多环芳烃致基因组不稳定性表型及相关基因的关联研究
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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。
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 levels
We 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 polymorphism
Multivariate 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]pyrene
Benzo[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.
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。
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 levels
We 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 polymorphism
Multivariate 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]pyrene
Benzo[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.
引文
1.IARC.Monographs on the evaluation of the carcinogenic risk of chemicals to humans.Polycyclic aromatic hydrocarbons,part 1,chemical,environmental and experimental data.France:International Agency for Research on Cancer;1983.
2.IARC.Monographs on the evaluation of the carcinogenic risk of chemicals to humans.Polycyclic aromatic hydrocarbons,part 3,industrial exposure in aluminium production,coal gasification,coke production,and iron and steel founding.France:International Agency for Research on Cancer.1984.
3.Unwin J,Cocker J,Scobbie E,et al.An assessment of occupational exposure to polycyclic aromatic hydrocarbons in the UK.Ann Occup Hyg,2006,50:395-403.
4.Schauer C,Niessner R,Poschl U.Polycyclic aromatic hydrocarbons in urban air particulate matter:decadal and seasonal trends,chemical degradation,and sampling artifacts.Environ Sci Technol,2003,37:2861-2868.
5.Liu Y,Tao S,Yang Y,et al.Inhalation exposure of traffic police officers to polycyclic aromatic hydrocarbons(PAHs) during the winter in Beijing,China.Sci Total Environ,2007,383:98-105.
6.Varlet V,Serot T,Monteau F,et al.Determination of PAH profiles by GC-MS/MS in salmon processed by four cold-smoking techniques.Food Addit Contam,2007,24:744-757.
7.Sanderson E,Kelly P,Farant JE Effect of Soderberg smelting technology,anode paste composition,and work shift on the relationship between benzo[a]pyrene and individual polycyclic aromatic hydrocarbons.J Occup Environ Hyg,2005,2:65-72;quiz D66-67.
8.Greenberg A,Hsu C,Rothman N,et al.PAH Profiles of Charbroiled Hamburgers:Pyrene/B[a]P Ratios and Presence of Reactive PAH Polycyclic Aromatic Compounds,1993 3:101-110.
9.杨洪彪,段小丽,zhang J,等.焦炉工人PAHs空气日均暴露浓度分析.环境监测管理与技术,2004,16:13-16.
10.陈波,郑力行,胡云平,等.焦炉工职业性多环芳烃暴露特征的研究 中华劳 动卫生职业病杂志,2004,22:327-330.
11.Zhivotovsky B,Kroemer G.Apoptosis and genomic instability.Nat Rev Mol Cell Biol,2004,5:752-762.
12.Lengauer C,Kinzler KW,Vogelstein B.Genetic instabilities in human cancers.1998,396:643-649.
13.Sen S.Aneuploidy and cancer.Curr Opin Oncol,2000,12:82-88.
14.Raptis S,Mrkonjic M,Green RC,et al.MLH1 -93G>A promoter polymorphism and the risk of microsatellite-unstable colorectal cancer.J Natl Cancer Inst,2007,99:463-474.
15.Bardelli A,Cahill DP,Lederer G,et al.Carcinogen-specific induction of genetic instability.Proc Natl Acad Sci U S A,2001,98:5770-5775.
16.田宝磊,孙志贤.PML与基因组稳定性 中国生物化学与分子生物学报,2006.22:349-254.
17.Charames GS,Bapat B.Genomic instability and cancer.Curr Mol Med,2003,3:589-596.
18.Eyfjord JE,Bodvarsdottir SK.Genomic instability and cancer:networks involved in response to DNA damage.Mutat Res,2005,592:18-28.
19.Swanton C,Tomlinson I,Downward J.Chromosomal instability,colorectal cancer and taxane resistance.Cell Cycle,2006,5:818-823.
20.Raptis S,Bapat B.Genetic instability in human tumors.Exs,2006:303-320.
21.Sieber O,Heinimann K,Tomlinson I.Genomic stability and tumorigenesis.Semin Cancer Biol,2005,15:61-66.
22.Bonassi S,Hagmar L,Stromberg U,et al.Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens.European Study Group on Cytogenetic Biomarkers and Health.Cancer Res,2000.60:1619-1625.
23.Rossner P,Boffetta P,Ceppi M,et al.Chromosomal aberrations in lymphocytes of healthy subjects and risk of cancer.Environ Health Perspect,2005,113:517-520.
24.Bonassi S,Znaor A,Ceppi M,et al.An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.Carcinogenesis, 2007,28:625-631.
25.Van Hummelen P,Gennart JP,Buchet JP,et al.Biological markers in PAH exposed workers and controls.Mutat Res,1993,300:231-239.
26.Burgaz S,Erdem O,Karahalil B,et al.Cytogenetic biomonitoring of workers exposed to bitumen fumes.Mutat Res,1998,419:123-130.
27.Kalina I,Brezani P,Gajdosova D,et al.Cytogenetic monitoring in coke oven workers.Mutat Res,1998,417:9-17.
28.Jarvholm B,Nordstrom G,Hogstedt B,et al.Exposure to polycyclic aromatic hydrocarbons and genotoxic effects on nonsmoking Swedish road pavement workers.Scand J Work Environ Health,1999,25:131-136.
29.Cavallo D,Ursini CL,Bavazzano P,et al.Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs.Ann Occup Hyg,2006,50:211-218.
30.Norppa H,Bonassi S,Hansteen IL,et al.Chromosomal aberrations and SCEs as biomarkers of cancer risk.Mutat Res,2006,600:37-45.
31.Boffetta P,van der Hel O,Norppa H,et al.Chromosomal aberrations and cancer risk:results of a cohort study from Central Europe.Am J Epidemiol,2007,165:36-43.
32.Bonassi S,Norppa H,Ceppi M,et al.Chromosomal Aberration Frequency in Lymphocytes Predicts the Risk of Cancer:Results from a Pooled Cohort Study of 22,358 Subjects in 11 Countries.Carcinogenesis,2008.
33.El-Zein RA,Schabath MB,Etzel CJ,et al.Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer risk.Cancer Res,2006,66:6449-6456.
34.Fenech M.The role of folic acid and Vitamin B12 in genomic stability of human cells.Mutat Res,2001,475:57-67.
35.Fenech M,Crott JW.Micronuclei,nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakage-fusion-bridge cycles in the cytokinesis-block micronucleus assay.Mutat Res,2002,504:131-136.
36.Downs JA,Lowndes NF,Jackson SP.A role for Saccharomyces cerevisiae histone H2A in DNA repair.Nature,2000,408:1001-1004.
37.Chen HT,Bhandoola A,Difilippantonio MJ,et al.Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX.Science,2000,290:1962-1965.
38.MacPhail SH,Banath JP,Yu TY,et al.Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays.Int J Radiat Biol,2003,79:351-358.
39.Rogakou EP,Pilch DR,Orr AH,et al.DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.J Biol Chem,1998,273:5858-5868.
40.Ismail IH,Wadhra TI,Hammarsten O.An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans.Nucleic Acids Res,2007,35:e36.
41.Rothkamm K,Lobrich M.Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.Proc Natl Acad Sci USA,2003,100:5057-5062.
42.Shivji MK,Venkitaraman AR.DNA recombination,chromosomal stability and carcinogenesis:insights into the role of BRCA2.DNA Repair (Amst),2004,3:835-843.
43.Pluth JM,Yamazaki V,Cooper BA,et al.DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells.DNARepair (Amst),2008,7:108-118.
44.O'Driscoll M,Jeggo PA.The role of double-strand break repair-insights from human genetics.Nat Rev Genet,2006,7:45-54.
45.Fenech M.Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability,mitotic dysfunction and cell death.Mutat Res,2006,600(l-2):58-66.
46.Fenech M.Cytokinesis-block micronucleus cytome assay.Nat Protoc,2007,2:1084-1104.
47.Thomas P,Umegaki K,Fenech M.Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay.Mutagenesis,2003,18:187-194.
48.Stewenius Y,Gorunova L,Jonson T,et al.Structural and numerical chromosome changes in colon cancer develop through telomere-mediated anaphase bridges,not through mitotic multipolarity.Proc Natl Acad Sci U S A,2005,102:5541-5546.
49.Fenech M.Biomarkers of genetic damage for cancer epidemiology.Toxicology,2002,181-182:411-416.
50.Fenech M.The in vitro micronucleus technique.Mutat Res,2000,455:81-95.
51.Fenech M,Chang WP,Kirsch-Volders M,et al.HUMN project:detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures.Mutat Res,2003,534:65-75.
52.Fenech M.Chromosomal biomarkers of genomic instability relevant to cancer.Drug Discov Today,2002,7:1128-1137.
53.Kryscio A,Ulrich Muller WU,Wojcik A,et al.A cytogenetic analysis of the long-term effect of uranium mining on peripheral lymphocytes using the micronucleus-centromere assay.Int J Radiat Biol,2001,77:1087-1093.
54.Touil N,Aka PV,Buchet JP,et al.Assessment of genotoxic effects related to chronic low level exposure to ionizing radiation using biomarkers for DNA damage and repair.Mutagenesis,2002,17:223-232.
55.Wold S,Albano C,Dunn Ⅲ W,et al.Multivariate data analysis in chemistry.Dordrecht,The Netherlands Reidel;1984.
56.Johnson R,Wichern D.Applied multivariate statistical analysis.New Jersey:Prentice-Hall Inc.,Englewood Cliffs;1992.
57.孙振球,徐勇勇.医学统计学.第二版∶人民卫生出版社;2005.
58.Umegaki K,Fenech M.Cytokinesis-block micronucleus assay in WIL2-NS cells:a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.Mutagenesis,2000,15:261-269.
59.Bonassi S,Neri M,Lando C,et al.Effect of smoking habit on the frequency of micronuclei in human lymphocytes:results from the Human MicroNucleus project.Mutat Res,2003,543:155-166.
60.Crott JW,Mashiyama ST,Ames BN,et al.The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro.Cancer Epidemiol Biomarkers Prev,2001,10:1089-1096.
61.Leopardi P,Marcon F,Caiola S,et al.Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes.Mutagenesis,2006,21:327-333.
62.Zhang S,Hunter DJ,Hankinson SE,et al.A prospective study of folate intake and the risk of breast cancer.Jama,1999,281:1632-1637.
63.Duthie SJ,Narayanan S,Blum S,et al.Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells.Nutr Cancer,2000,37:245-251.
64.Eto I,Krumdieck CL.Role of vitamin B12 and folate deficiencies in carcinogenesis.Adv Exp Med Biol,1986,206:313-330.
65.Blount BC,Mack MM,Wehr CM,et al.Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage:implications for cancer and neuronal damage.Proc Natl Acad Sci USA,1997,94:3290-3295.
66.Wang X,Thomas P,Xue J,et al.Folate deficiency induces aneuploidy in human lymphocytes in vitro-evidence using cytokinesis-blocked cells and probes specific for chromosomes 17 and 21.Mutat Res,2004,551:167-180.
67.Wagner C,Briggs WT,Home DW,et al.10-Formyltetrahydrofolate dehydrogenase:identification of the natural folate ligand,covalent labeling,and partial tryptic digestion.Arch Biochem Biophys,1995,316:141-147.
68.Frosst P,Blom HJ,Milos R et al.A candidate genetic risk factor for vascular disease:a common mutation in methylenetetrahydrofolate reductase.Nat Genet,1995,10:111-113.
69.Weisberg I,Iran P,Christensen B,et al.A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity.Mol Genet Metab,1998,64:169-172.
70.Kimura M,Umegaki K,Higuchi M,et al.Methylenetetrahydrofolate reductase C677T polymorphism,folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes.J Nutr,2004,134:48-56.
71.Kuschel B,Auranen A,McBride S,et al.Variants in DNA double-strand break repair genes and breast cancer susceptibility.Hum Mol Genet,2002,11:1399-1407.
72.West SC.Molecular views of recombination proteins and their control.Nat Rev Mol Cell Biol,2003,4:435-445.
73.Martin IV,MacNeill SA.ATP-dependent DNA ligases.Genome Biol,2002,3:REVIEWS3005.
74.Burma S,Chen BP,Chen DJ.Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.DNA Repair (Amst),2006,5:1042-1048.
75.Calsou P,Delteil C,Frit P,et al.Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.J Mol Biol,2003,326:93-103.
76.Leber R,Wise TW,Mizuta R et al.The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase.J Biol Chem,1998,273:1794-1801.
77.Fu YP,Yu JC,Cheng TC,et al.Breast cancer risk associated with genotypic polymorphism of the nonhomologous end-joining genes:a multigenic study on cancer susceptibility.Cancer Res,2003,63:2440-2446.
78.Lee KM,Choi JY,Kang C,et al.Genetic polymorphisms of selected DNA repair genes,estrogen and progesterone receptor status,and breast cancer risk.Clin Cancer Res,2005,11:4620-4626.
79.Leng S,Dai Y,Niu Y,et al Effects of genetic polymorphisms of metabolic enzymes on cytokinesis-block micronucleus in peripheral blood lymphocyte among coke-oven workers.Cancer Epidemiol Biomarkers Prev,2004,13:1631-1639.
80.Choi SW,Mason JB.Folate and carcinogenesis:an integrated scheme.J Nutr,2000,130:129-132.
81.Shen H,Spitz MR,Wang LE,et al.Polymorphisms of methylene-tetrahydrofolate reductase and risk of lung cancer:a case-control study.Cancer Epidemiol Biomarkers Prev,2001,10:397-401.
82.Shen H,Xu Y,Zheng Y,et al.Polymorphisms of 5,10-methylenetetrahydrofolate reductase and risk of gastric cancer in a Chinese population:a case-control study.Int J Cancer,2001,95:332-336.
83.Chen J,Ma J,Stampfer ML et al.Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer.Pharmacogenetics,2002,12:339-342.
84.Miller SA,Dykes DD,Polesky HF.A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res,1988,16:1215.
85.Lin J,Spitz MR,Wang Y,et al.Polymorphisms of folate metabolic genes and susceptibility to bladder cancer:a case-control study.Carcinogenesis,2004,25:1639-1647.
86.Carlson CS,Eberle MA,Rieder MJ,et al.Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans.Nat Genet,2003,33:518-521.
87.Wu X,Zhao H,Amos CI,et al.p53 Genotypes and Haplotypes Associated With Lung Cancer Susceptibility and Ethnicity.J Natl Cancer Inst,2002,94:681-690.
88.Ogino S,Wilson RB.Genotype and haplotype distributions of MTHFR677C>T and 1298A>C single nucleotide polymorphisms:a meta-analysis.J Hum Genet,2003,48:1-7.
89.朱慧萍,刘明珠,刀京晶.MTHFR基因第1298位核苷酸多态性与酶活性的关系.北京医科大学学报,2002,32:262-264.
90.孙耀峰,戴宇飞,程娟,等.亚甲基四氢叶酸还原酶基因单体型与焦炉作业工人染色体损伤易感性的关系.卫生研究,2006,35:387-390.
91.Le Marchand L,Donlon T,Hankin JH,et al.B-vitamin intake,metabolic genes,and colorectal cancer risk(United States).Cancer Causes Control,2002,13:239-248.
92.缪小平,邢德印,谭文,等.MTHFR基因C677T和A1298C多态与贲门癌的遗传易感性.中华医学杂志,2002,82:669-672.
93.van der Put NM,Gabreels F,Stevens EM,et al.A second common mutation in the methylenetetrahydrofolate reductase gene:an additional risk factor for neural-tube defects? Am J Hum Genet,1998,62:1044-1051.
94.Duthie SJ,Hawdon A.DNA instability(strand breakage,uracil misincorporation,and defective repair) is increased by folic acid depletion in human lymphocytes in vitro.Faseb J,1998,12:1491-1497.
95.Stem LL,Mason JB,Selhub J,et al.Genomic DNA hypomethylation,a characteristic of most cancers,is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene.Cancer Epidemiol Biomarkers Prev,2000,9:849-853.
96.Song C,Xing D,Tan W,et al.Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population.Cancer Res,2001,61:3272-3275.
97.Shen M,Rothman N,Berndt SI,et al.Polymorphisms in folate metabolic genes and lung cancer risk in Xuan Wei,China.Lung Cancer,2005,49:299-309.
98.Hung RJ,Hashibe M,McKay J,et al.Folate-related genes and the risk of tobacco-related cancers in Central Europe.Carcinogenesis,2007,28:1334-1340.
99.Hu J,Nyren O,Wolk A,et al.Risk factors for oesophageal cancer in northeast China.Int J Cancer,1994,57:38-46.
100.Ronnenberg AG,Goldman MB,Aitken IW,et al.Anemia and deficiencies of folate and vitamin B-6 are common and vary with season in Chinese women of childbearing age.J Nutr,2000,130:2703-2710.
101.Hao L,Ma J,Stampfer MJ,et al.Geographical,seasonal and gender differences in folate status among Chinese adults.J Nutr,2003,133:3630-3635.
102.UNICEF.Vitamin & mineral deficiency:a damage assessment report for CHINA.United Nations Children's Fund(UNICEF);2004.
103.齐军,缪小平,谭文,等.亚甲基四氢叶酸还原酶基因单核苷酸多态与乳腺癌风险.中华肿瘤杂志,2004,26:287-289.
104.Mu LN,Cao W,Zhang ZF,et al.Methylenetetrahydrofolate reductase(MTHFR)C677T and A1298C polymorphisms and the risk of primary hepatocellular carcinoma (HCC) in a Chinese population.Cancer Causes Control,2007,18:665-675.
105.Shen H,Newmann AS,Hu Z,et al.Methylenetetrahydrofolate reductase polymorphisms/haplotypes and risk of gastric cancer:a case-control analysis in China.Oncol Rep,2005,13:355-360.
106.Mu LN,Cao W,Zhang ZF,et al.Polymorphisms of 5,10-methylenetetralydrofolate reductase(MTHFR),fruit and vegetable intake,and the risk of stomach cancer.Biomarkers,2007,12:61-75.
107.Wang Y,Guo W,He Y,et al.Association of MTHFR C677T and SHMT(l) C1420T with susceptibility to ESCC and GCA in a high incident region of Northern China.Cancer Causes Control,2007,18:143-152.
108.Ma J,Stampfer MJ,Giovannucci E,et al.Methylenetetrahydrofolate reductase polymorphism,dietary interactions,and risk of colorectal cancer.Cancer Res,1997,57:1098-1102.
109.The International HapMap Consortium.The International HapMap Project.Nature.2003 Dec 18;Sect.789-796.
110.Kobayashi J,Antoccia A,Tauchi H,et al.NBS1 and its functional role in the DNA damage response.DNA Repair (Amst),2004,3:855-861.
111.Zhu XD,Kuster B,Mann M,et al.Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres.Nat Genet,2000,25:347-352.
112.Kobayashi J,Tauchi H,Sakamoto S,et al.NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain.Curr Biol,2002,12:1846-1851.
113.Hjorth JT,Gad J,Cooper H,et al.A zebrafish homologue of deleted in colorectal cancer (zdcc) is expressed in the first neuronal clusters of the developing brain.Mech Dev,2001,109:105-109.
114.Musak L,Soucek P,Vodickova L,et al.Chromosomal aberrations in tire plant workers and interaction with polymorphisms of biotransformation and DNA repair genes.Mutat Res,2008,641:36-42.
115.Medina PP,Ahrendt SA,Pollan M,et al.Screening of homologous recombination gene polymorphisms in lung cancer patients reveals an association of the NBSl-185Gln variant and p53 gene mutations.Cancer Epidemiol Biomarkers Prev,2003,12:699-704.
116.Sanyal S,Festa F,Sakano S,et al.Polymorphisms in DNA repair and metabolic genes in bladder cancer.Carcinogenesis,2004,25:729-734.
117.Zienolddiny S,Campa D,Lind H,et al.Polymorphisms of DNA repair genes and risk of non-small cell lung cancer.Carcinogenesis,2006,27:560-567.
118.Haber JE.DNA recombination:the replication connection.Trends Biochem Sci, 1999,24:271-275.
119.Yokoyama H,Sarai N,Kagawa W,et al.Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B,Rad51C,Rad51D and Xrcc2 protein complex.Nucleic Acids Res,2004,32:2556-2565.
120.Mohindra A,Bolderson E,Stone J,et al.A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks.Hum Mol Genet,2004,13:203-212.
121.Griffin CS,Simpson PJ,Wilson CR,et al.Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation.Nat Cell Biol,2000,2:757-761.
122.Cui X,Brenneman M,Meyne J,et al.The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.Mutat Res,1999,434:75-88.
123.Lopez-Cima MF,Gonzalez-Arriaga P,Garcia-Castro L,et al.Polymorphisms in XPC,XPD,XRCCl,and XRCC3 DNA repair genes and lung cancer risk in a population of northern Spain.BMC Cancer,2007,7:162.
124.Matullo G,Palli D,Peluso M,et al.XRCCl,XRCC3,XPD gene polymorphisms,smoking and (32)P-DNA adducts in a sample of healthy subjects.Carcinogenesis,2001,22:1437-1445.
125.Matullo G,Peluso M,Polidoro S,et al.Combination of DNA repair gene single nucleotide polymorphisms and increased levels of DNA adducts in a population-based study.Cancer Epidemiol Biomarkers Prev,2003,12:674-677.
126.Vodicka P,Kumar R,Stetina R,et al.Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates,chromosomal aberrations and single-strand breaks in DNA.Carcinogenesis,2004,25:757-763.
127.Tuimala J,Szekely G,Wikman H,et al.Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes:effects on levels of sister chromatid exchanges and chromosomal aberrations.Mutat Res,2004,554:319-333.
128.Ferguson DO,Sekiguchi JM,Chang S,et al.The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations.Proc Natl Acad Sci USA,2000,97:6630-6633.
129.Olive PL.The role of DNA single-and double-strand breaks in cell killing by ionizing radiation.Radiat Res,1998,150:S42-51.
130.Kim CH,Park SJ,Lee SH.A targeted inhibition of DNA-dependent protein kinase sensitizes breast cancer cells following ionizing radiation.J Pharmacol Exp Ther,2002,303:753-759.
131.Koike M.Dimerization,translocation and localization of Ku70 and Ku80 proteins.J Radiat Res (Tokyo),2002,43:223-236.
132.Dai Y,Kysela B,Hanakahi LA,et al.Nonhomologous end joining and V(D)J recombination require an additional factor.Proc Natl Acad Sci USA,2003,100:2462-2467.
133.Korabiowska M,Tscherny M,Stachura J,et al.Differential expression of DNA nonhomologous end-joining proteins Ku70 and Ku80 in melanoma progression.Mod Pathol,2002,15:426-433.
134.Zhu G,Duffy DL,Turner DR et al.Linkage and association analysis of radiation damage repair genes XRCC3 and XRCC5 with nevus density in adolescent twins.Twin Res,2003,6:315-321.
135.De Ruyck K,Wilding CS,Van Eijkeren M,et al.Microsatellite polymorphisms in DNA repair genes XRCC1,XRCC3 and XRCC5 in patients with gynecological tumors:association with late clinical radio sensitivity and cancer incidence.Radiat Res,2005,164:237-244.
136.Sadetzki S,Flint-Richter P,Starinsky S,et al.Genotyping of patients with sporadic and radiation-associated meningiomas.Cancer Epidemiol Biomarkers Prev,2005,14:969-976.
137.Barnes DE,Stamp G,Rosewell I,et al.Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice.Curr Biol,1998,8:1395-1398.
138.Frank KM,Sekiguchi JM,Seidl KJ,et al.Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV.Nature,1998,396:173-177.
139.Liu Y,Zhou K,Zhang H,et al.Polymorphisms of LIG4 and XRCC4 involved in the NHEJ pathway interact to modify risk of glioma.Hum Mutat,2008,29:381-389.
140.Sakiyama T,Kohno T,Mimaki S,et al.Association of amino acid substitution polymorphisms in DNA repair genes TP53,POLI,REV1 and LIG4 with lung cancer risk.Int J Cancer,2005,114:730-737.
141.Roddam PL,Rollinson S,O'Driscoll M,et al.Genetic variants of NHEJ DNA ligase IV can affect the risk of developing multiple myeloma,a tumour characterised by aberrant class switch recombination.J Med Genet,2002,39:900-905.
142.Mensing T,Marczynski B,Engelhardt B,et al.DNA adduct formation of benzo[a]pyrene in white blood cells of workers exposed to polycyclic aromatic hydrocarbons.Int J Hyg Environ Health,2005,208:173-178.
143.Divi RL,Beland FA,Fu PP,et al.Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts:validation by comparison with other methods,and use in human biomonitoring.Carcinogenesis,2002,23:2043-2049.
144.Ohnishi S,Kawanishi S.Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.Biochem Biophys Res Commun,2002,290:778-782.
145.Gao D,Luo Y,Guevara D,et al.Benzo[a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2'-deoxyguanosine via reactive oxygen species.Free Radic Biol Med,2005,39:1177-1183.
146.Shou M,Korzekwa KR,Crespi CL,et al.The role of 12 cDNA-expressed human,rodent,and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-dihydrodiol.Mol Carcinog,1994,10:159-168.
147.Richardson C.RAD51,genomic stability,and tumorigenesis.Cancer Lett,2005,218:127-139.
148.Ismail IH,Hendzel MJ.The gamma-H2A.X:is it just a surrogate marker of double-strand breaks or much more?Environ Mol Mutagen,2008,49:73-82.
149．曹佳．微核试验在中国的应用、发展与展望．遗传,2003,25：73～76．
150.Wilkening S,Stahl F,Bader A.Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.Drug Metab Dispos,2003,31:1035-1042.
151.Lehmann U,Kreipe H.Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies.Methods,2001,25:409-418.
152.Castedo M,Perfettini JL,Roumier T,et al.Cell death by mitotic catastrophe:a molecular definition.Oncogene,2004,23:2825-2837.
153.Goldsworthy TL,Conolly RB,Fransson-Steen R.Apoptosis and cancer risk assessment.Mutat Res,1996,365:71-90.
154.Mathias N,Johnson SL,Winey M,et al.Cdc53p acts in concert with Cdc4p and Cdc34p to control the Gl-to-S-phase transition and identifies a conserved family of proteins.Mol Cell Biol,1996,16:6634-6643.
155.Rudolph KL,Millard M,Bosenberg MW,et al Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nat Genet,2001,28:155-159.
156.Lindberg HK,Wang X,Jarventaus H,et al.Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes.Mutat Res,2007,617:33-45.
157.Shimizu N,Itoh N,Utiyama H,et al.Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.J Cell Biol,1998,140:1307-1320.
158.Shimizu N,Shimura T,Tanaka T.Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei.Mutat Res,2000,448:81-90.
159.Serrano-Garcia L,Montero-Montoya R.Micronuclei and chromatid buds are the result of related genotoxic events.Environ Mol Mutagen,2001,38:38-45.
160.Hyslop PA,Hinshaw DB,Scraufstatter IU,et al.Hydrogen peroxide as a potent bacteriostatic antibiotic:implications for host defense.Free Radic Biol Med,1995,19:31-37.
1.Schmid W.The micronucleus test.Mutat Res,1975,31:9-15.
2.Auletta AE,Dearfield KL,Cimino MC.Mutagenicity test schemes and guidelines:U.S.EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs.Environmental and molecular mutagenesis,1993,21:38-45;discussion 6-57.
3.Sofuni T.Japanese guidelines for mutagenicity testing.Ministry of Agriculture,Forestry and Fisheries;Ministry of Health and Welfare;Ministry of Labor.Environ Mol Mutagen,1993,21:2-7.
4.中华人民共和国卫生部药政局.新药(西药)临床研究指导原则汇编(毒理学).1993.
5.Fenech M.Cytokinesis-block micronucleus assay evolves into a "cytome" assay of chromosomal instability,mitotic dysfunction and cell death.Mutat Res,2006,600(1-2):58-66.
6.曹佳.微核试验在中国的应用、发展与展望.遗传,2003,25:73-6.
7.Fenech M,Chang WP,Kirsch-Volders M,et al.HUMN project:detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures.Mutat Res,2003,534:65-75.
8.曹佳,林真,余争平.微核试验-原理、方法及其在人群监测和毒性评价中的应用.北京:军事科学出版社;2000.
9.Miller B,Potter-Locher F,Seelbach A,et al.Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay:position of the GUM Working Group on the in vitro micronucleus test.Gesellschaft fur Umwelt-Mutations-forschung.Mutat Res,1998,410:81-116.
10.Miller B,Albertini S,Locher F,et al.Comparative evaluation of the in vitro micronucleus test and the in vitro chromosome aberration test:industrial experience.Mutat Res,1997,392:45-59,187-208.
11.曾延淑,熊亚隆,罗勇.焊工外周血淋巴细胞微核率和染色体畸变率观察.职业卫生与病伤,1995,10:102-4.
12.康玲,姚华,肖碧玉,等.新疆地方性砷中毒患者外周血淋巴细胞微核率及 染色体畸变研究 环境与健康杂志,2001,18:215-7.
13.闫莉,周宁,庞新侠,等.汞对作业者外周血淋巴细胞微核率的影响.劳动医学,2001,18:296-.
14.Leng S,Dai Y,Niu Y,等.Effects of genetic polymorphisms of metabolic enzymes on cytokinesis-block micronucleus in peripheral blood lymphocyte among coke-oven workers.Cancer Epidemiol Biomarkers Prev,2004,13:1631-9.
15.冷曙光,郑玉新,张文众,等.焦炉工外周血淋巴细胞遗传物质损伤水平的研究.中华劳动卫生职业病杂志,2004,22:29-32.
16.高峰,王宝梅,梁玉珍,等.职业性三苯接触对人外周血淋巴细胞染色体结构的影响.环境与职业医学,2005,22:359-60.
17.杨茜,李爱莲,周好乐,等.铅中毒病人外周血淋巴细胞微核率的观察.内蒙古医学院学报,2006,28:36-7.
18.文卫华,杨军,高绪芳,等.职业砷接触工人某些遗传毒性指标的变化.卫生研究,2007,36:340-2.
19.王立,韩志英,孙忠欢,等.甲醛接触工人的外周血淋巴细胞微核研究.癌变畸变 突变,1997,9:123-5.
20.韩存芝,荆洁线,孙桂训,等.环境污染对居民细胞遗传物质损伤的调查研究.中华流行病学杂志,1997,18:83-5.
21.郭桂枝,李烨,张荣.451名放射工作人员淋巴细胞微核的观察与分析.中国辐射卫生,2007,16:435-.
22.李建华,王林超,何伟.电离辐射对操作工淋巴细胞微核率和染色体畸变影响的研究.中国辐射卫生,2007,16:395-7.
23.Bonassi S,Znaor A,Ceppi M,et al.An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.Carcinogenesis,2007,28:625-31.
24.Sgura A,Antoccia A,Cherubini R,et al.Micronuclei,CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality.Int J Radiat Biol,2000,76:367-74.
25.Cicchetti R,Bari M,Argentin G.Induction of micronuclei in bone marrow by two pesticides and their differentiation with CREST staining:an in vivo study in mice. MutatRes,1999,439:239-48.
26.Migliore L,Cocchi L,Scarpato R.Detection of the centromere in micronuclei by fluorescence in situ hybridization:its application to the human lymphocyte micronucleus assay after treatment with four suspected aneugens.Mutagenesis,1996,11:285-90.
27.Huber R,Salassidis K,Kulka U,et al.Detection of centromeres in vinblastineand radiation-induced micronuclei of human lymphocytes using FISH with an alpha satellite pancentromeric DNA probe.Environmental and molecular mutagenesis,1996,27:105-9.
28.Miller BM,Nusse M.Analysis of micronuclei induced by 2-chlorobenzylidene malonitrile (CS) using fluorescence in situ hybridization with telomeric and centromeric DNA probes,and flow cytometry.Mutagenesis,1993,8:35-41.
29.Thomas P,Umegaki K,Fenech M.Nucleoplasm^ bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay.Mutagenesis,2003,18:187-94.
30.Rudolph KL,Millard M,Bosenberg MW,et al.Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nature genetics,2001,28:155-9.
31.Umegaki K,Fenech M.Cytokinesis-block micronucleus assay in WIL2-NS cells:a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.Mutagenesis,2000,15:261-9.
32.Lindberg HK,Wang X,Jarventaus H,et al.Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes.Mutat Res,2007,617:33-45.
33.Shimizu N,Itoh N,Utiyama H,et al.Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.The Journal of cell biology,1998,140:1307-20.
34.Shimizu N,Shimura T,Tanaka T.Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei.Mutat Res,2000,448:81-90.
35.Serrano-Garcia L,Montero-Montoya R.Micronuclei and chromatid buds are the result of related genotoxic events.Environmental and molecular mutagenesis,2001,38:38-45.
36.El-Zein RA,Schabath MB,Etzel CJ,et al.Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer risk.Cancer Res,2006,66:6449-56.
37.Fenech M.The in vitro micronucleus technique.Mutat Res,2000,455:81-95.
2.IARC.Monographs on the evaluation of the carcinogenic risk of chemicals to humans.Polycyclic aromatic hydrocarbons,part 3,industrial exposure in aluminium production,coal gasification,coke production,and iron and steel founding.France:International Agency for Research on Cancer.1984.
3.Unwin J,Cocker J,Scobbie E,et al.An assessment of occupational exposure to polycyclic aromatic hydrocarbons in the UK.Ann Occup Hyg,2006,50:395-403.
4.Schauer C,Niessner R,Poschl U.Polycyclic aromatic hydrocarbons in urban air particulate matter:decadal and seasonal trends,chemical degradation,and sampling artifacts.Environ Sci Technol,2003,37:2861-2868.
5.Liu Y,Tao S,Yang Y,et al.Inhalation exposure of traffic police officers to polycyclic aromatic hydrocarbons(PAHs) during the winter in Beijing,China.Sci Total Environ,2007,383:98-105.
6.Varlet V,Serot T,Monteau F,et al.Determination of PAH profiles by GC-MS/MS in salmon processed by four cold-smoking techniques.Food Addit Contam,2007,24:744-757.
7.Sanderson E,Kelly P,Farant JE Effect of Soderberg smelting technology,anode paste composition,and work shift on the relationship between benzo[a]pyrene and individual polycyclic aromatic hydrocarbons.J Occup Environ Hyg,2005,2:65-72;quiz D66-67.
8.Greenberg A,Hsu C,Rothman N,et al.PAH Profiles of Charbroiled Hamburgers:Pyrene/B[a]P Ratios and Presence of Reactive PAH Polycyclic Aromatic Compounds,1993 3:101-110.
9.杨洪彪,段小丽,zhang J,等.焦炉工人PAHs空气日均暴露浓度分析.环境监测管理与技术,2004,16:13-16.
10.陈波,郑力行,胡云平,等.焦炉工职业性多环芳烃暴露特征的研究 中华劳 动卫生职业病杂志,2004,22:327-330.
11.Zhivotovsky B,Kroemer G.Apoptosis and genomic instability.Nat Rev Mol Cell Biol,2004,5:752-762.
12.Lengauer C,Kinzler KW,Vogelstein B.Genetic instabilities in human cancers.1998,396:643-649.
13.Sen S.Aneuploidy and cancer.Curr Opin Oncol,2000,12:82-88.
14.Raptis S,Mrkonjic M,Green RC,et al.MLH1 -93G>A promoter polymorphism and the risk of microsatellite-unstable colorectal cancer.J Natl Cancer Inst,2007,99:463-474.
15.Bardelli A,Cahill DP,Lederer G,et al.Carcinogen-specific induction of genetic instability.Proc Natl Acad Sci U S A,2001,98:5770-5775.
16.田宝磊,孙志贤.PML与基因组稳定性 中国生物化学与分子生物学报,2006.22:349-254.
17.Charames GS,Bapat B.Genomic instability and cancer.Curr Mol Med,2003,3:589-596.
18.Eyfjord JE,Bodvarsdottir SK.Genomic instability and cancer:networks involved in response to DNA damage.Mutat Res,2005,592:18-28.
19.Swanton C,Tomlinson I,Downward J.Chromosomal instability,colorectal cancer and taxane resistance.Cell Cycle,2006,5:818-823.
20.Raptis S,Bapat B.Genetic instability in human tumors.Exs,2006:303-320.
21.Sieber O,Heinimann K,Tomlinson I.Genomic stability and tumorigenesis.Semin Cancer Biol,2005,15:61-66.
22.Bonassi S,Hagmar L,Stromberg U,et al.Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens.European Study Group on Cytogenetic Biomarkers and Health.Cancer Res,2000.60:1619-1625.
23.Rossner P,Boffetta P,Ceppi M,et al.Chromosomal aberrations in lymphocytes of healthy subjects and risk of cancer.Environ Health Perspect,2005,113:517-520.
24.Bonassi S,Znaor A,Ceppi M,et al.An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.Carcinogenesis, 2007,28:625-631.
25.Van Hummelen P,Gennart JP,Buchet JP,et al.Biological markers in PAH exposed workers and controls.Mutat Res,1993,300:231-239.
26.Burgaz S,Erdem O,Karahalil B,et al.Cytogenetic biomonitoring of workers exposed to bitumen fumes.Mutat Res,1998,419:123-130.
27.Kalina I,Brezani P,Gajdosova D,et al.Cytogenetic monitoring in coke oven workers.Mutat Res,1998,417:9-17.
28.Jarvholm B,Nordstrom G,Hogstedt B,et al.Exposure to polycyclic aromatic hydrocarbons and genotoxic effects on nonsmoking Swedish road pavement workers.Scand J Work Environ Health,1999,25:131-136.
29.Cavallo D,Ursini CL,Bavazzano P,et al.Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs.Ann Occup Hyg,2006,50:211-218.
30.Norppa H,Bonassi S,Hansteen IL,et al.Chromosomal aberrations and SCEs as biomarkers of cancer risk.Mutat Res,2006,600:37-45.
31.Boffetta P,van der Hel O,Norppa H,et al.Chromosomal aberrations and cancer risk:results of a cohort study from Central Europe.Am J Epidemiol,2007,165:36-43.
32.Bonassi S,Norppa H,Ceppi M,et al.Chromosomal Aberration Frequency in Lymphocytes Predicts the Risk of Cancer:Results from a Pooled Cohort Study of 22,358 Subjects in 11 Countries.Carcinogenesis,2008.
33.El-Zein RA,Schabath MB,Etzel CJ,et al.Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer risk.Cancer Res,2006,66:6449-6456.
34.Fenech M.The role of folic acid and Vitamin B12 in genomic stability of human cells.Mutat Res,2001,475:57-67.
35.Fenech M,Crott JW.Micronuclei,nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakage-fusion-bridge cycles in the cytokinesis-block micronucleus assay.Mutat Res,2002,504:131-136.
36.Downs JA,Lowndes NF,Jackson SP.A role for Saccharomyces cerevisiae histone H2A in DNA repair.Nature,2000,408:1001-1004.
37.Chen HT,Bhandoola A,Difilippantonio MJ,et al.Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX.Science,2000,290:1962-1965.
38.MacPhail SH,Banath JP,Yu TY,et al.Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays.Int J Radiat Biol,2003,79:351-358.
39.Rogakou EP,Pilch DR,Orr AH,et al.DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.J Biol Chem,1998,273:5858-5868.
40.Ismail IH,Wadhra TI,Hammarsten O.An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans.Nucleic Acids Res,2007,35:e36.
41.Rothkamm K,Lobrich M.Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.Proc Natl Acad Sci USA,2003,100:5057-5062.
42.Shivji MK,Venkitaraman AR.DNA recombination,chromosomal stability and carcinogenesis:insights into the role of BRCA2.DNA Repair (Amst),2004,3:835-843.
43.Pluth JM,Yamazaki V,Cooper BA,et al.DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells.DNARepair (Amst),2008,7:108-118.
44.O'Driscoll M,Jeggo PA.The role of double-strand break repair-insights from human genetics.Nat Rev Genet,2006,7:45-54.
45.Fenech M.Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability,mitotic dysfunction and cell death.Mutat Res,2006,600(l-2):58-66.
46.Fenech M.Cytokinesis-block micronucleus cytome assay.Nat Protoc,2007,2:1084-1104.
47.Thomas P,Umegaki K,Fenech M.Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay.Mutagenesis,2003,18:187-194.
48.Stewenius Y,Gorunova L,Jonson T,et al.Structural and numerical chromosome changes in colon cancer develop through telomere-mediated anaphase bridges,not through mitotic multipolarity.Proc Natl Acad Sci U S A,2005,102:5541-5546.
49.Fenech M.Biomarkers of genetic damage for cancer epidemiology.Toxicology,2002,181-182:411-416.
50.Fenech M.The in vitro micronucleus technique.Mutat Res,2000,455:81-95.
51.Fenech M,Chang WP,Kirsch-Volders M,et al.HUMN project:detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures.Mutat Res,2003,534:65-75.
52.Fenech M.Chromosomal biomarkers of genomic instability relevant to cancer.Drug Discov Today,2002,7:1128-1137.
53.Kryscio A,Ulrich Muller WU,Wojcik A,et al.A cytogenetic analysis of the long-term effect of uranium mining on peripheral lymphocytes using the micronucleus-centromere assay.Int J Radiat Biol,2001,77:1087-1093.
54.Touil N,Aka PV,Buchet JP,et al.Assessment of genotoxic effects related to chronic low level exposure to ionizing radiation using biomarkers for DNA damage and repair.Mutagenesis,2002,17:223-232.
55.Wold S,Albano C,Dunn Ⅲ W,et al.Multivariate data analysis in chemistry.Dordrecht,The Netherlands Reidel;1984.
56.Johnson R,Wichern D.Applied multivariate statistical analysis.New Jersey:Prentice-Hall Inc.,Englewood Cliffs;1992.
57.孙振球,徐勇勇.医学统计学.第二版∶人民卫生出版社;2005.
58.Umegaki K,Fenech M.Cytokinesis-block micronucleus assay in WIL2-NS cells:a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.Mutagenesis,2000,15:261-269.
59.Bonassi S,Neri M,Lando C,et al.Effect of smoking habit on the frequency of micronuclei in human lymphocytes:results from the Human MicroNucleus project.Mutat Res,2003,543:155-166.
60.Crott JW,Mashiyama ST,Ames BN,et al.The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro.Cancer Epidemiol Biomarkers Prev,2001,10:1089-1096.
61.Leopardi P,Marcon F,Caiola S,et al.Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes.Mutagenesis,2006,21:327-333.
62.Zhang S,Hunter DJ,Hankinson SE,et al.A prospective study of folate intake and the risk of breast cancer.Jama,1999,281:1632-1637.
63.Duthie SJ,Narayanan S,Blum S,et al.Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells.Nutr Cancer,2000,37:245-251.
64.Eto I,Krumdieck CL.Role of vitamin B12 and folate deficiencies in carcinogenesis.Adv Exp Med Biol,1986,206:313-330.
65.Blount BC,Mack MM,Wehr CM,et al.Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage:implications for cancer and neuronal damage.Proc Natl Acad Sci USA,1997,94:3290-3295.
66.Wang X,Thomas P,Xue J,et al.Folate deficiency induces aneuploidy in human lymphocytes in vitro-evidence using cytokinesis-blocked cells and probes specific for chromosomes 17 and 21.Mutat Res,2004,551:167-180.
67.Wagner C,Briggs WT,Home DW,et al.10-Formyltetrahydrofolate dehydrogenase:identification of the natural folate ligand,covalent labeling,and partial tryptic digestion.Arch Biochem Biophys,1995,316:141-147.
68.Frosst P,Blom HJ,Milos R et al.A candidate genetic risk factor for vascular disease:a common mutation in methylenetetrahydrofolate reductase.Nat Genet,1995,10:111-113.
69.Weisberg I,Iran P,Christensen B,et al.A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity.Mol Genet Metab,1998,64:169-172.
70.Kimura M,Umegaki K,Higuchi M,et al.Methylenetetrahydrofolate reductase C677T polymorphism,folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes.J Nutr,2004,134:48-56.
71.Kuschel B,Auranen A,McBride S,et al.Variants in DNA double-strand break repair genes and breast cancer susceptibility.Hum Mol Genet,2002,11:1399-1407.
72.West SC.Molecular views of recombination proteins and their control.Nat Rev Mol Cell Biol,2003,4:435-445.
73.Martin IV,MacNeill SA.ATP-dependent DNA ligases.Genome Biol,2002,3:REVIEWS3005.
74.Burma S,Chen BP,Chen DJ.Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.DNA Repair (Amst),2006,5:1042-1048.
75.Calsou P,Delteil C,Frit P,et al.Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.J Mol Biol,2003,326:93-103.
76.Leber R,Wise TW,Mizuta R et al.The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase.J Biol Chem,1998,273:1794-1801.
77.Fu YP,Yu JC,Cheng TC,et al.Breast cancer risk associated with genotypic polymorphism of the nonhomologous end-joining genes:a multigenic study on cancer susceptibility.Cancer Res,2003,63:2440-2446.
78.Lee KM,Choi JY,Kang C,et al.Genetic polymorphisms of selected DNA repair genes,estrogen and progesterone receptor status,and breast cancer risk.Clin Cancer Res,2005,11:4620-4626.
79.Leng S,Dai Y,Niu Y,et al Effects of genetic polymorphisms of metabolic enzymes on cytokinesis-block micronucleus in peripheral blood lymphocyte among coke-oven workers.Cancer Epidemiol Biomarkers Prev,2004,13:1631-1639.
80.Choi SW,Mason JB.Folate and carcinogenesis:an integrated scheme.J Nutr,2000,130:129-132.
81.Shen H,Spitz MR,Wang LE,et al.Polymorphisms of methylene-tetrahydrofolate reductase and risk of lung cancer:a case-control study.Cancer Epidemiol Biomarkers Prev,2001,10:397-401.
82.Shen H,Xu Y,Zheng Y,et al.Polymorphisms of 5,10-methylenetetrahydrofolate reductase and risk of gastric cancer in a Chinese population:a case-control study.Int J Cancer,2001,95:332-336.
83.Chen J,Ma J,Stampfer ML et al.Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer.Pharmacogenetics,2002,12:339-342.
84.Miller SA,Dykes DD,Polesky HF.A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res,1988,16:1215.
85.Lin J,Spitz MR,Wang Y,et al.Polymorphisms of folate metabolic genes and susceptibility to bladder cancer:a case-control study.Carcinogenesis,2004,25:1639-1647.
86.Carlson CS,Eberle MA,Rieder MJ,et al.Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans.Nat Genet,2003,33:518-521.
87.Wu X,Zhao H,Amos CI,et al.p53 Genotypes and Haplotypes Associated With Lung Cancer Susceptibility and Ethnicity.J Natl Cancer Inst,2002,94:681-690.
88.Ogino S,Wilson RB.Genotype and haplotype distributions of MTHFR677C>T and 1298A>C single nucleotide polymorphisms:a meta-analysis.J Hum Genet,2003,48:1-7.
89.朱慧萍,刘明珠,刀京晶.MTHFR基因第1298位核苷酸多态性与酶活性的关系.北京医科大学学报,2002,32:262-264.
90.孙耀峰,戴宇飞,程娟,等.亚甲基四氢叶酸还原酶基因单体型与焦炉作业工人染色体损伤易感性的关系.卫生研究,2006,35:387-390.
91.Le Marchand L,Donlon T,Hankin JH,et al.B-vitamin intake,metabolic genes,and colorectal cancer risk(United States).Cancer Causes Control,2002,13:239-248.
92.缪小平,邢德印,谭文,等.MTHFR基因C677T和A1298C多态与贲门癌的遗传易感性.中华医学杂志,2002,82:669-672.
93.van der Put NM,Gabreels F,Stevens EM,et al.A second common mutation in the methylenetetrahydrofolate reductase gene:an additional risk factor for neural-tube defects? Am J Hum Genet,1998,62:1044-1051.
94.Duthie SJ,Hawdon A.DNA instability(strand breakage,uracil misincorporation,and defective repair) is increased by folic acid depletion in human lymphocytes in vitro.Faseb J,1998,12:1491-1497.
95.Stem LL,Mason JB,Selhub J,et al.Genomic DNA hypomethylation,a characteristic of most cancers,is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene.Cancer Epidemiol Biomarkers Prev,2000,9:849-853.
96.Song C,Xing D,Tan W,et al.Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population.Cancer Res,2001,61:3272-3275.
97.Shen M,Rothman N,Berndt SI,et al.Polymorphisms in folate metabolic genes and lung cancer risk in Xuan Wei,China.Lung Cancer,2005,49:299-309.
98.Hung RJ,Hashibe M,McKay J,et al.Folate-related genes and the risk of tobacco-related cancers in Central Europe.Carcinogenesis,2007,28:1334-1340.
99.Hu J,Nyren O,Wolk A,et al.Risk factors for oesophageal cancer in northeast China.Int J Cancer,1994,57:38-46.
100.Ronnenberg AG,Goldman MB,Aitken IW,et al.Anemia and deficiencies of folate and vitamin B-6 are common and vary with season in Chinese women of childbearing age.J Nutr,2000,130:2703-2710.
101.Hao L,Ma J,Stampfer MJ,et al.Geographical,seasonal and gender differences in folate status among Chinese adults.J Nutr,2003,133:3630-3635.
102.UNICEF.Vitamin & mineral deficiency:a damage assessment report for CHINA.United Nations Children's Fund(UNICEF);2004.
103.齐军,缪小平,谭文,等.亚甲基四氢叶酸还原酶基因单核苷酸多态与乳腺癌风险.中华肿瘤杂志,2004,26:287-289.
104.Mu LN,Cao W,Zhang ZF,et al.Methylenetetrahydrofolate reductase(MTHFR)C677T and A1298C polymorphisms and the risk of primary hepatocellular carcinoma (HCC) in a Chinese population.Cancer Causes Control,2007,18:665-675.
105.Shen H,Newmann AS,Hu Z,et al.Methylenetetrahydrofolate reductase polymorphisms/haplotypes and risk of gastric cancer:a case-control analysis in China.Oncol Rep,2005,13:355-360.
106.Mu LN,Cao W,Zhang ZF,et al.Polymorphisms of 5,10-methylenetetralydrofolate reductase(MTHFR),fruit and vegetable intake,and the risk of stomach cancer.Biomarkers,2007,12:61-75.
107.Wang Y,Guo W,He Y,et al.Association of MTHFR C677T and SHMT(l) C1420T with susceptibility to ESCC and GCA in a high incident region of Northern China.Cancer Causes Control,2007,18:143-152.
108.Ma J,Stampfer MJ,Giovannucci E,et al.Methylenetetrahydrofolate reductase polymorphism,dietary interactions,and risk of colorectal cancer.Cancer Res,1997,57:1098-1102.
109.The International HapMap Consortium.The International HapMap Project.Nature.2003 Dec 18;Sect.789-796.
110.Kobayashi J,Antoccia A,Tauchi H,et al.NBS1 and its functional role in the DNA damage response.DNA Repair (Amst),2004,3:855-861.
111.Zhu XD,Kuster B,Mann M,et al.Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres.Nat Genet,2000,25:347-352.
112.Kobayashi J,Tauchi H,Sakamoto S,et al.NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain.Curr Biol,2002,12:1846-1851.
113.Hjorth JT,Gad J,Cooper H,et al.A zebrafish homologue of deleted in colorectal cancer (zdcc) is expressed in the first neuronal clusters of the developing brain.Mech Dev,2001,109:105-109.
114.Musak L,Soucek P,Vodickova L,et al.Chromosomal aberrations in tire plant workers and interaction with polymorphisms of biotransformation and DNA repair genes.Mutat Res,2008,641:36-42.
115.Medina PP,Ahrendt SA,Pollan M,et al.Screening of homologous recombination gene polymorphisms in lung cancer patients reveals an association of the NBSl-185Gln variant and p53 gene mutations.Cancer Epidemiol Biomarkers Prev,2003,12:699-704.
116.Sanyal S,Festa F,Sakano S,et al.Polymorphisms in DNA repair and metabolic genes in bladder cancer.Carcinogenesis,2004,25:729-734.
117.Zienolddiny S,Campa D,Lind H,et al.Polymorphisms of DNA repair genes and risk of non-small cell lung cancer.Carcinogenesis,2006,27:560-567.
118.Haber JE.DNA recombination:the replication connection.Trends Biochem Sci, 1999,24:271-275.
119.Yokoyama H,Sarai N,Kagawa W,et al.Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B,Rad51C,Rad51D and Xrcc2 protein complex.Nucleic Acids Res,2004,32:2556-2565.
120.Mohindra A,Bolderson E,Stone J,et al.A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks.Hum Mol Genet,2004,13:203-212.
121.Griffin CS,Simpson PJ,Wilson CR,et al.Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation.Nat Cell Biol,2000,2:757-761.
122.Cui X,Brenneman M,Meyne J,et al.The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.Mutat Res,1999,434:75-88.
123.Lopez-Cima MF,Gonzalez-Arriaga P,Garcia-Castro L,et al.Polymorphisms in XPC,XPD,XRCCl,and XRCC3 DNA repair genes and lung cancer risk in a population of northern Spain.BMC Cancer,2007,7:162.
124.Matullo G,Palli D,Peluso M,et al.XRCCl,XRCC3,XPD gene polymorphisms,smoking and (32)P-DNA adducts in a sample of healthy subjects.Carcinogenesis,2001,22:1437-1445.
125.Matullo G,Peluso M,Polidoro S,et al.Combination of DNA repair gene single nucleotide polymorphisms and increased levels of DNA adducts in a population-based study.Cancer Epidemiol Biomarkers Prev,2003,12:674-677.
126.Vodicka P,Kumar R,Stetina R,et al.Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates,chromosomal aberrations and single-strand breaks in DNA.Carcinogenesis,2004,25:757-763.
127.Tuimala J,Szekely G,Wikman H,et al.Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes:effects on levels of sister chromatid exchanges and chromosomal aberrations.Mutat Res,2004,554:319-333.
128.Ferguson DO,Sekiguchi JM,Chang S,et al.The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations.Proc Natl Acad Sci USA,2000,97:6630-6633.
129.Olive PL.The role of DNA single-and double-strand breaks in cell killing by ionizing radiation.Radiat Res,1998,150:S42-51.
130.Kim CH,Park SJ,Lee SH.A targeted inhibition of DNA-dependent protein kinase sensitizes breast cancer cells following ionizing radiation.J Pharmacol Exp Ther,2002,303:753-759.
131.Koike M.Dimerization,translocation and localization of Ku70 and Ku80 proteins.J Radiat Res (Tokyo),2002,43:223-236.
132.Dai Y,Kysela B,Hanakahi LA,et al.Nonhomologous end joining and V(D)J recombination require an additional factor.Proc Natl Acad Sci USA,2003,100:2462-2467.
133.Korabiowska M,Tscherny M,Stachura J,et al.Differential expression of DNA nonhomologous end-joining proteins Ku70 and Ku80 in melanoma progression.Mod Pathol,2002,15:426-433.
134.Zhu G,Duffy DL,Turner DR et al.Linkage and association analysis of radiation damage repair genes XRCC3 and XRCC5 with nevus density in adolescent twins.Twin Res,2003,6:315-321.
135.De Ruyck K,Wilding CS,Van Eijkeren M,et al.Microsatellite polymorphisms in DNA repair genes XRCC1,XRCC3 and XRCC5 in patients with gynecological tumors:association with late clinical radio sensitivity and cancer incidence.Radiat Res,2005,164:237-244.
136.Sadetzki S,Flint-Richter P,Starinsky S,et al.Genotyping of patients with sporadic and radiation-associated meningiomas.Cancer Epidemiol Biomarkers Prev,2005,14:969-976.
137.Barnes DE,Stamp G,Rosewell I,et al.Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice.Curr Biol,1998,8:1395-1398.
138.Frank KM,Sekiguchi JM,Seidl KJ,et al.Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV.Nature,1998,396:173-177.
139.Liu Y,Zhou K,Zhang H,et al.Polymorphisms of LIG4 and XRCC4 involved in the NHEJ pathway interact to modify risk of glioma.Hum Mutat,2008,29:381-389.
140.Sakiyama T,Kohno T,Mimaki S,et al.Association of amino acid substitution polymorphisms in DNA repair genes TP53,POLI,REV1 and LIG4 with lung cancer risk.Int J Cancer,2005,114:730-737.
141.Roddam PL,Rollinson S,O'Driscoll M,et al.Genetic variants of NHEJ DNA ligase IV can affect the risk of developing multiple myeloma,a tumour characterised by aberrant class switch recombination.J Med Genet,2002,39:900-905.
142.Mensing T,Marczynski B,Engelhardt B,et al.DNA adduct formation of benzo[a]pyrene in white blood cells of workers exposed to polycyclic aromatic hydrocarbons.Int J Hyg Environ Health,2005,208:173-178.
143.Divi RL,Beland FA,Fu PP,et al.Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts:validation by comparison with other methods,and use in human biomonitoring.Carcinogenesis,2002,23:2043-2049.
144.Ohnishi S,Kawanishi S.Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.Biochem Biophys Res Commun,2002,290:778-782.
145.Gao D,Luo Y,Guevara D,et al.Benzo[a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2'-deoxyguanosine via reactive oxygen species.Free Radic Biol Med,2005,39:1177-1183.
146.Shou M,Korzekwa KR,Crespi CL,et al.The role of 12 cDNA-expressed human,rodent,and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-dihydrodiol.Mol Carcinog,1994,10:159-168.
147.Richardson C.RAD51,genomic stability,and tumorigenesis.Cancer Lett,2005,218:127-139.
148.Ismail IH,Hendzel MJ.The gamma-H2A.X:is it just a surrogate marker of double-strand breaks or much more?Environ Mol Mutagen,2008,49:73-82.
149．曹佳．微核试验在中国的应用、发展与展望．遗传,2003,25：73～76．
150.Wilkening S,Stahl F,Bader A.Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.Drug Metab Dispos,2003,31:1035-1042.
151.Lehmann U,Kreipe H.Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies.Methods,2001,25:409-418.
152.Castedo M,Perfettini JL,Roumier T,et al.Cell death by mitotic catastrophe:a molecular definition.Oncogene,2004,23:2825-2837.
153.Goldsworthy TL,Conolly RB,Fransson-Steen R.Apoptosis and cancer risk assessment.Mutat Res,1996,365:71-90.
154.Mathias N,Johnson SL,Winey M,et al.Cdc53p acts in concert with Cdc4p and Cdc34p to control the Gl-to-S-phase transition and identifies a conserved family of proteins.Mol Cell Biol,1996,16:6634-6643.
155.Rudolph KL,Millard M,Bosenberg MW,et al Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nat Genet,2001,28:155-159.
156.Lindberg HK,Wang X,Jarventaus H,et al.Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes.Mutat Res,2007,617:33-45.
157.Shimizu N,Itoh N,Utiyama H,et al.Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.J Cell Biol,1998,140:1307-1320.
158.Shimizu N,Shimura T,Tanaka T.Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei.Mutat Res,2000,448:81-90.
159.Serrano-Garcia L,Montero-Montoya R.Micronuclei and chromatid buds are the result of related genotoxic events.Environ Mol Mutagen,2001,38:38-45.
160.Hyslop PA,Hinshaw DB,Scraufstatter IU,et al.Hydrogen peroxide as a potent bacteriostatic antibiotic:implications for host defense.Free Radic Biol Med,1995,19:31-37.
1.Schmid W.The micronucleus test.Mutat Res,1975,31:9-15.
2.Auletta AE,Dearfield KL,Cimino MC.Mutagenicity test schemes and guidelines:U.S.EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs.Environmental and molecular mutagenesis,1993,21:38-45;discussion 6-57.
3.Sofuni T.Japanese guidelines for mutagenicity testing.Ministry of Agriculture,Forestry and Fisheries;Ministry of Health and Welfare;Ministry of Labor.Environ Mol Mutagen,1993,21:2-7.
4.中华人民共和国卫生部药政局.新药(西药)临床研究指导原则汇编(毒理学).1993.
5.Fenech M.Cytokinesis-block micronucleus assay evolves into a "cytome" assay of chromosomal instability,mitotic dysfunction and cell death.Mutat Res,2006,600(1-2):58-66.
6.曹佳.微核试验在中国的应用、发展与展望.遗传,2003,25:73-6.
7.Fenech M,Chang WP,Kirsch-Volders M,et al.HUMN project:detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures.Mutat Res,2003,534:65-75.
8.曹佳,林真,余争平.微核试验-原理、方法及其在人群监测和毒性评价中的应用.北京:军事科学出版社;2000.
9.Miller B,Potter-Locher F,Seelbach A,et al.Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay:position of the GUM Working Group on the in vitro micronucleus test.Gesellschaft fur Umwelt-Mutations-forschung.Mutat Res,1998,410:81-116.
10.Miller B,Albertini S,Locher F,et al.Comparative evaluation of the in vitro micronucleus test and the in vitro chromosome aberration test:industrial experience.Mutat Res,1997,392:45-59,187-208.
11.曾延淑,熊亚隆,罗勇.焊工外周血淋巴细胞微核率和染色体畸变率观察.职业卫生与病伤,1995,10:102-4.
12.康玲,姚华,肖碧玉,等.新疆地方性砷中毒患者外周血淋巴细胞微核率及 染色体畸变研究 环境与健康杂志,2001,18:215-7.
13.闫莉,周宁,庞新侠,等.汞对作业者外周血淋巴细胞微核率的影响.劳动医学,2001,18:296-.
14.Leng S,Dai Y,Niu Y,等.Effects of genetic polymorphisms of metabolic enzymes on cytokinesis-block micronucleus in peripheral blood lymphocyte among coke-oven workers.Cancer Epidemiol Biomarkers Prev,2004,13:1631-9.
15.冷曙光,郑玉新,张文众,等.焦炉工外周血淋巴细胞遗传物质损伤水平的研究.中华劳动卫生职业病杂志,2004,22:29-32.
16.高峰,王宝梅,梁玉珍,等.职业性三苯接触对人外周血淋巴细胞染色体结构的影响.环境与职业医学,2005,22:359-60.
17.杨茜,李爱莲,周好乐,等.铅中毒病人外周血淋巴细胞微核率的观察.内蒙古医学院学报,2006,28:36-7.
18.文卫华,杨军,高绪芳,等.职业砷接触工人某些遗传毒性指标的变化.卫生研究,2007,36:340-2.
19.王立,韩志英,孙忠欢,等.甲醛接触工人的外周血淋巴细胞微核研究.癌变畸变 突变,1997,9:123-5.
20.韩存芝,荆洁线,孙桂训,等.环境污染对居民细胞遗传物质损伤的调查研究.中华流行病学杂志,1997,18:83-5.
21.郭桂枝,李烨,张荣.451名放射工作人员淋巴细胞微核的观察与分析.中国辐射卫生,2007,16:435-.
22.李建华,王林超,何伟.电离辐射对操作工淋巴细胞微核率和染色体畸变影响的研究.中国辐射卫生,2007,16:395-7.
23.Bonassi S,Znaor A,Ceppi M,et al.An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.Carcinogenesis,2007,28:625-31.
24.Sgura A,Antoccia A,Cherubini R,et al.Micronuclei,CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality.Int J Radiat Biol,2000,76:367-74.
25.Cicchetti R,Bari M,Argentin G.Induction of micronuclei in bone marrow by two pesticides and their differentiation with CREST staining:an in vivo study in mice. MutatRes,1999,439:239-48.
26.Migliore L,Cocchi L,Scarpato R.Detection of the centromere in micronuclei by fluorescence in situ hybridization:its application to the human lymphocyte micronucleus assay after treatment with four suspected aneugens.Mutagenesis,1996,11:285-90.
27.Huber R,Salassidis K,Kulka U,et al.Detection of centromeres in vinblastineand radiation-induced micronuclei of human lymphocytes using FISH with an alpha satellite pancentromeric DNA probe.Environmental and molecular mutagenesis,1996,27:105-9.
28.Miller BM,Nusse M.Analysis of micronuclei induced by 2-chlorobenzylidene malonitrile (CS) using fluorescence in situ hybridization with telomeric and centromeric DNA probes,and flow cytometry.Mutagenesis,1993,8:35-41.
29.Thomas P,Umegaki K,Fenech M.Nucleoplasm^ bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay.Mutagenesis,2003,18:187-94.
30.Rudolph KL,Millard M,Bosenberg MW,et al.Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.Nature genetics,2001,28:155-9.
31.Umegaki K,Fenech M.Cytokinesis-block micronucleus assay in WIL2-NS cells:a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.Mutagenesis,2000,15:261-9.
32.Lindberg HK,Wang X,Jarventaus H,et al.Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes.Mutat Res,2007,617:33-45.
33.Shimizu N,Itoh N,Utiyama H,et al.Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.The Journal of cell biology,1998,140:1307-20.
34.Shimizu N,Shimura T,Tanaka T.Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei.Mutat Res,2000,448:81-90.
35.Serrano-Garcia L,Montero-Montoya R.Micronuclei and chromatid buds are the result of related genotoxic events.Environmental and molecular mutagenesis,2001,38:38-45.
36.El-Zein RA,Schabath MB,Etzel CJ,et al.Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer risk.Cancer Res,2006,66:6449-56.
37.Fenech M.The in vitro micronucleus technique.Mutat Res,2000,455:81-95.
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