Estrogen receptor independent neurotoxic mechanism of bisphenol A, an environmental estrogen

Abstract

Bisphenol A (BPA), a ubiquitous environmental contaminant, has been shown to cause developmental toxicity and carcinogenic effects. BPA may have physiological activity through estrogen receptor (ER) -alpha and -beta, which are expressed in the central nervous system. We previously found that exposure of BPA to immature mice resulted in behavioral alternation, suggesting that overexposure of BPA could be neurotoxic. In this study, we further investigated the molecular neurotoxic mechanisms of BPA. BPA increased vulnerability (decrease of cell viability and differentiation, and increase of apoptotic cell death) of undifferentiated PC12 cells and cortical neuronal cells isolated from gestation 18 day rat embryos in a concentration-dependent manner (more than 50 microM). The ER antagonists, ICI 182,780, and tamoxifen, did not block these effects. The cell vulnerability against BPA was not significantly different in the PC12 cells overexpressing ER-alpha and ER-beta compared with PC12 cells expressing vector alone. In addition, there was no difference observed between BPA and 17-beta estradiol, a well-known agonist of ER receptor in the induction of neurotoxic responses. Further study of the mechanism showed that BPA significantly activated extracellular signal-regulated kinase (ERK) but inhibited anti-apoptotic nuclear factor kappa B (NF-kappaB) activation. In addition, ERK-specific inhibitor, PD 98,059, reversed BPA-induced cell death and restored NF-kappaB activity. This study demonstrated that exposure to BPA can cause neuronal cell death which may eventually be related with behavioral alternation in vivo. However, this neurotoxic effect may not be directly mediated through an ER receptor, as an ERK/NF-kappaB pathway may be more closely involved in BPA-induced neuronal toxicity.

Fig. 1

Effect of BPA on PC12 cell viability. (A) Cell viability of PC12 cells after 12, 24, 48, and 72 h treatments with/without several concentrations of BPA. Living cells were represented as a percentage of the total population of living and dead cells. (B) Photomicrographs of DAPI-stained PC12 cells treated for 24 h with several concentrations of BPA. Cells were stained with DAPI to visualize nuclear morphology. The percentage of apoptosis which presents a reduction in nuclear size, chromatin condensation, and nuclear fragmentation from two experiments was entered into a graph. (C) Photomicrographs of PC12 cells treated with the indicated materials for 5 days, respectively. PC12 cells were cultivated for 1 day in the condition of non-treated DMEM supplemented with Ham's F12 nutrient, 1% horse serum, 50 ng/ml NGF, 100 units/ml penicillin, and 100 µg/ml streptomycin, and were then cultured with/without several concentrations of BPA added to the medium. Control (Con), those cultured with/without BPA and NGF. ^*Significant difference from control (p < 0.05).

Fig. 2

Effect of BPA on cortical neuronal cell viability. (A) Cell viability of neuronal cells after 72 h treatments with/without various concentrations of BPA. Living cells were represented as a percentage of the total population of living and dead cells. (B) Photomicrographs of DAPI-stained neuronal cells treated for 24 h with various concentrations of BPA. Cells were stained with DAPI to visualize nuclear morphology. The percentage of apoptosis which presents a reduction in nuclear size, chromatin condensation, and nuclear fragmentation from two experiments was entered into a graph. (C) Photomicrographs of neuronal cells treated with BPA for 5 days, respectively. Neuronal cells were cultivated for 1 day in the condition of non-treated neurobasal medium supplemented with B 27 serum, and then cultured with/without several concentrations of BPA added to the medium. Control (Con), those cultured with/without BPA. ^*Significant difference from control (p < 0.05).

Fig. 3

BPA-induced PC12 cell death was not mainly mediated by estrogen receptor. PC12 cells were treated with ER antagonist 30 min prior to treatment with BPA. (A) Viability of PC12 cell cultures was determined after treatment with compounds for 3 days. (B) Photomicrographs of PC12 cells cultured with BPA with/without ER antagonists added to DMEM supplemented with Ham's F12 nutrient, 5% fetal bovine serum, 10% horse serum, 50 ng/ml NGF, 100 units/ml penicillin, and 100 µg/ml streptomycin for 5 days. ^*Significant difference from control (p < 0.05).

Fig. 4

BPA-induced neuronal cell death was not mainly mediated by estrogen receptor. Neurocortical (neuronal) cells were treated with ER antagonist 30 min prior to treatment with BPA. (A) Viability of neuronal cell cultures was determined after treatment with compounds for 3 days. (B) Photomicrographs of neuronal cell cultures cultured with BPA with/without ER antagonists added to neurobasal media supplemented with B 27 serum for 5 days. ^*Significant difference from control (p < 0.05).

Fig. 5

BPA toxicity in PC12, PC12/neo, PC12/ER-α, or β cells. (A) Expression of ERs in established PC12 cells expressing ER-α and β. (B) Comparison of four cell types according to a viability assay performed 72 h after BPA treatments. The four cell types were cultured with/without several concentrations of BPA. (C) Comparison between BPA and 17-β estradiol on cell viability according to concentration using the WST-1 method. Viability was assayed in PC12 cells and neuronal cells treated with different concentrations of BPA and 17-β estradiol for 72 h. ^*Significant difference from control (p < 0.05).

Fig. 6

Effect of BPA concentrations for activations of the MAP kinase family. Cells were treated with BPA, and after 2 h exposure to BPA, activation of MAP kinase family was determined by Western blotting. Similar patterns of expression were found from three individual experiments.

Fig. 7

Protective effects of ERK inhibitor on BPA toxicity. PC12 and neuronal cells were pretreated with MAP kinase inhibitors (PD 98,059, SB 203,580, and SP 600,125) followed by BPA (300 µM) treatment. After 72 h of exposure, we examined PC12 and neuronal cell survival using the WST-1 assay. Living cells were represented as a percentage of the total population of living and dead cells. ^*Significant difference from control (p < 0.05). ^#Significant difference from only BPA-treated group (p < 0.05).

Fig. 8

Effect of BPA, ER antagonists, and ERK inhibitor on NF-κB activation. PC12 cells (A) and cortical neuronal cells (B) were treated with 0, 10, 50, 100, and 200 µM (BPA) for 90 min with/without pretreatment of different doses of ER antagonists (tamoxifen and ICI 182,780) (C & D) or ERK inhibitor (E & F) 30 min prior to treatment with BPA. Nuclear extracts were prepared and assayed for NF-κB by EMSA from two individual experiments performed in duplicate.