Study On The Toxic Effects On The Liver And Brain Induced By Combined Treatment Of 1,2-dichloroethane And Ethanol In Mice And Their Related Mechanisms

Objective:1,2-dichloroethane(1,2-dichloroethane,1,2-DCE)as a colorless,tasteless,high volatile oily liquid at room temperature,is highly toxic.In industrial production,1,2-DCE is mainly used for the synthesis of PVC monomer.In addition,as organic solvents are also widely used in adhesives,degreasing agents and cleaning agents.As an organic solvent,1,2-DCE is very volatile,mainly through the respiratory tract into the human body,and was quickly absorbed into the blood.As a result of fat-soluble,1,2-DCE easily through the BBB into the brain tissue.Although there are more than 150 years of use history,but before 1990,domestic and international occupational exposure to 1,2-DCE lead to damage to the central nervous system and animal research data are very few.But since 1990,as a binder widely used in toys,plastics and footwear and other industries,From the beginning of Guangdong Province,in China’s many provinces and cities have occurred subacute 1,2-DCE occupational poisoning accidents,clinical manifestations are toxic encephalopathy and liver injury.At present,subacute 1,2-DCE occupational poisoning has become a serious harm to our workers’ health and the new occupational hazards of life.However,there is still a lack of information on the subacute toxicity of 1,2-DCE,in urgent need of further study.Studies have shown that cytochrome P450 2E1(CYP2E1)is a key enzyme that mediates low-molecular compounds in the body,especially the halogenated hydrocarbons,and that most halogenated hydrocarbons can promote CYP2E1 expression during metabolic processes Up.1,2-DCE eliminated through activemetabolism by CYP2E1 can produce more active chemical intermediates chloroacetaldehyde and 2-chloroethanol,the final metabolism of chloroacetic acid with the urine excreted.Compared with other CYP450 subtypes,CYP2E1 has stronger NADPH oxidase activity and is prone to electron transport uncoupling in the process of catalyzing the metabolism of substrates,producing reactive oxygen species(ROS),causing oxidative damage to cells.At present,CYP2E1 is a subtype of CYP450,mainly distributed in the central region of the liver lobular,accounting for about 7% of the total CYP450.However,a large number of studies have shown that CYP2E1 expression is also present in the extrahepatic tissues of human and experimental animals.Especially in the brain tissue olfactory bulb,hippocampus,cerebellum and brain frontal cortex and other regions of the neurons and glial cells have a relatively high level of CYP2E1 expression.Ethanol is a recognized CYP2E1 specific inducer.Studies have shown that acute and chronic ethanol exposure can lead to liver and brain tissue CYP2E1 protein levels and enzyme activity increased.Ethanol produces acetaldehyde and acetic acid under CYP2E1 catalysis.Acetaldehyde is the active intermediate in the metabolism of ethanol,which can react with biological macromolecules such as protein and DNA to produce adduct,and cause of tissue cell damage.Studies have shown that ethanol-induced brain injury is closely related to its induction of CYP2E1 overexpression,suggesting that ethanol metabolism in brain tissue is the main cause of neurotoxicity.Studies have shown that ethanol by raising the CYP2E1 protein levels and enzyme activity to promote chloroform and carbon tetrachloride and other halogenated hydrocarbon compounds metabolism,and increase the chemical-induced liver injury.Based on the above research results and our study,we hypothesized that exposure to ethanol and 1,2-DCE would increase the damage of liver and brain tissue,lead to increased susceptibility of alcoholics to 1,2-DCE toxicity.In this study,Kunming mouse are exposed to 1,2-DCE and ethanol in vivo.Ethanol and 1,2-DCE were given to explore the interaction between ethanol and 1,2-DCE in the damage caused by liver and brain tissue injury in mice,to reveal the health effects of 1,2-DCE on alcohol workers,and provide experimental reference data to 1,2-DCE occupational poisoning prevention and work control.Methods:1.Experimental animals 1.1 Exposure and grouping 1.1.1 Animal selection: healthy,sexually mature,clean female Kunming mice,weight 20-24 g.The experiment established different concentrations of ethanol combined with 1,2-DCE exposure model.In the model,the experimental animals were randomly divided into 6 groups: blank control group,ethanol control group,1,2-DCE simple exposure group and low,medium and high dose ethanol and 1,2-DCE combined exposure group,each group of only 10.1.1.2 Processing method(1)Pre treatment In experiment,3 days before exposure to 1,2-DCE: the combined exposure groups are given 0.75g/Kg,1.5g/Kg,3.0g/Kg ethanol gavage once a day,respectively;Ethanol group is given 3.0g/Kg ethanol;control group and 1,2-DCE group are given the same volume of double distilled water.(2)Exposure treatment The test animals were placed in 100 L of static exposure cabinet,1,2-DCE group and combined exposure groups are exposed to 1.0 mg/L 1,2-DCE for 3.5 h per day;ethanol group and control group are exposed to air for the same time,and before four hours of the 1,2-DCE and air exposure,all the groups receive the same treatment as before.Gas chromatography is used to monitor the 1,2-DCE concentration.Mice were sacrificed the next day after the end of the last exposure,and blood,liver and brain tissue were quickly removed,the following indicators were detected.2.Measurement 2.1 Effects of ethanol and 1,2-DCE combined exposure on brain organ coefficient and cerebral water content in mice After the exposure,mice were anesthetized with ether and decapitated,the brain was quickly removed for the following test.(1)Brain organ coefficient(%)= brain weight / body weight.(2)Brain water content(%)=(wet weight-dry weight)/ wet weight × 100%.2.2 Pathological observation of mouse brain tissue Take the side of the brain and liver tissue.After 72 hours’ formaldehyde fixation,tissues were ordinary embedded in paraffin,consecutive coronal slices,slice thickness 5 μm,HE staining,observed under ordinary optical microscope.2.3 Using the assay kits for detecting the activity of activity of glutamic-pyruvic transaminase(ALT),and activity of glutamic-oxalacetic transaminase(AST)in serum.2.4 Using the assay kits for detecting the activity of superoxide dismutase(SOD),and level of malondialdehyde(MDA),nonprotein sulfhydryl(NPSH),activity of glutamic-pyruvic transaminase(ALT),and activity of glutamic-oxalacetic transaminase(AST)in brain and liver.2.5 Protein expression of CYP2E1,ZO-1,Occludin,AQP4,Nrf2,HO-1,g-GCSc,GR,g-GCSm in brain were detected with Western Blot.2.6 Protein expression of CYP2E1,Nrf2,HO-1,g-GCSc,GR,g-GCSm in liver were detected with Western Blot.2.7 Real-time PCR was used to evaluate gene expression of CYP2E1,ZO-1,Occludin,AQP4,Nrf2,HO-1,g-GCSc,GR,g-GCSm in brain.2.8 Real-time PCR was used to evaluate gene expression of CYP2E1,Nrf2,HO-1,g-GCSc,GR,g-GCSm in liver.3.Statistical analysis Data were analyzed using SPSS for Windows,version 16.0.The significant difference among multiple groups was evaluated by the analysis of variance test(ANOVA).Post hoc tests were analyzed by Student-Newman-Keuls test(SNK).The statistical significance was defined as P < 0.05.Results:1.poisoning performance In the model,there were no obvious symptoms of poisoning in the control group,the ethanol control group and the 1,2-DCE group.But,some mice in the combined exposure groups appeared limb tremor and double forelimbs and / or double hind legs cross each other claws,limbs can not completely extend.The behavioral changes rate of brain injury in low,medium and high dose combined exposure group were 20%(2/10),50%(5/10),and 50%(5/10),respectively,and only the high dose combined exposure group emerge death mouse(20%).2.brain edema-correlated indicators In the model,compared with the control group,the brain organ coefficient in middle dose and high dose groups increased significantly(P < 0.05).The brain water content increased with the increase of the dose of ethanol exposure,but compared with the control group,there was not statistically significant(P > 0.05);in addition,mice brain tissue in middle and high dose combined exposure groups,cells showed interstitial loose around the nucleus,lacuna widened,lightly stained cytoplasm,cell body swelling degeneration,blurred edge the change of brain edema;the typical pathological part of cells and capillaries around the cavity expansion.3.liver injury correlated indicators In the model,low,middle and high dose combined exposure groups had pathological changes in liver injury;compared with the control group,AST activity of serum in the middle and high combined exposure group significantly increased(P < 0.05);compared with the control group,ethanol control group,ALT activity in combined exposure groups significantly increased(P < 0.05).4.liver and brain tissue oxidative stress-related indicators 4.1 liver tissue oxidative stress-related indicators In the model,compared with the control group,GSH content in combined exposure groups decreased significantly(P < 0.05);compared with the control group,ethanol control group,the content of MDA in combined exposure groups significantly increased(P < 0.05);There were no significant differences in SOD activity between groups.4.2 brain tissue oxidative stress-related indicators In the model,compared with the control group,ethanol control group,1,2-DCE simple exposure group,and low dose combined exposure group,MDA content in middle and high dose combined exposure groups decreased significantly(P < 0.05),Ethanol and 1,2-DEC have a synergistic effect on the content of MDA in brain injury;There were no significant differences in SOD activity and GSH content between groups.5.the combined related mechanisms of,2-dichloroethane and ethanol on the liver and brain 5.1 Effects of combined exposure of ethanol and 1,2-DCE on blood-brain barrier associated protein and m RNA in mice In the model,the levels of Occludin and ZO-1 proteins in the brain tissue of the combined exposure groups were decreased in different degree,the trend of middle and high dose combined exposure group was more obvious,the same trend was also detected at the m RNA level;Ethanol and 1,2-DEC have a synergistic effect on Occludin and ZO-1 protein in brain injury,and a synergistic effect on Occludin and ZO-1 m RNA in brain injury.5.2 Effects of combined exposure of ethanol and 1,2-DCE on AQP4 protein and m RNA in mice In the model,the levels of AQP4 proteins in the brain tissue of the combined exposure groups were decreased in different degree,the trend of middle and high dose combined exposure group was more obvious,the same trend was also detected at the m RNA level;Ethanol and 1,2-DEC have a synergistic effect on AQP4 m RNA in brain injury.5.3 The role of CYP2E1 in liver and brain injury in the model of ethanol and 1,2-DCE combined exposure In the model,the levels of CYP2E1 proteins in the brain and liver of the combined exposure groups were increased in different degree,the trend of middle and high dose combined exposure group was more obvious,the same trend was also detected at the m RNA level;Ethanol and 1,2-DEC have a synergistic effect on CYP2E1 proteins and m RNA in brain injury,and a synergistic effect on CYP2E1 m RNA in liver damage.5.4 The role of NRF2 signal path in liver and brain injury in the model of ethanol and 1,2-DCE combined exposure In the model,the levels of Nrf2,HO-1,γ-GCSc,GR proteins in the brain and liver of the combined exposure groups were increased in different degree,the trend of middle and high dose combined exposure group was more obvious,the same trend was also detected at the m RNA level,however,γ-GCSm was not detected significant changes in protein and gene level;Ethanol and 1,2-DEC have a synergistic effect on Nrf2 protein in brain damage,and a synergistic effect on Nrf2,γ-GCSc,GR protein in liver damage;Ethanol and 1,2-DEC have a synergistic effect on Nrf2,HO-1,γ-GCSc m RNA protein in brain and liver damage.Conclusions:1.Ethanol and 1,2-DCE combined exposure can cause liver and brain injury,and the damage induced by 1,2-dichloroethane enhanced with the ethanol exposure dose increase,which had a dose-effect relationship.2.Ethanol and 1,2-DCE combined with toxic effects can caused liver and brain injury,the possible mechanism might be related with ethanol enhancing 1,2-dichloroethane induced expression of CYP2E1,increasing dichloroethane metabolism,producing a large number of reactive oxygen free radicals,resulting in oxidative stress,causing liver and brain oxidative related damage.3.Ethanol and 1,2-DCE combined with toxic effects caused liver and brain oxidative,further activated the Nrf2 signaling pathway,the body expressed a large number of antioxidant enzymes HO-1,γ-GCSc,GR.