【作者】 孙毅；

【导师】 金泰廙； 朱国英； 孙东红； 雷立健；

【作者基本信息】 复旦大学 ， 卫生毒理学， 2008， 博士

【摘要】 铅(lead,Pb)是金属元素,是受WHO、IARC、美国ATSDR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。铅为已知的最易在体内蓄积的毒物之一,人体内90%～95%的铅储存于骨内,且铅在骨中的半衰期很长(5～20年)并可保持相对稳定。无论是从毒性还是蓄积作用来看,铅都是污染人类生存环境、威胁人类健康的第一个金属元素。骨骼是铅的主要靶器官之一,人群流行病学调查和动物实验显示,铅能导致骨软化及骨质疏松。本研究的目的是通过对职业铅接触人群的流行病学调查,分析铅接触对人群骨密度、骨代谢、骨质疏松、腰椎骨折的影响,并将基准剂量引入铅接触引起的骨质疏松危险度评价,以求得到人群接触铅对骨骼系统效应的剂量—反应关系。通过研究铅接触对肾功能的影响及其与骨损伤之间的关系,以及分析ALAD基因多态性与铅骨毒性的关系,来探讨职业性铅接触对铅的骨毒效应的影响因素。此外,通过大鼠成骨细胞的染毒实验在铅对成骨细胞作用的分子机理方面作初步的探索。本研究选取上海市某蓄电池厂298名工人作为铅接触组,其中男性254名,女性44名;选取无职业铅接触史的该厂办公室工作人员81名为对照组,其中男性56名、女性25名。接触组铅作业工龄为1～40年,平均为18.1年。两组在年龄、体重、身高、体质指数(BMI)、吸烟、饮酒等方面均衡可比。本研究使用手提式单光子骨密度仪测定骨密度,并以WHO Z评分判断骨量减少,测定了人群铅接触生物标志指标(血铅、尿铅)与反映骨代谢和肾功能的生物标志指标(血清BGP、血清ALP、血清BALP、尿HYP、尿NAG、尿ALB以及腰椎侧位X线片),计算了各效应生物标志指标的血铅和尿铅BMDL值,测定了ALAD基因型。通过铅对骨密度和骨代谢影响的研究发现:铅接触组工人的血铅、尿铅水平均高于对照组,其中男性两组之间差异显著。接触组的骨密度低于对照组,但差异不显著。接触组的BGP、HYP、ALP、BALP高于对照组,其中男性HYP、ALP、BALP差异显著。以对照组各指标第90百分位数作为上限值,得到人群各指标的异常率。接触组男性和总人群只有ALP、BALP的异常率显著高于对照组。将人群分别按血铅、尿铅水平分组后,骨密度在BPb＞30μg/dl组显著下降,BGP在BPb＞20μg╱dl组显著升高,HYP、ALP、BALP在BPb＞10μg/dl组即显著升高;骨密度在UPb＞5μg╱g Cr组显著下降,HYP在UPb＞5μg╱g Cr组显著升高,ALP、BALP在UPb＞10μg╱g Cr组显著升高,BGP在UPb＞20μg╱g Cr组显著升高。随着血铅、尿铅水平的升高,骨量明显减少的发生率和各骨代谢指标的异常率也随着升高,有显著性趋势。经过计算各骨效应指标的血铅、尿铅BMDL值后发现,ALP、BALP的BMDL值最低,男女之间的差异主要表现在Z评分和BGP的顺序,血铅、尿铅BMDL值之间的差异主要表现在BGP的顺序。与骨代谢指标相比,Z评分的血铅、尿铅BMDL值较高。通过铅对人群腰椎骨折影响的研究发现:铅接触组腰椎骨折发生率显著高于对照组。随着血铅、尿铅水平的升高,腰椎骨折的发生率也随着升高,男性和总人群有显著性趋势。随着骨量减少,腰椎骨折发生率有上升趋势,男性和总人群有显著性趋势。通过铅对肾功能的影响的研究发现:铅接触组工人尿NAG、尿ALB均高于对照组,男性差异显著。接触组男性和总人群NAG的异常率显著高于对照组,接触组总人群ALB的异常率显著高于对照组。将人群分别按血铅、尿铅水平分组后,NAG在BPb＞10μg╱dl组显著升高,ALB在BPb＞20μg╱dl组显著升高;NAG、ALB在UPb＞5μg/g Cr组显著升高。随着血铅、尿铅水平的升高,NAG、ALB的异常率也随着升高,有显著性趋势。经过计算NAG、ALB的血铅、尿铅BMDL值后发现,反映肾小管损伤的尿NAG具有较低的BMDL值,而Z评分的BMDL值介于尿NAG与尿ALB之间,反映肾小球损伤的尿ALB的BMDL值最高。对人群骨量明显减少和肾功能损伤的分层分析发现,骨量明显减少者肾功能损伤发生率显著高于非骨质疏松者,肾小管损伤者骨量明显减少发生率显著高于肾小管功能正常者,肾小球损伤者骨量明显减少发生率与肾小球功能正常者发生率无明显差异。通过ALAD基因多态性的研究发现:机体对铅毒性作用易感性与ALAD的遗传多态性有关,本研究采用聚合酶链式反应—限制性片段长度多态性(PCR-RFLP)技术分析ALAD基因多态性。ALAD1-1和ALAD1-2基因型频率分别为91.29%和8.71%,未发现ALAD2-2型纯合子,ALAD1和ALAD2等位基因频率分别为95.65%和4.35%。血铅、尿铅水平在铅接触组ALAD1-2基因型明显高于ALAD1-1基因型。铅接触组ALAD1-2基因型骨密度明显低于ALAD1-1基因型,BGP、HYP、ALP、BALP明显高于ALAD1-1基因型。而对照组不同基因型间血铅、尿铅、骨代谢指标比较无统计学差异。无论男女,ALAD1-2基因型各骨效应指标与血铅、尿铅的线性回归系数的绝对值均大于ALAD1-1基因型。同时,本研究分离培养了SD乳鼠头颅骨成骨细胞,观察不同浓度醋酸铅染毒对成骨细胞增殖活性及碱性磷酸酶活性的影响,并测定了染毒前后成骨细胞OPG、ODF、VDR、TGF-β、IGF-Ⅰ的表达水平。体外实验证实,铅在剂量＞50μmol/L后,能引起大鼠成骨细胞增殖活性和碱性磷酸酶活性的下降,与对照组比有统计学意义。铅在各个水平均未能诱导ODF mRNA的表达。铅在1、5μmol/L时显著上调OPG mRNA的表达水平,100、500μmol/L时则下调其表达水平。铅在5μmol/L时能上调VDR mRNA的表达水平,大于50μmol/L时则抑制其表达。对TGF-β与IGF-Ⅰ而言,铅能抑制其mRNA的表达,但分别在＞50μmol/L和＞100μmol/L的剂量时与对照组比才有显著性差异。以上结果表明:①人群在较高剂量长时间接触铅时能明显引起骨密度降低,导致铅接触人群发生骨量明显减少和骨代谢改变。通过各指标血铅、尿铅BMDL值的比较,可以认为Z评分是比较特异而敏感的反映铅接触人群骨损伤的效应指标,而ALP与BALP结合可作为筛选铅接触高危人群的指标,BGP与HYP则相对缺乏敏感性和特异性。②长期较高剂量接触铅可能会引起职业人群腰椎骨折发生率增高,腰椎骨折的发生可能与骨量减少有关。③铅对肾脏的损害可间接导致对骨的损害。铅致骨质疏松的出现可能晚于肾小管损伤,而早于肾小球损伤。④ALAD基因是铅毒性的一个易感基因,在相同的职业外暴露环境下,ALAD2等位基因能够增加个体的铅负荷水平,并有可能对铅致骨骼的毒性产生影响。ALAD基因多态性影响个体血铅、尿铅水平的程度随环境中铅接触水平的升高而增加。ALAD1-2型基因可能增加铅对机体的骨毒性,影响骨密度和骨代谢,增加骨质疏松和骨折发生的危险性。⑤体外实验表明,低剂量的铅对成骨细胞OPG、VDR mRNA的表达有上调作用,但高剂量的铅对成骨细胞的直接损伤比较明显,同时能下调多个细胞因子的mRNA表达。这也从一方面解释了铅要在高剂量下才能使人群出现明显的骨质疏松症状,时间上晚于铅致肾损伤的出现。更多还原

【Abstract】 Lead（Pb） is one of metal element which has been considered as a environmental harmful pollutant of world wide concern.Lead can cause toxicity on several organs in human.Lead is a toxicant most easily accumulated and deposited over time in bone with a half-life of about 20 years.Bone is one target organ of lead and osteoporosis is the most familiar syndrome.The purposes of the present study are:1) to investigate the effects of occupational lead exposure on bone mineral density,bone metabolism,osteoporosis, lumbar vertebral fracture using the method of epidemiological investigation and the dose-response relationship between lead exposure and the toxicity effects on bone using the method of benchmark dose（BMD）.2) to examine a possible relationship between lead nephropathy and its effects on the skeleton and the relationship betweenδ-aminolevulinic acid dehydratase（ALAD） gene polymorphism and lead toxicity on bone.and 3) to investigate the possible molecular mechanisms on osteoblasts using in vitro experiments of lead on the osteoblasts from calvarias of newborn SD rat.The target population comprised people who worked in a storage battery plant in Shanghai.The exposure group was consisted of the workers working in the workshop, who were occupationally exposed to lead for more than one year.The control group consisted of the office faculty,who were not exposed to lead at work.The exposure group included 298 subjects（male 254,female 44）,and the control group included 81 subjects（male 56,female 25）.The average length of lead works in the exposure group is 18.1 years（1～40 years）.Two groups are comparable in age,weight,height,body mass index（BMI）,smoking and drinking habits.We used the portable single-photon absorptiometry（SPA） to measure the bone mineral density of forearm radius in each subjects of study population.A socalled Z score recommended by WHO was used to define osteoporosis.Blood lead（BPb） and urinary lead（UPb） were measured as exposure biomarkers.Serum osteocalcin（BGP）, serum alkaline phosphatase（ALP）,serum bone specific alkaline phosphatase（BALP）, urinary hydroxyproline（HYP）,urinary N-acetyl-β-D-glucosaminidase（NAG）, urinary albumin（ALB） and lumbar vertebral X-ray were also measured as effect biomarkers.We estimated benchmark dose（BMD） and the lower confidence limit of the benchmark dose（BMDL） for the adverse effects of bone and renal systems.We measured the ALAD genotype for each subject.Effects of lead on bone mineral density and bone metabolism:It was showed that BPb and UPb levels in the exposure group were significantly higher than those in the control group in males.The average BMD levels in the exposure group were lower than those in control group with no statistically significant differences in both genders. The values of BGP,HYP,ALP,BALP in the exposure group were higher than those in control group,and there were statistically significant differences in males for HYP, ALP,BALP.We defined the abnormal value using the 90%upper limits of each index of control group.The abnormal rates of ALPand BALP were significantly higher in exposure group of male and total population.After dividing the participants into several groups according to UPb and BPb levels,respectively,bone mineral density significantly decreased when BPb was higher than 30μg/dl;BGP significantly increased when BPb was higher than 20μg/dl;HYP,ALP,BALP significantly increased when BPb was higher than 10μg/dl;bone mineral density significantly decreased when UPb was higher than 5μg/g Cr;HYP significantly increased when UPb was higher than 5μg/g Cr;ALP,BALP significantly increased when UPb was higher than 10μg/g Cr;BGP significantly increased when UPb was higher than 20μg/g Cr.The prevalence of osteoporosis and the bone metabolism indexes were significantly increasing following the increase of BPb and UPb.After benchmark dose calculation for Z score and indexes of bone metabolisms,we could find ALP and BALP had the smallest values.The difference between male and female was the sequence between Z score and BGP.The difference of the values of BMDL between BPb and UPb was the sequence of BGP.Compared with the indexes of bone metabolism,Z score had higher BMDL values.Effects of lead on lumbar vertebral fracture:It was showed that the prevalence of lumbar vertebral fracture in exposure group was significantly higher than that in control group.The prevalence of lumbar vertebral fracture was significantly increasing following the increase of BPb and UPb in male and total population.The prevalence of lumbar vertebral fracture was significantly increasing following the decrease of bone mass in male and total population.Effects of lead on renal function and the associations with bone effects:It was showed that UNAG and UALB levels in the exposure group were significantly higher than those in the control group in males.The abnormal rates of UNAG and UALB were significantly higher in exposure group.UNAG significantly increased when BPb was higher than 10μg/dl;UALB significantly increased when BPb was higher than 20μg/dl;UNAG and UALB significantly increased when UPb was higher than 5μg/g Cr.The abnormal rates of UNAG and UALB were significantly increasing following the increase of BPb and UPb.After benchmark dose calculation for UNAG and UALB,the BMDL values of UNAG were smaller than UALB,and Z score was ranged between UNAG and UALB.We also found the renal dysfunction might develop earlier than osteoporosis.Osteoporosis caused by lead was related to renal dysfunction.With respect to renal dysfunction,osteoporosis was especially related to tubular damage but not to glomerular damage.Study on ALAD gene polymorphism:It has been proved that the susceptibility to lead toxicity is related toδ-ALAD gene polymorphism.Polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） techniques were used in this study to analyseδ-ALAD genotype.The results revealed that the frequencies of ALAD_1-1 and ALAD_1-2 genotypes were 91.29%and 8.71%, respectively.No ALAD_2-2 homozygote was found.The frequencies of alleles of ALAD₁ and ALAD₂ were 95.65%and 4.35%,respectively.In the exposure group, BPb,UPb,BGP,HYP,ALP,BALP with ALAD_1-2 genotype were obviously higher than those of ALAD_1-1 genotype;bone mineral density with ALAD_1-2 genotype were significantly lower than that of ALAD_1-1 genotype.While in the control group,there was no remarkable difference between the two genotypes in blood lead,urinary lead and the indexes of bone metabolism.The absolute values of the linear regression coefficient of the indexes of bone metabolism with ALAD_1-2 genotype were larger than those of ALAD_1-1 genotype.In addition,the osteoblasts were obtained from calvarias of newborn SD rat and treated with lead acetate in order to understand the possible mechanisms on osteoblasts.In vitro experiments showed that lead acetate could decrease osteoblastic proliferation and the activity of ALP when the dose of lead acetate was higher than 50μmol/L.Lead acetate couldn’t induce the expression of ODF while low level（1, 5μmol/L） lead acetate could up-regulate gene expression of OPG and VDR.High level lead acetate（＞50μmol/L） could repress the gene expression of OPG,VDR, TGF-βand IGF-I.In conclusion,high level of lead exposure could decrease bone mineral density, induce osteoporosis and affect bone metabolism.Occupational lead exposure could increase the prevalence of lumbar vertebral fracture.The occurrence of lumbar vertebral fracture might be associated with the decrease of bone mass.Osteoporosis might occur later than renal damage related to lead exposure.Osteoporosis caused by lead was related to renal dysfunction.With respect to renal dysfunction,osteoporosis was especially related to tubular damage but not to glomerular damage.ALAD gene, a susceptible gene to lead toxicity,may increase potential risk of lead toxicity at certain level of lead exposure.The degree of BPb and UPb level that is affected by ALAD gene polymorphism increases along with the rising level of lead exposure. ALAD_1-2 genotype carriers had a high risk of osteoporosis and fracture than ALAD_1-1 genotype.Low level lead could stimulate some cytokines expression in osteoblast and when the level increased,lead became more and more toxic.更多还原