The Role Of CYP2E1 In Dimethylformamide-induced Acute Injury In Mice And Its Related Mechanisms

ObjectiveN,N-Dimethylformamide(DMF)is a kind of organic material with strong solubility.It has long been recognized as the "universal solvent".At room temperature,DMF could be soluble with water,ketone,ester,alcohol,chlorinated hydrocarbons,aromatic hydrocarbons and other organic matter in any proportion.In the industry,DMF could be used as a solvent in polyurethane artificial leather industry and synthetic leather processing,as well as be used as extraction agent in the pharmaceutical and dye production process.DMF has certain volatility,and could be taken into the body through breathing and skin contact to harm the digestive system,reproductive system and immune system.Liver is the target organ of DMF.DMF can lead to liver/body ratio index elevated,liver enzymes such as ALT and AST increased,and liver cell necrosis.As a metabolite of DMF,hemoglobin adduct(NMHb)could be used as a specific biomarker of DMF.In the metabolic process of DMF,cytochrome P450 2E1 enzyme plays a very important role.We establish Cyp2el gene knockout mice to investigate the acute hepatotoxicity induced by DMF in C57BL/6 mice.Acute liver toxicity effect models of mice with different genders and different genotypes are established to discuss the acute liver toxicity caused by DMF and to discuss CYP2El’s role in the DMF metabolic process.The levels of proteins related to oxidative stress and endoplasmic reticulum stress are detected to explore the mechanisms of liver injury.Methods1.Cyp2el gene knockout mice:Cyp2el gene knockout mice are established by TALEN.By microscopic injection the plasmid established by TALEN is injected to a fertilized egg off C57BL/6,which is further transplanted into a female C57BL/6 mouse.The knockout heterozygous mouse is obtained.By mating heterozygote male and female mice,the homozygous mice are obtained.Generation sequencing is used to identify genes in mice,and western blot is used to identify Cyp2el protein levels and LC-MS/MS is used in detection of Cyp2el enzyme activity.2.Acute liver toxicity effect model:Two genotype mice(wild type and knockout)were used,with 32 male and 32 female mice in each genotype mice.The mice in each genotype and gender were divided into four groups randomly:CON,24H,48H and 72H.All subjects were weighted before treatment and sacrificed.Groups of 24H,48H and 72H were sacrificed at 24 h,48 h and 72 h after oral DMF exposure of 1500 mg/kg bw,respectively.Group CON was a blank control and was sacrificed at 72 h.The liver/body ratio indexes of all subjects were calculated.The blood was collected by plucking the eyeball from 8 mice in each group and blood of 4 mice was used for blood biochemistry analysis,including alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),triglyceride(TG)and total bile acid(TBA).The histopathological changes of the liver were observed via H&E staining.3.NMHb and CYP2E1:The blood of another 4 mice was collected for NMHb detecting by LC-MS/MS.Cyp2el mRNA is detected by PCR.Cyp2el protein content and enzyme activity is detected by WB and LC-MS/MS,respectively.4.Oxidative stress and endoplasmic reticulum stress:GSH and MDA were the index of oxidative stress,SOD and GSH-Px were the index of antioxidant exzymes;GRP94 and GRP78 were the index of endoplasmic reticulum stress.Results1.Cyp2el gene knockout mice:A generation sequencing result suggested the genetype of mice is homozygous.The Cyp2el content and activity of homozygous mice are much lower than that of wild type.2.Acute liver toxicity effect model:In wild type mice,compared with control mice,significant increases in liver enzyme levels of ALT and AST were observed after the DMF treatment for 24h,48h and 72h,while no difference of liver enzyme levels was observed between control and DMF exposed mouse in Cyp2el gene knockout mice.The pathology of wild type mice showed hydropic degeneration,liver cell necrosis and inflammatory cells infiltration,while slight liver injury was observed in Cyp2el gene knockout mice.3.NMHb and CYP2E1:NMHb of all control mice was undetectable.NMHb is specificily detectable in DMF exposed C57BL/6 wild type mice.In male wild-type mice,NMHb detected after 24h treatment was as much as that of 48h and 72h treatment.Compared with male wild-type mice,much less NMHb was observed in female wild-type mice after 24h DMF treatment and the NMHb increase sharply after 48h treatment.In all the homozygous type mice,very small amouts of NMHb were observed.In western blot experiments,in the homozygous group,obviously Cyp2el stripe was not observed,while in Cyp2el wild-type mice,the Cyp2el protein level increased first and then deceased,which indicated that the inhibition of CYP2E1 may occur.4.DMF induced oxidative stress and endoplasmic reticulum stress:DMF exposure can lead to the decrease of GSH content in the liver,the increase of MDA content,and the activation of antioxidant enzymes such as SOD and GSH-Px in vivo.The levels of endoplasmic reticulum stress related protein GRP94 and GRP78 increased after DMF exposure.5.Gender differences:The cholesterol and triglycerides increased in female wild type mice after DMF exposure,which has no difference in male wild type.The liver histopathologic examination showed the liver cell necrosis in wild type mice.Male homozygous perform hydropic degeneration in liver and female homozygous perform steatosis.NMHb suggested that male mice metabolism DMF in 0-24h after exposure but female mice metabolism in 24-48h.Conclusions1.Cyp2el knockout mice is successfully constructed;2.CYP2E1 play an important role in DMF induced acute liver injury in mice by toxic metabolic activation and reactive oxygen species production.Oxidative antioxidant imbalance and endoplasmic reticulum stress is the possible mechanism of DMF-induced liver injuty.