The Comparison Of Arsenic Methylation And Genome-wide DNA Methylation Between C57BL/6J And 129X1/SvJ Mice

Objective: Arsenic is an environmental toxicant that seriously endangers human health.The reason for individual differences after arsenic exposure is not clear.The liver is the main target organ of arsenic metabolism,which is an important factor in determining the toxicity of arsenic.In recent years,the change of DNA methylation pattern is a research hotspot of arsenic poisoning mechanism.Genome-wide DNA methylation reflects the methylation state of the whole chromosome,and the occurrence risk of many diseases is closely related to it.It was found that the basal redox level of C57BL/6J(B6)and 129X1/SvJ(129)mice were significantly different,which could simulate the two populations and be used to explore the individual differences after arsenic exposure.Moreover,B6 mice showed obvious oxidative damage after arsenic exposure compared with 129 mice,indicating that there were differences between the two types of mice in susceptibility to arsenic poisoning,among which redox played a certain role,but other causes could not be denied.In this study,two inbred strains of B6 and 129 mice were used to study the effects of arsenic exposure on arsenic methylation and genome-wide DNA methylation in the liver and the differences between the two mouse lines,preliminarily exploring the causes of individual differences after arsenic exposure.Methods: 1.Establishment of mouse model of acute arsenic exposure: Healthy male B6 and 129 mice(weight 20-25 g)of clean grade aged 8 to 12 weeks were randomly divided into three groups by body weight,Veh(physiological saline,24 h),6 h and 24 h 5 mg/kg arsenic-treated groups(n = 6).5 mg/kg sodium arsenite was administrated by gavage once.2.Detection of liver injury: AST,ALT and MDA levels were detected by kit.3.Detection of arsenic metabolism: The content of arsenic and its metabolites were detected by hydride generation-ultra low temperature trapping-atomic absorption spectrophotometer.4.Detection of genome-wide DNA methylation: The genome-wide DNA methylation level in the liver was detected by 5-mC DNA ELISA Kit.5.Detection of the expression levels of enzymes related to arsenic metabolism: Arsenic metabolism-related enzymes includes 5,10-methylene tetrahydrofolic acid reductase(MTHFR),methionine synthase(MTR)and arsenic methyl transferase(CYT19).The mRNA levels of Mthfr,Mtr and Cyt19 in the liver were detected by Reverse Transcript Quantitative Polymerase Chain Reaction(RT-qPCR).The protein levels of MTHFR,MTR and CYT19 in the liver were detected by Western blot.6.Detection of the expression levels of DNA methyltransferases: DNA methyltransferase(DNMT)includes DNMT1,DNMT3 a and DNMT3 b.The mRNA levels of Dnmt1,Dnmt3 a and Dnmt3 b in the liver were detected by RT-qPCR.The protein levels of DNMT1,DNMT3 a and DNMT3 b in the liver were detected by Western blot.7.Statistical analysis: All data in this study were analyzed using GraphPad Prism 7.0 software.Results: 1.Effects of acute arsenic exposure on liver injury: There were no significant differences in AST and ALT.However,compared with the control group,the content of malondialdehyde(MDA)in the liver of B6 mice significantly increased at 24 h after acute sodium arsenite treatment,indicating that there was hepatic oxidative damage in B6 mice.However,there was no significant difference between infected group and the control group 2.Effects of acute arsenic exposure on arsenic metabolism: We examined arsenic metabolism in the liver including Inorganic arsenic(iAs),monomethylarsenic acid(MMA),dimethyl arsenic acid(DMA)and total arsenic(TAs).Notably,the levels of iAs and TAs in the liver of 129 mice was significantly lower than that of B6 mice at 6 h after arsenic treatment(P < 0.05),and was close to that of B6 mice at 24 h.There were lower iAs% and higher DMA% in the liver of 129 mice at 6 h and 24 h after acute arsenic treatment,compared with B6 mice.These data showed that arsenic methylation ability in the liver of 129 mice was better than of B6 mice after acute arsenic treatment.To further test this idea,we examined the concentration and distribution of arsenic in the blood.Data showed significantly higher MMA,DMA and TAs in the blood of 129 mice than that of B6 mice at 6 h and 24 h after acute arsenic treatment(P < 0.05).The iAs% in the blood of 129 mice was significantly lower that of B6 mice,MMA% was significantly higher than that of B6 mice at 6 h and 24 h(P < 0.05),and DMA% was also higher than that of B6 mice at 24 h.These data also showed that arsenic methylation was stronger in 129 mice.We measured the mRNA levels and protein levels of arsenic related enzymes in the liver.The results showed that the mRNA level of Mtr of 129 mice was significantly higher than that of B6 mice at the basal level(P < 0.05).The mRNA level of Cyt19 of 129 mice was significantly higher than that of B6 mice,at 6 h after acute sodium arsenite treatment(P < 0.05).Compared with the control group,the mRNA level of Mtr of 129 mice significantly decreased at 6 h after acute sodium arsenite treatment(P < 0.05).Protein levels of MTHFR and MTR at the basic level and protein level of MTHFR at 6 h after arsenite administration of 129 mice were significantly higher than B6 mice(P < 0.05).Compared with the control group,protein levels of MTHFR and MTR significantly increased of B6 mice at 24 h after arsenite administration(P < 0.05).We could think that arsenic methylation ability of 129 mice was better than of B6 mice after acute arsenic treatment.3.Effects of acute arsenic exposure on genome-wide DNA methylation in the liver: The genome-wide DNA methylation level in the liver of 129 mice was significantly higher than that of B6 mice at the basic level(P < 0.05).The genome-wide DNA methylation level in the liver of 129 mice was significantly lower than B6 mice at 24 h after arsenite administration(P < 0.05).Compared with the control group,the genome-wide DNA methylation level significantly decreased in the liver of 129 mice at 6 h after arsenite administration(P < 0.05),and significantly increased in the liver of B6 mice at 24 h after arsenite administration(P < 0.05).4.Effects of acute arsenic exposure on DNA methyltransferase in the liver: Protein levels of DNMT1,DNMT3 a and DNMT3 b at the basic level and the protein level of DNMT3 b at 24 h after arsenite administration in the liver of 129 mice were significantly higher than B6 mice(P < 0.05).Compared with the control group,protein levels of DNMT1 and DNMT3 a significantly decreased in the liver of 129 mice at 6 h after arsenite administration(P < 0.05).Compared with the control group,protein levels of DNMT1 and DNMT3 a significantly increased in the liver at 24 h and protein level of DNMT3 b significantly increased in the liver at 6 h of B6 mice after arsenite administration(P < 0.05).Conclusion: 1.After acute sodium arsenite poisoning,because the arsenic methylation ability of 129 mice was better than that of B6 mice,the acute liver injury in 129 mice was less severe than that in B6 mice.2.After acute sodium arsenite poisoning,DNA methyltransferase and genome-wide DNA methylation levels of B6 and 129 mice were different.