| BackgroundBisphenols(BPs),the main raw material of polycarbonate plastics and epoxy resins,are widely produced and used.As a typical representative of BPs,bisphenol A(BPA)can be detected in a variety of environmental matrices and human biological samples.Epidemiology studies and animal experiments have demonstrated that BPA exhibits multiple toxicities,thus,strict regulations were established to restrict or ban the application of BPA in many countries.As a kind of important alternative of BPA,bisphenol AF(BPAF)is widely used in fluoroelastomers,food packaging materials,and electronic materials.Similar with BPA,human exposure to BPAF occurs through a variety of pathways,and BPAF can be detected in multiple human biological samples.Because BPAF is an emerging kind of chemical,there are few studies on its neurotoxicity at present.Except that few animal experiments focus on the neurobehavior injury of prenatal or perinatal BPAF exposure,in terms of its neurotoxicity and mechanism in adolescence,no information is available.Adolescence is a crucial developmental period during the lifespan,the nervous system is sensitive to environmental exposure during this stage.According to the World Health Organization(WHO),one in seven(14%)10-19-year-olds adolescents suffer from a mental disorder on a global scale.Therefore,it is important to explore the effects of BPAF exposure on neurobehavior in adolescents.In this study,the nested case-control study was used to explore the association between BPAF exposure and neurobehavioral injury at the population level.In vivo animal assay and in vitro cellular experiments were performed to confirm the neurotoxicity and to elucidate the mechanism of BPAF.The present study provides epidemiology evidence and a theoretical basis for further exploring the neurotoxicity of BPAF.Part 1:Associations between Serum Bisphenol AF Concentration and Risk of Depressive Symptoms in Junior High School Students:A Nested Case-control Study in ChinaObjectivesPopulation epidemical research was used to describe serum bisphenol AF(BPAF)level in junior high school students,and to explore the associations between serum BPAF concentration and depressive symptoms.MethodsOur nested case-control study was based on an ongoing longitudinal prospective adolescent cohort.The baseline survey and serum collection of this cohort were conducted in 2019,and the first wave of follow-up was conducted in 2020.At the first wave of follow-up,175 eligible cases who have new-onset depressive symptoms were regarded as the case group.The same number of controls were selected from the population without depressive symptoms in a ratio of 1:1 matching with gender and age(±1).The baseline serum BPAF concentration was detected by using Ultra Performance Liquid Chromatography-triple Quadrupole Mass Spectrometry(UPLC-MS/MS),and descriptive analyses were performed.Serum BPAF raw concentration and its logarithmic transformed values were used for analysis.The association between serum BPAF level and risk of depressive symptoms was determined using conditional logistic regression.sex-stratified analyses also were conducted.The dose-response relation between serum BPAF level and risk of depressive symptoms was determined using restricted cubic spline(RCS).ResultsIn this study,the detection rate of serum BPAF was 100%,the median(interquartile range,IQR)serum BPAF concentration was 5.24(4.41-6.11)pg/m L in the case group and 4.86(4.02-5.77)pg/m L in the control group,and the serum BPAF level of case group was significantly higher than that of the control group(P=0.009).Serum BPAF raw concentration was enrolled in the conditional logistic regression model.After adjustment for all confounding factors,the results showed that serum BPAF exposure was a risk factor of depressive symptoms(OR=1.132,95%CI:1.013-1.264).Logarithmic transformed values of serum BPAF was categorized into tertiles(corresponding to low-exposure,middle-exposure,and high-exposure groups)based on the distribution among controls and were enrolled in the conditional logistic regression model.After adjustment for all for confounders,compared with the low-exposure group,the highest BPAF concentration were associated with a 2.806-fold increased risk of depressive symptoms(OR=2.806,95%CI:1.188–6.626).Stratified analysis by sex revealed that males were more vulnerable to BPAF exposure than females.After adjustment for all for confounders,compare with the low-exposure group,the relative risk of depressive symptoms in the high-exposure group was 3.858(95%CI:1.118-12.535)for males,however,no significant association between BPAF exposure and depressive symptoms was found in females.RCS analysis was showed that there was a marked linear association between BPAF exposure and risk of depressive symptoms in the total population and males.ConclusionsThe population in this study widespread exposure to the lower level of BPAF.A significant positive association was found between serum BPAF level and risk of depressive symptoms.The association was significantly modified by sex,males were more vulnerable to BPAF exposure.Part 2:Effects of Adolescent BPAF Exposure on Neurobehavior and Neuroplasticity in MiceObjectivesAnimal experiments were used to further confirm the effect of adolescent BPAF exposure on neurobehavior,to explore the blood-brain barrier(BBB)permeability of BPAF,and to explore the effect of BPAF exposure on synaptic plasticity in hippocampus and prefrontal cortex.MethodsExperiment 1:Mice were treated with BPAF(0.34 mg/kg)for different times(10,30,60,120,and 240 min)at PND 56.Plasma and fresh brains were obtained to measure the concentration of BPAF in different time points by using UPLC-MS/MS and BBB partition coefficients(Kp,brain)of BPAF in different time points was calculated.Experiment 2:The adolescent mice were divided into four groups.The four groups were given 0 mg/kg/day,0.034 mg/kg/day,0.34 mg/kg/day and 3.4mg/kg/day(correspond to Ctrl,low-,medium-and high-dose groups)BPAF exposure by oral gavage for 28 consecutive days(PND 28-PND 56),respectively.After BPAF exposure was finished,the sucrose preference test,the open-field test and the Morris water maze test were performed.Experiment 3:The adolescent mice were divided into four groups.The four groups were given 0 mg/kg/day,0.034 mg/kg/day,0.34mg/kg/day and 3.4 mg/kg/day(correspond to Ctrl,low-,medium-and high-dose groups)BPAF exposure by oral gavage for 28 consecutive days(PND 28-PND 56),respectively.After BPAF exposure was finished,the fresh brain was obtained and its coefficient was calculated.The morphology and synaptic plasticity in hippocampus and prefrontal cortex were detected by using HE stain,Nissl stain,and Golgi-Cox stain.The synaptic ultrastructure in hippocampus and prefrontal cortex was detected by using transmission electron microscopy,and related proteins in hippocampus and prefrontal cortex were detected by using western blotting.ResultsConcentrations of both free and total BPAF in brain and plasma reached the maximal levels after 30 min of BPAF treatment.The blood-brain barrier(BBB)partition coefficients(Kp,brain)of both free and total BPAF reached the maximal levels after 60 min of BPAF treatment,and the corresponding values of Kp,brainwere 8.79 for free BPAF and 0.50 for total BPAF,which suggested that free BPAF penetrates the BBB more readily.BPAF did not significantly impacts the body weight,food and water intake throughout the duration of the study.No obviously changed on brain and hippocampus weight and their coefficient after BPAF exposure.The result of sucrose preference test demonstrated that all dose BPAF exposure obviously decreased the sucrose consumption rate.The results of open-field test demonstrated that total distance of moving,distance on the central zone,central residence time,number of central zone entries,and number of zones crossing were significantly decreased in all BPAF groups compared to the control group.The results of Morris water maze test demonstrated that the swimming velocity of mice was not changed after BPAF exposure,however,all dose BPAF exposure significantly increased the escape latency and swimming distance,however,decreased the target quadrant residence time.The results of HE and Nissl staining demonstrated that all dose BPAF exposure could alter the neuronal morphology in hippocampus and prefrontal cortex of mice and reduced the number of surviving neurons in hippocampal CA1 region and prefrontal cortex of mice.The results of Golgi-Cox staining demonstrated that all dose BPAF exposure could reduce the dendritic arborization and complexity in hippocampal CA1 and DG regions and reduce the dendritic spine density in hippocampal CA1 region,DG region,and prefrontal cortex of mice.BPAF exposure could also damage the synaptic ultrastructure in hippocampus and prefrontal cortex of adolescent mice.The results of western blotting demonstrated that adolescent BPAF exposure obviously downregulated the expression of both PSD-95 and Synapsin-1 proteins in hippocampus and prefrontal cortex of mice compared with the control group.Adolescent BPAF exposure did not affect ER?and ER?proteins in hippocampus,however,the expression level of ER?protein in prefrontal cortex in the median-and high-dose BPAF groups were significantly increased.ConclusionsIn summary,our study demonstrated that free BPAF was more likely to crosses the BBB and accumulate in brain.Adolescent BPAF exposure induced depression-like and anxiety-like behavior and damaged cognitive function of mice.BPAF exposure damaged dendritic complexity,dendritic spine density,and synaptic ultrastructure and downregulated the expression levels of PSD-95 and Synapisin-1 in hippocampus and prefrontal cortex of mice.Taken together,BPAF crossing the BBB damaged neuronal morphology and neuroplasticity,which contribute to neurobehavioral impairment.Part 3:Role of ROS-mediated PERK/ATF4 Signaling Activation in BPAF-induced Neurobehavioral ImpairmentsObjectiveIn vitro and in vivo experiments were performed to investigate the role of ROS-mediated PERK/ATF4 pathway activation in BPAF-induced neurobehavioral impairments.MethodsIn vivo experiment:The adolescent mice were divided into four groups.The four groups were given 0 mg/kg/day,0.034 mg/kg/day,0.34 mg/kg/day and 3.4 mg/kg/day(correspond to Ctrl,low-,medium-and high-dose groups)BPAF exposure by oral gavage for 28 consecutive days(PND 28-PND 56),respectively.After BPAF exposure was finished,the fresh brain was obtained.The oxidative stress indicators(MAD and SOD)in brain tissue were detected.The Bdnf m RNA expression level in hippocampus and prefrontal cortex was detected by using RT-q PCR.The levels of p-CREB,CREB,BDNF,p-PERK,PERK,p-e IF2?,e IF2?,ATF4,NOX4,HO-1,and SOD2 proteins were detected by using western blotting.In vivo experiment was consisted of four parts.Experiment 1:SH-SY5Y cells were treated with BPAF(25?M)for different times(0,6,12 and 24 h).At the end of treatment,the cells were harvested.The BDNF m RNA expression level in SH-SY5Y cells was detected by using RT-q PCR.The expression levels of p-CREB,CREB,BDNF,p-PERK,PERK,p-e IF2?,e IF2?,and ATF4 proteins were detected by using western blotting.Experiment 2:SH-SY5Y cells were treated with BPAF(25?M)for different times(0,2,6 and 12 h).At the end of treatment,the cells were harvested.The ROS level in SH-SY5Y cells was detected by using DCFH-DA.The expression levels of NOX4,HO-1,and SOD2 proteins were detected by using western blotting.Experiment 3:GSK2606414(GSK),a kind of PERK activity inhibitor,was used in this part.SH-SY5Y cells were divided into four groups,namely control group,GSK-treated group,BPAF-treated group,and GSK+BPAF-treated group.At the end of treatment,the cells were harvested.The BDNF m RNA expression level in SH-SY5Y cells was detected by using RT-q PCR.The expression levels of p-CREB,CREB,BDNF,p-PERK,PERK,p-e IF2?,e IF2?,and ATF4 proteins were detected by using western blotting.Experiment 4:NAC,a kind of antioxidant,was used in this part.SH-SY5Y cells were divided into four groups,namely the control group,NAC-treated group,BPAF-treated group,and NAC+BPAF-treated group.At the end of treatment,the cells were harvested.The ROS level in SH-SY5Y cells was detected by using DCFH-DA.The BDNF m RNA expression level in SH-SY5Y cells was detected by using RT-q PCR.The expression levels of NOX4,HO-1,SOD2,p-CREB,CREB,BDNF,p-PERK,PERK,p-e IF2?,e IF2?,and ATF4 proteins were detected by using western blotting.ResultsThe result of RT-q PCR showed that BPAF exposure significantly downregulated the expression level of BDNF m RNA in hippocampus,prefrontal cortex,and SH-SY5Y cells.The western blotting analysis revealed that BPAF exposure significantly suppressed the BDNF and p-CREB proteins expression and upregulated PERK/ATF4 signal expression in hippocampus,prefrontal cortex,and SH-SY5Y cells.GSK obviously alleviated BPAF-activated PERK/ATF4 signal and significantly reversed the reduced expression levels of BDNF and p-CREB proteins by BPAF exposure.BPAF exposure leaded imbalance between pro-oxidant and antioxidant systems and resulted in oxidative damage in hippocampus and prefrontal cortex.BPAF exposure could also significantly increase ROS production in SH-SY5Y cells.NAC obviously alleviated BPAF-elevated level of ROS,inhibited the activation of PERK/ATF4 signal,and then reversed the reduced expression levels of BDNF and p-CREB proteins by BPAF exposure.ConclusionsBPAF exposure induced neurobehavioral impairment via ROS-mediated PERK/ATF4 signal activation downregulated CREB/BDNF pathway.SummaryOur epidemiological studies found that adolescent bisphenol AF exposure increase the risk of depressive symptoms in male junior high school students in a sex dependent manner.Animal experiments found that adolescent bisphenol AF exposure can lead to the impairment of neurobehavior in mice.Mechanism research found that BPAF exposure induce depression-like behavior through inducing oxidative stress,activating PERK/ATF4 signaling,inhibiting CREB/BDNF pathway,ultimately leading to the dendritic arborization and complexity impairment in hippocampus and prefrontal cortex. |
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