The Influence Of Bisphenol A On Androgen-Regulated Learning And Memory

Bisphenol-A (BPA) is an industrial chemical and is known to act as an endocrine disrupter, which is widely used in food and drink cans. It has been reported that BPA easily leaches from food packaging and dental sealants into saliva, dialysis to the surrounding ecological environment to endanger our health. BPA can alter steroid hormone synthesis and clearance, receptor expression and gene activity in the target tissue. Whereas earlier studies focused on the estrogenic properties of bisphenol-A. Our previous study found that exposure to BPA in perinatal, adolescent and adulthood sex-specifically impaired spatial memory and avoidance memory only in the male mice. Androgens are just as critical in the cognitive functions and synaptogenesis in males as estrogens are in females. Therefore, this study explores whether BPA sex-specifically impaired learning and memory is due to it interfered the regulation of androgen on learning and memory.Synaptic plasticity is closely related to learning and memory. It is well-known that gonadal hormones significantly affect cognition in human beings and laboratory animals through synaptic remodeling in the central nervous system (CNS), especially prefrontal cortex and the hippocampus. Reduced spine synapse density in area CA1 of gonadectomized (GDX) male rats can be reversed by replacement with either testosterone or the non-aromatizable androgen, DHT. The synaptic interface is a structure that is very sensitive to the chemical substance, the synaptic clefts, the length of active zone, the postsynaptic density and the synaptic interface curvaturecan be changed due to the stimulus. PSD is composed of many proteins such as cytoskeleton protein, scaffold proteins, receptors, calmodulin, ion channels and signaling molecules, the altered expression of these proteins can affect synaptic activity and plasticity, and also change the PSD of thickness.This study tested effects of BPA alone, and in combination with androgen, on learning and memory of GDX or gonadally intact adult male mice. We examined spine synapse formation and synaptic modification in the CA1 area of the hippocampus. Furthermore, Western blot analysis of the levels of PSD-95, synapsin I, NMDA receptor subunit NR2B and signaling pathway protein ERK1/2, p38 and phosphorylation of them to study the possible mechanisms of the effects of BPA on learning and memory and synaptic plasticity in male mice.The research methods:Eight-week-old male ICR mice were purchased from the Experimental Animal Center, Zhejiang Academy of Medical Science, and kept under standard laboratory conditions in a 12-h light,12-h dark cycle, feed room temperature control in 18-22℃, humidity from 50% to 60%, with free access to food and water. After acclimatization for 1 week, gonadectomy (GDX) or sham operation was performed under deep anesthesia with a Chloral hydrate (400mg/kg). Two weeks later, the mice were treated for 45 days, consisting of daily s.c. injections of testosterone propionate (TP,0.5 mg/kg/day), BPA (0.4,4 mg/kg/day), TP+BPA (0.4 or 4 mg/kg/day), or the sesame oil vehicle (50μL/day). Forty-five days later, fourteen mice from each group were received behavioral tests, and the rest were prepared for the hippocampal morphometric measurement (n=6) and western blot analyses of proteins (n=4) and 8 for the determination of serum and brain androgen levels.The data were expressed as the means±SEM and analyzed using SPSS 17.0 statistical software. Three-way repeated ANOVAs were used to assess surgery, treatment, and training (day 1-4) in Morris water maze. The rest data were analyzed by two-way ANOVAs. Multiple comparisons within significant interactions were performed with the Tukey’s HSD (Honestly Significant Difference) test. A statistical significance was accepted at p<0.05.The results of the study:1. GDX operation markedly decreased T level in the serum (p<0.001) and in the brain (p<0.001) when compared with vehicle-treated sham mice, whereas these decreases were completely reversed by TP treatment (p<0.001). BPA exposure (0.4, 4mg/kg/day) had no significant effects on T level in the serum and the brain of GDX mice, but significantly reduced T level in the serum (p<0.05;p<0.01) and in the brain (p<0.05;p<0.01) of sham mice and TP-treated GDX mice (p<0.05;p<0.05;p<0.01).2. Compared with vehicle-treated sham mice, GDX significantly prolonged the latency of male mice to locate the platform in training day 4 (p<0.05). TP supplement reversed GDX-induced extension of the latency (p<0.05), suggesting that androgens deprivation seriously impaired spatial memory. Exposure to BPA slightly extended the latency of locating the platform in GDX mice (p>0.05), but significant extended the latency of sham mice (4 mg/kg/day, p<0.01) and eliminated TP-induced shortening of the latency in GDX mice (0.4 mg/kg/day, p<0.05). BPA decreased the percentage of time in the target quadrant in sham (0.4 mg/kg/day, p<0.05) and eliminated the TP-treated reversion of that in GDX mice (p<0.001;p<0.01),but did not change that of GDX mice. These results suggest that BPA impaired spatial memory of sham mice and inhibited the rescue effect of androgen on GDX-induced spatial memory damage.3. GDX significantly decreased the numeric synaptic density in hippocampus CA1 of males (p<0.001), but that was reversed by TP supplement (p<0.001). Exposure to BPA decreased the numeric synaptic density of sham males (p<0.001; p<0.001) but did not change that of GDX males. Co-treatment of TP with BPA (0.4 or 4 mg/kg/day) inhibited TP-induced increase in the numeric synaptic density of GDX mice (p<0.01; p<0.001). GDX significantly shortened the length of active zone (p<0.01), thinned the PSD (p<0.001), and widened the synaptic cleft (p<0.001), but these changes were reversed by TP supplement (p<0.05; p<0.05; p<0.001). BPA exposure (0.4 and 4 mg/kg/day) had the similar effects on the structural parameters of the synaptic interface in sham and TP-treated GDX mice. These results suggest that BPA can inhibit androgen-induced synaptogenesis and synaptic structural modification of hippocampus.4. BPA, especially at 4 mg/kg/day, significantly decreased the expressions of synapsin I (p<0.05), PSD-95 (p<0.01), and NR2B (p<0.05) in sham mice. GDX significantly reduced the levels of these proteins, especially of PSD-95 (p<0.05) and NR2B (p<0.01), which was reversed by TP supplement (p<0.05; p<0.05). Co-treatment of BPA with TP inhibited the enhancement of TP on the level of these proteins (p<0.05; p<0.05), as well as synapsin I (p<0.01). Furthermore, we found that no significant changes in the levels of p38 and ERK1/2 were found in both GDX and treatment. However, BPA at 4 mg/kg/day significantly reduced phosphorylation of ERK1/2 (p-ERK1/2, p<0.05) but increased phosphorylation of p38 (p-p38, p<0.001) in sham mice. TP supplement reversed GDX-induced decreases of p-ERK1/2 (p<0.01) and increases of p-p38 (p<0.001), but BPA partially inhibited TP-induced enhancement of p-ERK1/2 (p<0.01) and reduction of p-p38 (p<0.01) in GDX mice. These results suggest that BPA inhibited androgen-enhanced levels of synaptic proteins and NMDA receptor subunit NR2B and changed the activity of MAPK/ERKs and MAPK/p38 signaling pathways in hippocampus.Conclusion:In conclusion, sex-specifical impairment of spatial memory and synaptic remodeling of hippocampal neurons in males after long-term BPA exposure is related to the anti-androgen effect of BPA. These effects of BPA may be associated with down-regulated synaptic proteins and NMDA receptor via inhibiting ERKs and promoting p38 pathways.