【作者】 段化伟；

【导师】 郑玉新；

【作者基本信息】 中国疾病预防控制中心 ， 卫生毒理学， 2008， 博士

【摘要】 焦炉工肺癌是我国八种职业肿瘤之一,也是我国职业肿瘤防治领域面临的一个重点问题。焦炉逸散物(Coke-oven emission,COE)中的致癌性多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是导致焦炉工肺癌的主要原因。焦化生产已被国际癌症研究署(International Agency for Research on Cancer,IARC)归为“1类致癌物”。基因组不稳定性(genomic instability)是肿瘤发生过程中的重要生物学事件,微核(micronucleus,MN)是基因组不稳定性的重要形态学特征,由于其来源不明和自发率高的特点使其在一些研究不能完全反映化学物致基因组损伤的状态。近年来,随着机理的深入研究胞质分裂阻滞法微核试验(Cytokinesis-blockmicronucleus,CBMN)已经发展成为研究基因组不稳定性和细胞毒性的综合手段,即胞质分裂阻滞法微核细胞组学试验(Cytokinesis-block micronucleus cytomeassays)。“细胞组学(cytome)”的概念意味着涂片系统内计数的每一个细胞都有其细胞学意义,包括:1)存活状态—坏死、凋亡;2)有丝分裂情况—单核、中期分裂相、后期分裂相、双核、多核;3)染色体不稳定和损伤特征—微核、核质桥(Nucleoplasmic bridge,NPB)、核芽(Nuclear bud,NBUD)和核内或微核内利用着丝粒探针信号反映的信息。其中的核质桥和核芽是新的评价染色体损伤的指标,分别代表染色体断裂/重组的标志和基因扩增的标志。利用新的效应性生物标志物评价PAHs职业暴露人群的基因组不稳定性状态,综合分析基因组不稳定性的指标,有助于理解PAHs致癌的生物学过程,提高危险度评价的精度。本研究采用横断面分子流行病学设计,收集158名PAHs暴露工人和69名非多环芳烃暴露者的职业史、年龄、性别、吸烟和饮酒等信息。利用CBMN试验涂片计数核质桥和核芽的发生率,评价PAHs暴露者的基因组不稳定性状态。核质桥和核芽数经平方根转换后呈正态分布。使用方差分析比较不同外暴露等级、焦化作业工龄和年龄工人核质桥率和核芽率的差异,两两比较时采用Bonferroni校正。使用Student’s t检验比较吸烟者和不吸烟者、不同性别和不同饮酒状况工人中核质桥和核芽数的差异。利用PHASE 2.1软件经Bayesian统计方法计算个体MTHFR基因的单体型;在校正可能影响外周血淋巴细胞核质桥和核芽发生的因素(年龄、性别等)后,使用多元协方差分析比较不同基因型或单体型之间外周血淋巴细胞核质桥率和核芽率的差异。利用具有代谢苯并[a]芘(B[a]P)能力的细胞,经B[a]P染毒后,利用γ-H2AX焦点检测DNA双链断裂情况,结合CBMN细胞组学试验验证人群的研究结果,为其发展成为效应标志物提供有力证据。一、多环芳烃暴露与基因组不稳定性表型的关系研究应用外周血淋巴细胞胞质分裂阻滞法微核试验制备涂片,计数核质桥和核芽发生率评价外周血淋巴细胞的染色体损伤水平。PAHs暴露工人外周血淋巴细胞核质桥率(9.41±3.73)高于非暴露组(1.88±1.49),差异有统计学意义(P＜0.001);其核芽率为(7.13±4.01)也明显高于非暴露组(2.20±1.73)(P＜0.001)。发现核质桥和核芽率与多环芳烃暴露等级具有明显的剂量-效应关系。尿1-OHPyr水平与外周血淋巴细胞核质桥率有良好的相关性(n=227,Pearson’s r=0.741;P＜0.001)。进一步分析,在PAHs暴露工人和非暴露组中分别研究尿1-OHPyr水平与核质桥率的关系,也都发现了明显的相关性(分别是n=158,Pearson’s r=0.245,P=0.003和n=69,Pearson’s r=0.279,P=0.02)。同样,也发现尿1-OHPyr水平与外周血淋巴细胞核芽率有良好的相关(n=227,Pearson’s r=0.639;P＜0.001)。在PAHs暴露工人和非暴露组中分别研究尿1-OHPyr水平与核芽率的关系,也都发现了明显的相关性(分别是n=158,Pearson’s r=0.294,P＜0.001和n=69,Pearson’s r=0.382,P=0.001)。主成分分析表明:核质桥和核芽与PAHs内外暴露水平均有较好的剂量—效应关系,这在微核分析中却没有发现。与男性PAHs暴露工人相比,女性PAHs暴露工人具有较少的核质桥(7.14±3.04比9.63±3.73,P=0.01)和核芽(4.71±2.73比7.36±4.05,P=0.02)发生率。校正年龄后,较长焦化作业工龄的暴露者的核芽发生率较高(P=0.01)。未发现年龄、吸烟和饮酒等对PAHs暴露工人的核质桥和核芽率的影响。二、PAHs暴露工人基因组不稳定性表型与相关基因的关联研究使用多元协方差分析校正了年龄、性别、尿中1-羟基芘后,在PAHs暴露组和非暴露组中分别比较MTHFR 677和1298两个位点与基因组不稳定性之间的关联。结果发现PAHs暴露工人中MTHFR 677位点TT基因型或CT+TT基因型个体的核质桥率显著高于CC基因型个体(P=0.03)。在非暴露组中也发现TT基因型个体的核质桥率显著高于CC基因型个体(P=0.002),但未发现CT+TT基因型有类似的关联。以核芽为表型的研究中,没有发现这两个位点的基因多态性与核芽率的显著性关联。经单体型分析,PAHs暴露工人中携带MTHFR 677C-1298C、677T-1298A、677T-1298C单体型个体的核质桥率均高于677C-1298A单体型个体,差异有统计学意义(P=0.03,P=0.005和P=0.002);677T-1298A单体型个体的核芽率显著高于677C-1298A单体型个体(P=0.02)。未发现非暴露组中不同MTHFR单体型与核质桥和核芽率的显著性关联。DNA双链断裂修复是维持基因组稳定性的重要机制。同源重组修复基因与基因组不稳定性的关联研究中发现,PAHs暴露工人中NBSl rs1805794、rs1805800位点,MREll rs13447623位点,RAD52 rs1051672,RAD54B rs3019148位点与核质桥的发生率相关。发现MREll rs1014666、rs11020799、rs13447623、rs622961位点和RAD54L rs10789488位点与核芽的发生率相关。非同源末端连接的修复基因的关联研究中发现XRCC4 rs1382373、rs1478485、rs4703951位点和XRCC5rs10182201、rs3821107、rs828699位点与核质桥的发生率相关;LIG4 rs1805388和XRCC4 rs4703951位点与核芽的发生率相关。提示这些位点的基因多态性可影响个体的基因组稳定性,是PAHs暴露工人染色体损伤的遗传易感因素之一。三、苯并[a]芘对基因组稳定性的影响利用转入CYP1A1的人支气管上皮细胞16HBE-CYP1A1和空载质粒细胞16HBEV,研究了苯并[a]芘对基因组稳定性的影响。结果发现,B[a]P作用后,高浓度染毒组均有γ-H2AX焦点形成,并具有剂量依赖性。利用CBMN细胞组学试验检测了B[a]P的细胞毒性,发现B[a]P染毒后,16HBE-CYP1A1细胞的双核细胞率和核分裂指数(NDI)发生降低,随着染毒剂量的增加,细胞坏死率不断升高,具有剂量—效应关系。而凋亡细胞随着B[a]P作用剂量的增加出现先增高后降低的趋势。在16HBEV细胞中,中低剂量组间(1—10μM组)的双核细胞率并没有发生明显的变化,在20μM组降至40%左右。活细胞的NDI值在高于10μM处理组的细胞中发生降低。细胞坏死率随染毒剂量增加而升高,但到高于10μM组后没有明显升高。而凋亡细胞随着B[a]P作用剂量的增加而增高,在20μM组又降为正常水平。基因组不稳定性分析中发现,在16HBE-CYP1A1细胞中,随B[a]P作用浓度的增加,核质桥和核芽发生率也随之增加,具有明显的剂量-效应关系。微核率在高于10μM组后不再发生增加。在16HBEV细胞中,B[a]P作用后,微核、核质桥和核芽的发生率都有增加趋势。但在高于5μM组后没有明显改变(P＞0.05)。研究发现,与微核相比,核质桥和核芽具有更好的剂量—效应关系。这些指标的增加可能与DNA双链断裂损伤和细胞凋亡受抑制有关。综上所述,本研究系统的分析了PAHs对基因组不稳定性的影响。在人群中发现核质桥和核芽可以作为基因组不稳定性的效应标志物。叶酸代谢酶和DNA双链断裂修复酶的遗传变异能够影响PAHs暴露工人的基因组不稳定性状态。体外研究发现,CYP1A1是B[a]P代谢的关键酶,胞质分裂阻滞法微核细胞组学试验可以综合评价B[a]P的细胞毒性和遗传毒性,证实了B[a]P致核质桥和核芽形成的剂量-效应关系,发现其遗传毒性可能与DNA双链断裂损伤和抑制凋亡有关。这些结果提示基因组不稳定性在PAHs致癌过程中扮演重要角色,综合分析其指标的改变有助于暴露人群的生物监测。更多还原

【Abstract】 Chemicals produced in the coal-coking process has been classified as class I carcinogens by IARC.The product coke is formed by blending and heating suitable grades of coals to 1000-1400℃in the absence of oxygen.Coking workers are regularly exposed to coke oven emissions,which are mainly comprised of polycyclic aromatic hydrocarbons(PAHs).Epidemiological studies have shown that workers with a long-term exposure to PAHs had a significantly higher risk of lung cancer. Lung cancer of coke-oven workers has been classified as one of the eight prescribed occupational cancers in China,and its incidence rate was about 10 times of that of the general population.Therefore,lung cancer of PAHs workers is still a critical issue in the field of prevention and control of occupational cancers in our country.An early key event in carcinogenesis is the induction of genomic instability, which facilitates a proliferative cell to progress into a cancer cell.There is strong evidence that micronucleus(MN) frequency in peripheral blood lymphocytes is predictive marker of cancer risk,and increased levels of MNi formation are associated with early events in carcinogenesis.However,its complicated formation mechanisms, relatively high baseline make it too difficult to estimate the genomic damage induced by chemicals.Over the past decades,Cytokinesis-block micronucleus(CBMN) test has evolved into a comprehensive method for measuring DNA damage,cytostasis and cytotoxicity,which called Cytokinesis-block micronucleus cytome assays.This method is now also to score Nucleoplasmic bridge(NPB),a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair,and to score nuclear bud(NBUD),a biomarker of gene amplification.The "cytome" concept implies that every cell in the system studied is scored cytologically for its viability status(necrosis,apoptosis),its mitotic status(monoucleated,bionucleated, multinucleated) and its chromosomal damage or instability status(presence of MNi, NPBs,NBUDs and number of centromere probe signals among nuclei or MNi if such molecular tools are used in combination with the assay).Based on the new effect biomarkers of genomic instability induced by PAHs,it can help us to elucidate the carcinogensis of PAHs,improve the accuracy of risk assessment on PAHs.In this cross-sectional molecular epidemiologically designed study,NPBs and NBUDs were used as the effect biomarkers of the genomic instability in peripheral blood lymphocytes among 158 PAH-exposed workers and 69 unexposed controls by the CBMN test.For multivariate analysis,the NPBs and NBUDs data were sqrttransformed to normalize the variance.One-way ANOVA analysis was used to compare the NPBs or NBUDs’ difference in different environmental exposure levels, different coking history levels and different age levels.The differences in sqrt-transformed NPBs or NBUDs data between each genotype of related genes were analyzed with multivariate analysis of covariance with adjustment for age,gender,and cigarettes per day in PAHs exposed workers and controls separately.The MTHFR haplotypes were estimated by Bayesian statistical method with the PHASE software. Indicators of genomic instability found in epidemiological study were validated in cell lines which benzo[a]pyrene can be metabolized in vitro.DNA double strand breaks (DSBs) were test byγ-H2AX foci formation.Part 1.Relationiship between biomarkers of genomic instability induced by PAHs and exposure levelsWe found that NPBs and NBUDs were significantly higher in PAH-exposed workers than in the controls(9.41±3.73,7.13±4.01 versus 1.88±1.49,2.20±1.73, respectively;P＜0.001 for both comparisons) in a dose-dependent manner.The dose-dependent increase was also observed by integrating variables of MNi,NPBs and NBUDs,although that for MNi not found in the same study.In all 227 subjects,a significantly positive correlation was found between urinary 1-OHP concentrations and the frequency of NPBs(Pearson’s r = 0.741,P＜0.001) and NBUDs(Pearson’s r = 0.64,P＜0.001).Combining with principal component analysis,it revealed that NPBs and NBUDs were more susceptible to PAHs exposure.Compared with male PAH-exposed workers,female workers had less NPBs or NBUDs.NBUDs were found higher in workers with longer coke-working history(P=0.01).No effects of age, smoking and alcohol using were found on frequencies of NPBs or NBUDs among PAH-exposed workers.Part 2.Association study between phenotypes of genomic instability and related genetic polymorphismMultivariate analysis of covariance revealed that PAHs exposed workers with the MTHFR 677 TT and CT-TT genotypes had significantly higher NPBs frequency(9.79±0.49,p=0.03 and 9.75±0.34,p=0.03) than those with CC genotype(7.97±0.56) with adjustment for covariates.Further haplotypes analysis of the MTHFR 677-1298, PAHs exposed workers carrying CC,TA,TC haplotype had significant higher NPBs frequency than those with CA(P = 0.03,P = 0.005 and P = 0.002 respectively).No significant association between C677T and A1298C polymorphism and NBUDs frequency was found in both groups.Based on haplotype analysis of MTHFR 677-1298,subjects carrying TA haplotype had significant higher NBUDs frequency than those with CA(P = 0.02) in PAHs exposed workers.No significant association between MTHFR gene haplotype and NPBs/NBUDs frequencies among controls was found.A DNA double-strand break(DSB) is a critical lesion that can promote genomic instability.Eukaryotic cells have developed two pathways to repair DNA DSBs, including the homologous recombination(HR) and the non-homologous end-joining (NHEJ) pathways.We performed genetic association studies in the PAH-exposed population study analyzing polymorphisms in genes involved in HR(NBS1,MRE11, RAD50,RAD52,RAD54L,RAD54B,XRCC2 and XRCC3) and NHEJ(XRCC4,LIG4 and XRCC5).In the single locus analysis,elevated NPB frequency statistically significantly associated with NBS1 rs1805794、rs1805800,MRE11 rs13447623, RAD52 rs1051672,RAD54B rs3019148(all P＜0.05),and elevated NBUD frequencies significantly associated with MRE11 rs 1014666,rs 11020799,rs 13447623, rs622961 and RAD54L rs10789488(all P＜0.05) in the HR pathway.In the NHEJ pathway,XRCC4 rs1382373,rs1478485,rs4703951 and XRCC5 rs10182201, rs3821107,rs828699(all P＜0.05)associated with NPB frequencies;LIG4 rs1805388 and XRCC4 rs4703951(all P＜0.05)associated with NBUD frequencies.Part 3.Genomic instability induced by benzo[a]pyreneBenzo[a]pyrene(B[a]P) can be activated in 16HBE-CYP1A1 cells which are human bronchial epithelial cell with CYP1A1 transformed.The vector control cell is 16HBEV in the study.Genomic instability induced by benzo[a]pyrene was tested by cytokinesis-block micronucleus cytome assays.DSBs were detected byγ-H2AX foci formation.After B[a]P treatment for 24h,higher concentration induced moreγ-H2AX foci in 16HBE-CYP1A1 and 16HBEV cells,but theγ-H2AX loci were less in 16HBEV cells than 16HBE-CYP1A1 cells at the same B[a]P treatment. Cytotoxicity of B[a]P tested by CBMN cytome assays,the values of binucleated cells (BNC) ratio and nuclear division index(NDI) decreased and those of necrosis increased in a dose-dependent manner in 16HBE-CYP1A1 cells.Apoptotic cells were increased at lower dose of B[a]P and rarely observed at the highest dose of B[a]P in 16HBE-CYP1A1 cells.In 16HBEV cells,no significant BNC ratio changed in lower than 10 taM B[a]P treatment;it was decreased 40%at 20μM group.NDI was decreased at higher than 10μM B[a]P treated 16HBEV cells.Necrosis and apoptosis increased in a dose-dependent manner lower than 10μM B[a]P treated 16HBEV cells; apoptotic cells were decreased to normal level at 20μM B[a]P treatment.The genomic instability was assessed by the frequencies of MNi,NPBs and NBUDs.NPBs and NBUDs increased in a dose-dependent manner,however,MNi dose-relationship only last to 10μM B[a]P treated group in 16HBE-CYP 1A1 cells.In 16HBEV cells,frequencies of MNi,NPBs and NBUDs were increased,but no significant difference of them was found between higher than 5 tam B[a]P treated groups.Those indicators of genomic instability were increased may be induced by DSBs and inhibition of apoptosis pathway. In summary,genomic instability induced by PAHs is investigated logically in both of population-based and laboratory studies.In PAH-exposed population,NPBs and NBUDs can be taken as effect biomarkers for chromosomal instability, polymorphism of MTHFR and DSBs repair genes are associated to biomarkers of genomic instability among PAHs-exposed workers.Based on laboratory study, CYP1A1 is key metabolized enzyme of B[a]P;CBMN cytome assays can be used to analyze cytotoxicity and genotoxicity of B[a]P comprehensively,validate the dose-effect relationship between PAHs and NPBs/NBUDs were validated in vitro, which found in population study.The genomic instability formation may be associated to DNA double strand breaks and inhibition of apoptosis pathway.These findings suggest that genomic instability play an important role in PAHs carcinogenesis,and comprehensive biomarkers can be applied to biomonitor exposed population.更多还原