| Arsenic(As)is a naturally occurring metalloid,it is also an environmental toxicant.i Ascontaminated drinking water has become one of the most public health-threatening problems.Chronic exposure to As via drinking water is known to cause multiple adverse health conditions,including neurodevelopmental disorders.Autism spectrum disorder(ASD)is a diffuse central nervous system(CNS)developmental disorder caused by multiple environmental factors and genetic factors.It is typically characterized by difficulties with communication and social interactions,restricted interests,and stereotyped repetitive behaviors.ASD is also generally accompanied with anxiety-like behaviors,learning and memory deficits,depression,attention deficits and other symptoms.The prevalence of ASD is increasing year by year,but the precise etiology remains unclear,and there is no cure for ASD by far.It has become a significant cause of mental illness in children,and ASD accounts for substantial burden to their families and the social.Therefore,we should deeply study the risk factors closely related to ASD,especially the role of environmental risk factors in the progression of ASD,and then take preventive measures.It is of great significance for ASD prevention and treatment.In recent years,emerging epidemiological studies support that As acts as a kind of environmental risk factor of ASD.However,more direct evidence of the involvement of developmental exposure to As in autism-like behaviors should be provided and further studies of the underlying mechanisms are needed.Objective: A mouse model of As exposure via drinking water during pregnancy and lactation was established to explore whether exposure to As via drinking water during pregnancy and lactation caused autism-like behaviors in male offspring.Furthermore,we observed the changes in the number and morphology of axons to clarify whether exposure to As via drinking water during pregnancy and lactation affects the growth and development of hippocampal neuron axons in the offspring.In addition,we analyzed the protein expression changes of AKT / GSK-3β / Kinesin-1 cascade to explore the relevant molecular mechanisms that cause changes in autism-like behaviors.Methods:1.The pregnant mice were randomly assigned into five different groups and exposed to different concentrations of As through drinking water(0.5 ppm,5 ppm,20 ppm,50 ppm).The dams were treated with i As-contaminated water from GD 0 to PND 28 and allowed to raise their own offspring until weaning at PND 28.In addition,male offspring were selected to enter the study.2.Levels of As species(i As,MMA,DMA and TMA)in urine and brain were determined by an atomic absorption spectrophotometer with an As speciation pretreatment system.3.To study the relationship between As exposure and autism-like behaviors,a battery of animal behavioral experiments in the laboratory setting were used to observe whether such exposure can lead to autism-like behaviors in offspring.Ultrasonic vocalizations test and Three-chamber social preference test were used to observe whether exposure to As via drinking water during pregnancy and lactation damages the social skills and preference for social novelty in male offspring.Self-grooming,Marble burying test and T-maze spontaneous alternation test were used to evaluate the behavioral changes of repetitive restriction in male offspring.Open field test was used to observe whether exposure to As via drinking water during pregnancy and lactation causes anxiety-like behaviors in male offspring.Furthermore,the Morris water maze test was used to observe whether exposure to As via drinking water during pregnancy and lactation damages the learning and cognitive ability in male offspring.4.Immunofluorescence staining was performed on the progeny hippocampus to observe axonal morphological changes.Western blot was used to detect the levels of axon associated neurofilament light chain(NFL).5.Western blot was used to detect the levels of axon associated protein AKT,p-AKT Ser 473,GSK-3β,p-GSK-3β Ser 9,KHC,KLC1.Results: 1.Effects of As exposure during pregnancy and lactation on body and brain weights of offspring.There were no significant differences in the body weights between control and Astreated pups.Furthermore,no noticeable changes in the weights of cortex,hippocampus and cerebellum were observed in offspring.2.Effects of As exposure during pregnancy and lactation on As levels in urine,cortex, hippocampus and cerebellum of offspring.On PND 28,the levels of t As,i As,MMA,and DMA in the urine of pups subjected to high dose As treatment(5 ppm,20 ppm and 50 ppm As treatment)were significantly higher than that of the control group(P< 0.05).Furthermore,the levels of t As,i As,MMA,and DMA in the urine of As-treated pups on PND 56 were significantly lower than that on PND 28,and there was no significant difference between the As-treated group and the control group.On PND 28,the levels of t As in the cortex,hippocampus and cerebellum of pups subjected to high dose As treatment were significantly higher than that of the control group(P < 0.05).Furthermore,the levels of t As in the cortex,hippocampus and cerebellum of As-treated pups on PND 56 were significantly lower than that on PND 28,and there was no significant difference between the As-treated group and the control group.3.Effects of As exposure during pregnancy and lactation on communication and social interaction of offspring.The results of Ultrasonic vocalizations(USV)test analysis showed that total calling number and times of the control group were higher than those of the 5 ppm,20 ppm and 50 ppm groups(P < 0.05).Additionally,the 20 ppm and 50 ppm As treatments resulted in significant increase in latency to first call of offspring compared to that in the control group,suggesting that high dose As exposure decreased the ability of communication in offspring.Three-chamber social preference test showed that the total social time of pups subjected to high dose As treatment was significantly lower than that of the control group(P < 0.05).In the session of social interaction(Ⅲ phase),the pups in the control group spent more time on the room(R1)where the unfamiliar sex-matched mouse(Stranger 1)compared with the pups in high dose groups(P < 0.05).These results suggest that high dose As treatment damages the ability of communication and social interaction of offspring.4.Effects of As exposure during pregnancy and lactation on social novelty preference of offspring.In the third phase of Three-chamber social preference test,there was no significant difference between the staying time of the As treated pups in the R1 with S1 and the staying time in the R2 with the empty cage.Furthermore,in the session of social novelty preference(Ⅳ phase),there was no significant difference between the staying time of the high dose As treated pups in the R1 with S1 and the staying time in the R2 with the novel mouse(Stranger 2).In addition,there is no significant difference between the time of the pups in the 20 ppm and 50 ppm groups communicating with S2 compared with the time of communicating with S1.These results suggest that high dose As treatment can cause the deficit of social novelty preferences in offspring.5.Effects of As exposure during pregnancy and lactation on repetitive behaviors of offspring.The significant increase of grooming time and number of repeated entries were displayed by pups subjected to high dose As treatment compared with those in control in self-grooming task and T maze spontaneous alternation task(P < 0.05).In marble burying task,the As-treated pups buried more marbles than that pups in control group.These results suggest that high dose As treatment can cause the repetitive stereotype behaviors in offspring.6.Effects of As exposure during pregnancy and lactation on anxiety-like behavior of offspring.The open field experiment was used to evaluate the anxiety-like behaviors of the offspring,and the results showed that the pups of the high-dose As treatment group spent obvious less time in central zone and exhibited fewer center entries compared with that in the control group,suggesting anxiety-like behaviors(P < 0.05).7.Effects of As exposure during pregnancy and lactation on spatial learning and memory ability of offspring.The results of Morris water maze test showed that time spent in platform quadrant and platform zone entries of the pups in high-dose exposure groups were significantly less than that in control group(P < 0.05),and the latency of pups in high dose As treatment groups to reach the platform was longer than that in the control group(P < 0.05).8.Effects of As exposure during pregnancy and lactation on axonal development in hippocampus of offspring.On PND 28,the results of immunofluorescence showed that the length and density of axons in the hippocampal CA1,CA3 and DG areas of the pups in As-treated groups were less than those in the control group.What’s more,on PND 56,the axonal length and density of the pups in As-treated groups didn’t return to normal levels. Western blot results showed that the expression of NFL in the hippocampus of pups in 5 ppm and 20 ppm group was significantly lower than that in the control group(P <0.05).9.Effects of As exposure during pregnancy and lactation on the AKT / GSK-3β / Kinesin-1 cascade in hippocampus of offspring.Perinatal exposure to As decreased the levels of p-AKT Ser 473,p-GSK-3β Ser 9,KHC,KLC1(P <0.05).Besides,no noticeable changes in the levels of AKT and GSK-3β were observed in As-treated offspring.Conclusion: 1.Perinatal exposure to As leads to a series of autism-like behaviors in male offspring.2.Perinatal exposure to As disturbs AKT / GSK-3β / Kinesin-1 cascade and leads to the impairment axon development in male offspring.This damage may be the molecular mechanism of autism-like behaviors. |
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