【作者】 李述刚；

【导师】 刘开泰；

【作者基本信息】 中国疾病预防控制中心 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 目的:研究砷代谢相关酶AS3MTG35991A、PNP第二外显子密码子20、密码子51、密码子57、GSTO1A140D、MTHFRA222V位点多态性与砷中毒易感性的关系;研究砷代谢相关酶AS3MTG35991A、PNP第二外显子密码子20、密码子51、密码子57、GSTO1A140D、MTHFRA222V位点多态性与砷甲基化水平的关系;研究砷代谢相关酶AS3MT、PNP、GSTO1、MTHFR基因mRNA相对表达与砷甲基化水平的关系,为探讨饮水型砷中毒的发病机制提供参考依据。方法:1.应用现场流行病学调查方法,对内蒙古自治区土左旗、托克托县、山西省山阴县、新疆生产建设兵团123团、131团砷中毒患者、病区对照人群和非病区对照人群进行流行病学调查;2.采用PCR-RFLP、PCR-CTPP方法检测了AS3MTG35991A、PNP第二外显子密码子20、密码子51、密码子57、GSTO1A140D、MTHFRA222V位点多态性;3.采用RT-PCR检测了外周血淋巴细胞AS3MT、PNP、GSTO1、MTHFR mRNA相对表达;4.采用高效液相色谱原子荧光法测定iAs~Ⅲ、iAs~Ⅴ、MMA、DMA 4种形态的砷含量,并计算iAs%、MMA%、DMA%、PMI、SMI等砷甲基化水平指标;5.采用秩和检验、t检验、logistic回归、多重线性回归分析砷代谢相关酶基因多态性及其mRNA表达与砷甲基化水平和砷中毒易感性的关系,采用Pearson相关法分析砷代谢相关酶mRNA表达与砷甲基化水平的关系。结果:1.砷代谢相关酶基因多态性与砷中毒易感性的关系(1)重度组、中度组、轻度组、病区对照和非病区对照组之间在PNP密码子51处G/G、G/A、A/A三种基因型频率分布差异有统计学意义,重度组、中度组携带G/A和A/A基因型频率显著高于非病区对照组和病区对照组;(2)性别对患砷中毒具有一定的影响作用,女性患砷中毒的危险约是男性的0.5倍;年龄对是否患砷中毒具有一定的影响作用,与20岁组相比,50岁组和60-70岁组患砷中毒的风险分别是其1.110(95CI%:1.023-1.522)倍和1.231(95CI%:1.072-1.739)倍;PNP密码子51位点多态性对是否患砷中毒有影响,携带G/A和A/A突变基因型人群患砷中毒的危险是携带G/G基因型人群的1.797倍(95CI%:1.198-4.049):(3)在调整年龄、性别、吸烟、饮酒因素后PNP密码子51位点多态性与砷中毒发病具有显著关联性,携带G/A和A/A基因型人群发生砷中毒的风险是携带G/G基因型人群的1.484倍(95CI%:1.197-5.189);(4)在调整性别、年龄、吸烟、饮酒因素后,携带PNP密码子51和GSTO1A140D的联合突变基因型(G/A+C/A或A/A+C/A)人群患砷中毒的风险是携带(G/G+C/C或G/G+C/A或G/A+C/C或A/A+C/C)基因型人群的2.694倍(95%CI:1.258-5.867):(5)在调整性别、年龄、吸烟、饮酒因素后,携带PNP密码子51和MTHFRA222V位点的突变基因型(G/A+C/T或G/A+T/T或A/A+C/T或A/A+T/T)人群患砷中毒的风险是携带(G/G+C/C或G/G+C/T或G/G+T/T或G/A+C/C或A/A+C/C)基因型人群的3.730倍(95%CI:1.352-6.515);2.砷代谢相关酶基因多态性与砷甲基化水平的关系(1)男性MMA%、PMI显著高于女性(t=2.909,P=0.004,t=2.461,P=0.015),DMA%显著低于女性(t=2.140,P=0.034);经多重线性回归分析可见,年龄对DMA%具有负向影响作用;(2)吸烟者MMA%、PMI显著高于不吸烟者(t=2.376,P=0.019,t=3.506,P=0.001),DMA%显著低于不吸烟者(t=2.776,P=0.006);饮酒者iAs%、MMA%、PMI显著高于不饮酒者(t=2.046,P=0.042,t=2.058,P=0.041,t=2.349,P=0.020);(3)重度组、中度组人群MMA%、PMI显著高于非病区对照和病区对照组;重度组人群DMA%水平显著低于病区对照和非病区对照组人群;(4)经秩和检验可见,AS3MTG35991A位点携带G/G和G/A基因型人群尿中iAs、MMA、DMA、TAs、iAs%、MMA%、DMA%、PMI、SMI差异无统计学意义;(5)PNP基因密码子51的G/G、G/A、A/A三种基因型人群相比,携带A/A、G/A变异基因型人群的尿MMA%显著高于携带G/G基因型人群;DMA%、SMI显著低于G/G基因型人群;PNP基因密码子20和密码子57的C/C、C/T、T/T三种基因型人群之间的iAs、MMA、DMA、TAs、iAs%、MMA%、DMA%、PMI、SMI差别无统计学意义;多重线性回归分析可见,PNP密码子51位点多态性对MMA%有正向影响作用;(6)GSTO1密码子140的C/C、C/A两种基因型人群尿中iAs、MMA、DMA、TAs、iAs%、MMA%、DMA%、PMI、SMI差别无统计学意义;多重线性回归分析可见,GSTO1A140D位点多态性对MMA%具有正向影响作用;(7)MTHFRA222V位点C/C、C/T、T/T三种基因型人群尿中iAs、MMA、DMA、TAs、MMA%、PMI、SMI差别无统计学意义;携带C/T、T/T基因型人群的iAs%显著高于携带C/C基因型人群,DMA%显著低于携带C/C基因型人群;经过多重线性回归分析可见,MTHFRA222V位点多态性对iAs%具有正向影响作用,对DMA%、PMI具有负向影响作用;(8)经秩和检验可见,重度组、中度组MMA%,PMI水平显著高于病区对照组和非病区对照组人群(χ~2=28.83,P＜0.001;χ~2=4.698,P=0.021),而DMA%水平显著低于病区对照组和非病区对照组人群(χ~2=5.519,P＜0.001)。3.砷代谢相关酶基因mRNA表达与砷甲基化水平的关系(1)对砷代谢相关酶mRNA表达分析可见,中重度组人群AS3MT、PNP、MTHFR、GSTO1mRNA表达显著高于非病区对照组和病区对照组人群:(2)砷代谢相关酶mRNA表达与砷甲基化水平相关性分析可见,AS3MT mRNA表达与MMA%(r=0.485,P=0.041)和PMI(r=0.476,P=0.046)呈显著正相关关系;PNPmRNA表达与MMA%(r=0.649,P=0.022)呈显著正相关关系;MTHFRmRNA表达与MMA%(r=0.511,P=0.010)、PMI(r=0.419,P=0.041)呈显著正相关关系。在男性中,MTHFRmRNA表达还与SMI(r=0.530,P=0.008)呈显著正相关关系。GSTO1mRNA表达与砷甲基化水平无显著关联。(3)经过多重线性回归分析可见,对MMA%有正向影响作用的有病情、AS3MTmRNA表达、MTHFR mRNA表达,其他因素对MMA%影响不明显;对PMI有正向影响的有年龄、AS3MTmRNA表达;对SMI有负向影响的有AS3MTmRNA表达,其他因素对SMI影响不明显;对iAs%有负向影响作用的因素是吸烟,而AS3MT、PNP、MTHFR、GSTO1mRNA表达对iAs%影响不明显:结论:1.PNP第二外显子密码子51位点多态性与砷中毒的发生具有显著关联性,在砷暴露人群中携带G/A和A/A突变基因型人群发生砷中毒的风险显著增加;PNP密码子51与GSTO1A140D联合突变基因型即携带(G/A+C/A、MA+C/A)基因型比携带(G/G+C/C、G/G+C/A、G/A+C/C、A/A+C/C)基因型人群患砷中毒的风险显著增加;PNP密码子51与MTHFRA222V联合突变基因型即携带(G/A+C/T、G/A+T/T、A/A+C/T、A/A+T/T)基因型比携带(G/G+C/C、G/G+C/T、G/G+T/T、G/A+C/C、A/A+C/C)基因型患砷中毒的风险显著增加;AS3MTG35991A、PNP第二外显子密码子20和密码子57位点多态性与砷中毒发生无显著关联;2.砷甲基化水平除受到性别、年龄、吸烟、饮酒的影响作用外还受到PNP第二外显子密码子51位点多态性和MTHFRA222V位点多态性的影响。在PNP第二外显子密码子51位点携带G/A和A/A基因型的人群MMA%增高;在MTHFRA222V位点携带C/T、T/T基因型人群DMA%水平显著降低;而AS3MTG35991A、PNP第二外显子密码子20(CAC→CAT)、密码子57(CCC→CCT)位点多态性与砷甲基化水平无显著关联;GSTO1A140D位点多态性可能与MMA%水平增高有关;3.慢性砷暴露可能引起AS3MT、PNP、MTHFR、GSTO1mRNA表达增高;AS3MTmRNA表达增高可能促进PMI水平增高,导致MMA产生过多;PNP mRNA表达增高与MMA%增高有关;MTHFR mRNA表达增高可能促进PMI和SMI水平增高,促进砷在体内进行更有效的甲基化;GSTO1mRNA表达可能与砷甲基化水平无显著相关性;4.砷中毒发病机制与砷的一甲基化水平增高、二甲基化水平降低有关。更多还原

【Abstract】 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.更多还原