【作者】 邵华；

【导师】 李会庆；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2007， 博士

【摘要】 甲醛（Formaldehyde）是一种常见的、被广泛应用于建筑，装饰材料和家用化学品的重要化学物质。常温下是一种无色，具有强烈刺激性的气体，易溶于水，其35％-40％的水溶液通称为福尔马林，常作为浸渍标本的防腐剂。长期接触低浓度甲醛，可引起人体免疫系统、呼吸系统、神经系统及皮肤、肝脏等器官的损害。高浓度甲醛是一种遗传毒性物质，美国职业卫生阈限值中将甲醛列为可疑人类致癌物（2A组）。甲醛具有正电性，是一种化学性质比较活泼的小分子醛类化合物，可以进攻亲核基团而造成大分子物质如蛋白质、核酸的损伤。苯乙烯（Styrene）是人类可疑致癌物（2B组），作为制造合成树脂、合成橡胶、塑料、油漆、药品和香料等的主要化工原料，用途十分广泛。苯乙烯主要通过呼吸道吸收，占总吸收量的89％，经皮肤和胃肠道可少量吸收。长期接触低浓度苯乙烯，对神经系统和肝脏有慢性毒作用，亦可引起血液改变和肾的损害，对心脏及生殖系统的毒性亦有报道，属于中等偏低毒性化合物。国内外对甲醛和苯乙烯的职业接触、在体内的吸收、代谢、排泄情况都进行了研究，基本搞清了两者的代谢规律，但这些研究仅局限在代谢机理、流行病学等领域，很少触及到对机体的损伤情况，生物监测方面也缺乏系统性，而且外部因素如饮酒、吸烟对生物监测的影响也难以定量消除。随着分子生物学理论和技术的迅速发展，生物标志物（Biomaker）的研究作为一个崭新的领域逐渐引起了国内外预防医学界的共同关注。生物标志物所反映的是生物体系与环境因子（化学的、物理的或生物学的）相互作用引起的生理、生化、免疫和遗传等多方面的分子水平上的改变，是环境医学发展到分子水平的重要里程碑，它的研究在分子流行病学、分子毒理学、劳动卫生学、环境医学等诸多领域中均具有极其重要的价值。DNA加合物是生物有效剂量（到达剂量或靶剂量）的生物标志物，它是毒性化合物进入人体后与细胞DNA相互作用而形成的。DNA加合物被认为是化学致癌、致畸、致突变过程的关键，它一旦逃避了自身修复就可能成为“三致”作用的最小启动因子。化学代谢物是内剂量生物标志物，它是毒物在人体内氧化、降解的产物，它反映了毒物在体内的吸收、代谢、排泄等情况。巯基尿酸是另一类内剂量生物标志物，亲电化合物在体内谷胱甘肽转移酶作用下可与谷胱甘肽形成巯基尿酸代谢物，其含量与DNA和蛋白加合物的浓度有良好的相关性。基因多态性是一类重要的易感性生物标志物，生物代谢酶的不同形态，在外源性化合物对机体毒性作用的过程中起着不同的作用，决定了机体对化学毒物的易感性，可以作为高危人群的重要筛选指标。生物标志物的检测不但考虑了空气中有毒物质浓度的变化和接触者在工作场所不同岗位间的移动，而且还考虑了多种吸收途径—呼吸道、皮肤、消化道，也考虑了职业和非职业因素以及各种理化的和毒代动力学的因素，如：工作负荷（影响吸收）、有毒物质代谢的能力（吸收、分布、生物转化）和个体易感性，因此比空气检测更能有效地代表接触程度，是一种理想的综合评价有毒物质接触情况的方法。【研究目的】以生物有效剂量（到达剂量或靶剂量）标志物—DNA加合物、内剂量生物标志物—化学代谢物及巯基尿酸代谢物、易感性生物标志物-生物代谢酶基因多态性为突破点，探讨甲醛、苯乙烯DNA化学损伤机制、研究生物标志物作为两者暴露监测因素的应用价值，为甲醛、苯乙烯接触人群的生物学监测、职业危害的预防及危险性评价、高危人群的筛选等提供科学的理论根据。【研究方法】1．以高效液相色谱法测定工作场所空气中甲醛、苯乙烯及其职业接触者尿中代谢产物的浓度依据《工作场所空气中毒物检测方法的研制规范》、《生物材料分析方法的研制准则（尿样及血样）》的要求，研究建立工作场所空气中甲醛的HPLC测定方法（包括现场采样方法、HPLC分析方法）、甲醛职业接触者尿中代谢产物（甲酸）的HPLC测定方法、苯乙烯职业接触者尿中代谢产物（苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸）的HPLC测定方法，为工作场所空气中甲醛的测定、甲醛和苯乙烯职业接触者的生物监测提供规范化方法。2．应用体外及体内培养、紫外吸收光谱移动法筛选、高效液相色谱分析等方法研究甲醛、苯乙烯DNA加合反应特性取小牛胸腺DNA（20.0μg／ml），加入不同浓度的甲醛、苯乙烯及其代谢产物，于37℃恒温水浴保持24h，用紫外吸收光谱移动法筛选可能发生加合反应的化合物。取四种单脱氧核苷酸在磷酸盐缓冲液（PH 7.2）中，分别与筛选化合物进行反应，37℃恒温下孵育，充N₂密封，18h之后进高效液相色谱测定，确定加合反应的位点，同时利用该反应体系研究DNA加合物的化学健型、加合反应的反应级数等。运用ficoll密度梯度离心技术，从健康成人外周血中分离出淋巴细胞，用培养液配成一定浓度的细胞悬液，分别加入不同浓度的筛选化合物染毒、孵育。用试剂盒将染毒的健康成人外周血淋巴细胞DNA提取出来，经蒸馏水稀释后，应用紫外吸收光谱移动法证实甲醛、苯乙烯是否可诱导人淋巴细胞DNA加合效应，从而对哺乳动物细胞有一定损伤作用。3．分别以高效液相色谱、PCR-RFLP方法研究甲醛职业接触人群接触生物标志物甲酸和易感性生物标志物ALDH2、CYP2E1的基因多态性，探讨甲酸作为甲醛暴露监测指标的可能性，寻找易感基因用于甲醛高危人群的筛选。以浸渍2，4-二硝基苯肼的硅胶管采集工作场所空气中的甲醛，高效液相色谱仪测定其浓度。用聚乙烯塑料瓶，采集甲醛接触者的晨尿或班后尿，加入磷酸盐缓冲液（PH7.6）和衍生剂1-溴甲基-五氟基苯，60℃水浴保温1h，正己烷萃取，高效液相色谱仪测定其中甲酸的浓度。应用HPLC法测定吸烟者尿中尼古丁、甲酸含量，探讨尿中尼古丁含量与甲酸代谢的相关关系，从而定量消除吸烟因素对甲酸测定的影响。采集甲醛接触者的静脉血样5 ml（经知情同意），装入EDTA抗凝管内混匀，冷藏保存，ficoll密度梯度离心法提取人外周血淋巴细胞，饱和苯酚／氯仿抽提法提取全血基因组DNA，PCR—RFLP法检测ALDH2、CYP2E1的遗传多态性。4．分别以高效液相色谱、PCR-RFLP方法研究苯乙烯职业接触人群接触生物标志物苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸和易感性生物标志物CYP2E1、GSTT1、GSTM1、EPHX1的基因多态性，探讨接触生物标志物作为苯乙烯暴露监测指标的可能性，寻找易感基因用于苯乙烯高危人群的筛选。以活性炭管采集苯乙烯接触者8小时个体接触剂量，高效液相色谱仪测定其浓度。用聚乙烯塑料瓶，采集苯乙烯接触者的晨尿或班后尿，以乙酸乙酯萃取，高效液相色谱仪测定其中苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸的浓度。采集苯乙烯接触者的静脉血样5 ml（经知情同意），装入EDTA抗凝管内混匀，冷藏保存，ficoll密度梯度离心法提取人外周血淋巴细胞，饱和苯酚／氯仿抽提法提取全血基因组DNA，PCR—RFLP法检测CYP2E1、6STT1、GSTM1、EPHX1的遗传多态性。【研究结果】1．本项研究建立的“工作场所空气中甲醛的HPLC测定方法”，样品采集应用浸有2，4-二硝基苯肼的硅胶管，易于稳定保存，甲醛的穿透容量＞0.1381mg，采样效率＞95％；甲醛的加标回收率＞95％，相对标准偏差＜0.88％；检出限达0.0053μg／ml，最低检出浓度达0.0015mg／m³；甲醛样品在室温下至少可保存7天。“甲醛职业接触者尿中代谢产物的HPLC测定方法”，甲酸的加标回收率＞90.0％，变异系数＜5.7％；检出限为0.15μg／ml；尿样在普通冰箱4℃保存，两周之内相对偏差＜8.93％。“苯乙烯职业接触者尿中代谢产物的HPLC测定方法”，苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸的加标回收率＞90％，变异系数＜6.3％，最低检出浓度分别为0.02、0.3、0.1mg／g肌酐；样品普通冰箱（4℃）放置一周后相对偏差＜8.3％。2．本研究结果表明，在体外试管反应条件下，一分子甲醛可以通过共价键与一分子dGMP相互结合，形成稳定的加合物；苯乙烯则通过在体内的活性亲电中间代谢产物环氧苯乙烯与四种单脱氧核苷酸中的dGMP及dAMP结合形成加合物，其中一分子环氧苯乙烯可以通过共价键与一分子dGMP结合，生成稳定的加合物，而环氧苯乙烯与dAMP形成的加合物相当微弱或不稳定。3．正常人淋巴细胞染毒试验表明，甲醛中活泼的醛基使得它们不需经过代谢就能攻击核酸碱基，甲醛浓度在40μmol／L时，可与淋巴细胞DNA发生微弱结合，甲醛浓度＞40μmol／L时，易与人淋巴细胞内DNA发生结合，活体细胞与小牛胸腺DNA的体外加合反应一致。苯乙烯在浓度较高时，可与淋巴细胞内DNA发生弱结合，活体细胞与小牛胸腺DNA的结合作用有差异；其活性中间体环氧苯乙烯具有亲核性，可与淋巴细胞内DNA发生稳定结合，活体细胞与小牛胸腺DNA的体外加合反应一致。提示苯乙烯与环氧苯乙烯均可导致DNA加合反应的发生，且环氧苯乙烯的作用强于苯乙烯。4．对某厂三个车间甲醛浓度和工人晨尿、班末尿中甲醛代谢产物甲酸含量检测结果表明，晨尿中甲酸增加量和甲醛接触剂量之间呈良好的相关性（R²=0.9881，P＜0.05），可作为甲醛接触者的生物标志物，同时根据实验结果推荐与现行甲醛的MAC值相配套的甲醛接触者晨尿中甲酸增加量的生物阈限值为19.7 mg／g肌酐。5．选取60名吸烟者和20名非吸烟者，所选吸烟者日吸烟量不等，烟龄均在一年以上，采集吸烟者尿样，以HPLC分析其中尼古丁、甲酸含量，双变量相关分析，可见吸烟者吸烟的多少与甲酸代谢增加量有显著性相关关系（R²=0.9303，P＜0.05）。吸烟能够影响甲醛代谢物的生物监测。6．以107名甲醛接触者外周血淋巴细胞为样本，测定ALDH2和CYP2E1基因型，结果发现，不同ALDH2基因型影响尿中甲酸增加量水平，ALDH2*1纯合子基因型对甲醛的代谢活性要高于ALDH2*2纯合子基因型（平均秩次相差11.38）；CYP2E1基因5′-frank区域RsaⅠ／PstⅠ位点多态性未影响尿中甲酸的含量，不影响甲醛的代谢活动。7．对某厂苯乙烯接触工人个体接触剂量、晨尿和班末尿中苯乙烯代谢产物苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸含量的检测结果表明，晨尿中苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸、（苯乙醇酸+苯乙醛酸）及班后尿中苯乙烯巯基尿酸可作为苯乙烯接触者的生物标志物，同时根据实验结果推荐与现行TWA相配套的苯乙烯接触者晨尿中苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸、（苯乙醇酸+苯乙醛酸）的生物阈限值的推荐值为—苯乙烯巯基尿酸：11 mg／g肌酐；苯乙醇酸：607mg／g肌酐；苯乙醛酸：386mg／g肌酐；（苯乙醇酸+苯乙醛酸）：992mg／g肌酐；班后尿中苯乙烯巯基尿酸的生物阈限值的推荐值为29 mg／g肌酐。8．以100名苯乙烯接触者外周血淋巴细胞为样本，测定CYP2E1、GSTM1、GSTT1和EPHX1基因型，结果发现，CYP2E1PstⅠ／RsaⅠC2C2基因型与苯乙烯代谢增毒过程显著相关，GSTMI（+）基因型、EPHXI高活性组基因型与苯乙烯代谢减毒过程显著相关，未发现GSTTI基因型与接触工人尿中的UMA含量有显著统计学意义。【研究结论】1．本项研究建立的“工作场所空气中甲醛的HPLC测定方法”，“甲醛职业接触者尿中代谢产物的HPLC测定方法”，“苯乙烯职业接触者尿中代谢产物的HPLC测定方法”，符合《工作场所空气中毒物检测方法的研制规范》、《生物材料分析方法的研制准则（尿样及血样）》的要求，可用于工作场所空气中甲醛的测定、甲醛及苯乙烯职业接触者尿中代谢产物的测定。2．甲醛中活泼的醛基使得其不需经过代谢就能攻击核酸碱基，一分子甲醛可以通过共价键与一分子dGMP相互结合，形成稳定的加合物，活体细胞与小牛胸腺DNA的体外加合反应一致，提示甲醛具有遗传毒性效应。苯乙烯则主要通过其活性中间体环氧苯乙烯攻击核酸碱基，一分子环氧苯乙烯可以通过共价键与一分子dGMP结合，生成稳定的加合物。活体细胞与小牛胸腺DNA的体外加合反应一致。提示苯乙烯也具有遗传毒性。3．晨尿中甲酸增加量可作为甲醛接触者的生物标志物，同时推荐与现行甲醛的MAC值相配套的甲醛接触者晨尿中甲酸增加量的生物阈限值为19.7 mg／g肌酐。通过检测尿中尼古丁含量可以算出吸烟导致甲酸增加的量，可用接触甲醛的吸烟工人尿中甲酸含量减去吸烟导致甲酸增加的量，来排除吸烟对甲酸生物监测的影响。CYP2E1不影响甲醛的代谢活动；ALDH2可作为甲醛易感性生物标志物，用于甲醛高危人群的筛选。4．晨尿中苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸、（苯乙醇酸+苯乙醛酸）及班后尿中苯乙烯巯基尿酸可作为接触苯乙烯工人的生物标志物，同时推荐与现行TWA相配套的苯乙烯接触者晨尿中苯乙烯巯基尿酸、苯乙醇酸、苯乙醛酸、（苯乙醇酸+苯乙醛酸）的生物阈限值为—苯乙烯巯基尿酸：11 mg／g肌酐；苯乙醇酸：607mg／g肌酐；苯乙醛酸：386mg／g肌酐；（苯乙醇酸+苯乙醛酸）：992mg／g肌酐；班后尿中苯乙烯巯基尿酸的生物阈限值为29 mg／g肌酐。GSTTI不影响苯乙烯的代谢活动；CYP2E1、GSTM1和EPHX1可作为苯乙烯易感性生物标志物，用于苯乙烯高危人群的筛选。【研究的特点及意义】本项课题选择生物标志物为突破点，在方法学上建立了空气中甲醛的测定方法、甲醛及苯乙烯生物标志物的测定方法，为两者提供了规范化的检测手段；在作用机理上，研究了甲醛、苯乙烯DNA加合物的形成、作用位点、化学键型及加合反应级数，为甲醛、苯乙烯的化学损伤机理的研究提供了理论基础；在生物学效应上，探讨了甲醛、苯乙烯在淋巴细胞中DNA加合反应的特性，初步证实两者可诱导人淋巴细胞DNA加合效应，对哺乳动物细胞有一定损伤作用；在生物监测效果的评估上，对甲醛、苯乙烯的五种生物标志物进行了同时监测比较，对代谢物作为暴露监测因素做出了综合评价，并推出生物阈限值，为生物监测结果的评估提供了定量依据；在易感性生物标志物的筛选上，对甲醛、苯乙烯的五个代谢酶的基因多态性进行了研究，发现了易感基因，为甲醛、苯乙烯高危人群的筛选、职业危害的一级预防提供了理论依据。更多还原

【Abstract】 Formaldehyde, a familier and important chemistry material, has been widely used in architecture, decorated material, household-use chemicals. It is a colorless and stimulative gas and easily to dissolve in water at ordinary temperature. Its aqueous solution （35%-40%） is called formalin which often act as the antiseptic to immerse specimen. Long-term exposure to low concentration of Formaldehyde can impair human immune system, respiratory apparatus, nervous system ,skin and liver, etc. High concentration of Formaldehyde has genetic toxicity. Formaldehyde has been listed as suspicious mankind’s carcinogen （2A group） in the American occupational health threshold limit value. Formaldehyde is mocromolecule aldehyde chemical compound that has electropositivity and vivid chemical property. It can attack nucleophilic group and result in the damage of giant molecular substance such as protein, nucleic acid.Styrene, a kind of suspect carcinogen （2B group） with wide use, is the main chemical material of synthetic resin, synthesizes rubber, plastics, coating, drug , perfume and so on. Styrene is absorbed mainly through respiratory tract （account 89% of total absorption）. The long-term exposure to low concentration of Styrene , has the chronic toxic action to the nervous system and liver. It is reported that Styrene, belonging to mid-class toxic chemical compound, also has the harmful fuction to the blood, kidney, heart and genital system.It is clear that the metabolic way of Formaldehyde and Styrene in domestic and international research. However, the metabolic mechanism of them is still on going research. It is also lacks the systematic study on biological monitoring and harm to organism, and it is difficult to quota eliminate the smoking influence to the biological monitoring.Along with the rapid development of theory and technology of molecular biology, the study of biomarker, a brand-new field, has gradually draw attention in the field of Preventive medicine throughout the world. What biomarker reflecting is the molecular changes of physiology, biochemical, immunity and heredity, etc. These changes are evoked by the interaction of biosystem and environmental factors-chemical, physical and biological factors. Biomaker is an important milestone that manifest environmental medicine has developed at molecular level. The research about it has vital value in the field of molecular epidemiology, molecular toxicology, labour hygiene, environmental medicine, etc.DNA adducts, a biomarker of biological effective dose （target dosage）, is formed when toxic compound enter a human body and interacte with DNA. DNA adducts may become the minimum factor to lead to carcinogenicity, malformation and mutation once it escapes the procedure of DNA repair.Chemical metabolites-the product of toxic compound oxidize and degradatiohn in the human body, is the biomarker of internal dose. It reflects the state of absorption, metabolism and excretion of toxic compound in body.Mercapturie acid is another biomarker of internal dose. Electrophilic compound and glutathion can form mercapturie acid metabolites under the effect of glutathione transferas in vivo. There are high dose-response relations between the concentration of DNA and proteinum adducts and mercapturie acid.Genetic polymorphism is an important biomarker of susceptibility. Different morphous of biological metabolic enzyme have different effect in the procedure of exogenous compound has toxic effect on organism, and determine the affectability of organism to chemical poison. As a result, it can be taken as an important screening index to occupational contraindication.The detection of biomarkers consides not only the variation of concentration of noxious substance in the air but also the ambulation of contact persons between different workplaces. Moreover, various absorptive path （ respiratory passage, skin, alimentary canal） , vocational factor , various physical and chemical factors such as workload （effect absorption）, the ability to metabolitic noxious substance （absorption, distribution, bioconversion）, individual affectability also be taken into account. For these reasons,the detection of biomarker represents exposure level more efficaciously than aerial detection. Consequently, it is an ideal means to estimate synthetic contact with noxious substance[Objective]With biomarkers of biological effective doses（ target dosage）-DNA adduct, biomarkers of internal doses-chemical metabolites, metabolites of mercapturie acid, biomarkers of susceptibility-genetic polymorphism for the breakthrough, we approached to the chemical damage mechanism of Formaldehyde, Styrene to DNA, studied the applied value of biomarker as a factor for exposure monitoring of both of them, and offered scientific theoretical basis for biological monitoring,precaution,and evaluation of the risk of population group exposed to Formaldehyde and Styrene.[Methods]1. To moitor Formaldehyde and Styrene in work place air and their metabolites in urine by HPLCAccording to "The development standard on the method of the moitoring intoxicant in work place air" and "The trituration standard of the method of creature material analysis （wet kind and blood kind）", to develop the monitoring method for Formaldehyde in the work place air（including the method of collecting sample in spot and the analytic method with HPLC）, the determinate method of the metabolite （formic acid ） in urine exposed to Formaldehyde and of the metabolites （MA, PGA and UMA） in Urine exposed to Styrene by HPLC, and applied a standardizational method for the measurement of Formaldehyde in the work place air and the biological monitoring of occupational exposed to Formaldehyde and Styrene.2.To study the DNA adduct characteristics of Formaldehyde and Styrene by culturing in vitro and in vivo, migration method of ultraviolet absorption spectrometry and HPLC methods. Takes the calf thymus DNA（20.0mg/ml）, drops in the different density of Formaldehyde, Styrene and their metabolites, maintains 24h in the 37℃water bath, screens compounds which occur chemical reaction with DNA by migration method of ultraviolet absorption spectrometryTakes four kind of single deaeration nucleotides in the phosphate buffer solution （PH 7.2）, carries on reaction with screened compounds separately, fills N₂ and seals tubes, maintains 18h in the 37℃water bath, Analysis intermixture by HPLC and determined the biding site. Simultaneously uses this reacting system to study the chemical bond type, chemical reaction progression of DNA adducts and so on.Separates the lymphocyte from the healthy adult circumference blood using the ficoll density gradient gentrifugalism technology, then configurates the cell suspending liquid with the nutrient fluid. Drops in the different density screened compounds to contaminate and fosters. Withdraws DNA form contaminated lymphocyte with the reagent box, and then conformed that screened compounds can induce DNA addiction reaction in human lymphocyte by using of migration method of ultraviolet absorption spectrometry, which is harmful to mammalian cells in a certain degree.3.To study on biological marker exposed to Formaldehyde- formic acid and biomarkers of susceptibility-ALDH2, CYP2E1 by HPLC and PCR-RFLP respectively. To discusse the possibility that formic acid took the monitoring index of Formaldehyde, and seek genes of susceptibility to use in the Formaldehyde high-risk group screening.To Sample Formaldehyde in work place air by silica gel tube soaked in 2,4-dinitrobenzene and determine it’s concentrations by HPLC.To Sample urine in the morning and the end of work shift from workers exposed to Formaldehyde with the polyethylene plastic bottle. Drops in the phosphate buffer solution （PH7.6） and the derivating agent 1-brominemethyl -5-fluorine benzene, maintains 1h in the 60℃water bath and extracting with n-hexane. determines formic acid by HPLC.To determine the nicotine and formic acid in urine from smokers using the HPLC, and to discusse the correlational relationship between nicotine and formic acid in urine, thus quota elimination influence that smoking factor to formic acid determination.To gather the venous blood samples from persons exposed to Formaldehyde, loads in the EDTA Anti-coagulate tube, mixes and Preserves in refrigerate. Separates the lymphocyte from the blood samples using the ficoll density gradient gentrifugalism technology, and extractes DNA in the whole blood by the saturated phenol/chloroform, Then analysis the polymorphism of ALDH2 and CYP2E1.4. To study on biological marker exposed to Styrene- UMA, MA and PGA and biomarkers of susceptibility- CYP2E1, EPHX1, GSTT1 and GSTM1 by HPLC and PCR-RFLP respectively. To discusse the possibility that UMA, MA and PGA took the monitoring index of Styrene, and seek genes of susceptibility to use in the Styrene high-risk group screening.To Sample Styrene in work place air by activated charcoal tube and determine it’s concentrations by HPLC.To Sample urine in the morning and the end of work shift from workers exposed to Styrene with the polyethylene plastic bottle, extracte with n-hexane, determine UMA, MA and PGA by HPLC.To gather the venous blood samples from persons exposed to Styrene, loads in the EDTA Anti-coagulate tube, mixes and preserves in refrigerate. Separates the lymphocyte from the blood samples using the ficoll density gradient gentrifugalism technology, and extractes DNA in the whole blood by the saturated phenol/chloroform, Then analysis the polymorphism of CYP2E1, EPHX1, GSTT1 and GSTM1.[Results]1. The method of measuring air Formaldehyde in workplace is estabolished in the study, samples are collected with silica gel dipping in DNPH to keep the samples stable during storage. The recovery rate of Formaldehyde is > 95% , and RSD <0.88%, and detecting limit is 0.0053μg/ml, and sampling efficiency >95%. The samples of air Formaldehyde is stable preserved in room temperature at lest 7 days. In the study on the method of measuring urinary metabolites of subjects exposured to Formaldehyde, the recovery rate of formic acid >90%, and CV<5.7%, and detecting limit is 0.15μg/ml. Preserving the urine samples in 4℃, RSD<8.93% in two weeks. In the study on the method of measuring urinary metabolites of subjects exposured to Styrene, the recovery rate of UMA, MA, PGA >90% and CV <6.3%. Preserving the urine samples in 4°C, RSD <8.3% in one week, and the detecting limits of UMA, MA and PGA are 0.02 0.3 and 0.1 mg/g creat.2. In vitro model system, one molecular Formaldehyde and one molecular dAMP form stable adduct through covalent bond. SO—the intermediate metabolite of Styrene—binds with dGMP and dAMP of four dNTPs. One molecular SO forms stable adducts binding with one dGMP through covalent bond,but the adduct is instable and weak formed by SO and dAMP.3. Health people peripheral lymphocytes contaminated by single Formaldehyde, Styrene or 7’8-SO indicate that active CHO- contained in Formaldehyde made them directly attack nucleate base. When 40μmol / L, Formaldehyde can weakly bind with human lymphocytes DNA. When >40μmol / L, Formaldehyde easily bind with human lymphocytes DNA, in accordance with the report that Formaldehyde bind with calf thymus DNA in vitro testing, which manifests that Formaldehyde is one genetoxin. In high concentration, Styrene can weakly bind with lymphocytes DNA in vitro cell culture, different from the response with calf thymus DNA. The intermediate metabolite -SO- stably binds with lymphocytes DNA because of its nucleophilicity, the same response with calf thymus DNA in vitro., which disclose Styrene and SO are both genetoxicant, and both covalent bind with DNA, but the binding reaction formed by SO is stronger than Styrene itself.4. Detecting the level of formic acid - one metabolite of Formaldehyde in vivo, in morning urine and the end of work shift and the concentration of air Formaldehyde in three different workshops, we may reguard the level of the increment of formic acid in morning urine as biomarker that subjects exposed to Formaldehyde through statistical analysis. Meanwhile, according to the experimental result, BTLV （biological threshold limit value） of the level of the increment of formic acid in morning urine for laborers exposed to Formaldehyde was recommended matching with current MAC of Formaldehyde as: 19.7 mg/g creat.5. Selects 50 smokers who smoke the diffirent quantity cigerattes and periode of smoking is over 1 year, and 20 non-smokers, gathers the smoker urine, determines nicotine and formic acid by HPLC. From the divariant quantity correlation analysis, we find that it has the significance correlational between the quantity cigerattes smoked and the increase of formic acid （R2 = 0.9303, P<0.05）. So smoking can affect the Formaldehyde metabolite in the biological monitor.6. Detecting the genotype of ALDH2 and CYP2E1 of 107 workers exposed to Formaldehyde through collecting peripheral blood lymphocytes, we can find the level of urine formic acid increment was influenced by genotypes of ALDH2. The metabolism of ALDH2 *1 homozygotic genotype to Formaldehyde is more active than ALDH2 *2 homozygotic genotype （the difference of the two mean rank is 11.38）, but the polymorphism of Rsa I/Pst I site of CYP2E1 5’-franking region Rsa I/Pst I site does not influence the level of urine formic acid.7. Detecting the level of the metabolites of Styrene, UMA, MA and PGA, in urine collected in morning and the end of work shift and detecting individual exposure dose of workers exposed to Styrene, through statistical analysis, we may reguard that UMA, MA, PGA and （MA + PGA） in morning urine and urinary UMA in the end of work shift as biomarkers of workers exposed to Styrene. Meanwhile, according to the experimental result, the recommended values of BTLV matching with current TWA of the UMA, MA, PGA, （MA + PGA） in morning urine of laborers exposed to Styrene are 11 mg/g creat for UMA, 607 mg/g creat for MA,386 mg/g creat for PGA, 992 mg/g creat for （MA + PGA）, 29 mg/g creat for UMA in the end of work shift.8. Detecting the genotype of CYP2E1, EPHX1, GSTT1 and GSTM1 of 100 workers exposed to Styrene through collecting peripheral blood lymphocytes, we find activity CYP2E1PstI/ RsaIC2C2 ,no-activity GSTM1（-） and EHPX1（-） can be taken as Styrene’s susceptible biomarker, can be used to screen Styrene occupational contraindication. That it has remarkable correlation between CYP2ElPstI/the RsaIC2C2 genotype and of Styrene metabolism in the increase poisonous process,and it has also remarkable correlation between GSTMI（+） genotype, the EPHXI high-activity group genotype and Styrene metabolism reduce the poisonous process. It is not discovered that it has the remarkable statistics significance between the GSTTI genotype and UMA in urine.[Conclusions]1. The method of measuring air Formaldehyde in workplace, the method of measuring urinary metabolites of subjects exposured to Formaldehyde, and the method of measuring urinary metabolites of subjects exposured to Styrene, is estabolished in the study. These methods fit the demands of "The development standard on the method of the moitoring intoxicant in work place air" and "The trituration standard of the method of creature material analysis （wet kind and blood kind）". They are available in determination of air Formaldehyde in workplace and measuring urinary metabolites of subjects exposured to Formaldehyde and Styrene.2. The lively aldehyde group in the Formaldehyde enable to attack the basic group in nucleic acid. One molecular Formaldehyde and one molecular dAMP form stable adduct through covalent bond. They have the same result that contaminated test with human lymphocytes DNA and chemical reaction test with calf thymus DNA in vitro, which manifests that Formaldehyde has the heredity poisonous effect.The Styrene,t hrough its active intermediate SO, attacks basic group in nucleic acid. One molecular SO forms stable adducts binding with one dGMP through covalent bond. They have the same result that contaminated test with human lymphocytes DNA and chemical reaction test with calf thymus DNA in vitro, which manifests that Styrene has also the heredity poisonous effect.3. The level of the increment of formic acid in morning urine enable to be biomarker that subjects exposed to Formaldehyde, BTLV （biological threshold limit value） of the level of the increment of formic acid in morning urine for laborers exposed to Formaldehyde was recommended matching with current MAC of Formaldehyde as: 19.7 mg/g creat.The increases of the formic acid caused by smoking may figure out from nicotine in urine. It is eliminated which smoking to the formic acid biology monitor influence from the urinary formic acid of smoking worker exposed to Formaldehyde subtract the increases of urinary formic acid caused by smoking.CYP2E1 does not affect the Formaldehyde metabolism activity. ALDH2 may take as the biomarker of susceptibility for Formaldehyde, using in the Formaldehyde high-risk group screening.4. UMA, MA, PGA and （MA + PGA） in morning urine and urinary UMA in the end of work shift may take as biomarkers of workers exposed to Styrene, the recommended values of BTLV matching with current TWA of the UMA, MA, PGA, （MA + PGA） in morning urine of laborers exposed to Styrene are 11 mg/g creat for UMA, 607 mg/g creat for MA,386 mg/g creat for PGA, 992 mg/g creat for （MA + PGA）, 29 mg/g creat for UMA in the end of work shift.GSTTI does not affect the Styrene metabolism activity. CYP2E1, GSTM1 and EPHX1 may take as the biomarkers of susceptibility for Styrene, using in the Styrene high-risk group screening.[Characteristics and significance]Choosing biomarkers as breakthrough point, in technology we developed determination methods of biomarkers and provided standard methods for biological monitoring of Formaldehyde and Styrene. In mechanism of action we studied the formation, conjoint position, type of conjoint chemical bond and reaction order of DNA adducts and gave the theoretical foundation for the mechanism of chemical impairment of Formaldehyde and Styrene. In biological effect we have discussed the characteristics of addition reaction between Formaldehyde, Styrene and DNA in human lymphocyte, and primarily certified both of them can induce the additive effect in human lymphocyte, consequently, it can impair mammalian cells. In the evaluation of biological monitoring, we studied the five biomarkers separately and judge the possibility of application of biomarkers, such as metabolites as exposure monitoring factors. We also promoted the biological threshold limit value, and provided the foundation for the quality evaluation of biological monitor. In screening for biomarkers of susceptibility, we studied the genetic polymorphism of five metabolic enzyme for Formaldehyde and Styrene, and find the susceptibilite gene. The findings provided the theoretical foundation to the high-risk group’s screening and primary prevention of occupational impairment for Formaldehyde and Styrene.更多还原