Exposure To Nonylphenol During Gestation And Lactation Causes Deficits In Social Behavior And Stereotyped/Repetitive Behavior In Offspring And The Possible Mechanisms

Objective: Nonylphenol(NP),a typical environmental endocrine disrupting chemicals(EDCs),is a class of strong surfactants that have been widely applied in cosmetics,household chemicals,leather and textile lining,and plastic products.Many lines of evidence have indicated that administration of NP induced long-term behavioral abnormalities such depression-like behaviors,cognitive impairment,abnormal ultrasonic vocalizations(USV),and anxiety-like behaviors,which are the most common psychiatric comorbidities found in most cases of autism spectrum disorder(ASD).Social behavior defects and stereotyped repetitive behaviors are the main behavioral manifestations of ASD.Recent evidence suggest environmental factors contributes up to 40-50% of variance in etiology and pathogenesis of ASD.Pregnancy and lactation are critical stages of neurodevelopment.Studies have shown that exposure to environmental pollutants during this critical period can cause serious nervous system damage.However,it is not clear whether exposure to NP during pregnancy and lactation lead to social behavior defects and repetitive / stereotyped behaviors in offspring.Therefore,in this study,typical social deficits and repetitive / stereotyped behaviors induced by gestational single valproic acid(VPA)treatment,a widely accepted ASD model,was defined as a positive control group.Furtherly,the impacts of neurobehavior on offspring following gestational and lactational exposure to NP were observed.To elucidate the possible mechanism,we explored the morphology of prefrontal cortex and cerebellar neurons and the related pathways.Methods: Pregnant rats were intragastrically administered with corn oil vehicle or NP in corn oil at a dose of 0,10,50 mg/kg/day from E0.5 to PN21.On E12.5,pregnant dams were administered 600?mg/kg VPA sodium salt resuspended in physiological saline or physiological saline by single intraperitoneal injection.From childhood to adolescence(PN24-40),the autistic-like behavior of male offspring was tested to observe whether there was autistic-like neurobehaviour in male offspring.The density of dendritic spines in medial prefrontal cortex(layer II/III)was observed by Golgi staining on PN42(adolescence)and PN80(adulthood).The synaptic morphology and mitochondrial morphology in the prefrontal cortex of male offspring at different stages were observed by transmission electron microscopy.Immunofluorescence staining was used to observe the expression levels of postsynaptic density-95(PSD-95),SHANK2,SHANK3,4-Hydroxynonenal,and endoplasmic reticulum stress marker protein BIP in medial prefrontal cortex neurons(layer II/III).Adenosine triphosphate(ATP)kit was used to detect ATP levels in medial prefrontal cortex.Immunoblotting was used to detect the expression levels of PSD-95,SHANK2,SHANK3,NFE2L2,cytochrome C and endoplasmic reticulum-related proteins.The primary cortical neurons of SD male rats were cultured in vitro and transfected with e GFP plasmid to observe the effects of different doses of NP on dendritic spines.Phenyl-N-tert-butylnitrone(PBN)and Mito TEMPO were used to observe the effect of oxidative stress on dendritic spine density in cortical neurons induced by NP treatment.The endoplasmic reticulum stress inhibitor 4-phenylbutyrate(4-PBA)was used to observe the effect of endoplasmic reticulum stress on dendritic spine density of cortical neurons induced by NP treatment.The effects of PBN,Mito TEMPO and 4-PBA on oxidative stress induced by NP treatment were determined by reactive oxygen species assay kit.The effects of PBN,Mito TEMPO and 4-PBA on endoplasmic reticulum stress induced by NP treatment were determined by immunofluorescence.Results: 1.Perinatal exposure to NP led to deficits in social interaction and social novelty preference in offspring.In the sociability session(III phase),offspring from control,NP-10,and NP-50 group spent significantly more time with the unfamiliar sex-matched rat(Stranger 1)than with the empty wired cup.However,the significantly different duration between the Stranger 1 and the empty wired cup was not exhibited in offspring exposed to VPA.Offspring exposed to 50 mg/kg NP or VPA spent less time on sniffing the stranger 1 than that in control group.The preference index based on chamber time in NP and VPA treatment groups were significantly reduced compared with that in the control group.In the session of social novelty preference(? phase),offspring in control group exhibited normal social novelty preference,as they spent more time interacting with the novel rat(Stranger 2)than with the familiar one(Stranger 1)2.Perinatal exposure to NP led to stereotyped,repetitive,and anxiety-like behaviors in offspring.The significant increase of grooming time and number of repeated entries were displayed by pups subjected to NP and VPA treatment compared with those in control in self- grooming task and T maze spontaneous alternation task.For marble burying task,the offspring exposed to 50 mg/kg NP and VPA treatment groups buried significant more marbles than that in control group.Offspring from 50 mg/kg NP and VPA treatment groups spent obvious less time in central zone and exhibited fewer center entries compared with that in the control group,suggesting anxiety-like behavior.3.Perinatal exposure to NP induced neuronal dendritic spine loss in m PFC of offspring.50 mg/kg NP and VPA treatment caused a reduction in total spine density of apical and basal dendrite in offspring compared to that in control group on PN42 and PN80.The total spine density of apical dendrite on PN80 was also significantly decreased in 10 mg/kg group,however,the reductions in total spine density of apical and basal dendrite on PN42 and basal dendrite on PN80 were not significant.4.Perinatal exposure to NP altered synaptic ultrastructure and protein expression of PSD-95 in m PFC of offspring.Thickness of post synaptic density in the 50 mg/kg NP-treated group was significantly reduced than those found in the control group on PN42 and PN80,respectively.The levels of PSD-95 was significantly downregulated in offspring from 50 mg/kg NP treatment group compared with that in control group on PN42 and PN80,respectively.The offspring from NP treatment groups showed obvious decrease of PSD-95 expression in neuron of m PFC compared to control offspring on PN42 and PN80,respectively.5.Perinatal exposure to NP decreased expression of SHANK2 and SHANK3 in m PFC of offspring.A significant downregulation of SHANK2 was found in offspring of the NP and VPA treatment groups on PN42 and PN80,compared with the control group.Pups exposed to 50 mg/kg NP and VPA showed lower SHANK3 levels than those in control group on PN42 and PN80.Significant decreases in levels of SHANK3 were observed in PFC of the pups in 10 mg/kg NP treatment groupcompared with that in the control group on PN80.Immunofluorescence results suggested all NP and VPA treatment groups tend to decrease in SHANK2 and SHANK3 levels in neuron of m PFC compared with control offspring on PN42 and PN80,respectively.6.Perinatal exposure to NP impaired mitochondrial morphology and induced oxidative stress in m PFC of offspring. TEM results shown several morphological abnormalities in NP and VPA treatment group on both PN42 and PN80.The pups in NP and VPA treatment groups also showed significant reduction in ATP content compared to control group on PN42 and PN80.The level of lipid peroxidation was obviously increased in offspring subjected to NP and VPA treatment compared to control group on PN42 and PN80.The offspring from 50 mg/kg NP and VPA treatment group showed significantly increased expression of NFE2L2 and cytochrome C compared to control group on PN42 and PN80.7.Perinatal exposure to NP leaded to activation of ER stress signals in m PFC of offspring.Immunoblotting results indicated that the levels of BIP,PERK,phosphorylated PERK,phosphorylated IRE1?,and ATF6 was significantly increased in offspring from 50 mg/kg NP and VPA treatment group compared with controls on PN42 and PN80.Immunofluorescence staining suggested that the positive BIP staining in neuron of m PFC was much stronger in the NP and VPA treatment groups than control group on PN42 and PN80.8.Role of oxidative stress in NP-induced neuronal dendritic spine reduction.In vitro results showed that treatment of NP(50,70 and 100 ?M)significantly reduced the dendritic spines of neurons.Antioxidants PBN and ito TEMPO significantly reduced the increase of ROS and the decrease of dendritic spines induced by NP.9.The role of endoplasmic reticulum stress in dendritic spine reduction induced by NP.Cellular immunofluorescence showed that compared with the control group,the fluorescence intensity of BIP is increased significantly after NP exposure.Antiendoplasmic reticulum stress drugs 4-PBA and antioxidants PBN and ito TEMPO significantly reduced the neuronal endoplasmic reticulum stress and dendritic spine reduction induced by NP.10.Perinatal exposure to NP impairs dendritic growth of cerebellar PCs in offspring.The total length of dendrites in pups subjected to NP treatment significantly decreased compared with that in the control group on PN21 and PN80.The 50 and 100 mg/kg NP treatment groups showed significantly decreased numbers of intersections compared with that in the control group on PN21 and PN80.The dendritic branch points of the PCs significantly decreased in all NP treatment groups compared to those in the control group on PN21 and PN80.The number of dendritic terminal ends in all NP treatment groups significantly decreased compared with the control group on PN80.11.Perinatal exposure to NP increases PKC? phosphorylation in cerebella of offspring only on PN21.A significant up-regulation of phospho-PKC? was found in offspring of the 50 and 100 mg/kg groups on PN21,compared with the control group.Phospho-PKC? levels were not significantly different between the NP treatment groups and the control group on PN80.Total expression of PKC? did not differ among any of the four groups on PN21 or PN80 12.Perinatal exposure to NP decreases phosphorylation of stathmin in cerebellar PCs of offspring.Immunoblotting results indicated phosphorylation of stathmin significantly decreased in all NP treated groups compared to the control groups on PN21 and PN80,respectively.The expression of total stathmin was not significantly different among the four groups on PN21 or PN80.Immunofluorescence staining suggested that the positive phospho-stathmin staining in Purkinje cell layer was much weaker in the NP treatment groups than that in the control group on PN21 and PN80 13.Perinatal exposure to NP decreases BDNF expression in cerebellar PCs of offspring.Immunoblotting results indicated the 50 and 100 mg/kg NP treatments resulted in significant decreases in BDNF expression in offspring cerebella compared to that in the control groups on PN21 and PN80,respectively.Immunofluorescence staining suggested all NP treatment groups showed obvious reductions in BDNF expression in cerebellar PCs compared with control pups on PN21 and PN80,respectively.14.Perinatal exposure to NP decreases phosphorylation of TrkB in cerebellar PCs of offspring.Immunoblotting results indicated the levels of phospho-TrkB were downregulated in pups subjected to 50 and 100 mg/kg NP treatment compared with that of the control group on PN21 and PN80.The expression of total TrkB was not significantly different among the four groups on PN21 or PN80.Immunofluorescence staining suggested NP treatment led to a visible reduction in phospho-TrkB in cerebellar PCs compared with control pups on PN21 and PN80,respectively.Conclusion: 1.Perinatal exposure to NP resulted in mild but specifically social behavior defects and repetitive / stereotyped behaviors in male offspring.2.Abnormal alteration of dendritic spines and synaptic morphology in the medial prefrontal cortex induced by perinatal exposure to NP may be associated with the specifically social behavior defects and repetitive / stereotyped behaviors in male offspring.3.Perinatal exposure to NP led excessive oxidative stress,endoplasmic reticulum stress,impairment of mitochondria.4.The decrease of dendritic spines due to endoplasmic reticulum stress mediated by oxidative stress,which was induced by NP treatment.5.Perinatal exposure to NP irreversibly inhibited dendritic growth of PCs in the cerebella of offspring,which may be involved in the specifically social behavior defects and repetitive / stereotyped behaviors.6.The irreversible damage to PC dendrites in the cerebella of offspring subjected to perinatal NP exposure may be due to increased stathmin activity mediated by BDNF-TrkB signaling.