Effects and regulatory mechanisms of bisphenol a on the increases apoptosis and decreases differentiation potential in mouse embryonic stem cells

Abstract

Bisphenol A has deleterious effects on reproductive, developmental, cell biological, and physiological functions. Here, we investigated the dosage effects of bisphenol A on the differentiation potential and apoptosis of mouse embryonic stem cells, and assessed some relevant regulatory mechanisms. Our results showed that bisphenol A at doses of 1–2 μmol/L triggers apoptotic processes without necrotic cell death in the ESC-B5 mouse embryonic stem cell line. No death effect was seen at treatment dosages of 0.5 μmol/L or less. Mechanistically, the application of 1–2 μmol/L bisphenol A directly increased the intracellular oxidative stress levels, significantly increased the cytoplasmic calcium and nitric oxide contents, decreased the mitochondrial membrane potential, activated caspases-9 and -3, and triggered programmed cell death. Interestingly, embryoid body formation assays showed that 0.5 μmol/L bisphenol A decreased the differentiation potential of ESC-B5 cells without inducing apoptotic processes. Together, our results indicate that treatment with 1–2 μmol/L bisphenol A induces apoptosis and triggers hazardous effects on the differentiation and developmental potential of mouse embryonic stem cells in vitro. These results provide important evidence that bisphenol A should be considered a potent cytotoxin that has dose-dependent impacts on differentiation and apoptosis in a mouse embryonic stem cell line.

Graphical Abstract

Graphical Abstract.

Introduction

Bisphenol A (BPA), which is a lipophilic member of the endocrine disrupting chemical (EDC) group, is used as an additive in the manufacturing of polycarbonate plastics and epoxy resins.^1,2 Many daily-use appliances and utensils commonly contain BPA, including food and water containers, baby bottles, toys, and (in particular) medical equipment, such as infusion bags.^3,4 The global production of BPA is estimated to be about 8 million metric tons per year; this production is expected to grow annually to a predicted production of 10.6 million metric tons by 2022.⁵ It is concerning that BPA production and use result in large-scale environmental contamination and humans being inevitably exposed to BPA on a daily basis. Indeed, BPA can be detected in various human body fluids, including blood, breast milk, sweat, urine, and (particularly) amniotic fluid and placenta.^6,7

Previous studies found that prenatal and perinatal exposure of rats to BPA can trigger precancerous and cancerous lesions and alter mammary gland development.^8,9 Moreover, BPA reportedly has potential hepatotoxic and neurotoxic effects.^10,11 The toxicity of BPA has been attributed to its ability to trigger endocrine disruption, developmental defects, and apoptosis.^12–14 Concerns around the health risks of BPA have prompted many countries to issue regulations restricting the use of BPA in the production of many containers, such as baby bottles.

Numerous chemical and physical treatments can trigger cell apoptosis mediated by the intracellular generation of reactive oxygen species (ROS).^15,16 BPA has been shown to induce apoptotic processes through ROS generation, and this can be prevented by the potent ROS scavenger, N-acetylcysteine (NAC).¹⁷ Nitric oxide (NO), which is synthesized from L-arginine and O₂ by nitric oxide synthase (NOS), is an important key second messenger involved in regulating tumor development, metastasis, and apoptosis.^18,19 The production of NO is largely catalyzed by Ca²⁺-sensitive mitochondrial NO synthase (NOS) in mitochondria.^20,21 During the NO production process, NOS may also regulate oxygen consumption and mitochondrial membrane potential via cytochrome c oxidase. The NOS-generated NO molecules react with superoxide to produce peroxynitrite, which acts on its target substrates to induce oxidative stress.^22,23 ROS and Ca²⁺ influx are important upstream regulators of mitochondrial NOS activity.^24,25 We previously reported that dihydrolipoic acid at concentrations of 50–100 μM can induce NO-, Ca²⁺-, and ROS-related mitochondria- and caspase-dependent apoptosis in the ESC-B5 mouse embryonic stem cell line.²⁴ Recent investigations further found that BPA and its analogs, bisphenol S (BPS) and bisphenol B (BPB), can induce intracellular ROS generation to trigger mitochondria- and caspase-dependent apoptotic processes in hippocampal and neuroblastoma cell lines.^26,27

Embryonic stem cells (ESCs) are isolated and derived from early-stage embryos. ESCs cultured in medium containing embryonic fibroblasts or leukemia inhibitory factor (LIF, an anti-differentiation agent) can proliferate and maintain their potential to differentiate into any type of cell in the body.^28,29 When cultured in anti-differentiation agent-free medium, ESCs will spontaneously differentiate and develop in a manner that recapitulates early embryogenesis.^24,30 ESCs differentiated in suspension culture form three-dimensional aggregate structures called embryoid bodies (EBs), which contain ectodermal, mesodermal, and endodermal tissues structures and resemble the egg-cylinder stage of an embryo. Given these properties, the analysis of EB formation potential is a powerful and useful method for analyzing early embryo development and differentiation processes in an in vitro system.

NO- and ROS-mediated apoptotic processes represent just one important apoptotic signaling cascade among many. Here, we show that BPA acts via ROS-, Ca²⁺-, and NO-involving regulatory mechanisms to dose-dependently induce apoptosis and affect cell differentiation potential in ESC-B5 cells.

Materials and methods

Chemicals and reagents

Bisphenol A (BPA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), sodium pyruvate, 2′,7′-dichlorofluorescin diacetate (DCF-DA), dihydrorhodamine 123 (DHR 123), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), N-acetyl cysteine (NAC), ethyleneglycol-bis (β-aminoethylether) tetraacetic acid (EGTA) and Anti-microtuble associated protein 2 (MAP-2) antibody were obtained from Sigma (St. Louis, MO, USA). CDP-Star™ (a chemiluminescent substrate for alkaline phosphatase) was purchased from Boehringer Mannheim (Mannheim, Germany). The bicinchoninic acid (BCA) protein assay reagent was purchased from Pierce (Rockford, IL, USA). DiOC6(3) and TMRE fluorescent dyes were from Invitrogen (Carlsbad, CA, USA). Anti-cleaved caspase-9 (Asp353) and anti-cleaved caspase-3 (Asp175) antibodies were from Cell Signaling (Beverly, MA, USA).

Cell culture and BPA treatment

ESC-B5 cells were cultured in DMEM supplemented with 20% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/mL streptomycin in a humidified 37 °C, 5% CO₂ incubator. BPA was dissolved in 95% ethanol in glass bottles; the final concentration of ethanol in all treatment and control solutions was 0.01%. ESC-B5 cells were incubated in culture medium containing the indicated concentrations of BPA or 0.01% ethanol for 24 h. The cells were then washed three times with ice-cold phosphate buffered saline (PBS) and lysed in 400 μL lysis buffer (20 mmol/L Tris–HCl, pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 1 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L NaF, 20 μmol/L sodium pyrophosphate, and 1 mmol/L sodium orthovanadate) for 15 min on ice. Cell lysates were sonicated three times (10 sec each) on ice and centrifuged at 15,000 × g for 20 min at 4 °C, and supernatants (cell extracts) were collected.

Measurement of cell viability by MTT assay

Cell survival rates were measured by the MTT (3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) test. In brief, cells were cultured in a 96-well plate for 24 h with or without BPA and treated with 100 μL of 0.45 g/L MTT solution for 1 h at 37 °C to allow color development. The reaction was stopped by the addition of 100 μL of 20% SDS in DMF:H₂O (1:1) solution. The plates were incubated overnight at 37 °C for solubilization of the formazan products, and cell viability was measured by spectrophotometry using an ELISA reader (Multiskan FC microplate photometer, Thermo Fisher Scientific) at a wavelength of 570 nm.

Assessment of apoptosis and necrosis

A TUNEL Cell Death Detection ELISA^plus kit (Roche Molecular Biochemicals, Mannheim, Germany) was used to measure oligonucleosomal DNA fragmentation as a marker of apoptosis. The TUNEL assay was performed according to the manufacturer’s protocol and spectrophotometric data were obtained using an ELISA reader (Multiskan FC microplate photometer, Thermo Fisher Scientific) at 405 nm. Cell necrosis was determined by monitoring the presence of lactate dehydrogenase (LDH) in the culture medium, as described in our previous studies.^31–33 In brief, cells (5 × 10⁴) were cultured in 96-well microtiter plates (100 μL medium/well), the LDH-Glo™ Cytotoxicity Assay Kit was used as directed by the manufacturer (Promega, Madison, WI), and LDH activity was measured with an ELISA reader (Multiskan FC microplate photometer, Thermo Fisher Scientific) at 490 nm. The absorption of cell-free medium lacking test substances was measured as a blank control.

Assessment of intracellular ROS level

DCF-DA or DHR 123 dyes were used to measure the intracellular ROS level. In brief, 1 × 10⁶ cells were incubated in 50 μL PBS containing 20 μmol/L DCF-DA or DHR123 for 1 h at 37 °C, and a fluorescence ELISA reader (Multiskan FC microplate photometer, Thermo Fisher Scientific) was used to measure relative ROS units (excitation 485 nm and emission 530 nm). The results were expressed as arbitrary absorbance units/mg protein.

Assessment of intracellular calcium concentration ([Ca²⁺]i)

Intracellular calcium concentration ([Ca²⁺]i) was detected with Fluo-3 AM fluorescence dye according to previously published reports.^24,34,35 In brief, cells were incubated with BPA for 24 h, harvested, washed, and loaded with 6 μmol/L Fluo-3 AM in standard medium (140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, 5.6 mmol/L glucose, 1.5 mmol/L CaCl₂, and 20 mmol/L HEPES, pH 7.4) for 30 mins. The cells were then washed three times with PBS and further resuspended in standard medium or Ca²⁺-free standard medium. A fluorescence spectrophotometer (F-2000; Hitachi, Tokyo, Japan) was used to measure the fluorescence intensity of Fluo-3 (excitation 490 nm and emission 526 nm).

Measurement of intracellular NO content

The intracellular NO contents were assessed with the DAF-2DA fluorescence dye according to a previously reported method.^24,35,36 Briefly, BPA-treated or untreated cells were collected and washed, and then incubated with 3 μmol/L DAF-2DA for 1 h. The cells were then further washed 3 times with PBS and the fluorescence intensity was measured with a F-2000 fluorescence spectrophotometer (Hitachi; excitation at 485 nm, emission at 515 nm).

Assessment of intracellular caspase activity

Caspase-9 activity was measured using a Colorimetric Caspase-9 Assay Kit (Calbiochem, San Diego, CA, USA). Caspase-3 activity was detected using the Z-DEVD-AFC fluorogenic substrate, according to previous reports.^24,37,38

Real-time RT-PCR assay

Total RNA was extracted from cells with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified with an RNeasy Mini kit (Qiagen) according to the provided protocols. Real-time PCR was executed with an ABI 7000 Prism Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The mRNA level of β-actin was detected as an endogenous control for normalization. The primers used for PCR were as follows: p53, 5′-CCC ATC CTC ACC ATC ATC AC-3′ and 5′-GTC AGT GGG GAA CAA GAA GTG-3′; p21, 5′-GCC GAA GTC AGT TCC TTG TGG A-3′ and 5′-GTG GGC GGA TTA GGG CTT-3′; β-actin, 5′-CTG TCC CTG TAT GCC TCT G-3′ and 5′-ATG TCA CGC ACG ATT TCC-3′.

siRNA knockdown

Lipofectamine was used to transfect ESC-B5 cells with 150 nmol/L of siRNA targeting p53 (5’-GACUCCAGUGGUAAUCUACTT-3′; sip53) or a scrambled control duplex (5’-GCGCGCUUUGUAGGAUUCG-3′; siScr). At 24 h post-transfection, the culture medium was replaced with fresh culture medium and the cells were incubated with the indicated concentration of BPA or vehicle for another 24 h.

EB formation

EB formation potential was assessed as described in our previous study.²⁴ Briefly, ESC-B5 cells were dissociated with trypsin–EDTA (0.25%) and cultured in leukemia inhibitory factor (LIF)-free stem cell medium for differentiation. Cell-suspension liquid cultures (<10⁴ cells/mL) were dispensed to 10-cm petri dishes (10 mL per dish). Each 10-μL droplet had about 100 ESC-B5 cells, and hanging-drop cultures were generated in dishes to initiate EB formation. The hanging-drop cultures of ESC-B5 cells were cultured for 48 h to allow for aggregation, and then transferred to liquid suspension culture.

Statistics

Data were evaluated and analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons, and are presented as means ± SD. All data were normally distributed and met the assumptions of the parametric ANOVA test. Different symbols in figures represent significant differences at P < 0.05.

Results

Cytotoxic effects of BPA on ESC-B5 cells

To investigate the potential cytotoxicity of BPA, the MTT assay was used to monitor cell viability in ESC-B5 cells exposed to 0.5–2 μmol/L BPA. Our results showed that 0.5 μmol/L BPA or less had no injury effect on cell viability, whereas the cell viabilities of the 1–2 μmol/L BPA-treated groups were significantly decreased compared to the vehicle (control)-treated group (Fig. 1A). The cell death types evident in BPA-treated ESC-B5 cells were investigated. Our results revealed that cells treated with 1–2 μmol/L BPA showed dose-dependent increases in the proportion of apoptotic cells (Fig. 1B), whereas there was no evidence of necrotic cell death in groups treated with up to 2 μmol/L BPA (Fig. 1C). Together, our results indicate that 0.5 μmol/L BPA or less do not have detectable cytotoxic effects in our ESC-B5 cell system, whereas treatment with 1–2 μmol/L BPA dose-dependently triggers apoptosis.

Fig. 1.

Effects of BPA on ESC-B5 cells. ESC-B5 cells were incubated with 0.5–2 μmol/L BPA or vehicle control (0.01% ethanol) for 24 h. A) Cell viability was measured using the MTT assay. B) Apoptosis was assessed with a TUNEL cell death detection ELISA kit. C) The activity of LDH released to the culture medium was measured as a means to detect cell necrosis. Experimental data are expressed as a percentage of the maximal level (max) of LDH activity measured under conditions of total cell lysis. Values are presented as means ± SD of six determinations. Different symbols indicate significant differences at P < 0.05.

Intracellular ROS generation in BPA-treated ESC-B5 cells

To detect ROS levels in ESC-B5 cells treated with BPA, we stained the cells with DCF-DA and DHR 123 fluorescent dyes. The ROS levels in the 1–2 μmol/L BPA-treated groups were ~ 2.3–4.5-fold higher than that in the vehicle-treated control group (Fig. 2A). Notably, BPA-induced ROS generation and apoptosis could be effectively prevented by pretreatment with the potent ROS scavenger, NAC (Fig. 2B and C). The results clearly indicate that intracellular ROS generation is an important event in the BPA-triggered apoptosis of ESC-B5 cells.

Fig. 2.

BPA induces intracellular ROS generation in ESC-B5 cells. A) ESC-B5 cells were incubated with 0.5–2 μmol/L BPA or vehicle control (0.01% ethanol) for 24 h. ROS generation was measured using DCF-DA (20 μmol/L) or DHR 123 (20 μmol/L). (B and C) ESC-B5 cells were pretreated with or without N-acetyl cysteine (NAC; 300 μmol/L) for 30 min, followed by incubation with the indicated concentrations of BPA or vehicle (control). DCF-DA was used to detect ROS generation. B) Apoptosis was measured with a TUNEL cell death detection ELISA kit. C) Data are representative of six independent experiments. Different symbols indicate significant differences at P < 0.05.

BPA-triggered apoptosis of ESC-B5 cells involves changes in the levels of intracellular Ca²⁺ and NO

To assess changes in intracellular Ca²⁺ concentration ([Ca²⁺]i), BPA- and vehicle-treated ESC-B5 cells were preincubated with Fluo-3 AM fluorescence dye. Our results revealed that treatment with 1–2 μmol/L BPA elicited increases in [Ca²⁺]i, with 2 μmol/L BPA-treated groups exhibiting a ~ 3-fold increase in [Ca²⁺]i relative to controls, regardless of whether they were cultured in Ca²⁺-containing or Ca²⁺-free medium (Fig. 3A). Thus, the observed increase in [Ca²⁺]i primarily arose via the release of Ca²⁺ from internal compartments, which might include the endoplasmic reticulum, mitochondria, nucleus, and/or calcium-binding proteins. Preincubation of cells with PTIO, an inhibitor of NOS and scavenger of NO, and L-NMMA, an inhibitor of NO synthase (NOS), had no significant effect on the increase of [Ca²⁺]i seen in the 2 μmol/L BPA-treated groups (Fig. 3B). In contrast, pretreatment of cells with NAC significantly suppressed this BPA-triggered increase of [Ca²⁺]i (Fig. 3B). These findings indicate that ROS generation is an important upstream regulator of the [Ca²⁺]i level in BPA-treated ESC-B5 cells, whereas NO is not. We next used the NO-sensitive dye, DAF-2DA, to assess changes in intracellular NO levels in BPA-triggered apoptosis. Our results revealed that intracellular NO levels were significantly increased in 2 μmol/L BPA-treated cells, and that this could be effectively prevented by pretreatment with the NOS inhibitor, L-NMMA, or the Ca²⁺ chelator, EGTA (Fig. 3C). Based on these findings, we suggest that [Ca²⁺]i acts as an important regulator for triggering NOS activation and increasing NO during the BPA-induced apoptosis of ESC-B5 cells.

Fig. 3.

Changes in intracellular calcium and NO content in BPA-treated ESC-B5 cells. A) ESC-B5 cells were incubated with 0.5–2 μmol/L BPA or vehicle control (0.01% ethanol) for 24 h. Intracellular Fluo-3 fluorescence intensity was measured in the presence/absence of extracellular Ca²⁺ in the culture medium, as described in the “Materials and Methods”. B) Intracellular Ca²⁺ levels in cells treated with 2 μmol/L BPA after preincubation with inhibitors of ROS (NAC: 300 μmol/L) or NO (L-NMMA: 400 μmol/L; PTIO: 20 μmol/L). C) ESC-B5 cells were preincubated with L-NMMA (400 μmol/L) or EGTA (500 μmol/L) for 30 min, and then treated with 2 μmol/L BPA for 24 h. Intracellular NO generation was detected using the DAF-2DA fluorescence dye. Data are representative of six independent experiments. Different symbols indicate significant differences at P < 0.05.

BPA alters mitochondrial membrane potential (MMP) and activates caspases, and these effects can be inhibited by PTIO

Next, we assessed the change in MMP, which is an important component of mitochondria-mediated intrinsic apoptosis. Using the fluorescent MMP-detecting dyes, DiOC6(3) and TMRE, we found that MMP was significantly decreased in cells treated with 2 μmol/L BPA (Fig. 4A). Moreover, caspase-9 and -3, which are important caspases involved in mitochondrial-mediated apoptosis, were activated under 2 μmol/L BPA-triggered apoptosis (Fig. 4B and C). Preincubation with 20 μmol/L PTIO could significantly prevent the MMP loss and caspase activation seen following treatment with 2 μmol/L BPA (Fig. 4A–C). Together, these results show that generation of intracellular NO may act as an upstream regulator of mitochondria-mediated apoptotic processes, including the decrease of MMP and activation of caspase-9 and -3, in BPA-treated ESC-B5 cells.

Fig. 4.

Changes of mitochondrial membrane potential (MMP) and caspase-9 and -3 activity in BPA-treated ESC-B5 cells. ESC-B5 cells were preincubated with PTIO (20 μmol/L) for 1 h and then treated with 2 μmol/L BPA or vehicle control (0.01% ethanol) for a further 24 h. A) MMP changes were measured using 40 nmol/L DiOC6(3) or 1 μmol/L TMRE. B) Caspase-9 activity was assessed by a colorimetric caspase-9 assay (upper panel) and immunoblotting with an antibody specific for activated caspase-9 (lower panel). C) For caspase-3 activity analysis, cell extracts (50 μg) were analyzed using Z-DEVD-AFC as the substrate (upper panel), and immunoblotted using anti-activated caspase-3 antibody (lower panel). Values are representative of five independent experiments. Different symbols indicate significant differences at P < 0.05.

p53 and p21 are upregulated in BPA-treated ESC-B5 cells

Our real-time quantitative RT-PCR analyses showed that the mRNAs encoding p53 and p21 were significantly upregulated in 2 μmol/L BPA-treated ESC-B5 cells, and that this was effectively blocked by pretreatment with NAC or PTIO (Fig. 5A and B). To further examine the roles of p53 and p21 in BPA-triggered apoptosis, we used specific siRNA knockdown. The transfection of siRNAs specific to p53 effectively decreased the expression levels of the p53 and p21mRNAs in 2 μmol/L BPA-treated cells (Fig. 6A). In addition, knockdown of p53 expression significantly decreased the apoptosis induced by 2 μmol/L BPA (Fig. 6B). These finding show that BPA increases ROS and NO levels to upregulate p53 and p21 expression in ESC-B5 cells, thereby stimulating apoptotic processes.

Fig. 5.

mRNA expression levels of p53 and p21 in BPA-treated cells. ESC-B5 cells were preincubated with or without NAC (300 μmol/L) and PTIO (20 μmol/L) for 1 h, and then treated with 2 μmol/L BPA or vehicle control (0.01% ethanol) for a further 24 h. Real-time PCR was used to assess the mRNA expression levels of p53 (A) and p21 (B). Data are representative of five independent experiments. Different symbols indicate significant differences at P < 0.05.

Fig. 6.

Knockdown of p53 blocks BPA-triggered apoptosis in ESC-B5 cells. siRNAs specifically targeting p53 was transfected into ESC-B5 cells. After 24 h, the cells were treated with 2 μmol/L BPA or vehicle control (0.01% ethanol) for a further 24 h. A) The mRNA levels of p53 and p21 were measured using real-time PCR. B) Apoptosis was detected as described in the “Materials and Methods” and the legend to Fig. 1. Values are representative of four independent experiments. Different symbols indicate significant differences at P < 0.05.

Impacts of BPA on the development and differentiation potential of ESC-B5 cells in vitro

To further measure the effects of BPA on early embryonic development potential, we used an in vitro assay model in which ESC-B5 cells were incubated with 0.5–2 μmol/L BPA or vehicle (control) for 24 h and analyzed for their ability to form EB in vitro. The EB formation percentage and average EB diameter were significantly and dose-dependently decreased among 0.5–2 μmol/L BPA-treated ESC-B5 cell groups compared to the vehicle control group (Fig. 7A–C). The addition of 50 ng/mL nerve growth factor (NGF) to the differentiation medium for 14 days prompted control cells to differentiate into nerve cells, as shown by immunoblotting for the nerve cell biomarker, microtubule-associated protein-2 (MAP-2) (Fig. 7D). Notably, pretreatment with 2 μmol/L BPA effectively blocked the NGF-induced expression of MAP-2 (Fig. 7D). Collectively, our findings indicate that 0.5 μmol/L BPA can decrease the differentiation potential of ESC-B5 cells without inducing evident apoptosis, whereas concentrations of 1–2 μmol/L BPA induce apoptosis and trigger hazardous effects on the differentiation and development potential of mouse embryonic stem cells in vitro.

Fig. 7.

Effects of BPA on the differentiation potential of ESC-B5 cells. ESC-B5 cells were incubated with 0.5–2 μmol/L BPA or vehicle control (0.01% ethanol) for 24 h. A) Cells were dissociated with trypsin–EDTA and cultured in LIF-free medium to induce differentiation. The hanging-drop method was employed for embryoid body (EB) formation, as described in the “Materials and Methods”. B) The sizes of EBs were observed under microscopy. C) Measurement and statistical analysis of EB diameters at day 10. D) ESC-B5 cells were incubated with 2 μmol/L BPA or vehicle control (0.01% ethanol) for 24 h, and further treated with 50 ng/mL nerve growth factor (NGF) for 14 days. Microtubule-associated protein 2 (MAP-2) was analyzed by immunoblotting with anti-MAP-2 antibodies. Values are representative of five independent experiments. Different symbols indicate significant differences at P < 0.05. Scale bar = 100 μm.

Discussion

Over the past 30 years, BPA has been found to cause various injury effects on development and reproductive and somatic health.^12,39,40 Moreover, accumulating evidence indicates that BPA analogs also can cause cellular and developmental injury effects.^41,42 Recent investigations found that several specific BPA analogs are even more estrogenic, potent and toxic than BPA itself.^41–43 BPA and its analogs, bisphenol S (BPS), bisphenol AF (BPAF), and tetramethyl bisphenol F (TMBPF), have been shown to dose-dependently trigger apoptosis in rat adipose-derived stem cells and human mesenchymal stem cells.^40,44 Moreover, treatment with a low dose of BPA analogs was found to induce apoptosis in nearly 100% of human stem cells through activation of the early-apoptosis indicator, caspase-6.⁴⁰ BPA, BPS, and BPB (bisphenol B) were found to trigger generation of excessive ROS and malondialdehyde (MDA) and induce oxidative stress- and mitochondria-dependent apoptotic cascades, thereby causing neurotoxicity in neuroblastoma cells.²⁷ In the present study, we investigated the effects and underlying regulatory mechanisms of BPA in mouse embryonic stem cells. Our results showed that BPA exerts cytotoxicity through the induction of ROS, Ca²⁺, NO, and mitochondria- and caspase-dependent apoptotic signaling. In addition, the deleterious impacts of BPA on ESC-B5 cells affected their differentiation potential. Based on the previous reports and our present results, it seems clear that BPA and its analogs can potently trigger programmed cell death (apoptosis) via a ROS- and mitochondria-dependent pathway.

Cells control intracellular ROS levels to regulate signaling pathways and transcription factors that govern cell fates under normal physiological conditions, with ROS levels acting as critical controls for a number of important biological and physiological functions in organisms.^45,46 However, overproduction of intracellular ROS due to an external stimulus or internal status change can overwhelm the ability of the endogenous redox buffering system to scavenge free radicals or ROS and return them to their normal ranges. This can disrupt important cellular structures or components, including cell membranes, proteins, DNA, and lipids, leading to physiological dysregulation and/or pathological changes.^47,48 Several recent investigations found that ROS is an important key regulator of intrinsic mitochondria-dependent apoptotic processes.^49–51 In the present study, we show that BPA induces apoptosis, not necrosis, and dose-dependently increases intracellular ROS levels at concentrations of 1–2 μmol/L, whereas these effects were not seen in groups treated with 0.5 μmol/L or less of BPA (Figs 1 and 2). The BPA-induced inductions of ROS generation and apoptosis were effectively prevented by the potent ROS scavenger, NAC (Fig. 2B and C). BPA exposure was previously reported to trigger intracellular ROS generation and increase MDA levels, leading to apoptosis in neuroblastoma cells.²⁷ In addition, several studies reported that antioxidant pretreatment could prevent BPA-induced apoptosis and deleterious effects in rat neuron cells and mouse myoblast C2C12 cells.^52,53 These previous research results and our present results collectively indicate that ROS is an important upstream regulator of the ability of BPA to trigger apoptosis and hazardous effects, as seen in both in vitro and in vivo assay models. However, the exact regulatory mechanisms and signaling cascades through which ROS impacts signaling and ultimately induces apoptosis under BPA treatment were previously unknown.

Previous investigations and our present study showed that the intracellular calcium level plays an important role in regulating programmed cell death cascades,^24,54 and that intracellular ROS generation is a key regulator of intracellular calcium and apoptosis.^24,49 Several studies recently reported that BPA exposure could trigger ROS generation and accumulation of Ca²⁺ in granulosa cells, leading to ovarian dysfunction.^55–57 Here, we report that BPA treatment and the subsequent increase of ROS increases intracellular Ca²⁺ in a mouse embryonic stem cell line via the mobilization of internal Ca²⁺ stores, and that this effect of BPA can be effectively prevented by NAC pretreatment (Fig. 3A and B). These findings indicate that BPA-triggered ROS generation is responsible for the influx of internal Ca²⁺ to the cytosol, thereby increasing the intracellular calcium concentration in ESC-B5 cells.

NO is an endogenous metabolic product of the catalysis of L-arginine by nitric oxide synthase (NOS). The generation of NO is an important regulator of cascades leading to apoptotic processes under several different types of stimuli.^24,58 The roles and regulatory actions of NO in the mitochondrial apoptotic signaling cascades have been well elucidated using in vitro and in vivo models.^20,24 In the present study, we show that the intracellular NO content was about 3.4-fold higher in the BPA-treated group than in the vehicle-treated group (Fig. 3C). Pretreatment with EGTA significantly attenuated this increase in the intracellular NO level (Fig. 3C), indicating that the increase of intracellular NO in BPA-treated ESC-B5 cells depends on the increase in intracellular Ca²⁺. However, the actions and regulatory roles of NO in apoptosis are complex, and the contributions of NO to apoptosis are modulated via different mechanisms in distinct cell types.^24,58,59 Importantly, we herein found that PTIO attenuates the loss of MMP and inhibits caspase activation in our system (Fig. 4), indicating that NO is an important mediator of apoptotic processes in BPA-treated ESC-B5 cells.

The activation of p53 gene expression is reportedly associated with NO-mediated apoptotic cascades occurring through p21^Waf1/Cip1 or Bax.^24,60,61 In addition, we previously showed that the expression levels of p53 and p21 are increased during the apoptotic signaling of methylglyoxal-treated human mononuclear cells, dihydrolipoic acid-treated ESC-B5 cells, and emodin-treated human neuroblastoma cells.^24,62,63 Here, we show that pretreatment of ESC-B5 cells with PTIO or NAC could significantly attenuate the BPA-induced upregulations of the mRNAs encoding p53 and p21 (Fig. 5). Damaged DNA triggers the accumulation of phosphorylated p53 proteins to promote cell cycle arrest and apoptosis. The p53 protein can activate specific genes, such as p21, which encodes a Cdk inhibitory protein (p21) that can trigger cell cycle arrest. Here, we show that siRNA-mediated knockdown of the p53 mRNA could block the BPA-triggered upregulation of p21 mRNA expression and prevent downstream apoptosis (Fig. 6A and B). These finding clearly indicate that both p53 and p21 proteins play important regulatory roles in BPA-triggered apoptosis. Based on our experimental data, we suggest that excess ROS triggers DNA damage to induce upregulation of p53 and p21 mRNA expression, thereby promoting downstream apoptotic signal cascades.

BPA is known to impair cell proliferation and stem cell differentiation, and this has been attributed to its ability to trigger ROS generation and promote mitochondria-dependent apoptotic events.^64,65 A recent study reported that exposure to relatively high-dose BPA could inhibit differentiation in cardiomyocytes but exhibited only weak cytotoxicity in embryonic stem cells.⁶⁶ This suggested that BPA has different capacities for exerting cytotoxicity versus impairing cell differentiation. However, we previously lacked information on the detailed regulatory mechanisms through which BPA impairs the development and differentiation of stem cells. Here, we demonstrate that BPA at doses of 0.5 μmol/L or less has no apoptotic effect in ESC-B5 cells but can decrease their differentiation potential (Fig. 7). Meanwhile, BPA at concentrations of 1–2 μmol/L can trigger hazardous effects on ESC-B5 cells, inducing apoptosis and decreasing cell differentiation. This finding provides the new insight that a low dosage (0.5 μmol/L or less) of BPA does not induce apoptosis but is capable of decreasing the differentiation potential of a mouse embryonic stem cell line, whereas higher concentrations of BPA (1–2 μmol/L) both cause apoptosis and decrease the differentiation potential. We thus show for the first time that different dosages of BPA can have different effects on differentiation, development, and apoptosis induction in a mouse embryonic stem cell line.

Taken together, our present results show that BPA directly induces ROS generation, which in turn triggers a Ca²⁺ influx from intracellular Ca²⁺ storage organelles, leading to increased intracellular Ca²⁺ concentrations and downstream stimulation of NO production to dose-dependently trigger apoptosis, but not necrosis, in the ESC-B5 mouse embryonic stem cell line. Pretreatment with NAC or PTIO blocks the BPA-induced upregulation of critical genes and rescues cell viability. In addition to triggering apoptosis, BPA also has dose-dependent injury impacts on the differentiation potential of ESC-B5 cells. These findings demonstrate that BPA is a potent chemical toxin that negatively affects stem cells by inducing apoptosis and decreasing the differentiation potential. Continuous exposure of humans to BPA is therefore likely to cause hazardous impacts on development and overall health.

Author contributions

Wen-Hsiung Chan conceived and designed the experiments; Cheng-Kai Lee, Chien-Hsun Huang, Fu-Ting Wang and Hsin-Ju Lin performed the experiments; all authors analyzed and interpreted the data; Wen-Hsiung Chan wrote the paper; all authors contributed to the final approval of manuscript.

Funding

This work was supported by grants from the Ministry of Science and Technology, Taiwan, ROC (MOST 109-2311-B-033-001 and MOST 110-2314-B-715-012).

Conflicts of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.