Bisphenol A inhibits proliferation and induces apoptosis in micromass cultures of rat embryonic midbrain cells through the JNK, CREB and p53 signaling pathways

doi:10.1016/j.fct.2012.10.033

Food and Chemical Toxicology

Volume 52, February 2013, Pages 76-82

https://doi.org/10.1016/j.fct.2012.10.033 Get rights and content

Abstract

Bisphenol A (BPA) has been widely used in the manufacture of polycarbonate plastic, water bottles and food containers. Previous studies have established that BPA could cause developmental toxicity by inhibiting the proliferation and differentiation of rat embryonic midbrain (MB) cells in vitro. However, the underlying mechanisms have not been well studied yet. In the current study, we examined the proliferation and differentiation of MB cells treated with 10⁻¹²–10⁻⁴ M BPA and found that only 10⁻⁴ M BPA inhibited proliferation and differentiation. Then, we investigated the cell cycle progression and apoptosis of MB cells; 10⁻⁴ M BPA caused an explicit S phase and G2/M phase arrest in the cell cycle and increased the percentage of apoptotic cells. Moreover, the phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic-AMP response binding protein (CREB) and the expression of some apoptotic regulatory genes were investigated. BPA (10⁻⁴ M) reduced the phosphorylation of C-Jun N-terminal kinase (JNK) and CREB, and increased the mRNA expression level of Bax and p53. Our findings demonstrated that BPA could cause developmental toxicity through anti-proliferation and pro-apoptosis in MB cells. Multiple signaling pathways, such as the JNK, CREB and p53-mitochondrial apoptosis pathways, participate in these effects.

Highlights

► Bisphenol A (BPA) inhibited proliferation and differentiation in rat midbrain (MB) cells. ► BPA induced cell cycle block and apoptosis in MB cells. ► BPA reduced the phosphorylation level of JNK and CREB in MB cells. ► BPA increased the mRNA expression level of Bax and p53 in MB cells.

Introduction

Bisphenol A (BPA) is a high production volume chemical used primarily in the manufacture of polycarbonate plastic, epoxy resins and dental sealants. Moreover, BPA can migrate into food and beverages from products made of polycarbonate plastic, such as baby bottles, tableware, food containers and water bottles (Vandenberg et al., 2007, The National Toxicology Program-Center for the Evaluation of Risks to Human Reproduction, 2007). Therefore, human exposure to BPA is widespread. The U.S. Centers for Disease Control have estimated that 95% of Americans have detectable levels of BPA in their urine (Calafat et al., 2005). Additionally, BPA can be detected in the blood of pregnant women, amniotic fluid, placental tissue and umbilical cord blood, indicating some degree of fetal exposure (Engel et al., 2006, Padmanabhan et al., 2008, Tan and Mohd, 2003).

Many studies indicate that the developing fetus is more sensitive to xenobiotics than the adult (Vandenberg et al., 2007). Although there is no direct evidence that human exposure to BPA adversely affects development, studies with laboratory rodents show that exposure to BPA during pregnancy and/or lactation can reduce survival, birth weight, and growth of offspring early in life. In an extensive review by the National Toxicology Program (NTP) Center in the US, the developmental toxicity of BPA for fetuses, infants and children, especially the effects on the brain and behavior were deemed worthy of “some” concern (Chapin et al., 2008). Our previous studies indicated that BPA induced brain and neural tube abnormalities in rat embryos cultured in vitro (Xing et al., 2010). BPA could also inhibit the proliferation and differentiation of rat embryonic midbrain cells (Xiao et al., 2011). However, the underlying mechanism is still unknown.

During embryonic development, cells proliferate rapidly and are vulnerable to chemicals. In addition, apoptosis functions to clear abnormal or redundant cells in developing embryos (Hardy et al., 2003). Apoptosis plays an important role in embryogenesis; several studies have confirmed the function of apoptosis in normal embryonic development (Chan and Shiao, 2008, Lotz et al., 2006). However, excessive apoptosis caused by chemicals could lead to developmental defects (Chan, 2011).

Mitogen-activated protein kinase (MAPK) cascades have been shown to play a key role in the transduction of extracellular signals to cellular responses. In mammalian cells, three MAPK families have been clearly characterized: extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase (JNK), and p38 kinase. The MAPK families play important roles in complex cellular programs such as proliferation, differentiation, development, transformation and apoptosis (Zhang and Liu, 2002). Many studies have indicated that the cyclic-AMP response binding protein (CREB) is upstream of Bcl-2 and that CREB phosphorylation could positively regulate the anti-apoptotic factor Bcl-2 and inhibit apoptosis (Lebesque et al., 2009, Kwak et al., 2008). In many cell types, the anti-oncogene p53 has the ability to activate transcription of various proapoptotic genes, including those encoding members of the Bcl-2 family such as Bax, Noxa and Puma, and finally regulate the induction of apoptosis (Amaral et al., 2010).

Therefore, we hypothesize that the developmental toxicity of BPA may be due to the inhibition of cell proliferation and promotion of apoptosis, and multiple signaling pathways may participate in the toxicity. In the present study, we employed micromass cultures of rat embryonic midbrain cells to investigate the effects of BPA on cell proliferation and apoptosis and then examined whether MAPK, CREB and p53 are involved in the effects of BPA.

Section snippets

Chemicals and antibodies

BPA (97% pure) was obtained from Sigma (St. Louis, MO, USA). BPA was dissolved in dimethyl sulfoxide (DMSO; Amresco Company, Solon, Ohio, USA) and added to the culture medium. The final volume of DMSO in medium was 0.1%. Polyclonal rabbit anti-rat ERK1/2, ERK1/2 phosphorylation, JNK, JNK phosphorylation, p38, p38 phosphorylation, CREB, CREB phosphorylation antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The β-actin antibody was purchased from Santa Cruz (Santa Cruz

BPA inhibited the proliferation and differentiation of MB cells

Although 10⁻¹²–10⁻⁶ M BPA showed no significant changes in cell proliferation and differentiation of MB cells, 10⁻⁴ M BPA decreased cell proliferation to 64%. In addition, the total area of foci containing neurocytes was reduced to 52% (Fig. 1). Microscopic images of micromass cultures are shown in Fig. 2.

Because BPA caused no remarkable changes at the low dose levels (10⁻¹²–10⁻⁶ M), 10⁻⁸ M was chosen as a representative low dose level for further study along with 10⁻⁴ M.

BPA induces cell cycle block in MB cells

To confirm the inhibition of

Discussion

In this study, the developmental toxicity of BPA was examined using a rat embryonic midbrain micromass model. Our results showed that 10⁻⁴ M BPA could inhibit the proliferation and foci differentiation of MB cells and concurred with our previous results (Xiao et al., 2011).

We further investigated the apoptosis of MB cells exposed to 10⁻⁴ M BPA using Annexin V-FITC/PI double staining and TUNEL staining and found that BPA could promote apoptosis. In addition to the proapoptotic effect of BPA, we

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grant from the National Natural Sciences Foundations of the People’s Republic of China [No. 30972501], Key Projects in the National Science and Technology Pillar Program in the Eleventh Five-year Plan Period [2006BKA02A02] and National Science and Technology Major Specific Project [2009ZX09301-010].

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Bisphenol A inhibits proliferation and induces apoptosis in micromass cultures of rat embryonic midbrain cells through the JNK, CREB and p53 signaling pathways

Abstract

Highlights

Introduction

Section snippets

Chemicals and antibodies

BPA inhibited the proliferation and differentiation of MB cells

BPA induces cell cycle block in MB cells

Discussion

Conflict of Interest

Acknowledgements

Mol. Cell

Toxicol. Lett.

Reprod. Toxicol.

Reprod. Toxicol.

Cell. Signal.

J. Craniomaxillofac. Surg.

Reprod. Toxicol.

Biochem. Biophys. Res.

Chem. Biol. Interact.

Talanta

Reprod. Toxicol.

Toxicol. Lett.

Reprod. Toxicol.

Toxicol. In Vitro

Biochem. Biophys. Res.

Toxicol. In Vitro

The role of p53 in apoptosis

Discov. Med.

Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population

Environ. Health Perspect.

Cytotoxic effect of CdSe quantum dots on mouse embryonic development

Acta Pharmacol. Sin.