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Reproductive Toxicology 32 (2011) 69– 76

Contents lists available at ScienceDirect

Reproductive Toxicology

jo u r n al hom epa ge: ww w.elsev ier .com/ locate / reprotox

ntiproliferative and proapoptotic effects of bisphenol A on human trophoblasticEG-3 cells

ucie Morice, Delphine Benaîtreau, Marie-Noëlle Dieudonné, Corinne Morvan, Valérie Serazin,hilippe de Mazancourt, René Pecquery, Esther Dos Santos ∗

aboratoire de Recherche, Université de Versailles-St-Quentin, Service de Biochimie et Biologie Moléculaire, UPRES-EA 2493, Faculté de Médecine Paris-Ile de France Ouest, PRESniversud Paris, Centre Hospitalier de Poissy-Saint Germain, Rue du Champ Gaillard 78303, Poissy Cedex, France

r t i c l e i n f o

rticle history:eceived 7 June 2010eceived in revised form 30 March 2011ccepted 12 May 2011vailable online 20 May 2011

a b s t r a c t

Different studies performed in rodents revealed that bisphenol-A (BPA), an environmental compound,altered early embryonic development. However, little is known concerning the direct effects of BPA onhuman implantation process. Thus, we decided to study in vitro BPA’s effects on proliferative capacitiesof the human trophoblastic cell line, JEG-3. For this purpose, we first have shown that JEG-3 cells expressthe specific BPA receptor, namely estrogen-related receptor �1 (ERR�1). Secondly, we demonstrated that

eywords:PArophoblastsEG-3roliferation

BPA did not exert any cytotoxic action in JEG-3 cells up to 10−6 M. Moreover [3H]-thymidine incorporationexperiments revealed that BPA significantly reduced cell proliferation. The results also showed that BPAinduced JEG-3 apoptosis capacity as reflected by DNA fragmentation experiments.

In conclusion, we describe here the direct impact of BPA on trophoblastic cell number mediated throughboth anti-proliferative and pro-apoptotic effects.

poptosis

. Introduction

Humans are exposed daily to a great number of chemicalompounds and their metabolites which are described as strongollutants [1]. Among them, bisphenol-A (BPA) is widely used inpoxy resins, polycarbonate plastics, and flame retardants. It isound in a broad range of products including toys, water pipes,rinking glasses, baby-bottles, and food beverage containers [2].PA is considered as a xenoestrogen product and is described as anndocrine-disrupting chemical that can potentially affect humanealth. In support of this concept, many epidemiological studiesuggest a link between BPA and the development of hormonal can-ers, obesity, and metabolic syndrome [3]. Like most endocrineisruptors, BPA accumulates in adipose tissue [4]. In addition, dif-erent studies have detected BPA in serum of pregnant women, inollicular fluid, in placental tissue, and in umbilical cord blood [5–7].n humans, blood levels of BPA range from to 1 to 20 nM [8]. More-ver, BPA concentrations increase 5-fold in amniotic fluid duringhe first-trimester suggesting early accumulation in human fetuses9]. Other studies have clearly described how BPA undergoes a rapid

ransport across the placental barrier to get into the cord bloodf fetuses [10,11]. Finally, in utero exposure of female rodents toaily doses of high BPA concentrations (10–50 mg/kg body weight)

∗ Corresponding author. Tel.: +33 01 39 27 54 65.E-mail address: dossantos.esther@orange.fr (E. Dos Santos).

890-6238/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2011.05.003

© 2011 Elsevier Inc. All rights reserved.

revealed different BPA effects including increased body weight[12,13], abnormal genital tract development [14], altered earlyembryonic development [10] and significantly reduced implanta-tion site number [15].

Some of BPA’s actions are attributed to its ability to bind as aweak estrogen agonist to the classical nuclear estrogen receptors(ER), ER� and ER� [16,17]. Furthermore, different studies revealedthat BPA interacts with sites distinct from the estradiol ligandbinding domain of the receptor, and differences have also beennoted in the recruitment of transcriptional coregulators [18] lend-ing support to the idea that BPA is not merely an estrogen mimic.Recent studies discovered that BPA can bind strongly to anotherhuman estrogen nuclear receptor, the estrogen-related receptor� (ERR�) which has long been considered as an orphan receptor[19,20]. Moreover, BPA is also able to bind to non-classical mem-brane ER as well as to the G-protein-coupled receptor 30 (GPCR30)and thus exerts rapid non genomic effects [21]. Indeed, there isnow convincing evidence that BPA is able to trigger rapid mem-brane activation of a variety of second-messenger-mediated signaltransduction pathways with possible implications on cell prolifer-ation, apoptosis, or survival. For example, BPA rapidly stimulatescAMP-dependent protein kinase (PKA), cGMP-dependent proteinkinase (PKG) and MAP kinase signaling pathways in some cell types

[22–24].

The goal of this study was first to evaluate possible cytotoxiceffects of BPA and secondly to describe BPA effects on proliferativeand apoptotic capacities of human trophoblastic cell line JEG-3.

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. Materials and methods

.1. Materials

DMEM/F12, penicillin, streptomycin, Hepes, leupeptin, aprotinin, 4-(2-minoethyl)-benzene-sulfonyl fluoride (AEBSF), NaF, bovine serum albumin (BSA)nd 2, 2-bis (p-hydroxyphenyl) propane (BPA) were purchased from Sigma Chem-cal Co. (Saint Louis, MO, USA). Cell culture dishes were purchased from DutscherBrumath, France). Fetal calf serum (FCS) was provided by Fisher Bioblock Scientificnd corresponded to lot number 41F0862K (Illkirch, France). Superscript II Rnase-RT was provided by Invitrogen corporation (Carlsbad, CA, USA), and RNAguard byharmacia Biotechnology (Uppsala, Sweden). [3H]-thymidine was purchased frommersham (Aylesbury, UK). Calcein AM was collected at BD Biosciences (Bedford, PA,SA). The in situ cell death detection kit fluorescein was obtained from Roche Molec-lar Biochemicals (Mannheim, Germany). The origins of the different antibodies areescribed in the paragraphs below.

.2. Tissue collection

This study was approved by the local ethical committee (CCPPRB) and eachonor gave informed consent before clinical sampling.

Human placental tissues from first trimester (4–8 weeks, n = 8) were collectedmong healthy pregnant women aged 18–35 and undergoing legal abortions. Pla-ental tissues were placed in HBSS supplemented with streptomycin (0.1 mg/ml),enicillin (100 U/ml), washed several times, and aseptically dissected to removeecidual tissues and fetal membranes.

Ovary tissues were obtained from women experiencing diagnostic hysteroscopyn = 3). The patients had a regular cycle without hormonal treatment and suspected

alignancy.

.3. Cell culture

JEG-3 and BeWo human placental choriocarcinoma cell lines were obtained frommerican Type Collection of Cells (Manassas, VA, USA). The culture media usedere phenol-red free DMEM/F12 and contained streptomycin (0.1 mg/ml), peni-

illin (100 U/ml), and 10% fetal calf serum (FCS) or 15% FCS for JEG-3 and BeWo cells,espectively. Cells were maintained under standard culture conditions under 5% CO2

tmosphere at 37 ◦C.

.4. Human cytotrophoblast isolation

Human primary extravillous and villous cytotrophoblasts were prepared fromips of placental terminal villi as previously described [25].

.5. Calcein AM assays

The calcein AM viability test is commonly used in toxicity assays to detect cellembrane integrity and to quantify the number of damaged cells. “Live” cells, i.e.

ells with an intact membrane in which esterase activity is present, are distinguishedy an intense uniform green fluorescence generated by the enzymatic hydrolysis ofalcein acetoxymethyl (calcein AM). JEG-3 cells were plated into 96-well cultureishes (2500 cells/well) in 10% FCS. After 24 h, cells were cultured in DMEM/F12edium supplemented with 5% charcoal-stripped FCS and various concentrations

f BPA (10−10–10−5 M) for different time periods (24, 48 and 72 h). DMSO was useds vehicle and control and its concentration in culture medium did not exceed 0.1%.or each time point, calcein AM at 8 �M was added to the culture medium for 1 h.luorescence was quantified using an infinite microplate fluorimeter (Tecan, VA,SA).

.6. Cell viability determination using LDH activity

The cellular toxicity of BPA was verified by measuring the lactate dehydrogenaseLDH) activity released into the culture medium as described previously [26].

.7. [3H]-thymidine incorporation

JEG-3 and BeWo cells were plated into 24-well culture dishes at a density of × 104 in 10% and 15% FCS, respectively. After 24 h, JEG-3 cells were cultured inMEM/F12 medium supplemented with 5% charcoal-stripped FCS and various con-entrations of BPA (10−10–10−6 M) during 24 or 48 h. Only one concentration of BPA10−8 M) was tested for BeWo cells. Five percent charcoal-stripped FCS was chosenecause previous experiments performed with 0%, 1% or 5% charcoal-stripped FCSevealed that 5% charcoal-stripped serum was the best compromise between stim-

latory effects of FCS and survival of the cell lines. For the last 6 h, [3H]-thymidine1 mCi/ml) was added to the culture medium. After washing them 3 times withaline buffer, cells were lysed during 5 min with 1% SDS and treated with 10%richloroacetic acid for 45 min at 4 ◦C. Radioactivity was counted after filtration onF/C filters (Whatman, Clifton, NY, USA).

xicology 32 (2011) 69– 76

2.8. Cell counting

Besides the experimental cell culture conditions described above only 10−8 Mand 10−7 M BPA concentrations were tested and, after the washing steps, cellswere harvested with calcium- and magnesium-free Hanks’ solution containing 0.2%trypsin. Finally, cells were stained during 5 min using crystal violet (0.5 mg/ml) andcounted in a hemocytometer before and after 24 h of treatment.

2.9. Apoptosis assay

JEG-3 cells were plated into 6-well plates at a density of 3 × 105 in 10% FCS.After 24 h, cells were cultured in DMEM/F12 medium supplemented with 5%charcoal-stripped FCS and two concentrations of BPA (10−8 and 10−7 M) during48 h. Attached cells were harvested by trypsinization, combined with floating cellsand suspended in PBS at a density of 106 cells/ml. Then, cells were fixed in 70%ethanol at −20 ◦C overnight and washed twice with PBS. Cells were labeled for DNAfragmentation by TUNEL according to the instructions provided by the manufac-turer. Apoptotic index was calculated after counting a minimum of 5000 eventsthrough flow cytometry using an EPICS flow cytometer (Coulter Electronics, Miami,FL, USA).

2.10. Immunocytochemistry

JEG-3 cells were plated (4 × 104 cells/well) in a labtech culture device (BD bio-sciences, San Jose, CA, USA) and cultured as described above. After 48 h of BPAtreatment, cells were washed three times in PBS buffer and fixed in methanol for10 min at +4 ◦C. Endogenous peroxidase activity was blocked by incubation with 1%H2O2 and non-specific IgG binding was blocked by incubation with normal blockerserum. Samples were then incubated with monoclonal primary mouse anti-humanM30-cytodeath antibody (Roche Diagnostics, Basel, Switzerland) for 1 h at roomtemperature. The slides were then rinsed and incubated with biotin-conjugatedsecondary antibody for 10 min at room temperature. Visualization was achievedusing DAB detection kit (Ventana Medical System, Tucson, AZ, USA). To determinethe extent of non-specific immunostaining, primary antibody was substituted withmouse IgG. The slides were counterstained with hematoxylin before mounting. Foreach culture condition, at least 1500 cells per section were randomly examinedusing the analysis software Histolab (version 5.2.3, Microvision instrument, Paris,France). The number of M30 positive cells relative to the total number of cells wasdetermined.

2.11. RNA interference for ERR�1

Three pairs of small-interfering RNAs (siRNAs) corresponding to differentregions of ERR�1 mRNA were chemically synthesized by Santa Cruz Biotechnol-ogy (sc-44704). A fluorescently labeled, non-silencing control siRNA (10 nM/well)was used for the optimization of transfection conditions and the control of non-specific silencing effects. A positive control consisting of a siRNA directed againsthuman MAP kinase RNA sequence (25 nM/well) was used concomitantly in orderto ensure that optimal conditions were maintained and to confirm experimen-tal results. For the knockdown experiments, JEG-3 cells were plated in 24-welldishes at 1 × 105 cells/well and were transfected with different siRNA concentra-tions (1, 5 or 10 nM/well) using a Lipofectamine RNAiMAX transfection reagentfrom Invitrogen (Carlsbad, CA, USA) according to the manufacturer’s instruc-tions. BPA (10−8 M and 10−7 M) was added 48 h after transfection. After 24 h ofculture, the [3H]-thymidine incorporation analysis was performed as describedabove.

2.12. Protein expression

Confluent JEG-3 cells were scrapped and sonicated on ice in buffer contain-ing 50 mM Tris, 120 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.5 mM desoxycholate,0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 100 �g/ml AEBSF, 30 mM �-glycerophosphate, 5 �g/ml aprotinin, 12.5 �g/ml leupeptin, and 10 mM NaF. Humanfirst-trimester placental tissues were also used as positive controls [27]. After cen-trifugation at 100,000 × g for 10 min at 4 ◦C, supernatants were diluted in Laemmli’sbuffer (v/v). Protein concentrations were measured according to Bradford [28] withBSA as a standard. Equal amounts (100 �g) of cellular extracts were subjected to SDS-PAGE (10%). Proteins were transferred to PVDF membrane and blocked in buffer A(20 mM Tris HCl, 137 mM NaCl and 0.1% Tween 20) with 2.5% gelatin during 2 h.Then, membranes were incubated overnight at room temperature with rabbit poly-clonal anti-ERR� (sc-32969) antibody (1:200, Santa Cruz Biotechnology) in bufferA with 2.5% gelatin. The resulting blots were extensively washed with buffer A,and incubated with the secondary antiserum coupled to peroxidase (1: 15,000 dilu-tion in buffer A) for 1 h at room temperature and washed. Finally, an enhanced

chemiluminescence (ECL) from Thermo Scientific (Rockford, IL, USA) was used forsignal detection. Finally, in order to confirm that all bands contain equal amountsof protein, membranes were re-blotted with a standard protein (rabbit polyclonalanti-�-actin (sc-130656)) antibody (1:200, Santa Cruz Biotechnology) in buffer Awith 2.5% gelatin.

L. Morice et al. / Reproductive To

Table 1Primer pairs used for RT-PCR.

Primer sets Sequence PCRproduct(bp)

ERR�1Sense 5′ TGG TCC CAA CTG GCT GTG CC 3′

129Antisense 5′ TCC ATG TGC GAC CGG CAA CC 3′

ERR�2/3Sense 5′ TAA AAT CCC CGA CGA CTC AC 3′

74Antisense 5′ GAA CCA GTA AAT TGT CAG CTC TTG 3′

C-MycSense 5′ GAC GCG GGG AGG CTA TTC TG 3′

236Antisense 5′ GAC TCG TAG AAA TAC GGC TGC ACC GAGTC 3′

P53Sense 5′ ACT AAG CGA GCA CTG CCC AA 3′

231Antisense 5′ ATG GCG GGA GGT AGA CTG AC 3′

�2microSense 5′ TGC TGT CTC CAT GTT TGA TGT ATC T 3′

86Antisense 5′ TCT CTG CTC CCC ACC TCT AAG T 3′

TBPSense 5′ TGC ACA GGA GCC AAG AGT GAA 3′

132Antisense 5′ CAC ATC ACA GCT CCC CAC CA 3′

RP13ASense 5′ TTG AGG ACC TCT GTG TAT TTG TCA A 3′

125Antisense 5′ CCT GGA GGA GAA GAG GAA AGA GA 3′

Cyclin D1Sense 5′ GAG TGG CGG CAG CGG C 3′

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69Antisense 5′ CAG TTC GCG GCT CAG CTG TT 3′

.13. Quantitative RT-PCR

Total RNA (0.1 �g) was extracted and reverse transcribed as described previ-usly [29]. Quantitative PCR was performed using a LightCycler480® instrumentrom Roche Diagnostics (Basel, Switzerland) with primer sets indicated in Table 1nd Sybergreen I master mix (Roche Diagnostics). Second derivative maximumethod was used to automatically determine the Cp for individual samples. Three

eference genes TBP, RP13A, and �2 microglobulin have been tested for their expres-ion stability in JEG-3 cells and in human placental tissues. Analysis using the

eNorm software revealed that TBP and �2 microglobulin genes were the mosttable genes to normalize data in these cells and tissues. For each sample, the con-entration ratio (target/reference mRNA) was calculated using the RelQuant Rocheoftware and expressed in arbitrary units. Calibration curves were log-linear over

ig. 1. ERR� mRNA and protein expression. (A) ERR� mRNA expression. Total RNA was end villous cytotrophoblasts, and human placental tissues. Total RNA was then analyzed

nalyzed through agarose gel electrophoresis. This figure shows one experiment represend BeWo cells. RT-PCR relative quantification of mRNA expressions. Results are the mytotrophoblasts and placental tissue. RT-PCR relative quantification of mRNA expressixpression. Cells lysates (100 �g) were subjected to Western Blot analysis using anti-Experiment representative of three separate experiments.

xicology 32 (2011) 69– 76 71

the quantification range with correlation coefficient r2 > 0.99 and efficiency rangingfrom 1.8 to 2. The intra-assay variability of duplicate crossing point (Cp) values neverexceeded 0.2 cycle and the inter-assay variability (CV value) ranged from 1 to 5% forthe five or seven runs of each transcript.

PCR products were separated on a 2% agarose gel in 90 mM Tris–borate, 2 mMEDTA buffer (TBE, pH 8.0), and visualized by staining with ethidium bromide andultraviolet transillumination.

2.14. Statistical analysis

All values were expressed as means ± SEM of four to eight separate experi-ments. Statistical analysis was performed using paired t test and post hoc analysis(Dunnett’s test) for comparison to control.

3. Results

3.1. Expression of specific BPA receptors in trophoblastic cell linesand in human placenta

Before investigating BPA effects on JEG-3 cells, we studied thepresence of the specific BPA receptor, ERR�1, in both human JEG-3and BeWo choriocarcinoma cells, in isolated extravillous and vil-lous cytotrophoblasts, and in first and third-trimester placenta.Using real time RT-PCR, we found that ERR�1 was expressedto a higher extent (2×) in JEG-3 cells compared to BeWo cells(Fig. 1B). For this reason, JEG-3 cells were chosen to study BPAeffects. Interestingly, these experiments also revealed that JEG-3cells and human isolated extravillous cytotrophoblasts approxi-mately expressed the same level of ERR�1 mRNA. Moreover, wehave shown that ERR�1 mRNA was expressed at a 20- to 25-foldlower level in JEG-3 cells than in human first-trimester placentaltissues used as positive control [27] (Fig. 1C). Finally, ERR�1 mRNAexpression was more abundant in third than in first-trimester pla-cental tissues (Fig. 1C).

According to the results shown in Fig. 1A, the two other ERR�subtypes (ERR�2 and ERR�3) were expressed neither in JEG-3 nor

in BeWo cells. In contrast, these two receptors were present in first-trimester human placental tissue.

Western blot analyzes confirmed: (i) the presence of ERR� inboth JEG-3 and BeWo cells as well as in first and third-trimester

xtracted from human JEG-3 cells, human BeWo cells, human primary extravillousby RT-PCR with primers as described in Table 1. PCR products after 45 cycles werentative of four separate experiments. (B) ERR�1 mRNA expression in human JEG-3eans ± SEM of 5-11 separate experiments. (C) ERR�1 mRNA expression in humanons. Results are the means ± SEM of 5–8 separate experiments. (D) ERR� proteinRR� and �-actin antibodies as described under Section 2. This figure shows one

72 L. Morice et al. / Reproductive To

Fig. 2. Effect of BPA on JEG-3 cell viability. Cells were cultured in DMEM/F12 mediumsupplemented with 5% charcoal-stripped FCS and various concentrations of BPA(10−10–10−5 M) or without BPA (CONT) for 48 h. Cell viability was assayed by LDHactivity assay. Data represent the mean ± SEM of 5 independent experiments and arenormalized as percentage of the control value (without BPA). In each experiment,vv

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lacenta extracts, (ii) that ERR� protein level is higher in JEG-3 cellsompared to BeWo cells and, (iii) that ERR� protein level is highern placental tissues compared to JEG-3 cells. Human ovary tissues

ere used as positive control tissues (Fig. 1D).

.2. Cell viability in BPA-treated JEG-3 cells

The viability of cells exposed to BPA was determined by bothDH assay and calcein assay. JEG-3 cells were cultured withifferent doses of BPA for 48 h. As shown in Fig. 2, cell viabil-

ty was unaffected by the different BPA concentrations tested−10 −6

10 –10 M) indicating that, under these experimental condi-

ions, BPA did not induce any cellular lysis (Fig. 2). However, at aigher dose (10−5 M) BPA appeared to exert a cytotoxic effect asssessed by cell viability quantification (+120 ± 43%) and calcein

ig. 3. Effect of BPA on JEG-3 cell proliferation. (A) Effect of BPA on JEG-3 DNA synthesiPA (10−10–10−5 M) in the presence of [3H]-thymidine as described under Section 2 for 2ormalized as percentage of the control value (without BPA): 2168 ± 245 cpm/24 h. In eaith control values, *p ≤ 0.01, Dunnett’s test. (B) Effect of BPA on c-myc and cyclin D1 mRMEM/F12 medium during 18 h and then incubated during 1 h or 6 h in the absence (COxtracted from these cells and subjected to RT-PCR to determine mRNA levels as describes percentage of control (without BPA). ns: non-significant compared with control values

xicology 32 (2011) 69– 76

assay (data not shown). In the following experiments, we decidedto use BPA concentration not exceeding 10−6 M.

3.3. Inhibition of JEG-3 proliferation by BPA treatment

Once the non toxic effects of BPA on JEG-3 cell viability wereverified, we studied the direct action of this compound on cell pro-liferation capacities. To do so, we measured the changes in the rateof DNA synthesis by [3H]-thymidine incorporation. As shown inFig. 3A, exposure to BPA for 24 h resulted in a significant reduc-tion of [3H]-thymidine incorporation in JEG-3 cells (−28.6 ± 3.1%,−59 ± 5%, and −54 ± 5% for BPA at 10−8, 10−7 and 10−6 M, respec-tively). Under our experimental conditions, we demonstrated thatthese effects still persisted after 48 h and until 72 h of BPA treat-ment (data not shown). The anti-proliferative action of BPA wasalso confirmed by direct counting of JEG-3 cells (−29.7 ± 7.9%and −36 ± 10% for BPA at 10−8 and 10−7 M, respectively). For thefollowing experiments we had chosen the same two intermedi-ate concentrations of BPA (10−8 and 10−7 M), which are in therange of the experiments performed in vitro by others investigators[24,30,31].

Surprisingly, under the same experimental conditions, [3H]-thymidine incorporation assays performed in human BeWo cellsdid not reveal any regulation by BPA (data not shown).

In order to delineate BPA-target genes in JEG-3 cells, the influ-ence of BPA on c-myc and cyclin D1 mRNA expressions wasmeasured using RT-PCR. As shown in Fig. 3B, in JEG-3 cells BPAsignificantly reduced c-myc mRNA expression (−29 ± 0.5% and−30 ± 0.6%, respectively). However, under the chosen experimen-

tal conditions BPA did not affect cyclin D1 mRNA expression.These findings indicate that the proto-oncogene c-myc couldbe a nuclear target of BPA in human trophoblastic JEG-3cells.

s. Growing JEG-3 cells were exposed or not (CONT) to different concentrations of4 and 48 h. Data represent the mean ± SEM of 6 independent experiments and arech experiment, values were determined in triplicate. ns: non-significant comparedNA expressions in JEG-3 cells. Growing JEG-3 cells were deprivated in serum-freeNT) or in presence of two BPA concentrations (10−8 and 10−7 M). Total RNA was

d under Section 2. Results are means ± SEM of five experiments and are expressed, *p ≤ 0.005, paired t test.

L. Morice et al. / Reproductive Toxicology 32 (2011) 69– 76 73

Fig. 4. Effect of BPA on JEG-3 cell apoptosis. (A) Effects of BPA on DNA fragmentation in JEG-3 cells. Confluent JEG-3 cells were cultured for 48 h in DMEM/F12 supplementedwith 5% charcoal-stripped FCS in the presence or absence (CONT) of BPA (10−8 and 10−7 M) or staurosporine (10−7 M). Cells were then analyzed by TUNEL staining and flowcytometry. Results are expressed as a percentage of control (without BPA). Each bar represents the mean ± SEM of four separate experiments. *p ≤ 0.02, **p ≤ 0.002, pairedt test. (B) Effects of BPA on p53 mRNA expression JEG-3 cells. Confluent JEG-3 cells were cultured for 8 h in DMEM/F12 supplemented with 5% charcoal-stripped FCS in thepresence or in the absence (CONT) of BPA (10−8 M or 10−7 M). Total RNA was extracted from these cells and analyzed by RT-PCR to determine p53 mRNA level as describedunder Section 2. Results are means ± SEM of five to eight experiments and are expressed as percentage of control (without BPA). *p ≤ 0.03, **p ≤ 0.01, paired t test. (C) Effectsof BPA on M30 immunostaining in JEG-3 cells. Growing JEG-3 cells were cultured for 48 h in DMEM/F12 supplemented with 5% charcoal-stripped FCS in the presence or int expreo

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he absence (CONT) of BPA (10−8 M or 10−7 M) or staurosporine (10−7 M). Results aref four separate experiments. *p ≤ 0.05, **p ≤ 0.01, paired t test.

.4. Induction of JEG-3 cell death by BPA treatment

To test the influence of BPA on cell apoptosis, TUNEL assays cor-esponding to the ultimate apoptosis step were performed in ordero determine the percentage of labeled apoptotic nuclei. As shownn Fig. 4A, BPA was able to induce significantly DNA fragmentationn human JEG-3 cells (+76 ± 30% and +132 ± 48% for BPA at 10−8

nd 10−7 M, respectively) after 48 h treatment. Under the sameonditions, exposure of cells to 10−7 M staurosporine (a knownro-apoptotic factor) was used as positive control also inducedpoptosis by 246 ± 45%.

In order to get insight on the molecular mechanisms wherebyPA induced JEG-3 apoptosis, we measured mRNA expression ofhe pro-apoptotic p53 gene. As shown in Fig. 4B, BPA significantly

nduced the expression of p53 (1.3×- and 1.5×-fold for BPA at0−8 and 10−7 M, respectively) after 8 h exposure. Moreover, thexpression of M30-cytodeath protein, which corresponded to theytokeratin 18 that is formed by early caspase cleavage in the apo-

ssed as a percentage of control (without BPA). Each bar represents the mean ± SEM

ptotic cells, was also a target for BPA since a 59% and 66% proteininduction for BPA at 10−8 and 10−7 M, respectively was observed(Fig. 4C). Under the same conditions, exposure of cells to 10−7 Mstaurosporine, used as positive control, strongly induced apoptosis(7×-fold).

Thus, exposure to BPA stimulated an in vitro pro-apoptoticresponse in human JEG-3 cells.

3.5. Effects of ERR� invalidation on BPA biological effects onJEG-3 cells

In these experiments, we have tested different ERR� siRNAconcentrations (1, 5 and 10 nM) and finally chosen 10 nM whichinduced a 60% decrease within 48 h after transfection (Fig. 5A and

B). Furthermore, as shown in Fig. 5C, the suppression of ERR� withsiRNA partially prevented the reduced [3H]-thymidine DNA incor-poration by BPA. These data indicated that biological actions of BPAin human JEG-3 cells required at least in part ERR�.

74 L. Morice et al. / Reproductive To

Fig. 5. Effect of BPA in ERR�-deficient JEG-3 cells. (A) ERR� mRNA level in trans-fected JEG-3 cells. Growing JEG-3 cells were treated 24 h post-plating with differentsiRNA concentrations (1, 5 or 10 nM) for 48 h. Total RNA was extracted from thesecells and analyzed by RT-PCR. Results are means ± SEM of three experiments andare expressed as percentage of control (non-silencing). ns: non significant, *p ≤ 0.05,paired t test. (B) ERR� protein expression in transfected JEG-3 cells. Growing JEG-3 cells were treated 24 h post-plating with 10 nM of ERR� siRNA for 48 h. Cellslysates (100 �g) were subjected to Western Blot analysis using anti-ERR� antibodyas described under Section 2. One experiment representative of three is shown.(C) Effect on BPA-reduced DNA synthesis in JEG-3 cells. ERR�-depleted cells with10 nM of siRNA were exposed or not (CONT) to BPA (10−7 M) for 24 h. Results aremeans ± SEM of five experiments and are expressed as percentage of control (with-oip

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ut BPA). Each bar represents the mean ± SEM of four separate experiments ands expressed as percentage of control (without BPA). ns: non significant, *p ≤ 0.05,aired t test.

. Discussion

It is now well established that humans are routinely exposedo BPA through ingestion, respiration or skin absorption [32,33].herefore, it is not surprising that BPA impacts on humans areresently under extensive investigations. For example, it has beenescribed that BPA induces premature puberty, altered mammaryevelopment, increased body weight, higher incidence of breastnd prostate cancer, and more particularly disrupted placentaevelopment [12,14,34]. Different studies clearly demonstrated

correlation between alterations in trophoblast development

differentiation, invasion, proliferation, apoptosis) and preg-ancy disorders. For example, restricted trophoblastic invasion isssociated to human preeclampsia and/or intra-uterine growthestriction [35,36]. In this context, it appears important to deter-

xicology 32 (2011) 69– 76

mine more precisely the environmental impacts of BPA on placentalfunction and fetal development.

In order to identify toxic concentrations of BPA in human pla-centa, we first analyzed the cell viability of JEG-3 cells, used asan appropriate in vitro system since they have preserved manycytotrophoblastic properties [37]. We found that BPA did not exertany cytotoxic effects at any dose tested up to 10−6 M. These resultsdisagree with those observed in human placental cells from third-trimester in which authors described a toxic effect of BPA atconcentrations close to 10−9 M [38]. These discrepancies could beexplained by the fact that both cell types do not express the samedensity of the specific BPA receptor ERR�1. In this study, we haveindeed found that JEG-3 cells expressed a considerably lower levelof ERR�1 compared to third-trimester placenta. Thus, it could behypothesized that ERR�1 expression level is critical in the cytotoxiceffects of BPA which would indicate higher susceptibility on third-trimester placenta. Otherwise, as already observed in BeWo cells[39], BPA at a higher concentration (10−5 M) significantly reducedcell viability in JEG-3 cells. Based on these findings, we selectedlower, non-toxic concentrations to study BPA actions on prolifera-tive capacities of trophoblastic cells.

Secondly, we have tested different BPA concentrations(10–100 nM) on proliferative capacities of two different tro-phoblastic cell lines, JEG-3 and BeWo. These concentrations areclose to the levels that have been detected in human biologicalfluids and could correspond to chronic BPA exposure [9]. Indeed,human exposure to BPA has been estimated by the European Com-mission to be 1.46 �g/kg body weigh/day for an adult and differentBPA measurements have revealed levels in plasma ranging from1.3 to 82.8 nM [5,6,9]. BPA has also been detected in umbilicalcord blood (0.9–40.3 nM), indicating transport of BPA across theplacental barrier, placental tissue (up to 459.4 nM) [5], and amni-otic fluid (0–36.7 nM) [6,9]. Our results revealed that BPA exertsdirect anti-proliferative effects in JEG-3 cells but not in BeWo cells.The differential effects of BPA on JEG-3 and BeWo cells may reflectgenetic differences between the cell lines, even though they bothare derived from choriocarcinoma. For example, BeWo cells are ableto fuse spontaneously [40] causing a blockage of cell cycle progres-sion, thus counteracting the anti-proliferative effect of BPA. On theother hand, JEG-3 cells are highly proliferative and invasive but fusepoorly [37].

Cell proliferation is the result of the balance between cell divi-sion and apoptosis. So, in order to gain further insight into themechanisms whereby BPA exposure decreases JEG-3 cell number,the expression of some specific cell cycle and apoptotic regulatorygenes that could be under the control of BPA was investigated. Indifferent tissue and cell types, BPA is able to induce G1-S phasecell cycle progression through increased expression of cyclin D1and some proto-oncogenes such as c-myc [41,42]. In our study, wehave shown that BPA reduced c-myc mRNA expression but did notaffect cyclin D1 mRNA expression. These results suggest that theinhibitory effect of BPA on JEG-3 cell growth is partly due to a reduc-tion of G1-S phase cell cycle progression. Concerning the apoptoticprocess, as previously reported in other cell types [43,44], we havealso observed that BPA was able to induce DNA fragmentation, thelast step of apoptosis. This stimulatory effect was accompanied bychanges in the expression profile of some apoptosis markers likethe M30-cytodeath protein and the pro-apoptotic gene p53, theexpressions of which were both increased.

According to our findings that JEG-3 cells expressed the spe-cific BPA receptor, ERR�1, we further investigated whether BPAaffects these cells directly through ERR�1. Invalidation of ERR�1 by

siRNA partially abolished BPA-reduced proliferation. These resultssuggest that BPA action on JEG-3 cells could be partly mediatedby ERR�1. Persistence of the BPA anti-proliferative effect aftera 60% invalidation of ERR�1 raises the possibilities that: (i) the

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0% remaining active receptors are sufficient to mediate the BPAffects and/or (ii) the action of BPA is mediated via alternativeolecular/signaling pathways. Previous studies reveal that BPAas able to rapidly stimulate diverse transduction pathways such

s p42/p44 MAP kinase, PI3 kinase, PKA, and PKG (for a review8,24,31]). However, there is little information available concern-ng the non-genomic effects of BPA on trophoblastic cells, and thessue of whether BPA exerts or not non-genomic effects in JEG-3ells remains obscure. Thus, additional experiments are currentlyn progress in order to delineate the pathway(s) through which BPAransduced its effects on trophoblastic cells.

In the present study we have described for the first time theresence of ERR�1 not only in two different human trophoblasticell lines (JEG-3 and BeWo) but also in human isolated extrav-llous and villous cytotrophoblasts from first-trimester placenta.

hile this manuscript evaluates BPA’s actions in an in vitro model,he authors are aware of the limitation of this model to fullyescribed the in vivo situation. However, these encouraging resultsllow us to consider a study on the actions of BPA on humanrimary cytotrophoblasts and more particularly on isolated extrav-

llous cytotrophoblasts, since these cells expressed the same ERR�1RNA levels as JEG-3 cells. Our laboratory is currently performing

xperiments on the effects of BPA on human trophoblast invasionapacities corresponding to first steps of embryo implantation.

Taken together, these results demonstrate that BPA throughRR�1 can affect proliferative process in trophoblastic cells in vitro.urthermore, our results imply a possible impact of BPA on humanlacental development which is essential for human fertility toccur.

cknowledgements

The authors express their sincere gratitude to Vincent RinchevalUniversité de Versailles/Saint Quentin, CNRS FRE-2445, Labora-oire de Génétique et Biologie cellulaire) for assistance in flowytometry analysis. The authors also gratefully acknowledge Eliseegedus for his helpful advice in correcting our manuscript.

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