Mutation Research 769 (2014) 29–33
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Bisphenol A induces oxidative stress-associated DNA damage in INS-1 cells Fei Xin a , Liping Jiang b , Xiaofang Liu a , Chengyan Geng b , Wenbin Wang c , Laifu Zhong a,b , Guang Yang a,b,∗∗ , Min Chen a,∗ a
Department of Food Nutrition and Safety, Dalian Medical University, No. 9 W. Lushun South Road, Dalian 116044, China Liaoning Anti-Degenerative Diseases Natural Products Engineering Technology Research Center, Dalian Medical University, Dalian 116044, China c School of Public Health, Shandong University, Jinan 250012, China b
a r t i c l e
i n f o
Article history: Received 24 October 2013 Received in revised form 13 February 2014 Accepted 5 April 2014 Available online 21 May 2014 Keywords: Bisphenol A INS-1 cells DNA strand-breaks ROS p53 p-Chk2 (T68)
a b s t r a c t Bisphenol A (BPA), an endocrine disruptor, is widely used to manufacture polycarbonate plastic and epoxy resins. Many studies have demonstrated that BPA can play a role in reproductive toxicity and affect the normal metabolic function. Recent research has shown that BPA can influence the function of pancreatic islets. In this study, our aim is to assess the DNA damage induced by BPA and to clarify the mechanism, by use of rat insulinoma INS-1 cells. INS-1 cells were exposed to different doses of BPA (0, 25, 50, 100 M). We conducted the single-cell gel electrophoresis (SCGE) assay to measure DNA damage, and studied proteins such as p53 and p-Chk2 (T68) by Western blotting, in order to verify the (geno)toxicity of BPA. Moreover, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) to discuss the possible mechanism of DNA damage. The results show that BPA caused an increased in DNA strand-breaks along with greater DNA migration from the nucleus into the comet tail. The expression of DNA damage-associated proteins (p53 and p-Chk2 (T68)) was significantly increased. The exposure to various doses of BPA caused a significant increase in intracellular ROS and a significant reduction in the level of GSH. N-Acetyl cysteine, an inhibitor of intracellular ROS formation, can significantly reduce the generation of intracellular reactive oxygen. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bisphenol A (BPA) is the common name for 2,2-bis-(4hydroxyphenyl)propane (C15 H16 O2 ). It is widely used to manufacture polycarbonate plastic and epoxy resins. BPA is a low-toxicity compound, with extensive application in plastic products, such as dental sealants, adhesives, food packaging, and plastic beverage containers [1]. In the recent years, it has become increasingly concerned about BPA, an endocrine disruptor, in view of its widespread presence in a variety of human tissues and body fluids [2]. It has been suspected that BPA is associated with various diseases including cancer, cardiovascular disease and reproductive disorders, but the results are controversial [3,4]. Some experimental studies in rodents on BPA
∗ Corresponding author. Tel.: +86 411 8611 0326; fax: +86 411 8611 0326. ∗∗ Corresponding author at: Department of Food Nutrition and Safety, Dalian Medical University, Dalian 116044, China. Tel.: +86 411 8611 0326; fax: +86 411 8611 0326. E-mail addresses: [email protected] (G. Yang), xinfei [email protected] (M. Chen). http://dx.doi.org/10.1016/j.mrgentox.2014.04.019 1383-5718/© 2014 Elsevier B.V. All rights reserved.
and its presence in human blood and urine, suggest that BPA may increase the risk of hormone-dependant cancers in humans [5]. However, in the cancer-bioassay report, the National Toxicology Program concluded that there was no convincing evidence that BPA is carcinogenic for rats or mice [6]. Experimental data have shown that BPA adversely affects the male reproductive system in general, and the testis in particular [7,8]. In addition, other studies reported that BPA could affect the function of the beta-cells and impair insulin-signaling in rat testis [9,10]. However, one study indicated that low-dose exposure to BPA in the diet does not adversely affect reproductive development in F(1) rat offspring [11]. Therefore, more studies are needed to further investigate whether there is a true association between exposure to BPA and various diseases. Recent epidemiological studies indicate that a concentrationdependent correlation exists between BPA exposure and the occurrence of diabetes [12]. The mechanism underlying this link remains unclear. It is suggested that BPA affects glucose metabolism through diverse pathways including insulin resistance, pancreatic -cell dysfunction, adipogenesis, inflammation and oxidative stress [13]. Considering that the pancreas may be a potential target site of BPA, we selected INS-1 cells as the experimental
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in vitro system in our study. INS-1 cells are derived from a rat insulinoma induced by X-radiation. These cells display many important characteristics of the pancreatic beta-cell, including a high insulin content and responsiveness to glucose [14]. Therefore, it can provide valuable information about physiological processes. With more extensive studies on BPA, people pay more attention to its genotoxicity and the ensuing DNA damage. It has been reported that BPA caused a significant increase in the frequency of micronuclei (MN) in polychromatic erythrocytes (PCEs), structural chromosome aberrations in bone-marrow cells, and DNA damage in blood lymphocytes [15]. Moreover, BPA can up-regulate clusterin expression in atrophic prostate epithelial cells and induce lipid peroxidation and DNA fragmentation in spermatozoa, while a BPA-related increase in DNA adducts was found in cultured human prostatic cells [16]. It has also been reported that germ-cell DNA of adult male rats was damaged after treatment with BPA [17]. Up to now, there are no data indicating that BPA can induce DNA damage in pancreatic cells. To the best of our knowledge, this is the first study to show oxidative stress-associated DNA damage induced by BPA in INS-1 cells. The aim of this research was to assess the toxicity effects of BPA in INS-1 cells and its possible mechanisms. Intracellular reactive oxygen species (ROS) were detected with 2,7-dichlorofluorescein diacetate (DCFH-DA). Glutathione (GSH) was detected with o-phthalaldehyde (OPT). We measured the DNA strand-breaks by use of the comet assay, and DNA damage-associated proteins were detected by Western blotting. 2. Materials and methods 2.1. Materials Bisphenol A (CAS No. 80-05-7; purity, 99%) was purchased from Sigma–Aldrich and dissolved in dimethyl sulfoxide (DMSO). Ethidium bromide (EB), DCFH-DA, o-phthalaldehyde (OPT) were purchased from Sigma (St. Louis, USA). Normalmelting-point (NMP) agarose and low-melting-point (LMP) agarose were purchased from Gibco BRL, Life Technologies (Paisley, UK). N-Acetyl cysteine (NAC) was purchased from Wako (Wako Pure Chemical Industries, Ltd., Japan). -Actin is a mouse monoclonal antibody was supplied by SANTA CRUZ, p53 polyclonal antibody was purchased from ptglab (Wuhan, China) and p-Chk2 (T68) was purchased from Bio-World Technology, Inc. Bicinchoninic acid (BCA) Protein Assay Kit, SDS-PAGE Sample Loading Buffer (5×), and Electrochemiluminescence (ECL) Plus were supplied by Beyotime (Shanghai, China). Goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Thermo Fisher Scientific. All tissue-culture reagents, i.e. Minimum Essential Eagle’s Medium (MEM), fetal bovine serum (FBS), antibiotics (penicillin and streptomycin) and trypsin–EDTA solution were supplied by Gibco BRL-Life Technologies (Grand Island, NY).
Table 1 DNA strand-breakage induced by BPA and BPA + NAC in INS-1 cells. Group
Tail length (m)
Control BPA (25 M) BPA (50 M) BPA (100 M) BPA (100 M) + NAC
3.00 3.53 8.67 17.28 8.18
± ± ± ± ±
0.00 1.55 0.85* 1.25* 0.78#
Tail DNA (%) 2.43 2.85 7.49 12.98 6.74
± ± ± ± ±
0.43 0.51 0.83* 1.59* 0.55#
Tail moment 0.96 1.27 6.18 10.53 6.51
± ± ± ± ±
0.36 0.43 0.76* 1.95* 0.76#
Cells were treated with BPA (0–100 M) for 24 h. To assess the effect of NAC, the cells were pretreated with NAC (10 mM) for 1 h. Results are expressed as mean ± S.D. of three independent experiments (n = 3). * P < 0.05 vs control. # P < 0.05 vs BPA (100 M).
2.4. Measurement of intracellular ROS The level of intracellular ROS was measured with the DCFH-DA method, with slight modifications [19]. Briefly, INS-1 cells were incubated with BPA (25, 50, 100 M) at 37 ◦ C for 24 h. The cells were trypsinized, harvested, and counted: the cell number was the same at different concentrations of BPA, which met the experimental requirements (5 × 105 cells). Cells were washed twice with cold PBS, suspended in PBS at 5 × 105 cells/ml, and incubated with DCFH-DA (5 M) at 37 ◦ C for 40 min in the dark. To evaluate the protective effect of NAC, INS-1 cells were pretreated with NAC (10 mM) at 37 ◦ C for 1 h, then incubated with BPA at the concentration of 100 M at 37 ◦ C for 24 h. The relative fluorescence intensity of the cell suspensions was measured. Excitation and barrier wavelengths were 485 nm and 530 nm, respectively. 2.5. Measurement of intracellular GSH Reduced glutathione (GSH) was measured by use of a modified method [20]. Briefly, INS-1 cells were incubated with BPA (25, 50, 100 M) at 37 ◦ C for 24 h. Then cells were trypsinized, harvested, washed twice with cold PBS, and treated with 5% trichloroacetic acid (TCA) at 4 ◦ C for 30 min. Finally, the supernatant was diluted with phosphate-EDTA buffer (pH 8.3) and OPT (50 g/ml) solution, and the mixture was incubated at 37 ◦ C for 15 min in the dark. Emission and excitation wavelengths were 350 nm and 420 nm, respectively. 2.6. Western-blot analysis A Western-blot analysis was performed to analyze the expression of p53 and p-Chk2 (T68). The INS-1 cells were exposed to BPA (25, 50, 100 M) for 24 h. The Nuclear and Cytoplasmic Protein Extraction Kit and the BCA Protein Assay Kit (Beyotime) were used for the extraction and quantification of protein. Nuclear protein of about 50 g was loaded per lane to detect p53, and 100 g was loaded to detect p-Chk2. The proteins were fractionated on a 12% sodium-dodecyl sulfate (SDS)-polyacrylamide gel, and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, the filters were incubated with the antibody-containing solution overnight at 4 ◦ C, washed twice, and incubated with a blocking solutioncoupled secondary antibody at 37 ◦ C for 2 h. 2.7. Statistical analyses
2.2. Cell culture and treatment The INS-1 cell line was obtained from China Center for Type Culture Collection (CCTCC), and cultivated in MEM containing 10% FBS and penicillin (100 UI/ml)–streptomycin (0.1 mg/ml). The cells were incubated in 5% CO2 at 37 ◦ C. BPA was dissolved in DMSO and a stock solution of 20 mM was stored at room temperature. The BPA stock was diluted to the concentration indicated with culture medium (without FBS), immediately before each experiment. The final DMSO concentration in culture medium was 0.1% (v/v).
All values were expressed as mean ± standard deviation (S.D.). The data were analyzed by means of one-way analysis of variance (ANOVA) and Student’s t-test (spss v 11.5 software). P < 0.05 and P < 0.01 were considered to correspond with statistical significance.
3. Results 3.1. DNA strand-breaks induced by BPA
2.3. Comet assay The comet assay was performed as described by Stephens and Singh [18], with slight modifications. The INS-1 cells were incubated with BPA (25, 50, 100 M) at 37 ◦ C for 24 h. The cells were trypsinized and harvested; the number of cells was the same for different concentrations of BPA, which met the experimental requirements (1 × 106 cells). To evaluate the protective effects of NAC, INS-1 cells were pretreated with or without NAC (10 mM) at 37 ◦ C, then incubated with BPA at the concentration of 100 M for 24 h at 37 ◦ C. To avoid artifacts resulting from apoptosis and necrosis, we used Hoechst Staining Kit (Beyotime) and trypan blue (50 g/ml) to detect apoptotic cells and assess cell viability. Only when there were no apoptotic cells and no less than 90% cell survival, cell suspensions were used to detect the DNA migration. Slides were viewed with a fluorescence microscope (Olympus BX-51) with a 549-nm excitation filter and a 590-nm barrier filter. Images with 50 randomly selected cells from each slide were subject to quantitative analysis with Comet Assay Software Project casp-1.2.2 (University of Wrocław, Poland).
DNA strand-breaks in INS-1 cells exposed to BPA (0–100 M) for 24 h were evaluated by means of the comet assay. BPA caused a significant increase in DNA migration compared with that observed with untreated cells (50–100 M, p < 0.05; see Table 1). When the cells were pretreated with NAC for 1 h, the DNA migration was significantly less than that in cells treated with BPA only (p < 0.05). 3.2. Increase of intracellular ROS The DCFH-DA fluorescent dye was used to evaluate the generation of ROS in INS-1 cells treated with BPA (0–100 M). A significant increase of the fluorescence intensity was observed in
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Fig. 2. Effect of BPA on GSH content. Cells were exposed to BPA (0–100 M) for 24 h. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control; **P < 0.01 vs control).
DNA-damage induction, the cells were pretreated with NAC for 1 h: the expression of p53 was significantly decreased compared with that in cells treated with BPA only (Fig. 4). 4. Discussion
Fig. 1. (A) Effect of BPA on ROS production. Cells were exposed to BPA (0–100 M) for 24 h. Each bar represents mean ± S.D. of three independent experiments (*P < 0.05 vs control; **P < 0.01 vs control). (B) Effect of NAC on ROS production. Cells were pretreated with NAC for 1 h and then exposed to BPA (100 M) for 24 h. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control; **P < 0.05 vs BPA).
cells treated with BPA at the concentrations of 50 and 100 M (p < 0.05 or p < 0.01) (Fig. 1A). To verify if oxidative stress plays a role in the mechanism of DNA-damage induction, we used NAC, a strong antioxidant compound, which was able to reduce intracellular ROS formation and attenuate GSH depletion. When the cells were pretreated with NAC for 1 h, the DCF fluorescence intensity in INS-1 cells was significantly lower than that seen in cells treated with BPA only (p < 0.05 or p < 0.01) (Fig. 1B). 3.3. Depletion of intracellular GSH by BPA In addition, we used OPT fluorescence dye to evaluate the depletion of GSH. Fig. 2 indicates a striking decrease of intracellular GSH at the concentrations of 25–100 M (p < 0.05 or p < 0.01). 3.4. Effect of BPA on the expression of p-Chk2 and p53 In order to investigate the molecular mechanisms underlying induction of DNA damage by BPA, we examined the expression of p-Chk2 and p53 in INS-1 cells by Western blotting. As illustrated in Fig. 3, the expression of these proteins was increased in BPAtreated cells at 25–100 M, in a dose-dependent manner. Similarly, in order to confirm the role of oxidative stress in the mechanism of
Bisphenol A is widely used to manufacture polycarbonate plastic and epoxy resins. Recently, more attention has been focused on its impact on human health. As we know, BPA is an environmental endocrine disruptor that can exert estrogen-like effects. In vivo and in vitro experiments both demonstrated that BPA could modulate or disrupt the function of the endocrine system [21]. It has been demonstrated that BPA and estradiol-17 (E2) have similar effects [22,23]. Many studies reported that BPA might be an important environmental risk factor for diabetes. Recent studies suggested that BPA has a direct effect on the function of pancreatic betacells, leading to insulin resistance, and BPA exposure could also be involved in effects on peripheral metabolic tissues [24–26]. In the present study, the genotoxicity of BPA was evaluated by the use of comet assay, which is a powerful tool for the assessment of DNA breakage. The comet assay revealed that BPA induced substantial DNA strand-breakage at high dose. It provided evidence of BPA-induced DNA damage in INS-1cells. Numerous studies have demonstrated that oxidative stress is a critical factor in the mechanism of DNA-damage induction. Oxidative stress occurs when excessive generation of ROS exceeds their catabolism, and it plays an important role in cell and organismal biology [27]. Intracellular ROS include hydrogen peroxide, ozone, organic peroxide, etc. [28]. In principle, chemicals that give rise to excess ROS production will cause different types of toxic effect, overwhelm the antioxidant mechanism of target cells and contribute to further tissue damage [29]. To explore the oxidative stress-induced DNA damage, we investigated the generation of ROS. DCFH was used as a fluorescent probe. Our findings show that BPA caused the content of intracellular ROS to increase significantly. Overproduction of ROS can damage DNA and lead to genotoxicity. We infer that ROS may play an important role in the induction of DNA damage by BPA. In addition, we considered the level of GSH in INS-1 cells after treatment with BPA. The function of GSH is to protect against injuries by oxidants, and it is a main component of the antioxidation system [30]. It is able to quench ROS and protect cells from toxic compounds, and depletion of GSH is a marker of oxidative stress. Our results show that the level of ROS was significantly increased while the level of GSH was significantly decreased in INS-1 cells. These data indicate that GSH, as a main intracellular
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Fig. 3. Effect of BPA on the expression of p-Chk2 (T68) and P53. Cells were exposed to BPA (0–100 M) for 24 h. Western blots were performed on the nuclear protein fractions of untreated and treated cells. -Actin was used as control for quantification. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control).
Fig. 4. Effect of NAC on the expression of P53. Cells were pretreated with NAC for 1 h and then exposed to BPA (100 M) for 24 h. Western blots were performed on the nuclear protein fractions of untreated and treated cells. -Actin was used as control for quantification. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control; # P < 0.05 vs BPA).
antioxidant, plays a vital role in the defense against toxic effects induced by BPA. NAC, as an intracellular ROS inhibitor, can prevent oxidative stress, and react directly with oxidative metabolites, or as a precursor of GSH indirectly scavenging ROS [31,32]. In our study, we observed that the level of ROS and DNA damage induced by BPA were significantly decreased when cells were pretreated with NAC. The results further confirm that oxidative stress was involved in DNA-damage induction and that NAC acts as a protective agent to reduce the genotoxicity of BPA. To further confirm BPA-mediated DNA damage, we observed the expression of some DNA damage-related proteins. When DNA damage occurs, the cells will prepare a response, and the expression of several proteins will change accordingly. Activation of DNA-dependent protein kinases include ATM (ataxia telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related). They both belong to PI3K (phosphatidylinositol-3-kinase), which through different pathways of the DNA-repair process plays an important role and participates in different forms of DNA-damage processing [33]. ATM has a variety of phosphorylated substrates, including Chk2, p53, BRAC1, etc. [34], and it activates many downstream effector proteins, enabling the repair of DNA damage. When DNA damage is sensed, ATM phosphorylates and activates Chk2 at Thr-68, which then inhibits cell-cycle progression [35]. Chk2 activation would normally lead to p53 stabilization and its accumulation in the nucleus, which enhances the specific DNA-binding capacity. Chk2 participates in sustained cell-cycle arrest, some forms of DNA repair, and promotes apoptosis when DNA damage cannot be repaired [36]. As in previous studies, we chose p-Chk2 (T68) and p53 to further assess repair of BPA-mediated DNA damage [37]. The expression of p-Chk2 (T68) and p53 was significantly increased, which suggests that these proteins are critically involved in BPA-induced DNA damage. We hypothesize that the DNA-damage checkpoint detects DNA damage and responds by activating the ATM/Chk2/p53 signaling
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Fig. 5. The proposed mechanism of action of BPA on INS-1 cells. BPA may increase the ROS level and decrease the GSH level, which causes DNA strand-breaks in INS-1 cells. When DNA damage is sensed, ATM activates Chk2, and Chk2 activation would lead to p53 stabilization and its accumulation in the nucleus.
pathway, which brings the cell cycle to a halt while the damage is repaired, or induces apoptosis if the damage cannot be repaired (Fig. 5). Moreover, after pretreatment with NAC, the expression of p53 was reduced. These results indicated that oxidative stress maybe the underlying mechanism of DNA-damage induction by BPA. In conclusion, results of our study indicate that BPA causes DNA damage in INS-1 cells. Apart from this, upon pretreatment with NAC, the intracellular level of ROS and the expression of the p53 protein were both decreased compared with those in cells exposed to BPA only. The data demonstrate that the DNA damage is probably associated with oxidative stress. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This investigation was supported by the Central Laboratory of Dalian Medical University (instrumental assistance). This work was supported by the National Natural Science Foundation of China (81102135). References [1] N. Hanioka, H. Jinno, T. Tanaka-Kagawa, T. Nishimura, M. Ando, Interaction of bisphenol A with rat hepatic cytochrome P450 enzymes, Chemosphere 41 (2000) 973–978. [2] L.N. Vandenberg, I. Chahoud, J.J. Heindel, V. Padmanabhan, F.J.R. Paumgartten, G. Schoenfelder, Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A, Environ. Health Perspect. 118 (2010) 1055–1070. [3] L.N. Vandenberg, M.V. Maffini, C. Sonnenschein, B.S. Rubin, A.M. Soto, Bisphenol-A and the great divide: a review of controversies in the field of endocrine disruption, Endocr. Rev. 30 (2009) 75–95. [4] R. Rezg, S. El-Fazaa, N. Gharbi, B. Mornagui, Bisphenol A and human chronic diseases: current evidences, possible mechanisms, and future perspectives, Environ. Int. 29 (2013) 83–90. [5] H. Rochefort, Bisphenol A and hormone-dependent cancers: potential risk and mechanism, Med. Sci. (Paris) 29 (2013) 539–544. [6] J. Huff, Carcinogenicity of bisphenol-A in Fischer rats and B6C3F1 mice, Odontology 89 (2001) 12–20. [7] R.E. Chapin, J. Adams, K. Boekelheide, L.E. Gray, S.W. Hayward, P.S.J. Lees, B.S. McIntyre, K.M. Portier, T.M. Schnorr, S.G. Selevan, NTP-CERHR expert panel report on the reproductive and developmental toxicity of bisphenol A, Birth Defects Res. B: Develop. Reprod. Toxicol. 83 (2008) 157–395. [8] O. Takahashi, S. Oishi, Testicular toxicity of dietarily or parenterally administered bisphenol A in rats and mice, Food Chem. Toxicol. 41 (2003) 1035–1044. [9] P. Alonso-Magdalena, S. Morimoto, C. Ripoll, E. Fuentes, A. Nadal, The estrogenic effect of bisphenol A disrupts pancreatic -cell function in vivo and induces insulin resistance, Environ. Health Perspect. 114 (2006) 106–112.
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Bisphenol A induces oxidative stress-associated DNA damage in INS-1 cells Fei Xin a , Liping Jiang b , Xiaofang Liu a , Chengyan Geng b , Wenbin Wang c , Laifu Zhong a,b , Guang Yang a,b,∗∗ , Min Chen a,∗ a
Department of Food Nutrition and Safety, Dalian Medical University, No. 9 W. Lushun South Road, Dalian 116044, China Liaoning Anti-Degenerative Diseases Natural Products Engineering Technology Research Center, Dalian Medical University, Dalian 116044, China c School of Public Health, Shandong University, Jinan 250012, China b
a r t i c l e
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Article history: Received 24 October 2013 Received in revised form 13 February 2014 Accepted 5 April 2014 Available online 21 May 2014 Keywords: Bisphenol A INS-1 cells DNA strand-breaks ROS p53 p-Chk2 (T68)
a b s t r a c t Bisphenol A (BPA), an endocrine disruptor, is widely used to manufacture polycarbonate plastic and epoxy resins. Many studies have demonstrated that BPA can play a role in reproductive toxicity and affect the normal metabolic function. Recent research has shown that BPA can influence the function of pancreatic islets. In this study, our aim is to assess the DNA damage induced by BPA and to clarify the mechanism, by use of rat insulinoma INS-1 cells. INS-1 cells were exposed to different doses of BPA (0, 25, 50, 100 M). We conducted the single-cell gel electrophoresis (SCGE) assay to measure DNA damage, and studied proteins such as p53 and p-Chk2 (T68) by Western blotting, in order to verify the (geno)toxicity of BPA. Moreover, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) to discuss the possible mechanism of DNA damage. The results show that BPA caused an increased in DNA strand-breaks along with greater DNA migration from the nucleus into the comet tail. The expression of DNA damage-associated proteins (p53 and p-Chk2 (T68)) was significantly increased. The exposure to various doses of BPA caused a significant increase in intracellular ROS and a significant reduction in the level of GSH. N-Acetyl cysteine, an inhibitor of intracellular ROS formation, can significantly reduce the generation of intracellular reactive oxygen. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bisphenol A (BPA) is the common name for 2,2-bis-(4hydroxyphenyl)propane (C15 H16 O2 ). It is widely used to manufacture polycarbonate plastic and epoxy resins. BPA is a low-toxicity compound, with extensive application in plastic products, such as dental sealants, adhesives, food packaging, and plastic beverage containers [1]. In the recent years, it has become increasingly concerned about BPA, an endocrine disruptor, in view of its widespread presence in a variety of human tissues and body fluids [2]. It has been suspected that BPA is associated with various diseases including cancer, cardiovascular disease and reproductive disorders, but the results are controversial [3,4]. Some experimental studies in rodents on BPA
∗ Corresponding author. Tel.: +86 411 8611 0326; fax: +86 411 8611 0326. ∗∗ Corresponding author at: Department of Food Nutrition and Safety, Dalian Medical University, Dalian 116044, China. Tel.: +86 411 8611 0326; fax: +86 411 8611 0326. E-mail addresses: [email protected] (G. Yang), xinfei [email protected] (M. Chen). http://dx.doi.org/10.1016/j.mrgentox.2014.04.019 1383-5718/© 2014 Elsevier B.V. All rights reserved.
and its presence in human blood and urine, suggest that BPA may increase the risk of hormone-dependant cancers in humans [5]. However, in the cancer-bioassay report, the National Toxicology Program concluded that there was no convincing evidence that BPA is carcinogenic for rats or mice [6]. Experimental data have shown that BPA adversely affects the male reproductive system in general, and the testis in particular [7,8]. In addition, other studies reported that BPA could affect the function of the beta-cells and impair insulin-signaling in rat testis [9,10]. However, one study indicated that low-dose exposure to BPA in the diet does not adversely affect reproductive development in F(1) rat offspring [11]. Therefore, more studies are needed to further investigate whether there is a true association between exposure to BPA and various diseases. Recent epidemiological studies indicate that a concentrationdependent correlation exists between BPA exposure and the occurrence of diabetes [12]. The mechanism underlying this link remains unclear. It is suggested that BPA affects glucose metabolism through diverse pathways including insulin resistance, pancreatic -cell dysfunction, adipogenesis, inflammation and oxidative stress [13]. Considering that the pancreas may be a potential target site of BPA, we selected INS-1 cells as the experimental
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in vitro system in our study. INS-1 cells are derived from a rat insulinoma induced by X-radiation. These cells display many important characteristics of the pancreatic beta-cell, including a high insulin content and responsiveness to glucose [14]. Therefore, it can provide valuable information about physiological processes. With more extensive studies on BPA, people pay more attention to its genotoxicity and the ensuing DNA damage. It has been reported that BPA caused a significant increase in the frequency of micronuclei (MN) in polychromatic erythrocytes (PCEs), structural chromosome aberrations in bone-marrow cells, and DNA damage in blood lymphocytes [15]. Moreover, BPA can up-regulate clusterin expression in atrophic prostate epithelial cells and induce lipid peroxidation and DNA fragmentation in spermatozoa, while a BPA-related increase in DNA adducts was found in cultured human prostatic cells [16]. It has also been reported that germ-cell DNA of adult male rats was damaged after treatment with BPA [17]. Up to now, there are no data indicating that BPA can induce DNA damage in pancreatic cells. To the best of our knowledge, this is the first study to show oxidative stress-associated DNA damage induced by BPA in INS-1 cells. The aim of this research was to assess the toxicity effects of BPA in INS-1 cells and its possible mechanisms. Intracellular reactive oxygen species (ROS) were detected with 2,7-dichlorofluorescein diacetate (DCFH-DA). Glutathione (GSH) was detected with o-phthalaldehyde (OPT). We measured the DNA strand-breaks by use of the comet assay, and DNA damage-associated proteins were detected by Western blotting. 2. Materials and methods 2.1. Materials Bisphenol A (CAS No. 80-05-7; purity, 99%) was purchased from Sigma–Aldrich and dissolved in dimethyl sulfoxide (DMSO). Ethidium bromide (EB), DCFH-DA, o-phthalaldehyde (OPT) were purchased from Sigma (St. Louis, USA). Normalmelting-point (NMP) agarose and low-melting-point (LMP) agarose were purchased from Gibco BRL, Life Technologies (Paisley, UK). N-Acetyl cysteine (NAC) was purchased from Wako (Wako Pure Chemical Industries, Ltd., Japan). -Actin is a mouse monoclonal antibody was supplied by SANTA CRUZ, p53 polyclonal antibody was purchased from ptglab (Wuhan, China) and p-Chk2 (T68) was purchased from Bio-World Technology, Inc. Bicinchoninic acid (BCA) Protein Assay Kit, SDS-PAGE Sample Loading Buffer (5×), and Electrochemiluminescence (ECL) Plus were supplied by Beyotime (Shanghai, China). Goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Thermo Fisher Scientific. All tissue-culture reagents, i.e. Minimum Essential Eagle’s Medium (MEM), fetal bovine serum (FBS), antibiotics (penicillin and streptomycin) and trypsin–EDTA solution were supplied by Gibco BRL-Life Technologies (Grand Island, NY).
Table 1 DNA strand-breakage induced by BPA and BPA + NAC in INS-1 cells. Group
Tail length (m)
Control BPA (25 M) BPA (50 M) BPA (100 M) BPA (100 M) + NAC
3.00 3.53 8.67 17.28 8.18
± ± ± ± ±
0.00 1.55 0.85* 1.25* 0.78#
Tail DNA (%) 2.43 2.85 7.49 12.98 6.74
± ± ± ± ±
0.43 0.51 0.83* 1.59* 0.55#
Tail moment 0.96 1.27 6.18 10.53 6.51
± ± ± ± ±
0.36 0.43 0.76* 1.95* 0.76#
Cells were treated with BPA (0–100 M) for 24 h. To assess the effect of NAC, the cells were pretreated with NAC (10 mM) for 1 h. Results are expressed as mean ± S.D. of three independent experiments (n = 3). * P < 0.05 vs control. # P < 0.05 vs BPA (100 M).
2.4. Measurement of intracellular ROS The level of intracellular ROS was measured with the DCFH-DA method, with slight modifications [19]. Briefly, INS-1 cells were incubated with BPA (25, 50, 100 M) at 37 ◦ C for 24 h. The cells were trypsinized, harvested, and counted: the cell number was the same at different concentrations of BPA, which met the experimental requirements (5 × 105 cells). Cells were washed twice with cold PBS, suspended in PBS at 5 × 105 cells/ml, and incubated with DCFH-DA (5 M) at 37 ◦ C for 40 min in the dark. To evaluate the protective effect of NAC, INS-1 cells were pretreated with NAC (10 mM) at 37 ◦ C for 1 h, then incubated with BPA at the concentration of 100 M at 37 ◦ C for 24 h. The relative fluorescence intensity of the cell suspensions was measured. Excitation and barrier wavelengths were 485 nm and 530 nm, respectively. 2.5. Measurement of intracellular GSH Reduced glutathione (GSH) was measured by use of a modified method [20]. Briefly, INS-1 cells were incubated with BPA (25, 50, 100 M) at 37 ◦ C for 24 h. Then cells were trypsinized, harvested, washed twice with cold PBS, and treated with 5% trichloroacetic acid (TCA) at 4 ◦ C for 30 min. Finally, the supernatant was diluted with phosphate-EDTA buffer (pH 8.3) and OPT (50 g/ml) solution, and the mixture was incubated at 37 ◦ C for 15 min in the dark. Emission and excitation wavelengths were 350 nm and 420 nm, respectively. 2.6. Western-blot analysis A Western-blot analysis was performed to analyze the expression of p53 and p-Chk2 (T68). The INS-1 cells were exposed to BPA (25, 50, 100 M) for 24 h. The Nuclear and Cytoplasmic Protein Extraction Kit and the BCA Protein Assay Kit (Beyotime) were used for the extraction and quantification of protein. Nuclear protein of about 50 g was loaded per lane to detect p53, and 100 g was loaded to detect p-Chk2. The proteins were fractionated on a 12% sodium-dodecyl sulfate (SDS)-polyacrylamide gel, and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, the filters were incubated with the antibody-containing solution overnight at 4 ◦ C, washed twice, and incubated with a blocking solutioncoupled secondary antibody at 37 ◦ C for 2 h. 2.7. Statistical analyses
2.2. Cell culture and treatment The INS-1 cell line was obtained from China Center for Type Culture Collection (CCTCC), and cultivated in MEM containing 10% FBS and penicillin (100 UI/ml)–streptomycin (0.1 mg/ml). The cells were incubated in 5% CO2 at 37 ◦ C. BPA was dissolved in DMSO and a stock solution of 20 mM was stored at room temperature. The BPA stock was diluted to the concentration indicated with culture medium (without FBS), immediately before each experiment. The final DMSO concentration in culture medium was 0.1% (v/v).
All values were expressed as mean ± standard deviation (S.D.). The data were analyzed by means of one-way analysis of variance (ANOVA) and Student’s t-test (spss v 11.5 software). P < 0.05 and P < 0.01 were considered to correspond with statistical significance.
3. Results 3.1. DNA strand-breaks induced by BPA
2.3. Comet assay The comet assay was performed as described by Stephens and Singh [18], with slight modifications. The INS-1 cells were incubated with BPA (25, 50, 100 M) at 37 ◦ C for 24 h. The cells were trypsinized and harvested; the number of cells was the same for different concentrations of BPA, which met the experimental requirements (1 × 106 cells). To evaluate the protective effects of NAC, INS-1 cells were pretreated with or without NAC (10 mM) at 37 ◦ C, then incubated with BPA at the concentration of 100 M for 24 h at 37 ◦ C. To avoid artifacts resulting from apoptosis and necrosis, we used Hoechst Staining Kit (Beyotime) and trypan blue (50 g/ml) to detect apoptotic cells and assess cell viability. Only when there were no apoptotic cells and no less than 90% cell survival, cell suspensions were used to detect the DNA migration. Slides were viewed with a fluorescence microscope (Olympus BX-51) with a 549-nm excitation filter and a 590-nm barrier filter. Images with 50 randomly selected cells from each slide were subject to quantitative analysis with Comet Assay Software Project casp-1.2.2 (University of Wrocław, Poland).
DNA strand-breaks in INS-1 cells exposed to BPA (0–100 M) for 24 h were evaluated by means of the comet assay. BPA caused a significant increase in DNA migration compared with that observed with untreated cells (50–100 M, p < 0.05; see Table 1). When the cells were pretreated with NAC for 1 h, the DNA migration was significantly less than that in cells treated with BPA only (p < 0.05). 3.2. Increase of intracellular ROS The DCFH-DA fluorescent dye was used to evaluate the generation of ROS in INS-1 cells treated with BPA (0–100 M). A significant increase of the fluorescence intensity was observed in
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Fig. 2. Effect of BPA on GSH content. Cells were exposed to BPA (0–100 M) for 24 h. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control; **P < 0.01 vs control).
DNA-damage induction, the cells were pretreated with NAC for 1 h: the expression of p53 was significantly decreased compared with that in cells treated with BPA only (Fig. 4). 4. Discussion
Fig. 1. (A) Effect of BPA on ROS production. Cells were exposed to BPA (0–100 M) for 24 h. Each bar represents mean ± S.D. of three independent experiments (*P < 0.05 vs control; **P < 0.01 vs control). (B) Effect of NAC on ROS production. Cells were pretreated with NAC for 1 h and then exposed to BPA (100 M) for 24 h. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control; **P < 0.05 vs BPA).
cells treated with BPA at the concentrations of 50 and 100 M (p < 0.05 or p < 0.01) (Fig. 1A). To verify if oxidative stress plays a role in the mechanism of DNA-damage induction, we used NAC, a strong antioxidant compound, which was able to reduce intracellular ROS formation and attenuate GSH depletion. When the cells were pretreated with NAC for 1 h, the DCF fluorescence intensity in INS-1 cells was significantly lower than that seen in cells treated with BPA only (p < 0.05 or p < 0.01) (Fig. 1B). 3.3. Depletion of intracellular GSH by BPA In addition, we used OPT fluorescence dye to evaluate the depletion of GSH. Fig. 2 indicates a striking decrease of intracellular GSH at the concentrations of 25–100 M (p < 0.05 or p < 0.01). 3.4. Effect of BPA on the expression of p-Chk2 and p53 In order to investigate the molecular mechanisms underlying induction of DNA damage by BPA, we examined the expression of p-Chk2 and p53 in INS-1 cells by Western blotting. As illustrated in Fig. 3, the expression of these proteins was increased in BPAtreated cells at 25–100 M, in a dose-dependent manner. Similarly, in order to confirm the role of oxidative stress in the mechanism of
Bisphenol A is widely used to manufacture polycarbonate plastic and epoxy resins. Recently, more attention has been focused on its impact on human health. As we know, BPA is an environmental endocrine disruptor that can exert estrogen-like effects. In vivo and in vitro experiments both demonstrated that BPA could modulate or disrupt the function of the endocrine system [21]. It has been demonstrated that BPA and estradiol-17 (E2) have similar effects [22,23]. Many studies reported that BPA might be an important environmental risk factor for diabetes. Recent studies suggested that BPA has a direct effect on the function of pancreatic betacells, leading to insulin resistance, and BPA exposure could also be involved in effects on peripheral metabolic tissues [24–26]. In the present study, the genotoxicity of BPA was evaluated by the use of comet assay, which is a powerful tool for the assessment of DNA breakage. The comet assay revealed that BPA induced substantial DNA strand-breakage at high dose. It provided evidence of BPA-induced DNA damage in INS-1cells. Numerous studies have demonstrated that oxidative stress is a critical factor in the mechanism of DNA-damage induction. Oxidative stress occurs when excessive generation of ROS exceeds their catabolism, and it plays an important role in cell and organismal biology [27]. Intracellular ROS include hydrogen peroxide, ozone, organic peroxide, etc. [28]. In principle, chemicals that give rise to excess ROS production will cause different types of toxic effect, overwhelm the antioxidant mechanism of target cells and contribute to further tissue damage [29]. To explore the oxidative stress-induced DNA damage, we investigated the generation of ROS. DCFH was used as a fluorescent probe. Our findings show that BPA caused the content of intracellular ROS to increase significantly. Overproduction of ROS can damage DNA and lead to genotoxicity. We infer that ROS may play an important role in the induction of DNA damage by BPA. In addition, we considered the level of GSH in INS-1 cells after treatment with BPA. The function of GSH is to protect against injuries by oxidants, and it is a main component of the antioxidation system [30]. It is able to quench ROS and protect cells from toxic compounds, and depletion of GSH is a marker of oxidative stress. Our results show that the level of ROS was significantly increased while the level of GSH was significantly decreased in INS-1 cells. These data indicate that GSH, as a main intracellular
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Fig. 3. Effect of BPA on the expression of p-Chk2 (T68) and P53. Cells were exposed to BPA (0–100 M) for 24 h. Western blots were performed on the nuclear protein fractions of untreated and treated cells. -Actin was used as control for quantification. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control).
Fig. 4. Effect of NAC on the expression of P53. Cells were pretreated with NAC for 1 h and then exposed to BPA (100 M) for 24 h. Western blots were performed on the nuclear protein fractions of untreated and treated cells. -Actin was used as control for quantification. Each bar represents mean ± S.D. of three independent experiments (n = 3) (*P < 0.05 vs control; # P < 0.05 vs BPA).
antioxidant, plays a vital role in the defense against toxic effects induced by BPA. NAC, as an intracellular ROS inhibitor, can prevent oxidative stress, and react directly with oxidative metabolites, or as a precursor of GSH indirectly scavenging ROS [31,32]. In our study, we observed that the level of ROS and DNA damage induced by BPA were significantly decreased when cells were pretreated with NAC. The results further confirm that oxidative stress was involved in DNA-damage induction and that NAC acts as a protective agent to reduce the genotoxicity of BPA. To further confirm BPA-mediated DNA damage, we observed the expression of some DNA damage-related proteins. When DNA damage occurs, the cells will prepare a response, and the expression of several proteins will change accordingly. Activation of DNA-dependent protein kinases include ATM (ataxia telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related). They both belong to PI3K (phosphatidylinositol-3-kinase), which through different pathways of the DNA-repair process plays an important role and participates in different forms of DNA-damage processing [33]. ATM has a variety of phosphorylated substrates, including Chk2, p53, BRAC1, etc. [34], and it activates many downstream effector proteins, enabling the repair of DNA damage. When DNA damage is sensed, ATM phosphorylates and activates Chk2 at Thr-68, which then inhibits cell-cycle progression [35]. Chk2 activation would normally lead to p53 stabilization and its accumulation in the nucleus, which enhances the specific DNA-binding capacity. Chk2 participates in sustained cell-cycle arrest, some forms of DNA repair, and promotes apoptosis when DNA damage cannot be repaired [36]. As in previous studies, we chose p-Chk2 (T68) and p53 to further assess repair of BPA-mediated DNA damage [37]. The expression of p-Chk2 (T68) and p53 was significantly increased, which suggests that these proteins are critically involved in BPA-induced DNA damage. We hypothesize that the DNA-damage checkpoint detects DNA damage and responds by activating the ATM/Chk2/p53 signaling
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Fig. 5. The proposed mechanism of action of BPA on INS-1 cells. BPA may increase the ROS level and decrease the GSH level, which causes DNA strand-breaks in INS-1 cells. When DNA damage is sensed, ATM activates Chk2, and Chk2 activation would lead to p53 stabilization and its accumulation in the nucleus.
pathway, which brings the cell cycle to a halt while the damage is repaired, or induces apoptosis if the damage cannot be repaired (Fig. 5). Moreover, after pretreatment with NAC, the expression of p53 was reduced. These results indicated that oxidative stress maybe the underlying mechanism of DNA-damage induction by BPA. In conclusion, results of our study indicate that BPA causes DNA damage in INS-1 cells. Apart from this, upon pretreatment with NAC, the intracellular level of ROS and the expression of the p53 protein were both decreased compared with those in cells exposed to BPA only. The data demonstrate that the DNA damage is probably associated with oxidative stress. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This investigation was supported by the Central Laboratory of Dalian Medical University (instrumental assistance). This work was supported by the National Natural Science Foundation of China (81102135). References [1] N. Hanioka, H. Jinno, T. Tanaka-Kagawa, T. Nishimura, M. Ando, Interaction of bisphenol A with rat hepatic cytochrome P450 enzymes, Chemosphere 41 (2000) 973–978. [2] L.N. Vandenberg, I. Chahoud, J.J. Heindel, V. Padmanabhan, F.J.R. Paumgartten, G. Schoenfelder, Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A, Environ. Health Perspect. 118 (2010) 1055–1070. [3] L.N. Vandenberg, M.V. Maffini, C. Sonnenschein, B.S. Rubin, A.M. Soto, Bisphenol-A and the great divide: a review of controversies in the field of endocrine disruption, Endocr. Rev. 30 (2009) 75–95. [4] R. Rezg, S. El-Fazaa, N. Gharbi, B. Mornagui, Bisphenol A and human chronic diseases: current evidences, possible mechanisms, and future perspectives, Environ. Int. 29 (2013) 83–90. [5] H. Rochefort, Bisphenol A and hormone-dependent cancers: potential risk and mechanism, Med. Sci. (Paris) 29 (2013) 539–544. [6] J. Huff, Carcinogenicity of bisphenol-A in Fischer rats and B6C3F1 mice, Odontology 89 (2001) 12–20. [7] R.E. Chapin, J. Adams, K. Boekelheide, L.E. Gray, S.W. Hayward, P.S.J. Lees, B.S. McIntyre, K.M. Portier, T.M. Schnorr, S.G. Selevan, NTP-CERHR expert panel report on the reproductive and developmental toxicity of bisphenol A, Birth Defects Res. B: Develop. Reprod. Toxicol. 83 (2008) 157–395. [8] O. Takahashi, S. Oishi, Testicular toxicity of dietarily or parenterally administered bisphenol A in rats and mice, Food Chem. Toxicol. 41 (2003) 1035–1044. [9] P. Alonso-Magdalena, S. Morimoto, C. Ripoll, E. Fuentes, A. Nadal, The estrogenic effect of bisphenol A disrupts pancreatic -cell function in vivo and induces insulin resistance, Environ. Health Perspect. 114 (2006) 106–112.
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