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Bisphenol-A impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells
Accepted Manuscript Title: Bisphenol-A impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells Authors: Sanjay Basak, Vilasagaram Srinivas, Asim K. Duttaroy PII: DOI: Reference:
S0890-6238(18)30233-8 https://doi.org/10.1016/j.reprotox.2018.10.009 RTX 7752
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
8-6-2018 11-10-2018 17-10-2018
Please cite this article as: Basak S, Srinivas V, Duttaroy AK, BisphenolA impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells, Reproductive Toxicology (2018), https://doi.org/10.1016/j.reprotox.2018.10.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Bisphenol-A impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells a
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Sanjay Basak a,b, Vilasagaram Srinivas b and Asim K. Duttaroy a * Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University
of Oslo, Oslo, Norway, National Institute of Nutrition, Hyderabad, India
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b
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*Corresponding Author
Department of Nutrition
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Institute for Basic Medical Sciences,
Norway
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N-0316 Oslo
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University of Oslo POB 1046 Blindern
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Tel: +47 22 85 15 47
Fax: +47 22 85 13 41 Email: [email protected]
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Professor Asim K. Duttaroy
2 Highlight BPA (0.1nM) at very low concentration significantly decreased cell proliferation without affecting growth and viability of the cells
BPA upregulated the expression of key glucocorticoid 11-β-hydroxysteroid dehydrogenase 2 in these cells
BPA impaired both basal- and VEGF-stimulated tube formation with concomitant downregulation of the expression of angiogenic growth factors
BPA down regulated CpG methylation of the gene promoters associated with metabolic stress and oxidative damage in first trimester trophoblast cells
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Abstract
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Humans are exposed to Bisphenol A (BPA) from the consumer products and plastic substances. However, impacts of low levels of BPA exposure on placental developmental processes such as first trimester trophoblast cell growth, angiogenesis and epigenetic modifications are not well studied. Low concentration of BPA (1nM) affected cell proliferation of human placental first trimester trophoblasts using a model cell, HTR8/SVneo. BPA abolished both basal- and vascular endothelial growth factor (VEGF)-stimulated tube formation in these cells. BPA significantly down regulated mRNA expression of VEGF, proliferating cell nuclear antigen, intercellular adhesion molecule 1 with concomitant upregulation of 11-β-hydroxysteroid dehydrogenase 2 mRNA and protein expression in HTR8/SVneo cells. BPA also lowered CpG methylation of gene promoter associated with metabolic and oxidative stress. This study demonstrated that BPA at 1nM not only affected cellular growth, development and angiogenic activities but also affected DNA methylation of stress response and down-regulation of angiogenic growth factors in first trimester trophoblast cells.
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Abbreviations: BPA, Bisphenol-A; ERRγ, estrogen related receptor gamma; VEGF, vascular growth endothelial factor; PCNA, proliferating cell nuclear antigen; ICAM1, Intercellular adhesion molecule 1; HSD11β2, 11-β-hydroxysteroid dehydrogenase 2; MTT, 3-(4,-
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dimethylthiazol-2-yl)-2,-diphenyl tetrazolium bromide
Key words: Bisphenol; Tube formation; Thymidine assay; VEGF; HTR8/SVneo; First trimester placenta; PCR-array; DNA methylation
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BPA (0.1nM) at very low concentration significantly decreased cell proliferation without affecting growth and viability of the cells
BPA upregulated the expression of key glucocorticoid 11-β-hydroxysteroid dehydrogenase 2 in these cells
BPA impaired both basal- and VEGF-stimulated tube formation with concomitant downregulation of the expression of angiogenic growth factors
BPA down regulated CpG methylation of the gene promoters associated with metabolic stress and oxidative damage in first trimester trophoblast cells
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1. Introduction
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Bisphenol-A (BPA), an endocrine disruptor, is widely integrated in plastic and other substances
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[1]. BPA can leach from these items in contact with food and drink. Given the ubiquity of BPA in human environments, it is not surprising that exposure to BPA is virtually universal. BPA is
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present in pregnant women and in newborns [2]. In US, exposure to BPA was reported as high
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as 96% of pregnant women [3]. BPA is found in concentrations of 1–18 ng/mL in the maternal serum, 1–10 ng/mL in amniotic fluid and cord serum taken at birth and up to 100 ng/g in
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placenta [2, 4-7]. Due to its estrogen-like activity, BPA is thought to affect human early developmental processes. In fact, early developmental exposures of low level of BPA resulted in
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numerous development abnormalities [8-12]. The higher incidence of premature delivery and intrauterine growth restriction was speculated due to an increased exposure of BPA [13]. BPA at
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environmental doses disturbed placental angiogenesis and morphology in mice [14]. BPA was also shown to affect hCG secretion by placental trophoblasts, and the apoptotic activity [15]. BPA downregulates expression of the steroidogenic genes such as CYP11A1 and CYP19 via the ERK signaling pathway thus suggested one of the possible mechanisms for observed BPA-
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mediated recurrent miscarriages in humans [16]. BPA inhibited invasion of decidual stromal cells by decreasing the expression of CXCL8 [17]. BPA can be transported across the human placenta, the trophoblast cell monolayer and thus make itself available for feto-placental exposure [15]. Accumulated BPA may induce damages
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in feto-placental growth and development [3, 18]. Placental estrogen-related receptor gamma (ERRγ) is shown to be responsible for the placental accumulation of BPA [19, 20].
Although the tolerable daily intake of BPA was reduced from 50 to 4 μg/kg (body weight)/day by the European Food Standards Agency in 2015, however, the lower levels of BPA exposure in human pregnancy are yet to be ascertained [7]. Exposure to BPA at even at lower doses may lead
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to a functional or structural insufficiency of the placenta that might ultimately affect the feto-
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placental outcome. BPA affects extra villous pathways of human trophoblast cells HTR8/SVneo
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but at relatively higher concentration than reported through environmental exposure [21]. BPA
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induces apoptosis and affects gene expression in placental cells at a high concentration [22]. BPA modulates gene expression with direct and trans-generational effects through epigenetic
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modification such as methylation of CpG island [23, 24]. However, studies of the mechanisms
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by which BPA affects the human placental cells are limited [3]. In contrast, BPA inhibits cell death under stress conditions in last trimester placental cells, BeWo cells [25]. Therefore, it is
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important to investigate the effects of BPA at low concentration on cell viability, proliferation and epigenetic changes of DNA methylation in first trimester placental trophoblast cells.
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The present study aimed to investigate the exposure of low concentrations of BPA on growth and
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development, and functionality of the first trimester placental trophoblasts.
2. Materials and Methods 2.1
Materials
The HTR8/SVneo trophoblast cell line was gifted by Dr. C.H. Graham, Queen’s University, Canada. Human vascular endothelial cells, EA.hy926 was purchased from ATCC, USA. Bisphenol A (#239658), Bisphenol S (#103039), Methyl thiazolyldiphenyl-tetrazolium bromide
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(MTT#5655), Trypsin-EDTA (#T3924), penicillin-streptomycin solution (#P4458), RPMI-1640 (# R0883), Fetal bovine serum (#F7524), L-glutamine (#G7513), VEGF receptor tyrosine kinase (SU5416) inhibitor were obtained from Sigma Aldrich, Germany. Matrigel (#356230) was procured from BD Biosciences, USA. Thymidine (3H]- (#NET027 X250UC) was purchased
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from PerkinElmer, USA.
Methods
2.2.1 Cell culture
Human first trimester trophoblast cells, HTR8/SVneo and human endothelial cells EA.hy926 were cultured in RPMI with 5% FBS supplemented with 2mM L-glutamine and 1% antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin)[26]. The cells were routinely maintained at
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37°C in a 5% CO2 atmosphere. The cells were sub-cultured using a trypsin-EDTA solution to
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suspend the cells. Assay media with 1% FBS (fetal bovine serum) was used for treating
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HTR8/SVneo cells with chemicals. Bisphenols (A & S) powders were dissolved in absolute alcohol and stored in dark glass container. Stock solution was diluted with working media prior
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to their treatment to the cells. 2.2.2 Cytotoxicity assay
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The cytotoxicity effect of BPA and BPS was determined by lactate dehydrogenase (LDH)
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releases in HTR8/SVneo cells after incubating these cells for 24h according to the instruction of the supplier (Cat. No.04744926 001, Roche, Germany)
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2.2.3 Cell viability by MTT assay Cell viability assay was performed in 96-well plate by using MTT dye. HTR8/SVneo and
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EA.hy926 cells (5x103/well/100µl) were seeded homogeneously using 8-channel pipette and loading reservoir in 5% FBS-RPMI. Cells were cultured overnight at 37°C in 5% CO2 and
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starved subsequently for 4h in low FBS (1%) media prior to add assay media (100µl/well). After 24-48h, assay media was replaced by FBS free media. Cells were incubated for 4h in presence of MTT (0.5ng/ml of DPBS) at 37°C in 5% CO2 followed by their incubation in solubilization buffer (10% SDS, 0.01M HCl) overnight. Optical density (OD) was measured at 562 nm using Synergy H1 hybrid multimode reader (Biotek Instrument, USA). 2.2.4 Cell proliferation by thymidine (3H) incorporation assay
7 Proliferation assay measured the incorporation of [3H] thymidine, into the newly synthesized DNA produced during cellular replication of HTR8/SVneo[26]. In brief, 60-70% confluent cells were pre-starved with 1% FBS RPMI for 4h followed by incubation with assay media for 24h. [3H] thymidine was added as 0.3µCi per 100ul @50ul per well. Cells were trypsinized to ensure
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homogeneous suspension and harvested with the help of Unifilter 96 (Laborel, Pakard) by transferring to unifilter 96 GF/B plate (Perkin Elmer #6005177). The incorporated radioactivity [3H] was counted using liquid scintillation counter (Topcount; Packard Instrument) after adding microsint LSC cocktail (20μl/well, PerkinElmer #6013611) to each well. Count per minute (cpm) is compared with cases and control after deducting blank value. 2.2.5 Tube formation assay
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Tube formation assay was performed in 96 well plate in matrigel that provides the substrate
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necessary to study angiogenesis in vitro [26]. Lower passage, 60-70% confluent, HTR8/SVneo
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cells were FBS starved for 48h. Plate was coated with liquid growth factor reduced matrigel (40µl/well) under chilling condition and incubated for solidification at 37°C for 30 min. Cells
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(5x104/well) were seeded in matrigel-coated well prior to add assay media and incubated for 78h at 37°C in 5% CO2. The wells were captured at least five different fields per well by an
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inverted microscope at 4X magnification (Nikon TS100F, Japan). Images were analyzed by
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Wimasis software (Onimagin Technologies SCA, Spain). 2.2.6 Analysis of gene expression by real-time PCR
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Total RNA was isolated from cells by using RNeasy mini kit (#74104, Qiagen,). cDNAs were synthesized from total RNA by using cDNA kit (#4368814, Applied Biosystems, USA). Gene
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expression was quantified on the basis of mRNA level expression of the target genes using ABI7900HT system. Predesigned SYBR green I primers, (# KSPQ12012 Sigma) were used
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(Sup.Table1) for this purpose. The Ct value of an endogenous control gene (TBP) was subtracted from the corresponding Ct value for the target gene resulting in the delta Ct value which was used for relative quantification of mRNA expression by the comparative Ct method (2-ΔΔCt method). 2.2.7 Analysis of DNA promoter methylation of human Stress & toxicity genes by real-time PCR array
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This method involves the restriction digestion of genomic DNA combined with real-time PCR to investigate promoter methylation status of genes key to human stress and toxicity pathway. It essentially estimates CpG island DNA methylation profiling of individual genes. 2.2.7.1 Restriction digestion
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In brief, genomic DNA was purified by DNeasy kit (#69504, Qiagen) from BPA (0nM and 1nM) exposed HTR8/SVneo cells. Each sample with equal amount of purified genomic DNA were digested in four separate setups consists of control digest (Mo), methylation sensitive (Ms), methylation dependent (Md) and double digests (Msd) by using DNA restriction kit (#335452, Qiagen). Genomic DNA (2µg) was diluted with 100µl restriction digestion buffer (5x) to make final volume till 470µl with RNase/DNase free water. Diluted DNA from each sample was
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divided into four reaction setup as shown in Sup. Table 2. Digestion was performed at 37ºC for
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using water bath and stored -20ºC for further use.
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6h in a heating block. Reaction was stopped by heat inactivation of enzyme at 65ºC for 20min
2.2.7.2 Real-time PCR to quantify DNA methylation
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This method ultimately quantified leftover digested DNA of each gene by real-time PCR using predesigned primers that flank promoter (gene) region of interest. Real-time PCR was performed
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in 384-well plate in ABI7900HT (Applied Biosystem) system using epitect® PCR array kit
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(#335222 EAHS-3580ZE, Qiagen). Each of 94-genes of this array was amplified with equal amount of four digested DNA (Mo, Ms, Md and Msd) with high specificity and amplification
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efficiency to detect promoter DNA methylation of genes linked with human stress & toxicity pathway. Realtime PCR setup was prepared for each of the four digestions (Mo, Ms, Md and
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Msd) of every sample as shown in Sup. Table 3. Final reaction mixture (10µl) was added carefully to the appropriate well of the 384-well array plate. PCR was programmed as : 95ºC for
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10 min with one cycle, annealing at 99ºC for 30sec followed by 72ºC for 1min for 3 cycles and denaturation at 97ºC for 15 sec followed by 72ºC for 1min for 40 cycles. 2.2.7.3 Data analysis Methylation PCR array data provided gene methylation status as percentage of unmethylated and methylated fraction of input genomic DNA. The relative fractions of methylated (M) and unmethylated (UM) DNA were determined by comparing the amount in each digest with that of
9 a control (no enzymes added) digest using a ΔΔCt method .SEC (methylation sensitive enzyme control) and DEC (methylation dependent enzyme control) were included in array as a quality control in digestion efficiency of methylation-sensitive enzyme A and methylation-dependent enzyme B, respectively. In general, the value ΔCt (Ms-Mo) ≥4 for SEC or ΔCt(Md-Mo)≥4 for
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DEC indicates more than 93.8% of control DNA molecules are digested where the restriction enzymes are active and digests DNA efficiently. When the refractory DNA percentage was greater than 12.5 percent (R > 12.5%), the digestions were incomplete and analysis was reported as a "Failure". 2.2.8 Immunoblotting
Expression of HSD11β1 and HSD11β2 proteins were measured by immunoblotting after pre-
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stimulation of the HTR8/SVneo trophoblast cells with BPA and BPS (1nM) for 24h.
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Immunoblotting was performed to determine the expression of proteins in similar way as
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described previously. However, this study used 10 µg of protein/lane to transfer to PVDF membrane [27]. After blocking, blots were incubated with GAPDH (1:5000 # PA534847,
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Thermo Scientific Pierce, USA), 11β-HSD2 (1:5000 # sc 20176) and 11β-HSD1 (1:5000 # sc20175) antibodies (Santacruz, USA). Blots were further incubated with HRP-conjugated
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2.2.9 Statistical analysis
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secondary antibodies and were processed as per the procedure described previously [27].
Data were analyzed by unpaired Student’s t-test to compare treatments over control using
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Graphpad prism 4. Statistical significance was considered when p value <0.05. Majority of the data are obtained at least from three independent experiments as indicated elsewhere and
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expressed as mean ± SEM.
3. Results
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3.1 BPA and BPS exposures decrease the numbers of viable HTR8/SVneo cells In order to assess the exposure of bisphenols on cell growth and viability, we performed doseresponse experiment in both HTR8/SVneo cells and non-trophoblast cells (EA.hy926 )using MTT assay. Compared to control, cell viability was decreased consistently when cells were exposed with 1-100nM of BPA and BPS in HTR8/SVneo cells (Fig.1A). BPS, the predominant replacement chemical for BPA[28] was used for comparison purpose.
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At 1nM concentration, viability of HTR8/SVneo trophoblast cells was decreased significantly by >15% of the total (control vs. BPA and BPS: 99.99 ± 0.041 vs. 81.16 ± 0.464 and 83.63 ± 0.411 percent; n=8). The concentration of 1nM was considered for subsequent assays since viability was not affected by more than 20% of the total. This concentration response study revealed that
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first trimester trophoblast cells are well viable in the ranges of 0.001 to 1nM exposure of BPA and BPS for 24h (Fig.1A). Cellular viability was further measured at higher concentration (1nM -100nM) with exposure of 48h and 72h in presence of BPA and BPS. Viability of these cells was decreased with increasing exposure duration and concentration (data not presented). Effect of bisphenols on cell growth and viability was also investigated in EA.hy926 cells after exposure with BPA and BPS for 24h. Unlike HTR8/SVneo cells, the cell viability was not significantly
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affected when human vascular endothelial EA.hy926 cells were exposed with 0.001-10nM of
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BPA or BPS, the exposure at much higher concentration (100nM BPA or BPS) only significantly
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reduced the cell viability (Fig.1B). These data suggest HTR8/SVneo cells are more sensitive to BPA or BPS compared with non-trophoblastic EA.hy926 cells.
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3.2 BPA and BPS exposures decrease proliferation of HTR8/SVneo cells Effects of BPA and BPS exposure on cellular proliferation of the HTR8/SVneo cells were
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investigated by measuring active DNA synthesis of the growing cells using [3H] -thymidine
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assay after exposing the cells with (0.1-10nM) of BPA and BPS for 24h. Compared to control, [3H] thymidine incorporation [3H] was decreased significantly by ~10-12% (control vs. 0.1nM
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and 1nM BPA: 52090 ± 1515cpm vs. 47220 ± 917cpm and 45890 ± 906 cpm, n=9) when these cells were exposed to low concentrations of BPA (0.1-1nM) for 24h (Fig.2). A concentration
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dependent decrease was recorded in the proliferation of HTR8/SVneo cells when these cells were exposed to environmentally relevant concentration of BPA (1nM). Despite the fact that BPS is
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considered as safer substitute of BPA, BPS (0.1-1nM) significantly decreased radioactive thymidine incorporation [3H] to a greater extent (~14-18%) as compared BPA (control vs. 0.1nM and 1nM BPS: 52090 ± 1515cpm vs. 44970 ± 488cpm and 42940 ± 599cpm, n=9). Both BPA and BPS exposures decreased proliferation of HTR8/SVneo cells. 3.3 BPA and BPS exposures inhibited basal- and VEGF-stimulated tube formation in HTR8/SVneo cells
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VEGF (10ng/ml) significantly increased tubular network (control vs. VEGF 10ng/ml: 24650 ± 193 pixel vs. 27360 ± 156 pixel; n=9) of HTR8/SVneo trophoblast cells (Fig.3 A-B). VEGFstimulated tube formation was decreased by 32%, 26% and 17% in presence of 150nM SU5416, 1nM of BPA and BPS, respectively (VEGF vs. VEGF+SU5416, VEGF+BPA and VEGF+BPS:
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27360 ± 156 pixel vs. 18600 ± 274 pixel, 20090 ± 226 pixel and 22630 ± 267 pixel; n=9). Both BPA and BPS partially disrupted tube formation induced by VEGF (Fig.3A). In addition, BPA (1nM) and BPS (1nM) by themselves also prevented the sprouting of spontaneous tube formation significantly by 31% and 22% respectively, (control vs. BPA and BPS: 23030 ± 548 pixel vs. 15700 ± 265 pixel, and 17820 ± 203 pixel; n=9, p<0.05 ) in HTR8/SVneo cells. Basal- and VEGF stimulated tube formation of the HTR8/SVneo cells were inhibited by the exposure of
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both BPA and BPA.
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3.4 Exposure of BPA and BPS altered mRNA expression of angiogenesis, endocrine and
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metabolic mediators in HTR8/SVneo cells
Expression of several angiogenesis, endocrine and metabolic mediators were significantly
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modulated after exposing HTR8/SVneo cells with BPA (1nM) and BPS (1nM) for 6h and 24h. Compared to 6h, mRNA expression yielded more stable and reproducible Ct values across the
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replicates at 24h (data not presented). Expression of mRNA those encode VEGFA and
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ANGPTL4, proliferation and cell adhesion mediators such as proliferating cell nuclear antigen (PCNA) and intercellular adhesion molecule 1 (ICAM1) were significantly down regulated upon
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24h exposure of BPA (1nM) and BPS (1nM) in the HTR8/SVneo cells (Table-1). Expression of metabolic factor such as fatty acid binding protein 4 (FABP4) and regulator of cellular
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cholesterol and phospholipid homeostasis such as ATP binding cassette subfamily A member 1 (ABCA1) were significantly decreased after such exposure. Concomitant to that, mRNA
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expression of glucocorticoid such as 11-β-hydroxysteroid dehydrogenase 2 (HSD11β2) was significantly up regulated in presence of both BPA (1nM) and BPS (1nM) exposure. It is observed that changes at the level of mRNA expression were more profound with BPS (1nM) exposure in vitro as compared with BPA (1nM). However, mRNA expression of CTGF, CDH1, KRT1, KRT7, HLAG, DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), ADRP, SLC2A1, SREBF2 were unaffected. All these data demonstrated that both BPA and BPS
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exposure inhibit proliferation and tube formation of the first trimester trophoblastic cells possibly by down regulating expression of growth promoting genes such as VEGF, PCNA, ICAM-1 in the first trimester trophoblast cells, HTR8/SVneo. 3.5 Protein expression of 11β-hydroxysteroid dehydrogenases 2 increased after BPA and
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BPS exposure Compared to control, exposing cells with BPA (1nM) and BPS (1nM) significantly up-regulated protein expression of HSD11β2 by ~80% (fold expression: control vs. BPA (1nM) and BPS (1nM): 0.990 ± 0.010 vs. 1.810 ± 0.013, and 1.831 ± 0.011; n=3) while changes in the expression of HSD11β1 (fold expression: control vs. BPA (1nM) and BPS (1nM): 0.998 ± 0.004 vs. 0.8053 ± 0.06, and 0.8345 ± 0.03; n=3) remained insignificant (Fig.4A-B).
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3.6 BPA exposure altered DNA promoter methylation of cellular stress mediators In order to investigate epigenetic changes due to BPA exposure, promoter DNA methylation of
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human stress and toxicity pathway were measured. The EpiTect Methyl II PCR system was used
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to compare methylation levels of CpG islands in the promoter regions of stress and toxicity gene
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pathway. QC reported a “failure” of total 8 and 12 genes out of 94 genes in control and BPA (1nM) group respectively (data not presented). DNA methylation percentage was compared
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between the cells in the absence and presence of BPA (1nM) in the heat map (Fig. 5). In general, percentage of promoter CpG methylation was comparatively higher in control (without BPA) as
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compared to BPA-exposed cells. The status of CpG methylation obtained using Epitect Methyl II Signature PCR array on human stress & toxicity is presented in Table 2. CpG promoter sites of
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BNIP3, MT3, RAD9A, SMC1A (cell cycle, proliferation and DNA replication) and HSPA1A, XPC, GPX7 (antioxidant and heat shock protein) genes were methylated predominantly in both
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control and BPA exposed cells but in a differential manner. In presence of BPA (1nM), percentage of CpG promoter methylation of these genes were down regulated by 12-48% while
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XRCC2,CREB3, RAD23A,VCP (cell cycle, proliferation and DNA replication) , GSR,DNAJA1, PRDX2, GPX3 (antioxidant and heat shock protein) and DDIT3, HERPUD1, INSIG1 & MBTPS1 (stress and metabolism) were down regulated by 50-99% as compared with those of control cells (Table 2). Overall, the percentages of promoter methylation of stress responsive genes were low in presence of BPA (1nM) as compared to its absence in HTR8/SVneo cells.
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4. Discussion The present study demonstrates that BPA at very low concentrations (0.01 nM-1 nM) affects cell growth, proliferation, tube formation and gene expression in first trimester trophoblast cells,
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HTR8/SVneo. The levels of BPA (0.01–1 nM; 1 nM BPA=0.228 ng/ml) used in this study are much lower than the average BPA concentrations as present in the body fluid [2, 4, 5]. A recent review of 16 studies indicates that the range of serum BPA concentrations varies between 0.5 and 2.5 ng/ml [29]. Early placentation process involving growth and tube formation of first trimester trophoblasts, a critical step for optimum feto-placental growth and development, may be affected by very low concentrations of BPA (0.01–1 nM; 1 nM BPA=0.228 ng/ml). Our data
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therefore justify further in-depth work in order to determine the safe levels of BPA in human
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reproductive health. Both BPS and BPA exposure affected cell viability and growth of the
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HTR8/SVneo cells. Interestingly, both BPA and BPS exposure also prevented tube formation in
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basal and VEGF-treated cells to a similar extent. Overall, bisphenols inhibit the tube formation via inhibiting expression of genes responsible for angiogenesis such as VEGF, PCNA and
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ICAM1 in these HTR8/SVneo cells. BPA exposure selectively affects VEGFA expression in
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cancer cells as compared to non-cancer cells. Earlier data also showed that BPA lowered VEGFA expression significantly in cancer cell lines [30]. HTR8/SVneo tube-like formation embodies
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trophoblast migration and differentiation toward an invasive phenotype, a physiologic process
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that takes place during early human pregnancy. The mechanisms responsible for BPA/BPSinduced inhibition of tube formation in these cells are not yet clearly known. Since BPA/BPS
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inhibited VEGF-induced tube formation and also significantly down-regulated VEGF mRNA expression in these cells suggesting that the inhibitory action may be mediated at least in part via VEGF. However, one also could argue that the inhibitory effect of BPA/BPS on tube formation could be also in part due to the decreased cell viability. However, it may be unlikely the case, as the tube formation was measured after 6-7h of incubation whereas cell viability (20% decreased)
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measured only after 24h of incubation with the BPA/BPS. Moreover, one has to consider the facts that tube formation phenotype may involve many cellular activities such as cell viability, proliferation, metabolic fitness, and other activities[31-33]. Further work is required in order to
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understand the detailed mechanisms of BPA effects in these cells. It is difficult to assume the individual contribution of each process in tube formation under any circumstances. This in vitro study raises the possibility that BPA exposure even at very low concentration may be associated with major complications of pregnancy. BPA at very high concentration (100nM) also altered the HTR-8/SVneo cell invasion capacity by altering migration of cells
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However, further studies are required in order to understand the detailed mechanisms responsible
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for BPA- induced inhibition of angiogenesis.
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Adequate placental angiogenesis is critical for the establishment of the placental circulation and
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thus for normal fetal growth and development. It is therefore reasonable to suggest that
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inappropriate placental expression of angiogenic factors by BPA may contribute to placental
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vascular defects and placental dysfunction and therefore could be an important cause of
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infertility and fetal growth retardation. With the recent spate of clinical work on regulators of angiogenesis, these observations lead us to believe that regulation of placental angiogenesis
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could become a novel and powerful method for ensuring positive outcomes for most pregnancies. BPA exposures in pregnancy may therefore preclude optimal feto-placental growth
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and development. Further work is required on the effects of bisphenols acids in feto-placental development.
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The HTR-8/SVneo cell line was developed from first trimester extra-villous trophoblast infected with SV40 Large T antigen. HTR8/SVneo cell line has been extensively used as a sufficient model of extravillous trophoblasts. Recently, Abou-Kheir et al. [35] reported that HTR8/SVneo cells are a mixed population of cells, this group did not perform any type of DNA fingerprinting to verify the identity of their line. HTR8/SVneo cells still closely resemble with
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first trimester placental genotypes[36] and undergo similar phenotypic changes as happen in vivo placentation process[37]. During pregnancy high levels of cortisol in human are prevalent in the maternal circulation [38].
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The placental HSD11β2 protects the fetus from exposure to high levels of maternal glucocorticoids [39]. The precise control of placental HSD11β2 is critical for normal fetal organ growth and maturation. The activity and expression of HSD11β2 within the placenta correlates with fetal birth weight [40, 41], and altered placental HSD11β2 gene expression was observed in intrauterine growth restriction [40, 42]. BPA at lower concentration increased the expression of both protein and mRNA of HSD11β2. The effect of BPA-induced increase in HSD11β2 level is
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not without precedent. BPA at (0.1–2 g/ml) for 24 h increased mRNA, protein and activity of
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HSD11β2 in placental last trimester trophoblasts cells [24]. It could be compensatory effects of
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these cells of BPA exposure or simply due to its estrogenic activity, however further work is
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required for definitive conclusions. Further work using animal model will be required to determine the effects of higher levels of HSD11β2 induced by BPA on the reproductive and
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endocrine systems. The human placenta is a dynamic endocrine organ producing a wide variety
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of hormones, factors and enzymes, which function in a concerted fashion to regulate maternal adaptation to pregnancy and parturition as well as fetal development. Placental trophoblast cells
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respond to variety of exogenous agents by activating stress pathways and epigenetic changes often regulate some of these responses. This study highlighted gene-specific methylation changes
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in stress response pathways upon in vitro exposure of the early trophoblast cells to BPA. DNA methylation profiling of first trimester trophoblast genomic DNA actually correlated CpG island
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methylation status of gene promoters key to stress and toxicological responses, including oxidative stress, inflammation, DNA damage, apoptosis, hypoxia, and the unfolded protein response. Percentage of promoter methylation was lowered by BPA exposure as compared with those of control cells. DNA methylation of gene promoters typically act to repress gene transcription of mRNA those encode genes for cellular stress and toxicity pathways and thus
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stress response genes were de-repressed in presence of BPA exposure. Methylated genomic DNA could be adequate to repress gene transcription of stress and toxicity mediators which otherwise unmethylated by BPA (1nM) indicating that activation of these pathways in response to BPA exposure. A significant number of CpG methylation changes of gene promoters were
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observed in BPA-exposed cells those are also associated with cellular proliferation indicates decreased proliferation could be due to promoter hypomethylation of the cell cycle, proliferation replication associated genes. This study is limited to measure BPA alone on CpG methylation of the first trimester trophoblast cells although BPS showed similar effect on proliferation as like BPA. Since BPA is predominant endocrine hazard at this moment, emphasis was given priority
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for CpG methylation to BPA over BPS. In order to confirm further, if methylation status could
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lead to repress transcription, measuring corresponding expression levels of genes between
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control and BPA exposure is required. Mechanism of these methylation sensitivities and their
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functional consequences in the first trimester placental cells warrants further investigation. In humans, studies on the relationship between prenatal exposure to BPA and fetal growth have
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shown mixed results [5, 43-45]. Our findings point out the relevance of further research on
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long‐term consequences of altered prenatal nutrition and programming related to BPA‐induced placental and embryo effects. In addition, our findings lend further support to different
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mechanisms being elicited according to the level of BPA exposure, which may result in non‐monotonic dose–response patterns across exposure levels of different orders of magnitude.
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For the first time, we demonstrated here that BPA exposure at low concentrations (0.01nM-1nM) affected cellular growth, proliferation and angiogenic activities possibly by affecting gene
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methylation of stress response and down-regulation of growth promoting factors such as VEGF, PCNA, ICAM-1 in the first trimester trophoblast cells. In addition to these effects, BPA upregulated HSD11β2 expression, the one is responsible for detoxifying the effects of BPA or similar molecules. 5. Conclusions
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The present study demonstrates that BPA exposure at concentrations much lower than those found in the human placenta, severely disrupts gene expression and tube formation in first trimester human placental trophoblast cells. If these in vitro findings extrapolated to human pregnancy in vivo, in that case, exposure to BPA could have a significant impact on placentation
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processes that may contribute to pregnancy complications, such as spontaneous abortion, preterm birth, fetal growth restriction, and preeclampsia.
Acknowledgements
We are grateful to Camilla Solberg for her help in thymidine assay. This study was supported by
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the grant from the Thune Holst Foundation and HRD fellowship, Department of Health Research
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References
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(Dr. Sanjay Basak), Government of India.
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[43] B.E. Lee, H. Park, Y.C. Hong, M. Ha, Y. Kim, N. Chang, B.N. Kim, Y.J. Kim, S.D. Yu, E.H. Ha, Prenatal bisphenol A and birth outcomes: MOCEH (Mothers and Children's Environmental Health) study, Int J Hyg Environ Health 217(2-3) (2014) 328-34. [44] M.S. Wolff, S.M. Engel, G.S. Berkowitz, X. Ye, M.J. Silva, C. Zhu, J. Wetmur, A.M. Calafat, Prenatal phenol and phthalate exposures and birth outcomes, Environ Health Perspect 116(8) (2008) 1092-7. [45] W.C. Chou, J.L. Chen, C.F. Lin, Y.C. Chen, F.C. Shih, C.Y. Chuang, Biomonitoring of bisphenol A concentrations in maternal and umbilical cord blood in regard to birth outcomes and adipokine expression: a birth cohort study in Taiwan, Environ Health 10 (2011) 94.
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Figure legends Fig.1 Effect of Bisphenols (BPA & BPS) on growth and viability of the first trimester HTR8/SVneo cells (Fig 1A), and human vascular endothelial EA.hy926 cells (Fig 1B). Cells
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were exposed to BPA and BPS (100nM, 10nM, 1nM, 0.01nM and 0.001nM) for 24h. Control cells (vehicle) were received 1% FBS RPMI with 0.1% ethanol. OD values were compared between control and treatment after normalized with blank values and expressed as percentage of control. Data are shown as mean ± SEM (n=8) *p<0.05,**p<0.01 and ***p<0.001vs. control (Student’s t test)
Fig.2 Effect of Bisphenols (BPA & BPS) on proliferation of the trophoblast cells,
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HTR8/SVneo.3H-Thymidine incorporation into the DNA of HTR8/SVneo cells was investigated
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by radioactive thymidine [3H] assay. Cells were pulsed with [3H] thymidine and harvested, and
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counted as described in the methods. Counts per minute (cpm) were compared with control after deducting blank value. Data are shown as mean ± SEM (n=9).*p<0.05,**p<0.01 and ***p<0.001
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vs. control (Student’s t test)
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Fig. 3. Effect of BPA and BPS on VEGF stimulated tube formation of the HTR8/SVneo
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cells Tube formation assay (in vitro angiogenesis) was performed in matrigel as described in ‘‘method’’ section. FBS starved cells (5x104 ) were incubated with or without VEGF (10ng/ml).
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SU5416 (150nM) was added 1h prior to addition of VEGF. BPA (1nM) and BPS (1nM) were added to VEGF treated cells; Control= 0.1% ethanol in 1% FBS media; Image was captured after
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7-8 h of incubation of BPA and BPS in 5X magnification. (A) Representative images of tubules formed after 8h where blue colour shows tubule network. (B) Tubule network length was quantified by Wimasis software and expressed as pixel as per their instruction. Data are shown as
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mean ± SEM (n=9). *p value < 0.05 versus control; # p value < 0.05 versus VEGF (10ng/ml) Fig 4. Effects of BPA and BPS on the expression of 11β-hydroxysteroid dehydrogenases (HSD) in HTR8/SVneo cells. Cells were pre-incubated with BPA (1nM) and BPS (1nM) for 24 h. Cellular proteins were harvested and HSD11β1 and HSD11β2 proteins were measured by immunoblotting (A) and their expressions were measured as described in the method (B). Data
22 of protein expression are reported as fold expression of mean ± SEM (n=3) after normalizing expression with GAPDH; * p<0.05 vs. control (Student’s t test).
Fig.5 Effect of BPA on promoter DNA methylation of cellular stress mediators in the first trimester trophoblast cells. HTR8/SVneo cells were exposed with or without BPA (1nM) for
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24h. Cellular genomic DNAs were restricted with methylation sensitive and methylation dependent enzyme followed by quantification of digested DNA by real-time PCR using 384-well epitect array kit as described in method section. Methyl-profiler data (DNA methylation percentage) of the gene promoters involved with human stress & toxicity pathway are plotted for the control and BPA group in the heat map. Percentage of DNA methylation index is showing as
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blue and green colors.
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26 Table
Type of genes
Gene symbol
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Table 1: Effect of Bisphenol (A & S) on modulation of gene expression in first trimester trophoblast HTR8/SVneo cells 1 Relative mRNA fold over unit control expression (Target gene/TBP) 2 BPA (1nM)
Angiogenesis & cell
VEGFA
0.59 ± 0.061 (-1.7)
0.48 ±0.033 (-2.0)
ANGPTL4
0.61±0.052 (-1.6)
0.46 ±0.055 (-2.1)
PCNA
0.45 ±0.031 (-2.2)
0.06 ±0.077 (-16.0)
ICAM1
0.11±0.011 (-9.0)
0.03±0.005 (-33.0)
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proliferation factors
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Endocrine & metabolic FABP4 ABCA1
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factors
BPS (1nM)
0.03 ±0.01 (-33.0)
0.29 ±0.07 (-3.4)
0.04 ±0.037 (-25.0)
7.75±0.813 (+7.7)
6.01 ±0.77 (+6.0)
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HSD11β2
0.22 ±0.003 (-4.5)
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mRNA expression was measured after incubating cells with BPA (1nM) and BPS (1nM) for 24h.Net fold expression are depicted in parenthesis by down (-) or up (+) regulation over control. 2
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The level of each mRNA expression of genes was quantified after normalized with endogenous control, TBP and calculated according to the ΔΔCt method. Data are expressed as mean of relative mRNA fold expression over control ± SEM, n=3. p < 0.05 vs. control
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Table 2: Percentage of CpG methylation of stress & toxicity genes in response to BPA exposure of the trophoblast cells, HTR8/SVneo Change (%)
BNIP3
Control 99.93
BPA(1nM) 81.97
17.97
MT3
92.6
81.73
11.74
RAD9A
79.69
50
37.26
SMC1A
96.61
50
48.25
GADD45G
59.17
74.36
25.67
XRCC2
50
7.6
84.80
CREB3
57.55
0.5
99.13
RAD23A
56.97
0.09
VCP
66.74
0.02
DNAJC15
99.52
HSPA1A
Gene function
a pro-apoptotic factor
8.17
97.58
85.43
12.45
Heat shock protein 70 Kd protein
XPC
68.06
50
26.54
GPX7
50
95.78
91.56
GSR
83.78
0.13
99.84
DNA damage recognition and repair factor Protects against oxidative DNA damage and double-strand breaks Cellular antioxidant defense
DNAJA1
82.01
0.02
99.98
PRDX2
81.45
0.94
98.85
GPX3
51.25
1.8
96.49
Stress and Metabolism
DDIT3
52.88
2.5
95.27
HERPUD1
68.83
0.06
99.91
INSIG1
93.69
0.08
99.91
MBTPS1
91.45
0.33
99.64
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99.84
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Antioxidant & heat shock protein
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91.39
Encodes protein for growth inhibitory factor Cell cycle checkpoint protein required for cell cycle arrest and DNA damage repair Structural maintenance of chromosomes 1A Growth arrest and DNA-damageinducible protein Maintain chromosome stability and repair DNA damage Regulator of cell proliferation and migration Encodes nucleotide excision repair protein DNA repair, replication and regulation of the cell cycle Heat shock protein 40 Kd protein
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Cell cycle, proliferation and DNA replication
Methylation (%)
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Gene symbol
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Gene pathways
99.97
Heat shock protein that prevents protein aggregation Cell protection against oxidative stress Protect cells against oxidative damage and associated with Preeclampsia Transcription factor in ER stress response Homocysteine-inducible ER stressinducible ubiquitin-like domain member 1 protein Regulates cholesterol metabolism, lipogenesis and glucose homeostasis Ubiquitously expressed membrane
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bound protease that regulates cholesterol or lipid homeostasis
S0890-6238(18)30233-8 https://doi.org/10.1016/j.reprotox.2018.10.009 RTX 7752
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
8-6-2018 11-10-2018 17-10-2018
Please cite this article as: Basak S, Srinivas V, Duttaroy AK, BisphenolA impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells, Reproductive Toxicology (2018), https://doi.org/10.1016/j.reprotox.2018.10.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Bisphenol-A impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells a
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Sanjay Basak a,b, Vilasagaram Srinivas b and Asim K. Duttaroy a * Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University
of Oslo, Oslo, Norway, National Institute of Nutrition, Hyderabad, India
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b
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*Corresponding Author
Department of Nutrition
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Institute for Basic Medical Sciences,
Norway
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University of Oslo POB 1046 Blindern
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Tel: +47 22 85 15 47
Fax: +47 22 85 13 41 Email: [email protected]
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Professor Asim K. Duttaroy
2 Highlight BPA (0.1nM) at very low concentration significantly decreased cell proliferation without affecting growth and viability of the cells
BPA upregulated the expression of key glucocorticoid 11-β-hydroxysteroid dehydrogenase 2 in these cells
BPA impaired both basal- and VEGF-stimulated tube formation with concomitant downregulation of the expression of angiogenic growth factors
BPA down regulated CpG methylation of the gene promoters associated with metabolic stress and oxidative damage in first trimester trophoblast cells
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Abstract
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Humans are exposed to Bisphenol A (BPA) from the consumer products and plastic substances. However, impacts of low levels of BPA exposure on placental developmental processes such as first trimester trophoblast cell growth, angiogenesis and epigenetic modifications are not well studied. Low concentration of BPA (1nM) affected cell proliferation of human placental first trimester trophoblasts using a model cell, HTR8/SVneo. BPA abolished both basal- and vascular endothelial growth factor (VEGF)-stimulated tube formation in these cells. BPA significantly down regulated mRNA expression of VEGF, proliferating cell nuclear antigen, intercellular adhesion molecule 1 with concomitant upregulation of 11-β-hydroxysteroid dehydrogenase 2 mRNA and protein expression in HTR8/SVneo cells. BPA also lowered CpG methylation of gene promoter associated with metabolic and oxidative stress. This study demonstrated that BPA at 1nM not only affected cellular growth, development and angiogenic activities but also affected DNA methylation of stress response and down-regulation of angiogenic growth factors in first trimester trophoblast cells.
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Abbreviations: BPA, Bisphenol-A; ERRγ, estrogen related receptor gamma; VEGF, vascular growth endothelial factor; PCNA, proliferating cell nuclear antigen; ICAM1, Intercellular adhesion molecule 1; HSD11β2, 11-β-hydroxysteroid dehydrogenase 2; MTT, 3-(4,-
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dimethylthiazol-2-yl)-2,-diphenyl tetrazolium bromide
Key words: Bisphenol; Tube formation; Thymidine assay; VEGF; HTR8/SVneo; First trimester placenta; PCR-array; DNA methylation
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Highlight
BPA (0.1nM) at very low concentration significantly decreased cell proliferation without affecting growth and viability of the cells
BPA upregulated the expression of key glucocorticoid 11-β-hydroxysteroid dehydrogenase 2 in these cells
BPA impaired both basal- and VEGF-stimulated tube formation with concomitant downregulation of the expression of angiogenic growth factors
BPA down regulated CpG methylation of the gene promoters associated with metabolic stress and oxidative damage in first trimester trophoblast cells
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1. Introduction
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Bisphenol-A (BPA), an endocrine disruptor, is widely integrated in plastic and other substances
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[1]. BPA can leach from these items in contact with food and drink. Given the ubiquity of BPA in human environments, it is not surprising that exposure to BPA is virtually universal. BPA is
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present in pregnant women and in newborns [2]. In US, exposure to BPA was reported as high
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as 96% of pregnant women [3]. BPA is found in concentrations of 1–18 ng/mL in the maternal serum, 1–10 ng/mL in amniotic fluid and cord serum taken at birth and up to 100 ng/g in
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placenta [2, 4-7]. Due to its estrogen-like activity, BPA is thought to affect human early developmental processes. In fact, early developmental exposures of low level of BPA resulted in
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numerous development abnormalities [8-12]. The higher incidence of premature delivery and intrauterine growth restriction was speculated due to an increased exposure of BPA [13]. BPA at
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environmental doses disturbed placental angiogenesis and morphology in mice [14]. BPA was also shown to affect hCG secretion by placental trophoblasts, and the apoptotic activity [15]. BPA downregulates expression of the steroidogenic genes such as CYP11A1 and CYP19 via the ERK signaling pathway thus suggested one of the possible mechanisms for observed BPA-
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mediated recurrent miscarriages in humans [16]. BPA inhibited invasion of decidual stromal cells by decreasing the expression of CXCL8 [17]. BPA can be transported across the human placenta, the trophoblast cell monolayer and thus make itself available for feto-placental exposure [15]. Accumulated BPA may induce damages
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in feto-placental growth and development [3, 18]. Placental estrogen-related receptor gamma (ERRγ) is shown to be responsible for the placental accumulation of BPA [19, 20].
Although the tolerable daily intake of BPA was reduced from 50 to 4 μg/kg (body weight)/day by the European Food Standards Agency in 2015, however, the lower levels of BPA exposure in human pregnancy are yet to be ascertained [7]. Exposure to BPA at even at lower doses may lead
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to a functional or structural insufficiency of the placenta that might ultimately affect the feto-
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placental outcome. BPA affects extra villous pathways of human trophoblast cells HTR8/SVneo
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but at relatively higher concentration than reported through environmental exposure [21]. BPA
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induces apoptosis and affects gene expression in placental cells at a high concentration [22]. BPA modulates gene expression with direct and trans-generational effects through epigenetic
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modification such as methylation of CpG island [23, 24]. However, studies of the mechanisms
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by which BPA affects the human placental cells are limited [3]. In contrast, BPA inhibits cell death under stress conditions in last trimester placental cells, BeWo cells [25]. Therefore, it is
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important to investigate the effects of BPA at low concentration on cell viability, proliferation and epigenetic changes of DNA methylation in first trimester placental trophoblast cells.
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The present study aimed to investigate the exposure of low concentrations of BPA on growth and
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development, and functionality of the first trimester placental trophoblasts.
2. Materials and Methods 2.1
Materials
The HTR8/SVneo trophoblast cell line was gifted by Dr. C.H. Graham, Queen’s University, Canada. Human vascular endothelial cells, EA.hy926 was purchased from ATCC, USA. Bisphenol A (#239658), Bisphenol S (#103039), Methyl thiazolyldiphenyl-tetrazolium bromide
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(MTT#5655), Trypsin-EDTA (#T3924), penicillin-streptomycin solution (#P4458), RPMI-1640 (# R0883), Fetal bovine serum (#F7524), L-glutamine (#G7513), VEGF receptor tyrosine kinase (SU5416) inhibitor were obtained from Sigma Aldrich, Germany. Matrigel (#356230) was procured from BD Biosciences, USA. Thymidine (3H]- (#NET027 X250UC) was purchased
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from PerkinElmer, USA.
Methods
2.2.1 Cell culture
Human first trimester trophoblast cells, HTR8/SVneo and human endothelial cells EA.hy926 were cultured in RPMI with 5% FBS supplemented with 2mM L-glutamine and 1% antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin)[26]. The cells were routinely maintained at
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37°C in a 5% CO2 atmosphere. The cells were sub-cultured using a trypsin-EDTA solution to
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suspend the cells. Assay media with 1% FBS (fetal bovine serum) was used for treating
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HTR8/SVneo cells with chemicals. Bisphenols (A & S) powders were dissolved in absolute alcohol and stored in dark glass container. Stock solution was diluted with working media prior
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to their treatment to the cells. 2.2.2 Cytotoxicity assay
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The cytotoxicity effect of BPA and BPS was determined by lactate dehydrogenase (LDH)
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releases in HTR8/SVneo cells after incubating these cells for 24h according to the instruction of the supplier (Cat. No.04744926 001, Roche, Germany)
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2.2.3 Cell viability by MTT assay Cell viability assay was performed in 96-well plate by using MTT dye. HTR8/SVneo and
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EA.hy926 cells (5x103/well/100µl) were seeded homogeneously using 8-channel pipette and loading reservoir in 5% FBS-RPMI. Cells were cultured overnight at 37°C in 5% CO2 and
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starved subsequently for 4h in low FBS (1%) media prior to add assay media (100µl/well). After 24-48h, assay media was replaced by FBS free media. Cells were incubated for 4h in presence of MTT (0.5ng/ml of DPBS) at 37°C in 5% CO2 followed by their incubation in solubilization buffer (10% SDS, 0.01M HCl) overnight. Optical density (OD) was measured at 562 nm using Synergy H1 hybrid multimode reader (Biotek Instrument, USA). 2.2.4 Cell proliferation by thymidine (3H) incorporation assay
7 Proliferation assay measured the incorporation of [3H] thymidine, into the newly synthesized DNA produced during cellular replication of HTR8/SVneo[26]. In brief, 60-70% confluent cells were pre-starved with 1% FBS RPMI for 4h followed by incubation with assay media for 24h. [3H] thymidine was added as 0.3µCi per 100ul @50ul per well. Cells were trypsinized to ensure
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homogeneous suspension and harvested with the help of Unifilter 96 (Laborel, Pakard) by transferring to unifilter 96 GF/B plate (Perkin Elmer #6005177). The incorporated radioactivity [3H] was counted using liquid scintillation counter (Topcount; Packard Instrument) after adding microsint LSC cocktail (20μl/well, PerkinElmer #6013611) to each well. Count per minute (cpm) is compared with cases and control after deducting blank value. 2.2.5 Tube formation assay
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Tube formation assay was performed in 96 well plate in matrigel that provides the substrate
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necessary to study angiogenesis in vitro [26]. Lower passage, 60-70% confluent, HTR8/SVneo
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cells were FBS starved for 48h. Plate was coated with liquid growth factor reduced matrigel (40µl/well) under chilling condition and incubated for solidification at 37°C for 30 min. Cells
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(5x104/well) were seeded in matrigel-coated well prior to add assay media and incubated for 78h at 37°C in 5% CO2. The wells were captured at least five different fields per well by an
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inverted microscope at 4X magnification (Nikon TS100F, Japan). Images were analyzed by
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Wimasis software (Onimagin Technologies SCA, Spain). 2.2.6 Analysis of gene expression by real-time PCR
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Total RNA was isolated from cells by using RNeasy mini kit (#74104, Qiagen,). cDNAs were synthesized from total RNA by using cDNA kit (#4368814, Applied Biosystems, USA). Gene
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expression was quantified on the basis of mRNA level expression of the target genes using ABI7900HT system. Predesigned SYBR green I primers, (# KSPQ12012 Sigma) were used
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(Sup.Table1) for this purpose. The Ct value of an endogenous control gene (TBP) was subtracted from the corresponding Ct value for the target gene resulting in the delta Ct value which was used for relative quantification of mRNA expression by the comparative Ct method (2-ΔΔCt method). 2.2.7 Analysis of DNA promoter methylation of human Stress & toxicity genes by real-time PCR array
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This method involves the restriction digestion of genomic DNA combined with real-time PCR to investigate promoter methylation status of genes key to human stress and toxicity pathway. It essentially estimates CpG island DNA methylation profiling of individual genes. 2.2.7.1 Restriction digestion
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In brief, genomic DNA was purified by DNeasy kit (#69504, Qiagen) from BPA (0nM and 1nM) exposed HTR8/SVneo cells. Each sample with equal amount of purified genomic DNA were digested in four separate setups consists of control digest (Mo), methylation sensitive (Ms), methylation dependent (Md) and double digests (Msd) by using DNA restriction kit (#335452, Qiagen). Genomic DNA (2µg) was diluted with 100µl restriction digestion buffer (5x) to make final volume till 470µl with RNase/DNase free water. Diluted DNA from each sample was
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divided into four reaction setup as shown in Sup. Table 2. Digestion was performed at 37ºC for
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using water bath and stored -20ºC for further use.
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6h in a heating block. Reaction was stopped by heat inactivation of enzyme at 65ºC for 20min
2.2.7.2 Real-time PCR to quantify DNA methylation
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This method ultimately quantified leftover digested DNA of each gene by real-time PCR using predesigned primers that flank promoter (gene) region of interest. Real-time PCR was performed
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in 384-well plate in ABI7900HT (Applied Biosystem) system using epitect® PCR array kit
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(#335222 EAHS-3580ZE, Qiagen). Each of 94-genes of this array was amplified with equal amount of four digested DNA (Mo, Ms, Md and Msd) with high specificity and amplification
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efficiency to detect promoter DNA methylation of genes linked with human stress & toxicity pathway. Realtime PCR setup was prepared for each of the four digestions (Mo, Ms, Md and
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Msd) of every sample as shown in Sup. Table 3. Final reaction mixture (10µl) was added carefully to the appropriate well of the 384-well array plate. PCR was programmed as : 95ºC for
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10 min with one cycle, annealing at 99ºC for 30sec followed by 72ºC for 1min for 3 cycles and denaturation at 97ºC for 15 sec followed by 72ºC for 1min for 40 cycles. 2.2.7.3 Data analysis Methylation PCR array data provided gene methylation status as percentage of unmethylated and methylated fraction of input genomic DNA. The relative fractions of methylated (M) and unmethylated (UM) DNA were determined by comparing the amount in each digest with that of
9 a control (no enzymes added) digest using a ΔΔCt method .SEC (methylation sensitive enzyme control) and DEC (methylation dependent enzyme control) were included in array as a quality control in digestion efficiency of methylation-sensitive enzyme A and methylation-dependent enzyme B, respectively. In general, the value ΔCt (Ms-Mo) ≥4 for SEC or ΔCt(Md-Mo)≥4 for
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DEC indicates more than 93.8% of control DNA molecules are digested where the restriction enzymes are active and digests DNA efficiently. When the refractory DNA percentage was greater than 12.5 percent (R > 12.5%), the digestions were incomplete and analysis was reported as a "Failure". 2.2.8 Immunoblotting
Expression of HSD11β1 and HSD11β2 proteins were measured by immunoblotting after pre-
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stimulation of the HTR8/SVneo trophoblast cells with BPA and BPS (1nM) for 24h.
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Immunoblotting was performed to determine the expression of proteins in similar way as
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described previously. However, this study used 10 µg of protein/lane to transfer to PVDF membrane [27]. After blocking, blots were incubated with GAPDH (1:5000 # PA534847,
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Thermo Scientific Pierce, USA), 11β-HSD2 (1:5000 # sc 20176) and 11β-HSD1 (1:5000 # sc20175) antibodies (Santacruz, USA). Blots were further incubated with HRP-conjugated
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2.2.9 Statistical analysis
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secondary antibodies and were processed as per the procedure described previously [27].
Data were analyzed by unpaired Student’s t-test to compare treatments over control using
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Graphpad prism 4. Statistical significance was considered when p value <0.05. Majority of the data are obtained at least from three independent experiments as indicated elsewhere and
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expressed as mean ± SEM.
3. Results
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3.1 BPA and BPS exposures decrease the numbers of viable HTR8/SVneo cells In order to assess the exposure of bisphenols on cell growth and viability, we performed doseresponse experiment in both HTR8/SVneo cells and non-trophoblast cells (EA.hy926 )using MTT assay. Compared to control, cell viability was decreased consistently when cells were exposed with 1-100nM of BPA and BPS in HTR8/SVneo cells (Fig.1A). BPS, the predominant replacement chemical for BPA[28] was used for comparison purpose.
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At 1nM concentration, viability of HTR8/SVneo trophoblast cells was decreased significantly by >15% of the total (control vs. BPA and BPS: 99.99 ± 0.041 vs. 81.16 ± 0.464 and 83.63 ± 0.411 percent; n=8). The concentration of 1nM was considered for subsequent assays since viability was not affected by more than 20% of the total. This concentration response study revealed that
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first trimester trophoblast cells are well viable in the ranges of 0.001 to 1nM exposure of BPA and BPS for 24h (Fig.1A). Cellular viability was further measured at higher concentration (1nM -100nM) with exposure of 48h and 72h in presence of BPA and BPS. Viability of these cells was decreased with increasing exposure duration and concentration (data not presented). Effect of bisphenols on cell growth and viability was also investigated in EA.hy926 cells after exposure with BPA and BPS for 24h. Unlike HTR8/SVneo cells, the cell viability was not significantly
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affected when human vascular endothelial EA.hy926 cells were exposed with 0.001-10nM of
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BPA or BPS, the exposure at much higher concentration (100nM BPA or BPS) only significantly
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reduced the cell viability (Fig.1B). These data suggest HTR8/SVneo cells are more sensitive to BPA or BPS compared with non-trophoblastic EA.hy926 cells.
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3.2 BPA and BPS exposures decrease proliferation of HTR8/SVneo cells Effects of BPA and BPS exposure on cellular proliferation of the HTR8/SVneo cells were
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investigated by measuring active DNA synthesis of the growing cells using [3H] -thymidine
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assay after exposing the cells with (0.1-10nM) of BPA and BPS for 24h. Compared to control, [3H] thymidine incorporation [3H] was decreased significantly by ~10-12% (control vs. 0.1nM
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and 1nM BPA: 52090 ± 1515cpm vs. 47220 ± 917cpm and 45890 ± 906 cpm, n=9) when these cells were exposed to low concentrations of BPA (0.1-1nM) for 24h (Fig.2). A concentration
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dependent decrease was recorded in the proliferation of HTR8/SVneo cells when these cells were exposed to environmentally relevant concentration of BPA (1nM). Despite the fact that BPS is
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considered as safer substitute of BPA, BPS (0.1-1nM) significantly decreased radioactive thymidine incorporation [3H] to a greater extent (~14-18%) as compared BPA (control vs. 0.1nM and 1nM BPS: 52090 ± 1515cpm vs. 44970 ± 488cpm and 42940 ± 599cpm, n=9). Both BPA and BPS exposures decreased proliferation of HTR8/SVneo cells. 3.3 BPA and BPS exposures inhibited basal- and VEGF-stimulated tube formation in HTR8/SVneo cells
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VEGF (10ng/ml) significantly increased tubular network (control vs. VEGF 10ng/ml: 24650 ± 193 pixel vs. 27360 ± 156 pixel; n=9) of HTR8/SVneo trophoblast cells (Fig.3 A-B). VEGFstimulated tube formation was decreased by 32%, 26% and 17% in presence of 150nM SU5416, 1nM of BPA and BPS, respectively (VEGF vs. VEGF+SU5416, VEGF+BPA and VEGF+BPS:
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27360 ± 156 pixel vs. 18600 ± 274 pixel, 20090 ± 226 pixel and 22630 ± 267 pixel; n=9). Both BPA and BPS partially disrupted tube formation induced by VEGF (Fig.3A). In addition, BPA (1nM) and BPS (1nM) by themselves also prevented the sprouting of spontaneous tube formation significantly by 31% and 22% respectively, (control vs. BPA and BPS: 23030 ± 548 pixel vs. 15700 ± 265 pixel, and 17820 ± 203 pixel; n=9, p<0.05 ) in HTR8/SVneo cells. Basal- and VEGF stimulated tube formation of the HTR8/SVneo cells were inhibited by the exposure of
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both BPA and BPA.
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3.4 Exposure of BPA and BPS altered mRNA expression of angiogenesis, endocrine and
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metabolic mediators in HTR8/SVneo cells
Expression of several angiogenesis, endocrine and metabolic mediators were significantly
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modulated after exposing HTR8/SVneo cells with BPA (1nM) and BPS (1nM) for 6h and 24h. Compared to 6h, mRNA expression yielded more stable and reproducible Ct values across the
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replicates at 24h (data not presented). Expression of mRNA those encode VEGFA and
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ANGPTL4, proliferation and cell adhesion mediators such as proliferating cell nuclear antigen (PCNA) and intercellular adhesion molecule 1 (ICAM1) were significantly down regulated upon
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24h exposure of BPA (1nM) and BPS (1nM) in the HTR8/SVneo cells (Table-1). Expression of metabolic factor such as fatty acid binding protein 4 (FABP4) and regulator of cellular
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cholesterol and phospholipid homeostasis such as ATP binding cassette subfamily A member 1 (ABCA1) were significantly decreased after such exposure. Concomitant to that, mRNA
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expression of glucocorticoid such as 11-β-hydroxysteroid dehydrogenase 2 (HSD11β2) was significantly up regulated in presence of both BPA (1nM) and BPS (1nM) exposure. It is observed that changes at the level of mRNA expression were more profound with BPS (1nM) exposure in vitro as compared with BPA (1nM). However, mRNA expression of CTGF, CDH1, KRT1, KRT7, HLAG, DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), ADRP, SLC2A1, SREBF2 were unaffected. All these data demonstrated that both BPA and BPS
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exposure inhibit proliferation and tube formation of the first trimester trophoblastic cells possibly by down regulating expression of growth promoting genes such as VEGF, PCNA, ICAM-1 in the first trimester trophoblast cells, HTR8/SVneo. 3.5 Protein expression of 11β-hydroxysteroid dehydrogenases 2 increased after BPA and
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BPS exposure Compared to control, exposing cells with BPA (1nM) and BPS (1nM) significantly up-regulated protein expression of HSD11β2 by ~80% (fold expression: control vs. BPA (1nM) and BPS (1nM): 0.990 ± 0.010 vs. 1.810 ± 0.013, and 1.831 ± 0.011; n=3) while changes in the expression of HSD11β1 (fold expression: control vs. BPA (1nM) and BPS (1nM): 0.998 ± 0.004 vs. 0.8053 ± 0.06, and 0.8345 ± 0.03; n=3) remained insignificant (Fig.4A-B).
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3.6 BPA exposure altered DNA promoter methylation of cellular stress mediators In order to investigate epigenetic changes due to BPA exposure, promoter DNA methylation of
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human stress and toxicity pathway were measured. The EpiTect Methyl II PCR system was used
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to compare methylation levels of CpG islands in the promoter regions of stress and toxicity gene
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pathway. QC reported a “failure” of total 8 and 12 genes out of 94 genes in control and BPA (1nM) group respectively (data not presented). DNA methylation percentage was compared
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between the cells in the absence and presence of BPA (1nM) in the heat map (Fig. 5). In general, percentage of promoter CpG methylation was comparatively higher in control (without BPA) as
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compared to BPA-exposed cells. The status of CpG methylation obtained using Epitect Methyl II Signature PCR array on human stress & toxicity is presented in Table 2. CpG promoter sites of
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BNIP3, MT3, RAD9A, SMC1A (cell cycle, proliferation and DNA replication) and HSPA1A, XPC, GPX7 (antioxidant and heat shock protein) genes were methylated predominantly in both
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control and BPA exposed cells but in a differential manner. In presence of BPA (1nM), percentage of CpG promoter methylation of these genes were down regulated by 12-48% while
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XRCC2,CREB3, RAD23A,VCP (cell cycle, proliferation and DNA replication) , GSR,DNAJA1, PRDX2, GPX3 (antioxidant and heat shock protein) and DDIT3, HERPUD1, INSIG1 & MBTPS1 (stress and metabolism) were down regulated by 50-99% as compared with those of control cells (Table 2). Overall, the percentages of promoter methylation of stress responsive genes were low in presence of BPA (1nM) as compared to its absence in HTR8/SVneo cells.
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4. Discussion The present study demonstrates that BPA at very low concentrations (0.01 nM-1 nM) affects cell growth, proliferation, tube formation and gene expression in first trimester trophoblast cells,
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HTR8/SVneo. The levels of BPA (0.01–1 nM; 1 nM BPA=0.228 ng/ml) used in this study are much lower than the average BPA concentrations as present in the body fluid [2, 4, 5]. A recent review of 16 studies indicates that the range of serum BPA concentrations varies between 0.5 and 2.5 ng/ml [29]. Early placentation process involving growth and tube formation of first trimester trophoblasts, a critical step for optimum feto-placental growth and development, may be affected by very low concentrations of BPA (0.01–1 nM; 1 nM BPA=0.228 ng/ml). Our data
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therefore justify further in-depth work in order to determine the safe levels of BPA in human
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reproductive health. Both BPS and BPA exposure affected cell viability and growth of the
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HTR8/SVneo cells. Interestingly, both BPA and BPS exposure also prevented tube formation in
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basal and VEGF-treated cells to a similar extent. Overall, bisphenols inhibit the tube formation via inhibiting expression of genes responsible for angiogenesis such as VEGF, PCNA and
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ICAM1 in these HTR8/SVneo cells. BPA exposure selectively affects VEGFA expression in
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cancer cells as compared to non-cancer cells. Earlier data also showed that BPA lowered VEGFA expression significantly in cancer cell lines [30]. HTR8/SVneo tube-like formation embodies
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trophoblast migration and differentiation toward an invasive phenotype, a physiologic process
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that takes place during early human pregnancy. The mechanisms responsible for BPA/BPSinduced inhibition of tube formation in these cells are not yet clearly known. Since BPA/BPS
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inhibited VEGF-induced tube formation and also significantly down-regulated VEGF mRNA expression in these cells suggesting that the inhibitory action may be mediated at least in part via VEGF. However, one also could argue that the inhibitory effect of BPA/BPS on tube formation could be also in part due to the decreased cell viability. However, it may be unlikely the case, as the tube formation was measured after 6-7h of incubation whereas cell viability (20% decreased)
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measured only after 24h of incubation with the BPA/BPS. Moreover, one has to consider the facts that tube formation phenotype may involve many cellular activities such as cell viability, proliferation, metabolic fitness, and other activities[31-33]. Further work is required in order to
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understand the detailed mechanisms of BPA effects in these cells. It is difficult to assume the individual contribution of each process in tube formation under any circumstances. This in vitro study raises the possibility that BPA exposure even at very low concentration may be associated with major complications of pregnancy. BPA at very high concentration (100nM) also altered the HTR-8/SVneo cell invasion capacity by altering migration of cells
[34].
However, further studies are required in order to understand the detailed mechanisms responsible
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for BPA- induced inhibition of angiogenesis.
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Adequate placental angiogenesis is critical for the establishment of the placental circulation and
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thus for normal fetal growth and development. It is therefore reasonable to suggest that
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inappropriate placental expression of angiogenic factors by BPA may contribute to placental
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vascular defects and placental dysfunction and therefore could be an important cause of
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infertility and fetal growth retardation. With the recent spate of clinical work on regulators of angiogenesis, these observations lead us to believe that regulation of placental angiogenesis
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could become a novel and powerful method for ensuring positive outcomes for most pregnancies. BPA exposures in pregnancy may therefore preclude optimal feto-placental growth
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and development. Further work is required on the effects of bisphenols acids in feto-placental development.
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The HTR-8/SVneo cell line was developed from first trimester extra-villous trophoblast infected with SV40 Large T antigen. HTR8/SVneo cell line has been extensively used as a sufficient model of extravillous trophoblasts. Recently, Abou-Kheir et al. [35] reported that HTR8/SVneo cells are a mixed population of cells, this group did not perform any type of DNA fingerprinting to verify the identity of their line. HTR8/SVneo cells still closely resemble with
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first trimester placental genotypes[36] and undergo similar phenotypic changes as happen in vivo placentation process[37]. During pregnancy high levels of cortisol in human are prevalent in the maternal circulation [38].
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The placental HSD11β2 protects the fetus from exposure to high levels of maternal glucocorticoids [39]. The precise control of placental HSD11β2 is critical for normal fetal organ growth and maturation. The activity and expression of HSD11β2 within the placenta correlates with fetal birth weight [40, 41], and altered placental HSD11β2 gene expression was observed in intrauterine growth restriction [40, 42]. BPA at lower concentration increased the expression of both protein and mRNA of HSD11β2. The effect of BPA-induced increase in HSD11β2 level is
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not without precedent. BPA at (0.1–2 g/ml) for 24 h increased mRNA, protein and activity of
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HSD11β2 in placental last trimester trophoblasts cells [24]. It could be compensatory effects of
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these cells of BPA exposure or simply due to its estrogenic activity, however further work is
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required for definitive conclusions. Further work using animal model will be required to determine the effects of higher levels of HSD11β2 induced by BPA on the reproductive and
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endocrine systems. The human placenta is a dynamic endocrine organ producing a wide variety
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of hormones, factors and enzymes, which function in a concerted fashion to regulate maternal adaptation to pregnancy and parturition as well as fetal development. Placental trophoblast cells
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respond to variety of exogenous agents by activating stress pathways and epigenetic changes often regulate some of these responses. This study highlighted gene-specific methylation changes
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in stress response pathways upon in vitro exposure of the early trophoblast cells to BPA. DNA methylation profiling of first trimester trophoblast genomic DNA actually correlated CpG island
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methylation status of gene promoters key to stress and toxicological responses, including oxidative stress, inflammation, DNA damage, apoptosis, hypoxia, and the unfolded protein response. Percentage of promoter methylation was lowered by BPA exposure as compared with those of control cells. DNA methylation of gene promoters typically act to repress gene transcription of mRNA those encode genes for cellular stress and toxicity pathways and thus
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stress response genes were de-repressed in presence of BPA exposure. Methylated genomic DNA could be adequate to repress gene transcription of stress and toxicity mediators which otherwise unmethylated by BPA (1nM) indicating that activation of these pathways in response to BPA exposure. A significant number of CpG methylation changes of gene promoters were
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observed in BPA-exposed cells those are also associated with cellular proliferation indicates decreased proliferation could be due to promoter hypomethylation of the cell cycle, proliferation replication associated genes. This study is limited to measure BPA alone on CpG methylation of the first trimester trophoblast cells although BPS showed similar effect on proliferation as like BPA. Since BPA is predominant endocrine hazard at this moment, emphasis was given priority
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for CpG methylation to BPA over BPS. In order to confirm further, if methylation status could
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lead to repress transcription, measuring corresponding expression levels of genes between
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control and BPA exposure is required. Mechanism of these methylation sensitivities and their
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functional consequences in the first trimester placental cells warrants further investigation. In humans, studies on the relationship between prenatal exposure to BPA and fetal growth have
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shown mixed results [5, 43-45]. Our findings point out the relevance of further research on
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long‐term consequences of altered prenatal nutrition and programming related to BPA‐induced placental and embryo effects. In addition, our findings lend further support to different
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mechanisms being elicited according to the level of BPA exposure, which may result in non‐monotonic dose–response patterns across exposure levels of different orders of magnitude.
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For the first time, we demonstrated here that BPA exposure at low concentrations (0.01nM-1nM) affected cellular growth, proliferation and angiogenic activities possibly by affecting gene
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methylation of stress response and down-regulation of growth promoting factors such as VEGF, PCNA, ICAM-1 in the first trimester trophoblast cells. In addition to these effects, BPA upregulated HSD11β2 expression, the one is responsible for detoxifying the effects of BPA or similar molecules. 5. Conclusions
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The present study demonstrates that BPA exposure at concentrations much lower than those found in the human placenta, severely disrupts gene expression and tube formation in first trimester human placental trophoblast cells. If these in vitro findings extrapolated to human pregnancy in vivo, in that case, exposure to BPA could have a significant impact on placentation
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processes that may contribute to pregnancy complications, such as spontaneous abortion, preterm birth, fetal growth restriction, and preeclampsia.
Acknowledgements
We are grateful to Camilla Solberg for her help in thymidine assay. This study was supported by
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the grant from the Thune Holst Foundation and HRD fellowship, Department of Health Research
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References
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(Dr. Sanjay Basak), Government of India.
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[43] B.E. Lee, H. Park, Y.C. Hong, M. Ha, Y. Kim, N. Chang, B.N. Kim, Y.J. Kim, S.D. Yu, E.H. Ha, Prenatal bisphenol A and birth outcomes: MOCEH (Mothers and Children's Environmental Health) study, Int J Hyg Environ Health 217(2-3) (2014) 328-34. [44] M.S. Wolff, S.M. Engel, G.S. Berkowitz, X. Ye, M.J. Silva, C. Zhu, J. Wetmur, A.M. Calafat, Prenatal phenol and phthalate exposures and birth outcomes, Environ Health Perspect 116(8) (2008) 1092-7. [45] W.C. Chou, J.L. Chen, C.F. Lin, Y.C. Chen, F.C. Shih, C.Y. Chuang, Biomonitoring of bisphenol A concentrations in maternal and umbilical cord blood in regard to birth outcomes and adipokine expression: a birth cohort study in Taiwan, Environ Health 10 (2011) 94.
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Figure legends Fig.1 Effect of Bisphenols (BPA & BPS) on growth and viability of the first trimester HTR8/SVneo cells (Fig 1A), and human vascular endothelial EA.hy926 cells (Fig 1B). Cells
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were exposed to BPA and BPS (100nM, 10nM, 1nM, 0.01nM and 0.001nM) for 24h. Control cells (vehicle) were received 1% FBS RPMI with 0.1% ethanol. OD values were compared between control and treatment after normalized with blank values and expressed as percentage of control. Data are shown as mean ± SEM (n=8) *p<0.05,**p<0.01 and ***p<0.001vs. control (Student’s t test)
Fig.2 Effect of Bisphenols (BPA & BPS) on proliferation of the trophoblast cells,
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HTR8/SVneo.3H-Thymidine incorporation into the DNA of HTR8/SVneo cells was investigated
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by radioactive thymidine [3H] assay. Cells were pulsed with [3H] thymidine and harvested, and
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counted as described in the methods. Counts per minute (cpm) were compared with control after deducting blank value. Data are shown as mean ± SEM (n=9).*p<0.05,**p<0.01 and ***p<0.001
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Fig. 3. Effect of BPA and BPS on VEGF stimulated tube formation of the HTR8/SVneo
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cells Tube formation assay (in vitro angiogenesis) was performed in matrigel as described in ‘‘method’’ section. FBS starved cells (5x104 ) were incubated with or without VEGF (10ng/ml).
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SU5416 (150nM) was added 1h prior to addition of VEGF. BPA (1nM) and BPS (1nM) were added to VEGF treated cells; Control= 0.1% ethanol in 1% FBS media; Image was captured after
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7-8 h of incubation of BPA and BPS in 5X magnification. (A) Representative images of tubules formed after 8h where blue colour shows tubule network. (B) Tubule network length was quantified by Wimasis software and expressed as pixel as per their instruction. Data are shown as
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mean ± SEM (n=9). *p value < 0.05 versus control; # p value < 0.05 versus VEGF (10ng/ml) Fig 4. Effects of BPA and BPS on the expression of 11β-hydroxysteroid dehydrogenases (HSD) in HTR8/SVneo cells. Cells were pre-incubated with BPA (1nM) and BPS (1nM) for 24 h. Cellular proteins were harvested and HSD11β1 and HSD11β2 proteins were measured by immunoblotting (A) and their expressions were measured as described in the method (B). Data
22 of protein expression are reported as fold expression of mean ± SEM (n=3) after normalizing expression with GAPDH; * p<0.05 vs. control (Student’s t test).
Fig.5 Effect of BPA on promoter DNA methylation of cellular stress mediators in the first trimester trophoblast cells. HTR8/SVneo cells were exposed with or without BPA (1nM) for
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24h. Cellular genomic DNAs were restricted with methylation sensitive and methylation dependent enzyme followed by quantification of digested DNA by real-time PCR using 384-well epitect array kit as described in method section. Methyl-profiler data (DNA methylation percentage) of the gene promoters involved with human stress & toxicity pathway are plotted for the control and BPA group in the heat map. Percentage of DNA methylation index is showing as
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blue and green colors.
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26 Table
Type of genes
Gene symbol
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Table 1: Effect of Bisphenol (A & S) on modulation of gene expression in first trimester trophoblast HTR8/SVneo cells 1 Relative mRNA fold over unit control expression (Target gene/TBP) 2 BPA (1nM)
Angiogenesis & cell
VEGFA
0.59 ± 0.061 (-1.7)
0.48 ±0.033 (-2.0)
ANGPTL4
0.61±0.052 (-1.6)
0.46 ±0.055 (-2.1)
PCNA
0.45 ±0.031 (-2.2)
0.06 ±0.077 (-16.0)
ICAM1
0.11±0.011 (-9.0)
0.03±0.005 (-33.0)
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proliferation factors
A
Endocrine & metabolic FABP4 ABCA1
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factors
BPS (1nM)
0.03 ±0.01 (-33.0)
0.29 ±0.07 (-3.4)
0.04 ±0.037 (-25.0)
7.75±0.813 (+7.7)
6.01 ±0.77 (+6.0)
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HSD11β2
0.22 ±0.003 (-4.5)
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mRNA expression was measured after incubating cells with BPA (1nM) and BPS (1nM) for 24h.Net fold expression are depicted in parenthesis by down (-) or up (+) regulation over control. 2
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The level of each mRNA expression of genes was quantified after normalized with endogenous control, TBP and calculated according to the ΔΔCt method. Data are expressed as mean of relative mRNA fold expression over control ± SEM, n=3. p < 0.05 vs. control
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Table 2: Percentage of CpG methylation of stress & toxicity genes in response to BPA exposure of the trophoblast cells, HTR8/SVneo Change (%)
BNIP3
Control 99.93
BPA(1nM) 81.97
17.97
MT3
92.6
81.73
11.74
RAD9A
79.69
50
37.26
SMC1A
96.61
50
48.25
GADD45G
59.17
74.36
25.67
XRCC2
50
7.6
84.80
CREB3
57.55
0.5
99.13
RAD23A
56.97
0.09
VCP
66.74
0.02
DNAJC15
99.52
HSPA1A
Gene function
a pro-apoptotic factor
8.17
97.58
85.43
12.45
Heat shock protein 70 Kd protein
XPC
68.06
50
26.54
GPX7
50
95.78
91.56
GSR
83.78
0.13
99.84
DNA damage recognition and repair factor Protects against oxidative DNA damage and double-strand breaks Cellular antioxidant defense
DNAJA1
82.01
0.02
99.98
PRDX2
81.45
0.94
98.85
GPX3
51.25
1.8
96.49
Stress and Metabolism
DDIT3
52.88
2.5
95.27
HERPUD1
68.83
0.06
99.91
INSIG1
93.69
0.08
99.91
MBTPS1
91.45
0.33
99.64
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99.84
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Antioxidant & heat shock protein
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91.39
Encodes protein for growth inhibitory factor Cell cycle checkpoint protein required for cell cycle arrest and DNA damage repair Structural maintenance of chromosomes 1A Growth arrest and DNA-damageinducible protein Maintain chromosome stability and repair DNA damage Regulator of cell proliferation and migration Encodes nucleotide excision repair protein DNA repair, replication and regulation of the cell cycle Heat shock protein 40 Kd protein
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Cell cycle, proliferation and DNA replication
Methylation (%)
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Gene symbol
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Gene pathways
99.97
Heat shock protein that prevents protein aggregation Cell protection against oxidative stress Protect cells against oxidative damage and associated with Preeclampsia Transcription factor in ER stress response Homocysteine-inducible ER stressinducible ubiquitin-like domain member 1 protein Regulates cholesterol metabolism, lipogenesis and glucose homeostasis Ubiquitously expressed membrane
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bound protease that regulates cholesterol or lipid homeostasis