The Study On The Relationship Between The Genetic Polymorphisms And MRNA Expression Of Arsenic Metabolic Genes And The Susceptibility Of Arsenism And Arsenic Methylation

Objective:To investigate the correlation between the genetic polymorphisms of arsenic metabolic enzyme genes AS3MTG35991A,PNP codon 21,codon51,codon 57,GSTO1A140D,MTHFRA222V and the susceptibility of arsenic poisoning.To study on the relationship between the genetic polymorphisms of arsenic metabolic enzyme genes AS3MTG35991A,PNP codon 21,codon51,codon 57,GSTO1A140D, MTHFRA222V and the level of the arsenic methylation metabolism.To research on the association between the gene mRNA expression of AS3MT,PNP,MTHFR, GSTO1 and the level of the arsenic methylation metabolism.To provide reference for the mechanism of arsenic poisoning.Methods:1.The arsenisms,controls in arsenicosis district and controls in non-arsenicosis district were invested with the field epidemiology method in Innor Mongolia Autonomous Rigeon,Shan Xi Province and Xin Jiang Production and Construction Corps.2.The genetic polymorphisms of AS3MTG35991A,PNP codon 21,codon51,codon 57,GSTO1A140D,MTHFRA222V were detected by the PCR-RFLP and PCR-CTPP.3.The mRNA expression of AS3MT,PNP,GSTO1,MTHFR were detected by RT-PCR. v4.The concentration of the 4 forms of iAs~Ⅲ,iAs~Ⅴ,MMA,DMA in urine and water were detected by the high efficiency liquid chromatogram HG-AFS.The value of iAs%,MMA%,DMA%,PMI and SMI were calculated by the concentration of iAs~Ⅲ,iAs~Ⅴ,MMA,DMA in urine.5.The noparamatric test,t test,logistic regression model,multiple linear regression model were used to analyze the relationship between the genetic polymorphisms of the arsenic metabolic genes,the level of the arsenic methylation metabolism and the susceptibility of the arsenic poisoning.The Pearson correlation methods were used to elucidate the association between the mRNA expression of arsenic metabolic genes and the level of the arsenic methylation metabolism.Results:Ⅰ.The correlation between the genetic polymorphisms of the arsenic metabolic enzymes and the suceptibiliy of arsenism(1) There was a significant difference on the genotypes frequences distribution of PNP codon51 among the groups of the arsenism with sever,moderate,slight symptoms and the control groups in arsenism district and the nonarsenism district. The frequencies of the genotype G/A,A/A of PNP codon 51 of the arsenism group with sever and moderate symptoms were significant higher than the controls in arsenism district and nonarsenism district.(2) The gender could affect the susceptibility of arsenic poisoning.Female was significantly easier to be arsenic poisoning than male(OR=0.5,95%CI:0.251-0.996). The age also was an affecting factor for the susceptibility of arsenism.Compared with 20-yeare-old group,the possibility of the 50-,60-70 group were much higer (OR=1.110,95%CI:1.023-1.522,OR=1.231,95%CI:1.072-1.739).Individuals who carried with the genotypes of G/A,A/A of PNP codon 51 got much possibility than that of those who carried with the genotype of G/G(OR=,95%CI:1.198-4.049).(3) Under control of age,gender,smoking,alcohol using,the genetic polymorphisms of PNP codon 51 had a significant association with the susceptibility of arsenism.The hazard of individuals who carried with the genotypes of G/A,A/A was 1.484 times of those who carried with the genotype of G/G of PNP codon 51(OR=1.484,95%CI:1.197-5.189).(4) After controling the factors of gender,age,smoking,alcohol using,the results of gene combination analysis of logistic indicated that the individuals who carried with the genotype of(G/A+C/A or A/A+C/A) of PNP codon 51 and GSTO1 codon 140 had higher hazard of being arsenic poisoning than those who carried with the genotype of(G/G + C/C or G/G+C/A or G/A+C/C or A/A+C/C)(OR=2.694,95%CI: 1.258-5.867).(5) Under adjusting the gender,age,smoking,alcohol using,the logistic regression results indicated that individuals who carried with the genotypes of(G/A+C/T or G/A+T/T or A/A+C/T or A/A+T/T) of PNP codon 51 and MTHFRA222V had significant risk of being arsenic poisoning than those who carried with the genotype of(G/G+C/C or G/G+T/T or G/A+C/C or G/A+C/C or A/A+C/C)(OR=3.730, 95%CI:1.352-6.515).Ⅱ.The correlation between the genetic polimorphisms of the enzymes related with arsenic metabolism and the level of arsenic metabolism(1) The level of MMA%and PMI in urine of male were significantly higher than that of female(t=2.909,P=0.004;t=2.461,P=0.015),while the level of DMA%was significantly lower than that of female(t=2.140,P=0.034);According to the multiple linear regeression,age played a negative role of the level of DMA%.(2) The level of MMA%,PMI in urine of the smoking population were significantly higher than that of nonsmoking population(t=2.376,P=0.019;t=3.506,P=0.001), while the level of DMA%in urine was significantly lower than that of nonsmoking population(t=2.776,P=0.006).The level of iAs%,MMA%,PMI in urine of the alcohol using population were significantly higher than that of nonalcohol using population(t=2.046,P=0.042;t=2.058,P=0.041;t=2.349,P=0.020).(3) The level of MMA%,PMI in urine of arsenism with sever and moderate symptoms were significantly higher than that of the control population in arsenic poisoning district and nonarsenic poisoning district.The level of DMA%in urine of the sever arsenic poisoning population was significantly lower than that of the control population in arsenic poisoning district and nonarsenic poisoning district.(4) According to the nonparametric test,the level of iAs,MMA,DMA,TAs,iAs%, MMA%,DMA%,PMI,SMI in urine of individuals who carried with the genotype G/G were not significantly different from that of the ones who carried with the genetype G/A ofAS3MTG35991A.(5) The level of MMA%in urine of the population who carried with the genotypes G/A,A/A was much higher than that of the population who carried with the genotype G/G of PNP codon 51.While the level of iAs,MMA,DMA,TAs,iAs%, MMA%,DMA%,PMI,SMI were not significantly different among the three groups who carried with the three genotypes C/C,C/T,T/T of PNP codon 20 and codon 57. According to the multiple linear regeression,the genetic polymorphisms of PNP codon 51 impacted the level of the MMA%positively.(6) There were no significant difference between the two genotype groups who carried with C/C,C/A of GSTO1 codon 140 on the level of iAs,MMA,DMA,TAs, iAs%,MMA%,DMA%,PMI,SMI in urine.According to the multiple linear regeression,the genetic polymorphisms of GSTO1A140D impacted the level of the MMA%positively.(7) Compared with the group who carried with C/C of MTHFRA222V,the genotypes of C/T,T/T groups had significantly higher iAs in urine and lower DMA%. While there was no significant difference on the level of iAs,MMA,DMA,TAs, MMA%,PMI,SMI in urine among the three genotypes groups.According to the multiple linear regeression,the genetic polymorphisms of MTHFRA222V impacted the level of iAs%negatively,while the level of MMA%,PMI positively.(8) According to the rank test,the level of MMA%,PMI of the arsnicisms with sever and moderate symptoms were significantly higher than those controls in arsenism district and nonarsenism distrit(χ~2=28.83,P＜0.001χ~2=4.698,P=0.021),while the level of DMA%was significantly lower(χ~2=5.519,P＜0.001).Ⅲ.The correlation between the mRNA expression of the arsenic metabolic enzymes and the level of the arsenic metabolism.(1) According to the analysis of mRNA expression of the arsenic metabolic eczymes, The mRNA expression of AS3MT,PNP,MTHFR,GSTO1 of arsenism with moderate and sever symptoms were significantly higher than that of the controls group in arsenism district and nonarsenism district.(2) Based on the correlation analysis between the mRNA expression of arsenic metabolic enzymes and the arsenic methylation level,There was a significant positive correlation between the mRNA expression of AS3MT and the level of MMA% (r=0.485,P=0.041),PMI(r=0.476,P=0.046) in urine.There was a positive correlation between the mRNA expression of PNP and MMA%(r=0.649,P=0.022) in urine.There was a positive correlation between the mRNA expression of MTHFR and the level of MMA%(r=0.511,P=0.010),PMI(r=0.419,P=0.041) in urine.The positive correlation between the mRNA expression of MTHFR and SMI(r=0.530,P=0.008) was found only in males.The positive association between the mRNA expression of GSTO1(r=0.606,P=0.037) and PMI was also observed in males.(3) According to the multiple linear regression,smoking is an affecting factor to the level of iAs in urine,while the mRNA expression ofAS3MT,PNP,MTHFR,GSTO1 were not significant affecting factors to the level of iAs.The severity of the arsenism, the mRNA expression ofAS3MT,MTHFR were significant positive affecting factors to the level of MMA%,age,the mRNA expression of AS3MT were significant positive affecting factors.The mRNA expression of AS3MT and alcohol using were significant negative affecting factors to the level of SMI;Conclusions:1.The level of arsenic methylation was affected by the following factors:gender,age, smoking,alcohol using,the genetic polymorphisms of PNP codon 51 and MTHFRA222V.The level of MMA%in urine of the individuals who carried with the genotype of G/A,A/A of PNP codon 51 was significantly higher than those who carried with the genotype of G/G.The level of DMA%in urine of the individuals who carried with the genotype of C/T,T/T of MTHFRA222V was significantly lower than those who carried with the genotype of G/G.There were no significant association between the genetic polymorphisms of AS3MTG35991A,PNP codon 20,PNPcodon 57 and the level of arsenic methylation.The genetic polymorphisms of GSTO1A140D was possiblely related with the increase of MMA%,it needs further research.2.Chronic arsenic exposure could induce the overexpression of mRNA of AS3MT, PNP,MTHFR,GSTO1.The overexpression of mRNA of AS3MT possibly improve the increasing of the level of PMI,then induce the overcomposed MMA.The overexpression of mRNA of PNP also could induce the overcompose of MMA in urine.The overexpression of MTHFR could increase both the leve of PMI and SMI, which promoted the much effiency arsenic methylation.While there was no significant correlation between the expression of mRNA of GSTO1 and the level of arsenic methylation.3.The genetic polymorphisms of PNP codon 51 was significantly associated with the susceptibility of arsenism.With the exposure of arsenic,the individuals who carried with the genotypes of G/A,A/A had significantly higher hazard of arsenism than those who carried with the genotype of G/G of PNP codon 51.The combination of genes also could increase the susceptibility of arsenism.The combination of the genetic polymorphisms of PNPcodon 51 and GSTO1A140D,PNP codon51 and MTHFRA222V could significantly increase the susceptibility of arsenic poisoning.4.The arsenism was possibly associated with the increasing of the level of primary methylation and the decreasing level of second methylation of the arsenic.The severity of arsenism could affect the level of arsenic methylation in body.