【作者】 王红；

【导师】 邬堂春；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2006， 博士

【摘要】 焦炉工人暴露于含有PAH的焦炉逸散物中，流行病学表明，多环芳烃(Polycyclic aromatic hydrocarbons，PAH)是导致焦炉工肺癌的主要原因。在职业流行病学癌症危险度的研究中，暴露标志物(又分为内剂量和生物有效剂量标志物)可以提供暴露于致癌因子的信息，早期效应标志物则可以反应致癌因子导致的早期改变。但是，在焦炉工人中，暴露标志物和早期效应生物标志物之间的联系未得到很好阐明。PAH的终致癌物二氢二醇环氧苯并[α]芘(Benzo[α]pyrene-7，8dihydrodiol-9，10-epoxide，BPDE)可以与白蛋白和DNA结合形成BPDE-白蛋白加合物和BPDE-DNA加合物，白蛋白加合物可作为PAH暴露的生物有效剂量标志物。而单细胞凝胶电泳(又称彗星实验)是在单细胞水平上检测DNA损伤的方法，可作为焦炉工人DNA损伤的早期生物效应标志物。就我们所知，尽管BPDE-白蛋白加合物和DNA损伤这两个标志物在焦炉工人中的应用都分别有报道，但还未有焦炉工人BPDE-白蛋白加合物和DNA损伤水平之间相关关系的报道。因此，本研究试图评估焦炉逸散物的职业暴露是否导致BPDE-白蛋白加合物和DNA损伤水平的升高，并且探讨两者之间的相关关系。遗传易感性会影响个体对外来化合物诱发的癌症或者外来化合物诱发的暴露或效应标志物易感，而遗传易感性取决于个体对外来化合物的代谢或修复的差异。外来化合物的致癌作用主要由DNA损伤所启动。大量证据显示，PAH的致癌过程中，一方面有活性氧(Reactive oxygen species，ROS)卷入其中，导致氧化性损伤的产生。另一方面有DNA加合物和单链断裂在其代谢激活的过程中形成，卷入其致癌过程。DNA损伤的修复处于基因的调控之下，DNA修复基因在保持基因组的完整性和预防癌症发展中有重要作用。修复基因多态性导致的个体之间修复能力的差异与癌症的危险度相关联。核苷酸切除修复(Nucleotide excision repair，NER)机制是PAH引起的DNA损伤的重要修复途径。NER功能缺失或下降是多种癌症发生的重要原因。NER途径涉及几十种蛋白，其中由XPC(Xerodcrma pigmentosum group C，XPC)基因编码的XPC修复蛋白在NER途径中能识别受损DNA位点，在启动NER机制上发挥着关键的作用。切除修复交叉互补集团1(Excision Repair Cross Complementation Group 1，ERCC1)基因通常和XPF结合形成复合物共同发挥作用，在NER过程中负责在DNA损伤位点的5′端发挥切除作用。本研究选择了氧化性损伤相关基因AKR1C3以及核苷酸切除修复基因ERCC1、XPC，探讨这些基因的单核苷酸多态性位点与焦炉工人外周血淋巴细胞DNA损伤易感性之间的关系。第一部分流行病学调查及作业环境致癌性PAH浓度测定选取232名焦炉工人构成暴露组；127名对照选自同厂机关管理人员以及医务人员。两组调查对象在年龄、工龄、吸烟和饮酒方面差异无显著性(P＞0.05)。在炼焦作业区和非炼焦作业区进行空气采样。采用Waters公司550型高效液相色谱方法测定苯并[α]芘、苯并[α]蒽、屈、苯并[α]荧蒽、苯并[k]荧蒽、二苯[α，h]蒽、苯并[g，h，i]苝、茚并[1，2，3-cd]芘8种致癌性PAH浓度。结果显示，焦炉作业环境空气中苯并[α]芘等8种致癌性PAH浓度总和的中位数高于对照区域的8种致癌性PAH总和(P＜0.01)，焦炉作业区苯并[α]芘浓度的中位数也高于对照区域(P＜0.05)。第二部分焦炉工人血浆BPDE-白蛋白加合物与外周血淋巴细胞DNA损伤水平的关系探讨焦炉工人BPDE-白蛋白加合物与DNA损伤水平之间的关系。采用反相高效液相色谱法以及单细胞凝胶电泳分别测定血浆中BPDE-白蛋白加合物浓度和外周血淋巴细胞DNA损伤水平。结果显示，焦炉工人的血浆BPDE-白蛋白加合物浓度和外周血淋巴细胞DNA损伤水平显著高于对照组(P＜0.01)。使用logistic回归分析校正了个体的工龄、吸烟和饮酒状况后，焦炉工人BPDE-白蛋白加合物浓度增高的危险度是对照组的1.72倍(P＜0.05)，焦炉工人DNA损伤水平增高的危险度是对照组的1.96倍(P＜0.05)。BPDE-白蛋白加合物与DNA损伤水平在焦炉工人中存在显著的正相关关系(Spearman偏相关系数=0.235，P＜0.01)，但在对照组中不存在相关关系(Spearman偏相关系数=0.093，P＞0.05)。表明焦炉工人的生物有效剂量标志物BPDE-白蛋白加合物和早期生物效应标志物DNA损伤之间存在显著相关。第三部分代谢酶AKR1C3多态性与焦炉工人DNA损伤水平的关系PAH通过代谢酶激活后产生具有遗传毒性的中间产物，AKR1C3属于醛酮还原酶家族(Aldo-keto reductases，AKRs)，是代谢PAH中间产物-反式二氢二醇的一个关键酶，产生的DNA加合物和ROS导致DNA的氧化性损伤，但是，目前在流行病学研究中，还没有AKR1C3基因多态对DNA损伤水平的影响，我们假设AKR1C3在焦炉工人氧化性损伤中扮演重要角色，探讨AKR1C3的单核苷酸多态性和DNA损伤水平之间的关联。用碱性单细胞凝胶电泳评价个体外周血淋巴细胞DNA损伤，采用7900序列检测系统和等位基因识别程序测定AKR1C3的单核苷酸多态性，分析AKR1C3不同基因型与BPDE-白蛋白加合物水平和DNA损伤水平的关系。结果显示，在焦炉工人中，携带AKR1C3-His/His+His/Gln基因型组的BPDE-白蛋白加合物水平高于AKR1C3-Gln/Gln基因型，但差异无显著性(P＞0.05)。携带AKR1C3-His/His+His/Gln基因型组的外周血淋巴细胞DNA损伤水平高于AKR1C3-Gln/Gln基因型，差异有显著性(P＜0.05)。表明携带AKR1C3-His等位基因可增加焦炉工人PAH暴露导致的DNA损伤水平。第四部分核苷酸切除修复基因ERCC1和XPC基因多态性与DNA损伤水平的关系研究外源性化学物DNA损伤修复酶基因ERCC1和XPC多态性与焦炉作业工人外周血淋巴细胞DNA损伤易感性的关系。用碱性单细胞凝胶电泳评价个体外周血淋巴细胞DNA损伤水平，采用7900序列检测系统和等位基因识别程序检测ERCC1(rs3212948、rs2298881)和XPC(rs3729587、rs2607775)修复酶基因的四个位点的单核苷酸多态性，分析ERCC1和XPC基因的不同基因型与DNA损伤的关系。结果显示，仅在焦炉工人中发现：ERCC1-rs2298881位点CC基因型个体的DNA损伤水平显著高于AA+AC型个体。其它位点多态性与DNA损伤水平差异无统计学意义。得出结论，ERCC1-rs2298881CC基因型可能增加焦炉工人PAH暴露导致的外周血淋巴细胞DNA损伤水平。进一步探讨ERCC1的2个SNP位点的单倍型与焦炉工人DNA损伤水平之间的关系，结果发现：携带ERCC1的CC/GC、GC/GC单倍型对的焦炉工人DNA损伤水平高于CC/GA型，差异有显著性(P＜0.05)。以上结果提示携带ERCC1某些特定基因型或单倍型的个体在机体DNA修复能力上存在差异，这些基因型与DNA损伤水平之间的关系值得进一步研究。本研究的创新之处在于：(1)探讨了焦炉工人生物有效剂量标志物BPDE-白蛋白加合物和早期生物效应标志物DNA损伤作为焦炉工人生物标志物的可行性，并进一步探讨了两类标志物之间的相关关系。发现BPDE-白蛋白加合物与DNA损伤水平在焦炉工人中存在显著的正相关关系。(2)探讨了AKR1C3的单核苷酸多态性和DNA损伤水平之间的关联，在焦炉工人中发现，携带AKR1C3-His等位基因者，PAH暴露导致的DNA损伤水平显著升高。(3)发现ERCC1基因上rs2298881位点CC基因型的焦炉工人DNA损伤水平显著高于AA+AC型个体。对ERCC1基因rs2298881和rs3212948两个SNP位点进一步进行单倍型分析发现，焦炉工人携带CC/GC、GC/GC单倍型对的个体DNA损伤水平显著增高。更多还原

【Abstract】 Coke oven workers are exposed to polycyclic aromatic hydrocarbons (PAH). Epidemiological studies have shown an increase in cancer incidence among workers exposed to PAH, especially for the risk of developing lung cancer. In the exposure-to-disease pathogenic pathway, biomarkers that can provide information of exposure to carcinogenic agents (biomarkers of exposure) and early changes caused by the agents (biomarkers of effect) are needed in epidemiological studies of cancer risk. The albumin adducts are thought to be biologically effective-dose biomarkers of PAH. DNA damage (adducts and strand breaks) represents an early, detectable and critical step in the chemical carcinogenesis process and thus, may serve as an internal dosimeter for carcinogens. A wide variety of non-bulky base damage and single-strand breaks are formed during metabolic activation of PAH and involved in PAH carcinogenesis, and these types of DNA damage can be detected by comet assay and used as a marker of early biological effects of DNA-damaging agents in the living environments and occupational workplaces. To our knowledge, there was no reported investigation on the correlation between BPDE-Alb adduct concentrations and DNA damage in lymphocytes in coke oven workers, although these two biomarkers were individually applied in some occupational biomonitoring. Therefore, we further assessed whether occupational exposure to COEs resulted in high concentrations of BPDE-Alb adducts and DNA damage and their possible correlation in coke oven workers exposed to COEs.It became very clear that genetic predisposition may affect individual susceptibility to carcinogen-induced cancer and carcinogen-induced biomarkers of exposure and effect. Genetic susceptibility can be due to variation in genes for carcinogen metabolizing enzymes and in genes for repair enzymes. The carcinogenicity induced by PAH compounds is believed to be initiated by DNA damage. An increasing number of evidences demonstrate the involvement of reactive oxygen species (ROS) in the production of cancers by PAH. These oxygen species may lead to the formation of oxidative DNA damage. A wide variety of non-bulky base damage and single-strand breaks are formed during metabolic activation of PAH and involved in PAH carcinogenesis. Repair of DNA damage is under genetic control, and DNA repair genes may play a key role in maintaining genome integrity and preventing cancer development. Polymorphisms in DNA repair genes resulting in variation of DNA repair efficiency may therefore be associated with cancer risk. It is generally assumed that bulky DNA adducts, such as those induced by PAH, are repaired by the nucleotide excision repair pathway. XPC is essential in the initial step of damage recognition. ERCC1 in association with the XPF protein incises DNA on the 5’ side of the damaged site. The present study evaluated the influence of four polymorphisms of NER genes, together with DNA damage levels detected by comet assay of coke oven workers occupationally exposed to PAH. Our aim was to find potential susceptibility factors on a genetic basis capable of modulating individual responses to PAH genotoxic exposure and the consequent risk of cancer in coke oven workers. Part I Study design and airborne carcinogenic PAH monitoringIn this study, we recruited 232 male workers exposed to COEs and 127 controls not exposed to COEs in the same steel plant in northern China. Prior to blood sampling, a questionnaire to elicit the workplace description, smoking habits, medical history, age and diet was performed for each exposed worker. Questionnaires were completed in a personal interview. The distribution of age, worktime, and smoking and drinking status was similar between coke oven workers and controls. We selected four working sites for exposed group and three working sites for control group. Quantitative chemical analysis of eight carcinogenic PAH (benzo(a)pyrene (B[a]P), benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, dibenz[a,h]anthracene, benzo[g,h,i] perylene, indeno[1,2,3-cd]pyrene) were performed by high performance liquid chromatography with fluorescence detectors according to the methods 5506 published by the National Institute for Occupational Safety and Health. The results of airborne monitoring showed that the median value of the sum of eight carcinogenic PAH in the air of the work places was significantly higher than the control group (P<0.01). The airborne median level of B[a]P alone was significantly higher than the control group (P<0.05).Part II Association between plasma BPDE-Alb adduct concentrations and DNA damage of peripheral blood lymphocytes among coke oven workersCoke oven emissions containing PAH can induce both protein and DNA adducts, such as benzo[a]pyrene-r-7, t-8, t-9,c-10-tetrahydotetrol-albumin (BPDE-Alb) adducts and BPDE-DNA adducts. However, the relationship between these biomarkers for early biological effects is not well documented in coke oven workers. We measured BPDE-Alb adduct concentrations in plasma with reverse-phase high-performance liquid chromatography and DNA damage in peripheral blood lymphocytes with alkaline comet assay. Our results showed that the levels of BPDE-Alb adducts and the Olive tail moment (Olive TM) in exposed group were significantly higher than that in control group (P<0.05). Multivariate logistic regression analysis revealed that the odds ratio (OR) for BPDE-Alb adduct and Olive TM associated with the exposure were 1.72 and 1.96, respectively. Our results showed that there were significant correlations between the concentrations of BPDE-Alb adduct and Olive TM levels in exposed group (r_s=0.235, P<0.01) but not in control group (r_s=0.093, P>0.05). In conclusions, the relationships between biomarkers of biological effective and early biological effects were significantly among coke oven workers..Part III Association of AKR1C3 polymorphisms and DNA damage in peripheral lymphocytes of coke oven workersAKR1C3 is a member of the aldo-keto reductases (AKRs) superfamily, and has been shown experimentally to produce PAH metabolites that form DNA adducts or reactive oxygen species (ROS) leading to oxidative DNA damage. AKR1C3 is a particularly important enzyme in metabolizing potent trana-dihydrodiols containing more than two rings. SNPs in the AKR1C3 gene itself have not yet been examined for their influence on levels of DNA damage in an epidemiology study. We suggested that SNPs in the oxidative stress related-genes AKR1C3 may play a role in the repair of oxidative damage in coke oven workers. DNA damage was detected by alkaline comet assay. The polymorphism of AKR1C3 was analyzed using the ABI Prism 7900 sequence detection system. Data were analyzed using Allelic Discrimination Program. Our results showed that the DNA damage levels of AKR1C3-His/His+His/Gln genotype were higher than the AKR1C3-Gln/Gln genotype among coke oven workers.Part IV Association of ERCC1 and XPC polymorphisms and DNA damage in peripheral lymphocytes of coke oven workersTo investigate the association of polymorphisms of DNA repair enzyme genes and DNA damage in peripheral lymphocytes in coke oven workers, DNA damage was detected using alkaline comet assay and the polymorphisms were analyzed using the ABI Prism 7900 sequence detector. Data were analyzed using Allelic Discrimination Program. Our results showed that the coke oven workers with ERCC1-rs2298881 C/C genotype had higher levels of DNA damage levels compared with ERCC1-rs2298881 A/A+ A/C genotype carriers (P<0.05). ERCC1-rs3212948, XPC-rs2607775 and XPC-rs3729587 polymorphisms were not associated with DNA damage levels (P>0.05). Our results suggest that the coke oven workers with ERCC1-rs2298881 C/C genotype may be susceptible to DNA damage.We further analyzed the correlations of DNA damage levels in peripheral lymphocytes to the hapolotypes of two ERCC1 SNPs. Results showed that the DNA damage levels in workers with ERCC1-CC/GC and CA/CA haplotype pairs was higher than those with ERCC1-CC/GA haplotype pairs(P<0.05) only in coke oven workers, however, there were no significant associations of the DNA damage levels and haplotype pairs (P>0.05) in coke oven workers and controls. The results suggested that there was difference of the DNA damage levels in the coke oven workers with some genotypes or haplotypes in ERCC1 gene, however, these relationships among these genotypes, haplotypes, DNA damage and repair warrant further investigation.更多还原