A Study Of Chlorpyrifos-induced Oxidative Stress Toxicity To Early Embryonic Cerebrum Of Mice

As a kind of organophosphorus pesticide,chlorpyrifos(CPF)has been used widely in agriculture and construction industry for its low-price and broad effectiveness against most pests.It has been verified that CPF has neurodevelopmental toxicity,and the toxicity in offspring showed gender difference in adulthood.However,from when the gender difference began to show is still unknown.This study was aimed to investigate the oxidative stress toxicity of early prenatal CPF exposure on the embryonic brain and its gender differences.Embryos of ICR mice at Geststionsal 12 Days(GD 12)were used for experiment.Firstly,the morphological profile of the timed embryos was characterized,including external morphology,body and head weight of the embryo.We also did statistics of total numbers of embryos from each pregnant mouse and identified the gender of each embryo.Male-specific Sry gene on Y chromosome was used as the gender identification gene,and Myog on autosomal chromosome was used as the reference gene.We subsequently carried out CPF exposure experiment was subsequently carried out to investigate the reaction of the fetuses to oxidative stress.In detail,various doses of CPF(0,1,3,5 mg/kg body weight of pregnant mice)were subjected to pregnant mice from GD7 to GD11 daily by subcutaneous injections.The dams were sacrificed at GD12 by cervical dislocation,and embryos were then dissected and counted.Embryos were separated into body and head parts and measured separately.Body parts were used for gender identification,and head parts were used for detecting following indexes,including enzyme activity of ACh E,SOD,GPX,and CAT,MDA contents,and the expressing level of Sod1,Sod2,Cat,Gpx1,Gpx2 and Gsta1.Finally,data of each index were analyzed to determine whether difference to oxidative stress exist in embryos of different gender.Our results showed that limb structure and facial features are recognizable in GD12 fetuses.The average body weight of female and male embryos was 78.2±8.4mg and 76.6±8.8 mg,with the average head weight of 35.5±3.1 mg and 35.1±3.3 mg,respectively.The difference in body and head weight of males and females was not significant.The average number of embryos in each pregnant mouse was 12.3 and the ratio of females : males was 1.04:1.In the subsequent toxicological study,we found that CPF exposure during GD7-11 had no significant effect on the head and body weight of GD12 embryos.The activity of ACh E in the all treatment group was inhibited,and this inhibition was gradually enhanced as exposure dose increased.Statistical analysis showed a significant correlation between ACh E activity and CPF exposure dosage,indicating that under the exposure dose of 1 mg/kg/d to 5 mg/kg/d,CPF produced a toxic effect on fetal brain.The inhibition rates of ACh E activity in female and male fetal brain were 5.1% and 2.0% in the 1mg/kg/d group,14.5% and 8.9% in the 3 mg/kg/d group,and 20.4% and 18.9% in the 5 mg/kg/d group,respectively.Statistical analysis showed that there was no gender difference in the Ach E inhibition induced by CPF.In the case of activities of antioxidant enzymes functioned in oxidative stress reaction,we found that CPF exposure had a mild activation effect on SOD and GPX activity,while no significant effect on CAT activity was observed.MDA content was also not significantly affected.In comparison of the activity of the enzymes between males and females,no significant difference was observed either in control or treatment group.m RNA expression levels of genes related to oxidative stress in fetal brain were also investigated.Our results showed that CPF exposure during GD7-11 significantly increased the expression level of Sod1,Cat,Gpx1 and Gpx2 in fetal brain,and the increase was greatest at 3 mg/kg/d,both in females and males.The increase in gene expression fell back at 5 mg/kg/d,consistent with the change trend of the enzymes activity.In addition,no influence on Sod2 and Gsta1 gene expression was observed in the study.As of the difference in gene expression level between males and females,statistical analysis showed that neither control nor CPF treatment group showed significant difference.Taken above results together,exposure to CPF at early development stage could cause mild ACh E inhibition and oxidative stress in GD12 fetal brain,including activation of antioxidative metabolic enzymes and increase in the gene expression levels of relevant enzymes,while it does not affect fetal body or head weight significantly.In addition,no significant gender difference in the reaction of GD12 fetal brain to oxidative stress exist.Considering that the sexual gene expression was still in the start-up stage at GD12,the results of this study suggest that the neurodevelopmental toxicity of organophosphorus pesticides has not yet begun to produce gender differences.