Effect Of Dextromethorphan And Riluzole On Neurotoxicity Induced By Methyl Mercury

IntroductionMethylmercury(MMC)is a kind of neurotoxicity of environmental pollutants. It can be harmful to human through the food chain by biomagnification.The main part of where it damages are occipital lobethe brain's(calcarine visual hub area) and the cerebellum. It causes the adult memory loss as well as the children a shortage of language and memory abilities.At present, the studies on the mechanisms of methylmercury neurotoxicity were mostly used to be on the immediate-early genes,peptides and monoaminergic transmitters. Glutamate is the most important excitatory amino acid in mammalian brains.Some studies showed that there were some relationships between methylmercury chloride and the disfuction of glutamate metablism and transporters. However, the mechansims about it are still unknown.Although there is no glutamate metablism-related enzyme in synaptic cleft, glutamate is converted to glutamine by glutamine synthesis.The high-affinity glutamate transporters in astrocytes play a crucial role in the re-uptake of glutamate and maintaining the balance of glutamate metabolism. Therefore, the neurotoxicity caused methylmercury is likely to effect the function of the transporters as well as the content and activity of glutamine synthetase.in the meantime, some studies have shown that excessive glutamate can induce Ca2＋influx and Ca2＋overload can stimulate the astrocytes to bring more active oxygen. Thus, there is some relationship between glutamate metabolism and oxidative damage which result from methylmercury.Dextromethorphan is an antagonist of non-competitive NMDA receptor.It showed that dextromethorphan can prohibited the NMDA receptors from excessive activation by glutamate and inhibit calcium influx Riluzole is a sodium ion channel inhibitors and the inhibitor of glutamate release.The mechanisms of riluzole may be related to the promotion of glutamate uptake and the enhance of its integration with the glutamate transporters. However,the applications of dextromethorphan and riluzole to resist the nerve damage caused by methylmercury chloride have been reported rarely.The in vivo and in vitro experiments were used to determine the effects of dextromethorphan and riluzole respectively on the methylmercury chloride-induced neurotoxicity of cerebral cortex and the disturption of astrocytic glutamate metablism and transporters after methylmercury chloride exposure.Materials and Methods1. In vivo animal experimentsThe weight of 40 wistar rats from the Experimental Animal Center of China Medical University is 180±10 g.The number of female and male is the same.They were fed under the temperature of 17-23℃, relative humidity 45-55%.Before the experiment,they were fed one week and then were divided into 5 groups by weight at random, 8 animals for each group. The first group was the control group and the second and third groups were methyl-mercuric chloride (MMC) groups. The fourth and fifth groups were DM and riluzolegroup respectively.All the former three groups were subcutaneously (sc) injected with 0.9% NaCl.The fourth and fifth groups were sc injected with 13.5μmol/kg DM and 21.35μmol/kg respectively every other day. Two hours later, the animals in the control group were intraperitoneally(ip) given the injection of 0.9%NaCl, the second group was injected with 4.0μmol/kg MMC and from the third to the fifth groups were injected with 12.0μmol/kg MMC. The administration of MMC above was given five times a week, lasting 4 weeks as well as the administrati-on of DM and riluzole every other day before injected with MMC.24 hours after the last injection, 8 rats in each group sacrificed after paralyzed.Observe the contenrs of Hg, glutamate, glutamine and Malondialdehyde(MDA) as well as the activities of glutamine synthetase(GS), phosphate activated glutaminase(PAG), Superoxide Dismutase(SOD)and glutathione peroxidase(GSH-Px) in the rat cortex when they were injected the increasi-ng MMC dosage, DM and riluzole.2. In vitro primary astrocytesPrimary cultured astrocytes isolated and cultured from cerebral cortex of newborn (1 day old) Sprague-Dawlery rats.The cells would be used after more than 95% of the cells stained positively for the astrocytic marker glial fibrillary acidic protein (GFAP) immunocytochemically.Cellular proliferation activity was detected by MTT method. we studied the cytotoxicity of methylmercury chloride to astrocytes and the effects of riluzole on disfunction of glutamate metabolism and transporters.And we chose the proper dosages and time during the experiments.The damage of fibroblasts was observed with inverted phase contrast microscope.This research made use of the RT-PCR to analyze the expression of GS, GLT-1, EAAC1 mRNA and the Western blotting to analyze the expression of GS,GLT-1, EAAC1 protein.The changes of intensity of mRNAs and proteins were normalized using the intensity obtained in the internal control bands(G3PDH andβ-actin respectively).3. Statistical analysisMean and standard deviation were calculated for all measurements. All statistical analyses were performed using the SPSS software, version 11.5. Data were analyzed using one-way analysis of variance followed by the Students-Newman-Keuls, SNK. Statistical significance was set at P<0.05.Results1. In vivo animal experimentsCompared with the control group, in MMC group, the contents of Hg, Glu and MDA increased. The content of Gln was decreased. Moreover, the activities of GS and SOD,GSH-Px of the rat cortex were decreased. On the contrary,the activity of PAG increased. Compared with 12.0μmol/kg MMC group, in riluzole group, the contents of Hg,Glu,and MDA fell down.The content of Gln rose. Moreover, the activities of GS and SOD,GSH-Px of the rat cortex.2. In vitro primary astrocytesCompared with the control group, the level of the damage of the morphology of astrocyte and the destruction of membranous integrity are deteriorating with the increaing doses of methylmercury chloride (0-40μM,4-24h).There was no significant difference of GSmRNA and protein expression among the methylmercury chloride (0-20μM) treated group. Nevertheless, the EAAT1 and GLT-1 mRNA and protein expression of the methylmercury chloride (0-20μM) decreased.Observe the effects of riluzole (50-200μM) pretreatment 4 to 24 hours after destruction of astrocytes caused by 20μM methylmercury chloride exposure in cell morphology. Compare with the chloride methylmercury (0-20μM) treated group, riluzole group GLAST, and GLT-1 mRNA and protein expression increased which was treated with the chloride methylmercury (0-20μM).Conclusions1.The rat model of MMC was perfectly established by intraperitoneally injection by methylmercury chloride.The research showed that MMC could be conveyed through 'blood-brain barrier', disturb the 'glutamate-glutamine cycle' and indued oxidative damage.2.By animal experiments,it was showed that dromethorphan and riluzole may have the certain antagonistic effect on exitotoxicity and oxidative damage of MMC.3.By primary astrocytes cultures, we found that MMC could cause cytotoxicity,and induced the disorders of glutamate metabolism and transporters.4.Riluzole may have the certain antagonistic effect on glutamate metabolism and transporters of astrocytes by methylmercury chloride exposure.