BeWo cells were grown in Ham's F-12K (Kaighn's Modification) Medium cell culture media supplemented with 10% FBS, 30 nM selenium (as sodium selenite), 100 units mL−1 penicillin, and 100 μg mL−1 streptomycin at 37°C and 5% CO2. All experiments were performed with cells that were thawed from the same passage number, and all replicate experiments were performed with cells from different aliquots of the same source of cells in order to ensure and evaluate reproducibility. Cells were thawed and plated in T-75 flasks, media was changed every 48 h, and following the initial growth period cells were passaged and transferred to T-182 flasks for growth (1:3 passage ratio used; passage at 80% confluency/every 96 h).</p><!><p>Cells were passaged and transferred to cell culture dishes and seeded at a density of 1.0 × 106 cell cm−2. Cells were allowed to grow for 36 hours, after which point the growth media was replaced with the appropriate dosing media (cell culture growth media + inhibitor dissolved in DMSO). After the allotted exposure period (1, 6, 12, or 24 h), the growth media was collected and stored for TH analysis and verification of dosing concentrations. Cell culture dishes were rinsed with Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride. Cell culture dishes were then loaded with 1 mL of phosphate sucrose buffer (KPO4 buffer containing 0.25 M sucrose and 1 mM EDTA) and cells were stored at −80°C for future analysis.</p><p>The low concentration for 2,4,6-TBP, BDE-47, and BDE-99 was 0.05 μM (50 nM) to approximately reflect the 95th percentile of human serum concentrations measured in the United States15. Similarly, the low concentration selected for 3-OH BDE-47 and 6-OH BDE-47 was 0.001 μM (1 nM) in order to represent levels approximating the 95th percentile of human serum concentrations measured in the United States16. The high and medium concentrations were the same for all BFR compounds tested (1.0 μM and 0.5 μM, respectively). All dosing compounds were dissolved in DMSO at 1,000 times higher than the desired final concentration in the cell culture media to ensure a concentration of 0.1% DMSO. The concentrations of the compounds in the dosing media were verified using GC-MS and LC-MS/MS methods17,18.</p><!><p>Cytotoxicity was assessed using a resazurin cell viability assay19. Cells were exposed to dosed media (low, medium, and high concentrations) for 24 hours and then the reduction of resazurin to resorufin over 60 minutes was analyzed using a plate reader. Control cells were grown with and without DMSO as a comparison to treated cells.</p><!><p>Basal SULT activity was measured in BeWo cells after exposure (1, 6, 12, or 24 h) to low, medium, or high concentrations of BFRs, as well as in DMSO controls. The high and medium concentration used for all analytes was 1 μM and 0.5 μM. The low concentration for 2,4,6-TBP, BDE-47, and BDE-99 used was 0.05 μM, while the low concentration for 3-OH BDE-47 and 6-OH BDE-47 was 0.001 μM. Following exposure, cells were scraped from tissue culture dishes into 1 mL of KPO4 buffer containing 0.25 M sucrose and 1 mM EDTA. The 1 mL of phosphate buffer with BeWo cells was split into two aliquots: 500 μL and 250 μL aliquots. The 250 μL aliquot was frozen at −80°C for future mRNA extraction, while the 500 μL aliquot was sonicated for 10 seconds on ice before being stored at −80°C for future use in the basal SULT activity assay. The 3,3′-T2 SULT assays were performed using whole cellular homogenates. The SULT assays were performed using a previously published method from our laboratory8. After completing the SULT assay, the samples were spiked with 5 ng of stable isotope-labelled surrogate standard ([13C6]-3,3′-T2). The reaction mixtures were extracted using Agilent OPT Solid Phase Extraction tubes and analyzed using LC-MS/MS following previously published analytical methods8. The total mass of 3,3′-T2-sulfate (T2S) formed in each sample was normalized to the value of DMSO controls within each experiment, and all values are presented as a 3,3′-T2 SULT activity percent relative to control.</p><!><p>The concentrations of T4, T3, rT3, and 3,3′-T2 were measured in aliquots of cell culture media from each exposure dish to determine if there were any significant changes in TH levels. In addition, aliquots of the cell culture media were analyzed for the formation of sulfated metabolites of the BFRs. More detailed information on the extraction and analysis can be found in Supporting Information.</p><!><p>BeWo cells were grown under normal conditions and harvested after reaching >80% confluency. To harvest, cells culture flasks were rinsed twice with 3 mL of Dulbecco's phosphate buffered saline (no calcium chloride, no magnesium chloride). Following the saline rinse, 2 mL of 0.25% trypsin-EDTA (Gibco Life Technologies) was added to each flask and mixed to cover the entire flask bottom surface area. The flasks were then incubated at 37°C for five minutes to allow for adequate cell dissociation. Following trypsinization, flasks were rinsed with 10 mL of growth media and cells were transferred to a 15 mL centrifuge tube. Cells were pelleted at 200 g for three minutes, after which the remaining trypsin-media supernatant was poured off. Cells were then resuspended in 6 mL of cold 0.1 M phosphate sucrose buffer. At this time, the suspended cells were separated into 1 mL aliquots and sonicated (7 seconds sonication, 3 second pause, 7 second sonication) on ice. The sonicated BeWo cell homogenates were then recombined, aliquoted, and stored at −80°C for future use in the SULT assays.</p><p>Enzyme inhibition assays were performed using our previous methods8. Assays were performed using 0.1 M potassium phosphate buffer (pH 7.2) with 50 μM PAPS as the cofactor and 1 μM 3,3′-T2 as the substrate (total volume of 200 μL). Stock solutions of competitors were prepared in DMSO at concentrations 1000 times higher than the desired dosing concentrations in order to ensure a DMSO concentration of less than 0.1% of the incubation volume. For these experiments, the following BFR analytes were tested for SULT enzyme inhibition: 2,4,6-TBP, BDE-47, BDE-99, 3-OH BDE-47, and 6-OH BDE-47. A wide range of inhibitor concentrations were tested in order to fully examine inhibition potential. Briefly, 2,4,6-TBP was tested at 15 concentrations (range: 0.0001 μM – 100 μM), BDE-47 and BDE-99 were tested at ten different concentrations (range: 0.01 μM – 100 μM), 3-OH BDE-47 was tested at nine different concentrations (range: 0.001 μM – 2.0 μM), and 6-OH BDE-47 was tested at six different concentrations (range: 0.01 μM – 1.0 μM). All reactions were spiked with 1 μL of the corresponding inhibitor dosing stock (1 μL of DMSO spiked into control samples). All inhibitor concentration levels were performed in duplicate sets, while each set of DMSO control samples was performed in sets of four. Assay reactions were initiated with the addition of the BeWo cell homogenate (approximately 800 μg of protein/mL) and vials were incubated at 37°C for 30 minutes in a shaking water bath. Following incubation, the reactions were stopped with the addition of 800 μL of 0.1 M HCl and samples were then spiked with [13C6]-3,3′-T2 (5 ng) as the internal standard. Sample extracts were then cleaned using SampliQ OPT SPE cartridges (Agilent Technologies) as described above. The total 3,3′-T2-sulfate conjugate product formation (ng) was quantified for each vial. The mean value for the DMSO control samples (n = 4) was calculated for each experimental batch, and all inhibitor concentration levels were normalized to the control values to yield a final value of 3,3′-T2-sulfate formation as a percent value relative to control for each vial. All experiments were repeated at least two times total using different batches of cells in order to evaluate reproducibility between different cell populations and day-to-day experimental variability. No significant differences were observed among replicates, and the replicate experiments were combined to produce a single inhibition curve, where each data point represents the mean value of percent inhibition (n = 4; error bars represent SEM). The inhibition curves and half maximal inhibitory concentrations (IC50) for each analyte were calculated using four-parameter logistic model in Sigma Plot 12.0.</p><p>Michaelis-Menten kinetics of 3,3′-T2 sulfation were also investigated in BeWo cell homogenates. The protein concentration (mg/mL) of the BeWo cell homogenate used in each experiment was determined using the Bradford assay. Data were calculated and expressed as pmol of 3,3′-T2-sulfate formed per milligram of protein per minute (pmol T2S/mg protein/min). 3,3′-T2 sulfation showed typical Michaelis-Menten enzyme kinetics and the kinetics curves and parameters (Km and Vmax) were calculated using Sigma Plot 12 using the following equation: v = Vmax [S]/Km + [S]; where v is the reaction rate, Vmax is the maximal reaction rate, S is the substrate concentration, and Km is the substrate concentration at 50% of the maximal reaction rate.</p><!><p>Total RNA was extracted from a 250 μL aliquot of the scraped cells (25% of the total cells grown in the dish) using the Quick-RNA MicroPrep kit from Zymo Research (Irvine, CA) and RNA content was quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific). Total RNA was converted to cDNA using the High Capacity cDNA reverse transcription kit from Life Technologies (Carlsbad, CA) according to the manufacturer's methods. Approximately 10 ng of cDNA was analyzed in 20 μL qPCR reactions using TaqMan Gene Expression Assays (Life Technologies) and an Applied Biosystems 7300 Real-Time PCR system (Foster City, CA). The genes investigated in this study included sulfotransferases and several genes regulated by thyroid hormones: DIO3 (Hs00956431_s1), TR-α (Hs00268470_m1), TR-β (Hs00230861_m1), SULT1A1 (custom assay), SULT1C2 (Hs00602560_m1), SHBG (Hs01050182_g1), RPL13A (Hs04194366_g1), and β-actin (Hs01060665_g1). The threshold cycles (Ct) of RPL13A and β-actin were compared in an initial experiment to determine the best reference gene, and β-actin was selected as the internal reference gene due to its stable expression between control cells and different treatment groups. Expression values are reported as the expression ratio relative to control samples within each experiment normalized to β-actin using the 2−ΔΔCt method.</p><!><p>ANOVA tests were performed using Sigma Plot 12 to test for significant effects of treatment and experimental day. Significant effects were further tested using Tukey's post-hoc test. All experiments were performed with 2 – 4 samples and repeated on a separate day. When there was no significant effect of the experimental day, the results from the individual experiments were combined for a total of 4 – 8 samples per treatment group. The results for each experiment were first normalized to the control values within that experiment and then combined with the results from the corresponding repeated experiment.</p><p>The mRNA expression results were analyzed using ΔΔCt values and reported graphically as the 2−ΔΔCt expression ratios.</p><!><p>Initial experiments were first conducted using BeWo cell homogenates to examine inhibition of SULT activity by the brominated compounds. Inhibition of T2 SULT activity was assessed through co-incubation with BDE-47, BDE-99, 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. BDE-47 and BDE-99 displayed no inhibition of SULT activity at concentrations up to 100 uM; however, the remaining analytes did inhibit activity (Figure 1). 2,4,6-TBP was the most potent inhibitor tested. The calculated IC50 values for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47 are 11.6 nM (95% CI: 9.22 – 14.0), 48.9 nM (95% CI: 46.6 – 51.2) and 395.5 nM (95% CI: 391.6 – 399.4), respectively.</p><!><p>As 2,4,6-TBP and 3-OH-BDE-47 were found to be the most potent inhibitors of 3,3′-T2 SULT activity, further experiments were conducted to examine the specific Michaelis-Menten kinetics and attempt to determine the type of enzyme competition (i.e. direct or indirect competition). Interestingly, inhibition was observed at the highest substrate concentration tested (5 μM 3,3′-T2). This type of substrate inhibition is characteristic of SULT enzyme kinetics20. The apparent Km for the 3,3′-T2 sulfation reaction in the control BeWo cell homogenates was 0.61 ± 0.10 μM and the Vmax was 4.52 ± 0.27 pmol T2S/mg protein/min. The apparent Km for the 3,3′-T2 sulfation reaction in the 2,4,6-TBP exposed cells was 1.46 ± 0.20 μM and the Vmax was 2.29 ± 0.15 pmol T2S/mg protein/min. The apparent Km for the 3,3′-T2 sulfation reaction in the 3-OH BDE-47 exposed cells was 1.29 ± 0.16μM and the Vmax was 3.42 ± 0.20 pmol T2S/mg protein/min. The type of inhibition (competitive vs noncompetitive) was investigated by comparing these calculated Michaelis-Menten parameters. Both the Vmax and Km for 2,4,6-TBP and 3-OH BDE-47 showed a statistically significant difference from the control parameters. The Vmax for both compounds was significantly lower than the controls, while the Km for both compounds was significantly higher (Figure S1). This observed decrease in Vmax and increase in Km is indicative of mixed type inhibition in the BeWo cell homogenates for 2,4,6-TBP and 3-OH BDE-47.</p><!><p>Cells were exposed for 24 hours to media containing 2,4,6-TBP, BDE-47, BDE-99, 3-OH BDE-47, and 6-OH BDE-47. Cytotoxicity was assessed for all dosing concentrations of BFRs tested and no significant cytoxicity was observed (data not shown). Basal SULT activity was measured after 24 hours and data are presented as the percent of 3,3′-T2-sulfate formation relative to control (no BFRs). Results from the 24 h cell culture exposure experiments are summarized in Figure 2A. There was a statistically significant reduction in basal 3,3′-T2 SULT activity for all BFR analytes in the high and medium concentrations. Additionally, there was a statistically significant reduction in basal 3,3′-T2 SULT activity for all concentrations of 2,4,6-TBP, ranging from 17.0 ± 0.93% reduction in the low concentration to a 86.0 ± 0.28% reduction at the highest concentration tested. Treatment of the BeWo cells with BDE-99 significantly decreased the 3,3′-T2 SULT activity by 26.4 ± 3.7% to 38.4 ± 2.4%, in the medium and high concentrations, respectively. And exposure to 6-OH BDE-47 and 3-OH-BDE-47 significantly decreased the 3,3′-T2 SULT activity by 23.0 ± 3.5% to 41.5 ± 3.3%, and 26.0 ± 4.9% to 35.8 ± 7.5%, respectively, in the medium and high concentration exposures. There was no effect on basal 3,3′-T2 SULT activity in BeWo cells following exposure to BDE-47 for 24 hours.</p><p>Similar experiments were also conducted with a 12 h exposure duration. Following the 12 hour exposure, a statistically significant reduction in basal 3,3′-T2 SULT activity was observed only in the high concentrations for 3-OH BDE-47 (36.4 ± 2.6% decrease) and in the medium and high concentration for 2,4,6-TBP (58.3 ± 4.4% to 75.0 ± 1.5% decrease). There was no observed change in basal 3,3′-T2 SULT activity following 12 hour exposures to BDE-99 or 6-OH BDE-47 at any concentration tested. Results from the 12 h cell culture exposure experiments are summarized in Figure 2B.</p><p>Given the observed inhibition from exposure to 2,4,6-TBP at 12 hours, additional experiments were performed for exposure durations of 1 and 6 hours (Figure 3). Treatment of the BeWo cells with 2,4,6-TBP for 6 hours significantly decreased the 3,3′-T2 SULT activity measured in the cell homogenate by 34.4 ± 4.2% and 56.8 ± 3.1%, in the medium and high concentrations, respectively. Treatment of the BeWo cells with 2,4,6-TBP for 1 hour significantly decreased the 3,3′-T2 SULT activity measured in the cell homogenate by 50.5 ± 7.8% and 62.7 ± 5.0% in the medium and high concentrations, respectively. There was no observed effect on basal 3,3′-T2 SULT activity in BeWo cells at the low concentration for 1 hour or 6 hour exposure durations.</p><!><p>THs (T4, T3, rT3, and 3,3′-T2) were measured in BeWo cell growth media following 24 hr exposure to the various BFR chemicals. There were no observed effects of treatment on T4, T3, rT3, or 3,3′-T2 (data not shown).</p><!><p>Given that several of the BFRs contain a phenolic moiety, it was possible that they were acting as substrates for the SULTs and forming sulfated metabolites. To explore this possibility, the growth media was evaluated for potential sulfate metabolites of the BFRs using LC/MS-MS. Suggestive peaks were observed in the cell culture growth media isolated from 3-OH BDE-47 exposures, which were absent in all the control samples (Figure 4). The MRM transitions 580.8 to 81 and 580.8 to 501.8 ([M]− to [Br]−, and [M]− to [M-Br]−) were monitored, the same fragmentation patterns used to quantify 3,3′-T2S. Due to the lack of an authentic, pure standard, quantification of this metabolite could not be performed, and matrix effects were too great to collect a full mass spectrum. No suggestive peaks were observed in the growth media from the 2,4,6-TBP or 6-OH BDE-47 exposed cells.</p><!><p>Gene expression of SULT1A1 and several TH regulating genes was measured in cells exposed to all analytes in the 24-hour experiments. Additional expression analyses were also conducted in cells exposed to 2,4,6-TBP for 1 and 12 hours. Sex-hormone binding globulin (SHBG) and SULT1C2 were poorly expressed in preliminary evaluations of BeWo cells (Ct > 38) and were excluded from future analyses. DIO3, SULT1A1, TR-α, and TR-β were measured in all samples. However, DIO3 expression was highly stochastic and variable, even within the control treatment groups, and was excluded from statistical analysis. SULT1A1, TR-α, and TR-β expression was measured in all BeWo cell experimental groups; however, no significant changes in mRNA expression were observed for any BFR treatment groups (data not shown).</p><!><p>This is the first study to evaluate the effects of BFR exposure on TH SULT activity in the BeWo cell line. SULT enzyme inhibition was observed for 2,4,6-TBP and both OH-BDEs in cell homogenates co-incubated with the compounds, but no inhibition was observed with BDE-47 or BDE-99. 2,4,6-TBP was the most potent inhibitor of 3,3′-T2 SULT activity with an IC50 value of 11.6 nM, followed by 3-OH BDE-47 with an IC50 value of 48.9 nM, and the least potent inhibitor was 6-OH BDE-47 with an IC50 value of 395.5 nM. The PBDE congeners BDE-47 and BDE-99 did not exhibit any SULT inhibition in the BeWo cell homogenates. These results are consistent with a previous in vitro evaluation of BFR inhibition on TH SULT activity using pooled human liver cytosol8. Both studies found no effect on SULT activity from exposure to BDE-47 and BDE-99, and both studies found 2,4,6-TBP to be the most potent inhibitor of TH SULT activity with IC50 values in the low nM range. Additionally, both studies show 3-OH BDE-47 to be a more potent inhibitor of TH SULT activity than 6-OH BDE-47. However, these two studies used different tissues sources and types for the SULT enzymes (liver versus placenta; cytosol versus whole-cell homogenate) and different tissue types will likely have different SULT isoform profiles and expression levels that contribute to the observed differences in enzyme inhibition (ie. the observed differences in calculated IC50 values for 6-OH BDE-47 between hepatic and placental tissues).</p><p>The presence of the hydroxyl group (-OH) on the molecule appears to be necessary for 3,3′-T2 SULT inhibition. These results are supported by previous research that showed that only OH-PCBs inhibited TH SULT activity in rat liver cytosolic fractions (low μM range), while the parent PCBs (non-hydroxylated) did not21. Additionally, it has been shown that 3-OH BDE-47 and tetrabromobisphenol A (TBBPA) can bind to the substrate binding pocket and inhibit the activity of human estrogen sulfotransferase (SULT1E1) 22.</p><p>It has been shown that some chemicals are able to bind SULT enzymes so well that they act as substrates and become sulfated, implicating the role of direct competitive inhibition26. For example, urinary concentrations of sulfated conjugates of TBBPA have been measured in humans and rats following oral administration27,28. Such results are supported in this present study, as the detection of a chemical that is likely a sulfated metabolite of 3-OH BDE-47 was observed in cell culture dosing media following exposure. Interestingly, no mass spectrometry peaks were detected in the media to suggest sulfation of 6-OH BDE-47 and 2,4,6-TBP had occurred. The presence of sulfated conjugates in the cell culture growth media following exposure indicate that 3-OH BDE-47 is capable of binding SULT enzyme binding pockets and favorably interacting with the active site in such a way as to allow for sulfation of the hydroxyl moiety.</p><p>It was interesting to note that following a 24-hour exposure to each compound, basal 3,3′-T2 SULT activity in the cell cultures was significantly reduced for all BFR analytes, except BDE-47. BDE-99 showed significant effects at the high and medium concentrations following the 24-hour exposure; however, BDE-99 showed no inhibitory effect in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a mechanism of action other than direct enzyme inhibition. One possibility is that BDE-99 was metabolized (e.g. forming OH-BDEs), which then mediated the observed reduction in basal SULT activity. To investigate this, the growth media was extracted and analyzed for OH-BDEs following the 24-hour exposure; however, there were no OH-BDEs detected in the BDE-99 dosing media, indicating that oxidative metabolism and formation of OH-BDEs was likely not occurring in the BeWo cells. This suggests that the observed effects on basal 3,3′-T2 SULT activity were not mediated by OH-BDEs, and that BDE-99 may elicit its action via indirect mechanisms, such as effects on SULT protein post-translational processes.</p><p>It was also interesting to note the differences in inhibition following various exposure durations. For example, basal 3,3′-T2 SULT activity was significantly reduced after 12 hours of exposure to 3-OH BDE-47, yet there was no observed effect on basal 3,3′-T2 SULT activity following 12 hour exposures to BDE-99 or 6-OH BDE-47. The reasons for these differences are unclear, but it may be that the cellular machinery (ex. transport proteins, metabolizing enzymes, etc.) require more time to transport these BFR compounds into cells. One factor that might contribute to these differences in binding and transport are the physicochemical properties of the compounds. For example, 2,4,6-TBP is significantly smaller and less lipophilic than OH-BDE compounds and may have a greater capacity for transport into and within cells, contributing to its higher potency. Another possible explanation may be that these compounds impact SULT enzymes by upregulating proteasomal degradation pathways and lowering the cellular half-lives of SULT enzymes. As a result, the pool of SULT enzymes present in the BeWo cells may not be sufficiently degraded after a 12-hour exposure period, and require a 24-hour exposure period in order to elicit an effect on basal SULT activity. The ability of 2,4,6-TBP to disrupt basal SULT activity at all exposure durations ranging from 1 hour to 24 hours indicates a rapid mechanism of action. The mechanisms by which 2,4,6-TBP, as well as the other BFRs tested in the study, impact SULT activity in BeWo cells following exposure remain to be fully determined. One possibility that we were unable to explore in this study is that the activity and expression of sulfatases could have been increased in these exposures. Sulfatases hydrolyze sulfate esters, and can reverse the sulfation of thyroid hormones. To our knowledge, no one has investigated effects of BFRs on sulfatase activity.</p><p>We originally hypothesized that effects on basal 3,3′-T2 SULT activity would lead to changes in TH levels in the media. For example, samples with significant reductions in basal 3,3′-T2 SULT activity would be expected to have higher media concentrations of T3 as a result of decreased metabolism of T3 in the cells. However, there was no significant effect of exposure on media TH concentrations observed by treatment. These results may be reflective of the maintenance of TH levels in BeWo cells during BFR exposure due to compensatory and self-regulating action of TH transport proteins, TH binding proteins, sulfatases, and/or TH metabolizing enzymes such as DI or uridine diphosphate glucuronosyltransferase (UDPGT). So while BFR exposure may have impacted TH concentrations through SULT inhibition in cells, there were sufficient regulatory mechanisms that maintained TH concentrations within a narrow concentration window, and detection of changes in TH concentrations in the growth media were effectively masked. However, it is important to note that SULTs metabolize other endogenous chemicals such as estrogen, fatty acids, and other lipophilic environmental contaminants. Therefore, the effects of SULT enzyme inhibition may have biological consequences for other signaling pathways outside of TH regulation.</p><p>The mRNA expression of TH responsive and regulating genes were also measured in the cell culture BFR exposure experiments. There was no observed effect of treatment for any of the genes investigated. However, DIO3, exhibited highly variable and stochastic gene expression at all treatment levels, including the DMSO control groups. This is in contrast to the consistent results observed for TR-α, TR-β, and SULT1A1, which show non-significant deviation from the control group. It is unclear why the DIO3 mRNA expression was so variable in the BeWo cells, however, this variability excluded DIO3 from additional analysis. There was no observed effect of treatment on the mRNA expression profiles of TR-α, TR-β, and SULT1A1. These results are interesting because there was a significant decrease in basal 3,3′-T2 SULT activity in BeWo cells following these BFR exposures.</p><p>Inhibition of SULT enzymes in the human placenta has the potential to elicit downstream biological effects. Not only is sulfation an important Phase II metabolic pathway in adults, but sulfation is the primary metabolic pathway within the placenta and developing fetus29. As a result, disruption of SULT activity within the placenta is likely to impact a wide range of metabolic pathways outside of TH homeostasis. For example, reductions in basal SULT activity may lead to lower rates of sulfation of environmental contaminants, effectively increasing tissue concentrations of these chemicals due to lack of clearance and further perpetuating SULT enzyme inhibition within the placenta. Another potential outcome for SULT inhibition in the placenta, is the disruption of other sulfation pathways for endogenous compounds. Examples of other endogenous substrates that utilize sulfation include estradiol, testosterone, dopamine, cholesterol, and cortisol30. Notably, estradiol signaling is very important in mediating gestational development within the placenta during pregnancy, as well as in modulating cellular communication between the fetus, placenta, and mother31,32. Disruption of estradiol sulfation may lead to a perturbation of estradiol homeostasis, but further research is needed to verify this hypothesis.</p><p>This study demonstrated that BFR compounds can disrupt TH SULT activity within placental cells, and adds to our growing understanding of the effects of BFR exposure on TH metabolism within the placenta. Future work should aim to use purified and individual SULT isoforms in order to determine the exact mechanistic and kinetic characteristics of BFR action in BeWo cells, and perhaps consider effects on sulfatase activity. In addition, the PAPS co-factor used in this experiment was not entirely pure. Concerns have been raised that some PAPS may contain the de-sulfated isoform (PAP), which could potentially inhibit SULT activity. However, the amount of PAPS used in each sample in this experiment was the same, thus any inhibitory effect would have been equal in all samples. Despite this we still observed significant inhibition by several BFRs and their metabolites, and suggests that if PAP was present, the observed activity is a conservative estimate. Overall, the ability of BFRs, especially the hydroxylated compounds, to disrupt TH SULT activity in BeWo cells warrants further investigation into the mechanisms of action underlying these effects, as well as the biologically relevant downstream effects of exposure.</p>