Toxicology in Vitro 28 (2014) 373–380

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Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Estrogen receptors are involved in polychlorinated biphenyl-inducedapoptosis on mouse spermatocyte GC-2 cell line

0887-2333/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tiv.2013.10.024

⇑ Corresponding authors. Address: State Key Laboratory of ReproductiveMedicine, Institute of Toxicology, Nanjing Medical University, No. 818Tianyuan East Road, Nanjing, Jiangsu, China. Tel./fax: +86 25 86868427.

E-mail addresses: aihuagu@njmu.edu.cn (A. Gu), xrwang@njmu.edu.cn(X. Wang).

Jianhua Qu a,b,c, Wei Liu a,c, Cong Huang a,c, Cheng Xu a,c, Guizhen Du a,c, Aihua Gu a,c,⇑, Xinru Wang a,c,⇑a State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 211166, Chinab School of Public Health, NanTong University, 9 Seyuan Road, Nantong 226019, Chinac Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China

a r t i c l e i n f o

Article history:Received 17 July 2013Accepted 23 October 2013Available online 8 November 2013

Keywords:Aroclor 1254Estrogen receptorsSpermatocytesApoptosis

a b s t r a c t

Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants which havebeen shown to have reproductive toxicity and to disturb spermatogenesis. But the precise mechanismis not clear. A mouse pachytene spermatocyte-derived cell line, GC-2 cells were used in the present studyto investigate the toxic effect of PCBs (Aroclor 1254) and explore the underlying molecular mechanism.Results showed that Aroclor 1254 inhibited cell proliferation, caused the arrest of cells in G0/G1 phaseand induced apoptosis which might be partly explained by the decreased expression of Bcl-2 and cellcycle regulator cyclin D1 together with the activation of caspase-3. Besides, the treatment of Aroclor1254 decreased the protein expression of estrogen receptor (ER)-a while increasing that of ERb. Thenthe administration of selective ERa agonist PPT partly reversed Aroclor 1254-induced alteration inBcl-2, caspase-3 and cyclin D1 protein expression while selective ERb agonist DPN accelerated it. Theseresults suggest that Aroclor 1254, working through ERa and ERb, interferes with the expression ofproteins involved in the balance between cellular apoptosis and proliferation.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Polychlorinated biphenyls (PCBs), containing 209 possible PCBcongeners with differentially numbered and positioned chlorines,are members of halogenated aromatic hydrocarbons (HAHs).Although the use and production of PCBs have been strictly prohib-ited for decades in most countries, they remain a major globalenvironmental problem because of their chemical stability andbio-characteristic (Conka et al., 2005). Several recent studies havedemonstrated the persistence of PCBs in the surface soils, seawater,river sediments, air and some animal foods through which thegeneral population can be exposed to. Accordingly, measurablelevels of serum PCBs are detected in the majority of generalpopulations (Aksoy et al., 2012; Diamanti-kandarakis et al., 2009;Ferrante et al., 2010).

From studies in humans and other species it is well known thatexposure to PCBs results in a broad range of deleterious effectsincluding reproductive impairment (Aly, 2013; Fielden et al.,2001; Meeker and Hauser, 2010; Zhou and Zhang, 2005). In adultrats, exposure to PCBs caused decreased testicular size and

accessory sex organ weight (Aly et al., 2009). In contrast, enlargedtestis weights and increased sperm production were observed inneonatal male rats exposed to PCBs (Kim, 2001). In cocks, PCBsexposure resulted in a marked decrease in serum testosterone leveland the testicular weight (Zhang and Qiao, 2004). In human, epide-miological studies revealed that the levels of serum PCBs were in-versely associated with the serum testosterone levels (Goncharovet al., 2009). The increasing attention suggests that the harmful ef-fect on male reproductive system caused by PCBs remain a greatconcern.

Existing studies have suggested that testicle is a sensitive targetfor disruption by PCBs. Aroclor 1254, a widely used commercialPCBs mixture containing 54% chlorine by weight, has been shownto cause reproductive disorders and decrease epididymal spermcount in cocks, mice and rats (Cai et al., 2011; Krishnamoorthyet al., 2007; Zhang and Qiao, 2004). The experimental study byAly has also reported impaired sperm production in rats after thetreatment of Aroclor 1254 (Aly et al., 2009). It is known that nor-mal spermatogenesis is a complex and coordinated process of celldifferentiation which depends on a balance of cell apoptosis andproliferation. In this process, apoptosis plays an undoubtedly crit-ical role and an increase or decrease in apoptosis rate may result indefects in spermatogenesis (Shukla et al., 2012; Tripathi et al.,2009). Therefore, considering the deleterious effects of PCBs onspermatogenesis, in this study we investigated the effects of Aroc-lor 1254 on spermatocyte apoptosis using an immortalized mouse

Fig. 1. Effects of Aroclor 1254 on cell viability in GC-2 cells. Cells were treated withvarious concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for 24 hor 48 h, cell proliferation was evaluated by MTT assay as described in Section 2.Each data point was normalized to the control (DMSO) and represents themean ± S.E. from three independent experiments. � Indicates significant differencewhen the values were compared to that of the control (p < 0.05).

374 J. Qu et al. / Toxicology in Vitro 28 (2014) 373–380

pachytene spermatocyte-derived cell line, GC-2spd (ts) cells (GC-2cells).

In the testis, germ cell apoptosis is a complicated process whichcan be influenced by various biochemical factors such as hormonelevels, receptors for hormones and pathway-specific apoptoticgenes and proteins (Chausiaux et al., 2008; Ruwanpura et al.,2008). Recently, several lines of evidence have indicated that estro-gen receptors (ERs) are also involved in the regulation of apoptosis(Fatima et al., 2012). It has also been reported that exogenouschemicals might induce apoptosis in MCF-7 cells by interferingwith the crosstalk between ERs and other signal pathways (Menget al., 2012). In fact, ERs are ligand-activated transcription factorswhich are involved in regulating gene expression and cell growth(Poelzl et al., 2000). Two ERs including ERa and ERb have beenidentified. Immunohistochemical data have shown the expressionof both ERs in testicular cells including germ cells in several species(Carreau and Hess, 2010). Therefore, the second aim of this studywas to investigate the effect of Aroclor 1254 on ERs expression inspermatocyte apoptosis.

Fig. 2. Effects of Aroclor 1254 on cell apoptosis in GC-2 cells. Cells were treatedwith various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for24 h, then cell apoptosis was analyzed by flow cytometry as described in Section 2.(A) Representative images of flow cytometry analysis in GC-2 cells. (B) Percentageof apoptotic cells in GC-2 cells. Each data point represents the mean ± S.E. fromthree independent experiments. � Indicates significant difference when the valueswere compared to that of the control (p < 0.05).

2. Materials and methods

2.1. Chemicals and reagents

Aroclor 1254 (CAS number: 11097-69-1), a commercial mixtureof PCBs, was purchased from Sigma–Aldrich (St. Louis, MO, USA).The GC-2 cells were obtained from Cell Institute of Shanghai(Shanghai, China). Antibodies to ERa, ERb and cyclinD1 were pur-chased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2,3-Bis(4-hydroxyphenyl)-propionitrile diarylpropionitrile (DPN),4,40,400-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) andICI182780 were from Tocris (Ellisville, MO, USA). Rabbit anti-mouse Bcl-2, glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) and cleaved caspase-3 were from Cell Signaling Technology(Beverly, MA). Dulbecco’s Modified Eagle Medium: Nutrient Mix-ture F-12 (DMEM/F-12) and 3-[4,5-dimethylthiazole-2-yl]-2,5-Diphenyltetrazolium bromide (MTT) were from Sigma–Aldrich(St. Louis, MO, USA).

2.2. Cell culture and treatments

The GC-2 cells, an immortalized mouse pachytene spermato-cyte-derived cell line (Hofmann et al., 1992), were maintained inDMEM/F-12 supplemented with 10% fetal bovine serum (FBS)and 1% antibiotics (100 lg/ml streptomycin and 100 U/ml penicil-lin). Cells were seeded in 96-well plates (2 � 104/well) for cell via-bility assay in which treatments were performed in quadruplicatesand 6-well plates (5 � 105/well) for apoptosis, cell cycle and wes-tern blot analysis. After fasted for 24 h, the cells were washed twice

with phosphate buffer saline (PBS). Then various concentrations ofAroclor 1254 (0, 30, 60, 90 lmol/L) in the conditioned mediumwere added and incubated for 24 or 48 h. For the ER signaling path-way study, cells were treated with ICI 182780 (10 lM), PPT (1 lM)or DPN (1 lM) for 30 min prior to, and for the duration of the expo-sure to Aroclor 1254 (Sirianni et al., 2008). The cells were main-tained in a humidified atmosphere containing 95% air and 5%CO2 at 37 �C.

2.3. Cell viability assay

Cell viability was determined using a MTT assay. For the limitedsolubility of Aroclor 1254 in water, it was dissolved in dimethylsulfoxide (DMSO) and then diluted to various concentrations withcell culture medium. The final concentration of DMSO never ex-ceeded 0.1% and had no significant effect on cell viability. At theend of the various treatment, 100 ll of 0.5 mg/ml MTT solutionwas added to each well of 96-well plates and incubated for 4 h at37 �C. Then 100 ll dimethylsulfoxide was added. Optical densityat 490 nm was measured. The number of viable cells wasexpressed as percentage of control cells.

Fig. 3. Effects of Aroclor 1254 on cell cycle distribution in GC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for24 h, then cell cycle was analyzed by flow cytometry as described in Section 2. (A) Representative images of flow cytometry analysis in GC-2 cells. (B) Percentage of sub-G1cells. Each data point represents the mean ± S.E. from three independent experiments. � Indicates significant difference when the values were compared to that of the control(p < 0.05).

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2.4. Apoptosis analysis

Cells undergoing apoptosis were assessed using the annexinV-FITC apoptosis detection kit (Beyotime Institute of Biotechnology,Shanghai, China) as described in the manufacturer’s manual. Inbrief, after treatment, cells were washed twice with PBS, suspendedin binding buffer and then incubated with Annexin V-FITC conju-gated for 15 min. After that, the staining profiles were determinedwith FACScan and analyzed with the Cell-Quest software.

2.5. Cell cycle analysis

The cell cycle was analyzed by propidium iodide (PI) stainingusing flow cytometry. Briefly, after treatment, cells were washedwith cold phosphate-buffered saline (PBS) and fixed with 70%ice-cold ethanol and placed at -20 �C overnight. The cells were cen-trifuged at 300g for 5 min and then suspended with PBS. After that,

cells were treated with RNase and stained with PI (Sigma–Aldrich,St. Louis, MO, USA) solution. For each sample, at least 10,000 cellswere collected. Cell cycle distribution was analyzed with a FACScanflow cytometer and Cell-Quest software.

2.6. Western blot analysis

Proteins (30 lg) extracted from cells with different treatmentswere denatured in sodium dodecyl sulfate (SDS) sample bufferand loaded onto a 12.3% polyacrylamide–SDS gel. The proteinswere separated by electrophoresis and then transferred onto apolyvinylidene difluoride (PVDF) membrane (Millipore, Billerica,MA, USA). The protein transfer was performed at 100 V for 2 h.After that, the membrane was blocked with 5% skimmed milkand then incubated with primary antibodies against ERa (1:200),ERb (1:200), cyclin D1 (1:200), Bcl-2 (1:1000), cleaved caspase-3(1:1000) and GAPDH (1:1000) overnight at 4 �C. Incubation of

Fig. 4. Effects of Aroclor 1254 on cleaved caspase-3 and Bcl-2 protein expression inGC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60,90 lM) or DMSO as control for 24 h, then the expression of cleaved caspase-3 (A)and Bcl-2 (B) was measured by Western blot. The upper panel shows representativeWestern blot. The lower panel represents the mean ± S.E. of cleaved caspase-3 (orBcl-2) integrated optical density normalized to that of GAPDH. Each data pointrepresents the mean ± S.E. from three independent experiments. � Indicatessignificant difference when the values were compared to that of the control(p < 0.05).

Fig. 5. Effects of Aroclor 1254 on cyclin D1 protein expression in GC-2 cells. Cellswere treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSOas control for 24 h, then the expression of cyclin D1 was measured by Western blot.The upper panel shows a representative Western blot. The lower panel representsthe mean ± S.E. of cyclin D1 integrated optical density normalized to that of GAPDH.Each data point represents the mean ± S.E. from three independent experiments. �Indicates significant difference when the values were compared to that of thecontrol (p < 0.05).

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membranes with secondary antibodies was done at room temper-ature for 1 h and immunoreactive bands were visualized with theenhanced chemiluminescence (ECL) Western blotting detection re-agents (Millipore, Billerica, MA, USA). The band densitometry wasquantified using Image Lab software (BioRad laboratories, Hercu-les, CA, USA). The protein expression levels were expressed relativeto GAPDH levels.

2.7. Statistical analysis

All experiments were performed at least three times and theresults were from representative experiments. Values wereexpressed as mean ± S.E. for all the experiments. Statistical

significance between the control and the treatments were deter-mined by one-way ANOVA, followed by post hoc analysis compar-ing least significant differences. A p value of <0.05 was defined asstatistically significant.

3. Results

3.1. Effects of Aroclor 1254 on cell viability of GC-2 cells

The effect of Aroclor 1254 on GC-2 cell viability was determinedusing MTT assay. (Fig. 1). Results showed that Aroclor 1254 de-creased the viability of GC-2 cells in a dose- and time-dependentmanner. Significant reduction of cell viability was observed at60 lM and maximal reduction was observed at 90 lM Aroclor1254. These results showed the inhibitory effect of Aroclor 1254on GC-2 cell proliferation.

3.2. Effects of Aroclor 1254 on apoptosis and cell cycle of GC-2 cells

Next, we examined whether the anti-proliferative effect ofAroclor 1254 on GC-2 cells reflects the regulation of apoptosisand cell cycle. As shown in Fig. 2, a significant increase in the num-ber of apoptosis cells was observed after 60 lM and 90 lM of Aroc-lor 1254 treatment. The mean cell number in the sub-G1 phase ofthe cell cycle was also significantly higher in Aroclor 1254 treatedcells (60 lM and 90 lM) compared with non-treated cells (Fig. 3).These results suggested that Aroclor 1254 treatment induced GC-2cells apoptosis and cell cycle arrested in sub-G1 phase.

3.3. Effects of Aroclor 1254 on expression of cleaved caspase-3, Bcl-2and cyclinD1

Then we questioned whether apoptosis and cell cycle arrest in-duced by Aroclor 1254 were associated with the alteration of relatedprotein levels. Western blot was used to detect the expression ofapoptosis-related proteins. Results showed that Aroclor 1254

Fig. 6. Effects of Aroclor 1254 on ERa and ERb protein expression in GC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO ascontrol for 24 h, then the expression of ERa and ERb was measured by Western blot. (A) The panel shows a representative Western blot. (B) The panel represents themean ± S.E. of ERa and ERb integrated optical density normalized to that of GAPDH. Each data point represents the mean ± S.E. from three independent experiments.� Indicates significant difference when the values were compared to that of the control (p < 0.05).

Fig. 7. Effects of Aroclor 1254 combined with ER agonist/antagonist on cell viabilityin GC-2 cells. Cells were treated with Aroclor 1254 (90 lM) or DMSO as control for24 h, ICI 182780 (ICI) (10 lM), PPT (1 lM) or DPN (1 lM) was added to the cells for30 min prior to, and for the duration of the exposure to Aroclor 1254. Cellproliferation was evaluated by MTT assay. Each data point was normalized to thecontrol (DMSO) and represents the mean ± S.E. from three independent experi-ments. � Indicates significant difference when the values were compared to that ofthe control (p < 0.05); �� Indicates significant difference when the values werecompared to that of Aroclor 1254-treated (p < 0.05).

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(60 lM and 90 lM) induced an increase in the level of cleaved cas-pase-3 as well as a decrease in Bcl-2 protein expression (Fig. 4).

Similarly, the expression of a cell cycle-related protein was de-tected. As shown in Fig. 5, 60 lM and 90 lM Aroclor 1254 signifi-cantly decreased the expression of cyclin D1.

3.4. Effects of Aroclor 1254 on expression of ERa and ERb

It has been recently reported that ERa and ERb are involved inthe regulation of apoptosis (Fatima et al., 2012). Since we found

that Aroclor 1254 induced GC-2 cell apoptosis, we further exam-ined the effect of Aroclor 1254 on ERa and ERb protein expression.Results showed that Aroclor 1254 treatment decreased the expres-sion of ERa while increasing that of ERb significantly in a dose-dependent manner and 60 lM Aroclor 1254 was the minimumdose to have significant effect (Fig. 6). These results indicated thatthe effects on GC-2 cells induced by Aroclor 1254 might be medi-ated by ERa and ERb.

3.5. Effects of Aroclor 1254 on cell viability in the presence of ERantagonist/agonist

To verify ERs’ involvement in Aroclor 1254-induced cell prolif-eration inhibition, ICI 182780 (ERs antagonist), PPT (ERa specificagonist) and DPN (ERb specific agonist) were added to the mediumseparately. Then the ability of GC-2 cells to proliferate was evalu-ated using the MTT assay. Results showed that the addition ofPPT attenuated cytotoxicity of 90 lM Aroclor 1254 while DPN en-hanced the cytotoxicity of this toxicant. The addition of ICI 182780had no significant effect on GC-2 cell viability in the present ofAroclor 1254 compared to the control (Fig. 7).

3.6. Effects of Aroclor 1254 on expression of cleaved caspase-3, Bcl-2and cyclinD1 in the presence of ER antagonist/agonist

To further confirm the role of ERs in Aroclor 1254-induced GC-2cell apoptosis and cell cycle arrest, we tested effect of Aroclor 1254on the expression of several related proteins in the absence/pres-ence of ICI 182780, PPT or DPN. As shown in Fig. 8, DPN enhancedAroclor 1254-induced caspase-3 cleavage while PPT partially re-versed this change. Similarly, the decrease of Bcl-2 and cyclin D1protein expression induced by Aroclor 1254 was accelerated bythe addition of DPN while attenuated by PPT. The addition of ICI182780 did not alter the expression of these proteins significantly.

Fig. 8. Effects of Aroclor 1254 combined with ER agonist/antagonist on theexpression of cleaved caspase-3, Bcl-2 and cyclin D1 protein in GC-2 cells. Cellswere treated with Aroclor 1254 (90 lM) or DMSO as control for 24 h, ICI 182780(ICI) (10 lM), PPT (1 lM) or DPN (1 lM) was added to the cells for 30 min prior to,and for the duration of the exposure to Aroclor 1254. Then the expression of cleavedcaspase-3, Bcl-2 and cyclin D1 were measured by Western blot. (A) The panel showsa representative Western blot. (B) The panel represents the mean ± S.E. of cleavedcaspase-3, Bcl-2 and cyclin D1 integrated optical density normalized to that ofGAPDH. Each data point represents the mean ± S.E. from three independentexperiments. � Indicates significant difference when the values were compared tothat of the control (p < 0.05); �� Indicates significant difference when the valueswere compared to that of Aroclor 1254-treated (p < 0.05).

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4. Discussion

PCBs are ubiquitous environmental pollutions, which have beenshown to reduce testis weight and sperm count in several species(Aly et al., 2009; Krishnamoorthy et al., 2007). However, the mech-anisms behind the impaired spermatogenesis induced by PCBs arepoorly understood. In the present study, Aroclor 1254 inhibitedproliferation of GC-2 cells in a dose-dependent manner, suggestingits potent cytotoxic activity toward spermatocyte GC-2 cells. Apop-tosis of GC-2 cells was observed with a marked increase after Aroc-lor 1254 treatment. These findings suggested that Aroclor 1254interfered with the dynamic balance between germ cell prolifera-tion and apoptosis and the disrupted balance might result in de-fects in spermatogenesis (Shaha et al., 2010). Furthermore, ourstudy found that Aroclor 1254 induced an accumulation of GC-2cells in G0/G1 phase of cell cycle. Several lines of evidence haveshowed that some cell cycle-related genes regulate cell prolifera-tion and apoptosis (Wang et al., 2012a). So the G0/G1 arrest ofGC-2 cells induced by Aroclor 1254 might partly explain the inhib-ited cell proliferation and increased apoptosis.

The process of apoptosis involves two pathways: the cell deathreceptor pathway and the mitochondrial pathway (Elumalai et al.,2012; Mehmet, 2000). Both pathways are reported been involvedin testicular germ cell apoptosis (Moreno et al., 2011). To investi-gate more detail in the mechanism of Aroclor 1254-induced GC-2cell apoptosis, we evaluated the expression of apoptosis-relatedproteins. Results from western blot assay in this study showed thatBcl-2 expression was down-regulated and cleaved caspase-3expression was up-regulated significantly after the treatment ofAroclor 1254. It has been reported that Bcl-2 regulates apoptosisby affecting mitochondrial permeability and cleaved caspase-3participates in apoptosis execution (Mehmet, 2000). Therefore,Aroclor 1254 might induce apoptosis of GC-2 cells through themitochondrial pathway and caspase-3 activation. In addition, pre-vious studies have showed that caspase-3 and Bcl-2 play a crucialrole in spermatogenesis. It has also been reported that transgenicmice overexpressing Bcl-2 which are infertile display disruptedspermatogenesis. (Rodriguez et al., 1997). Thus, our findings thatAroclor 1254 interfered with Bcl-2 and caspase-3 expression maypartly contribute to the mechanism of impaired spermatogenesis byAroclor 1254 reported previously (Aly et al., 2009; Krishnamoorthyet al., 2007). In fact, an increase in factors linked to apoptoticpathways is often associated with a decrease in cell cycle-relatedcyclins expression. So we examined the expression of cyclin D1in GC-2 cells since Aroclor 1254 induced G0/G1 cell cycle arrestin the present study. Results showed that Aroclor 1254 treatmentinhibited cyclin D1 expression significantly in GC-2 cells. Thesefindings support previous observation that underexpression ofcyclinD1 was associated with decreased Bcl-2 expression and in-creased caspase-3 expression (Wang et al., 2012a,b), which mightthen result in inhibited cell proliferation and promoted apoptosis.

Estrogen receptors (ERs), belonging to the nuclear receptor fam-ily, are described in most of the testicular cells including germ cells(Bois et al., 2010). Studies on ER knock-out mice have showed theimportant role ERs play in spermatogenesis and both ERa and ERbare important for germ cell development and function in testis. ERbknockout mice show increased number of spermatogonia whereasERa knockout mice show decreased spermatogonial cell numberand increased germ cell apoptosis (Gould et al., 2007; Korach,1994). In this study, Aroclor 1254 treatment elevated the proteinexpression of ERb and decreased that of ERa, which are in agree-ment with the the opposing action of ERa and ERb on cell apopto-sis, ERa being the inhibitor and ERb the activator (Selva et al.,2004). This effect suggests that ERa and ERb might be involvedin Aroclor 1254-induced GC-2 cell apoptosis. In addition, it has

been reported that many chemicals can bind ERs and then interferewith ERs-induced effects through affecting ERs transcriptional

J. Qu et al. / Toxicology in Vitro 28 (2014) 373–380 379

activity, decreasing/increasing ERs phosphorylation or other possi-ble modes of action (Ko et al., 2011). So further studies are requiredto examine the exact action mode whereby Aroclor 1254 interfereswith ERs-induced effects in GC-2 cells.

In order to further confirm the involvement of ERs in Aroclor1254-induced apoptosis in GC-2 cells, we examined cell prolifera-tion, apoptosis and the expression of some related proteins in thepresence of various agonists or antagonists of ERs. Our datashowed that the inhibitory effect of Aroclor 1254 on GC-2 cell pro-liferation was mediated by both ERa and ERb, since ERa specificagonist PPT reversed this inhibitory effect partly while ERb specificagonist DPN accelerated it. Similar results were observed when weexamined the expression of two apoptosis-related proteins includ-ing caspase-3 and Bcl-2 in GC-2 cells. These results confirmed theinvolvement of both receptors in Aroclor 1254-induced GC-2 cellapoptosis. Considering the strict relationship between cyclins andcell apoptosis (Li et al., 2013), we further examined the expressionof cyclin D1 in response to Aroclor 1254 after the addition of ago-nists or antagonist of ERs. Results showed that Aroclor 1254 de-creased cyclin D1 expression and PPT attenuated this actionwhile DPN accelerated it. This results correlate well with those ob-tained on caspase-3 and Bcl-2 after the same treatment, leading usto conclude that ERa and ERb can be involved in the regulation of abalance between cell proliferation and apoptosis. The results ob-tained in this study support previous suggestion that ERs mightregulate cell apoptosis by interfering with the expression of someproteins such as caspase-3 and cyclin D1 (Chimento et al., 2012).But the exact pathway through which ERs act on caspase-3 and cy-clin D1 expression in Aroclor 1254-induced apoptosis still needsfurther investigation. Besides, the addition of ICI 182780 (a pureER antagonist) did not alter the expression of these proteins signif-icantly. One possible explanation for this finding might be theopposing role ERa and ERb play in the regulation of cell prolifera-tion and apoptosis (Wang et al., 2012b).

5. Conclusion

In conclusion, Aroclor 1254 inhibits proliferation and inducesapoptosis of GC-2 cells. This action might be mediated by bothERa and ERb, followed by activation of apoptotic pathway involv-ing Bcl-2 down-regulation, caspase-3 activation and a concomitantreduction of cyclin D1 levels, which provides new informationabout the mechanism of Aroclor 1254-induced spermatogenicimpairment.

6. Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by Grants National Basic ResearchProgram of China (973 Program, 2009CB941703), The key Projectof National Natural Science Foundation of China (No. 30930079),A Project Funded by the Priority Academic Program Developmentof Jiangsu Higher Education Institutions (PAPD), Scientific andTechnological Planning Project of Nantong (BK2012066).

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