Rapid Effect Of Bisphenol A On Calcium Level In Hippocampal Neurons

Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. This compound is a building block of polycarbonate plastics often used for food and beverage storage, and it is also a component of epoxy resins that are used to line food and beverage containers. BPA is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system,and has been recognized to have wide adverse effects on the brain development and behavior. Because BPA leaches out of plastic food and drink containers, as well as the BPA containing plastics used in dental prostheses and sealants, considerable potential exists for human exposure to this compound. Previous studies regarding the biological and toxicological effects of BPA on the human body have focused on it’s estrogenic action.This is because BPA seems to mimic some estradiol-induced biological actions such as vaginal cornification and growth and differentiation of the mammary glands.In addition to the effects on reproductive tissues, BPA is likely to alter the morphological and functional properties of neuronal cells in the central nervoussystem (CNS). BPA inhibits estradiol induced hippocampal synaptogenesis and suppresses the estradiol-induced enhancement of long-term potentiation in the hippocampal CA1 area. Besides the classical estrogen action via the regulation of gene transcription in nuclei, estrogen has rapid and gene transcription-independent effects (non-genomic effects) via putative membrane receptors. Intracellular Ca2+ signaling system mediated by NMDARs(N-methyl-D-aspartate receptors)plays an essential role in synaptic plasticity, such as neurotransmitter release and induction of long-term potentiation (LTP). Until the present study, little is known about the rapid effect of low dose BPA (at nanomolar level) in cellular Ca2+ regulation in hippocampal neurons.NMDARs (N-methyl-D-aspartate receptors) are critical for synaptic function throughout the CNS(central nervous system). NMDAR-mediated Ca2+ influx is essential for neuronal differentiation, neuronal migration, synaptogenesis, synaptic remodelling, long-lasting changes in synaptic efficacy, such as LTP (long-term potentiation) and LTD (long-term depression), and cognitive functions such as learning and memory. Some researches collectively provide strong evidence that alterations in Ca2+ influx through NMDA receptors and L-type voltage-gated Ca2+ channels mediate homeostatic intrinsic plasticity in hippocampal neurons in response to prolonged activity changes.And recent evidence indicates that Ca2+ permeability of neuronal NMDARs, NMDAR mediated Ca2+ signalling in spines and induction of NMDAR-dependent LTP (long-term potentiation) at hippocampal CA1 synapses are under control of the ERK1/2 signalling cascade.But whether BPA affect Ca2+ influx and the downstream pathway is still not clear. Here we investigated the mechanisms of rapid Ca2+ signaling induced by a low dose BPA application in cultured hippocampal neurons.Research contents Effects of different concentrations of bisphenol A on the changes of intracellular calcium levels induced by glutamate in hippocampal neurons; By the combination of estrogen receptor blocking ICI182 780 and Flutamide to determine that wether the changes of the Ca2+ signaling induced by BPA were mediate by estrogen receptor or androgen receptor;In addition, the ERK1/2 and p38 phosphoryla-tion level was detected to determine whether the ERK1/2 or p38 signaling pathway was involved in the regulation of the level of Ca2+ signaling by BPA.Research Method Hippocampal neurons were separated from postnatal 24 h-old rat and 8-10 days after the neurons were cultured in the medium, after Fluo 4-AM special pakcaging(Calcium fluorescent probe)were loaded for 30 min, BPA was added into the medium for 30 min. The final concentrations of BPA were 1,10,100 and 1000 nM by dissolved in dimethylsulfoxide (DMSO), DMSO (the final concentration was controlled in 0.001%) was also used as vehicle to dissolve the chemicals in the positive control cultures. In this study, we use 17p-estradiol (17β-E2) and dihydrotestosterone (DHT) for positive control. The medium containing ICI 182,780, Flutamide, U0126, or SB203580 were respectively pre-treated to the cultures prior to BPA,17β-E2 or DHT for 30 min to investigate the antagonism of ER antagonist, AR antagonist, a mitogen-activated ERK1/2-activating kinase (MEK1/2) inhibitor, or a p38 MAPK inhibitor, to BPA’s effects.We use Fluo-4/AM to test the Ca2+ signaling induced by glutamate.In order to further investigate the molecular mechanisms of BPA exposure on neuronal Ca2+signaling, western blot method was used to detect the protein expression of ERK, p-ERK, p38, p-p38 to explore whether ERK and p38 signaling pathway were involved in acute exposure of BPA on neuronal development.Research results1. Effect of glutamate on Ca2+ level. In this study, the most common excitatory amino acid transmitter in the brain was used as a trigger for calcium influx.The basic intensity of calcium was almost unchanged, but the fluorescence transient enhanced when glutamate was added. After that, the fluorescence intensity of calcium remained relatively stable. According to the result, glutamate was use to induced changes in the intracellular calcium level of neurons in the following experiments.2. In order to examine whether BPA alters [Ca2+]i, we tested the changes of [Ca2+]j after applicated BPA for 30 min induced by glutamate. The result showed that after applicated BPA for 30 min, [Ca2+]j was remarkably increased by 46% induced by glutamate compared with control(P＜0.001). Then we found that the [Ca2+]i maintained at a high level within 5 min.Application of different concentrations of BPA, we found BPA at 1,10,100 nM have significant effect on [Ca2+], and BPA at 10 nM has the most remarkable effect, but BPA at 1000 nM significantily decreased [Ca2+]i induced by glutamate compared with the control(P<0.01).3. For 17β-E2 and DHT, we tested different concentration (1,10,100 nM) of them, it showed that both of 17β-E2 and DHT at 10 nM have the most significant effect on [Ca2+]i compared with control(P<0.01). And we also found the level of BPA (10 nM) increased [Ca2+]i was similar to 17β-E2 (10 nM) and DHT (10 nM). Because the treatment with 10 nM 17β-E2,10 nM DHT or 10 nM BPA were most effective forcalcium influx, these concentration was used in the following investigations unless specified.4.To investigate whether the BPA-induced changes on [Ca2+]i were mediated by ERs and AR, the cultures were pre-treated with an ER antagonist (ICI 182,780) or AR antagonist (Flutamide) for 30 min before exposure to BPA (10 nM),17β-E2 (10 nM) or DHT (10 nM) respectively. The results showed that both ICI 182,780 and Flutamide completely eliminated the promoted effects of BPA,170-E2 and DHT on [Ca2+]i respectively. Note that the application of ICI 182,780 or Flutamide alone has no significant effect on [Ca2+], compared with control. In order to further proved ERs and AR mediate the effects of BPA on [Ca2+]i, we examined the [Ca2+]i of BPA co-treated with 17β-E2 (10 nM) or DHT (10 nM). The result showed that treatment of BPA at 10 nM co-treated with lOnM 17β-E2 for 30 min, the elevation of [Ca2+]i induced by glutamate has no obvious difference with control. Similarly, the [Ca2+]i has no obvious difference with control when 10 nM BPA co-treated with 10 nM DHT. But only treated with 10 nM BPA,10 nM 17β-E2 or 10 nM DHT, [Ca2+]i significantly increased compared with control(P<0.001, P<0.01, P<0.05).5. Next we investigated kinase signaling pathways involved in the BPA-induced change of [Ca2+]i levels by Western blot. Western blot analyses showed that treatment of BPA 10 nM did not influence the expressions of ERK1/2 in hippocampus, but significantly increased the level of p-ERK1/2(P< 0.001). However, co-treatment of BPA with ICI (10 μM) or Flutamide (1 μM), the antagonist of ERs or AR, significantly blocked the BPA-induced increase in the levels of p-ERK1/2. U0126 pretreatment significantly inhibited glutamate induced neuronal [Ca2+]i, BPA can not reversed the inhibition of the response to glutamate, but BPA pre-treatment can partially antagonized the the inhibition of U0126 on glutamate induced neuronal [Ca2+]i. Application of 1μM SB203580, a p38 MAPK inhibitor, also prevented the effect by BPA and [Ca2+]i has no obvious change compared with control. And the application of SB203580 alone has no significant effect on [Ca2+]i compared with control. The result suggested that ERK1/2 and p38 MAPK may mediate the effects of BPA on [Ca2+], induced by glutamate.Conclusion:BPA at 1,10,100 nM have rapid effect on [Ca2+]i and BPA at 10 nM has the most remarkable effect, but BPA at 1000 nM significantily decreased [Ca2+]i induced by glutamate compared with the control, suggesting that BPA has the effect of inhibition at low concentration and promotion at high concentration. ICI182 780 and Flutamide can blocked BPA’s rapid effect on [Ca2+]i, suggesting ERs and AR may mediate the effects of BPA on [Ca2+]i induced by glutamate in neurons. Furthermore, BPA can up-regulated the expression of p-ERK and p-p38, suggesting that BPA induced rapid effect on [Ca2+]imay be mediated by ERK1/2 and p38 MAPK.