Effect Of Tris(?-chloroethyl)phosphate On Learning And Memory In Mice And Its Underlying Mechanism

Tris(P-chloroethyl)phosphate(TCEP)is an excellect flame retardant and plasticizer.To effectively improve the safety performance in products,TCEP is widely used in various living products,such as furniture,electronic products,food packaging bags and building materials,etc.As an additive,because of the weaker combination of non-covalent manner with material,it can enter into the environment by abrasion and volatilization and has a certain environmental persistence.It can be intaken into body through breathing,diet and other ways,then enriches in the organisms.Studies have shown that TCEP had the potential toxicity and induce oxidative stress,endocrine disruption,and had certain carcinogenic,reproductive and developmental toxicity.In addition,some evidences showed that when the rats were exposed to high doses of TCEP for a long time,the neurons were loss in CA1 area of the hippocampus.The hippocampus is closely related to learning and memory function.However,the effect of TCEP on learning and memory and its mechanism is not clearly.In this study,we choosen male Kunming mice as experimental subjects through gastric infusion to study effect of TCEP on learning and memory ability.Firstly,mice anderwent two different dose TCEP treatment for consecutive three weeks,75mg/kg TCEP group and 150mg/kg TCEP group respectively;TCEP+TBSA group was given 20mg/kg Tubastatin A(TBSA)cotreatment after 150mg/kg TCEP treatment for one week;The control group was given solvent treatment of the two drugs.Then we used Morris water maze,Novel object recognition and Electrical step-down to detect the the effects of TCEP on learning and memory abilities.We further test the potential molecular mechanism by Western blotting and Immunohistochemistry.The results showed that:(1)After the treatment at a dose of 75 mg/kg,150 mg/kg TCEP and 20 mg/kg TBSA,there was no acute reaction and significant change in body weight among the four groups.(2)In the Morris water maze test:the escape latency was prolonged at 75 m/kg TCEP group compared with the control group(p<0.05 for day 2;p<0.05 for day 5);and 150 mg/kg TCEP group was significant prolonged(p<0.05 for day 4;p<0.001 for day 5);the escape latency was significantly shorter(p<0.01 for day 4;P<0.05 for day 5)at TCEP+TBSA group compared with 150mg/kg TCEP group.In the space exploration phase,the time spent in the target quadrant and number of platform area crossing at 150 mg/kg TCEP group were significantly reduced(p<0.05,respectively)compared with the control group.Swimming trajectories for looking for hidden platform at the two different dose TCEP treatment groups are aimless and messy.(3)In the novel object recognition test:the total exploration time was slightly increased at 150 mg/kg TCEP group compared with the control group(p<0.05);the time exploring the novel object was significantly decreased at 75 mg/kg TCEP group and 150 mg/kg TCEP groups compared with the control group(p<0.05 respectively).(4)In Eelectrical step-down test:Compared with the control group,the step down latency were significantly lower at 75 mg/kg TCEP group and 150 mg/kg TCEP groups(p<0.05 respectively),while the error times increased significantly(p<0.05 for day 2-5 respectively).(5)We first examined the effect of Akt/GSK-3?signaling pathway by immunohistochemistry and found that there was no change in the each total proteins,but their phosphorylation levels had some differences.Compared with the control group,the p-Akt(Ser 473)was significantly lower in these two TCEP groups(75 mg/kg TCEP group,p<0.05;150 mg/kg TCEP group,p<0.001)and p-GSK-3(3(Ser 9)was significantly decreased at 150 mg/kg TCEP group(p<0.05),and indicating that TCEP regulates Akt/GSK-3? pathway through phosphorylation.(6)There was no significant difference in HDAC6 protein level among the four groups.However,the protein level of ac-tubulin,a deacetylated substrate of HDAC6,was significantly decreased in the mice hippocampus at 150 mg/kg TCEP group(p<0.05)compared with the control group.We further examined the microtubules of neuronal axonal in hippocampal CA1 pyramidal neurons by immunohistochemistry,and revealed that the microtubules at the control group were long and tidy;and 75 mg/kg TCEP group were fewer,shorter,and cluttered;150 mg/kg TCEP group were short and dotted,and suggesting that the TCEP treatment resulted in the depolymerization of microtubules to being shortened,and even disintegratied.(7)Synaptic protein expression closely related to memory in the hippocampus:compared with the control group,Synapsin-1 was significantly decreased(75 mg/kg TCEP group,p<0.05;150 mg/kg TCEP group,p<0.05);PSD-95 was significantly decreased at 150 mg/kg TCEP group(p<0.05).We further observed the protein-rich area in the hippocampus by immunohistochemistry.Compared with the control group,Synapsin-1 had lighter staining in CA1 and CA3 area at 75 mg/kg TCEP group and 150mg/kg TCEP group,and PSD-95 showed lighter staining in CA3 region,indicating that the protein expression in the region is less after TCEP treatment.The staining was deeper at TCEP+TBSA group compared with 150 mg/kg TCEP group,indicating that these proteins expression increased in this area.The above results indicate that TCEP treatment can impaired spatial learning and memory,novel recognition memory and emotional memory in mice.The TBSA treatment significantly ameliorate the memory impairment by TCEP in the above behavioral changes.Further biochemical date suggested Akt/GSK-3? pathway,HDAC6,microtubule stability and synaptic proteins play a potential role in cognitive impairment induced by TCEP.This study provides a certain experimental basis for the impairment of learning and memory related to central nervous system induced by TCEP,and offer the references for the application of Tubastatin A in preventing the congitive disorders caused by TCEP.