Toxicology in Vitro 28 (2014) 373–380
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Estrogen receptors are involved in polychlorinated biphenyl-induced apoptosis on mouse spermatocyte GC-2 cell line 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, China School of Public Health, NanTong University, 9 Seyuan Road, Nantong 226019, China c Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China b
a r t i c l e
i n f o
Article history: Received 17 July 2013 Accepted 23 October 2013 Available online 8 November 2013 Keywords: Aroclor 1254 Estrogen receptors Spermatocytes Apoptosis
a b s t r a c t Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants which have been shown to have reproductive toxicity and to disturb spermatogenesis. But the precise mechanism is not clear. A mouse pachytene spermatocyte-derived cell line, GC-2 cells were used in the present study to 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 phase and induced apoptosis which might be partly explained by the decreased expression of Bcl-2 and cell cycle regulator cyclin D1 together with the activation of caspase-3. Besides, the treatment of Aroclor 1254 decreased the protein expression of estrogen receptor (ER)-a while increasing that of ERb. Then the administration of selective ERa agonist PPT partly reversed Aroclor 1254-induced alteration in Bcl-2, caspase-3 and cyclin D1 protein expression while selective ERb agonist DPN accelerated it. These results suggest that Aroclor 1254, working through ERa and ERb, interferes with the expression of proteins involved in the balance between cellular apoptosis and proliferation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Polychlorinated biphenyls (PCBs), containing 209 possible PCB congeners with differentially numbered and positioned chlorines, are members of halogenated aromatic hydrocarbons (HAHs). Although the use and production of PCBs have been strictly prohibited for decades in most countries, they remain a major global environmental problem because of their chemical stability and bio-characteristic (Conka et al., 2005). Several recent studies have demonstrated the persistence of PCBs in the surface soils, seawater, river sediments, air and some animal foods through which the general population can be exposed to. Accordingly, measurable levels of serum PCBs are detected in the majority of general populations (Aksoy et al., 2012; Diamanti-kandarakis et al., 2009; Ferrante et al., 2010). From studies in humans and other species it is well known that exposure to PCBs results in a broad range of deleterious effects including reproductive impairment (Aly, 2013; Fielden et al., 2001; Meeker and Hauser, 2010; Zhou and Zhang, 2005). In adult rats, exposure to PCBs caused decreased testicular size and ⇑ Corresponding authors. Address: State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, No. 818 Tianyuan East Road, Nanjing, Jiangsu, China. Tel./fax: +86 25 86868427. E-mail addresses: [email protected] (A. Gu), [email protected] (X. Wang). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.10.024
accessory sex organ weight (Aly et al., 2009). In contrast, enlarged testis weights and increased sperm production were observed in neonatal male rats exposed to PCBs (Kim, 2001). In cocks, PCBs exposure resulted in a marked decrease in serum testosterone level and the testicular weight (Zhang and Qiao, 2004). In human, epidemiological studies revealed that the levels of serum PCBs were inversely associated with the serum testosterone levels (Goncharov et al., 2009). The increasing attention suggests that the harmful effect on male reproductive system caused by PCBs remain a great concern. Existing studies have suggested that testicle is a sensitive target for disruption by PCBs. Aroclor 1254, a widely used commercial PCBs mixture containing 54% chlorine by weight, has been shown to cause reproductive disorders and decrease epididymal sperm count in cocks, mice and rats (Cai et al., 2011; Krishnamoorthy et al., 2007; Zhang and Qiao, 2004). The experimental study by Aly has also reported impaired sperm production in rats after the treatment of Aroclor 1254 (Aly et al., 2009). It is known that normal spermatogenesis is a complex and coordinated process of cell differentiation which depends on a balance of cell apoptosis and proliferation. In this process, apoptosis plays an undoubtedly critical role and an increase or decrease in apoptosis rate may result in defects in spermatogenesis (Shukla et al., 2012; Tripathi et al., 2009). Therefore, considering the deleterious effects of PCBs on spermatogenesis, in this study we investigated the effects of Aroclor 1254 on spermatocyte apoptosis using an immortalized mouse
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Fig. 1. Effects of Aroclor 1254 on cell viability in GC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for 24 h or 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 the mean ± S.E. from three independent experiments. Indicates significant difference when the values were compared to that of the control (p < 0.05).
pachytene spermatocyte-derived cell line, GC-2spd (ts) cells (GC-2 cells). In the testis, germ cell apoptosis is a complicated process which can be influenced by various biochemical factors such as hormone levels, receptors for hormones and pathway-specific apoptotic genes and proteins (Chausiaux et al., 2008; Ruwanpura et al., 2008). Recently, several lines of evidence have indicated that estrogen receptors (ERs) are also involved in the regulation of apoptosis (Fatima et al., 2012). It has also been reported that exogenous chemicals might induce apoptosis in MCF-7 cells by interfering with the crosstalk between ERs and other signal pathways (Meng et al., 2012). In fact, ERs are ligand-activated transcription factors which are involved in regulating gene expression and cell growth (Poelzl et al., 2000). Two ERs including ERa and ERb have been identified. Immunohistochemical data have shown the expression of both ERs in testicular cells including germ cells in several species (Carreau and Hess, 2010). Therefore, the second aim of this study was to investigate the effect of Aroclor 1254 on ERs expression in spermatocyte apoptosis. 2. Materials and methods 2.1. Chemicals and reagents Aroclor 1254 (CAS number: 11097-69-1), a commercial mixture of 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 purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2,3Bis(4-hydroxyphenyl)-propionitrile diarylpropionitrile (DPN), 4,40 ,400 -(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and ICI182780 were from Tocris (Ellisville, MO, USA). Rabbit antimouse Bcl-2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cleaved caspase-3 were from Cell Signaling Technology (Beverly, MA). Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) and 3-[4,5-dimethylthiazole-2-yl]-2,5Diphenyltetrazolium bromide (MTT) were from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Cell culture and treatments The GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line (Hofmann et al., 1992), were maintained in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 lg/ml streptomycin and 100 U/ml penicillin). Cells were seeded in 96-well plates (2 104/well) for cell viability assay in which treatments were performed in quadruplicates and 6-well plates (5 105/well) for apoptosis, cell cycle and western blot analysis. After fasted for 24 h, the cells were washed twice
Fig. 2. Effects of Aroclor 1254 on cell apoptosis in GC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for 24 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) Percentage of apoptotic cells in GC-2 cells. 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).
with phosphate buffer saline (PBS). Then various concentrations of Aroclor 1254 (0, 30, 60, 90 lmol/L) in the conditioned medium were added and incubated for 24 or 48 h. For the ER signaling pathway 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 exposure to Aroclor 1254 (Sirianni et al., 2008). The cells were maintained 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 limited solubility of Aroclor 1254 in water, it was dissolved in dimethyl sulfoxide (DMSO) and then diluted to various concentrations with cell culture medium. The final concentration of DMSO never exceeded 0.1% and had no significant effect on cell viability. At the end of the various treatment, 100 ll of 0.5 mg/ml MTT solution was added to each well of 96-well plates and incubated for 4 h at 37 °C. Then 100 ll dimethylsulfoxide was added. Optical density at 490 nm was measured. The number of viable cells was expressed as percentage of control cells.
J. Qu et al. / Toxicology in Vitro 28 (2014) 373–380
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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 for 24 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-G1 cells. 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).
2.4. Apoptosis analysis Cells undergoing apoptosis were assessed using the annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China) as described in the manufacturer’s manual. In brief, after treatment, cells were washed twice with PBS, suspended in binding buffer and then incubated with Annexin V-FITC conjugated for 15 min. After that, the staining profiles were determined with FACScan and analyzed with the Cell-Quest software. 2.5. Cell cycle analysis The cell cycle was analyzed by propidium iodide (PI) staining using flow cytometry. Briefly, after treatment, cells were washed with cold phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol and placed at -20 °C overnight. The cells were centrifuged 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 cells were collected. Cell cycle distribution was analyzed with a FACScan flow cytometer and Cell-Quest software. 2.6. Western blot analysis Proteins (30 lg) extracted from cells with different treatments were denatured in sodium dodecyl sulfate (SDS) sample buffer and loaded onto a 12.3% polyacrylamide–SDS gel. The proteins were separated by electrophoresis and then transferred onto a polyvinylidene 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 milk and 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
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Fig. 5. Effects of Aroclor 1254 on cyclin D1 protein expression in GC-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 cyclin D1 was measured by Western blot. The upper panel shows a representative Western blot. The lower panel represents the 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 the control (p < 0.05).
significance between the control and the treatments were determined by one-way ANOVA, followed by post hoc analysis comparing least significant differences. A p value of
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Estrogen receptors are involved in polychlorinated biphenyl-induced apoptosis on mouse spermatocyte GC-2 cell line 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, China School of Public Health, NanTong University, 9 Seyuan Road, Nantong 226019, China c Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China b
a r t i c l e
i n f o
Article history: Received 17 July 2013 Accepted 23 October 2013 Available online 8 November 2013 Keywords: Aroclor 1254 Estrogen receptors Spermatocytes Apoptosis
a b s t r a c t Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants which have been shown to have reproductive toxicity and to disturb spermatogenesis. But the precise mechanism is not clear. A mouse pachytene spermatocyte-derived cell line, GC-2 cells were used in the present study to 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 phase and induced apoptosis which might be partly explained by the decreased expression of Bcl-2 and cell cycle regulator cyclin D1 together with the activation of caspase-3. Besides, the treatment of Aroclor 1254 decreased the protein expression of estrogen receptor (ER)-a while increasing that of ERb. Then the administration of selective ERa agonist PPT partly reversed Aroclor 1254-induced alteration in Bcl-2, caspase-3 and cyclin D1 protein expression while selective ERb agonist DPN accelerated it. These results suggest that Aroclor 1254, working through ERa and ERb, interferes with the expression of proteins involved in the balance between cellular apoptosis and proliferation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Polychlorinated biphenyls (PCBs), containing 209 possible PCB congeners with differentially numbered and positioned chlorines, are members of halogenated aromatic hydrocarbons (HAHs). Although the use and production of PCBs have been strictly prohibited for decades in most countries, they remain a major global environmental problem because of their chemical stability and bio-characteristic (Conka et al., 2005). Several recent studies have demonstrated the persistence of PCBs in the surface soils, seawater, river sediments, air and some animal foods through which the general population can be exposed to. Accordingly, measurable levels of serum PCBs are detected in the majority of general populations (Aksoy et al., 2012; Diamanti-kandarakis et al., 2009; Ferrante et al., 2010). From studies in humans and other species it is well known that exposure to PCBs results in a broad range of deleterious effects including reproductive impairment (Aly, 2013; Fielden et al., 2001; Meeker and Hauser, 2010; Zhou and Zhang, 2005). In adult rats, exposure to PCBs caused decreased testicular size and ⇑ Corresponding authors. Address: State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, No. 818 Tianyuan East Road, Nanjing, Jiangsu, China. Tel./fax: +86 25 86868427. E-mail addresses: [email protected] (A. Gu), [email protected] (X. Wang). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.10.024
accessory sex organ weight (Aly et al., 2009). In contrast, enlarged testis weights and increased sperm production were observed in neonatal male rats exposed to PCBs (Kim, 2001). In cocks, PCBs exposure resulted in a marked decrease in serum testosterone level and the testicular weight (Zhang and Qiao, 2004). In human, epidemiological studies revealed that the levels of serum PCBs were inversely associated with the serum testosterone levels (Goncharov et al., 2009). The increasing attention suggests that the harmful effect on male reproductive system caused by PCBs remain a great concern. Existing studies have suggested that testicle is a sensitive target for disruption by PCBs. Aroclor 1254, a widely used commercial PCBs mixture containing 54% chlorine by weight, has been shown to cause reproductive disorders and decrease epididymal sperm count in cocks, mice and rats (Cai et al., 2011; Krishnamoorthy et al., 2007; Zhang and Qiao, 2004). The experimental study by Aly has also reported impaired sperm production in rats after the treatment of Aroclor 1254 (Aly et al., 2009). It is known that normal spermatogenesis is a complex and coordinated process of cell differentiation which depends on a balance of cell apoptosis and proliferation. In this process, apoptosis plays an undoubtedly critical role and an increase or decrease in apoptosis rate may result in defects in spermatogenesis (Shukla et al., 2012; Tripathi et al., 2009). Therefore, considering the deleterious effects of PCBs on spermatogenesis, in this study we investigated the effects of Aroclor 1254 on spermatocyte apoptosis using an immortalized mouse
374
J. Qu et al. / Toxicology in Vitro 28 (2014) 373–380
Fig. 1. Effects of Aroclor 1254 on cell viability in GC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for 24 h or 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 the mean ± S.E. from three independent experiments. Indicates significant difference when the values were compared to that of the control (p < 0.05).
pachytene spermatocyte-derived cell line, GC-2spd (ts) cells (GC-2 cells). In the testis, germ cell apoptosis is a complicated process which can be influenced by various biochemical factors such as hormone levels, receptors for hormones and pathway-specific apoptotic genes and proteins (Chausiaux et al., 2008; Ruwanpura et al., 2008). Recently, several lines of evidence have indicated that estrogen receptors (ERs) are also involved in the regulation of apoptosis (Fatima et al., 2012). It has also been reported that exogenous chemicals might induce apoptosis in MCF-7 cells by interfering with the crosstalk between ERs and other signal pathways (Meng et al., 2012). In fact, ERs are ligand-activated transcription factors which are involved in regulating gene expression and cell growth (Poelzl et al., 2000). Two ERs including ERa and ERb have been identified. Immunohistochemical data have shown the expression of both ERs in testicular cells including germ cells in several species (Carreau and Hess, 2010). Therefore, the second aim of this study was to investigate the effect of Aroclor 1254 on ERs expression in spermatocyte apoptosis. 2. Materials and methods 2.1. Chemicals and reagents Aroclor 1254 (CAS number: 11097-69-1), a commercial mixture of 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 purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2,3Bis(4-hydroxyphenyl)-propionitrile diarylpropionitrile (DPN), 4,40 ,400 -(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and ICI182780 were from Tocris (Ellisville, MO, USA). Rabbit antimouse Bcl-2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cleaved caspase-3 were from Cell Signaling Technology (Beverly, MA). Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) and 3-[4,5-dimethylthiazole-2-yl]-2,5Diphenyltetrazolium bromide (MTT) were from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Cell culture and treatments The GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line (Hofmann et al., 1992), were maintained in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 lg/ml streptomycin and 100 U/ml penicillin). Cells were seeded in 96-well plates (2 104/well) for cell viability assay in which treatments were performed in quadruplicates and 6-well plates (5 105/well) for apoptosis, cell cycle and western blot analysis. After fasted for 24 h, the cells were washed twice
Fig. 2. Effects of Aroclor 1254 on cell apoptosis in GC-2 cells. Cells were treated with various concentrations of Aroclor 1254 (30, 60, 90 lM) or DMSO as control for 24 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) Percentage of apoptotic cells in GC-2 cells. 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).
with phosphate buffer saline (PBS). Then various concentrations of Aroclor 1254 (0, 30, 60, 90 lmol/L) in the conditioned medium were added and incubated for 24 or 48 h. For the ER signaling pathway 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 exposure to Aroclor 1254 (Sirianni et al., 2008). The cells were maintained 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 limited solubility of Aroclor 1254 in water, it was dissolved in dimethyl sulfoxide (DMSO) and then diluted to various concentrations with cell culture medium. The final concentration of DMSO never exceeded 0.1% and had no significant effect on cell viability. At the end of the various treatment, 100 ll of 0.5 mg/ml MTT solution was added to each well of 96-well plates and incubated for 4 h at 37 °C. Then 100 ll dimethylsulfoxide was added. Optical density at 490 nm was measured. The number of viable cells was expressed as percentage of control cells.
J. Qu et al. / Toxicology in Vitro 28 (2014) 373–380
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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 for 24 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-G1 cells. 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).
2.4. Apoptosis analysis Cells undergoing apoptosis were assessed using the annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China) as described in the manufacturer’s manual. In brief, after treatment, cells were washed twice with PBS, suspended in binding buffer and then incubated with Annexin V-FITC conjugated for 15 min. After that, the staining profiles were determined with FACScan and analyzed with the Cell-Quest software. 2.5. Cell cycle analysis The cell cycle was analyzed by propidium iodide (PI) staining using flow cytometry. Briefly, after treatment, cells were washed with cold phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol and placed at -20 °C overnight. The cells were centrifuged 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 cells were collected. Cell cycle distribution was analyzed with a FACScan flow cytometer and Cell-Quest software. 2.6. Western blot analysis Proteins (30 lg) extracted from cells with different treatments were denatured in sodium dodecyl sulfate (SDS) sample buffer and loaded onto a 12.3% polyacrylamide–SDS gel. The proteins were separated by electrophoresis and then transferred onto a polyvinylidene 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 milk and 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
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Fig. 5. Effects of Aroclor 1254 on cyclin D1 protein expression in GC-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 cyclin D1 was measured by Western blot. The upper panel shows a representative Western blot. The lower panel represents the 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 the control (p < 0.05).
significance between the control and the treatments were determined by one-way ANOVA, followed by post hoc analysis comparing least significant differences. A p value of
