Activation of Peroxisome Proliferator-Activated Receptor Gammaand Disruption of Progesterone Synthesis of 2‑Ethylhexyl DiphenylPhosphate in Human Placental Choriocarcinoma Cells: Comparisonwith Triphenyl PhosphateWenxin Hu,† Fumei Gao,† Hong Zhang,† Youhei Hiromori,‡,§ Shuhei Arakawa,‡ Hisamitsu Nagase,‡

Tsuyoshi Nakanishi,‡ and Jianying Hu*,†

†MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871,China‡Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Gifu501-1196, Japan§Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science 3500-3, Minamitamagaki, Suzuka, Mie 513-8670, Japan

*S Supporting Information

ABSTRACT: 2-Ethylhexyl diphenyl phosphate (EHDPP), anorganophosphate flame retardant (OPFR), is frequentlydetected in human blood. In this study, the sensitive dual-luciferase reporter gene assay and molecular docking were usedto investigate the activation of EHDPP to human peroxisomeproliferator-activated receptor gamma (PPARG). Results showthat EHDPP exhibited stronger PPARG activation (EC20: 2.04μM) than triphenyl phosphate (TPhP) (EC20: 2.78 μM).EHDPP upregulated the gene expression of 3β-hydroxysteroiddehydrogenase type 1 (3β-HSD1) in human placentalchoriocarcinoma cells in a dose-dependent manner, and thelowest observable effective concentration was 10 μM, lower thanthat of TPhP (20 μM). EHDPP significantly alteredprogesterone secretion at a lower concentration (10 μM) thanthat of TPhP (20 μM), and both EHDPP and TPhP significantly promoted human chorionic gonadotropin (hCG) production at20 μM. Furthermore, inactivation of PPARG by either a pharmacological inhibitor (GW9662) or small interfering RNA (siRNA)abolished the change in progesterone secretion and gene expression in the cells exposed to EHDPP, suggesting that the PPARGsignaling pathway plays a role in the upregulation of progesterone by the two OPFRs. This is the first report to show that OPFRscan alter the biosynthesis of progesterone in the placenta, which could affect female reproduction and fetal development.

■ INTRODUCTION

There is growing concern about the possible health threatposed by anthropogenic compounds. Increasing evidence hasshown that substances found in the environment, food, andconsumer products can interfere with hormone biosynthesis,metabolism, and activity, resulting in deviation from normalhomeostatic control or reproduction.1 The wide variety ofpollutants reported to disrupt endocrine function includespesticides, polycyclic aromatic hydrocarbons, phthalate plasti-cizers, polychlorinated biphenyls, dioxins, furans, alkylphenols,and synthetic steroids.2

Of all environmental chemicals showing reproductivetoxicity, organophosphate flame retardants (OPFRs) are ofparticular concern. OPFRs are used as replacements forbrominated flame retardants (BFRs) such as polybrominateddiphenyl ethers (PBDEs), thus their use has increasedsignificantly in recent years, with annual global production

currently reaching approximately 2,000,000 tons.3 OPFRs arewidely used as plasticizers in various consumer products,building materials, and baby products4−6 and are thereforeubiquitous in various environmental media such as house dust,sediment, sludge, river water, biota, and even in human bloodand placenta.7−13 There is increasing evidence that OPFRsexposure can disrupt hormonal metabolism and biosynthesis inanimals and humans. Tris(1,3-dichloro-2-propyl) phosphate(TDCIPP) and triphenyl phosphate (TPhP) have been shownto reduce fecundity by significantly increasing plasma estradiollevels and inhibiting androgen levels in zebrafish.14 A similarphenomenon has also been observed in the stably transfectedhuman breast cancer cell line MVLN and the human

Received: February 16, 2017Accepted: March 10, 2017Published: March 10, 2017

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adrenocortical carcinoma cell line (H295R).15 One recentepidemiological study showed that OPFRs in house dust wereassociated with serum free thyroxin (T4), prolactin, anddecreased semen quality in men.16 In addition to alteration ofhormonal metabolism and biosynthesis, OPFRs can alsointeract with various nuclear receptors (NRs) such asconstitutive androstane receptor (CAR), pregnane X receptor(PXR), estrogen receptor (ER), androgen receptor (AR),glucocorticoid receptor (GR), and peroxisome proliferator-activated receptor gamma (PPARG).17,18 Of these NRs,PPARG is abundantly expressed in human placenta and servesas an essential regulator of endocrine functions that play a vitalrole in maintaining pregnancy.19−22 Thus, it would beinteresting to determine whether OPFRs can disrupt endocrinefunctions in human placenta. Among various OPFRs, onlyTPhP and tributyl phosphate (TBP) have been identified asagonists of PPARG,17 and for a broad range of OPFRs withdiverse structures the activity to PPARG has not beenevaluated. Particularly, 2-ethylhexyl diphenyl phosphate(EHDPP), tris(2-chloroethyl) phosphate (TCEP), and tris-2-chloroisopropyl phosphate (TCPP) have been detected in theblood of the Chinese population, with median concentrationsof EHDPP (1.22 ng/mL) and TCPP (0.71 ng/mL) found to behigher than that of TPhP (0.43 ng/mL).13 To date, however,their activation to PPARG remains to be elucidated.In this study, TCEP, TCPP, and EHDPP PPARG activation

was determined using the dual-luciferase reporter assay. TheTBP and TPhP PPARG activation was also determined toenable a direct comparison of the unknown PPARG activationby TCEP, TCPP, and EHDPP based on the same assay. Wealso investigated their effects on the synthesis of progesteroneand human chorionic gonadotropin (hCG) in the humanplacental choriocarcinoma cell line JEG-3, which retainscharacteristics of normal pregnancy trophoblast cells. Finally,the potential role in promotion of progesterone production wasalso investigated by coexposure with a pharmacologicalinhibitor of PPARG (GW9662) and small interfering RNA(siRNA) targeting PPARG in JEG-3 cells.

■ MATERIALS AND METHODSChemicals and Reagents. The standards of TCEP, TCPP,

TBP, EHDPP, and TPhP were purchased from TokyoChemical Industry Co (Tokyo, Japan). Rosiglitazone,GW9662, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), and dimethyl sulfoxide (DMSO) werepurchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzolwas obtained from Invitrogen (Carlsbad, CA, USA).Cell Cultures and Treatments. The human placental

choriocarcinoma cell line JEG-3 was cultured in DMEM-F12medium (Hyclone, Logan, UT, USA) supplemented with 10%fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) andincubated at 37 °C in a humidified, 5% CO2 atmosphere. TheJEG-3 cells were plated in 24-well plates in DMEM-F12medium and allowed to become confluent (2−3 d). Todetermine the effects of EHDPP, TPhP, TCEP, TBP, andTCPP on synthesis-related gene expression and production ofprogesterone and hCG, the JEG-3 cells were seeded,precultured for 24 h, and then treated with variousconcentrations of EHDPP, TPhP, TCEP, TBP, or TCPP inDMSO or with vehicle-control (0.1% DMSO).Reporter Assay. The 293T cells were seeded in 24-well

plates in 500 μL of DMEM-F12 containing 10% FBS. Afterincubation for 24 h, the cells showed 70−80% confluence and

were then transiently transfected with pBIND-PPARG-LBD,GAL4-pGL4-luc (provided by Shuyi Si, Institute of MedicinalBiotechnology, Chinese Academy of Medical Sciences andPeking Union Medical College), and pGL4.74 (Promega,Madison, WI, USA) or GAL4-RXR, pG5-luc, and pGL4.74using Lipofectamine 2000 Transfection Reagent (Invitrogen,Carlsbad, CA, USA) according to the manufacturer’sinstructions.23 Following 24 h of incubation, the medium wasreplaced, and the cultures were treated with DMSO (0.1%),EHDPP, TPhP, TCEP, TBP, or TCPP (0.05−25 μM) for 24 h.The extracts were prepared and assayed for firefly luciferase(LUC) activity using the Dual-Luciferase Reporter AssaySystem (Promega, Madison, WI, USA) and a LB 941 TriStarMultimode Microplate Reader (Berthold Technologies, BadWildbad, Germany) according to the manufacturer’s instruc-tions. In brief, the cells were collected with phosphate bufferedsaline (PBS), and then passive lysis buffer was added. Thesupernatant was collected to test the firefly LUC and RenillaLUC activities. Results were expressed as the average relativefirefly LUC activity of at least triplicated samples.

Molecular Docking. Molecular simulation software Sci-gress (Ultra Version 3.0.0, Fujitsu, USA) was used to dockflexible ligands into a rigid protein active site. In brief, thePPARG ligand binding domain (PDB ID code 2PRG) wasobtained from the Protein Data Bank (PDB, http://www.rcsb.org/). Explicit hydrogen atoms were created, and heteroatomssuch as water, ions, and cofactors were removed. Dockingcalculations were evaluated using a genetic algorithm with a 15× 15 × 15 Å grid box with 0.3 Å grid spacing, which containedthe active site for the original ligand. The procedure was set torun for 20000 generations with an initial population size of 100,elitism of 8, crossover rate of 0.8, mutation rate of 0.5, andconvergence of 1.0. Other parameters were set at their defaultvalues. The Potential of Mean Force (PMF) scores, aknowledge-based approach that extracts pairwise atomicpotentials from the structure information on known protein−ligand complexes contained in the Protein Data Bank, wasdetermined and used to score the binding activity of a chemicalto the active site of human PPARG. The PMF of rosiglitazone,the native ligand in the complex, was −91.15 kcal/mol whendocked into the binding site. The root-mean-square errorbetween the predicted conformation and the actual con-formation from the crystal structure was 1.78 Å, smaller thanthe X-ray crystallography resolution (2.20 Å).24 Thus, theparameter set used for the docking simulation was consideredto be reasonable.

Plasmid Construction. The pBIND-PPARG-LBD mutantconstruct, carrying a Ser342 to Ala mutation, was generated bysite-directed mutagenesis of pBIND-PPARG-LBD. The se-quences of the mutagenic primers are 5′-GGTTCTCATAGC-AGAGGGCCAAGGCTTCATGAC-3′ and 5′- CTTGGC-CCTCTGCTATGAGAACCCCATCTTTAT-3′.

Cell Viability Assays. Confluent JEG-3 cultures weretreated with vehicle-control (DMSO, 0.1%), EHDPP, TPhP,TCEP, TBP, or TCPP (5−40 μM) for 48 h. Cytotoxicity wasmeasured using the MTT assay. Briefly, JEG-3 cells wereseeded in 96-cell plates for 24 h and then treated with differentconcentrations of OPFRs for 48 h. At the end of treatment, 20μL of MTT was added, and the solution was incubated for anadditional 4 h. The formazan crystals were then dissolved in200 μL of DMSO. After shaking the plate for 10 min, cellviability was assessed by measuring the absorbance at 490 nmusing a spectrophotometer (Bio-Rad Model 550; Bio-Rad

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Laboratories, Inc., Hercules, CA, USA). Cell viability was alsoassessed by CellTiter-Glo Reagent (Promega, Madison, WI,USA) according to the manufacturer’s instructions. In brief,cells were seeded, precultured for 24 h, and then treated withvarious concentrations of EHDPP, TPhP, TCEP, TBP, orTCPP in 0.1% DMSO or with vehicle-control (0.1% DMSO)for 48 h. After 100 μL of CellTiter-Glo Reagent was added toeach well, the plate and its contents were equilibrated at roomtemperature for approximately 30 min. The luminescence wasdetermined using the LB 941 TriStar Multimode MicroplateReader. Absorbance or luminescence in the experimental wellswere normalized by dividing the absorbance or luminescence inuntreated cultures and reported as “Fold Change fromMedium”. Results were expressed as the average relative fireflyLUC activity of at least triplicated samples.Determination of hCG and Progesterone Production.

The JEG-3 cells were plated in 24-well plates. After 24 h ofculture, the JEG-3 cells were treated with various concen-trations of EHDPP, TPhP, TCEP, TBP, or TCPP for a further48 h. After incubation, the culture supernatants were collected,and the concentrations of hCG and progesterone weredetermined by radioimmunoassay (RIA) with a ProgesteroneDirect RIA Kit and hCG Direct RIA Kit (ICN Biomedicals,Irvin, CA, USA) according to the manufacturer’s protocols.Quantitative RT-PCR. Total RNA was extracted using

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according tothe manufacturer’s protocols and digested by DNaseI (TaKaRaBiotechnology, Dalian, China) in case of genomic DNAcontamination. The concentration and quality of RNA wereanalyzed using a Nanovue Plus spectrophotometer (GEHealthcare Life Science, Little Chalfont, Buckinghamshire,England). The purity of each sample was between 1.8 and 2.0(A260/A280 nm ratio). Total RNA was reverse transcribedwith MMLV reverse transcriptase in the presence of oligo (dT)and dNTP (TaKaRa Biotechnology, Dalian, China). Thecomplete reaction mixture was incubated at 37 °C for 50min, followed by 95 °C for 5 min to stop the reaction. SYBRGreen PCR Kits (TOYOBO, Osaka, Japan) were used for Q-PCR analysis. A real-time PCR profile was used: first, enzymeactivation at 95 °C (10 min), 40 cycles at 95 °C (30 s percycle), and 60 s at 58−62 °C, depending on the targettranscript, followed by postrun melt curve analysis (65 °C).Real-time fluorescence detection was carried out using theSTEP ONE PLUS sequence detection system (AppliedBiosystems, Foster City, CA. USA). Relative gene expressionwas evaluated by the 2-ΔΔCt method, as suggested by AppliedBiosystems. The primers sequences used are shown in TableS1.RNA Interference Assay. Sequence-specific small interfer-

ing RNA (siRNA) targeting PPARG (siPPARG) shown inTable S2 was purchased from Dharmacon/GE Healthcare(Little Chalfont, Buckinghamshire, England). AllStars NegativeControl siRNA purchased from Qiagen (Valencia, CA) wasused for the negative control siRNA. JEG-3 cells (3 × 105 cells/well) were seeded in 6-well plates and precultured at 37 °C for24 h. The cells then were transfected with siRNA duplexes (20nM/well) by using Lipofectamine RNAiMAX (Invitrogen/Thermo Fisher Scientific, Grand Island, NY) in accordancewith the manufacturer’s instructions.Statistical Analysis. Results were presented as means ±

standard errors of the mean (SEM) and tested for statisticalsignificance by analysis of variance (ANOVA) followed by posthoc Dunnett’s test using SPSS 16.0 (SPSS Inc., Chicago, IL,

USA). The number of replicates is indicated in each figurelegend. Dose response curves were performed with Prism 5(Graphpad Inc., La Jolla, CA). A p-value <0.05 was consideredstatistically significant.

■ RESULTS AND DISCUSSIONHuman PPARG Activation. In this study, we determined

the activation of TCEP, TCPP, and EHDPP to PPARG using

the sensitive dual-luciferase reporter assay, as shown in FigureS1 (half effective concentration of rosiglitazone is 0.01 μM).Although TPhP and TBP PPARG activation has been reportedpreviously,25 we show their PPARG activation in Figure 1. The20% effective concentrations (EC20) were 2.78 μM and 5.96μM for TPhP and TBP, respectively, slightly lower than thosefor TPhP (3.27 μM) and TBP (13.35 μM) reportedpreviously.25 Among the three OPFRs newly determined inthis study, TCEP and TCPP showed no significant PPARGactivation under the testing concentrations, whereas EHDPPshowed significant PPARG activation compared with thevehicle-control, with a EC20 value of 2.46 μM, showingstronger PPARG activation than that of TPhP. The EC20value of EHDPP was similar to that of mono(2-ethylhexyl)phthalate (2.12 μM) and 3-hydroxide-2,2′,4,4′-tetrabromodi-phendyl ether (3-OH-BDE47) (2.99 μM) but higher than thatof tetrabromobisphenol A (TBBPA) (0.41 μM) and 3,5,3′,5′-tetrachlorobisphenol A (TCBPA) (0.47 μM).25 Although usedas a replacement for PBDE, it should be noted that theactivation of EHDPP to PPARG was stronger than that ofBDE47 (7.00 μM).25

Molecular docking was performed to understand thestructural basis for the observed activation of OPFRs toPPARG using Scigress. The PMF values of EHDPP, TPhP, andTBP were calculated to be −54.134, −44.288, and −32.534kcal/mol, respectively, consistent with their PPARG activation.As shown in Figure 2, hydrophobicity of TBP, EHDPP, andTPhP plays an important role in human PPARG activation, as

Figure 1. Ability of tributyl phosphate (TBP), triphenyl phosphate(TPhP), and 2-ethylhexyl diphenyl phosphate (EHDPP) to activatePPARG. The 293T cells were transfected with pBIND-PPARG-LBDand GAL4-pGL4-luc and then treated with TBP, TPhP, or EHDPP atvarious concentrations. pGL4.74 was transfected as the control fornormalization of Renilla LUC activity. Data are expressed relative tothe levels of vehicle-treated cells; these levels were set to 1. Results areexpressed as means ± SEM. This experiment was repeated at leastthree times.

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the nine important amino acid residues (Leu333, Phe347,Ile341, Met348, Phe352, Leu270, Phe287, Ile281, and Phe363)present in the inner surface of the ligand-binding domain(LBD) of human PPARG form the hydrophobic agonistpocket. EHDPP and TPhP both showed π−π interactionbetween their benzene rings and Phe287 of PPARG-LBD (faceto face). In contrast to TPhP, an important feature of theEHDPP interaction was the formation of hydrogen bondsbetween EHDPP and the residue Ser342 of PPARG-LBD(2.217 Å), which might contribute to the relatively highPPARG activation by EHDPP. Such differences in bindingmode might also explain the differences in PPARG activation ofthe three chemicals. To verify the involvement of the hydrogenbonds in the activation of EHDPP to PPARG, we substitutedSer342 of PPARG with alanine and then carried out PPARGactivation of EHDPP using the dual-luciferase reporter assay.The activation of EHDPP to the PPARG mutant was found tobe about 2.5-fold lower than that of wild type (Figure 3),demonstrating the importance of the hydrogen bond inPPARG activation by EHDPP.To further demonstrate the PPARG activity of OPFRs, three

main downstream genes of PPARG, hCG, pregnancy-associatedplasma protein-A (PAPP-A), and mucin gene Muc1, were alsoquantified in human placental choriocarcinoma JEG-3 cells.26,27

Confluent JEG-3 cells showed no change in cell viability or cellproliferation after treatment with EHDPP, TPhP, TCEP, TBP,

or TCPP at 5−40 μM for 48 h, and therefore theconcentrations of 5, 10, 20, and 40 μM were used in thefollowing experiments (Figure S2). EHDPP and TPhP

Figure 2. Binding modes of 2-ethylhexyl diphenyl phosphate (EHDPP) (A), triphenyl phosphate (TPhP) (B), and tributyl phosphate (TBP) (C) toPPARG LBD.

Figure 3. Ability of EHDPP to activate wild type and Ser342A mutantsof PPARG. The 293T cells were transfected with pBIND-PPARG-LBD or pBIND-PPARG-LBD (SER342A) and GAL4-pGL4-luc andthen treated with EHDPP at various concentrations. pGL4.74 wastransfected as the control for normalization of Renilla LUC activity.Data are expressed relative to the levels of vehicle-treated cells; theselevels were set to 1. Results are expressed as means ± SEM. Thisexperiment was repeated at least three times.

Figure 4. Quantitative analysis of expressions of human chorionicgonadotropin (hCG), mucin gene Muc-1, and pregnancy-associatedplasma protein-A (PAPP-A) in JEC-3 cells treated with tris(2-chloroethyl) phosphate (TCEP), tris-2-chloroisopropyl phosphate(TCPP), tributyl phosphate (TBP), triphenyl phosphate (TPhP), or2-ethylhexyl diphenyl phosphate (EHDPP) at various concentrations.Data are expressed relative to the levels of vehicle-treated cells; theselevels were set to 1. Results are expressed as means ± SEM of triplicatecultures. * (p < 0.05) indicate values significantly different fromvehicle-control value.

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significantly upregulated Muc1 and hCG expression butsignificantly down-regulated PAPP-A gene expression comparedwith the control (Figure 4), further demonstrating PPARGactivation of TPhP and EHDPP in human placenta.Disruption of Progesterone Secretion. Since hCG

governs the production of progesterone in the corpus luteumduring the first trimester,26,28 significantly upregulated hCGalterations may disrupt progesterone secretion. In addition tothe gene expression of hCG, its production alterations werealso determined. Both TPhP and EHDPP significantlyincreased hCG levels in the 20 and 40 μM groups comparedwith the control (Figure 5).To investigate whether the five target OPFRs could disrupt

progesterone biosynthesis, JEG-3 cells were grown toconfluence and treated with OPFRs at different concentrations,and concentration of progesterone was determined. Among theOPFRs tested, EHDPP and TPhP induced progesterone in adose-dependent manner, while no changes in progesteronelevel were observed in cells exposed to TBP, TCEP, or TCPP(Figure 5). The progesterone concentration was significantly

upregulated in the 20 and 40 μM TPhP exposure groups, with1.29 ± 0.09 and 1.28 ± 0.07-fold increases, respectively, relativeto that of the control (p < 0.01). As for EHDPP, upregulationsin progesterone levels were observed in the 10, 20, and 40 μMEHDPP exposure groups by 1.51 ± 0.02, 1.54 ± 0.04, and 2.0± 0.32-fold (p < 0.01), respectively, thus showing higherpotency than that of TPhP, consistent with their PPARGactivation results. In humans, after 7 weeks of gestation,progesterone in circulation is synthesized by the placenta andstrictly regulated for normal parturition.29 The secretion ofprogesterone by the placenta plays a vital role in theimplantation of the embryo and placental development formaintaining early pregnancy.30−32 Placental developmentaldisorders in early pregnancy are the most common causes ofmiscarriages, premature births, stunted growth, and intrauterinefetal death.31 Excessive ingestion of progesterone duringpregnancy is associated with a risk of hypospadias,33 and afunctional withdrawal of progesterone is critical for theinitiation of labor and normal parturition.28 Thus, humanexposure to TPhP and EHDPP might pose a risk to normal

Figure 5. Relative hormone production of hCG and progesterone and quantitative analysis of expression of 3β-HSD1 in JEG-3 cells treated withrosiglitazone (Rosi), tris(2-chloroethyl) phosphate (TCEP), tris-2-chloroisopropyl phosphate (TCPP), tributyl phosphate (TBP), triphenylphosphate (TPhP), or 2-ethylhexyl diphenyl phosphate (EHDPP) at various concentrations. Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means ± SEM of triplicate cultures. * (p < 0.05) and ** (p < 0.01) indicate valuessignificantly different from vehicle-control values. This experiment was repeated at least three times.

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parturition and fetal development due to the increase ofprogesterone.3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1) is an

important enzyme for the synthesis of progesterone in humanplacenta.26 The upregulation of progesterone production inhuman placental choriocarcinoma cells exposed to EHDPP andTPhP might be due to a change in 3β-HSD1 gene expression.To investigate whether OPFRs can change 3β-HSD1 geneexpression, we determined the gene expression of 3β-HSD1. Ofthe five OPFRs tested, alteration of 3β-HSD1 gene expressionwas only observed for EHDPP and TPhP. In the 20 and 40 μMTPhP exposure groups, 3β-HSD1 was significantly upregulatedby 1.30 ± 0.06 to 1.53 ± 0.13 fold (p < 0.01) relative to thecontrol (Figure 5). In the 10, 20, and 40 μM EHDPP exposuregroups, 3β-HSD1 was significantly upregulated by 1.29 ± 0.10to 2.94 ± 0.34 fold (p < 0.01) relative to the control (Figure 5).Such dose responses are similar to the progesterone

production, suggesting that EHDPP can increase the geneexpression of 3β-HSD1 at lower concentrations than that ofTPhP.

Inactivation of PPARG Pathway. To well understandwhether EHDPP or TPhP can disrupt progesterone productionvia the PPARG pathway in human placental choriocarcinomacells, inactivation of PPARG by pharmacological inhibitorGW9662 (5 μM) coexposed to EHDPP or TPhP wasinvestigated. After coexposure to GW9662 with EHDPP orTPhP, no change in JEG-3 cell viability or cell proliferation wasobserved (Figure S3). EHDPP and TPhP alone significantlyincreased the level of hCG and progesterone in humanplacental choriocarcinoma cells; both hCG and progesteronelevels were reversed to levels comparable to the DMSO controlwhen the cells were coexposed to GW9662 with EHDPP orTPhP (Figure 6).The gene expression of endogenous PPARG in JEG-3 cells

was further knocked-down by siRNA. The transfection ofsiPPARG led to an approximate 70% decrease in PPARG geneexpression compared with that of the control, and correspond-ingly PPARG knockdown significantly reversed EHDPP-induced 3β-HSD1 and hCG gene expression change comparedwith the control (Figure 7), further indicating that EHDPP candisrupt progesterone production in human placental chorio-carcinoma cells by activation of the PPARG pathway. It hasbeen reported that the activation of PPARG requiresheterodimerization with retinoid X receptor (RXR),34 and thePPARG-RXR heterodimer can be activated by either PPARG orRXR agonist.34 A RXR agonist, LG100268, can promoteprogesterone production in human placental choriocarcinomacells.19 Considering RXR’s role in regulating progesteroneproduction, we also tested the RXR activation of EHDPP andTPhP and found that EHDPP and TPhP did not elicit RXRactivation (Figure S4). Thus, we can conclude that EHDPP andTPhP induced 3β-HSD1 via PPARG and therefore disruptingprogesterone production.OPFRs have been detected in human blood and

placenta.12,13 In Chinese men and women, the concentrationsof TBP, TPhP, and EHDPP in blood were up to 758 ng/mL,1.21 ng/mL, and 3.13 ng/mL, respectively.13 In humanplacenta, the concentrations of TBP, TPhP, and EHDPPhave been detected at concentrations of 100 ng/g lw, 215 ng/glw, and 145 ng/g lw.13 In the present study, the levels of TBPand TPhP were expressed as EHDPP equivalent (EQEHDPP) bycomparing with the EHDPP PPARG activation. The

Figure 6. Hormone change in JEC-3 cells treated with EHDPP orTPhP (40 μM) or coexposed with GW9662 (5 μM). Relativehormone production of hCG and progesterone in JEG-3 cells treatedwith EHDPP or TPhP at various concentrations. Data are expressedrelative to the levels of vehicle-treated cells; these levels were set to 1.Results are expressed as means ± SEM of triplicate cultures. *(p <0.05) and **(p < 0.01) indicate values significantly different fromvehicle-control values. This experiment was repeated at least threetimes.

Figure 7. Effects of PPARG knockdown on the gene expression change induced by EHDPP in JEG-3 cells. (A) Quantitative analysis of expression ofPPARG in negative-control JEG-3 cells (control siRNA) and PPARG-knockdown JEG-3 cells (PPARG siRNA). (B) and (C) Quantitative analysis ofexpressions of 3β-HSD1 and hCG in JEG-3 cells treated with Rosi (100 nM) or EHDPP (20 μM). Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means ± SEM of triplicate cultures. * (p < 0.05) and ** (p < 0.01) indicate valuessignificantly different from vehicle-control values. This experiment was repeated at least three times.

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equivalency factors of PPARG activation were 0.62 for TPhPand 0.33 for TBP, and thus the EQEHDPP in human placenta wasabout 0.86 μM. The significant increase in progesterone in cellsexposed to EHDPP at 10 μM was about 10-fold higher thanthat of the concentration in human placenta. There are obviouslimitations to this parallel, but considering this study focused onthe effects of short-term exposure to OPFRs, whereas humansare exposed over a life-long period, OPFRs might disrupthuman placental choriocarcinoma cells and further produceadverse health issues at environmentally relevant levels.This study demonstrated, for the first time, that EHDPP is a

PPARG agonist with the most potent PPARG activation amongOPFRs well-detected in humans. The disruption of hCG andprogesterone synthesis in human placental choriocarcinomacells via the PPARG pathway implicated its potentialreproductive toxicity to pregnant women. As replacements forPBDE, the PPARG activation of some OPFRs, such as EHDPPand TPhP, is stronger than that of PBDE, which raises concernthat replacement chemicals should be tested for their relativesafety prior to being promoted as alternatives.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.7b00872.

Figures addressing the dose−response curve of thepositive agonist of PPARG, cell viability, RXR activationof TPhP and EHDPP, and primers used in theQuantitative RT-PCR and RNA Interference Assay(PDF)

■ AUTHOR INFORMATIONCorresponding Author*Phone/Fax: 86-10-62765520. E-mail: hujy@urban.pku.edu.cn.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study received financial support from the National NaturalScience Foundation of China [41330637, 21577001], theInternational S&T Cooperation Program of China(2016YFE0117800), and FY2016 Strategic InternationalCollaborative Research Program (SICORP) from Japan Scienceand Technology (JST) Agency.

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