【作者】 张轩；

【导师】 朱益民；

【作者基本信息】 浙江大学 ， 流行病与卫生统计学， 2011， 硕士

【摘要】 铬是自然界中广泛存在的一种元素,主要以三价铬和六价铬的形式存在。三价铬参与人和动物体内的糖与脂肪的代谢,是人体必需的微量元素；六价铬则是明确的有害元素。流行病学研究已经证实,过去因从事铬酸盐化学品制造及电镀工艺等使用铬酸的工作而接触高水平六价铬可导致呼吸道癌症的发病率增加,其潜伏期可长达15年以上。1987年我国已将铬酸盐制造业所致肺癌列入职业肿瘤名单。1990年国际癌症研究机构(IARC)将六价铬化合物定为人类确定致癌物。六价铬能够通过S042-和HPO42这类等电和同构阴离子转运通道穿过细胞膜,三价铬由于不能进入细胞,毒性低于六价铬。六价铬是很强的氧化剂,在氧化过程中可被还原成中间产物(Cr(V)和Cr(IV)),最终形成稳定的三价态。生物体的还原剂如抗坏血酸和硫醇等和六价铬反应时可能会产生有机自由基,从而导致氧活化形成活性氧,而过量的活性氧则会对细胞内蛋白质和DNA造成损伤,产生氧化应激的状态。镀铬的传统电解工艺借电化学作用,将铬金属沉积到钢铁、铜和铜合金等制件的表面上,用于提高抗蚀性、耐磨性和硬度以及增加反光性和美观等。这一工艺会释放出一种含有铬酸的薄雾,如果不使用铬雾抑制剂或没有有效的工程控制措施消除铬雾,工人即可能会接触到铬雾。近年来随着电镀企业防护措施的改善,高浓度铬酸雾已不多见,而低浓度铬酸雾对人体的远期效应(致癌,致畸,致突变)越来越引起人们的关注。铬酸溶剂电镀工艺与肺癌患病风险率的升高存在联系,皮肤溃疡及鼻中隔穿孔也可能发生在该行业,并且还存在着皮肤和呼吸道过敏的可能性。因此,寻找可靠的生物学指标,综合评价铬盐的暴露水平和健康效应十分必要。本次调查中铬接触组选择浙江省杭州市25家电镀企业镀铬工人157名,对照组选择浙江省杭州市某企业无任何毒物和重金属接触史的工人93名。肝肾功能异常者、患有其他慢性疾病者均在排除之列。使用统一设计的健康状况调查表分别对铬接触工人和对照人群进行流行病学调查,并对铬作业岗位进行现场监测,采样参照GBZ159-2004工作场所空气中有害物质监测采样规范。由于红细胞内铬含量能够真实可靠地反映相对较低水平时的铬接触情况,采用平台石墨炉原子吸收光谱法测定红细胞铬,基体改进剂Triton X-100的加入使血液发生溶血作用,以释放出红细胞中的铬。通过碱性彗星实验检测个体DNA链断裂水平,8-羟基脱氧鸟嘌呤(8-OHdG)作为DNA氧化损伤的生物标记物。本研究选择了碱基切除修复、核苷酸切除修复及代谢酶基因中的九个SNP位点研究六价铬所致DNA损伤的遗传易感性,对XRCC1 Arg194Trp和XRCC1 Arg399Gln采用聚合酶链反应扩增,对OGG1 Ser326Cys、ERCC1 C8092A、ERCC6 Gly399Asp、ERCC5 His1104Asp、XPD Lys751Gln、XPC Lys939Gln、GSTP1 Ile105Val采用TaqMan探针法基因分型。调查发现杭州市电镀企业车间规模较小,设备简陋,自动化水平低下,车间通风不良,可使工人受到铬酸盐的危害。此外,镀铬作业工人文化程度较低(初中文化程度以下占85.35%),大部分工人缺乏个人防护意识。铬接触工人红细胞内铬含量的中位数为4.41(0.93-14.98),约为对照组的2倍(1.54(0.14-4.58),P<0.001)。按性别、年龄、吸烟、饮酒分层后,除了小于30岁的人群(P=0.11),其余各组都具有统计学差异(P<0.05)。铬接触工人尾长中位数为11.77(3.46-52.19)、尾DNA%中位数为3.69(0.65-16.20), Olive尾距中位数为1.13(0.14-6.77),与对照相比均具有统计学差异(P<0.001),尿8-OHdG含量为13.65(3.08-66.30)μg/g肌酐,与对照8.31(2.94-30.83)μg/g肌酐相比差异也具有统计学意义(P<0.001)。按性别、年龄、吸烟、饮酒分层分析后,两组间尾长、尾DNA%, Olive尾距和尿8-OHdG含量差异仍然存在(P<0.05)。当校正了性别、年龄、吸烟、饮酒等因素后,职业铬(Ⅵ)暴露对红细胞中铬含量,尾长、尾DNA%、Olive尾距和尿8-OHdG含量的影响具有统计学意义(P<0.001),此外,红细胞中铬含量与尾长、尾DNA%. Olive尾距之间呈显著正相关(P<0.05),而红细胞中铬含量对尿8-OHdG含量的影响则未发现统计学意义(P>0.05)。SNP分析结果显示,XRCC1Arg399Gln突变型携带者DNA断裂水平显著高于野生型纯合子携带者,其基因多态性也与Olive尾距显著相关。铬暴露与ERCC6-399突变型的交互作用对Olive尾距的影响具有统计学意义(P<0.05),OGG1Ser326Cys与ERCC1-8092突变型、ERCC5-1104与ERCC6-399突变型、XPD751分别与XRCC1-194、ERCC5-1104、ERCC6-399突变型间的交互作用分别对尿8-OHdG含量的影响有统计学意义(P<0.05)。本次研究发现电镀作业环境中空气中铬浓度明显高于国家职业卫生标准(0.05mg/m3),铬接触工人红细胞内铬含量显著高于正常人群。铬电镀作业存在六价铬污染。铬接触工人DNA链断裂水平和尿8-OHdG含量均高于正常人群,说明职业性六价铬暴露能够引起DNA损伤。XRCC1Arg399Gln是铬接触工人的遗传易感性生物标志物,铬暴露与核苷酸切除修复基因的交互作用以及DNA切除修复通路与代谢酶基因的交互作用在铬接触工人DNA损伤修复中也发挥了重要作用。更多还原

【Abstract】 Chromium (Cr) is one of the most important metal elements existing in the environment, which occurs predominantly in two valence states:trivalent chromium (Cr(Ⅲ)) and hexavalent chromium (Cr(Ⅵ)). Cr(Ⅲ) is involved in the metabolism of sugar and fat in human, and is the essential trace element. Cr(Ⅵ) is recognized as harmful element. Epidemiological studies have shown that exposure to Cr (Ⅵ) significantly increases the risk of respiratory tract cancer, and the incubation period can be more than 15 years. The lung cancer caused by occupational chromate manufacture industry was listed as one of occupational tumors by China in 1987. And Cr (Ⅵ) was classified as a human carcinogen (group 1) by the international Agency for the Research on Cancer (IARC) in 1990.Cr(Ⅵ) compounds can actively penetrate the cells membrane through channels to transfer isoelectric and isostructural anions such as SO42- and HPO42-channels. Cr(Ⅲ) compound, because of its poor membrane permeability, has been considered to have lower toxicity than Cr(Ⅵ). Cr(Ⅵ) is a strong oxidizing agent, and is reduced through short lived Cr intermediates (Cr(Ⅴ) and Cr(Ⅳ)) to the ultimate stable trivalent species (Cr(Ⅲ)). The reactions of Cr(Ⅵ) with biological reductants, such as ascorbate and thiols, might be accompanied by formation of organic radicals, which in turn cause O2 activation and formation of reactive oxygen species (ROS), and excessive quantities of ROS generated by these reactions would cause damage to cellular proteins and DNA leading to a state known as oxidative stress.Electroplating traditional electrolysis process is depositing chromium metal on the surface of steel, copper and alloys through electrochemical function to improve corrosion resistance, abrasion resistance and hardness, and increase the reflective ablity and beautifulness, etc. This process can release a kind of mist containing chromic acid, so if we do not use inhibitors or effective control measures to eliminate chromium fog, workers may be exposed to chromium. In recent years, with protective measures of improving, high concentration of chromic acid mist has been rare, but long-term effect such as cancer, teratogenic and mutation which caused by low concentration chromic acid mist has attracted people’s attention. Electroplating has relation with increased risk rate of lung cancer, skin ulcer and septal perforation can also occur in this industry, and the possibility of skin and respiratory tract allergy still exists. So, this study was to evaluate the potential health effects related to chronic Cr(VI) exposure.157 electroplating workers from 25 electroplating factories and 93 non-chromate exposure subjects without exposure to other known physical or chemical genotoxic agents were recruited in Hangzhou, China. Subjects with abnormal liver or kidney function and suffering from other chronic diseases were excluded in the study. Epidemiological investigation questionnaires were conducted on electroplating workers and control subjects, respectively. Short-term sampling in the electroplating workplace was conducted according to a Standard of Industrial Hygiene in China (GBZ159-2004). Cr (VI) concentration in erythrocytes was measured by graphite furnace atomic absorption spectrometry, and Triton X-100 was used to make the red blood cells to release chromium. DNA damage was evaluated with an alkaline single cell gel electrophoresis (comet assay) in peripheral lymphocytes and concentration of 8-OHdG in urine was determined with ELISA kit, which is one of the major oxidative adducts formed by radical induced damage to DNA. Nine SNPs were included in base excision repair genes, nucleotide excision repair genes and metabolic enzyme gene to study the relation of DNA damage and genetic susceptibility. Genotypes of XRCC1 Arg194Trp and Arg399Gln were identified by PCR-RFLP, genotypes of OGG1 Ser326Cys、ERCC1 C8092A、ERCC6 Gly399Asp、ERCC5 His1104Asp、XPD Lys751Gln、XPC Lys939Gln and GSTP1 Ile105Val were determined by the TaqMan probes.The workshops of electroplating enterprises in this investigation were poorly equipped. And poor ventilated workshops could be harmful to electroplating workers. Educational level of electroplating workers culture level was low (85.35% below middle cultural degree), and most workers were lack of awareness of self protection. In electroplating workers, the median of Cr(Ⅵ) concentration (μg/L) in erythrocytes was 4.41(0.93,14.98), which was about two times higher than that in control subjects (1.54(0.14,4.58), P＜0.001). After stratified by potential confounding factors such as gender, age, smoking status and alcohol consumption, significant differences were still existed between electroplating workers and control subjects, except for the subjects of age less than 30 years old (P=0.11).The median of tail length in electroplating workers was11.77 (3.46-52.19), the median of tail DNA% was 3.69(0.65-16.20), the median of Olive tail moment wasl.13 (0.14-6.77), and the differences between electroplating workers and control subjects were significant (P＜0.001). Urinary 8-OHdG concentration was 13.65 (3.08,66.30)μg/g creatinine in electroplating workers and 8.31 (2.94,30.83)μg/g creatinine in control subjects. Their difference was also significant (P<0.001). After stratified by gender, age, smoking status, and alcohol consumption, the differences in tail length, tail DNA%, Olive tail moment and urinary 8-OHdG concentration still existed between two groups(P<0.05). After controlled for gender, age, smoking status, and alcohol consumption, the occupational Cr(Ⅵ) exposure was significantly positively associated with the Cr(Ⅵ) concentration in erythrocytes (μg/L), tail length, tail DNA%, Olive tail moment and urinary 8-OHdG concentration (μg/g creatinine) (P＜0.001). The Cr (Ⅵ) concentration in erythrocytes (μg/L) was positively associated with tail length, tail DNA%, Olive tail moment (P＜0.05). However, statistically significant association between Cr(VI) concentration in erythrocytes and urinary 8-OHdG was not found.The results showed level of DNA strand breaks of mutation type carriers of XRCC1Arg399Gln was higher than that in wild type, and SNPs of XRCC1Arg399Gln had significantly positively correlation with Olive tail moment. The effect of interaction between exposure and ERCC6-399 mutation type had statistical significant on Olive tail moment (P<0.05). The effect of interaction between OGG1Ser326Cys and ERCC1-8092 mutation type, ERCC5-515 and ERCC6-399 mutation type, XPD751 respectively with XRCC1-194, ERCC5-515、ERCC6-399 mutation type had statistical significant on urinary 8-OHdG(P<0.05).These results showed that the average level of chromium concentration in the air of occupational environment was higher than the national occupational health standards (0.05mg/m3), and Cr(VI) concentration in erythrocytes of electroplating workers was higher than that in control subjects. Hexavalent chromium pollution existed in electroplating process. Level of DNA strand breaks and concentration of urinary 8-OHdG of electroplating workers were higher than those in control subjects, so occupational hexavalent chromium exposure could induce DNA damage. XRCC1 Arg399Gln was the genetic susceptibility biomarker of electroplating workers. The interaction between exposure and NER, and the interaction between DNA excision repair pathways and metabolic enzyme genes might play important roles in DNA damage repair of electroplating workers.更多还原