氯乙烯接触工人遗传损伤及其修复能力研究

发布时间：2018-04-21 14:37

本文选题：氯乙烯 + 染色体损伤　；参考：《复旦大学》2013年博士论文

【摘要】：氯乙烯单体(vinyl chloride monomer, VCM)是一种重要的化工原料,主要用于合成聚氯乙烯。2011年全球PVC总产能约5100万吨,而我国PVC产能达2162.2万吨,已成为世界第一大生产国和第一大消费国。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/m3, PEL),而我国VCM时间加权平均容许浓度(PC-TWA)为10mg/m3,短时间接触容许浓度为(PC-STEL)25mg/m3,高于西方国家卫生标准数倍。长期和低水平接触氯乙烯导致的健康危害已引起广泛关注,寻找敏感、有效的生物标志物对预防氯乙烯接触导致的有害效应十分必要。在我国现行氯乙烯职业卫生标准下,能否对接触工人的遗传物质产生显著损伤尤为引人关注。而且在同样接触条件下,并不是所有个体都会产生遗传物质损伤；遗传物质即便发生损伤,由于机体具有DNA修复能力(DNA repair capacity,DRC)而得到修复。因而DNA修复能力在维持机体基因组稳定和正常功能方面发挥重要作用,如果DRC功能低下或缺陷,将不能及时和有效地修复受损的DNA,从而导致基因突变甚至细胞癌变,增加个体罹患肿瘤的危险。本课题通过横断面调查和追踪研究,探讨染色体损伤与氯乙烯累积接触剂量的关系,同时应用本课题组发展的基于微核的DNA损伤修复能力检测方法,研究氯乙烯作业工人DNA损伤修复能力,并评价其用于预测氯乙烯致染色体损伤风险的价值,为保护易感作业工人提供科学依据。通过氯乙烯作业工人DNA细胞周期调控基因多态与VCM致遗传损伤之间的关系研究,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。本课题研究对象为某化工企业VCM接触工人309例(男224例,女85例),该企业行政和后勤人员(内对照)共149例(男61例,女88例),以及健康对照人群(外对照)共25例(男14例,女11例)；利用胞质分裂阻滞微核试验方法(简称CB微核试验)对染色体损伤情况进行检测, VCM接触组外周血淋巴细胞微核细胞率(简称微核率)明显高于对照组FR=3.24(95%CI2.80-3.75), p0.001,并存在剂量-反应关系,提示在目前职业性VCM接触水平较低的情况下,外周血淋巴细胞微核率可以作为评价低浓度VCM接触的效应指标。在2004,2007年VCM接触工人中选取重复样本91例,采用基准剂量(BMD)法分析微核发生率与氯乙烯累积接触剂量的关系。结果表明,按照氯乙烯累积接触剂量计算,其基准剂量95%可信区间下限(BMDL)在2004年男性和女性中分别为0.99mg/m3-year和3.38ng/m3-year;在2007年男性和女性中分别为0.63mg/m3-year和1.27mg/m3-year。提示当总接触剂量达到BMDL值时即引起微核损伤,为制定氯乙烯接触限值提供了参考。 yH2AX是目前国内外研究细胞DNA损伤应激反应的公认的敏感指标,当DNA双链发生断裂时,组蛋白H2A家族成员中的H2AX第139位丝氨酸残基迅速磷酸化,形成磷酸化的H2AX,即γ-H2AX。本研究应用流式细胞术(FCM)对上述追踪观察的55例氯乙烯接触工人,21例内对照和25例外对照个体外周血淋巴细胞进行DNA损伤(yH2AX)和细胞凋亡检测。结果显示不同VCM累积接触水平的作业工人外周血淋巴细胞DNA损伤率和几何平均荧光强度明显高于对照组,差异有统计学意义(p0.05)；以对照组淋巴细胞凋亡率95%上限(4.84)为界值判断淋巴细胞凋亡阳性,结果显示对照组、低、高VCM累积接触组淋巴细胞凋亡阳性率随剂量升高而升高(p0.05)。本次研究结果表明VCM接触可导致接触工人外周血淋巴细胞DNA损伤和凋亡率升高。提示外周血淋巴细胞DNA损伤率和凋亡率可以作为评价低浓度VCM接触的效应指标。本研究还对上述追踪观察的部分氯乙烯接触工人应用基于微核的DNA损伤修复能力检测方法开展染色体损伤修复能力研究,其中接触组43名,同时以17名该厂行政和后勤人员作为对照。按照氯乙烯累积接触剂量分为2组(累积接触剂量≤10984mg为低接触组；10984mg为高接触组),研究显示对照组、低、高接触组的3AB指数分别为0.37±0.17,0.28±0.11,0.18±±0.10,存在显著统计学差异p0.05),提示氯乙烯接触会影响DNA损伤修复能力,氯乙烯累积接触剂量越高,DNA损伤修复能力越低；另按3AB指数分成2组(3AB指数≤0.24为低损伤修复能力组；0.24为高损伤修复能力组),结果显示,损伤修复能力高组的微核率(2.28±±2.61)低于低损伤修复能力组(4.04±2.76),存在显著统计学差异p0.05),提示DNA损伤修复能力与氯乙烯作业工人染色体损伤的发生关系密切,修复能力差,染色体损伤高。通过对DNA损伤修复能力研究表明,DNA损伤修复能力与氯乙烯接触剂量有关,可作为氯乙烯接触的效应指标,同时又可用于易感人群的筛选,具有易感性指标的特点,提示DNA损伤修复能力具有重要的应用价值和前景。本课题还应用PCR-RFLP和CRS-RFLP法对VCM接触工人细胞周期调控相关基因的多态位点进行检测,VCM接触工人309例,对照组149例。应用Poisson回归分析各基因多态位点与染色体损伤之间的关系。选择参与细胞周期调控的常见基因p53, p21, mdm2, Gadd45a和p14ARF,对其10个常见多态位点(p53intron3,p53Ex4+119CG, P53IVS6+62GA,p21Ex3+7CT, mdm2Dell518, mdm2IVSl+309TG, Gadd45a g.1260TC, p14ARF g.9792GT, p14ARF g.22008GA,p14ARFg.26294GA)进行检测和分析。调整年龄、性别、累积接触剂量、吸烟、饮酒等因素后,结果显示p14ARFg.22008基因的突变位点是遗传损伤的保护因素,与野生纯合型比较,FR=0.77(95%CI0.63-0.93,P0.05)；而p53intron3,p21Ex3+70CT和mdm2IVS1+309TG三个基因的突变位点为VCM致染色体损伤的危险因素,与野生纯合型比较FR=1.47(95%CI1.09-1.98), FR=1.36(95%CI1.13-1.64)和FR=1.74(95%CI1.43-2.13),均具有显著意义(p0.05)。综上所述,在我国现行职业卫生标准下,氯乙烯接触仍能产生遗传损伤,且存在剂量-反应关系,据此计算得到的基准剂量远低于现行职业卫生标准；氯乙烯接触工人的DNA损伤修复能力降低；VCM致染色体损伤与细胞周期调控基因多态有关。
[Abstract]:Vinyl chloride monomer (VCM) is an important chemical raw material. It is mainly used in the synthesis of polyvinyl chloride (PVC) of about 51 million tons of global PVC production capacity in.2011, while our country's PVC capacity reaches 21 million 622 thousand tons. It has become the world's largest producer and the largest consumer country.VCM is a definite human carcinogen, which can lead to multisystem, multiple organ tumors. The carcinogenesis mechanism of VCM is mainly due to the initiation of carcinogenesis by the damage of genetic material induced by active metabolites.
At present, the limited value of professional contact of VCM in developed countries is 1ppm (2.79mg/m3, PEL), and the VCM time weighted mean permissible concentration (PC-TWA) is 10mg/m3, and the allowable concentration of short time contact is (PC-STEL) 25mg/m3, higher than that of the western national health standard. The health hazards caused by long and low exposure to vinyl chloride have attracted wide attention and look for the sensitivity. Therefore, effective biomarkers are necessary to prevent harmful effects caused by vinyl chloride contact.
Under the current standard of occupational hygiene of vinyl chloride in our country, it is particularly concerned about whether the genetic material of the exposed workers can be significantly damaged. And under the same contact conditions, not all individuals can produce genetic material damage. Even if the genetic material is damaged, the body has the ability of DNA repair (DNA repair capacity, DRC). To repair. Therefore, DNA repair ability plays an important role in maintaining the body's genomic stability and normal function. If DRC is ineffective or defective, the damaged DNA will not be repaired in time and effectively, resulting in gene mutation and even cell canceration, increasing the risk of cancer in the individual.
Through cross-sectional investigation and tracing study, the relationship between chromosome damage and the cumulative exposure dose of vinyl chloride was investigated. At the same time, the DNA damage repair ability of vinyl chloride workers was studied by using the microkernel based DNA damage repair ability detection method, and the risk of chloroethylene induced chromosomal damage was evaluated. In order to provide scientific basis for the workers of susceptible workers, the relationship between DNA cell cycle regulation gene polymorphism and genetic damage induced by VCM is studied to find the susceptibility biomarkers of VCM genetic damage, and to provide a scientific basis for clarifying the mechanism of its toxic action and assessing the risk of health.
The subjects were 309 cases of VCM contact workers in a chemical enterprise (224 males and 85 females), 149 cases (61 men, 88 women), and 25 cases (14 men and 11 cases) of the administrative and logistic staff (61 women), and the micronucleus test of cytoplasmic division block (CB micronucleus test) was used for chromosomal damage. The rate of micronucleus cells in peripheral blood lymphocyte in VCM contact group was significantly higher than that of the control group FR=3.24 (95%CI2.80-3.75), p0.001, and there was a dose response relationship. It was suggested that the micronucleus rate of the external Zhou Xuelin cell can be used to evaluate the effect of low concentration VCM contact in the present occupational VCM contact level. Index.
The relationship between the incidence of micronucleus and the cumulative exposure dose of vinyl chloride was analyzed by the baseline dose (BMD) method in 20042007 years of VCM workers. The results showed that the baseline dose 95% confidence interval lower limit (BMDL) was 0.99mg/m3-year and 3.38ng in men and women in 2004, respectively. /m3-year; in 2007, both men and women were 0.63mg/m3-year and 1.27mg/m3-year., respectively, suggesting that when the total contact dose reached BMDL value, it caused micronucleus damage, providing a reference for the formulation of the perchloroethylene contact limit.
YH2AX is a recognized sensitive indicator of DNA damage stress response at home and abroad. When DNA double strand breaks, H2AX 139th serine residues in histone H2A family members are rapidly phosphorylated to form phosphorylated H2AX, that is, gamma -H2AX. this study applied flow cytometry (FCM) to the trace of chloroethylene contact observed above. DNA injury (yH2AX) and cell apoptosis were detected in 21 cases of internal control and 25 exceptions in the peripheral blood lymphocytes. The results showed that the DNA damage rate and the geometric mean fluorescence intensity of peripheral blood lymphocytes of workers with different VCM accumulative contact levels were significantly higher than those of the control group (P0.05). The apoptosis rate of 95% (4.84) was the boundary value to determine the lymphocyte apoptosis positive. The results showed that the positive rate of lymphocyte apoptosis in the control group was low and high VCM accumulated contact group increased with the increase of dose (P0.05). The results of this study showed that VCM exposure could lead to the increase of DNA damage and apoptosis rate of peripheral blood gonorrhea cells in contact workers. DNA damage rate and apoptosis rate can be used as an indicator for evaluating low concentration VCM exposure.
This study also carried out a study on the ability to repair chromosomal damage by using micronucleus based DNA damage repair ability detection method based on microkernel based DNA damage repair. In this study, the contact group was divided into 2 groups (cumulative exposure dose < 10). 984mg was a low exposure group; 10984mg was a high contact group. The study showed that the 3AB index of the control group was 0.37 + 0.17,0.28 + 0.11,0.18 + 0.10, respectively, and there was a significant difference of P0.05. It suggested that vinyl chloride contact would affect the ability to repair DNA damage. The higher the dose of chloroethylene accretion contact, the lower the ability to repair DNA damage; and 3A The B index was divided into 2 groups (3AB index < 0.24 is low damage repair ability group and 0.24 is high damage repair ability group). The result showed that the micronucleus rate of high repair ability group (2.28 + 2.61) was lower than that of low injury repair ability group (4.04 + 2.76), and there was significant difference P0.05. It suggested that the repair ability of DNA injury and the chromosome of vinyl chloride workers The damage repair ability is close, the repair ability is poor and the chromosome damage is high. Through the study of DNA damage repair ability, the ability of DNA damage repair is related to the dose of vinyl chloride contact, which can be used as the effect index of vinyl chloride contact, and can also be used for screening of susceptible population, with the characteristics of susceptibility index, suggesting the ability of DNA damage repair. There is an important application value and prospect.
The PCR-RFLP and CRS-RFLP methods were used to detect the polymorphic loci of the cell cycle regulation related genes in VCM exposed workers, 309 workers in VCM contact and 149 in the control group. The relationship between polymorphic loci and chromosome damage was analyzed by Poisson regression. The common gene p53, p21, MDM2, Gadd participating in the regulation of cell cycle was selected. 45A and p14ARF were tested and analyzed for 10 common polymorphic loci (p53intron3, p53Ex4+119CG, P53IVS6+62GA, p21Ex3+7CT, mdm2Dell518, mdm2IVSl+309TG, Gadd45a g.1260TC, p14ARF g.9792GT). The results showed that the factors such as age, sex, cumulative exposure dose, smoking, drinking and other factors were shown. The mutation site of the 008 gene is a protective factor for genetic damage, compared with the wild homozygous type, FR=0.77 (95%CI0.63-0.93, P0.05), and the mutation sites of the three genes of p53intron3, p21Ex3+70CT and mdm2IVS1+309TG are the risk factors for chromosomal damage caused by VCM, compared with the wild homozygous type FR=1.47 (95%CI1.09-1.98), FR=1.36 (95%CI1.13-1.64) and FR=. 1.74 (95%CI1.43-2.13) was significant (P0.05).
To sum up, under the current standard of occupational health, chloroethylene exposure can still produce genetic damage, and there is a dose response relationship. According to this, the calculated baseline dose is far lower than the current occupational health standard; the DNA damage repair ability of vinyl chloride exposed workers is reduced; VCM induced chromosomal damage and cell cycle regulation genes are polymorphic. Close.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R135.1

【参考文献】