| Purpose Exploring the toxicity of low-dose organophosphorus pesticide chlorpyrifos (CPF) and its metabolites chlorpyrifos oxon(CPO) on cerebral cortex neurons of rat. Investigating secure use of pesticides among rural children’s guardians so that providing new idea for protecting children from pesticides.Methods1. Primary cerebral cortex neurons of fetal and astrocyte of neonatal SD rat have been cultured. Cellular immunofluorescence is used to identify the purity of neurons and astrocyte. Colorimetry is used to describe cells growth curve. The cells in logarithmic phase of high purity are selected. Firstly, exploring the sensibility of neurons and astrocytes to dimethyl sulfoxide(DMSO), which is the menstruum of chlorpyrifos and its metabolite.0.5%,1.0%,5.0%,10.0%DMSO are added to primary neurons, while0.25%,0.50%,1.00%.5.00%,10.00%DMSO are added to primary astrocytes. Control group use equal-dose culture medium instead. The concentration and acting time are determined by observation of cell morphology, calculation of live cells and measurement of O.D value at the time of12h,24h,48h.2. Add5μM、20μM、80μM、100μM、400μM chlorpyrifos and its metabolite chlorpyrifos oxon to primary cortex neurons and astrocytes. Control group use equal-dose DMSO instead. At the time of12h,24h,48h, observating cell and nucleus morphology, calculating live cells and use cell counting kit-8(CCK-8) measure O.D value of formaza, which is the metabolite of water-soluble tetrazolium-8(WST-8)in mitochondria.3. The number of526children’s guardians are chosen as object in Xinhua of Hunan province through the method of cluster random sampling. The questionnaire are including condition of family and behavior of secure use of pesticides. Software of epiData3.1is used to set databas. SPSS19.0is used to statistic analysis. Results1. Successfully cultured cortex neurons of fetal rat and astrocyte of neonatal SD rat. The purity of primary neurons is92.7%±3.1%, of astrocyte is95.1%±1.2%. Cells which are in logarithmic phase can be used to examination.2. There is no difference between group0.5%DMSO and control in the number of cells and cell morphology. Neurons morphology changes in group1.0%DMSO at12h, and aggravation can be observed at24h and48h. When more than1.0%DMSO are added to neurons, decrease of the number of cells and O.D value is significantly by increase of concentration(P<0.05). At the same concentration, with the extension of time, the number of neurons and O.D value are decrease, meanwhile, morphological change significantly(P<0.05). Almost neurons have been dead with exposure to10.0%DMSO for48h.3.0.25%DMSO intervention to48h did not see number and morphology of astrocytes change obviously. Concentration of0.50%,1.00%DMSO with astrocyte24h,48h, more intense, the increase in the number of cells adhered to the upper material particle samples, cell morphology has no obvious change, significantly enhance the cell vitality (P<0.01).5.00%DMSO24h after intervention, the decrease in the number of astrocytes, cell in the body cavity, the majority of cell shrinkage, vision there were more cell residue, as the extension of time and the increase of concentration of the above change, the vigor significantly reduced (P<0.01).4. Compared with control group, there are no significant differences with number, morphological, and O.D value of neurons with exposure to5μM、20μM CPF for12h,24h,48h. At the concentration of80μM, although there is no significant change with number and morphological, O.D value descend27%compared with12h group (P<0.05). With the increase of concentration higher than80μM, number of nuerons has no obvious change, but deformation of nucleus are more and O.D value descend significantly (P<0.05). At the same concentration of CPF, morphlogical of neurons have changed and O.D value reduce significantly P<0.01)5.5μM、20μM chlorpyrifos processing each time point are not seen the number of astrocyte with a significant change.80μM chlorpyrifos processing after48h astrocytes decreased (P<0.01), occasional intracytoplasmic vacuoles formation and deformation of nuclei.100mu M chlorpyrifos processing48h, astrocytes decreased, more cells formed vacuoles in cytoplasm and cell shrinkage, with the increase of concentration and the extension of time, the number of astrocyte continue to reduce, most cell shrinkage, nuclear deformation and energy decreased significantly (P<0.01).6. Conmpared with control group, there are no obviously change with neurons’numnber, form and O.D value with5μM、20μM CPO exposure for each point of time. At the concentration of80μM for48h, although there is no significant change with number and morphological, O.D value descend22%compared with12h group. When the concentration of CPO is more than80μM, O.D value decrease significantly with the increase of concentration and the extension of time (P<0.05). The decrease of number has been observed at the concentration of400μM for48h.7. CPO under80μM concentration of chlorpyrifos three time points after processing are not seen astrocytes quantity, morphology and dynamic change obviously. Greater than100microns oxidation chlorpyrifos dealing with24h, with the increase of concentration and the extension of time, the number of astrocyte significantly reduced (P0.01), cell aggregates, free cell in the body bubble increase, deformation of nuclei increased, activity decreased significantly (P<0.01).8. The O.D value of CPF group is significantly lower than CPO group when the concentration is more than100μM for48h (P<0.01)9. Chlorpyrifos concentration in more than80μM exposure each point in time, astrocytes O.D values are lower than the same concentration and time of oxidation of chlorpyrifos exposure group (P0.05).10. The left-behind children’s guardian’s secure use of pesticides score is (12.1±2.6), while the control group is (17.3±1.5)(P<0.01).Conclusion1. Chlorpyrifos and oxidation of chlorpyrifos on the original generation of cultured fetal rat cortex neurons and newborn rat cortical astrocytes have toxic effects, performance on the cell morphology, quantity and the influence of dynamic, and the toxic effect in a certain concentration range (80μM or higher) is dose dependent and time, with the increase of the concentration and the extension of time, the decrease in the number of cells, the morphological change of rising rates of cells, cell viability decreased.2. With the same condition, chlorpyrifos and oxidation of chlorpyrifos on the original generation of astrocyte morphology, quantity and the influence of dynamic more neurons is more apparent, prompt astrocytes may be its toxic effect of target cells.3. Chlorpyrifos metabolism for the oxidation of chlorpyrifos on the original generation of neurons and astrocytes after the toxic effect of weakening.4. At room temperature, using dimethyl sulfoxide as solvent of organophosphorus pesticides, the concentration of the role in the generation of fetal rats cortex neuron is less than0.5%, the role of the original generation of newborn rat cortical astrocytes concentration less than0.25%.5. Investigation of secure use of pesticides based on the view of children’s guardians indicate that left-behind children guardians’action are substandard, so that there is hidden danger with the children’s safety and health. |
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