| Growing studies showed EAAs was involved in Organophosphrus Pesticides (Ops) Poisoning, cerebral ischemia, toxic encephalopathy and degenerative nervous disorder, such as Parkinson's disease. The synthesis, release and elimination of EAA in brain are complicated process, in which "Glutamate-glutamine cycle" and NMDA receptor pathway all are the key point. So, further exploring the alteration of EAAs in rat cortical brain caused by dimethoate and verifying its role in Ops-caused neurobehavioral impairments will be helpful to clarify non-cholinergic mechanism in Ops Poisoning.Besides, although numerous studies focus the link between Ops and neurobehavioral disorder, sensitive and specific biomarker still need to be further explored. Neuron specific enolase(NSE), Glial fibrillary acidic (?)rotein(GFAP), Calcium-binding (?)rotein S1OOβare brain-specific (?)rotein which located in neuron and astrocytes. In recent years, they were often used in the diagnosis of acute brain injury or disease, so they are called brain injury-related (?)rotein. It was reported that the increase of NSE or S100βin blood serum or cerebrospinal fluid highly correlated with the severity and prognosis of disease. In this study, feasibility of NSE, GFAP, S100βbeing biomarker of neurotoxicity caused by dimethoate was investigated in vivo and in vitro, which will help diagnosis and prognosis of neurobehavioral disease resulting from Ops.In a single dimethoate administration (38.9mg/kg·bw,83.7mg/kg·bw and 180.0mg/kg·bw by gavage) to SD rats experiment,24h later, compared with control group, mRNA transcriptional level of muscarinic cholinergic receptor M1 decreased while that of NR1 increased (P<0.01); mRNA transcriptional level of cortical NSE, MAP-2, GFAP and S100B has no statistical significance differences with control group level.After 13 weeks of repeated oral exposure to dimethoate (5mg/kg·bw,10mg/kg·bw and 20mg/kg·bw by gavage), five days a week except weekends, compared with control group, muscarinic cholinergic receptor M1 transcriptional level decreased to 61.2% of control level (P<0.05) in low dosage group; GS activity decreased while mRNA level of NR1/NR2B increased in middle and high dosage groups (P<0.01); NSE transcription level increased in middle and high dosage groups (P<0.01); MAP-2 mRNA level increased in three dosage groups (P<0.05, P<0.05, P<0.01); GFAP, S100B mRNA level extremely increased in high dosage group. After pretreated with 0.05mg/kg·bw and 0.1mg/kg·bw MK801 to 10.0mg/kg·bw dimethaote(D10) for 30 min, transcriptional level of NR1 and GS activity increased in high dosage intervention group compared with D10 group and control group (P<0.01); mRNA level of four brain-specific protein decreased with dose-dependent manner compared with D10 group (P<0.01)Primary pure-cultured cortical neuron was administered 0,10-6M,10-5M, 10-4M dimethoate for 48h, compared with control group, concentration of EAA increased in high dosage group; ROS in neuron increased to 2.1,2.28 and 2.47 fold of control level in three dosage groups (P<0.01); intensity of TUNEL staining was 1.18,1.36 and 1.40 fold of control level (P<0.01),which means apoptosis increased with dose-dependent manner; transcription level of NSE increased to 1.053 and 1.048 fold of control level in low and middle dosage groups (P<0.01) and 1.35 times of control level in high dosage group (P<0.01). After treated with 50μM and 100μM MK801 to 10-4M dimethoate group, concentration of EAA decreased in both intervention group (P<0.01); the level of ROS decreased to 88.9% and 74.8% of 10-4M dimethoate group (P<0.01), while still higher than that in control group (P<0.01); transcription level of NR2B increased to 1.59 and 2.22 fold of 10-4M dimethoate group (P<0.01) and has big difference with control group(P<0.01); mRNA level of NSE decreased to 75.7% and 73.2% of 10-4M dimethoate group (P<0.01) and has no difference with control level; Protein level of NSE decreased to 73.4% and 72.8% of 10-4M dimethoate group (P<0.01) while extremely down to the control level (P<0.01), which suggest neuron death showing up.With pure-cultured cortical astrocytes,10-6M,10-5M,10-4M dimethoate was treated for 48h, there is no significant difference in GS activity, transcription level of NR2B and GLT-1 compared with control level; mRNA level of GLAST decreased to 67.8%,68.6% and 76.2% of control level (P<0.05); Concentration of EAA decreased compared with control group in high dosage group (P<0.01); The transcription and protein level of GFAP and S100B increased with dose-dependent manner (P<0.01) After intervened by 50μM and 100μM MK801 to 104M dimethoate group, mRNA level of GLT-1,GLAST increased in two intervention group(P<0.01), but the increase has no difference with control level; Transcription level of NR2B increased with no difference compared to 10-4M dimethoate group while which was higher than control level with statistically significance in two intervention group (P<0.05, P<0.01); Concentration of Glu increased by 1.81 fold of 104M dimethoate group in high dosage intervention group (P<0.01); Transcription and protein level of GFAP decreased but still higher than control level; S100B protein level in low doasage intervention group increased obviously compared with control group (P<0.01)With neuron and glail cell mix-cultured nerve cell model, AChE activity, ChAT activity, GS activity and EAA concentration decreased in 10-6M,10-5M, 10-4M dimethoate group (P<0.01); mRNA level of NR2B increased by 1.82 fold of control level (P<0.05) in high dosage group; NSE protein level increased by 1.46,1.60 and 1.98 fold of control level (P<0.01) in three groups; Transcription level of GFAP and S100B increased to 1.97 and 2.15 fold of control level in high dosage group (P<0.05); GFAP and S100B protein level increased obviously in middle and high dosage group (P<0.01). After given 50μM and 100μM MK801 to 10-4M dimethoate group, mRNA level of NR2B decreased to 65.5% of 10-4M dimethoate group level in high dosage intervention group (P<0.05); Concentration of Asp and Glu increased 2.35 and 1.78 fold in both intervention groups compared with 10-4M dimethoate group level (P<0.01) while no difference with control level in high dosage intervention group; Protein level of NSE decreased to 96.2% of 10-4M dimethoate group level which is significantly higher than control level in high dosage intervention group(P<0.01); Transcription level of GFAP decreased to 88.8% and 83.4% of 10-4M dimethoate group in both MK801 group (P<0.01, P<0.01) and has no statistically difference with control level; Transcription and protein level of S100B decreased in both MK801 group compared with 10-4M dimethoate group (P<0.01)The results of this study which based on animal and cell culture model demonstrated that dimethoate has effect on EAAs through disturbing related metabolism enzyme and NMDA receptor pathway, and perturbed NSE, GFAP,S100B transcription and protein level, finally leading to impairments of rat spatial learning and memory. After intervened by MK801, which blocks NMDAR pathway, alteration of EAAs almost back to control level, and NSE, GFAP, S100B transcription and protein level also decrease or increase to control level, meanwhile, data from animal study showed MK801 improved neurobehavior disorder caused by dimethoate. This suggested that central EAAs disorder contribute to neurotoxicity of dimethoate; the transcription and protein level alteration highly correlated with rat cognitive impairment which suggest they can be neurotoxic biomarker when treated with dimethoate; intervention of MK801 has positive effect to neurotoxicity caused by dimethoate, but its clinic use still need to be further explore.
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