| Object:Arsenic is a known environmental toxicant and human carcinogen.Long-term exposure to arsenic lead to increased risk or mortality of lung,skin,liver,bladder,kidney,and prostate cancers.Due to species differences,the animal models of arsenic carcinogenesis have not been established.Epidemiological evidence suggests that inorganic arsenic exposure in early life has been associated with an increased incidence of cancer and an increased mortality later in life,but the mechanism is still unclear.Nuclear factor erythroid-2 related factor 2(Nrf2),a member of the CNC-bZIP family,is an important transcription factor that regulates the antioxidant defense,and has dual roles in preventing and promoting cancer.At present,the role and mechanism of Nrf2 in enhanced susceptibility to hepatic tumors after exposed to inorganic arsenic in early life is still unclear.Therefore,the purpose of this study is to explore the changes of Nrf2 in the process of increasing the susceptibility to hepatic tumors in the later life with environmental-related doses of arsenic.The characteristics and effects provide basic data for the study of the carcinogenic mechanism of arsenic.Accordingly,in the first part of this study,to explore the effect of early-life inorganic arsenic exposure-DEN treatment on the susceptibility of liver cancer in mice and to explore the changes and characteristics of Nrf2,wild-type C57BL/6 mice were used to establish a mouse model of hepatic tumor induced by arsenic-Diethylnitrosamine(DEN)in early life in drinking water.In the second part of this study,to explore the mechanism of Nrf2 in the process of enhancing the susceptibility ofhepatic tumor in late life by exposure of inorganic arsenic to environment-related doses in early life,Nrf2 knockout(Nrf2-KO)and wild-type(Nrf2-WT)mice on C57BL/6 background were used to establish a drinking water type arsenic exposure-DEN induced hepatic tumor mouse model.The carcinogenic effect of DEN relies on the metabolic activation mediated by the typical P450 s family member(CYP2E1).Therefore,we speculated that Nrf2 may play an important role in P450 s mediated toxicant metabolism represented by CYP2E1.Based on this,we established thioacetamide(TAA)-induced mouse liver fibrosis model in this part of the study,and further explored the potential role of Nrf2 in CYP2E1-mediated chemical liver injury.In recent years,changes in epigenetics(such as DNA methylation,miRNA expression,histone modification,etc.)have been considered to be closely related to increased susceptibility to cancer caused by inorganic arsenic in early life.A large amount of scientific evidences show that miRNAs play an important role in Nrf2-Keap1 antioxidant pathway.In summary,in the third part of this study,we used the experimental method of combining wild-type C57BL/6 mice and primary hepatocytes in vivo and in vitro to explore the miRNA changes that play a key role in regulation of nrf2-mediated adaptive oxidation response by inorganic arsenic exposure in early life.The purpose of this study was to investigate the role of Nrf2 in early exposure to environment-related doses of inorganic arsenic to enhance susceptibility of hepatic tumor,and to provide data for the study of carcinogenic mechanisms of arsenic.Methods:1.Using wild-type C57BL/6 mice,Nrf2-KO and Nrf2-WT mice on C57BL/6 background as research objects respectively,to establish arsenic and DEN-induced mouse hepatic tumor model by drinking water.C57BL/6 mice were randomly divided into control group(double distilled water),and arsenic treatment group(the arsenic concentration in drinking water was 0.5 ppm and 5 ppm,respectively),mice were free to feed and drink.The Nrf2 knockout mouse model uses Nrf2 knockout heterozygote(Nrf2-HET)mice as parents,grouping principle and method of arsenic exposure are the same as C57BL/6 mice,and the offspring of Nrf2-WT and Nrf2-KO mice are collected as research objects.After weaning(4-week-old),non-tumor model male pups ate normally,drank double distilled water,and monitored the basic physiological indexes(body weight,organ coefficient,body fat composition and blood glucose)and liver oxidative stress state of pups(4-and 8-weekold).Two weeks after the birth of male pups of the tumor model,the control group and each arsenic-exposed group were randomly divided into a blank group(Vehicle,Veh)and a DEN group.DEN group mice were given intraperitoneal injection of DEN(25 mg/kg BW),Veh group mice were given intraperitoneal injection of the same volume of normal saline.After weaning,the mice in each group were normally fed and observed to 40-weekold to evaluate the formation of liver tumor.2.The liver of 4-week-old mice from 0.5 ppm inorganic arsenic group and the control group were collected.The differences of miRNA expression profiles between the two groups were analyzed by high-throughput miRNA array.The miRNAs interacting with Nrf2-3'-UTR were screened by target gene prediction software combined with literatures.Primary hepatocytes were extracted,and miRNA mimics and inhibitors were transiently transfected to up-or down-regulate miRNA expression,and control and transfection groups were established to detect the expression of Nrf2 and its downstream genes in primary hepatocytes.3.Nrf2-KO and Nrf2-WT mice on C57BL/6 background were used to establish TAA-induced liver injury and liver fibrosis models.The male 8-12-week-old mice were randomly divided into control group(intraperitoneal injection of normal saline)and TAA group(intraperitoneal injection of TAA).Acute experiment: TAA dose of 50 mg/kg BW,observe mouse response and record 60 h survival curve;TAA dose of 30 mg/kg BW,analyze liver injury;subacute experiment: TAA 50,100,200 and 300 mg/kg BW with escalating dosing regimen,three times a week for 5 weeks.The liver function level,TAA metabolite level,mRNA and protein levels of liver injury and fibrosis-related genes were measured.The primary hepatocytes were treated with TAA(20 ?M)to observe the CYP2E1 protein level.Results: 1.Effects of inorganic arsenic exposure in early life on basic physiological parameters of pups.There was no statistically significant difference in the weight,blood glucose,and organ coefficients of 4-week-old weaning male pups after exposed to inorganic arsenic in early life(P > 0.05).The body wight of 8-week-old male pups was decreased after exposed to arsenic in early life(P < 0.05),but there was no statistically significant difference in organ coefficients and blood glucose(P > 0.05).2.Exposure to inorganic arsenic in early life aggravates DEN-induced liver tumors in mice.Tumor burden assessment was performed on 40-week-old pups,and DEN treatment alone could induce the occurrence of liver tumors in mice.There is no significant difference of incidence of liver tumors in 40-week-old pups between DEN-treated group and arsenic exposure in early-life combined with DEN group(P > 0.05).However,the tumor volume was larger in arsenic and DEN treatment group than DEN group.There was a dose-response relationship(P < 0.05).According to immunohistochemical staining,the number of PCNA positive cells in the liver of mice exposed to arsenic combined with DEN treatment(DEN + 0.5 ppm As and DEN + 5 ppm As)was higher than that in DEN treatment alone.3.Nrf2 was activated in the liver tumors of mice induced by DEN.Compared with control liver tissue,the level of NRF2 protein in the cytoplasm of liver tumor tissues did not change significantly,but the nuclear enrichment of NRF2 in tumor tissues increased significantly(P < 0.05).In addition,the mRNA levels of Nrf2 and its downstream genes(Gs,Homx1,and Nqo1)were significantly increased in liver tumor tissues(P < 0.05).4.Nrf2 activation exists in human liver cancer tissues and may be related to poor prognosis of liver cancer.Gene expression profiling of human liver cancer tissues revealed that NRF2 expression was higher in human liver cancer tissues than in normal liver tissues.Survival analysis of liver cancer patients found that the overall survival rate of patients with NRF2 overexpressing liver cancer was lower than that of patients with low expression of NRF2.The high expression of NRF2 in liver cancer tissue may be related to poor prognosis of liver cancer(P = 0.09).The matrix diagram was used to compare the expression of downstream genes of NRF2 in human liver cancer tissues.It was found that the expression levels of downstream genes of NRF2(GSS,GSR,SQSTM1,GCLM,GCLC,HMOX1 and NQO1)were showed an increasing trend in liver tumor tissues compared with normal liver tissues,and NRF2 and its downstream genes showed a good correlation in liver cancer tissues.5.Inorganic arsenic exposure in early life induces Nrf2 activation in the liver from 4-week-old pups.Compared with control group,the levels of glutathione(GSH)and total glutathione(GSH + GSSG)in whole blood of 4-week-old pups exposed to arsenic in early life were showed an increasing trend.Compared with control group,the ratios of GSH/GSSG was increased in 5 ppm arsenic-exposed group as well as the levels of GSH and(GSH + GSSG)(P < 0.05).The mRNA and protein levels of Nrf2 in the liver of mice exposed to arsenic in early life were significantly higher than that in control group.The mRNA levels of Nrf2 downstream genes Gs,Gr,and Hmox1 were higher than that of the control group(P < 0.05);the protein levels of GS,GR,p62,GCLC and GCLM in the liver of mice exposed to arsenic in the early life were higher than that of the control group(P < 0.05).6.Confirmation of Nrf2-global knockout mouse model.The expressions of Nrf2 and its downstream genes(Gr,Gs,p62,Nqo1,Gclc,and Hmox1)in the liver of mice exposed to inorganic arsenic in early life were detected by qPCR and Western blot.The mRNA levels of Nrf2,Gr,Gs,p62 and Nqo1 in the liver of Nrf2-KO mice were significantly reduced in the arsenic-exposed group(5 ppm)(P < 0.05).The protein level was significantly reduced in the arsenic-exposed group(0.5 ppm)(P < 0.05).7.Nrf2 deficiency attenuates DEN-induced liver tumors after inorganic arsenic exposure in early life.Analysis of tumor formation in 40-week-old mice found that the incidence of liver tumors in Nrf2-KO mice treated with DEN alone,DEN + 0.5 ppm arsenic exposed,and DEN + 5 ppm arsenic exposed were lower than that in Nrf2-WT mice.The tumor index and tumor burden of mice from DEN + 5 ppm arsenic exposure group were significantly higher than that from the DEN alone treatment group(P < 0.05).In addition,the liver tumor index and tumor burden of Nrf2-KO mice in the DEN + 0.5 ppm arsenic exposure group and DEN + 5 ppm arsenic exposure group were decreased compared with Nrf2-WT mice(P < 0.05).Compared with the control group,the tumor burden of Nrf2-KO mice was lower than that of Nrf2-WT mice in DEN + arsenic exposure group(P < 0.05).8.Inorganic arsenic exposure in early life has potential impact on P450 s.According to the results of mRNAseq,KEGG enrichment analysis found that many differential genes were enriched to arachidonic acid metabolism,P450-mediated exogenous chemical metabolism and chemical carcinogenic pathways after arsenic exposure in early life.9.Nrf2 deficiency inhibits TAA metabolism activation and attenuates liver injury in mice by promoting CYP2E1 protein degradation.HE staining and Sirius red staining showed that compared with Nrf2-WT mice,liver fibrosis in Nrf2-KO mice was reduced and the protein level of COL1?1 in liver tissue of Nrf2-KO mice was lower than that of Nrf2-WT mice after TAA treatment(P < 0.05).After 50 mg/kg TAA treatment for 60 h,the survival rate of Nrf2-WT mice was 57%,while the survival rate of Nrf2-KO mice was 86%.After 30 mg/kg TAA treatment,the plasma ALT and AST levels of Nrf2-WT and Nrf2-KO mice were significantly increased(P < 0.05);and the plasma ALT levels of Nrf2-KO mice were significantly lower than that of Nrf2-WT at 48 h mice(P < 0.05);HE staining showed that both Nrf2-WT and Nrf2-KO mice had acute liver injury.After TAA treatment for 24 h,the ratio of TAASOx/(TAA+TAASOx)in the urine of Nrf2-KO mice was significantly lower than that of Nrf2-WT mice(P < 0.05),and the protein level of Ac-K in liver of Nrf2-KO mice was significant lower than Nrf2-WT mice(P < 0.05).Further extraction of mouse primary hepatocytes revealed that the half-life of CYP2E1 protein in primary hepatocytes of Nrf2-WT and Nrf2-KO mouse were 29.4 h and 22.6 h after TAA treatment,respectively.10.Effects of inorganic arsenic exposure on liver microRNAs in offspring.Early-life exposure to inorganic arsenic caused significant changes in liver microRNA(miRNA)profiles in 4-week-old pups.Using target gene prediction software and combing the literature to screening miRNAs that directly target Nrf2,and verifying the expression of the target miRNA obtained through the above screening and its effect on Nrf2 expression and activity.It was found that miR-126-3p,miR-677-3p,miR-7664-3p,miR-3087-5p,miR-144-3p,and miR-27-3p were significantly decresed after exposed 0.5 ppm arsenic in early life(P < 0.05).11.miR-144-3p can regulate the expression and activity of Nrf2 in primary hepatocytes.According to miRNA expression abundance(CT = 24-28)and conservation,4 miRNAs were selected for further functional verification.Functional verification in vitro revealed that miR-144-3p mimic(50 nM,48 h)was transfected into primary hepatocytes and found that the mRNA and protein levels of Nrf2 were significantly reduced(P < 0.05).When miR-144-3p inhibitor(100 nM,48 h)was transfected into primary mouse hepatocytes,the protein level of NRF2 increased significantly(P < 0.05),and the mRNA levels of Nrf2 and its downstream genes(Gs,Gr,p62,Gclc,Gclm,Nqo1 and Hmox1)were significantly increased(P < 0.05).12.miR-3087-5p has potential effect on Nrf2 expression.Further in vitro experiments were performed for functional verification of miR-7664-3p,miR-27-3p,and miR-3087-5p inhibitors after transfected into primary mouse hepatocytes,separately.As a result,it was found that miR-7664-3p and miR-27-3p inhibitors(100 nM,48 h)did not affect Nrf2 expression in primary hepatocytes.After treatment with miR-3087-5p inhibitor(100 nM,48 h),the mRNA level of Nrf2 did not change significantly(P > 0.05),but protein level of NRF2 was significantly increased(P < 0.05).The mRNA levels of Nrf2 downstream genes Gr and Gclm were decreased(P < 0.05).Conclusion:1.Arsenic exposure in early life at environmental levels induced Nrf2 activation and enhanced DEN-induced susceptibility to hepatic tumors in pups.2.Nrf2-mediated adaptive antioxidant was involved in the enhancement of susceptibility to hepatic tumors in mice induced by arsenic exposure in early life,and Nrf2 dificiency significantly inhibits the enhanced susceptibility to hepatic tumors induced by arsenic exposure in early life.3.Nrf2 deficiency inhibit the stability of CYP2E1 protein,thereby reducing the liver injury effect caused by CYP2E1-mediated metabolic activation of chemicals.4.Arsenic exposure in early life could affect Nrf2 expression and activity through microRNA: miR-144-3p has regulatory effect on Nrf2 expression and activity in primary hepatocytes,miR-3087-5p has potential effect on Nrf2 expression and activity in primary hepatocytes. |
PDF Full Text Request