Correlation Between The Intrinsic Activity Of CYP2E1 And Liver Fibrosis Induced By Diethylnitrosamine In Rats

Liver fibrosis is a pathological condition of abnormal proliferation and accumulation of ductile fibrous connective tissue in the liver.Liver fibrosis is the development of chronic liver disease to the stage of liver cancer.Liver cancer is a common malignant tumor,a serious threat to human life safety.The formation of liver cancer is very complex,epidemiological studies have shown that nitrosamines,hepatitis B virus,aflatoxin,alcohol damage,family genetic disease are the factors that induce liver cancer.Diethylnitrosamine is a typical representative of the nitrosamines family,and has a carcinogenic effect on humans and animals.Diethylnitrosamines are pre-carcinogens,CYP2E1 metabolism by acetaldehyde and alkylation products,product and DNA adduction induced gene mutation,induced liver injury fibrosis,and ultimately the formation of liver cancer.This study was found earlier that the activity of CYP2E1 in liver fibrosis tissue of patients with hepatocellular carcinoma was significantly increased.Suggesting that elevated CYP2E1 activity may be related to the formation of liver fibrosis,which has not been reported in the existing studies,so we hope to explore the relationship between the intrinsic activity of CYP2E1 and the hepatic fibrosis in rats by establishing rat model of hepatic fibrosis.In this study,rat rat model was established.The rat model of hepatic fibrosis was established by analyzing the correlation between the toxicokinetic parameters of diethylnitrosamine in rats and the indexes of hepatic fibrosis and the degree of hepatic fibrosis to investigate the correlation between the intrinsic activity of CYP2E1 and the hepatic fibrosis induced by diethylnitrosamine in rats.1 Method 1.1 Rat liver fibrosis model 1.1.1 Experimental animalsThirty adult healthy male SD rats weighing about 170±10 g were fed for about one week to accommodate the new environment.Rats were randomly divided into blank control group(n=10)and hepatic fibrosis model group(n=20).1.1.2 Dosing regimen Rats in the first 4 weeks were injected with diethylnitrosamine 50 mg/kg intraperitoneally twice a week for 5 to 12 weeks.The rats in the control group were injected with the same volume of saline and the rats were sacrificed at 12 weeks.1.1.3 Determination of liver fibrosis related indexesThe levels of aspartate transaminase(AST)and alanine aminotransferase(ALT)in rat plasma were determined by the kit,the positive rate of Proliferating Cell Nuclear Antigen(PCNA),the positive rate of Ki67 and the optical density of GST-P were measured by immunohistochemistry.1.1.4 Liver fibrosis scoreLiver slices were stained by HE and Masson staining.Pathological results were scored according to the Ishak score.The severity of liver fibrosis was divided into 0 ~ 6 points.1.2 The toxicokinetics of diethylnitrosamine in ratsRats were injected intraperitoneally with 50 mg/kg diethylnitrosamine at 0,1,4, 10,25 min and 1,2.5,4.5,7,10,24 h after orbital blood collection.The concentration of nitrosamine was determined by HPLC-UV method.The toxicokinetic parameters were obtained by DAS2.0 software at different time points.1.3 Correlation between the intrinsic activity of CYP2E1 and the degree of liver fibrosis in ratsThe toxicokinetic parameters of diethylnitrosamine in the first week of liver fibrosis in rats showed their CYP2E1 Inherent activity.The levels of AST and ALT in the plasma of liver fibrosis rats and the PCNA,Ki67 positive rate and GST-P optical density and Ishak score in liver tissue can reflect the degree of liver fibrosis.The relationship between the intrinsic activity of CYP2E1 and the degree of hepatic fibrosis in rats with hepatic fibrosis can be obtained by correlating the data.1.4 Statistical analysisThe concentration of diethylnitrosamine was analyzed at different time points to obtain the toxicokinetic parameters using DAS2.0 software,and the peak concentration(Cmax)and peak time(Tmax)was expressed as the measured value.SPSS17.0 statistical software was used to analyze the data.The test level ? is 0.05.2 Results2.1 Liver fibrosis rat model2.1.1 The success rate of liver fibrosis rat modelAt the 12 th week,all the rats in the model group developed fibrosis.The success rate of the liver fibrosis model was 100%.2.1.2 Body weight and liver indexThe average body weight was(336.6±43.6)g in the model group at the 12 th week,which was significantly lower than the control group(P<0.05),the body weight in the model group was(468.9±46.0)g.The liver index of the model group was(4.4±1.0)%,significantly higher than the same period of the control group(P<0.05),the liver index in the control group was(2.8±0.3)%.2.1.3 Liver fibrosis related indicatorsThe AST content in the plasma of the rats in the model group was(58.61±15.50)U/L,and the AST content in the control group was(14.64±3.47)U/L.The plasma AST content in the model group which was significantly higher than that in the control group(P<0.001).The plasma ALT content in the plasma model group was(27.76±8.76)U/L,and the plasma ALT content in the control group was(11.05±2.24)U/L,The plasma ALT content in the model group was significantly higher than that in the control group(P<0.001).The positive rate of PCNA in the liver of the fibrosis model group was(52.10±17.82)%,the positive rate of PCNA in the control group was(12.70±10.22)%,the positive rate of PCNA in the liver of the model group was significantly higher than that in the control group(P<0.001).The positive rate of Ki67 in the liver of the model group was(70.30±20.83)%,and the positive rate of Ki67 in the liver of the control group was(14.30±8.56)%,the positive rate of Ki67 in the liver of the model group was significantly higher than that in the control group(P<0.001).The optical density of GST-P in the liver of the model group was 67.71±16.13,and the GST-P optical density in the liver of the control group was 45.20±10.32,the optical density of GST-P in the liver of the model group was significantly higher than that in the control group(P<0.001).2.2 Toxicokinetics of diethylnitrosamine in rats2.2.1 The toxicokinetics parameters of diethylnitrosamine at the first weekIn the model group,the CL was(0.12±0.05)L/h,AUC0-t was(451.37±135.06)mg/L*h,AUC0-? was(494.88±190.62)mg/L*h,t1/2 was(5.62±2.70)h,Cmax was(59.83±7.04)mg/L,Tmax was(5.62±2.70)h,Vd was(0.75±0.10)L/kg.2.2.2 The toxicokinetics parameters of diethylnitrosamine at the 12 th weekIn the control group,the CL was(0.18±0.03)L/h,AUC0-t was(254.26±22.07)mg/L*h,AUC0-? was(285.04±43.45)mg/L*h,t1/2 was(2.88±0.85)h,Cmax was (57.36±3.84)mg/L,Tmax was(0.15±0.10)h,Vd was(0.72±0.13)L/kg.In the model group,the CL was(0.12±0.05)L/h,AUC0-t was(451.37±135.06)mg/L*h,AUC0-? was(494.88±190.62)mg/L*h,t1/2 was(5.62±2.70)h,Cmax was(59.83±7.04)mg/L,Tmax was(5.62±2.70)h,Vd was(0.85±0.25)L/kg.2.2.3 Differences in toxicokinetics parameters of diethylnitrosamineAt 12 th week,the CL in the model group was significantly lower than the control group(P<0.001),the t1/2 in the model group was significantly higher than the control group(P<0.01),the AUC0-t and AUC0-? in the model group was significantly higher than the control group(P<0.001),Cmax and Tmax and Vd were not significantly different from those in the control group(P>0.05).There was no significant difference in the toxicokinetics parameters of diethylnitrosamine between the control group and the model group at first week(P>0.05).2.3 The correlation between the toxicokinetic parameters of diethylnitrosamine and the degree of hepatic fibrosis in ratsThe toxicokinetic parameters of diethylnitrosamine expressed the metabolic capacity of CYP2E1 for diethylnitrosamine,and the toxicokinetic parameters of diethylnitrosamine in the first week of liver fibrosis in rats showed their CYP2E1 Inherent activity.The levels of AST and ALT in the plasma of liver fibrosis rats and the PCNA,Ki67 positive rate and GST-P optical density and Ishak score in liver tissue can reflect the degree of liver fibrosis.Correlation analysis of toxicokinetic parameters and Ishak score of liver fibrosis in the first week of liver fibrosis model group,AUC0-t and AUC0-? were negatively correlated with the liver fibrosis Ishak score(P<0.05).Analysis of the correlation between toxicokinetic parameters and AST in the first week of rats with liver fibrosis,CL was positively correlated with AST(P<0.01),AUC0-t and AUC0-? were negatively correlated with the AST(P<0.001).Analysis of the correlation between toxicokinetic parameters and ALT in the first week of rats with liver fibrosis,CL was positively correlated with ALT(P<0.05),AUC0-t and AUC0-? were negatively correlated with the ALT(P<0.05).The results showed that the CL of diethylnitrosamine in rats was positively correlated with the degree of liver fibrosis,and the AUC0-t and AUC0-? was negatively correlated with the degree of liver fibrosis,pre-carcinogen diethylnitrosamine metabolism as a carcinogenic material increased,leading to more severe liver fibrosis.3 Conclusion3.1 The toxicokinetics of diethylnitrosamine in normal rats and liver fibrosis rats were reported for the first time.3.2 The toxic effects of liver fibrosis rat CYP2E1 metabolism of diethylnitrosamine activity decreased.3.3 The intrinsic activity of CYP2E1 in rats was significantly correlated with hepatic fibrosis.