| Chlorpyrifos(CPF)is one of the most widely used organophos phorus pesticides in the world.It has environmental persistence and accumulation.Acute high-dose CPF poisoning is mainly manifested as cholinergic symptoms caused by the accumulation of acetylcholine in the synapse.In recent years,studies have found that chronic low-level CPF exposure(no visible symptoms of poisoning to the naked eyes)may have long-term effects on the structure and function of the nervous system.Our research group has been committed to the study of the neurotoxic mechanism of chronic low-level CPF exposure,and it has been found that CPF associated with lipopolysaccharide can cause long-term spatial memory impairment in rats,and neonatal rats exposed to low levels of CPF are significantly more likely to develop anxiety and amnesia in adulthood.And it has also been found that low-level exposure to CPF can induce neuroinflammation in the substantia nigra and amygdala of the neonatal rats through the HMGB1/TLR/NF-κB signaling pathway;PINK1/Parkin-mediated autophagy can alleviate the apoptosis of neurons induced by CPF.This study will further explore the neurodevelopmental toxicity of CPF on the basis of previous studies.Analyze the impact of CPF exposure on synaptic plasticity in vivo and in vitro;and then use a cross-sectional survey of children in rural China to explore the relationship between CPF exposure in children and the common neurodevelopmental disabilities attention deficit hyperactivity disorder(ADHD).And try to find possible protective factors for ADHD.Part 1 Low-level exposure to chlorpyrifos affects the synapticplasticity and Wnt/β-catenin pathway in hippocampal neurons Objective: The aim of this study was to investigate the effects of CPF on synaptic plasticity in hippocampal neurons and to explore the cellular mechanism under them.Methods: Establish a neonatal SD rat model with low-level exposure to CPF.Observe the effect of CPF exposure on the neurons and their synaptic morphology in various regions of the hippocampus by HE staining.The effect of CPF exposure on the expression level of postsynaptic protein(PSD-95)was detected by immunofluorescence.The primary hippocampal neuron model exposed to low-level CPF was established.Neurons were identified by cellular immunofluorescence on the 6th day;the changes in cell viability of primary hippocampal neurons were measured by CCK-8;the changes in cell and synapse morphology between the experimental group and the control group were observed by an inverted microscope;Through Western-Blot and RT-PCR to detect the expression levels of neuronal presynaptic protein(Synaptophysin),postsynaptic protein(PSD-95),ion glutamate receptors(NMDAR1,Glu R1)and Wnt7a/β-catenin pathway after CPF exposure.Lentiviral transfection was carried out on the 6th day of in vitro culture.After the exposure to CPF,the expression levels of presynaptic protein(Synaptophysin)and postsynaptic protein(PSD-95)in the overexpressionβ-catenin transfection group and the empty virus transfection group were detected by Western-Blot.Result: 1.In vivo,after exposure to CPF,the soma of neurons in the hippocampus of rats decreased slightly,the synapses became irregular,and the expression of postsynaptic protein PSD-95 in the hippocampus decreased significantly.2.In vitro,After exposure to low level of CPF,there were no significant changes in the number and viability of neurons in the primary hippocampal neurons.The synaptic network of neurons could still be formed,but the synapses of neurons were shortened after exposure to CPF.The connections between the cells become sparse.The expression level of the protein and m RNA of Syp and PSD-95 in hippocampal neurons after CPF treatment was significantly decreased(P<0.05),and the expression level of the protein and m RNA of NMDAR1 and Glu R1 in synaptic excitatory glutamate receptor was also significantly decreased(P<0.05).3.The results showed that the expression of WNT7 A and activeβ-catenin decreased significantly after low-level CPF exposure(P<0.05),but the expression of presynaptic and postsynaptic proteins and related glutamate receptor did not increase after over expression of β-catenin in primary hippocampal neurons(P>0.05).Summary: This study shows that low-level exposure to CPF can cause changes in the synaptic morphology of hippocampal neurons,and affect the expression levels of synaptic proteins and excitatory synaptic receptors in hippocampal neurons.On the other hand,low-level exposure to CPF will affect the Wnt7a/β-catenin pathway,but overexpression ofβ-catenin does not improve the effect of CPF on pre-synaptic proteins,post-synaptic proteins and synaptic receptors.Part 2 The relationship between micronutrients and the risk of ADHD in children exposed to chlorpyrifos Objective: To investigate the relationships between urinary CPF levels,blood micronutrient levels,and ADHD risk in children living in rural areas of China.And try to find out feasible protective factors.Methods: According to the geographical features of China,this study selected three counties which represent mountain,hill and plain district respectively.CPF in soil and water was measured in 3 villages randomly selected from each county,and CPF was measured by mass spectrometry in urine of all children aged 1-6 years old in the villages.ADHD was assessed using the diagnostic criteria of “Diagnostic and Statistical Manual of mental disorders” in the United States,and sociological and demographic data were collected using questionnaires.The concentrations of micronutrients(zinc,iron,calcium,copper,magnesium,lead,and 25-hydroxy vitamin D)were measured in peripheral venous blood.SPSS21.0 was used for statistical description and single-factor and multiple-factor regression analysis.Mplus 8.0 was used to do the path analysis.P <0.05 was statistically significant.Results:1.152 soil samples and 148 water samples were tested for chlorpyrifos around the environment where children lived.It was found that 95 soil samples(62.5%)and 55 water samples(37.%)could detect chlorpyrifos residues.2.A total of 738 children met the selection criteria in the cross-sectional survey.A total of 65 people did not agree to the survey or the children were unable to cooperate due to various reasons.673children(673/738,91.2%)were eventually included in the study,and all675 people were involved.A basic questionnaire survey and urine CPF test were conducted in 673 people.672 people collected blood samples,and 651 people completed the ADHD assessment.Though the result of mass spectrometry of the CPF in children’s urine,we found that there were 144 children exposed to low level CPF,accounting for 21.4%(144/673).And the rate of ADHD among the 651 children was 7%(45/651).3.Through logistic analysis,we found that CPF exposure,family income,and the level of vitamin D are all influencing factors of ADHD.4.CPF had a direct effect on ADHD risk(estimate = 0.197,P <0.05)as well as an indirect effect via vitamin D levels(estimate =-0.031,P <0.05).The mediating effect of urinary CPF levels on ADHD risk via vitamin D was 18.67%.Summary: 1.Chinese rural children aged 1-6 years are potentially exposed to the residual CPF in their living environment.2.The higher the exposure level of CPF in children,the higher the risk of ADHD is;the higher the vitamin D level in micronutrients,the less the risk of ADHD is.3.Increase vitamin D level may reduce the risk of ADHD caused by CPF exposure.Conclusion1.Low-level exposure to CPF affects the the proteins associated with synaptic plasticity and the Wnt7a/β-catenin signaling pathway in hippocampal neurons,which suggest that CPF affects synaptic plasticity and Wnt/β-catenin signaling pathway may be one of the possible mechanisms.2.Chinese rural children aged 1-6 are exposed to the living environment where CPF remains,and CPF exposure is one of the risk factors for ADHD.3.High vitamin D levels in micronutrients are a protective factor for ADHD in children,and can reduce the risk of ADHD due to CPF exposure. |
PDF Full Text Request