| This project is a part of the China National Scientific Supporting Program (Technical Research for Evaluating Combined Action of Pesticides or Endocrine Discrupting Substances)(2006BAK02A02).It firstly focused on study of chlorpyrifos (CPF) withdrawal-induced cytotoxicity after a period of exposure in hippocampal neurons,and tolerance of neurons to CPF,which will help to explain the mechanism of organophosphate induced chronic neurotoxicity(OPICN);It secondly focused on study of the effects of carbamate carbofuran pretreatment on organophosphorus CPF-induced cytotoxicity,and role of oxidative stress in this process,which will provide information about combined action of organophosphorus pesticide and carbamate pesticide.1.Chlorpyrifos withdrawal induces cytotoxicity in primary hippocampal neurons related to inhibition of ERK1/2 phosphorylation and cholinergic deficitOrganophosphate-induced chronic neurotoxicity(OPICN) usually delayed-occurs and perisists long,its mechanism is not very clear.In present study,the cytotoxicity and related mechanisms after CPF withdrawal were studied in primary rat hippocampal neurons.The results showed that 10μM CPF induced no detectable cytotoxicity during 96 h continuous exposure,but 10μM CPF withdrawal after 48 h exposure induced evident cytotoxicity,as indexed by decreased MTT metabolism, increased loss of neurons immunostained by neuron-specific enolase(NSE) antibody, and increased TUNEL positive cell rate in the following 24 h and 48 h incubation in the absence of CPF.AChE activity was inhibited by 10μM CPF during exposure,it slightly recovered after CPF withdrawal,suggesting CPF withdrawal-induced cytotoxicity is not direct consequence of inhibited AChE.ERK1/2 activation by phosphorylation was found,it persisted hours and then resumed to normal level during CPF exposure,while CPF withdrawal after 48 h exposure led to inhibition of ERK1/2 phosphorylation.Carbacol and NGF which activated ERK1/2 protected the neurons after CPF withdrawal,while atropine and PD98059 which inhibited ERK1/2 exacerbated the cytotoxicity after CPF withdrawal,indicating inhibition of ERK1/2 phosphorylation played critical role in CPF induced withdrawal-induced cytotoxicity. The effects of carbacol which is a cholinergic agonist,and atropine which is a cholinergic antagonist,also suggest cholinergic deficit involve in the mechanism of CPF withdrawal-induced cytotoxicity.In conclusion,10μM CPF shows noncytotoxic during 96 h exposure,but CPF withdrawal after 48 h exposure induces cytotoxicity in cultured neurons,which is not related to AChE inhibition,but is associated with ERK1/2 inhibition and cholinergic deficit.The results suggest CPF withdrawal-induced cytotoxicity is a kind of withdrawal effect,and OPICN may be related to withdrawal syndrome.2.Tolerance study of hippocampal neurons to CPFIn Part 1 study,the results showed non-cytotoxic CPF withdrawal after a period of exposure induced cytotoxicity,and suggested CPF induce withdrawal effect.Since tolerance is prerequisite of withdrawal effect,we studied whether CPF could induce tolerance in cultured hippocampal neurons to further elucidate the mechanism of CPF withdrawal-induced cytotoxicity.Method:the cultured hippocampal neurons were pretreated with 10μM CPF for 48 h,then,the neurons were exposed to high concentration of CPF(30,60,100μM CPF) for the following 48 h incubation,and cytotoxicity was measured.Result:the survival rate measured by MTT assay increased,the LDH leakage decreased,and Tunel positive rate decreased in noncytotoxic CPF pretreatment groups,compared with corresponding groups without CPF pretreatment.Conclusion:CPF pretreatment induces tolerance of neurons, confirming the cytotoxitiy after CPF withdrawal is related to withdrawal effect,and suggesting OPICN be related to withdrawal syndrome.3.Effects of carbamate carbofuran pretreatment on organophosphate chlorpyrifos-induced cytotoxicity in cortical neural cellsOrganophosphorus pesticides and cabarmate pesticides are used extensively in agriculture,resulting in pervasive human exposure,sequential or simultaneous exposure to the two kinds of pesticides leads combined action.To investigate the combined action of organophosphorus pesticide and cabarmate pesticide,in this study, the effect of carbofuran pretreatment on CPF-induced cytotoxicity and related mechanism were studied.Methods:primarily cultured cortical neural cells were exposed to various doses of carbofuran for 72 h,cytotoxicity was measured by MTT method,and a noncytotoxic level was determined.The cultured cells were pretreated with noncytotoxic level of carbofuran for different period,followed by exposure to various concentration of CPF(with CBF presence) for 48 h,and the cytotoxicity were measured.The effect of carbofuran pretreatment on CPF-induced ERK1/2 activation was examed by western blot assay.Results:10μM carbofuran was found nontoxic during 72 h exposure.10μM carbofuran pretreatment attenuated CPF-induced cytotoxicity,8 h carbofuran pretreatment showed the maximum effect;Carbofuran activated ERK1/2,and the ERK1/2 activation lasted shortly,while carbofuran 2-24 h pretreatment inhibited the CPF-induced ERK1/2 activation,which was corresponding to the cytotoxicity attenuation.While concomitant exposure of carbofuran (pretreatment time 0 h) and CPF induced no significant effect on CPF induced cytotoxicity and ERK1/2 activation.Conclusion:Carbofuran pretreatment for certain time antagonize CPF-induced cytotoxicity,which is related to the inhibition of ERK1/2 activation,but carbofuran does not antagonize the cytotoxicity induced by CPF when the two pesticides exposed simultaneously,suggesting the mode of combined action is affected by the exposure interval between the two pesticides.4.Effects of carbamate carbofuran pretreatment on organophosphorus chlorpyrifos-induced oxidative stress in cortical neural cellsIn part 3 study,10μM carbofuran pretreatment for 8 h antagonized the cytotoxicity induced by CPF,since oxidative stress is involved extensively in the toxicity of pesticides,we study the role of oxidative stress in the process of carbofuran pretreatment antagonizing CPF toxicity.Method:the neural cells were pretreated with 10μM carbofuran for 8 h,the oxidative stress indices MDA,SOD, GSH and GSH-PX were measured.After 8 h carbofuran pretreatment,the cells were treated with different concentration(20,40,60,80μM) of CPF for another 24 h,and the same indices as previous were measured.Result:Carbofuran treatment for 8 h inceased MDA level sightly but significantly,increased SOD and GSH-PX activity, and induced no obvious change in GSH level;CPF exposure inceased MDA level, and decreased SOD activity,GSH-PX activity and GSH level with a concentration—response mannner;CPF with carbofuran pretreatment decreased MDA level,and increased SOD activity,GSH-PX activity and GSH level,compared to that without carbofuran pretreatment.Conclusion:carbofuran pretreatment antagonizes CPF induced lipid peroxidative damage,the mechanism may be related to that carbofuran induces slightly oxidative stress and increases the anti-oxidative enzymes activity.Conclusion:1.Noncytotoxic concentration of CPF can induce tolerance of hippocampal neurons,and induces cytotoxicity after withdrawal,while ERK inhibition and cholinergic deficit are related to the mechanism of CPF withdrawal induced cytotoxicity,suggesting OPICN be related to withdrawal symptom.2.Carbofuran pretreatment can antagonize cytotoxicity induced by chlopyrifos in neronal cells,with the mechansim involving that carbofuran pretreatment reduces ERK1/2 activation and oxidative stress induced by CPF,suggesting the mode of combined action is affected by the exposure interval between the two pesticides.
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