Phenanthrene Induces Autism-like Behavior In Mice By Promoting Oxidative Stress And MTOR Pathway Activation

Phenanthrene(Phe)makes up a relatively high proportion of the low-ring polycyclic aromatic hydrocarbons(PAHs).It can enter the human body through bioaccumulation and other effects,and can affect human health.Autism is a generalized developmental disorder,which is thought to be associated with environmental and genetic factors.Studies have explored the toxic effects of polycyclic aromatic hydrocarbon mixtures on the neurodevelopment of young mice,but there is no published study focusing on the singular effect of phenanthrene on autism-like behavior,nor on the underlying mechanisms.In this study,we selected 3-week-old male Kunming mice to be adaptively reared and exposed to three concentrations of phenanthrene for 22 days.To explore the underlying mechanism,rapamycin(RAPA)was used to block the mTOR pathway,and vitamin E was used to inhibit oxidative stress.The experimental animals were divided into 7 groups with 10 animals in each group.(1)Control;(2)0.005 mg/kg/d Phe;(3)0.05 mg/kg/d Phe;(4)0.5 mg/kg/d Phe;(5)2 mg/kg/2d RAPA;(6)2 mg/kg/2d RAPA+0.5 mg/kg/d Phe;(7)100 mg/kg/d Vitamin E+0.5 mg/kg/d Phe.The open field test and the three-chambered social test were used to explore whether phenanthrene can cause autism-like behavior in mice.Observe the pathological changes of the mouse brain through brain pathological tissue sections.In addition,oxidative stress related indicators such as reactive oxygen species(ROS),reduced glutathione(GSH)and malondialdehyde(MDA)in mouse brain tissue homogenate were detected.We use fluorescence quantitative PCR to detect the expression levels of autism-related proteins such as Shank3 and Nlgn-1.The phosphorylation level of Akt and mTOR were analyzed by ELISA assays and immunohistochemistry.The transcription level of eIF4E was analyzed by fluorescence-based quantitative PCR.We also used quantitative analysis of the expression levels of autophagy-related genes Beclin1 and the expression levels of inflammatory factors such as tumor necrosis factor α(TNFα).Results:(1)A certain concentration of phenanthrene can affect the neurobehavioral and social activities of mice.Exposure to phenanthrene induced a marked decrease in the activity of the mice in the central area in the open field test(p<0.01),and caused a decrease in communication with unfamiliar mice,in the three-chambered social test(p<0.05).(2)The hippocampal tissue of a mouse exposed to Phe showed dose dependent pathological changes such as cells gradually becoming sparsely arranged.(3)Exposure to phenanthrene induced an increase in the levels of ROS(p<0.01)and a decrease in levels of glutathione(p<0.05).Expression of inflammatory factor TNF-α was enhanced in the brains of exposed mice(p<0.05).(4)Exposure to phenanthrene caused a significant decrease in the expression levels of the autism-related proteins Shank3(p<0.01)and Beclin1(p<0.05).(5)Exposure to phenanthrene can activate the mTOR pathway,and induced a significant increase in the transcription level of eIF4E(p<0.01).(6)Administering Rapamycin or vitamin E,inhibited oxidative stress and the activation of the mTOR pathway induced by Phe exposure,The level of ROS and MDA decreased(p<0.05),and the phosphorylation level of mTOR decreased(p<0.05).And they can alleviate autistic-like behaviors in exposed mice.Conclusion:Oral exposure to a certain concentration of phenanthrene will affect the brain development of mice,leading to autism-like behavior.The underlying mechanism involves oxidative stress and the mTOR pathway.This will disrupt the activity of related synapses and the development of related neurons and autophagy,causing abnormal brain development in mice and causing changes in the expression of autism-related proteins.It then leads to autism-like behavior in mice.