【作者】 文卫华；

【导师】 衡正昌；

【作者基本信息】 四川大学 ， 劳动卫生与环境卫生学， 2007， 博士

【摘要】 砷是影响人类健康的重要元素，为一种常见的环境毒物和确认的人类致癌物。已有的动物实验和人群流行病学研究表明，砷接触可引起不同程度的皮肤损伤、腱反射异常、肝功能损伤、心血管及神经系统病变等多方面的疾病，严重危害人体健康。砷与皮肤癌、膀胱癌、肺癌、肝癌、胃癌、前列腺癌及直肠癌等多种肿瘤的发病率升高有密切相关。但迄今为止，砷的遗传毒性和致癌机制研究还没有取得令人信服的进展。动物致癌模型难以建立和流行病学研究中的暴露评价不能严格控制混杂因素的干扰是主要原因。以上说明砷对人类健康危害非常严重，深入探讨其致病机制难度较大。Braman开始分别测定生物体内所含各种砷化合物后，对砷代谢研究有了迅速的发展。越来越多的研究认为三价甲基砷酸和三价二甲基砷酸比相应的无机砷毒性更强，可能是砷遗传毒性和致癌作用的关键物质。这与以前普遍认同的无机砷毒性强于有机砷观念有很大的差别，砷在人体代谢转化过程和／或代谢转化产物的遗传毒性可能是砷遗传毒性和致癌机制研究今后的主要方向。理想的砷致癌动物模型一直不能建立起来，可能与人类和多数动物对砷的代谢转化途径及体内各种产物含量存在巨大差异有关。流行病学研究在砷的遗传毒性和致癌机制探讨中具有关键的作用，但只有很好控制各种混杂因素才可能得到具有重要价值的研究结果。云南某砒霜厂是国内最大的专业生产厂家之一，存在的危害因素较为单一。其职业暴露特点为探讨砷的职业危害及剂量-效应关系提供了极为有利的条件，也具有解决目前研究比较难以解决的物种间、剂量间外推问题的良好条件。本课题选择云南某砒霜厂为研究现场，通过工程分析和职业卫生调查寻找存在的主要职业病危害因素，进行职业性健康监护体检和生命质量评价以掌握职业危害现状。在此基础上，研究职业砷接触对人群DNA和染色体损伤的影响，定量检测基因特异性损伤，寻找早期生物学效应标志物，深入探讨砷的遗传毒性和致癌机制。第一部分云南某砒霜厂工程分析和职业卫生调查云南某砒霜厂是生产砒霜、金属砷的专业厂家，采用反射炉熔炼法生产三氧化二砷；以94％左右的三氧化二砷为原料，木炭为还原剂生产金属砷。课题通过物料平衡计算、生产工艺流程分析和职业卫生调查等方法探讨该厂存在的主要职业病危害及可能的影响因素。在课题进行中，采用多种方法进行职业病危害调查。深入砒霜生产车间，了解生产工艺流程和主要生产设备，分析生产过程中可能产生和／或存在的主要职业病危害因素种类、分布及危害程度、职业病防护措施等。测定原辅材料、产品、熔炼渣及熔炼气等的成分和含量，计算生产环境存在的砷、铁、硫、碳等元素的物料平衡，掌握其来源与去处。职业卫生调查显示，职业环境砷化合物污染非常严重，是导致工人健康损害的主要原因，尤其在遗传毒性和致癌效应方面，其他职业危害因素影响较小。第二部分职业砷接触人群健康监护体检和生命质量评价课题研究中，对所有研究对象使用统一的表格、统一的方法严格按照《职业健康监护管理办法》及国家相关职业病诊断标准进行职业性健康监护体检，检测尿中无机砷和有机砷。同时，采用WHO推荐的生命质量评价量表(SF-36中文版)对所有研究对象进行量表自行调查。职业性健康监护体检表明，高接触组工人皮肤损伤、腱反射减弱、心电图异常等发病率高，而这些疾病在低暴露组和对照组发生频率很低；所有职业砷接触人群尿砷均显著高于美国政府工业卫生学家会议(ACGIH)最新制定的尿总砷生物接触限值(BEI)，即35ug／L。高接触组工人平均浓度超标10倍以上。工人尿中有机砷和无机砷均很高。生命质量评价显示，与对照人群相比，职业砷接触人群躯体健康得分较高，但躯体疼痛和总体健康、活力、心理功能对角色的影响和心理健康等多纬度得分较低(P＜0.05)。可以认为，部分职业砷接触人群身体处于亚健康状态。第三部分职业砷接触人群DNA损伤检测酶联免疫实验检测人群尿中8-羟基脱氧鸟苷(8-OHdG)水平和微量全血彗星实验检测外周血淋巴细胞DNA损伤实验操作方便、不需要特殊仪器、具有较高的敏感性，非常适合流行病学现场研究。本研究采用这两个实验对职业砷接触人群DNA损伤进行检测。研究发现，尿8-OHdG水平测定结果男性高暴露组明显高于对照组，低暴露组男性低于女性(P＜0.05)。砷暴露人群尿8-OHdG水平随有机砷含量增加而升高，存在相关关系(spearman correlation coefficient，r_s=0.279，P=0.019)。职业砷接触人群微量全血彗星实验彗星平均拖尾率和尾长均显著高于对照人群(P＜0.01)。第四部分职业砷接触人群基因特异性损伤与砷的代谢转化实时荧光定量PCR定量检测基因特异性损伤的理论依据是，活细胞DNA损伤将直接影响Taq聚合酶，导致PCR反应达到一定产物量需要的反应循环数更多(即C_T值更大)，基因特定片段损伤指数升高(即PCR扩增效率(E)下降)。本课题建立了实时荧光定量PCR定量检测人群外周血淋巴细胞p53基因第5和第8外显子损伤的实验方法，并应用于职业砷接触人群分子流行病学研究。研究发现，高接触组、低接触组和对照组之间p53基因第5外显子损伤指数存在显著差异(P＜0.05)，高接触组高于对照组(P＜0.05)。Pearson相关分析(Pearson’correlation coefficient=0.351，p=0.012)和多元逐步回归分析(Standardized coefficient=0.648，P=0.018)均显示，职业砷接触人群p53基因第5外显子损伤指数随尿中有机砷浓度的对数升高有显著的上升趋势。第五部分职业砷接触人群的染色体损伤检测采用常规培养法检测职业砷接触人群外周血淋巴细胞微核形成。现场采集研究对象静脉血进行培养，进行培养法微核实验。研究发现，职业砷接触人群外周血淋巴细胞微核细胞率和微核率均显著高于对照人群(P＜0.01)。相关分析显示，职业砷接触人群外周血淋巴细胞微核细胞率和微核率与尿有机砷浓度工龄乘积之间存在正相关关系(r_s=0.356，p=0.024；r_s=0.347，p=0.028)，微核细胞率和微核率随工龄、尿有机砷乘积增加而升高。长期职业砷接触可导致外周血淋巴细胞染色体损伤，染色体损伤随砷代谢转化作用累积而加重，无机砷在人体内代谢转化长期作用引起细胞遗传学改变可能是砷的致癌途径之一。更多还原

【Abstract】 Arsenic is a familiar environmental toxicoid that affects our health, and it isaffirmed that arsenic can induce cancer on human being. It was reported thatarsenic could induce skin damage, tendon hyporeflexia, liver function damage,cardivascular and neuropathy system pathological changes by both animalexperiment and epidemiology survey. In addition, arsenic exposure wasassociated with skin cancer, urinary bladder cancer, lung cancer, stomach cancer,liver cancer, prostate cancer and rectum cancer. But genotoxicity and carcinogeni-city mechanism is not clear, and a long way is needed to go. It may be greatreason that ideal animal model can not been established, and the interferentialfactors of exposure evaluation on epidemiology can not been strictly controlled.This will harm people health greatly, and it is of great difficult to study themechanism how arsenic induce damage to human beng.Since different arsenic compounds in bodies were mensurated respectivelyby Braman, the study on metabolism of arsenic developes quickly. There aremore and more researches sustain that monomethylarsonous acid (MMA) anddimethylarsinous acid (DMA) are more toxic than inorganic arsenic. At the sametime, MMA and DMA may play key role in inducing genotoxicity andcarcinogenesis. It is not consistent with the previous ubiquitous idea thatinorganic arsenic was more toxic than organic arsenic. The process of metabolismof arsenic, and/or metabolites came from it in human being may inducegenotoxicity and carcinogenesis, which will be a important aim that need further arsenic in animals and human beings may be one of the reason why ideal animalmodel on genotoxicity and carcinogenesis of arsenic could not been established.So epidemiology is great important for us to further understand the toxicmechanism of arsenic. But only when interferential factors are controlled strictly,valuable results could be gained.The research field we chose is a special mill that produces arsenic in YunNan province. The main factor that affect worker’s health is arsenic compounds.It is in favor of the research on occupational health hazard, dose-effectrelationship, and also offer a good chance to resolve the problem of extrapolatebetween different species and doses, which is very difficult to most investigation.In this study, we chose an arsenic mill in YunNan province as investigationfield, and search the primary factors that affect workers’ health by engineeringanalysis and occupational health investigation for arsenic exposure, and studyoccupational harm actuality by occupational health ward examination andestimation of quality of life. Then, the DNA and chromosome damage in workersexposed to arsenic were investigated, and gene-specific damage was measuredquantificationally. Forepart effect biomarkers for arsenic exposure were explored,and the mechanism on genotoxicity and carcinogenesis of arsenic were studieddeeply.PartⅠEngineering analyse and occupational health investigation of arsenic mill in YunNan provinceIt is a special mill for arsenic trioxide and metal arsenic production. Arsenictrioxide was produced by reverberator smelting, and materials are arsenic trioxidewhich purity is over 94%, and charcoal as reducing agent to produce metalarsenic. The primary occupational harm and possible influence factors werestudied by material balance calculation, production technics flow analyse andoccupational health investigation.In this investigation, many means were used to investigate occupationalharms including going into arsenic producing workshop, looking in production technics flow and main equipments, and analyzing the sort, distribution andextent of main occupational harm factors caming from production process, andoccupational defend measures. The components and contents of former andsupplement materials, arsenic trioxide, metal arsenic, smelting residues andexhaust gas were measured to calculate material balance of arsenic, iron, sulfur,carbon and so on, and understand their source and whither. The results comingfrom occupational health investigation show that pollution of arsenic compoundwas severe in workshop, and this is the primary reason that harmful to workers,especially in genotoxicity and carcinogenesis, and there were few interferentialfactors.PartⅡThe occupational health examination and quality life evaluation of workers exposed to arsenicIn this study, all objects were examined by uniform table and one meansaccording to the administrant ways of occupational health ward, nationaloccupational diagnosis standard strictly, and organic and inorganic urinary arsenicwere measured. At the same time, the quality life of all objects were selfexamined using quality life scale SF-36 recommended by WHO.Results coming from physical examination showed that the incidence rate ofskin damage, tendon hyporeflexia, cardivascular system pathological changeswere high in workers, but low in the others. Total urinary arsenic in allindividuals exposed occupationally to arsenic was more than 35ug/L, which is theBiological Exposure Indices (BEIs) on total urinary arsenic constituted up to dateand recommended by American Conference of Government Industrial Hygienists(ACGI). The concentrations of total urinary arsenic in high exposure workerswere as over 10 times as the the data’s, and organoic and inorganic urinaryarsenic of individuals exposed occupationally to arsenic were all very high.Results of quality life evaluation of individuals exposed to arsenic showedthat scores on physical function were higher, but that on Pain, General health,Vitality, Role-emotional, Mental Health were lower than references’ (P＜0.05). Vitality, Role-emotional, Mental Health were lower than references’ (P＜0.05)It could be thought that individuals exposed occupationally to arsenic were ininferior health partly.PartⅢDNA damage measure of individuals exposed occupationally to arsenicIt is easy operation, not needing especial instrument, holding highsensitivity, and is good for epidemiology to measure urinary 8-OHdG by ELISAmethod, and to detect DNA damage of lymphocyte in peripheral blood by ouncewhole blood comet assay. Those assays were used to measure DNA damage ofindividuals exposed occupationally to arsenic.Results showed that high exposure individuals hold more urinary 8-OHdGthan references’ (P＜0.05) in male, and the contents in male workers were lowerthan that in females in low exposure (P＜0.05). The increascd tendencies ofurinary 8-OHdG levels with the organic arsenic concentration were found inworkers (spearman’correlation coefficient, r_s=0.279, P=0.019). The comet rateand tail lengthof of lymphocyte of workors were all higher than references’ (P＜0.01).PartⅣGene-specific damage and arsenic metabolism in individuals exposed occupationally to arsenicThe theory to measure quantificationally gene-specific damage usingreal-time quantitative PCR is that DNA damage of alive cell will disturb Tappolymerization enzyme, which results in needing more reaction circles in PCR toreach one output (this means Ct value of PCR enlarging), and damage indexs ofcertain segment of specific gene increasing (this means expanding efficiency ofPCR decreasing). The assay to measure quantificationally the damage to exon 5and 8 of p53 gene of lymphocyte in peripheral blood using real-time quantitativePCR were established, and used to molecular epidemiology for individualsexposed occupationally to arsenic. The damage index of high exposure individuals were higher than references’ (P＜0.05). The increased tendencies of damage indexs of exon 5 of p53 gene withthe logarithm value of organic arsenic concentration was found in objects usingboth pearson correlation (pearson’ correlation coefficient=0.351, P=0.012)and stepwise linear regression analyzing (Standardized coefficient=0.648, P=0.018).PartⅤChromosome damage measure of individuals exposedoccupationally to arsenicMicronucleus rates of lymphocyte coming from peripheral blood inindividuals exposed occupationally to arsenic were measured by routine cultureassay. Vein blood of all objects were adopted and cultured on the spot, andperformed routine culture assay immediately.Results show that both micronucleated cell and micronucleus frequency oflymphocyte were higher than references’ (P＜0.01). The increased tendencies ofmicronucleied cells and micronucleus frequency with the product of organicarsenic concentration and service length were found in workers (r_s=0.356, P=0.024. r_s=0.347, P=0.028). Chromosome damage in individuals exposedoccupationally to arsenic for a long time were induced, and the increasedtendencies of chromosome damage with the accumulation of metabolism inarsenic were found. The cytogenetic induced by the metabolism of inorganicarsenic were one route of the carcinogenesis of arsenic in human for a long time.更多还原