【作者】 田丽婷；

【导师】 金泰廙；

【作者基本信息】 复旦大学 ， 毒理学， 2010， 博士

【摘要】 铅(lead, Pb)是金属元素,是受WHO、IARC、美国ATDSR关注的重金属环境污染物,对多种人体器官皆具有毒害作用。肾脏是铅毒性作用的主要靶器官,铅进入人体后大部分经肾脏排泄。血铅能较好地反映机体近期铅接触情况,当血铅浓度>1.9μmol/L (400μg/L)时,即可出现肾损害,长期接触低浓度铅的作业工人肾病检出率较高。本研究的目的是通过对职业铅接触人群的流行病学调查,分析铅接触对人群肾小管和肾小球的损伤,并将基准剂量引入铅接触引起的肾损伤危险度评价,以求得到人群短期、高剂量接触铅对肾损伤效应的剂量-反应关系。通过研究金属硫蛋白与铅接触标志和效应标志之间的关系来探讨将尿金属硫蛋白作为铅致肾损伤效应参考指标的可能性；进而分析金属硫蛋白基因多态性对铅致肾损伤的修饰作用并探讨其作用机制,积极寻找短期内高浓度铅接触所致肾损伤的易感人群,在不同性别人群与铅相互作用后出现的不同程度肾损伤方面进行初步的机制探讨。研究选取浙江省某蓄电池厂工人239人作为接触组,其中男性172人、女性67人；选取同地区某厨具厂工人76人作为对照(所招募工人无铅作业接触史)；其中男性50人、女性26人。接触组铅作业工人工龄为<1年-5年,平均为1.23年。两组在工龄、年龄、身高、体重、血压、吸烟、饮酒等方面均衡可比。本研究收集工厂2005年-2007年的空气铅资料,估算每一位工人所接触的铅外剂量,同时测定了人群铅接触生物标志(血铅和尿铅)与反映肾功能的生物标志(尿肌酐、尿NAG、尿ALB)以及待评价的铅接触参考指标(尿金属硫蛋白),分析了接触标志和效应标志之间的关系,计算了各效应标志的血铅和尿铅的基准剂量；选取金属硫蛋白1A亚型和2A亚型基因上的4个位点,采用实时荧光定量PCR(RTPCR)方法分别测定其基因型,分析了各种基因型在铅接触并致肾损伤过程中的修饰作用,初步探讨其可能的机制。通过对研究对象进行铅接触评估,研究发现：接触组空气铅接触剂量远高于对照组,空气铅在男性工作场所明显高于女性工作场所；外剂量与内剂量相关性良好。接触组工人铅的内剂量都较高,无论男、女,其血铅、尿铅水平均高于对照组,差异显著。在同一个组内,男女性血铅差异无统计学意义,女性尿铅则高于男性,差异显著。年龄和工龄对血铅、尿铅水平的影响无统计学意义;在铅接触剂量较低的情况下(如对照组),吸烟对铅接触有一定的影响。通过铅对肾小管和肾小球功能影响的研究发现：按照研究对象的外剂量水平分别分组后,UNAG、UALB水平在空气铅接触指数高于1mg·month的两个组均高于低剂量组,差异显著,尤其是女性。根据工人体内的血铅水平和尿铅水平分别将工人分为4个剂量组,工人在高于或略低于GBZ2-2002中规定的铅吸收标准时,UNAG和UALB水平即出现明显增加的现象。UANG在血铅、尿铅各剂量组均出现递增的趋势,UALB在尿铅各组出现递增的趋势,在血铅各组虽有增加,但差异无统计学意义。以对照组各指标第95百分位数作为上限值,得到人群各指标的异常率。UNAG异常率在空气铅、血铅、尿铅各组随着暴露剂量升高,异常率也随之升高,有显著性趋势。而女性和总体的UALB异常率在空气铅、血铅、尿铅各组随着暴露剂量升高,异常率也随之升高,有显著性趋势。经过计算UANG、UALB的血铅、尿铅BMDL值后发现,男性、女性、总体的UNAG的血铅BMDL分别为402.03μg/L、264.67μg/L和362.56μg/L,尿铅BMDL分别为81.80μg/gCr、71.52μg/gCr和78.79μg/gCr;男性、女性、总体的UALB的血铅BMDL分别为962.44μg/L、285.44μg/L和607.76μg/L,尿铅BMDL分别为153.37μg/gCr、75.71μg/gCr和117.79μg/gCr。UNAG的BMDL值总是低于UALB,而女性各指标的BMDL,总是低于男性。金属硫蛋白水平与铅接触关系研究发现：UMT与血铅和尿铅之间有着密切的正相关关系。其水平随着内剂量增加也逐渐递增。以对照组各指标第95百分位数作为上限值,得到人群UMT的异常率。UMT异常率在空气铅、血铅、尿铅各组均逐渐增加,趋势显著。经过计算UMT的血铅、尿铅BMDL值后发现：男性、女性和总体的血铅BMDL分别为411.85μg/L、310.99μg/L和398.60μg/L,尿铅BMDL分别为76.96μg/gCr、87.29μg/gCr和87.37μg/gCr;各人群UMT的血铅BMDL均高于UNAG,女性UMT的血铅BMDL高于UALB,女性UMT的尿铅BMDL高于UNAG和UALB,男性UMT的尿铅BMDL均低于UNAG和UALB。通过MT1A、MT2A亚型4个位点基因多态性的研究发现：机体对铅吸收、铅致肾损伤的易感性均与MT的遗传多态性有关。4个位点各自的3种基因型均在研究人群中找到。MT1A-rs11076161的基因多态性对研究对象总体的血铅、尿铅均有显著的修饰作用(P<0.01),AA基因携带者表现出铅吸收较其它基因型的人为低的趋势,这种趋势在低接触组男性工人中较为明显。rs28366003的GG型与其它两种基因型的组合相比,低接触组血铅值较高,携带rs28366003-AA型的女性血铅较高。携带rs11076161-AA型的高接触组男性尿铅较低而女性并未有这种变化。携带rs10636GG基因型的人在高接触组血铅和尿铅值较低即铅吸收较少,差异显著,尤其是男性。携带rs1610216-CC型的高接触组男性铅吸收较低,而同样携带这种基因型的女性则相反,铅吸收较高。通过研究MT基因多态性与铅致肾损伤之间的关系发现：携带rs11076161-AA基因型的女性肾小管和肾小球损伤标志的水平均较其它基因型携带者为低。rs10636-GG基因型携带者的UNAG值较低,这种现象在高接触组中更为明显,尤其是男性。携带rs28366003-AA型的高接触组女性尿NAG水平较高,携带rs28366003-GG型的高接触组女性尿ALB水平较高。携带rs1610216-CC基因型的高接触组男性尿NAG水平较低,而携带同样基因型的女性却有着较高的尿NAG和ALB水平,差异显著,同时吸烟和饮酒对所研究各研究群体的各基因位点多态性的修饰作用均无影响。以上结果表明：①空气铅可以作为高浓度铅作业工人铅接触的一个参考指标。②人群在高剂量铅作业时即使是短期作业也能明显引起肾小管、肾小球损伤。通过各指标血铅、尿铅BMDL值的比较,可以认为肾小管损伤早于肾小球损伤。③尿金属硫蛋白与铅的内剂量有剂量-效应关系和剂量-反应关系,提示其可以作为铅致肾损伤的一个参考指标。④女性接触的空气铅低于男性,而血铅水平与男性相似,尿铅高于男性；同时女性的肾损伤指标的血铅、尿铅BMDL均低于男性,说明女性易蓄积铅,对铅致肾损害更加敏感。⑤MT家族中的MTIA亚型和MT2A亚型是铅毒性的易感基因。在相同的职业外暴露环境下,MT1A-rs11076161的AA基因型携带者表现出铅吸收较其它基因型的人为低的趋势；铅接触量较低时MT2A-rs28366003的GG基因型携带者体内铅吸收水平较高；研究对象总体中MT2A-rs10636-GG基因型和MT2A-rsl610216-CC基因型携带者肾小管损害程度较低。⑥MT基因多态性对不同性别工人的铅毒性有不同的修饰作用：在铅接触剂量较低时,MT1A-rs11076161-AA基因型携带者中男性其铅在尿液中蓄积较多,这种基因型携带者中的女性肾小管和肾小球受损程度较低。在铅接触剂量较高时,MT2A-rs10636-GG基因型携带者中的男性铅负荷水平较低且肾小管损害程度较低；MT2A-rs1610216-CC基因型携带者中,男性铅负荷水平较低而女性铅负荷水平较高,同时男性肾小管损害程度较低而女性肾小管和肾小球损害程度均较严重。⑦MT2A-rs10636对研究总体、不同性别的铅吸收、肾损伤均有不同程度的修饰作用,携带CC+CG基因型的人群与GG型相比,UNAG水平明显较高,经比较,两个人群的血铅BMDL分别为300.88、378.68μg/L,说明前者对铅致肾损伤较为易感。前者人群在总人群中占45.05%,近一半,提示人群中有近一半的人对铅致肾损伤较为易感,因此,该位点多态性有较大的实际意义。更多还原

【Abstract】 Lead (Pb), is one of heavy metal elements, which has been considered as a harmful pollutant of world wide concern. Lead is excreted through kidney, so it is the major target organ of lead. Blood lead can reflects the recent exposure to lead, for example, people subject to renal impact when their blood lead climbed up to over 1.9μmol/L (400μg/L), on the other hand, it has high renal dysfunction mortality when it turned to low-dose and long-term lead exposureThe purpose of this study are:To investigate renal dysfunction in lead exposed workers using epidemiological study, particularly estimating bench mark dose in order to characterize the relationship between lead exposure dose and lead induced renal dysfunction, to examine the the possibility using urinary metallothionein as the biomarker in lead exposed workers and to identify the role of metallothionein gene polymorphism on the modification of renal dysfunction in the whole course.The target population comprised people who worked in a storage battery plant and a kitchenware factory in Zhejiang Province. The exposure group consisted of 237 workers (male 172, female 67); the control group consisted of 76 workers (male 50, female 26), who have not joined the job exposed to lead. The lead worker has lead related work history from less than 1 year to 5years,1.23 years averagely. Two groups are comparable in age, work history, height, weight, BP and daily habits (smoking, alcohol).We collected the documents of air lead (2005～2007) of the storage battery plant, according to which the external dose of lead was calculated for every worker. The lead exposure indicators (blood lead, urinary lead) and effect biomarkers (UNAG, UALB), plus UMT were measured at the same time. Then the relationship among the parameters mentioned above were analyzed and bench mark dose (BMD), the lower confidence limit of the bench mark dose (BMDL) were estimated for the adverse effect of renal system. After choosing 4 SNPs in MT-1A and MT-2A gene, the genotype for each subject was detected using real time PCR technique, following which the modification of each SNPs were evaluated.According to the exposure assessment, it was found that:the external dose was well correlated to internal dose, it was much higher in exposure group than that in control group and was higher in male workers than that in the female. The internal dose, both blood lead and urinary lead, was significantly higher in both men and women of exposure group than that in control group. Blood lead was similar in men to women when they were in a same group, while their urinary lead was totally different, it was significantly higher in women than that in men. In addition, it seems that age could not influence internal dose, but length of working history was correlated to lead body burden and smoking could affect the status when people exposure to lower lead dose.Concerning renal dysfunction, when the air lead was over lmg-month, the level of UNAG raised up then, especially for women. Once the body burden climbed over the GB for lead absorption, both of UNAG and UALB increased significantly. In each group of blood lead or urinary lead, UNAG increased gradually, UALB showed the same trend in each group of urinary lead, while this trend was not obvious in groups f blood lead. We then defined the cutoff point based on the 95th percentile value for UNAG, and UALB. The cutoff points of UNAG and UALB of subjects were 10 U/g Cr and 25mg/g Cr, respectively. The prevalence of hyperNAGuria and hyperALBuria at internal dose of lead were calculated in all subjects. In general, the results showed the significantly increased prevalence of hyperNAGuria and hyperALBuria. The prevalence of hyperNAGuria of subjects showed significant increasing trend with the increase of of BPb in lead exposed groups. The prevalence of hyperALBuria has significant increasing trend in total group and the female. As far as BMD was concerned, the B-Pb BMDL of UNAG in men, women and the total were 402.03、264.67、362.56μg/L respectively, and 81.80、71.52、78.79μg/gCr for U-Pb BMDL; in terms of UALB, they were 962.44、285.44、607.76μg/L respectively for B-Pb and 153.37、75.71、117.79μg/gCr for U-Pb. It was obvious that BMDL of UANG was always lower than that of UALB, while the BMDL of UALB and UNAG were lower in women than those in men.The study of metallothionein in urine told us:urinary metallothionein (UMT) was well correlated with lead body burden. Its level increased following the increased exposure. The cutoff point was also calculated for it in the same way that mentioned above. The prevalence of hyperMTuria showed significant increasing trend in external and internal dose of lead exposed group. BMDL value was also calculated for UMT. The B-Pb BMDL of UNAG in men, women and the total were 411.85,310.99, and 398.60μg/L, respectively, and 76.96,87.29 and 87.37μg/gCr for U-Pb BMDL. The B-Pb BMDL of UMT was higher than UNAG for men, women and the total and women’s B-Pb BMDL of UMT was higher than UALB, but men’s B-Pb BMDL of UMT was lower than UALB; on the other hand, women’s U-Pb BMDL of UMT was higher than UNAG and UALB while it demonstrated opposite feature in men.In the research of MT1A and MT2A polymorphism, we found that:people’s susceptibility to lead absorption and lead induced renal dysfunction could be modified by polymorphism of MT gene. Three genotype of each SNPs (totally 4 SNPs) were all found in our target population. SNP of MT1A-rs11076161 had modification to B-Pb and U-Pb for the whole population because people who carried with genotype AA showed lower body burden than the remains. When the population was divided into two groups according to their exposure level, the results became as follows:in the lower exposure group, workers with rs28366003-GG got higher B-Pb than AA+AG, while women who took rs28366003-AA had higher B-Pb and men who took rs11076161-AA had lower U-Pb. As a whole group, workers in higher exposure had lower body burden when they carried with rs10636-GG. But it was different in different gender:men with rs10636-GG or rs1610216-CC had lower body burden while women had not got this feature, if they carried with these genotype, they showed higher body burden yet. When it came to the relationship between MT gene polymorphism and lead induced renal dysfunction, it was found that:the level of UNAG was lower in workers with rs10636-GG or rs1610216-CC for the whole subjects. In lower exposure group, the level of UNAG and UALB in women with rs11076161-AA was lower than the remains. In higher exposure group, workers as a whole had lower UNAG if they carried with rs10636-GG, and it showed the same result in male workers but not in female workers. Women got higher UNAG if they carried with rs28366003-AA while they would get higher UALB when they carried with rs28366003-GG. Men with rs1610216-CC got lower UANG level while women with the same genotype didn’t show the same phenomenon. In the course of analysis, smoking and drinking were considered in the model, but neither of which influenced the modification of the 4 SNPs.It could be concluded:①the air lead could be used as a reference parameter of lead exposure for short term-high level lead workers.②people could got renal damage when they expose to high dose of leadwith short term. In the course of damage, tubular dysfunction was earlier than glomerular.③UMT showed significant dose-effect and dose-response relationship with internal dose of lead, which implied that it can be considered as an biomarker for lead induced renal dysfunction.④in the condition of higher air lead for women, they got similar blood lead as men and much higher urinary lead than men, at the same time, they even had higher renal dysfunction indicators than men, both of which implied that women was inclined to collect lead and be more sensitive to lead.⑤MT1A and MT2A subtypes were susceptible genes in MT gene family. In the same external exposure of lead, MT1A-rs11076161-AA could modify the lead body burden, its takers had lower lead body burden than the others. MT2A-rs28366003-GG could increase the level of lead absorption under lower lead exposure. For the subjects as a whole, MT2A-rs10636-GG and MT2A-rs1610216-CC could protect body from the impact of tubular impact of lead.⑥the modification of MT gene polymorphism was different to different gender. Under lower lead exposure, MT1A-rs11076161-AA could increase the storage of lead in urine for men while it may decrease the renal dysfunction indicator’s level for women. Under lower lead exposure, MT2A-rs10636-GG could release men from both lead absorption and renal impact to some extent. MT2A-rs1610216-CC could release the body burden for men but increase that for women; furthermore, this genotype could also protect men’s kidney but would make kidney impact more serious for women.⑦the lead absorption and kidney impact induced by lead in this target population and different workers were modified by MT2A-rs10636, workers who took CC+CG had higher UNAG level than GG. After further comparison, the B-Pb BMDL of the two groups were 300.88、378.68μg/L respectively, which indicated the former group was susceptible to renal dysfunction induced by lead. What’s more, the former group account 45.05% in the population, which implied almost half of our whole population susceptible to lead nephropathy. As a result, this genotype has great practical significance for occupational work.更多还原