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OBJECTIVE: To study the effect of aquaporin 4(AQP4) in regulating the permeability of blood-brain barrier(BBB) induced by subacute 1,2-dichloroethane(1,2-DCE) inhalation. METHODS: Specific pathogen free healthy CD-1 male Aqp4 genetically engineered mice(Aqp4~(+/+)and Aqp4~(-/-)) were randomly divided into control and low-, medium-and high-dose groups. The mice were exposed to 1,2-DCE at the dosages of 0.00, 100.00, 350.00 and 700.00 mg/m~3 for 6 hours per day for consecutive 28 days by systemic dynamic inhalation. After the end of 1,2-DCE exposure, the BBB permeability was evaluated by Evans blue staining. Real-time fluorescence quantitative polymerase chain reaction method was used to detect the mRNA expression of genes related to BBB tight junction protein(Tjp)1, Tjp2, Tjp3, claudin(Cldn)3, Cldn5, Cldn11, occludin(Ocln), matrix metalloproteinase(Mmp)2, Mmp9 and Na-K-Cl cotransporter-1(Nkcc1). RESULTS: The BBB permeability in mice showed significant change with 1,2-DCE dose and Aqp4 genotype(P<0.01). The BBB permeability of Aqp4~(+/+) genotype mice was higher in low-, medium-and high-dose groups than that of control group(all P values were <0.05). The permeability of BBB was lower in Aqp4~(+/+) genotype mice in the control group than that of Aqp4~(-/-) genotype mice in the same group(P<0.05), but BBB permeability was higher in Aqp4~(+/+) genotype mice in the three dose groups than that of Aqp4~(-/-) genotype mice in the same group(all P values were <0.05). The Cldn3 and Olcn mRNA relative expression in the brain cortex had statistical difference in mice with different genotype(all P values were <0.01). The mRNA relative expressions of Cldn3 and Olcn in the brain cortex were higher in Aqp4~(-/-) genotype mice than that of Aqp4~(+/+) genotype mice(all P values were <0.01). The relative mRNA expression levels of Tjp1, Tjp2, Tjp3, Cldn5, Cldn11, Mmp2, Mmp9 and Nkcc1 in the cerebral cortex of mice were not statistically significant in aspect of 1,2-DCE exposure dose and genotype(all P values were >0.05). CONCLUSION: Exposure to 1,2-DCE can increase BBB permeability in mice, and the mechanism may be associated with 1,2-DCE-induced down-regulation of Aqp4 and up-regulation of mRNA expression of the cerebral cortex TJP-related molecules Cldn3 and Ocln.
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OBJECTIVE: To explore the correlation between endothelial microparticles(EMPs) and subacute 1,2-dichloroethane(1,2-DCE) toxic encephalopathy. METHODS: A total of 24 patients with subacute 1,2-DCE toxic encephalopathy were selected as the case group, and 24 healthy individuals were selected as the control group using a convenient sampling method. Blood plasma was collected from the fasting venous blood of patients in these two groups, and the level of EMPs in the plasma was detected by flow cytometry. RESULTS: The levels of plasma EMPs of patients in the control group and the case group were(692.0±174.4) ×10~3/L and(839.8±155.8) ×10~3/L respectively. The levels of plasma EMPs in patients with mild, moderate and severe case subgroups were(691.6±101.9) ×10~3/L,(900.6±46.6) ×10~3/L and(1 026.8±69.8)×10~3/L respectively. The EMPs level of patients in the case group was higher than that of the control group(P<0.01). The level of EMPs in the moderate and severe case subgroups was higher than that of the control group and mild case subgroup(P<0.01). CONCLUSION: Endothelial injury was found in patients with subacute 1,2-DCE toxic encephalopathy and endothelial injury is related to the severity of poisoning.
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OBJECTIVE: To establish a method for detecting dichloromethane,trichloromethane and 1,2-dichloroethane in blood by gas chromatography-mass spectrometry. METHODS: Using static headspace analysis, three halogenated hydrocarbons in blood samples were separated by DB-5 MS elastic capillary column and detected by gas chromatographymass spectrometry. RESULTS: There was a good linear relationship in the selected range of dichloromethane,trichloromethane and 1,2-dichloroethane in blood. The linear correlation coefficient was greater than 0. 999 8. The detection limit and the lower limit of quantitation was 0. 19-0. 28 and 0. 64-0. 93 μg/L,respectively. The average recovery rate was 95. 1%-106. 6%. The within-run and between-run relative standard deviation was 2. 9%-4. 9% and 5. 0%-7. 0%,respectively. The samples could be preserved at room temperature or 4 ℃ for 3 days and at-8 ℃ or below for7 days. CONCLUSION: With the features of high sensitivity,precision,accuracy,easy operation and less interference,this method is suitable for detecting dichloromethane,trichloromethane and 1,2-dichloroethane in the blood.
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OBJECTIVE: To investigate the effect of 1,2-dichloroethane(1,2-DCE) acute inhalation exposure on the differential gene expression of phase Ⅰ metabolic enzymes. METHODS: The specific pathogen free SD rats were randomly divided into control group(16 rats), low-and high-dose groups(24 rats in each group, half males and half females). Low-and high-dose group were given daily 600, 1 800 mg/m~(3 ) of 1,2-DCE, and the control group given the fresh air by dynamic inhalation for 8 hours per day for consecutive 7 days. After the end of exposure, the relative mRNA expression of cytochrome P450 2 E1(CYP2 E1), alcohol dehydrogenase(ADH1) and acetaldehyde dehydrogenase 3 alpha 1(ALDH3α1) in the liver tissue was detected by real-time fluorescence quantitative polymerase chain reaction. RESULTS: The relative expression of CYP2 E1 in male high-dose group was higher than that in male low-dose group and female high-dose group(P<0.05). The relative expression of ADH1 in male low-and high-dose groups was higher than that in male control group(P<0.05). The relative expression of ADH1 in male high-dose group was higher than that in male low-dose group and female high-dose group(P<0.05). The relative expression of ALDH3α1 in high-dose group was higher than that in control group and low-dose group(P<0.05). CONCLUSION: High dose 1,2-DCE could increase the gene expression of phase Ⅰ metabolic enzymes in rat liver. The 1,2-DCE has more obvious effect in male rats than in female rats.
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OBJECTIVE: To investigate the effects of 1,2-dichloroethane(1,2-DCE) subacute exposure on depression in rats as well as the relevant mechanism of monoamine neurotransmitters. METHODS: The specific pathogen free male SD rats were randomly divided into control group, low-, medium-, and high-dose groups, with 10 rats in each group. The rats in these 4 groups were intra-gastrically administered with 1,2-DCE(diluted in corn oil) at the dose of 0, 20, 40, 80 mg/kg body weight, every other day for 14 times. After exposure, the behavior change of rats was observed by open-field test, sucrose preference test and forced swim test. The levels of the monoamine neurotransmitters including 5-hydroxytryptamine(5-HT), noradrenaline(NA) and dopamine(DA) in prefrontal cortex, hippocampus, and striatum of rats were analyzed by high performance liquid chromatography-electrochemical detection method. RESULTS: The number of rearing, time and distance of central area, sucrose preference index of mice in medium and high dose groups were decreased(P<0.05), while immobility time of forced swim test was increased(P<0.05) when compared with the mice in control group. The levels of 5-HT, NA and DA in prefrontal cortex, hippocampus, and striatum decreased with the increase of 1,2-DCE exposure(P<0.05), showing a dose-effect relationship. The levels of 5-HT, NA and DA in prefrontal cortex, hippocampus, and striatum in the high-dose group were lower than that of control group(P<0.05). CONCLUSION: The subacute exposure of 1,2-DCE can induce depression-like behavior in rats. The mechanism might be related to the reduction of monoamine neurotransmitters in striatum, hippocampus and prefrontal cortex.
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OBJECTIVE: To explore the molecular mechanism underlying 1,2-dichloroethane(1,2-DCE) induced apoptosis by screening differentially expressed proteins in human astrocytes( HAs). METHODS: HAs were cultured in complete medium with 1,2-DCE at various concentrations of 0-80 or 0-40 mmol/L. After 24 hours,apoptosis of HAs was evaluated using flow cytometry and staining with annexin Ⅴ-fluoresce in isothiocyanate and propidium iodide. An AAH-APO-1-2 protein chip was used to screen differentially expressed proteins and quantitative real-time polymease chain reaction(qRT-PCR) was used to verify related differentially expressed genes(DEGs). RESULTS: At 1,2-DCE concentrations of0-80 mmol/L,the total apoptosis rate of HAs increased with 1,2-DCE concentrations in a dose-dependent manner( P <0. 01). Seven different kinds of proteins were screened out by apoptotic protein chip. Among them,the expression of insulin-like growth factor-binding protein( IGFBP)-1,IGFBP-4 and cytochrome C( Cyto C) were up-regulated,while the expression of P27,cysteine aspartic acid specific protease-3( Caspase-3),B-cell lymphoma-2 interacting mediator of cell death( BIM) and BH3 interacting domain death agonist( BID) were down-regulated compared with the control group. The result of DEGs verified by qRT-PCR showed that the expression of mRNA of IGFBP-1,IGFBP-4 and Cyto C at 1,2-DCE concentrations of 40 mmol/L was up-regulated. This result was in consistent with the trend of target expression in the protein chip. The mRNA expression of Caspase-3,BIM and BID was also up-regulated. CONCLUSION: 1,2-DCE induces apoptosis of HAs through mitochondrial pathway.
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OBJECTIVE: To investigate the effects of subacute systemic inhalation exposure of 1,2-dichloroethane(1,2-DCE) on learning and memory in NIH mice. METHODS: Forty-five specific pathogen free healthy 7-week-old NIH mice were randomly divided into control,low-dose and high-dose groups with 5 female mice and 10 male mice in each group. The mice were exposed to 1,2-DCE at dosages of 0. 00,100. 00 and 350. 00 mg/m3 for 6 hours per day for consecutive 28 days by dynamic systemic inhalation. The neurobehavioral tests of mice were performed before and after the first to fourth weeks of exposure using the Morris water maze test. RESULTS: There was no significant difference in body weight and swimming speed among the three groups of mice( P > 0. 05). The navigation experiment results showed that the escape latency of mice in both low-and high-dose groups were longer than that of the control group at the same time point(P < 0. 05) during 1-4 weeks after exposure. In the control group,the escape latency was shorter than that of the same group before exposure( P < 0. 05). The escape latency of high-dose group prolonged with the increase of exposure time,and in the 4 th week the escape latency was significantly higher than that of the same group before exposure( P < 0. 05).The experiment results of space exploration indicated that the first time of crossing platform in low-and high-dose groups were longer than that of the control group at the second to the fourth week( P < 0. 05). The target quadrant retention time and the number of crossing the platform in the low-and high-dose groups were lower than those in the control group( P <0. 05). CONCLUSION: Subacute inhalation exposure of 1,2-DCE can impair the learning and memory ability of NIH mice.The high-dose exposure may reduce learning ability in mice in a time-effect manner.
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OBJECTIVE: To explore the effect of 1,2-dichloroethane(1,2-DCE) induced apoptosis on the expression of related proteins in human neuroblastoma cells(SH-SY5 Y cells). METHODS: SH-SY5 Y cells were cultured in complete medium with 1,2-DCE at final concentrations of 0,10,20,30,40,50,60,70 and 80 mmol/L. After being cultured for24 hours,the apoptosis of SH-SY5 Y cells was tested by flow cytometry using annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. Western blot was used to detect the protein expression of P53,B cell lymphoma/leukmia-2(BCL-2)and BCL-2 associated X protein(BAX). RESULTS: At 1,2-DCE concentrations of 0-80 mmol/L,the total apoptosis rate of SH-SY5 Y cells increased with 1,2-DCE concentrations in a dose-dependent manner(P < 0. 01). At 1,2-DCE concentrations of 30-80 mmol/L,the early apoptosis rate and total apoptosis rate of SH-SY5 Y cells increased significantly than the control group(P < 0. 05). Compared with the other groups,the protein expression of P53 was the lowest when the1,2-DCE concentration was 20 mmol/L(P < 0. 05),and the protein expression of BCL-2 and the BCL-2/BAX ratio were the lowest when the 1,2-DCE concentration was 70 mmol/L(P < 0. 05). There is no dose-response relationship in the1,2-DCE concentrations and the protein expression levels of P53,BCL-2 and BAX,and BCL-2/BAX ratio. Linear multiple regression analysis revealed that the total apoptosis rate of SH-SY5 Y cells treated with 1,2-DCE was associated with the protein expression of P53 and BCL-2,and BCL-2/BAX ratio(P < 0. 05). CONCLUSION: 1,2-DCE could inhibit the apoptosis of SH-SY5 Y cells. The mechanisms may be related to the changes of P53 and BCL-2 protein expression,and BCL-2/BAX relative amount.
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OBJECTIVE: To establish the cell model of human neuroblastoma cell( SH-SY5Y cell) exposed to1,2-dichloroethane( 1,2-DCE) in vitro and to explore the mechanism of 1,2-DCE-induced toxicity in SH-SY5Y cells.METHODS: SH-SY5Y cells were collected in their logarithmic growth phase and cultured in complete medium that had final concentrations of 1,2-DCE in 0,10,20,30,40,50,60,70 and 80 mmol / L for 24 hours. Cell morphology was observed and cell survival rate was examined by CCK-8 assay. Using chemical colorimetric method, the activity of lactic dehydrogenase( LDH) in the cell culture supernatant,and the intracellular level of malondialdehyde( MDA),the intracellular activities of superoxide dismutase( SOD) and adenosine triphosphate( ATP) enzymes were detected. RESULTS: With the increasing exposure concentrations of 1,2-DCE,the cell density of SH-SY5Y cells gradually decreased,the synapse became shorter,the membrane ruptured,cytoplasm condensed and cytoplasmic contents overflowed increased.With the increasing concentration of 1,2-DCE,the cell survival rate decreased( P < 0. 01),the activity of LDH in the cell culture supernatant increased( P < 0. 01). These changes had a dose-effect correlation. Intracellular MDA level,and activities of SOD,Na~+-K~+-ATP enzyme,Ca~(2+)-Mg~(2+)-ATP enzyme and total ATP enzyme increased at first and then decreased. The activity of LDH in the cell culture supernatant and cell survival rate was negatively correlated( the correlation coefficient is- 0. 907,P < 0. 01). CONCLUSION: 1,2-DCE could inhibit the proliferation of SH-SY5Y cells.The mechanism may be related to the permeability change of cell membrane,cellular damage from excessive free radicals,the decrease of free radical scavenging capacity,ATP enzyme activity and calcium overloading. SH-SY5Y cells can be used as a common cell line for 1,2-DCE cytotoxicity analysis.
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OBJECTIVE: To investigate the effects of 1,2-dichloroethane( 1,2-DCE) on myelin basic protein( MBP),neuron specific enolase( NSE) and S100 protein in the plasma of SD rats. METHODS: Forty-eight specific pathogen free adult SD rats were randomly divided into control group,low-dose group and high-dose group,with 8 females and 8 males in each group. Rats were given 1,2-DCE orally at the dose of 0,27 and 79 mg / kg body weight every other day( every Wednesday,Monday and Friday) for 4 weeks. After 1,2-DCE administration,8 survived rats( half male and female) were randomly selected in each group. The plasma levels of MBP,NSE and S100 protein were measured using enzyme-linked immunosorbent assay. The blood and urinary samples were collected to assess the concentration of 1,2-DCE and its main metabolites( 2-chlorideacetic acid, 2-chlorideacetaldehyde and 2-chlorideethanol) by gas chromatography. The pathological changes of cerebrum and cerebellum were observed through optical microscope,and the expression of MBP was detected by immunohistochemistry. RESULTS: Rats in high-dose group showed abnormal behavior from the third day of1,2-DCE exposure and 6 rats( 2 females,4 males) died from 1,2-DCE intoxication. Rats in low-dose group and control group appeared normal and no death was observed. MBP level in the plasma of high-dose group was higher than that in the control group( P < 0. 05),but the levels of NSE and S100 protein in each group did not show significant statisticaldifference( P > 0. 05). 1,2-DCE and 2-chloroethanol in the urine were detected in the high-dose group,and were below detection limit in the other two groups. 2-Chloroacetic acid level in high dose-group was significantly higher than that in the low-dose group( P < 0. 05),and was below detection limit in the control group. 2-Chloroacetaldehyde in the urine of each group was below detection limit. 1,2-DCE and its 3 kinds of metabolites were not detected in the plasma of all rats. There was no obvious structural damage,bleeding,edema or necrosis found in the cortex and white matter of cerebrum and cerebellum. The expression of MBP in the choroid plexus epithelial cells were significantly enhanced in the lateral ventricle and the fourth ventricle of rats in the high-dose group,and slight enhanced in rats in the low-dose group. CONCLUSION: MBP may play a role in the toxic effect of 1,2-DCE.
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OBJECTIVE: To explore the effects of aquaporin 4( APQ4) in rat toxic brain edema induced by subacute 1,2-dichloroethane( 1,2-DCE) exposure. METHODS: Thirty-two specific pathogen free healthy adult female SD rats were randomly divided into control( 8 rats),low-dose( 12 rats) and high-dose( 12 rats) groups. The treatment groups were exposed to 1,2-DCE( low-dose: 600 mg / m3; high-dose: 1 800 mg/m3,nose-only) and the control group was exposed to fresh air by dynamic inhalation for 8 hours per day for consecutive 7 days. After exposure,histopathologic changes were examined in the cerebral cortex. Real-time polymerase chain reaction was used to detect the mRNA relative expression of matrix metalloproteinase 2( MMP2),Na-K-Cl cotransporter-1( NKCC1) and AQP4. The Western blotting was used to detect the expression of AQP4 protein in the cerebral cortex. RESULTS: The pathological results showed that the cerebral cortex tissues were loose around the peripheral vessels and the vessels tissue space appeared widen in low-dose exposure group. The pathological change was more serious in high-dose group than low-dose group,with obvious loosen vessels and vacuole. Compared with those of the control group and the low-dose group,the relative expression level of MMP2 mRNA in the high-dose group increased significantly[( 1. 07 ± 0. 41) vs( 1. 56 ± 0. 55),( 1. 21 ± 0. 59) vs( 1. 56 ± 0. 55),P <0. 05],while the the relative expression level of AQP4 mRNA in the high-dose group significantly decreased [( 1. 03 ±0. 25) vs( 0. 81 ± 0. 12),( 1. 00 ± 0. 20) vs( 0. 81 ± 0. 12),P < 0. 05]. The relative expression levels of NKCC1 mRNA in all groups showed no statistical difference [( 1. 03 ± 0. 31) vs( 1. 14 ± 0. 43) vs( 1. 36 ± 0. 50),P > 0. 05]. The relative expression level of AQP4 protein in the high-dose group was lower than that of the control group [( 0. 80 ± 0. 25) vs( 1. 19 ± 0. 42),P < 0. 05]. CONCLUSION: The brain edema induced by subacute inhalation of 1,2-DCE is of mixed types with vasogenic edema as its main symptom. Its pathogenesis is related to the changes of AQP4 expression.
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OBJECTIVE: To explore the toxicity of 1,2-dichloroethane( 1,2-DCE) and its metabolites on human astrocytes( HAs). METHODS: Different doses of 1,2-DCE( 5. 00,10. 00,25. 00,50. 00 and 100. 00 mmol/L),2-chlorohydrins( 5. 00,25. 00,50. 00,100. 00 and 200. 00 mmol/L),2-chloroacetaldehyde( 1. 00,5. 00,10. 00,20. 00 and 50. 00 mmol / L) and chloroacetic acid( 0. 01,0. 05,0. 10,0. 50 and 1. 00 mmol / L) were used for treating HAs in vitro during their logarithmic phase. After 24 hours of culture,the morphology of HAs was observed by fluorescent inverted phase contrast microscope. The survival rate and the inhibition ratio of HAs were detected by CCK-8 colorimetry to estimate the50% inhibiting concentration in 24 hours( 24 h-IC50). The apoptosis of HAs was tested by double-labeling and flow cytometry using Annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. RESULTS: The morphology of HAs changed in varying degrees after 24 hours exposure to 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid. The changes included smaller size of cells,pseudopodia tapering,increased intracellular particles and suspension of circular cells and decreased transparency of cells. With the increasing does of 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid exposure,the survival rates of HAs decreased( P < 0. 01),while its inhibition ratios increased( P <0. 01). They all showed dose-effect relationship. 24 h-IC50 of the above 4 chemicals were 56. 25,235. 00,26. 43 and1. 38 mmol / L,respectively. The 1,2-DCE,2-chlorohydrins and chloroacetic acid could induce the apoptosis of HAs and the apoptosis rate of HAs was positively correlated with the 3 kinds of chemicals( P < 0. 01). CONCLUSION: 1,2-DCE and its metabolites 2-chloroacetaldehyde,2-chlorohydrins and chloroacetic acid can lead to toxic damage and induce the apoptosis of HAs. Chloroacetic acid has the strongest toxicity among the metabolites.