The Effects Of Environmental Endocrine Disrupter Bisphenol A On Learning-memory And Synaptic Structure Of Adult Mice

Bisphenol A (BPA), one of the most common environmental endocrine disrupters, can bind to estrogen receptors (ERs) to interfere with the regulation of endogenous estrogen on the central nervous system. The aim of the present study was to investigate the effects of long-term exposure to BPA on the learning and memory behavior. The adult mice at age of10weeks were exposed to BPA (0.4,4, and40mg/kg/day) or sesame oil for12weeks.In open field test, BPA increased the frequencies of rearing and grooming of the males, but reduced the frequency of rearing in the females. Exposure to BPA (0.4or40mg/kg/day) extended the escape pathlength to find the hidden platform in Morris water maze and shortened the step-down latency24h after footshock of the males, but no changes were found in the females. Meanwhile, BPA induced a reduced numeric synaptic density and a negative effect on the synaptic structural modification, including the enlarged synaptic cleft and the reduced length of active zone and PSD thickness in the hippocampus of the males. Western blot analyses further indicated that BPA down-regulated the expressions of synaptic proteins (Synapsin I and PSD-95) and NMDA receptor subunit NR1in the hippocampus of the males. These results suggest that long-term exposure to the low levels of BPA in adulthood sex-specifically impaired spatial and passive avoidance memory of mice. These effects may be associated with the higher susceptibility of hippocAMPA1synaptic plasticity processes, such as remodeling of spinal synapses and the expressions of synaptic proteins and NMDA receptor, to BPA in the adult males.Methods:8weeks ICR mice, the male weight30-35g, female weight25-30g, bought zhejiang province YaoJianSuo in jinhua. Male and female mice were fed separately, raising environment for a period of12h alternating day and night, feed room temperature control in18to22℃, humidity from50%to60%, free food and water intake. Mice adapt to2weeks, randomly divided into BPA exposure groups (0.4,4,40mg/kg/day (ShengGong BPA,99.8%, Shanghai biological engineering technology service co., LTD.) and solvent control group (golden dragon fish oils). At8o’clock every morning to fill the stomach infected for12weeks. Infected over three days later, the opening behavior was detected according to the order of water maze and passive avoidance. Another electron microscopic technique was done respectively observed in mice hippoc AMPA synaptic1interface structure, protein immunoblot (western blot) technology to detect hippoc AMPA and NMDA receptor NR1GluRl and AMPA receptor subunits expression and synaptic proteins expression of Synapsin1and PSD-95; Detection of body weight, serum levels of estrogen, androgen levels at the same time.Results:BPA exposure (10weeks of age) in mice body weight before there was no significant gender differences, male mice weight growth acceleration, then to22.5weeks, male mice body weight significantly higher than the female (F (1,88)=548.323, P<0.001). BPA exposure by gender in mice body weight is significantly influenced by the interaction (F (3,82)=5.764, P=0.001), and the exposure factor has no effect on body weight (F (3,86)=0.221, P=0.882).0.4group compared with the same sex control group, and40mg/kg/day after12weeks of BPA exposure significantly increases the weight of males (p<0.05). BPA exposure on female and male mice reproductive organ/body weight ratio, female mice and serum estradiol levels in the brain were not significantly affected, but the0.4and4mg/kg/day BPA significantly reduced testosterone of male mice serum and brain (p<0.05).Two-factor analysis of variance showed that BPA exposure in mice by gender interaction is the number1minutes and5min run without significant influence, but significant impact on stand and grooming times [F (3,88)=5.012, p=0.003;F(3,88)26.913, p<0.001, n=90). Gender factors for1minutes and5min run bars, standing and the number of grooming has significant effect, and respectively F(1,88)=18.630, p<0.001; F (1,88)=5.174, p=0.025; F (1,88)=5.269, p=0.024; F (1,88) =18.521, p<0.001). Among them, the normal male mice run1minute case number significantly higher than female mice (p<0.05),4mg/kg/day BPA to eliminate the gender difference. Exposure factors significantly influence run for1minutes (F (3,86)=3.225, P=0.026) and grooming (F (3,86)=12.694, P<0.001). Compared with the same sex control group, BPA, a significant increase in male mice of grooming and stand number (p<0.05, p<0.01), but lower the stand number of females (0.4mg/kg/day, p<0.05), and thereby inducing sex differences in males than females (p<0.05, p<0.01). These results suggest that long-term exposure to BPA to promote male mice, inhibit female inquiry activity, increase sample status of anxiety in the male mice in conflict situations.In four days of water maze training, each group of mice average swimming speed was no significant difference, but every day to explore the average distance of the platform is characterized by along with the increase of training days shorten, that each group has a certain ability of spatial learning and memory in mice, but normal female exploration platform required is much bigger than the average distance of the male mice (3,4days, P<0.05), suggesting that the normal males have better spatial learning and memory ability. Multifactor analysis of variance showed that BPA exposure×gender×training time (F (9,313)=2.582, P=0.007), BPA exposure by gender (F (3,337)=2.807, P=0.040), sex (training time (F (3,337)=3.339, P=0.020) the interaction of the mice were significantly affect the average distance of search platform. Gender and training time factor has obvious influence on the average distance of mice search platform (F (1,343)=15.034, P<0.001); F (3,341)=81.016, P<0.001). BPA exposure significantly extend the average distance of male mice search platform (P<0.05), but has no effect on female mice. Suggests that long-term exposure to BPA gender specificity to damage in adult male rats spatial memory ability, and thus eliminate the sex differences in spatial learning and memory.Electric shock, before there is no difference between groups of mice on the platform of the incubation period, all can be jumped off the platform within5SEC. Electric shock after24h, the type on the platform were significantly prolong the incubation period, and showed gender differences (P<0.001, male mice> female). Two-factor analysis of variance showed that BPA exposure by the interaction of gender has no effect on incubation period after the shock, but BPA exposure, and gender factors have significant impact on male mice were on incubation period of after shocks (F(3,89)=2.747, P=0.048; F (1,91)=4.026, P=0.046). Compared with the same sex control group,40mg/kg/day BPA markedly shortened the male rats after electric shock on the incubation period (P<0.05); Suggests that long-term exposure to BPA gender specificity to damage in adult male rats of passive avoidance memory ability, and thus eliminate the gender difference.Aimed to analyze the effect of long-term exposure to BPA on learning and memory behavior and relationship of synapse formation and structure, this study examined closely associated with learning and memory of CA1area pyramidal cell number density of synapses and synaptic structure modification. Two-factor analysis of variance showed that BPA exposure by the interaction of gender, exposure to BPA and gender factor has a significant influence on synaptic number density (F (3,40)=4.351, P=0.01; F (3,40)=3.887, P<0.05); F (1,78)=4.907, P<0.01).0.4and40mg/kg/day BPA exposure significantly reduced the number of male rat hippoc CA1area of synaptic density (P<0.01), and thereby inducing sex differences in adult mice synaptic density CA area (P<0.01, the males (females). Two-factor analysis of variance showed that BPA exposure by the interaction of gender, BPA exposure, and gender factors on synaptic activity with the length and the width of synaptic cleft were not affected, but BPA exposure by gender synaptic cleft width is significantly influenced by the interaction (F (3,712)=3.535, P=0.015) and the thickness of postsynaptic density body (PSD, F(3,712)=3.912, P<0.001), BPA exposure factors also have the same effect (synaptic cleft, F (3,712)=4.491, P<0.001; the PSD, F(3,712)=3.812, P=0.01). Compared with the same sex control group, BPA markedly widened male rats synaptic cleft width (P<0.05, P<0.01) and shorten the length of the active zone (0.4mg/kg/day, P<0.05), and reduce the thickness of the PSD (0.4,40mg/kg/day, P<0.05), but has no effect on female mice. Above results showed that long-term exposure to BPA mainly affects the synapse formation and structure of the adult male mice, the result is consistent with the BPA’s impact on memory behavior.BPA damage that in order to further analysis and synapse formation mechanism of learning and memory, this research adopts the Western blot analysis after long-term exposure to BPA, synaptic proteins and glutamate NMDA and AMPA receptors expression changes. Results showed that the gender specificity of BPA exposure to inhibit the expression of synaptic proteins and receptors, main show is3doses of BPA were cut in male mice of Synapsin Ⅰ expression and PSD-95(P<0.05, P<0.01) and40mg/kg/day BPA NMDA receptor NR1subunits expression (P<0.05, P<0.01), but there had no significant effect to females of these proteins. The above results suggest that adult male rats synaptic proteins and NMDA receptors sensitive to BPA, the results with the BPA caused by consistent synapse formation and structural change.Conclusion:Adulthood long-term exposure to low doses of BPA can gender selective damage of male rats learning and memory behavior and negative change in synaptic plasticity of neurons, synaptic proteins and NMDA receptor expression levels may participate in the above process.