| IntroductionPyrethroids (PD) are structural derivatives of pyrethrins that have greater potency and environmental stability. Pyrethroids are used in a wide array of indoor and outdoor applications, including veterinary, agricultural usages and home pest control. Pyre-throids act primarily on the nervous system. The commonly accepted mechanism-of-action of pyrethroids is disruption of excitatory neurotransmitter release, but the exact mechanism of neurotoxicity of PD is not well understood.Glutamate (Glu) is one of the most important neurotransmitter in the central nervous system. The glutamate-glutamine (Glu-Gln) cycle participates in cell metab-olism, and has important relevance to normal and pathological functions. When this cycle does not function properly, central nervous system functional damage can appear, and even cellular death can be produced. To accomplish the transformation of glutamate into glutamine, it depends on the activity of glutamine synthetase (GS) and phosphate-activated glutaminase in astrocytes in the brain. This is associated with correct function of the glutamate transporters-1 (GLT-1), to provide an adequate uptake, release and metabolism process of glutamate. Therefore, the importance of correct function of the glutamate-glutamine cycle during this detoxifying step in brain is clear. And the activity of GS and GLT-1 could be the target of neurotoxicity of PD.The major objective of the present study was to analyze the alterations to glutamate-glutamine cycle of brain in rat which was administrated with beta-cypermethrin (β-CP). It may be possible to understand more clearly the defense mechanism of the brain against the toxic substance. It is believed that the theoretical basis yielded from this experiment will be a great contritution to the prevention and treatment of pesticide poisoning in the future.Materials and methods1. Animal grouping and administration 40 healthy adult male and female Wistar rats were randomly assigned to control (1 group, n=10) and treatment groups (3 groups, n=10) and housed under standard laboratory conditions. Rats were fed a standard pellet diet and drinking water ad libitum. And the 4 dosages were 0,20,40 and 80mg/kg respectively. P-CP was dosed orally for once with edible salad oil as control in volume of 0.5ml per 100g body weight. The rats were subsequently euthanized by ether after 4 hours and the brain tissue were collected for neurochemical analysis.2. Observation and determination(1) Observation of symptoms:Continuously observed and recorded the symptoms after administration, including the emergence of time, poisoning and death of the performance.(2) Determination of glutamate and glutamine:Glutamate and glutamine were determined according to manufacturer's direction of kit.(3) Determination of activity of GS and the expression of GLT-1:1% homogenate was made from the brain tissue with cold saline solution. After ultrasonic treatment, we determined the activity of GS according to direction of the corresponding literature. Protein content was determined by using coomassie brilliant blue method. GS activity was expressed as nmolγ-gluta mylhydroxamate and per min-ute per milligram of protein.Total protein was extracted from 100mg brain tissue using RIPA reagent. The protein was transferred to polyvinylidene fluoride membrane, then incubated with the primer antibodies and the following antibodies, and photographed under a imaging camera analyzer compared with the ratio of P-actin gene expression as relative content.(4) Determination of expression of GS and GLT-1 mRNA:Total mRNA was extracted from 100mg brain tissue using TRIzol reagent. The reverse transcription-polymerase chain (RT-PCR) was performed to assess the mRNA levels of GS and GLT-1, as described by Takara RNA kit, then ran on 2% agarose gel electrophoresis and photographed under a gel imaging camera analyzer. Compare with the ratio ofβ-actin gene expression as relative content.3. Statistical analysisData were expressed as mean±standard deviation. Statistical analysis was performed by one-way ANOVA using SPSS13.0 statistical software. P＜0.05 is considered as significant.Results1. Observation of symptomsWe observed that all rats appeared the excitement symptoms of licking, anxiety and scratch after one hour, and then appeared the poisoning symptoms of salivation, convulsions, ataxia after two hours in high dose (80mg/kg). During the experiment a female appeared close to death. In the dose of 40mg/kg, individual rats appeared mild excitement after two hours. And in the dose of 20mg/kg, the rats had no change. No animals died during the entire experiment.2. The level of glutamate and glutamineβ-CP dose-dependently increased extracellular glutamate levels. The dose of 80 mg/kgβ-CP led to a marked elevation of glutamate release (P＜0.05). At the doses of 20, 40mg/kg it produced a mild but no insignificant increase (P＞0.05). There is no significant difference in glutamine among all groups (P＞0.05).3. The activity of GS and the expression of GLT-1We found that the activity of GS decreased in the brain of rats, as compared to control animals.β-CP dose-dependently decreased the activity of GS. A significant decrease was found in high dose (80mg/kg). The doses of 20,40mg/kg caused decrease, but there is no insignificant difference (P＞0.05). There is no significant difference in expression of GLT-1 among all groups (P＞0.05).4. The expression of GS and GLT-1 mRNAThere is no significant difference in GS and GLT-1 mRNA expression among all groups (P＞0.05).ConclusionsIn conclusion, this study shows thatβ-CP could interfere with Glu-Gln cycle of brain in rats, and increased the level of Glu. It showed that GS activity may be the target for neurotoxicity of PD.
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