【作者】 黄国香；

【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， 2011， 博士

【摘要】 多环芳烃(polycyclic aromatic hydrocarbons, PAHs)是焦炉逸散物(coke oven emission,COE)中最主要且最重要的致癌成分,可导致机体DNA损伤,而DNA损伤是焦炉工肺癌发病的主要原因。PAHs与多环芳烃受体(aryl hydrocarbon receptor, AhR)结合,导致AhR空间结构发生变化,入核后诱导多种外源性化学物代谢酶的合成,如I相代谢酶细胞色素P4501A1(cytochrome P4501A1, CYP1A1)、细胞色素P4501B1(cytochrome P4501B1, CYP1B1)、细胞色素P4502B6(cytochrome P4502B6, CYP2B6)、细胞色素P4502E1(cytochrome P4502E1, CYP2E1)。PAHs必须经过代谢酶的代谢活化之后才具有致癌性,其毒性除与本身的理化性质有关外,还与个体间遗传变异有关。在职业流行病学癌症危险度的研究中发现,作为体内致癌物PAHs代谢的关键酶CYPs,其亚家族成员CYP1A1、CYP1B1、CYP2B6和CYP2E1催化PAHs中重要致癌物苯并芘(benzo[a]pyrene, B[a]P)经CYPs催化代谢为终产物苯并(a)芘二氢二醇环氧化物(benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, BPDE),此终产物可与DNA和血浆中的白蛋白形成共价加合物,导致DNA的损伤。据我们所知,目前国内外研究均采用1-OHP作为PAHs职业暴露标志物,由于1-OHP生物半衰期比较短,只能反映短期内的PAHs的暴露水平,且绝大部分经尿液排出,参与DNA损伤的量极少,因此存在局限性。血浆BPDE-白蛋白加合物在血中含量较高,生物半衰期较长,可反映PAHs长期暴露水平,尽管血浆BPDE-白蛋白加合物在焦炉工人中的应用已有报道,但还未见有关研究其作为PAHs暴露标志物的报道,因此,本研究试图通过代谢酶与焦炉工血浆BPDE-白蛋白加合物及DNA损伤的关联研究,探讨焦炉工血浆BPDE-白蛋白加合物作为PAHs暴露标志物的可行性,以及在遗传学的基础上找到与焦炉工PAHs暴露与癌症风险的潜在敏感因素,为焦炉作业工人的职业暴露危害防治提供科学依据。本研究共分以下三个部分:第一部分研究对象基本情况采用职业流行病学调查方法,选取202例男性焦炉工人作为暴露组,96例来自同一工厂的男性机关管理人员以及医务人员作为对照组。通过问卷调查获得研究人群的一般资料,如年龄、工龄、吸烟及饮酒等。采用反相高效液相色潽法(reverse phase-high performance liquid chromatography, RP-HPLC)测定血浆中BPDE-白蛋白加合物水平以及碱性单细胞凝胶电泳实验检测彗星尾距(Olive Tail Moment, OTM)。结果显示,两组研究人群在年龄、工龄、吸烟及饮酒方面没有显著性差异(P>0.05)。焦炉作业工人血浆BPDE-白蛋白加合物水平(经自然对数转换后),在暴露组为(3.55( 2.37-4.11 ))显著高于对照组(3.06(1.47-2.85)),差异有显著性意义(P<0.01);外周血淋巴细胞OTM值(经自然对数转换后),在暴露组为(1.25±1.09)显著高于对照组(0.55±0.93),差异有显著性意义(P<0.01)。本部分结果提示焦炉工人中PAHs暴露标志物血浆BPDE-白蛋白加合物水平以及外周血淋巴细胞OTM值与对照组比较具有显著性差异。第二部分代谢酶基因多态性与血浆BPDE-白蛋白加合物的关联性研究焦炉工血浆BPDE-白蛋白加合物具有化学特性,因此其形成可能受诸多因素如PAHs外环境暴露水平、体内代谢酶的作用、以及吸烟、饮酒、饮食和生活方式等的影响。本部分通过对影响焦炉工血浆BPDE-白蛋白加合物形成的因素、代谢酶基因多态性与血浆中BPDE-白蛋白加合物水平的关联研究,探讨焦炉工血浆BPDE-白蛋白加合物作为PAHs的暴露标志物的可行性。根据国际人类基因组单体型图计划(International HapMap project, HapMap)数据库挑选了I相代谢酶中4个关键酶基因共12个标记性单核苷酸多态性(tagging single nucleotide polymorphism, tagSNPs),采用基质辅助激光解析电离飞行时间质谱技术,对298名焦炉工人进行基因分型,结果发现经校正了年龄、工龄、吸烟和饮酒因素后,在暴露组中,CYP2B6基因rs3760657位点GA+AA基因型携带者血浆BPDE-白蛋白加合物水平(3.31(2.01-4.07))显著低于GG基因型携带者(3.59(2.67-4.27)),差异有显著性意义(P<0.05)。单体型对分析结果显示,在对照组中,CYP2E1基因GC/GT单体型对携带者血浆BPDE-白蛋白加合物水平最低为(3.00(0.96-3.73)),与人群中分布最广泛的CG/CG单体型对相比(3.63(2.97-4.25)),差别有显著性意义(P<0.05)。本部分结果提示代谢酶基因的遗传变异是影响焦炉工血浆中BPDE-白蛋白加合物水平的主要因素,可将焦炉工血浆BPDE-白蛋白加合物作为PAHs的暴露标志物。第三部分代谢酶基因多态性与焦炉工DNA损伤的关联性研究体内的致癌物PAHs(以致癌性最强的B[a]P为代表),一般先由I相代谢酶CYPs氧化、还原、水解等作用,改变毒物的功能基团,使其降解或转变为亲电子极团而具致癌作用,其代谢产物BPDE与体内DNA和/或蛋白质形成共价加合物,造成DNA损伤。然而,目前流行病学研究资料中,有关代谢酶基因遗传变异与DNA损伤关联性研究甚少,且各结论间存在争议。因此,我们假设代谢酶基因遗传变异在焦炉工DNA损伤过程中起着重要的作用,探讨代谢酶基因遗传变异与DNA损伤的关联。利用第二部分的代谢酶基因12个tagSNPs的分型资料,按经对数转换后的血浆BPDE-白蛋白加合物水平的三分位数分为低暴露组、中暴露组和高暴露组3组。结果发现在高暴露组中,CYP2B6基因rs1042389位点TC基因型携带者外周血淋巴细胞OTM值(1.04±1.06)显著低于TT基因型携带者(1.51±1.05),差异有显著性意义(P<0.05)。CYP1A1基因、CYP1B1基因和CYP2E1基因的遗传变异与焦炉工DNA损伤程度无显著关联。综上所述,本研究结果提示,焦炉工人暴露于PAHs后其暴露标志物血浆BPDE-白蛋白加合物水平均明显增高,且代谢酶基因多态性是影响其形成的主要因素,因此,我们认为可以将焦炉工血浆BPDE-白蛋白加合物作为PAHs的暴露标志物;与PAHs相关的代谢酶对焦炉工人DNA损伤效应有重要关联,个体易感性差异对遗传效应有重要影响。本研究的创新之处在于:(1)首次发现代谢酶基因尤其是CYP2B6基因多态性可降低血浆中BPDE-白蛋白加合物的水平;(2)探讨了代谢酶基因的遗传变异与DNA损伤程度的关联性,发现CYP2B6基因遗传变异显著降低了焦炉工DNA的损伤程度。本课题局限性以及有待深入研究的方面有:(1) PAHs的代谢过程复杂,涉及到多种代谢酶基因,而这些基因可能都存在有大量SNPs,本次研究仅是初步探讨了I相代谢酶CYP1A1、CYP1B1、CYP2B6和CYP2E1四个基因的12个SNPs位点与PAHs致DNA损伤之间的关联,但其他代谢酶基因多态性对PAHs致机体的影响,如何评价焦炉工人的整体代谢过程,基因与基因的联合作用、基因与环境之间的复杂联合作用等,都需要进一步研究。因此我们目前所探讨的代谢酶基因的多态性对PAHs致机体效应的风险影响是不足的;(2)随着全基因组关联研究(genome-wide association studies, GWAS)的实施,本次少数基因少数位点的研究在解释科学假设时是不够的,因此有必要在更大的样本中验证本研究获得的阳性结果;(3)此外,对SNPs位点的细胞功能学研究不足,如可采用报告基因转染,凝胶迁移滞后实验等技术深入验证启动子区域SNPs的功能改变。更多还原

【Abstract】 Polycyclic aromatic hydrocarbons (PAHs) may cause DNA damage, which is the main cause for lung cancer of coke oven workers. Epidemiological studies have showed that coke oven workers had different DNA damage levels with a similar environmental PAHs exposure levels, suggesting that genetic variations may play an important role in the individual’s genetic toxicity. PAHs are regulated by aryl hydrocarbon receptor (AhR). Upon ligand activation, the AhR translocates into nucleus, induce xenobiotics metabolic enzymes synthesis, including cytochrome P4501A1 (CYP1A1), cytochrome P4501B1 (CYP1B1), cytochrome P4502B6 (CYP2B6) and cytochrome P4502E1 (CYP2E1).Although urinary 1-hyrdroxyprene (1-OHP) was used as PAHs exposure markers in numerous studies, there are limitations because urinary 1-OHP is used to assess recent exposure to PAHs only. Plasma BPDE-Alb adducts may be considered as PAHs exposure markers in coke oven workers because of its longer half-life. Therefore, we attempted to discuss the feasibility about using plasma BPDE-Alb adducts as PAHs exposure markers and hypothesized that the single nucleotide polymorphisms (SNPs) which exist in the CYP1A1 gene, CYP1B1 gene, CYP2B6 gene and CYP2E1 gene may affect the DNA damage in coke oven workers. This study aimed to identify the associations between genetic variants of CYP1A1, CYP1B1, CYP2B6 and CYP2E1 genes and plasma BPDE-Alb adducts, and DNA damage levels of coke oven workers, which would identify exposure markers and find the cancer risk potential sensitive factors for occupational exposure risk prevention.PartⅠEpidemiology study of research subjectsA total of 298 male subjects were recruited from a steel plant in Taiyuan, northern China, of whom 202 coke oven workers were defined as exposure groups, the other 96 non coke oven workers from the same plant were used as control groups. We detected the plasma BPDE-Alb adducts levels using reverse phase-high performance liquid chromatography (RP-HPLC), and measured Olive Tail Moment (OTM) by alkaline single cell gel electrophoresis experiment. The results showed that there were no significant difference of age, length of work, smoking and drinking between the exposure and control groups (P>0.05). The in-transformed plasma BPDE-Alb adducts concentration (median, 25-75 percentile) in coke oven workers (3.55(2.37-4.11)) was significantly higher than control groups (3.06(1.47-2.85)) (P<0.01). The in-transformed Olive Tail Moment value in coke oven workers (1.25±1.09) was higher than control groups (0.55±0.93) (P<0.01). These results demonstrated that there were significant differences of plasma BPDE-Alb adduct levels and OTM values between coke oven workers and control groups. PartⅡAssociations of genetic variations in metabolism enzymes genes and plasma BPDE-Alb adducts in coke oven workersThere are many factors including PAHs environmental exposure levels, metabolic enzyme function, smoking and drinking may influence plasma BPDE-Alb adducts levels. In this part, we discussed the feasibility of using plasma BPDE-Alb adducts as PAHs exposure markers in coke oven workers. According to the Han Chinese Beijing data in the HapMap project, we selected 12 tagSNPs in metabolic enzymes gene. We genotyped the selected tagSNPs in 298 study subjects by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques. Our results indicated in exposed groups CYP2B6 gene rs3760657 loci GA+AA genotype carriers had significantly lower plasma BPDE-Alb adducts (3.31(2.01-4.07)) compared with GG genotype carriers (3.59(2.67-4.27)) (P<0.05). The diplotype analysis revealed that the CYP2E1 GC/GT diplotype carriers had the lowest plasma BPDE-Alb adducts levels (3.00(0.96-3.73)) compared with that of the most widely distributed CG/CG diplotype carriers (3.63(2.97-4.25)) (P<0.05) in control groups. The results of this part suggested that the main factor affect plasma BPDE-Alb adducts levels was the genetic variations of CYP2B6 gene. The results of this part warrant validation by larger populations. PartⅢAssociations of genetic variations in metabolism enzymes genes and DNA damage in coke oven workersCarcinogens PAHs are oxidated, reducted, hydrolyzed and changed toxic function first commonly by I phase metabolic enzymes CYPs. DNA damage is induced by the end products BPDE of PAHs through direct DNA and/or protein binding. Therefore, we hypothesized that the genetic variations in CYPs genes play an important role in DNA damage in coke oven workers, and explored the associations of genetic variations in CYPs genes and DNA damage. We divided the sujects into three groups according to plasma BPDE-Alb adducts. The results showed that CYP2B6 gene rs1042389 loci TC genotype carriers DNA damage levels (1.04±1.06) was lower than TT genotype carriers (1.51±1.05) in high exposure groups (P<0.05). However, there were no associations between the other genes (CYP1A1 gene, CYP1B1 gene and CYP2E1 gene) and DNA damage levels in the three groups. The results of this part suggested that genetic variations in CYP2B6 gene may prevent coke oven workers from DNA damage.In summary, we selected 12 tagSNPs in the CYP1A1, CYP1B1, CYP2B6 and CYP2E1 genes and detected their associations with plasma BPDE-Alb adducts levels and DNA damage. There are some advantages in this study, to our knowledge, it was the first study using plasma BPDE-Alb adducts as PAHs exposure markers; Our study provided the first evidence that genetic variations in CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage.There were several limitations in our study that need to be addressed. First, it was insufficient only discussed the association among CYP1A1, CYP1B1, CYP2B6 and CYP2E1 genes and DNA damage, for the complex process of PAHs metabolism. Second, the association between SNPs in the CYP2B6 gene and plasma BPDE-Alb adducts and DNA damage should be validated by larger population studies. Finally, there were no functional investigations of positive SNPs sites in this study.更多还原