Subchronic Arsenic Exposure On Cerebellum Neurotoxicity And Study On Its Mechnism

Background and ObjectiveArsenic (As) is a metalloid that ubiquitously exists in both organic and inorganic form in the environment, and environmental exposure of As has become a global health problem affecting millions of people. In As-contaminated areas, As concentration in drinking water or groundwater ranges from 0.25 to 2.1 ppm and even reaches> 3.0 ppm in some severely contaminated areas. The three most affected areas in the world are Bangladesh, India and China. The exposed population of As overed 3 millions in china, which is not including the pollution of coal-smoke.It has been reported that As cause a wide array of adverse health effects including respiratory, gastrointestinal, hematologic, hepatic, renal, dermic, immunologic effects and neurologic effects. Epidemiological studies revealed that chronic exposure to inorganic As via drinking water resulted in a dose-dependent reduction in intellectual functions in children. In animals exposed to As, deficits in learning tasks as well as behavioral alterations have been observed. It indicated that As produced neurotoxicity to central nervous system including impairment in learning and memory. It was reported that As exposure caused defects in cerebellum. Our previous study also showed that As inflicted damage to cerebellar neurons. It was found that the concentration of As was significantly higher in cerebellum of the exposed mice subchronically than that in control group and increased in a dose-response manner. It indicated that As can accumulate in cerebellum and the accumulated level of As may be responsible for its neurotoxicity to cerebellum. Some literatures have documented that cerebellum contributes not only to motor function but also to some sensory, cognitive, linguistic, and emotional aspects of behavior. The researches above indicated that cerebellum may be involved in the impairment of learning and memory by As. However, the molecular mechanisms by which As affects adversely learning and memory through disturbing the function of cerebellum have been poorly understood. It has been reported that many neurotoxicants such as lead, aluminum and fluoride etc. can disturb gene expression of important receptors or signaling molecules of brain, and the impaired expression of these genes might be involved in neurotoxicity including the impairment of learning and memory induced by them. This suggests that the genes encoding many proteins expressed may be targets of neurotoxins. However, studies on toxic effect of As on expression of genes in the cerebellum are very limited.The microarray technology, which are used widely on area of the biological, allows for the simultaneous measurement of the gene expressions on a genomewide scale。The use of microarray could reveal the possible biological mechnisms of neurotoxicity, through analysis the gene expression after a stimumate of environment toxin. Therefore, it is important to analysis the gene expression profile of the cerebellum, for exploring the possible mechanism of arsenic-induced neurotoxicity.In this study, we chose adlut mice as study model, each of them were exposed to arsenic through drinking water for two month. To screening the differentially expressed genes in the mice cerebellar exposed to As, the expression profiles of genes in cerebellum of mice exposed to As were analyzed by GeneChip. Moreover, long-term memory pathway was found out via pathways analysis. The differentially expressed genes related to neurotoxicity were screened out. We found out that the genes encoding proteins at postsynaptic density were affected by arsenic, including CaMKII, Grial, Gria2, Grin1, Itpr1, Grm1, and PLCβ4. Because these genes are played key roles in long-term memory, we observed the ultrastructure of the PSD in cerebellum. The genes expression was confirmed. The Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), is the important regulators related to the LTP or LTD. Therefore, the expressions of CaMKII and the downstreames proteins were further confirmed at the protein level. This study aimed at screening the differentially expressed genes in the mice cerebellar exposed to As and providing evidences for exploring the mechanism that As affects adversely the function of learning and memory through disturbing the cerebellar function. Our findings could point to some of the underlying cellular and molecular mechanisms involved in the neurotoxicity caused by arsenic.Methods1. Sixty four Specific pathogen free mice (age,4 weeks), weighing 22.3-26.4 g were purchased from Experimental Animal Center, Dalian Medical University. All the animals were randomly assigned to four groups according to their body weight. Group 1 orally received double distilled water alone as control. Groups 2-4 were given double distilled water containing AS2O3 orally at the dose of 1,2, and 4 ppm for 60 days, respectively. The amount of daily drinking water of each mouse was recorded.2. The level of arsenic in cerebellum was determined using ICP-MS.3. The cerebellar tissus was stained with HE and observed via fluorescence microscope.4. Neurobehavioral tests:Morris Water Maze test was used to examine ability of learning and memory of exposed. Conditioned eyeblink response in mice was used to examine the function of cerebellum.5. The expression profiles of genes in cerebellum of mice exposed to 4 ppm As were analyzed by GeneChip. Moreover, the web tool DAVID was used to analyze the significantly enriched gene ontology (GO) and KEGG pathways in the differentially expressed genes.6. The expression of seven PSD genes (CaMKII, Grial, Gria2, Grin1, Itpr1, Grm1, and PLCβ4) related to LTP and LTD were measured by the means of RT-PCR. The cerebellums were collected from the mice exposed to arsenic for two months.7. Ultrastructure observation:Ultrastructure of Synapses in cerebellum of mice was observed by transmission electron microscopy (TEM) (JEM-2000EX, Olympus, Japan). Moreover, the thickness of the postsynaptic density (PSD) was also measured using a Guldner’s method.8. To investigate the effect of arsenic exposure on the CaMKII signaling pathway, we detected several important molecular. Protein expression of CaMKII, p-CaMKII, pRas, pRaf, p-ERK1/2 and ERK1/2 were detected by Western blot.Result1. General appearance and physical condition of arsenic-exposed and control mice were observed and no obvious difference was found. There were no differences in food intake between groups during the study. The body weights of mice were no significantly change exposed to 1 ppm As2O3 than that in control. Compared to the mice in control or exposed to 1 ppm As2O3, the body weights of mice exposed to 4 ppm As2O3 showed significantly differences change, started at the 36th day to the end of the study. At the end of two months, the body weights of mice exposed to 2 ppm As2O3 also showed significantly differences change, compared to the mice in control or exposed to 1 ppm As2O3.2. The arsenic content in brain increased with then concentration of arsenic in drinking water. The statistical significances were present between individual experiment group and control for three exposure time respectively (P<0.05). The results indicated that we have successfully raised the arsenic poisoning mice.3. The Purkinje cells and granule cells were observed under microscope:at high doses group of arsenic, cerebellum molecular cell:the staining of cytoplasm is uneven, the fibers is uneven distribution, the cytoplasm is pale, the cell is shrinkage; Purkinje cell layer:the cytoplasmic is stained, the layer is thinner, the fibers become larger and uneven, and the cytoplasm is vacuolated, the nuclei is shrinkage; Granular cell layer: the number of cell is decreased.4. Impact of subchronic arsenic exposure on learning and memory ability: compared to the control group, the escape latency in 2 ppm and 4 ppm groups is significant longer during the observation period.Impact of arsenic exposure on conditioned eyeblink response in mice:4 ppm groups is significantly lower than the control group (P<0.05).5. We observed that 1597 genes in total were differentially expressed between As-treated and control group mice. Of the 1597 genes, there were 1046 down-regulated genes as well as 551 up-regulated genes in As-treated mice of 4 ppm.6. Go analysis showed that 12 genes at PSD was significantly affected by As. Analysis of KEGG pathway showed that 20 differentially expressed genes were associated with the pathway of LTP and LTD exposed to As in mice. The mRNA expressions of Grinl and Grml in cerebellum were significantly lower in groups exposed to As than that in controls (p<0.05). The mRNA expressions of PLCP4, CaMKII, Itprl and Gria2 in cerebellum were significantly lower in groups exposed to 2 and 4 ppm As2O3 than that in control (p<0.05). The mRNA expression of Grial was significantly lower in groups exposed to 4 ppm As2O3 than that in control (p<0.05).7. Observation of ultrastructure showed that thickness of the cerebellar PSD was reduced in mice exposed to As.9. The expression of CaMKII protein in cerebellum decreased in groups exposed to As. Especially, there were significant differences between the groups exposed to 2 or 4 ppm As2O3 and controls (p<0.05), which were no significantly change exposed to 1 ppm As2O3 than that in controls. Moreover, the expression of CaMKII protein in cerebellum of mice exposed to As decreased in a dose-dependent manner. The protein expression of p-Ras and ERK1/2 were not affected after subchronic arsenic exposure. After arsenic exposure, the proteins expression of p-CaMKII, p-Raf, and p-ERK1/2 were significantly decreased compared to the control (p<0.05).Conclusion1. Subchronic arsenic exposure causes the abnormal morphology, and impairs the learning and memory ability.2. Subchronic arsenic exposure causes the arsenic accumulation in cerebellum, and the accumulation was dependent on the dose.3. Subchronic arsenic exposure disturbed the gene expression related to learning and memory.4. Our results indicate that the 7 differentiated genes including NR1、Gria1、Gria2、 mGluR1、Itpr1、PLCβ4、CaMKⅡ at the cerebellar PSD of mice, Observation of ultrastructure showed that thickness of the cerebellar PSD was reduced in mice exposed to As.5. Lower expression of p-CaMKII, pRaf and p-ERK1/2 protein after arsenic exposure, these results could inhibit the induction and maintenance of LTP or LTD. These changes maybe partly account for the molecular mechanism of learning and memory impairment caused by arsenic exposure.