Effects Of Bisphenol-A On Cultured Hippocampal Dendritic Development And Its Molecular Mechanism

Bisphenol A (BPA) is one of the typical environmental endocrine disruptors, which has a weak estrogen activity. It is widely used to manufacture food and beverage packaging materials such as polycarbonate plastic, epoxy resin, dental prostheses and sealants, and other related products. At present, the effects of BPA on the central nervous system has been taken more and more attention. BPA can not only affect the brain estrogen synthesis and receptor expression, but also the neurotransmitter systems and the morphologyical development of the brain. During the development of brain dendritic development are important processes. Its structure and characteristics determine the efficiency of synaptic trasmission. Motile dendritic filopodial processes are thought to participate in synaptogenesis, dendritic branching and the development of spines. Excessive growth of dendritic branches may lead to nervous system dysfunction. During the early development, dendritic branches grow and stuctural change rapidly. Excessive growth of dendritic branches will be disappeared for the loss of domination of afferent fibers. Endogenous estrogen can affect the dendritic development and synaptogenesis, BPA which has estrogenic activity may affect the dendritic development. N-methy-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor. It is invoved in a wide variety of processes in the central nervous system (CNS) including learning and memory process, neuronal survival, axon and dendrites stuctural development, and synaptogenesis and synaptic plasticity. Studies showed that NMDA receptor involved in the effect of dendritic development induced by estrogen. Because the important role of NMDA in the dendritic development, we use cultured hippocampal neurons to illuminate the effects of BPA. In this study, we dectect the rapid and slow effects of BPA on the dendritic morphology, the expression of NMDAR subunits NR1, NR2B and phosphorylation of NR2B, and the expression of Estrogen Receptor beta (ERβ).Methods:During the morphological detection of hippocampal neurons, we use adenovirus recombinants encoding EGFP (Ad-EGFP) to infect the cultured hippocampal neurons (5 days in virto),24-48 h after the infection EGFP can be detected. We set five groups:control and BPA (1,10,100,1000 nM BPA,0.01%DMSO as a vehicle). After application of BPA, image of live cells were captured every 2 min for 20 min, using an inverted fluorescent microscope. All images were analysised using Image Pro-Plus software. We measured filopodial motility and dendritic filopodia density and the total length of the dendritic. In order to analyze whether estrogen receptor regulation was involved in the rapid effect of BPA, cultures were pretreated by 10μM antagonist of estrogen receptor ICI 182,780 30 min prior the application of BPA. In order to analyze the interaction the BPA and E2, cultures were co-treated by BPA and E2.For further analysis of the mechanism of BPA effect on dendritic development, the detection of protein was carried out on cultured hippocampal neurons (7 DIV). The methods of BPA application and exprimental group were consistent with the above. For immunocytochemical staining, primary antibody NR1, NR2B, p-NR2B (phosphorylated NR2B) and ERβwere used to dectect the expression of protein. Image Pro-Plus software was used to analysis the ptical density of the neurons.Results:1.0.5 h acute exposure to BPA (10～1000 nM) promoted the motility and density of dendritic filopodia (P＜0.05, P＜0.01 or P＜0.001), and this effect can be inhibited by antagonist of ERs (P＜0.001); 17β-E2(10 nM) treatment significantly increased motility and density of dendritic filopodia, when the hippocampal cultures was co-treated by BPA (100 nM) with E2 (10 nM), the increased of filopodial motility induced by 17β-E2 was obviously attenuated (P＜0.01).24 h exposure to BPA,100 nM BPA promoted the motility and density of dendritic filopodia (P＜0.05, P＜0.01 or P＜0.001), and also the total length of the dendritic (P＜0.05), antagonist of ERs completely inhibited the increases in the filopodial motility, the density of filopodia and the total length of the dendritic induced by BPA (P＜0.05); E2 (10 nM) treatment significantly increased motility and density of dendritic filopodia, and also the total length of the dendritic, when the hippocampal cultures was co-treated by BPA (100 nM) with 17β-E2 (10 nM), the increased of filopodial motility and density and total length of the dendritic induced by 17β-E2 was obviously attenuated (P＜0.05).2.0.5 h treatment with BPA (10～1000 nM) did not change the immunoreactive intensity of NRl and NR2B, but significantly increased the immunoreactive intensity of phosphorylated NR2B (p-NR2B; P＜0.05, P＜0.01, P＜0.001, P＜0.001), the increasement was completely eliminated by antagonist of ERs (P＜0.01); while co-treatment of BPA (100 nM) with 17β-E2 (10 nM) markedly suppressed the immunoreactive intensity of phosphorylation of NR2B induced by 17p-E2 (P＜0.01).24 h treatment with BPA (10 to 1000 nM) significantly decreased the expression of NR1 and NR2B (P＜0.001), but antagonist of ERs did not change the depression of NR1 and NR2B induced by BPA; while co-treatment of BPA (100 nM) with 17β-E2 (10 nM) markedly suppressed the immunoreactive intensity of NRl and NR2B induced by 17β-E2.3.0.5 h acute exposure to BPA did not change the immunoreactive intensity of ERβ. But 24 h treatment with BPA (10～1000 nM) significantly increased the expression of ERβ(P＜0.001), antagonist of ERs markedly suppressed the immunoreactive intensity of ERβinduced by 17β-E2 (P＜0.01).17β-E2(10 nM) increased the expression of ERβ, when co-treatment of BPA (100 nM) with 17β-E2(10 nM) increased the immunoreactive intensity of ERp.Conclusions:Acute exposure to BPA promoted the motility and density of dendritic filopodia as well as the phosphorylation of NR2B, but not affect the expressions of NR1, NR2 and ERβ. For the effect of nuclear estrogen receptor needs more time, suggesting that rapid modulation of BPA involvement of the membrane-associated ER.24 h slowly exposure to BPA promoted the motility and density of dendritic filopodia and the total length of dendritic, and changed the expression of NRl, NR2 and ERβ. These results indicated involvement of ER mediation, but BPA affect NRl, NR2B expression may be through other ways.Acute or slowly exposure to BPA, similar to E2, can promoted the motility and density of dendritic filopodia as well as phosphorylation of NR2B. While co-treatment of BPA (100 nM) with 17β-E2 (10 nM), a suppression was observed. While co-treatment of BPA (100 nM) with 17β-E2 (10 nM), the increased of filopodia motility and density and phosphorylation of NR2B was markedly suppressed.In conclusion, BPA can rapidly effect the development of dendritic filopodia, and this rapid modulation may involve the membrane-associated ER. Slowly exposure to BPA not only effect the development of dendritic filopodia, but also change the expression of NMDA receptor, and then exert an important impact on the brain development.