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Toxicology Letters 226 (2014) 81–89

Contents lists available at ScienceDirect

Toxicology Letters

j our na l ho me page: www.elsev ier .com/ locate / tox le t

nvolvement of activating ERK1/2 through G protein coupled receptor0 and estrogen receptor �/� in low doses of bisphenol A promotingrowth of Sertoli TM4 cells

i-Chen Gea,1, Zhuo-Jia Chenb,1, Han-Yan Liuc, Kun-Shui Zhangd, Hao Liua,ong-Bin Huangb, Ge Zhanga, Chris K.C. Wonge, John P. Giesy f,

un Dua,∗, Hong-Sheng Wanga,∗

Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, No. 132 Waihuandong Road,niversity Town, Guangzhou 510006, ChinaDepartment of Pharmacy, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center forancer Medicine, Guangzhou 510060, ChinaCenter for Reproductive Medicine, Third Affiliated Hospital of Guangzhou Medical University, Key Laboratory for Major Obstetric Diseases of Guangdongrovince, Guangzhou 510150, ChinaDepartment of Pharmacy, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, ChinaDepartment of Biology, Hong Kong Baptist University, Kowloon, Hong Kong SAR, Kowloon Tong ChinaDepartment of Veterinary Biomedical Sciences & Toxicological Center, University of Saskatchewan, Saskatoon, SK, Canada

i g h l i g h t s

10−9 and 10−8 M BPA significantlystimulated the proliferation of TM4cells.GPR30/EGFR/ERK signal is involved inBPA-induced TM4 cell proliferation.BPA (1 nM) up regulated mRNA andprotein expression of GPR30.

g r a p h i c a l a b s t r a c t

Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor�/� (ER�/�), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30and ER�/� were involved in the observed effects of BPA.

r t i c l e i n f o

rticle history:eceived 14 August 2013

a b s t r a c t

Sertoli cells play a pivotal role in supporting proliferation of germ cells and differentiation during sper-matogenesis in mammals. Nanomolar concentrations of Bisphenol A (BPA) can significantly stimulate the

eceived in revised form 14 January 2014

ccepted 27 January 2014vailable online 2 February 2014

eywords:PR30ertoli cell

proliferation of mouse immature Sertoli (TM4) cells. However, mechanisms by which BPA caused theseeffects were still unclear. In the present study, an inverse U-shaped curve was observed when treatingTM4 cells with increasing doses of BPA: 1 to 10 nM BPA significantly stimulated the proliferation of TM4cells and increased the proportion of cells in S phase; >1 �M BPA caused lesser proliferation of cells. Expo-sure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor�/� (ER�/�), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30

∗ Corresponding authors at: Sun Yat-sen University, Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, No. 132 Waihuandong Road,niversity Town, Guangzhou 510006, China. Tel.: +86 20 39943024; fax: +86 20 39943022..

E-mail addresses: dujun@mail.sysu.edu.cn (J. Du), whongsh@sysu.edu.cn, whongsh@mail.sysu.edu.cn, hswang2000@gmail.com (H.-S. Wang).1 These authors contributed equally to this work.

378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2014.01.035

82 L.-C. Ge et al. / Toxicology Letters 226 (2014) 81–89

TM4Estrogen receptor �/� (ER�/�)MaleReproduction

and ER�/� were involved in the observed effects of BPA. Furthermore, exposure to BPA caused rapid(5 min) activation of ERK1/2 via both GPR30 and ER�/�. Blocking the GPR30/EGFR signal transductionpathway by antagonists suppressed both phosphorylation of ERK and BPA-induced cell proliferation. BPAup-regulated mRNA and protein expression of GPR30 in a concentration-dependent manner. In summary,the results reported here indicated that activating ERK1/2 through GPR30 and ER�/� is involved in lowdoses of BPA that promoted growth of Sertoli TM4 cells. The GPR30/EGFR/ERK signal is the downstreamtransduction pathway in BPA-induced proliferation of TM4 Sertoli cells.

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. Introduction

Bisphenol A (BPA) is large production volume chemical used toanufacture food and beverage containers, cigarette filters, ther-al (carbonless) receipts, and paper currency (Vandenberg et al.,

009). Currently, over 2.7 million metric tons of BPA are producednnually, primarily for use in manufacturing of epoxy resins andolycarbonate plastics. BPA can leach into food and beverages fromlastic containers and therefore accumulate in bodies of humansShin et al., 2004). Recently, BPA has been widely detected in variousuman tissues such as blood, fetal serum during pregnancy, amni-tic fluid, follicular fluid, placental tissue, umbilical cord blood, andrine (Lee et al., 2008; Vandenberg et al., 2010; Wan et al., 2010).or example, BPA has been detected in 100% of urine samples fromhinese children with a mean concentration of 3.00 ng/mL (Li et al.,013), and in 96% of urine samples from American college studentsith a mean concentration of 1.3 ng/mL (Carwile et al., 2009).

BPA has been implicated as a potential endocrine disruptinghemical (EDC), primarily as a weak estrogen receptor �/� (ER�/�)gonist (Vandenberg et al., 2009). Recently, more and more studiesave revealed that BPA is correlated with health outcomes such asiabetes, cardiovascular disease, breast cancer, increased inflam-ation, and oxidative stress (Rubin, 2011). Among these health

ssues, increasing concern has been focused on the risk of BPAo male fertility (Salian et al., 2009, 2011). In vivo studies haveocumented that prenatal and neonatal exposure of male ratso environmentally relevant concentrations of BPA significantlympairs spermatogenesis (Salian et al., 2011).

Sertoli cells, one of the somatic constituents of the testis, are therimary supporting cells creating the structural and physiologicalnvironments necessary for development of cells during spermato-enesis (Kopera et al., 2010). In mammals, Sertoli cells proliferateuring only two periods of life, in fetal or neonatal life and in theeri-pubertal period (Sharpe et al., 2003). Results of recent studies

ndicated that exposure to BPA affects fertility of males by affectingfficiency of spermatozoa, but there was limited data on effects ofPA on proliferation and functions of Sertoli cells.

Results of previous studies indicated that the binding affinityf BPA to estrogen receptor-� (ER�) or ER� is 10,000-fold lesshan that of the endogenous estrogen, estradiol (E2) (Kuiper et al.,998). This affinity is insufficient to explain why nanomolar dosesf BPA have been reported to cause E2-mediated effects (Watsont al., 2005; Welshons et al., 2006). Recently, G protein-coupledeceptor 30 (GPR30), an orphan G protein-coupled receptor (GPCR,lso known as G protein-coupled estrogen receptor, GPER), whichas been proposed as a nonclassical estrogen receptor, has beeneported to mediate estrogenic effects of estrogens, phytoestro-ens, xenoestrogens, and environmental EDCs (Prossnitz andarton, 2011). GPR30 was recently shown to mediate BPA-inducedroliferation of various types of cells (Bouskine et al., 2009; Shengnd Zhu, 2011). However, whether GPR30 is involved in BPA-nduced impairment of fertility of males and the mechanism by

hich these effects occurred remained unclear.

In the present study, effects of BPA on proliferation of Sertoli

M4 cells was studied. Specifically, the objectives of this studyere to: (1) investigate effects of BPA, particularly at nanomolar

© 2014 Elsevier Ireland Ltd. All rights reserved.

concentrations, on proliferation of TM4 cells; (2) determine the roleof GPR30 and ER�/� in BPA-induced proliferation of Sertoli TM4cells, and (3) evaluate changes in downstream signal pathways.

2. Materials and methods

2.1. Reagents

ICI 182,780 (ICI, ER antagonist) was purchased from Santa Cruz Biotechnol-ogy (Santa Cruz, CA, USA). PD 98059 (PD, MAPK/ERK kinase agonist) and AG 1478(AG, EGFR antagonist) were purchased from Selleck Chemicals (Houston, TX, USA).E2, G-1 (GPR30 agonist), G15 (GPR30 antagonist) and other chemicals were pur-chased from Sigma Chemical Co. (St. Louis, MO, USA). Antibody against GPR30was purchased from Abcam Plc (Abcam, UK). Antibodies against ERK1/2, Bcl-2,PCNA, Caspase 3, Bim and GAPDH were purchased from Cell Signaling Technol-ogy Inc. (Beverly, MA, USA). Antibodies against p-ERK1/2, and Cyclin D1 werepurchased from Bioworld Technology, Inc (Minneapolis, MN, USA). Horseradishperoxidase-conjugated secondary antibody was purchased from Santa Cruz Biotech-nology (Santa Cruz, CA, USA). All compounds were solubilized in dimethyl sulfoxide(DMSO). Steroid-free medium containing DMSO was used as the control. Concen-trations of DMSO in the assay were less than 0.5% (v/v).

2.2. Culture of cells

TM4 cells (mouse Sertoli cell line; American Type Culture Collection, Manas-sas, VA, USA) were cultured in phenol red-free Dulbecco’s modified Eagle’s mediumnutrient mixture F-12 HAM (Sigma-Aldrich, St. Louis, MO, USA) containing 5% heat-inactivated fetal bovine serum (FBS), 5% heat-inactivated horse serum and 1.2 g/Lsodium bicarbonate supplemented with 10 �g/mL penicillin–streptomycin. Thecells were incubated at 37 ◦C in a 5% CO2 atmosphere. Both the plastic items usedfor the experiments and the water used to prepare the reagents were pretreatedby enhanced sonochemical degradation to reduce any potential background BPA(Petrier et al., 2010; Sheng and Zhu, 2011).

2.3. Cell viability assays

Viability of cells were detected by use of previously described procedures (Wanget al., 2012). Briefly, viability of TM4 cells was evaluated by use of the CCK-8 kit(Dojindo Molecular Technologies, Gaithers burg, MD, USA). CCK-8 allows for con-venient assays by utilizing Dojindo’s highly water-soluble tetrazolium salt. WST-8produces a water-soluble formazan dye upon reduction in the presence of an elec-tron carrier. Cells were seeded in 96-well plates at a cell density of 1 × 104 per well.After treatment with BPA for 24, 48, or 72 h, 10 �L of CCK-8 solution was addedto each well. The plates were incubated for another 2 h, and the absorbance wasmeasured at 450 nm using a microplate reader. Experiments were repeated threetimes.

2.4. Analysis of cell cycle

Cells were plated at a density of 1 × 106 per well on six-well plates. After treat-ment with BPA, both the suspension and the adherent cells were collected intoflow cytometry tubes and centrifuged at 2000 rpm for 5 min to obtain cell pel-lets. Cells were washed with phosphate-buffer saline (PBS), and fixed with 70%ethanol overnight at 4 ◦C. Fixed cells were washed with PBS and incubated withRNAase A (0.1 mg/mL) for 30 min followed by incubation with propidium iodide (PI,50 �g/mL) at room temperature for 30 min. Analysis of the cell cycle was performedwith a Coulter Epics XL Flow Cytometry System (Beckman-Coulter, Miami, USA).In each analysis, 10,000 events were recorded. The percentage of cells with sub-G1, G0/G1, S, and G2/M DNA content was calculated using EXPO32 ADC analysisSoftware (Beckman-Coulter, Miami, USA).

2.5. Western blotting analysis

Western blotting was performed as previously described (Jiang et al., 2013).

Briefly, cells were lysed in cell lysis buffer, and then lysates were cleared by cen-trifugation and denatured by boiling in Laemmli buffer. Aliquats of protein wereseparated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels and elec-trophoretically transferred to nitrocellulose membranes. Following blocking with

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% non-fat milk at room temperature for 2 h, membranes were incubated with therimary antibody at 1:1000 dilution overnight at 4 ◦C and then incubated with aorseradish peroxidase-conjugated secondary antibody at 1:1000 dilution for 2 ht room temperature, and detected with the Western Lightning Chemiluminescentetection reagent (Perkin-Elmer Life Sciences, Wellesley, MA).

.6. Quantitative real-time PCR

TM4 cells (1 × 106) were plated on 6-well plates. After exposure to BPA for 24 h,M4 cells were washed twice with ice-cold PBS. Total mRNA was extracted withRIZOL reagent. First strand of cDNA was generated from 2 �g total RNA by use ofligo-dT primer and Superscript II Reverse Transcriptase (GIBCO BRL, Grand Island,Y, USA). Quantitative Real-Time PCR was run on an iCycler (Bio-rad, Hercules, USA)sing validated primers and SYBR Premix Ex Taq II (Takara, Japan) for detection.he cycle number when the fluorescence first reached a preset threshold (Ct) wassed to quantify the initial concentration of individual templates for expression ofRNA of genes of interest. Transcripts of the housekeeping gene GAPDH in the same

ncubations were used for internal normalization. Primer pairs were as follows:APDH, forward 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse 5′-TGG TGA AGAGC CAG TGG A-3′; GPR30, forward 5′-CCT GGA CGA GCA GTA TTA CGA TAT C-3′ andeverse 5′-TGC TGT ACA TGT TGA TCT G-3′; p53, forward 5′-CCG CAG TCA GAT CCTGC G-3′ and reverse 5′-AAT CAT CCA TTG CTT GGG ACG-3′; p21, forward 5′-CCTTC ACT GTC TTG TAC CCT-3′ and reverse 5′-GCG TTT GGA GTG GTA GAA ATC T-3′;GFR, forward 5′-ATA CGC GGC AGG ACC AAG-3′ and reverse 5′-GGA GCG TAA TCCAA GGA TGT-3′ .

.7. Statistical analysis

All values were reported as mean ± SD of three independent experiments unlesstherwise specified. Data were analyzed by two-tailed unpaired Student’s t-testetween two groups and by One-Way ANOVA followed by Bonferroni test for mul-iple comparison involved. Normality was confirmed by the Kolmogorov–Smirnovest and homogeneity of variance was confirmed by use of Levine’s test. The statis-ical analyses were performed using SPSS 17.0 for Windows. A p-value of <0.05 wasonsidered to be statistically significant.

. Results

.1. Effects of BPA on proliferation of TM4 cells

Proliferation of TM4 cells was determined by use of CCK-8ssay after exposure to concentrations of BPA ranging from 10−8

o 10−3 M for 48 or 72 h (Fig. 1), respectively. Proliferation was

20% more than control in cells exposed to 10−8 M BPA for 48 h,ut decreased at higher concentrations. The IC50 of BPA on TM4ell for 48 and 72 h was 5.0 × 10−5 and 9.2 × 10−5 M, respectively.hese results demonstrated that proliferation of TM4 cells was

ig. 1. Effects of increasing concentrations of BPA on the proliferation of TM4 cells. (A) Cells

nd then cell viability was assessed by CCK-8 kit. Data are presented as means ± SD of thrnd high (10−4 M) concentrations of BPA on the TM4 morphology after exposure for 48egend, the reader is referred to the web version of this article.)

ters 226 (2014) 81–89 83

stimulated by BPA at nanomolar concentrations of BPA whileinhibited at concentrations greater than micromolar.

3.2. Nanomolar concentrations of BPA stimulate proliferation ofTM4 cells

CCK-8 assay revealed that exposure to 10−9 or 10−8 M BPA for24 h stimulated proliferation of Sertoli cells by 30.5% and 37.6%,relative to controls (Fig. 2A), respectively, and for 48 h by 19.3% or12.4% (Fig. 2B), respectively. Furthermore, exposure of TM4 cells to10−9 or 10−8 M BPA for 48 h resulted in a significantly greater pro-portion of cells in S phase of the cell cycle, compared with untreatedcontrols (Fig. 2C). These results demonstrated that nanomolar con-centrations of BPA stimulated proliferation of TM4 Sertoli cellsand caused cells to enter the cell cycle. Effects of BPA on expres-sion of cell cycle and apoptosis related proteins were detected byWestern-blotting (Fig. 2D). Exposure to 10−8 M BPA significantly(p < 0.05) increased expression of anti-apoptotic Bcl-2 protein andproliferating cell nuclear antigen (PCNA), while decreased expres-sion of Cyclin D1 and Bim protein. Expressions of mRNAs for bothp21and p53 were down-regulated in TM4 cells exposed to 10−9 or10−8 M BPA for 24 h (Fig. 2E). These findings suggested that down-regulation of p21 and p53 might mediate the observed greaterproliferation caused by nanomolar concentrations of BPA. Takentogether, these results provided strong evidence for stimulatoryeffects of concentration of 1–10 nM BPA on proliferation of TM4cells.

3.3. Involvement of GPR30 and ER˛/ ̌ in BPA-induced TM4proliferation

Both GPR30 and ER�/� were examined to determine if theywere involved in changes in proliferation of TM4 cells exposedto BPA. Exposure to 1 nM BPA or the presence of G-1, a spe-cific agonist of GPR30, resulted in stimulation of proliferation ofTM4 cells (Fig. 3). Both E2, the GPR30 antagonist G15, and theER�/� antagonist ICI 182,780 inhibited proliferation of cells. Both

ICI and G15 blocked the stimulation effects of 10−9 M BPA onproliferation of TM4 cells. Furthermore, the combination of BPA,ICI 182,780, and G15 significantly (p < 0.01) decreased prolifera-tion, which suggested an additive or synergistic effect between

were treated with various concentrations (10−8 to 10−3 M) of BPA for 48 and 72 h,ee independent experiments (n = 3). (B) Effects of low (10−8 M), medium (10−6 M),

and 72 h, respectively. (For interpretation of the references to color in this figure

84 L.-C. Ge et al. / Toxicology Letters 226 (2014) 81–89

Fig. 2. Nanomolar concentrations of BPA stimulated the proliferation of TM4 cells and induced cells to enter cell cycle. Cells were treated with nanomolar concentrations (10−9

and 10−8 M) of BPA for 24 (A) and 48 h (B), respectively, and then cell viability was assessed by CCK-8 kit. (C) Percentages of TM4 cells treated with 10−9 and 10−8 M of BPAfor 48 h at different cell-cycle phases. (D) Effects of BPA on the expression of cell cycle and apoptosis related proteins. TM4 cells were treated with 10−8 M BPA for 24 h, thenCyclin D1, Bcl-2, PCNA, Caspase 3, and Bim protein expression levels were analyzed by Western blotting. (E) RT-PCR analysis mRNA expression of p21 and p53 of cells treatedwith 10−9 and 10−8 M BPA for 24 h. GAPDH was used as internal control to ensure that equal amounts of gene were loaded in each lane. Data represent results from threeindependent experiments. Bands from three independent experiments (n = 3) quantified by densitometry, with results (mean ± SD) normalized to GAPDH expression in eachsample. The control value was set as 1 or 100%. * p < 0.05 compared with control; ** p < 0.01 compared with control.

L.-C. Ge et al. / Toxicology Let

Fig. 3. Effects of BPA and E2 on TM4 proliferation in vitro. Cell proliferation is assessedafter 24 h exposure to different compounds: estradiol 17� (E2) 1 nM, BPA 1 nM, G-1(a specific agonist of GPR30) alone (10 nM), ICI 182,780 (ICI, a specific antagonist ofER�/�, 1 �M) or G15 (a specific antagonist of GPR30, 1 �M), or to BPA 1 nM after a 90-min pretreatment with ICI or G15 singly or combined. Values shown are expressed inthe percent change in cell number compared to control (steroid-free medium) givenas the mean ± SD of three independent experiments (n = 3). Cell proliferation wasanalyzed by use of CCK-8 kit. * p < 0.05 compared with control; ** p < 0.01 comparedwith control. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

Fig. 4. Induction of ERK1/2 phosphorylation (p-ERK) by BPA through GPR30 and ER˛/ ̌ in TM4periods. (B) Cells were treated with vehicle (control) or 10−8 M BPA for increasing time per15 min: estradiol 17� (E2) 1 nM, BPA 1 nM, G-1 (a specific agonist of GPR30) alone (10 nM)1 �M) or G15 (a specific antagonist of GPR30, 1 �M), respectively; values shown represencontrol; ** p < 0.01 compared with control.

ters 226 (2014) 81–89 85

GPR30 and ER�/�. These findings indicated that both GPR30 andER�/� were involved in proliferation of TM4 cells stimulatedby BPA.

3.4. BPA rapidly stimulates phosphorylation of ERK1/2 via GPR30and ER˛/ˇ

Activation of ERK1/2 was investigated by exposing TM4 cellsto BPA with increasing time. The results revealed that ERK can berapidly activated by 10−9 or 10−8 M BPA in a non-genomic manner.Nanomolar concentrations of BPA caused rapid (5 min) phosphory-lation (activation) of ERK1/2 in TM4 cells (Fig. 4A and B). Maximumphosphorylation of ERK1/2 was reached at 10–15 min. BPA-inducedERK1/2 phosphorylation was dependent on estrogen receptor �/�(ER�/�) and GPR30, since both ICI and G15 completely abolishedBPA-induced ERK1/2 phosphorylation (Fig. 4C). Furthermore, thecombination of BPA, ICI 182,780, and G15 caused significantly(p < 0.01) decreased phosphorylation of ERK1/2, which suggestedan additive or synergistic effect between GPR30 and ER�/� forERK1/2 activation.

3.5. Involvement of GPR30/EGFR/ERK1/2 in BPA induced TM4

proliferation

Roles of GPR30 signaling in activation of ERK1/2 and prolifera-tions of cells were investigated. Exposure to either BPA (10−9 M)

cells. (A) Cells were treated with vehicle (control) or 10−9 M BPA for increasing timeiods. (C) ERK1/2 phosphorylation in TM4 cells exposure to different compounds for

or after a 90-min pretreatment with ICI 182,780 (ICI, a specific antagonist of ER�/�,t the mean ± SD of three independent experiments (n = 3). * p < 0.05 compared with

86 L.-C. Ge et al. / Toxicology Letters 226 (2014) 81–89

Fig. 5. Involvement of GPR30/EGFR/ERK1/2 in BPA induced TM4 proliferation. Cell proliferation is assessed by CCK-8 kit after 24 h exposure to different compounds: BPA 1 nM,G-1 (a specific agonist of GPR30) alone (10 nM) or after a 90-min pretreatment with G15 (a specific antagonist of GPR30, 1 �M), PD 98059 (an ERK1/2 antagonist, 10 �M),AG-1478 (a potent antagonist of EGFR, 10 �M), or the mixture (G15, PD 98059, and AG-1478). (A) Cells were treated with vehicle (control), 10−9 M BPA alone or plus otherantagonists with the concentrations mentioned above; (B) Cells were treated with vehicle (control), 10−8 M G-1 alone or plus other antagonists with the concentrationsm or plu1 ent coe SD of

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entioned above; (C) Cells were treated with vehicle (control), 10−9 M BPA alone �M; AG-1478, 1 �M); (D) ERK1/2 phosphorylation in TM4 cells exposure to differxpressed in the percentage of control (steroid-free medium) given as the mean ±

ompared with control.

r G-1 for 24 h induced proliferation of TM4 cells. When thepidermal growth factor receptor (EGFR) antagonist AG 147810 �M) or the mitogen-activated protein kinase MEK antagonistD 98059 (10 �M) or the GPR30-specific antagonist G15 (1 �M)ere added, no stimulatory effect on proliferation of cells was

bserved in TM4 cells treated with antagonist alone or combina-ion with BPA (Fig. 5A and B). Inhibition by mixture of antagonists

as greater than that of any individual antagonist. To avoid the

ytotoxicity of inhibitors, the concentration which had no inhi-ition effect for cell proliferation was used for G15 (100 nM), PD8059 (1 �M), and AG-1478 (1 �M) before treatment with BPA.

s other antagonists with no cytotoxicity concentrations (G15, 100 nM; PD 98059,mpounds with the concentrations mentioned above for 15 min. Values shown are

three independent experiments (n = 3). * p < 0.05 compared with control; ** p < 0.01

All inhibitors successfully abolished stimulatory effects of 10−9 MBPA (Fig. 5C). This result suggested that an additive or synergis-tic effect is possibly involved and that the GPR30/EGFR/ERK1/2signal transduction pathway was involved in stimulation of prolif-eration of TM4 cells by BPA or G-1. Furthermore, phosphorylationof ERK1/2 caused by exposure to BPA was abolished in thepresence of G15, PD 98059, or AG 1478 alone or in combina-

tion of all three antagonists together (Fig. 5D), suggesting thatGPR30 and EGFR signaling is required for ERK1/2 activation afterexposure to BPA and consequently for BPA-induced TM4 cellproliferation.

L.-C. Ge et al. / Toxicology Letters 226 (2014) 81–89 87

Fig. 6. Low-concentration BPA induces gene and protein expression of GPR30 but not EGFR in TM4 cells. RT-PCR analysis mRNA expression of GPR30 (A) and EGFR (B) of cellstreated with 10−9 and 10−8 M BPA for 24 h (mean ± SD of three independent experiments). Western blot (C) analysis protein expression of GPR30 of cells treated with 10−12

to 10−8 M BPA for 24 h. GAPDH was used as internal control to ensure that equal amounts of gene or protein were loaded in each lane. Data represent results from threei ified bs compa

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ndependent experiments. Bands from three independent experiments (n = 3) quantample. The control value was set as 1. * p < 0.05 compared with control; ** p < 0.01

.6. BPA stimulates the mRNA and protein expression of GPR30 inM4 cells

BPA has been found to significantly induce expression of mRNAs well as transcription of GPR30 protein in mouse spermatogo-ia GC-1 cells through the GPR30-EGFR-ERK-c-Fos, ER-�, and PKGathways (Sheng et al., 2013). Exposure to 10−9 or 10−8 M BPAesulted in significant up-regulation of mRNA of GPR30 in TM4ells (Fig. 6A), but not of EGFR (Fig. 6B). GPR30 protein measuredy Western-blotting demonstrated that it can be induced by BPA in

concentration-dependent manner (10−11 to 10−8 M) in TM4 cellsFig. 6C). Collectively, nanomolar concentrations of BPA can elevatehe mRNA and protein expression of GPR30.

. Discussion

Results of the present study revealed, for the first time,hat nanomolar concentrations of BPA can significantly increaseroliferation of immature mouse Sertoli TM4 cells. Small (pM) con-entrations of BPA have also been reported to trigger a nongenomicroliferative effect in the pancreatic islet, endothelium, breast,permatogonial and pituitary gland by initiating rapid responsesLaPensee et al., 2009; Sheng and Zhu, 2011; Wetherill et al., 2007).n the present study, a comparable representative nongenomicroliferative effect was observed for 10−9 to 10−8 M BPA, whichesulted a greater proportion of cells being in S phase and down-

egulation of expression of p21 and p53 gene (Fig. 2). p53 hashe ability to activate transcription of various proapoptotic genes,ncluding genes encoding members of the Bcl-2 family (Meek,009). The p21 gene is responsible for inhibition of proliferation

y densitometry, with results (mean ± SD) normalized to GAPDH expression in eachred with control.

(Chen et al., 2009). Inhibition of expression of mRNA of p53 andp21 by nanomolar concentrations of BPA in TM4 cell were con-sistent with that small concentrations of BPA can have significantgrowth-stimulation response to TM4 cells. Accordingly, 10−8 MBPA up-regulated the protein expression of Bcl-2 and PCNA, bothof which are markers of cell proliferation.

Stimulatory effects of BPA on proliferation of Sertoli cells haveimportant biological significances. Regulation of spermatogenesisby Sertoli cells is dependent on its functions and cell number. Thephysical support, junctional barriers, and biochemical stimulationof Sertoli cells, can ultimately determine fertility and sperm effi-ciency (Salian et al., 2009). The number of Sertoli cells is stable inadults with no proliferation once adult numbers have been reached(Johnson et al., 2008), therefore dysfunction of proliferation of TM4cells might be one reason responsible for male infertility influencedby BPA. Furthermore, these effects might also result in malignantgerm cell transformation/carcinoma in situ and then testicular germcell cancer, the most frequent cancer of young men, with increas-ing incidence. However, in the current study Sertoli TM4 cells areimmature (for preweaning mice), therefore the consequences ofeffects of BPA on development of Sertoli cells on adult sperm qual-ity, fertility and malignancy requires further study (Royer et al.,2012).

Concentrations of BPA in blood, fetuses, urine, saliva, and varioustissues have been reported to be in the range of 0.4–44 nM (Doergeet al., 2010; Vandenberg et al., 2010) are in the same range as those

used in the present study. However, Doerge et al. (2010) suggestedthat circulating concentrations of BPA aglycone were quite smallfollowing oral administration (<1% of total), which implies that tox-icological effects observed in rats from early postnatal exposures to

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PA might over-predict those possible in primates of the same age.erum TOTBPA concentrations (TOTBPA = conjugated BPA + BPA) waseported to rang from less than or equal to limit of detection (LOD,.3 nM) to 5.7 nM and were, on average, 42 times lower than urineoncentrations (Teeguarden et al., 2011). Therefore, accurate deter-ination of concentration of unconjugated (active) BPA in human

issues such as blood and urine remains be to further illustratednd compared with the concentration (10−9 and 10−8 M) used inhe current study.

Estrogens and xenoestrogens classically mediate their actionsfter binding to estrogen receptors �/� (ER�/�) that act asranscription factors to modulate activities of target genes bynteracting with several DNA response elements. Recently, novelstrogen receptor GPR30 has been shown to potentially mediateapid E2-dependent proliferation in cancer cells (Albanito et al.,007, 2008; Revankar et al., 2005). Nongenomic signals mediatedy GPR30 can directly or indirectly modulate cellular responsesuch as calcium mobilization, kinase activation, and nitric oxideroduction with a slower time course (Prossnitz et al., 2008).PR30 was recently shown to mediate proliferative effects of E2 inC-1 cells (Sirianni et al., 2008) and spermatogonial cells (Shengnd Zhu, 2011). In the present study, both GPR30 and ER�/� areound to be involved in proliferation of TM4 cells and ERK1/2hosphorylation induced by nanomolar BPA, since antagonists ofPR30 (G15) or ER�/� (ICI 182,780) were able to block the effectsf BPA on proliferation of TM4 cells. These results are similar tohose of another study (Sheng and Zhu, 2011) which revealedhat BPA induces proliferation of mouse spermatogonia cells byPR30 and ER-�. Furthermore, both GPR30 and ER�/� are also

nvolved in BPA-induced ERK1/2 phosphorylation, which was cor-esponding to its stimulatory effect on TM4 cell proliferation. BPAan stimulate rapid phosphorylation of ERK1/2 are consistent withhe results of previous studies performed in cancer cells (Gaot al., 2011; Prossnitz and Barton, 2011). BPA might still stimu-ate proliferation of cells and active ERK1/2 in the combinationreatment with antagonists, because inhibition caused by antag-nists were much stronger and therefore, the stimulatory effects ofPA were not seen. Furthermore, other estrogen signaling recep-ors such as estrogen related receptor (ERR �/�/�) might also

ediate proliferation of cells induced by BPA and need furthertudy (Okada et al., 2008). The results of the study which is pre-ented here suggested that both ER�/� and GPR30 are involvedn BPA-stimulated TM4 cell proliferation and ERK phosphoryla-ion.

GPR30/EGFR transduction pathway and GPR30 up regulationere involved in BPA-induced proliferation of TM4 cells and phos-horylation of ERK1/2 observed in the current study. Results of aecent study indicated that expression of GPR30 is regulated byhe EGF/EGFR signaling pathway (Migliaccio et al., 2010). Previoustudies indicated that estrogens acting via GPR30 are capable oftimulating adenylyl cyclase activity, which in turn leads to PKA-ediated suppression of EGFR-ERK (Prossnitz and Barton, 2011).

ased on the present data and available literature, BPA may actia GPR30 to activate EFGR-ERK pathways (Prossnitz and Barton,011), finally leading to the stimulation of a mitogenic signalingetwork and cell proliferation (Alonso-Magdalena et al., 2012). PD,G, and mixture of G15 + PD + AG also inhibited the phosphoryla-

ion of ERK1/2, suggesting that BPA is still stimulating activationf ERK1/2 in the combination treatment but the inhibitions causedy the antagonists were much stronger. The results of the currenttudy demonstrated that low-concentration BPA (10−11 M) inducedPR30 mRNA and protein expression, while it had no effect on

xpression of mRNA of EGFR. BPA was previously reported to up-egulate expression of GPR30 via an EGFR-ERK-c-Fos pathway inpermatogonial cell (Sheng et al., 2013), and a similar pathway maylso exist in Sertoli TM4 cells.

ters 226 (2014) 81–89

In the model system studied here, nanomolar concentrations ofBPA significantly increased proliferation of cells and both GPR30and ER�/� were involved in this process. Furthermore, GPR30 areable to activate ERK via EGFR pathway after stimulated by BPA.In future research, the in vitro experiments on which we reporthere will be expanded to an in vivo study using rats chronicallyexposed to environmentally relevant concentrations of BPA. Over-all, our results provide a novel insight regarding the potential roleof GPR30 in mediating a growth stimulatory action of low nM levelsof BPA in immature Sertoli cells.

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (Grant No. 31101071 and No. 81302317),the National Basic Research Program of China (973 Program, No.2011CB9358003), the Fundamental Research Funds for the CentralUniversities (Sun Yat-sen University) (No. 12ykpy09), the Scienceand Technology Planning Project of Guangdong Province, China(No. 2012B031500005), and the Seed Collaborative Research Fundfrom the State Key Laboratory in Marine Pollution (SCRF0003).The research was supported, in part, by a Discovery Grant fromthe National Science and Engineering Research Council of Canada(Project No. 326415-07). Prof. Giesy was supported by the pro-gram of 2012 “High Level Foreign Experts” (No. GDW20123200120)funded by the State Administration of Foreign Experts Affairs, theP.R. China to Nanjing University and the Einstein Professor Programof the Chinese Academy of Sciences.

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