【作者】 王威；

【导师】 夏昭林； 金锡鹏；

【作者基本信息】 复旦大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 氯乙烯单体(vinyl chloride monomer,VCM)是一种重要的化工原料,约95%用于合成聚氯乙烯。我国目前年产量约占全球产量的1/10,已成为世界聚氯乙烯生产大国。因此,庞大的职业接触氯乙烯人群的健康问题值得关注。VCM是确定的人类致癌剂,可导致多系统、多器官肿瘤的发生。对其致癌机制的研究认为主要是VCM活性代谢产物导致遗传物质损伤而启动致癌过程。目前,发达国家VCM职业接触限值多为1ppm(2.79mg/m~3,PEL),而我国VCM时间加权平均容许浓度(PC-TWA)为10mg/m~3,短时间接触容许浓度为(PC-STEL)25mg/m~3,高于西方国家卫生标准数倍。在我国现行卫生标准接触浓度情况下,能否对接触工人产生健康损害特别是遗传损伤十分必要进行探讨。毒物引起的遗传损伤与个体易感性有关,特别是其代谢活化差异和DNA修复能力不同相关,该方面的研究有助于阐明其毒作用机制和进行健康风险评估。本研究通过健康体检和遗传损伤检测评价VCM接触工人健康状况,并通过VCM工人累积接触剂量的估算评价VCM暴露和遗传损伤之间的关系,研究氯乙烯致遗传损伤的剂量-反应关系,为VCM职业卫生标准的修订提供参考和依据。通过人群代谢酶基因和DNA修复基因多态性及其与VCM致遗传物质损伤的关系研究,寻找VCM遗传损伤的易感性生物标志物,为阐明其毒作用机制和进行健康风险评估提供科学依据。本次研究对VCM接触工人进行常规体检和胞质分裂阻滞微核实验调查其健康状况。微核检测结果表明VCM接触组损伤情况高于对照组,VCM暴露与微核率之间存在剂量反应关系,即随着VCM暴露量的增加微核率升高,同时发现高龄和女性为微核率升高的危险因素。本研究提示外周血淋巴细胞微核率作为评价低浓度VCM暴露的效应指标具备一定的推广应用价值。健康体检结果未发现接触工人肝功能和肝B超等指标高于对照组人群。采用BMD法对氯乙烯暴露总量与微核损伤关系进行研究,分析表明男性和女性氯乙烯暴露总剂量BMDL分别为3.35和2.43g,表明在总暴露剂量达到BMDL值时将引起微核损伤。该结果为我国VCM职业卫生标准修订提供了参考价值。应用PCR和RFLP技术对VCM接触工人的代谢酶基因和DNA修复基因多态性进行检测,包括氯乙烯代谢相关的酶类ADH2、ALDH2、GSTT1、GSTM1、GSTP1、CYP2E1和CYP2D6等基因,DNA修复类基因OGG1、MGMT、XRCC1和P53等11个基因共18个多态,并应用PHASE 2.0.2软件分析分析XRCC1和P53基因多态的双体型。应用Poisson回归分析多态及相关因素对微核率的影响。多态检测方法研究中采用CRS-PCR-RFLP方法建立了MGMT基因84位点多态检测方法,该方法较传统PCR-RFLP方法降低了实验费用;而根据同一原理建立的MGMT基因143、160与178位点的检测方法,进行一次PCR联合三次酶切即可检测三个位点多态,该方法可以减少标本用量、实验耗材和时间;根据多重PCR和PCR-RFLP原理建立的XRCC1基因194和399位点的检测方法,亦达到了相似的效果。多因素Poisson逐步回归分析微核危险因素表明对照组年龄、ADH2、OGG1和MGM784为影响因素,而接触组中年龄、性别、ADH2、GSTT1、GSTP1、CYP2E1和MGMT84为影响因素,其他多态以及吸烟和饮酒等与微核无关,未发现各危险因素之间存在交互作用。研究表明对照人群ADH2 G等位基因可视为保护因素,每增加一个等位基因相应危险度为原来的0.61(0.39-0.94)。而接触组结果显示G等位基因为危险因素,其FR为1.10(0.99-1.23)。可能与氯乙烯接触而诱导酶活性有关,其机制有待研究。而ALDH2多态性在本次研究中未发现与微核变化有关。研究表明接触组微核率随着CYP2E1突变型等位基因C的增加而升高,其FR为1.75(1.28-2.33)。研究还发现接触组中GSTT1缺失型为保护因素,而GSTP1 G等位基因为危险因素。对于DNA修复相关基因研究表明,对照组中OGG1(GC+GG)个体相比CC基因型危险度为3.07(1.44-7.96),而接触组中却未发现该现象,有待进一步研究。对照组中MGMT84CT个体相对CC个体危险度为0.53(0.25-0.98),而接触组MGMT84 CT个体相对CC个体危险度为0.85(0.71-1.02),表明MGMT84位点T等位基因为保护因素。分析XRCC1 194、280、399位点和P53内含子3、外显子4及内含子6位点双体型与微核的关系,结果表明XRCC1三位点双体型与微核无关,而对照组P53 AAA/ABA与AAA/AAA相比,微核率较低(P=0.086)。结果表明微核有随年龄增高的趋势,且40岁以上年龄组微核率升高明显,且与是否存在VCM暴露无关。而女性在VCM接触组中表现为易感者,对照组差异无统计学意义。总之,本研究说明在我国现行职业卫生标准下,VCM可引起遗传损伤,当前职业卫生标准有进一步修订的必要。VCM代谢酶类基因和DNA修复基因多态对VCM接触工人染色体损伤具有修饰作用。更多还原

【Abstract】 Vinyl chloride monomer（CH₂=CHCl,VCM） is widely used in industry,almost 95%in polymerization to polyvinyl chloride（PVC）.China is one of the important PVC production countries,and its annual production accounts for about 10%of the global production,necessitating research regarding the health of VCM-exposed workers.VCM is a human carcinogen that has been proven to have multi-organ and multi-system effects.The mechanism of carcinogenesis was presumed to be related to the genetic damage induced by electrophilic metabolites of VCM.At present,the permissible exposure limit（PEL） of VCM in developed countries is 1 ppm （2.79mg/m³）,and the STEL and TWA in China are 25mg/m³ and 10mg/m³, respectively.The permissible exposure limit is higher in China than that those in developed countries,indictating that more research is needed to investigate the health effect and genetic toxicity caused by VCM-exposure under this condition.Since genetic damage induced by toxic substances was related with individual susceptibility, especially with polymorphisms in genes encoding metabolic enzymes and DNA repair proteins.Studies on relationship between genetic damage in VCM workers and polymorphisms in metabolizing enzymes and/or repair genes have been useful for revealing mechanism of toxicology and evaluation to health risk.Routine health examination and cytokinesis-block micronucleus（CB-MN） assay were performed to investigate the health conditions of VCM-exposed workers.The CB-MN results showed that adverse effects in the exposed group were significantly higher than that of the control group,and that there is a dose-response relationship between VCM-exposure and the frequency of MN.Also,great age and female were risk factors for the frequency of MN.Therefore,the frequency of MN of peripheral blood lymphocyte can be used as an effect biomarker under low-level VCM exposure. According to the data of routine health examination,adverse effects in liver function and liver ultrasonography in the exposed group were not significantly higher than that of the control group.The relationship between cumulative exposed dose of VCM and chromosomal damage in VCM workers was analysed by BMDS soft,the result showing that BMDL of cumulative exposed dose of male and female were 3.35 and 2.43g,respectively.It means MN damage will occurr when cumulative exposed dose achieved to BMDL. This result can give reference to adjust the occupational standard of VCM in our country.Next,PCR-RFLP was used to detect polymorphisms of metabolizing enzymes and DNA repair genes.Using Poisson regression analysis,we investigated the relationship between polymorphisms in DNA repair genes and/or metabolizing enzyme genes and the frequency of MN.The PHASE 2.0.2 software was used to obtain maximum-likelihood estimates of the diplotype frequencies.In this study,we detected the 18 polymorphisms in 11 genes:ADH2,ALDH2,GSTT1,GSTM1,GSTP1, CYP2E1,and CYP2D6,which participate in the metabolism of VCM,and OGG1, MGMT,XRCC1,and P53,which participate in the process of DNA repair.A proper assay was developed for identifying SNP in 84 site of the MGMT gene, applying Creating Restriction Site and PCR-RFLP principle,and this assay is more economical than the old assasy.Also,a new assay was developed to detect the SNPs in sites 143,160,178 of MGMT gene using the same principle,and this assay can detect three SNPs by one PCR and three RFLP.The merit of this method include economy,speed and sample conservation.We additionally designed a new method to detect SNPs in site 194 and 399 of XRCC1 gene based on the principle of multiplex PCR and PCR-RFLP;this method is also effective.Poisson analyze showed that age,ADH2,OGG1 and MGMT84 were important factors in control group,while age,gender,ADH2,GSTT1,GSTP1,CYP2E1 and MGMT84 was impact factors in exposure group.There was no association between frequency of MN and other gene polymorphism,smoking,and alcohol consumption, nor interactions between these factors.This study showed that allele G of AHD2 gene was a protect factor,and the risk was 0.61（0.39-0.94） when one allele G increased in the site,in normal group.On the contrary,it was showed a risk factor in exposed group（FR=1.10[0.99-1.23]）.Thereis a need for more study,as the mechanism was unknown.The polymorphism of ALDH2 had no relationship with changes in MN.The frequency of MN increased along with the increased prevalence of mutant allele C in CYP2E1 gene,in the exposed group（FR=1.75[1.28-2.33]）.Also the result showed that GSTT1 null type had a protect effect,while allele G in GSTP1 had an opposite effect. The risk of OGG1（GC+GG） was 3.07（1.44-7.96） in control group compared with genotype CC and no same result in exposed group,a result need more studies. However,the risk of MGMT84CT was 0.53（0.25-0.98） in control group compared with CC genotype,while the value is 0.85（0.71-1.02） in exposure group.This result showed allele T in MGMT84 had a protect effect.The study showed that there is no relationship between MN frequency and diplotype of 194,280,399 sites of XRCC1 gene whether in the control and exposed group.Diplotype analysis of P53 intron3, exon4 and intron 6 demonstrated that the MN frequency in subjects with AAA/ABA（A:wild allele;B:variant allele） was significantly lower than that in subjects with AAA/AAA in control group（P=0.086）.Results showed that frequency of MN increased accompany with age,especially at age≥40,with or without contact with chemical substances.Female gender was a susceptibility factor in the VCM group,while the fact that no similar result occurred in control group may due to limited samples.In conclusion,VCM can induce chromosomal damage even when the exposure level is lower than the national occupational health standard in China;some of the polymorphisms of DNA repair genes and metabolizing enzyme genes may be associated with chromosomal damage in VCM-exposed workers.更多还原