Estrogen-Like Response to p-Nonylphenol in Human First Trimester Placenta and BeWo Choriocarcinoma Cells

ToxSci Advance Access published June 21, 2006

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Estrogen-like response to p-nonylphenol in human first trimester placenta and BeWo choriocarcinoma cells Nicoletta Bechi*, Francesca Ietta*, Roberta Romagnoli*, Silvano Focardi†, Ilaria Corsi†, Carlo Buffi‡, and Luana Paulesu*. *

Department of Physiology, University of Siena, via A. Moro, Siena 53100, Italy; †Department of

Environmental Sciences, University of Siena, via A. Mattioli, Siena 53100, Italy; ‡Obstetrics and Gynecology Division, USL 7, Hospital, Campostaggia, Siena 53036, Italy.

[email protected]; Silvano Focardi†, [email protected]; Ilaria Corsi†, [email protected],

Short title: Effects of p-nonylphenol on human placenta Address correspondence to: Luana Paulesu, Department of Physiology, University of Siena, Via Aldo Moro 53100 Siena, Italy. E-mail: [email protected] Phone +39 0577 234224, Fax +39 0577 234219,

Acknowledgments This study was supported by research grants from the University of Siena, Siena (Italy) and from Fondazione Monte dei Paschi di Siena, Siena (Italy). The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration. © The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: [email protected]

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Nicoletta Bechi*, [email protected]; Francesca Ietta*, [email protected]; Roberta Romagnoli*,

2 Abstract p-Nonylphenol (p-NP) is a metabolite of alkylphenol ethoxylates used as surfactants in the manufacturing industry. Although it is reported to have estrogenic activity and to be transferred from the mother to the embryo, no data is available on its effects on the development of the human placenta. In the present study, we investigated Estrogen Receptors (ERs) expression in the first trimester human placenta. Using an in vitro model of chorionic villous explants, we then compared the effects of p-NP and 17β-estradiol (17β-E2). Finally, a trophoblast-derived choriocarcinoma cell line, BeWo, was used as a model of trophoblast cell differentiation.

kDa and one ERβ isoform of 55 kDa. Immunohistochemistry revealed expression of ERα in the villous cytotrophoblast, whereas ERβ was mainly expressed by the syncytiotrophoblast. Treatment of explant cultures with p-NP (10-9 M) and 17β-E2 (10-9 M) significantly increased β-hCG secretion and cell apoptosis but did not modify ERs expression. After 72 hours of exposure, hormone release was significantly higher in p-NP- than 17β-E2-treated explant cultures. By this time, cleavage of caspase-3 was evident in cultures treated with 17β-E2 and p-NP. In BeWo cells, a caspase-3 band of 20-16 kDa was evident after 1 hour of treatment with p-NP and after 24 hours of treatment with 17βE2 or forskolin. These findings suggest that the human trophoblast may be highly responsive to p-NP and raise concern about maternal exposure in early gestation.

Key words: p-nonylphenol; endocrine disruptors; estrogen receptors; human placenta; human chorionic gonadotropin; trophoblast apoptosis.

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Our results showed that the first trimester placenta expresses three ERα isoforms of 67, 46 and 39

3 Introduction Increasing evidence suggests that exposure to certain man-made chemicals present in the environment may interfere with endocrine function in humans and wildlife (Safe, 1995 Weiss, 1998). Alkylphenol ethoxylates are a class of non-ionic surfactants introduced in the 1940s and used in detergents, paints, pesticides, personal care products and plastics (Soto et al., 1991; White et al., 1994). The most commonly used alkylphenol ethoxylates in consumer products have nine carbon atoms with branched alkyl chains and are thus known as nonylphenol ethoxylates. These chemicals are discharged into aquatic environments with urban and industrial wastewater. Here they

intermediate nonylphenol (Rudel et al., 2003; White et al., 1994). These metabolites are also used as intermediates in the chemical industry (Müller and Schlatter, 1998). Human exposure to p-nonylphenol (p-NP) may occur by cutaneous absorption, ingestion of contaminated food or water and inhalation (Guenther et al., 2003; Monteiro-Riviere et al., 2000). Great concern has been expressed about this compound, after Soto et al., (1991), accidentally found that p-NP, released by polystyrene, induced progesterone receptor and cell proliferation in estrogendependent breast cancer cells (MCF-7). Estrogenic activity of p-NP has been then investigated in a number of in vitro and in vivo studies. By binding to Estrogen Receptors (ERs), p-NP may disrupt normal endocrine function, promoting reproductive failure and carcinogenesis in estrogen-sensitive tissues (Blair et al., 2000; Sonnenschein and Soto, 1998). p-NP acts directly via the ERs, displacing 17β-estradiol (17β-E2) from human cell line estrogen receptors (Soto et al., 1995; White et al., 1994) and specifically inhibiting binding of 17β-E2 to ERs (Kwack et al., 2002). Interestingly, in vivo studies have shown that maternal exposure to p-NP resulted in an increase in uterine calbinding-D 9k (CaBP-9k) expression in maternal and neonatal rat uteri (Hong et al., 2004; Kwack et al., 2002). These and other studies suggest that p-NP can cross the placenta and cause reproductive and developmental toxicity. Reports on several endocrine disruptor chemicals (bromodichloromethane, organochlorine compounds) describe the effect of these substances on

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are broken down microbially into nonylphenol ethoxylate by-products and the final degradation

4 human trophoblast (Chen et al., 2003; Chen et al., 2004; Hamel et al., 2003) while, no functional studies regarding the effects of p-NP on the placenta have yet been reported. Previous studies show that the placenta is an estrogen-target tissue and that estrogens have important physiological roles in regulating functional differentiation of the placental villous trophoblast (Bukovsky et al., 2003a; Cronier et al., 1999; Rama et al., 2004). Expression of estrogen receptor (ER) α and β protein was recently demonstrated in the human placenta at term (Bukovsky et al., 2003b). Expression of ERα, β and γ mRNA was also recently reported to increase in the placenta throughout gestation (Fujimoto et al., 2005).

human placentas; b) to compare the effects of p-NP and 17β-E2 on trophoblast differentiationapoptosis by an in vitro model of chorionic villous explants; c) to verify the role of p-NP in a trophoblast-derived choriocarcinoma cell line, BeWo. We demonstrate that first trimester placenta, like the placenta at term, expresses estrogen receptor proteins α and β and that p-NP exerts estrogen-like activity on trophoblast, inducing hCG secretion and caspase-3 activity, two main markers of trophoblast differentiation and apoptosis (StraszewskiChavez et al., 2005).. Unexpectedly, at equimolar concentrations of 10-9 M, the effect of p-NP was significantly greater and longer lasting than that of 17β-E2.

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The aim of the present study was: a) to investigate protein expression of ERs in first trimester

5 Materials and Methods Tissue collection. First trimester placentas (n=25) were obtained after elective terminations of pregnancy at weeks 7-12 of gestation, with the consent of patients and approval of the hospital Ethics Committee (Siena, May 2004). A portion of each was immediately snap-frozen and stored at -80°C for protein analysis; another portion was fixed in 10% buffered formalin and embedded in paraffin for immunohistochemical analysis. The rest was rinsed in cold phosphate buffered saline (PBS) to remove excess blood and processed for explant cultures within 2 hours. Human term placental tissues (n=5) were snap-frozen immediately after collection and stored at -80°C for

Isolation and treatment of chorionic villous explants. Chorionic villous explant cultures were established from 7-9 week human placentas. Villous explants were dissected as described by Caniggia et al., (1997). Briefly, small fragments of villous tips (15–20 mg wet weight) were placed on Millicell CM culture dish inserts (Millipore Corp, Bedford, MA), previously coated with 180 µL undiluted matrigel (Collaborative Research, Inc., Bedford, MA) and inserted in 24-well plates. Explants were cultured in serum-free DMEM/F12 without phenol red (Gibco, Grand Island, NY) supplemented with 100 U/mL penicillin/streptomycin and 2 mM L-glutamine (Sigma Chemical Co, St. Louis, MO). Explant cultures were incubated overnight at 37°C in 5% CO2 for attachment to the matrigel. The next day the culture medium was replaced with medium supplemented with 17βestradiol (10-9 M) (Sigma Chemical Co) or p-NP (10-9 M), both dissolved in 0.1% ethanol, or with medium plus ethanol (controls). This concentration was selected far below the levels found in human samples (Kawaguchi et al. 2004; Inoue et al. 2000; Tan and Nohd, 2003). At predetermined incubation times (4, 24, 48, 72 hours), explants were removed from matrigel and washed in PBS. Part of each was frozen and stored at -80°C until processing for western blot analysis for ERs and caspase-3 expression. Culture medium was stored at -80°C until assay for βhCG.

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western blot analysis.

6 BeWo cell culture and treatment. BeWo cells (Istituto Zooprofilattico Sperimentale, Brescia, Italy) were cultured in Ham’s F-10 without phenol red (Sigma Chemical Co) supplemented with 10% FBS (Biochrom, Berlin, Germany), 100 U/mL penicillin/streptomycin and 2 mM glutamine (Sigma Chemical Co) in 75 cm2 flasks (Becton Dickinson Labware, Franklin Lakes, NJ) in a humidified atmosphere of 20% air and 5% CO2 at 37 °C until 70-80% confluence. After overnight storage in serum-free conditions, BeWo cells were exposed to 17β-estradiol (10-9 M) or p-NP (10-9 M). Cell differentiation was induced by addition of 10 µM Forskolin (Sigma Chemical Co). Control cells were cultured in serum-free medium containing vehicle (0.1% ethanol). Cells were harvested at 1, 6

Western blot. Placental tissue, villous explants and BeWo cells were homogenized at predetermined times of incubation in ice-cold lysing buffer (Tris-HCl 50 mM, NaCl 50 mM, Triton X 100 1%, Na deoxycholate 1%, SDS 0.1%) containing sodium orthovanadate 100 mM and a protease inhibitor cocktail containing 4-(2-aminoethyl benzenesulfonyl fluoride), pepstatin A, E-64, bestatin, leupeptin and aprotinin (Sigma Chemical Co.). Protein lysates were clarified by centrifuging at 13,000 x g for 15 minutes at 4°C. Protein concentration was determined by Quick Start Bradford Protein Assay (Biorad Laboratories, Hercules, CA). Proteins (50 µg for placenta tissue, 75 µg for villous explants and 100 µg for BeWo cells) were separated on 10% and 12% polyacrylamide gel for ERs (ERα, ERβ) and caspase-3, respectively, in the presence of SDS and β-mercaptoethanol. After electrophoresis the gel was removed and equilibrated in transfer buffer (20 mM Tris, 190 mM glycine and 20% [v/v] methanol pH 8.3) for 5 minutes at room temperature. Proteins were transferred to nitrocellulose filters (Hybond-C, Amersham International, Little Chalfont, UK) for 1.5 hours. The blot was incubated in blocking solution (5% [wt/v] powdered milk in 10 mM PBS, 0.1% Tween 20) for 1 hour and exposed to rabbit anti-human ERα antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:2000, rabbit anti-human ERβ antibody (Affinity Bioreagent, Golden, CO) diluted 1:1000 and goat anti-human caspase-3 antibody (R&D Systems, Abingdon, UK) diluted 1:1000 with overnight incubation at 4°C. The nitrocellulose filter was

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and 24 hours and processed for western blot analysis of caspase-3 expression.

7 washed three times with PBST (0.1% Tween 20 in PBS 10 mM) and exposed for 1 hour to the swine anti-rabbit antibody for ERs (dilution 1:3000) or rabbit anti-goat antibody for caspase-3 (dilution 1:1000), respectively, labeled with peroxidase (Biorad Laboratories) at room temperature. The blot was washed three times with PBST and visualized with a chemiluminescence kit (ImmunStar Chemiluminescent Kit) (Biorad Laboratories) according to the manufacturer' s instructions. Equal loading of the proteins was confirmed by staining the blots with a 10% (v/v) Ponceau S solution (2% Ponceau S in 30% trichloroacetic acid/30% sulfosalicylic acid, Sigma Chemical Co). Immunohistochemistry. Immunohistochemical staining was performed on formalin fixed, paraffin

streptavidin-biotin method with specific antibodies for ER and ER : rabbit anti-human ER (Santa Cruz Biotechnology) and rabbit anti-human ER (Affinity Bioreagent) diluted 1:200 and 1:20, respectively. Sections were dewaxed, rehydrated and washed in TBS (Tris-buffer saline, 20 mM Tris-HCl and 150 mM NaCl pH 7.6). Antigen retrieval was carried out by incubating sections in sodium citrate buffer (10 mM pH 6.0) in a microwave oven at 750 W for 15 min. Slides were preincubated with normal swine serum (Dako, Copenhagen, Denmark) and incubated overnight at 4°C with the anti-ERα and -ERβ primary antibodies. After washing with TBS for 5 minutes, the slides were incubated with a swine anti-rabbit antibody (Dako) labeled with biotin (dilution 1:500). The reaction was revealed using streptavidin-biotin complex (Dako). Sections were not counterstained. Slides were mounted with an aqueous mounting (Merk, Darmstadt, Germany) and examined by light microscope. For each stain a negative control was carried out, replacing the specific antibody with non-immune serum immunoglobulins at the same concentration as the primary antibody.

β-hCG assay. The concentration of β-hCG in the explant culture medium was assessed by a commercial immunoenzymometric assay (IEMA) (Radim SpA, Pomezia, Italy) and expressed in relation to protein concentration, determined from explant lysates by Quick Start Bradford Protein Assay (Biorad Laboratories) at 4, 24, 48 and 72 hours of incubation.

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embedded first trimester placental tissue. Tissue sections (4 µm) were processed by the

8 Statistical analysis. Values are means ± SEM. Data were compared by ANOVA followed by Fisher’s least significant difference post hoc test as appropriate. p values 0.05 were considered statistically significant.

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9 Results Expression of estrogen receptors in human placental tissue Western blot analysis performed in first trimester (7-12 weeks) and term placental tissues revealed expression of ERα corresponding to the three known isoforms of 67 kDa, 46 kDa and 39 kDa (Denger et al., 2001) (Figure 1A). Analysis using antibody against ERβ showed one band of 55 kDa (Figure 1B). Immunohistochemistry in first trimester placenta sections showed strong expression of ERα in most chorionic villi, predominantly in the villous cytotrophoblast layer (Figure 2A). Weak staining for ERα was also observed in some stromal cells of the villous core while no staining was

and in only few cells of the cytotrophoblast; no staining was observed in the stromal region (Figure 2B). Effects of p-NP and 17β-E2 on first trimester villous explant cultures a. ER expression. Treatment of first trimester villous explant cultures with p-NP or 17β-E2 did not produce significant differences compared to untreated cultures (controls) in the expression of ERs at 24, 48, 72 hours of incubation (Figure 3A). Western blot analysis showed expression of three bands for ERα at 67 kDa, 46 kDa and 39 kDa and a single band of 55 kDa for ERβ (Figure 3B). b. β-hCG concentration. β-hCG concentrations increased throughout incubation in treated (17β-E2 or p-NP) and untreated cultures (controls) (Figure 4). A significant increase in β-hCG production with respect to control cultures was observed at 48 hours, in 17β-E2 (p 0.05) and in p-NP (p 0.05) treated cultures. Although a reduced effect for 17β-E2 was observed at 72 hours, a massive response was observed in p-NP treated cultures at the same time (p 0.001, p-NP treated versus control cultures and p 0.05, p-NP treated versus 17β-E2 treated cultures). c. Trophoblast apoptosis. The increased levels of β-hCG associated with p-NP treatment suggested differentiation of cytotrophoblast into syncytiotrophoblast with an increase in apoptosis (Huppertz et al., 2002). As cleaved caspase-3 expression has been documented as a marker of trophoblast

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observed in the syncytiotrophoblast (Figure 2A). ERβ was localized in the syncytiotrophoblast layer

10 apoptosis, we assessed its expression by western blot analysis in explants treated with p-NP or 17βE2 or medium alone (control cultures) after 24, 48 and 72 hours of incubation. The three bands of the cleaved form of caspase-3 (20 kDa, 18 kDa and 16 kDa) were detected in p-NP and 17β-E2 treated explant cultures after 72 hours of incubation (Figure 5). None of these bands were detected in control cultures (Figure 5). Apoptosis in BeWo cells The cleaved forms of caspase-3 were found in p-NP treated cultures after one hour of treatment, whereas the caspase pathway only started to be activated in Forskolin and 17β-E2 cultures after 24

NP-treated cultures after 6 hours. This 32 kDa band disappeared at 24 hours, suggesting complete cleavage of the protein (Figure 6).

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hours of incubation (Figure 6). A sharp decrease in the pro-caspase form was also observed in p-

11 Discussion Placental development is a key event for the success of pregnancy (Cross et al., 1994). Abnormalities in this event may lead to embryo developmental defects and death (Chaddha et al., 2004). It is therefore important to identify factors that may interfere with the processes characterizing the first phases of pregnancy. In our study we investigated whether p-NP, a metabolite of alkylphenol ethoxylates, may interfere with placental development. We demonstrated that the first trimester human placenta is an estrogentarget tissue expressing ERα and ERβ proteins. Using an in vitro model of chorionic villous

Unlike cultures of isolated cells, this model has the advantage of preserving the topology of chorionic villi and maintaining the paracrine relations between the different cell components i.e. cyto- and syncytio-trophoblast, trophoblast and stromal cells (Genbacev et al., 1992). This is why it appears to be a good model for studying substances interfering with the homeostasis of placentation (Miller et al., 2005). Using explant cultures from first trimester placentas, we demonstrated that pNP mimics the effects of estrogens, increasing hCG secretion and cell apoptosis. Surprisingly, at equimolar concentrations of 10-9 M, the effect of p-NP on hCG secretion was significantly greater than that of 17β-E2. Moreover, hCG secretion induced by p-NP increased up to 72 hours, whereas that induced by 17β-E2 peaked at 48 hours and was much lower by 72 hours. Evidence of cell apoptosis, shown by caspase-3 fragmentation, was detected at 72 hours in 17β-E2- and p-NPtreated tissues. Since the trophoblast is not the only cell component in villous explant cultures, we used a trophoblast-derived choriocarcinoma cell line, BeWo, to verify the effects of p-NP on cell differentiation, syncytialization and apoptosis. Previous reports showed that 17β-E2 treatment of BeWo cells induced differentiation by formation of multinucleate syncytial structures (Rama et al., 2004). In this study we demonstrated that like 17β-E2 and forskolin, an inducer of cell differentiation, p-NP induces apoptosis in BeWo cells by cleavage of caspase-3. This effect was

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explants, we then compared the effects of p-NP and 17β-E2 on first trimester human trophoblast.

12 observed after one hour of treatment with p-NP but not until 24 hours of treatment with 17β-E2 or forskolin. The apoptotic effect of p-NP by the caspase-3 pathway was recently demonstrated by Kudo et al., (2004), in neural stem cells. Taken together, the present findings indicate that p-NP exerts estrogen-like effects on first trimester placentas, affecting trophoblast differentiation and apoptosis. It is noteworthy that p-NP activity was greater and longer lasting than that of 17β-E2, suggesting that metabolism of p-NP in the trophoblast produces more stable and possibly more active metabolites. Reports on in vitro systems e.g. recombinant yeast (Gaido et al., 1997), primary trout hepatocytes

cells (White et al., 1994), have shown that p-NP is about 1000 to 10 000 times less potent than 17βE2. These studies also showed that the lowest effective concentrations of p-NP ranged from 100 nM to 1 µM. Our findings showing that p-NP is more potent than 17β-E2 at 10-9 M, a concentration 100-1000 times less than that reported in other in vitro systems and at least than 1000 times lower that levels found in humans (Kawaguchi et al. 2004; Inoue et al. 2000; Tan and Nohd, 2003). In fact concentration of 10-9 M used in the present study is about 220.36 ng/L while the levels of p-NP detected in human samples vary from 0.3 to 221.7 ng/mL in plasma and blood samples (Kawaguchi et al., 2004; Inoue et al., 2000) and 15.17ng/mL in human cord boold samples (Tan and Nohd, 2003) suggesting that the trophoblast is extremely sensitive to this chemical. In a recent paper, Bukovsky et al., (2003b), showed that cell nuclei distant from the syncytiotrophoblast exhibited many ERα, while cell nuclei associated with the syncytiotrophoblast showed fewer ERα and some ERβ. The same authors also showed that estrogens may play a role, via ERα, in the stimulation of cytotrophoblast differentiation into syncytiotrophoblast (Bukovsky et al., 2003a). In line with Bukovsky et al., (2003b), we showed here that expression of ERα is mainly localized in the cytotrophoblast whereas that of ERβ is predominantly distributed in the

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(Flouriot et al., 1995), MCF-7 cell line (Blom et al., 1998; Nagel et al., 1997) and transfected avian

13 syncytiotrophoblast of the chorionic villi. Further studies are necessary to investigate the exact role of each isoform and the mechanism of action of estrogen-like compounds. Differentiation of trophoblast into syncytiotrophoblast is a physiological event in placental development: the internal cytotrophoblast cells aggregate and their plasma membranes fuse (Potgens et al., 2002). The syncytiotrophoblast, the external layer of villi, is an endocrine tissue producing hCG and placental lactogen. It also exchanges nutrients and gases between mother and fetus. The physiological turnover of placental epithelium produces syncytial knots which are shed apoptotically into the maternal bloodstream (Huppertz et al., 2002; Straszewski-Chavez et al.,

disorders including pre-eclampsia (Allaire et al., 2000; Crocker et al., 2004;). Our finding of increased hCG secretion and cell apoptosis after exposure to the endocrine disruptor p-NP suggest that maternal exposure to this chemical may lead to aberrant or adaptive placental cell turnover in the uterus. This could cause early termination of pregnancy, gestational pathologies and fetal growth defects. In conclusion, our data show that p-NP induces an estrogen-like response in first trimester human placenta by increasing trophoblast differentiation and apoptosis. The power of p-NP on early placenta even at low concentrations raises considerable concern about the implications of exposure to this chemical for the fetus and pregnancy.

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2005). Abnormal apoptosis, leading to faulty placentation, is involved in several gestational

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70-76. Potgens, A.J., Schmitz, U., Bose, P., Versmold, A., Kauffman, P., and Frank, H.G. (2002). Mechanism of syncytial fusion: a review. Placenta 23, 107-113. Rama, S., Petrusz, P., and Rao, A.J. (2004). Hormonal regulation of human trophoblast differentiation: a possible role for 17 -estradiol and GnRH. Mol. Cell. Endocrinol. 218, 9-94. Rudel, R.A., Camann, D.E., Spengler, J.D., Korn, L.R., and Brody, J.G. (2003). Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrine-disrupting compounds in indoor air and dust. Environ. Sci. Technol. 37, 4543-4553. Safe, S.H. (1995). Environmental and dietary estrogens and human health. Is there a problem? Environ. Health. Perspect. 103, 346-351. Sonnenschein, C., and Soto, A.M. (1998). An update review of environmental estrogen and androgen mimics and antagonists. J. Steroid. Biochem. Mol. Biol. 65, 143-150. Soto, A.M., Justicia, H., Wray, J.W., and Sonnenschein, C. (1991). p-Nonylphenol: an estrogenic xenobiotic released from “modified” polystyrene. Environ. Health. Perspect. 92, 167-173. Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., and Serrano, F.O. (1995). The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health. Perspect. 103, 113-122.

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18 Straszewski-Chavez S.L., Abrahams V.M., Mor G.(2005). The role of apoptosis in the regulation of trophoblast survival and differentiation during pregnancy. Endocr Rev. 26, 877-897.Tan B.L.L., and Nohd M.A. (2003). Analysis of selected pesticides and alkylphenols in human cord blood by gas chromatography-mass spectrometer. Talanta 61, 385-391. Weiss, B. (1998). A risk assessment perspective on the neurobehavioral toxicity of endocrine disruptors. Toxicol. Ind. Health. 14, 341-359. White, R., Jobling, S., Hoare, S.A., Sumpter, J.P., and Parker, M.G. (1994). Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135, 175-182. Downloaded from http://toxsci.oxfordjournals.org/ by guest on June 1, 2013

19 Figure legends Figure 1. Representative western blot for Estrogen Receptors (ERα α and ERβ) β) in total placental lysates at first trimester (7, 10, 12 weeks) and term pregnancy. Three bands at molecular weights about of, 67, 46 and 39 kDa were obtained for ERα (Α); a single band at 55kDa was obtained for ERβ (Β). Expression of estrogens receptor is the same in both, term placenta (term) and first trimester human placenta (7, 10, 12 weeks). Ponceau staining shows equal protein loading. Figure 2. Immunohistochemical localization of ERα α and ERβ β in placental tissues at 8 weeks of

human ERα and ERβ polyclonal antibodies. Reddish staining represents immunopositivity. (A) Strong immunoreactivity for ERα was present in the villous cytotrophoblast (arrow) and in some stromal cells of the villous core (arrow head); the syncytiotrophoblast was negative. (B) ERβ immunoreactivity was expressed by the syncytiotrophoblast (arrow) and by only few cells of the cytotrophoblast (arrow head). Original magnification x20. Figure 3. Representative western blot analysis for ERα α and ERβ β in treated (E2 or p-NP) and untreated (C) chorionic villous explants at different times of incubation. Treatment with 17β estradiol (E2) or p-nonylphenol (p-NP), on first trimester explants, did not produce any significant change in ERα (A) or ERβ (B) expression during different times of incubation. Ponceau staining shows equal protein loading. Figure 4. β -hCG concentrations, detected by immunoenzymometric assay (IEMA) in culture medium of treated (E2 or p-NP) and untreated (C) chorionic villous explants at different times of incubation. The values are the mean ± SEM of five separate experiments. Results are expressed in mIU/µg of total proteins. Significantly higher levels of β-hCG were detected in 17-β estradiol (E2) and p-nonylphenol (p-NP) treated cultures when compared to controls, at 48 hours of incubation (*p 0.05, E2 vs C; ** p 0.05, p-NP vs C). Note that β-hCG concentrations were

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pregnancy. Immunohistochemistry was performed by the streptavidin-biotin method and anti-

20 significantly higher in p-NP than in E2 treated cultures at 72 hours (°p 0.05, p-NP vs C; °° p 0.001, p-NP vs E2). Figure 5. Representative western blot for caspase-3 in treated (E2 or p-NP) and untreated (C) chorionic villous explants at different times of incubation. Note that caspase-3 fragments of about 20-16 kDa were obtained in 17-β estradiol (E2)- and p-nonylphenol (p-NP)- treated cultures at 72 hours of incubation. Ponceau staining shows equal protein loading. Figure 6. Representative western blot for caspase-3 in treated (E2, p-NP, or Fk) and untreated (C) BeWo cells. Note that caspase-3 fragments were obtained at 20-16 kDa after 1 hour of

Forskolin (Fk)- treated cultures. The pro-caspase-3 band at 32 kDa was drastically reduced at 6 hours in p-NP- treated cultures. Ponceau staining shows equal protein loading.

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incubation in p-nonylphenol (p-NP)- treated cultures and at 24 hours in 17-β estradiol (E2)- and

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