Effects Of Arsenic And Its Metabolites On Nrf2 Pathway Gene Expression And Regulation

Objective:The effects of arsenic and treatment factors on gene expression of Keap1-Nrf2-ARE pathway and its relationship with arsenic metabolism were studied in rabbits exposed to low,medium and high doses of mixed arsenic(As³⁺and As⁵⁺)and treated with 5-aza-2'-deoxycytidine?5-Aza-DC?and sulforaphane?SFP?.Methods:90clean-grade rabbits were divided into blank control group,low dose arsenic group(0.075mg kg^-11 w^-1),low dose arsenic+5-Aza-DC group,low dose arsenic+SFP group;medium dose arsenic group(0.15 mg kg^-11 w^-1),medium dose arsenic+5-Aza-DC group,hmedium dose arsenic+SFP group;high dose arsenic group(0.75 mg kg^-11 w^-1),high dose arsenic+5-Aza--DC group and high dose arsenic+SFP group,total of 10 groups,9in each group.Rabbits were exposed to arsenic and water freely for 14 weeks.The urine of rabbits at the 9th week and the urine,skin,blood of rabbits at the 14th week were collected for experiment.1.After 14 weeks of arsenic exposure,the epidermal skin of the same part of the rabbit's back was taken and the morphological changes of skin tissue were observed by electron microscopy.2.The contents of inorganic arsenic?iAs III,iAs V?,DMA and MMA in urine and skin of rabbits were determined by high performance liquid chromatography?HPLC?-Hydride Generation Atomic spectrophoto metry?HGAFS?,and PMI and SMI were calculated.3.Methylation-specific PCR?MSP?was used to detect the methylation of Nrf2,Keap1,Bach1,As3MT and N6AMT1 genes in rabbit skin and blood.4.Western blotting was used to detect the expression of phosphorylated Nrf2?pNrf2?protein in skin and blood of rabbits in each group.Results:1.The outcomes of electron microscopy of rabbit skin indicated that arsenic could basal cell layer abnormality and aggravate skin keratosis.The addition of 5-Aza-DC slightly aggravated basal cell layer abnormality and SFP slightly improved skin tissue injury.2.Metabolic level of arsenic methylation:?14 weeks of skin and urine?Compared with blank control group,,skin As³⁺,DMA,MMA,total arsenic high dose arsenic group,urine As³⁺,DMA,total arsenic low,medium and high dose arsenic group,the above load levels were different?all P<0.05?.Compared with the low-dose arsenic group:skin,urine As³⁺,DMA,MMA,As⁵⁺,total arsenic high-dose arsenic group,urine PMI high-dose arsenic group;skin As³⁺low-dose arsenic+SFP group,urine As³⁺,DMA,MMA,total arsenic low-dose arsenic+SFP group and As³⁺,MMA,total arsenic,PMI low-dose arsenic+5-Aza-DC group,the above load levels were different?P<0.05?.Compared with the medium dose arsenic group,the skin MMA medium dose arsenic+SFP group,urine As³⁺,DMA,MMA,total arsenic,PMI medium dose arsenic+SFP group and medium dose arsenic+5-Aza-DC group,the above load levels were different?P<0.05?.Compared with the high dose group,urine DMA,As⁵⁺,total arsenic,PMI high dose arsenic+SFP group,As⁵⁺,PMI high dose arsenic+5-Aza-DC group,the above load levels were different?P<0.05?.Compared with the low dose arsenic+5-Aza-DC group,skin As³⁺,DMA,MMA,As⁵⁺,total arsenic,PMI arsenic+5-Aza-DC group,urine As³⁺,DMA,MMA,total arsenic,PMI medium and high dose arsenic+5-Aza-DC group the above load levels were different?all P<0.05?.9-week urine:Compared with the blank control group,PMI low-dose arsenic group,As³⁺,DMA,MMA,total arsenic and PMI high-dose arsenic groups,the above load levels were different?all P<0.05?;compared with low-dose group,PMI medium-dose arsenic group,As³⁺,DMA,MMA,total arsenic high-dose arsenic group,the above load levels were different?all P<0.05?;compared with medium-dose group,As³⁺,DMA,MMA,total arsenic,PMI high dose arsenic group,the above load levels were different?all P<0.05?.9 weeks and 14 weeks:Compared with 9 weeks low dose group,As³⁺,MMA,As⁵⁺,total arsenic group increased in 14 weeks low dose arsenic group?t=-16.82,t=-4.43,t=-6.16,t=-3.45,t=11.45,t=20.15,all P<0.05?,while that of PMI and SMI group decreased in 14 weeks low dose arsenic group?t=11.45,t=20.15,all P<0.05?.3.Gene methylation level:Compared with blank control group,the methylation rate of Keap1 gene in high-dose arsenic-exposed skin group and medium,high-dose arsenic-exposed blood group was higher than that in high-dose arsenic-exposed whole?skin?²=8.96,P=0.003;blood?²=6.13,P=0.013?.Compared with the same-dose arsenic group,the blood high-dose arsenic+SFP group,high-dose arsenic+5-Aza-DC group and skin high-dose arsenic+5-Aza-DC group the methylation rate of Keap1 gene showed a decreasing trend??²=6.13,P=0.013;?²=11.03,P=0.001;?²=8.416,P=0.004?;the methylation rate of Bach1 gene in medium-dose arsenic+5-Aza-DC group showed an increasing trend??²=6.13,P=0.013?,while that in high-dose arsenic+5-Aza-DC group showed a decreasing trend??²=6.13,P=0.013?.4.pNrf2 protein level:Compared with the blank control group,the expression of arsenic protein in the middle and high dose arsenic groups of skin was different?all P<0.001?,and the expression of arsenic protein in the low and middle dose arsenic groups of whole blood was different?P<0.001,P=0.023?.Compared with low dose arsenic group,the expression of protein in skin,whole blood medium and high dose arsenic group was different?skin P<0.001,blood P<0.001,P<0.001,P=0.005?.Compared with high dose group,the expression of protein in skin,whole blood high dose arsenic+SFP group and skin high dose arsenic+5-Aza-DC group were different?skin P=0.001,P=0.005;blood P=0.001?.Compared with the low dose arsenic+5-Aza-DC group,the expression of protein in skin and whole blood with high dose arsenic+5-Aza-DC group was different?skin P=0.005;blood P=0.006?.Conlusions:Low arsenic exposure promotes the expression of pNrf2 protein and activates Nrf2 pathway;high arsenic exposure inhibits the expression of pNrf2 protein and inhibits Nrf2 pathway in a dose-dependent manner,and interacts with arsenic metabolism.The methylation rate of Keap1 gene in rabbit skin and whole blood was increased by arsenic exposure;the methylation rate of Keap1 gene in whole blood was decreased by SFP;the methylation rate of Keap1 and Bach1 gene in skin and whole blood was decreased by 5-Aza-DC;and the regulation of gene expression of Nrf2 pathway might affect the process of arsenic methylation metabolism in skin and urine.