Bisphenol A Interference The Modulation Of Androgen On The Morphological Development Of Dendrite And Synapse In Hippocampal Neurons

Bisphenol A(BPA),one of the most representative phenolic compounds in environmental endocrine disruptors(EEDs),is widely used in the manufacture of polycarbonate,epoxy resins,fungicide,antioxidant and the packing material of food and drink.EEDs can mimic or antagonism the role of endogenous hormones,and then disturb the normal functions of endocrine system.The chemical structural formula of BPA is similar to diethylstilbestrol,therefore BPA have affinity to estrogen receptor.Presently,studies about the toxic effect of BPA was focus on its estrogen-like activity.But,the toxic effect of BPA on central nervous system turn into a focus in recent years,Previous studies of our laboratory indicated that BPA impair passive avoidance memory and spatial learning memory in male rats were more remarkable than female,no matter the exposed time was at puberty or mature period.BPA intensify the depressive state more significance in male rats.Thus,we speculate that the effect of BPA exposure on male’s brain and behavior may be refer to the interference the modulation of androgen in brain.Synaptic plasticity is necessary for cognitive function,mainly includes formation of synapse,morphological structure of synapse,and the changing ability of synaptic functions concomitant external stimuli.Androgen receptors(AR)are highly expressing at forebrain and hippocampus in adult male animals.In male rats,the synaptic plasticity and learning memory are mainly regulated by androgen not estrogen.Lack of androgen can not maintain the normal structure and function of hippocampus in male rats,but the normal level of androgen contribute to the stabilization of hippcampal circuit.Hence,by means of culture hippocampal neurons in vitro,we investigated the effect of acute exposure concentration gradient of BPA and DHT on the development of dendrite and synapse.BPA and DHT were co-incubated on hippocampal neurons for investigate the interaction of BPA and DHT.BPA and 17β-E2 were co-treated on hippocampal neurons for investigate the interaction of BPA and 17β-E2.Next,androgen receptor antagonist,flutamide(Flu)and estrogen receptor inhibitor(ICI 182,780)were used to study that whether AR or ER can mediate the impact of BPA on neuronal plasticity.The effect molecular mechanism of acute exposure BPA on the development of hippocampal neurons was studied with Western Blot technology by detect the expression of synapsin I,PSD-95 and AR;For further research,MEK 1/2 inhibitor(U0126)and p38 inhibitor(SB 203,580)was pretreatment before BPA to the analysis the signal pathway about the role of BPA on synapse density and spines density.Experimental method:The neonatal hippocampus were isolated from 24 h-old suckling Spraque-Dawley rats,hippocampal neurons were acute exposured BPA after cultured for 12 days in vitro.Primarily,vehicle control group,concentration gradient of BPA(1-1000 nM)group and concentration gradient of DHT(1-100 nM)group were exposured to investigate the dose-effect relationship of acute impact.In order to detect polymerized filamentous actin(F-actin,red fluorescence),the cultures were incubated with rhodamine-conjugated phalloidin.Images were captured with laser confocal scan microscope(LCSM;Leica TCS-SP5,Germany)and thorns density and synapses density were calculated 30μm length from the start of each secondary dendrite by MetaMoph Microsoft.Next,immunocytochemistry was proceed with the specificity combination of primary antibodies(Syn I)and secondary antibodies with green fluorescence marked.The location of synapse(yellow fluorescence)was con-located by Syn I(green fluorescence)and F-actin(red fluorescence).Next,the most significance dose-effect of BPA(100 nM)and DHT(10 nM)or 17β-E2(10 nM)were co-incubated to investigate the interference effects of BPA on DHT or 17β-E2 modulated the formation of synapses and thorns.Next,flutamide(Flu,1 μM)and estrogen receptor inhibitor(ICI 182,80,10 μM)were used to study that whether AR or ER can mediate the impact of BPA on neuronal plasticity.Eventually,MEK 1/2 inhibitor(U0126,25 μM)and p38 inhibitor(SB 203,580,10 μM)was pretreatment before BPA to the analysis the signal pathway about the role of BPA on synapses density and spines density.At the same time,the effects of molecular mechanism by acute exposure BPA on the development of hippocampal neurons were studied with Western Blot technology by detect the expression of synapsin I,PSD-95 and AR.Experimental result:(1)Acute exposure BPA(1nM-1μM)for 1 h showed that 10 nM BPA and 100 nM BPA significant enhancement the density of synapses and spines(P<0.001),but the promotion disappered at 1000 nM BPA.The effect curve of BPA showing inversion "U" shape,it reminds that BPA play a dose dependency role on the morphology development of dendrite.Dose dependency showed that low dosage(10 nM,100 nM)promote the morphology development of dendrite while high dosage(1000 nM)the promotion disappered,and the most effective dose is 100nM BPA;(2)The results showed that co-treated of BPA(100 nM)with DHT(10 nM)completely eliminated the promoted effects of DHT or BPA on the density of synapses and spines(P<0.001),it hints that BPA play a antagonism role to androgen.Meanwhile,cultures pretreated with androgen receptor antagonist,flutamide(Flu,1μM)for 0.5 h before BPA didn’t eliminated the increase effects of BPA,which indicates that AR isn’t the intermediary which mediate the promotion of low dosage BPA on the density of synapses and spines;(3)The results showed that co-treated of BPA(100 nM)with 17β-E2(10 nM)completely eliminated the promoting effects of 17β-E2 or BPA on the density of spines(P<0.001)and the density of synapses(P<0.05).Cultures were pre-treated with estrogen receptor inhibitor(ICI 182,780)for 0.5 h before exposure to BPA completely declined the promoting effects of BPA on the density of spines and the density of synapses(P<0.001).Above results investigate that ER act as the intermediary which mediate the promotion of low dosage BPA on the density of synapses and spines;(4)Although incubation with U0126 or SB 203,5 80 alone did not showed any affect of the the density of synapses and.spines in hippocampal neurons,but pre-treated with a MEK 1/2 inhibitor(U0126)or p38 inhibitor(SB 203,580)for 0.5 h before exposure BPA completely declined the promoting effects of BPA on the density of spines(P<0.001)and the density of synapses(P<0.001).Above results investigate that MEK 1/2 or p38 MAPK signal pathway is the intermediary which mediate the promotion of low dosage BPA on the density of synapses and spines;(5)The results of Western Blot experiments showed that treatment of BPA 100 nM enhanced the expression of presynaptic protein Syn I(P<0.01)and postsynaptic density protein 95(PSD-95)(P<0.01),but co-treated of BPA(100 nM)with DHT(10 nM)completely eliminated the expressions of Syn I(P<0.01)and PSD-95(P<0.01).Above results investigate that BPA acute exposure promoted the formation of dendrites and synapses involves the expression of synaptic proteins(Syn I and PSD-95).Conclusion:The influence of BPA on the morphology development of dendrites and synapses in hippocampal neurons were bidirectional dose-dependent,low dose showed that promoting the development of dendrite and synapse while the promotion disappered at high dose.The promotion of BPA was mediated through ER not AR,which was proceeding in the MEK 1/2 or p38 MAPK signal pathway.BPA antagonized the promotion of androgen or estrogen on the development of dendrites and synapses in the environment of androgen or estrogen.BPA acute exposure promoted the formation of dendrites and synapses involve the expression of synaptic proteins,such as Syn I and PSD-95.