【作者】 张毅；

【导师】 仲来福；

【作者基本信息】 大连医科大学 ， 劳动卫生与环境卫生学， 2010， 硕士

【摘要】 前言：溴酸钾(potassium bromate, PB)是饮用水臭氧消毒的副产物,在食品工业中曾广泛用作面粉处理剂,用来改善面粉的品质。因此,PB主要通过饮食而被人体摄入。鉴于研究发现PB存在致突变性和致癌性,我国和其他一些国家已禁止将其作为面粉处理剂继续应用。2007年7月1日实施的《生活饮用水卫生标准》中,我国首次将溴酸盐纳入饮用水国家标准,规定在使用臭氧消毒时,水质溴酸盐含量的限值为0.01毫克／升,与世界卫生组织的制定标准一致。国际癌症研究机构(International Agency for Research on Cancer,IARC)早在1987年就将PB归为2B类致癌物。PB是一种可增加人类患癌症风险的化学物。此外,有研究发现显示PB具有遗传毒性,但有关遗传毒性机制的研究不多。研究表明,PB可导致细胞中活性氧类(ROS)生成增多,也能引起细胞内主要的抗氧化物质——还原型谷胱甘肽(GSH)含量降低。另外,有研究发现,溶酶体膜稳定性改变和线粒体膜电位变化也与遗传毒性机制有关。因此,我们研究了PB遗传毒性与细胞内ROS与GSH含量以及溶酶体线粒体的关系,旨在探讨PB遗传毒性的氧化应激机制。PB经消化道途径进入体内,肝脏作为PB代谢的最初部位,因此本研究选用人肝癌细胞系(HepG2)作为试验系统。HepG2细胞保留了人类正常肝实质细胞的许多功能,还保留了生物转化Ⅰ相酶和Ⅱ相酶的活性,被认为是检测外来化合物遗传毒性的理想细胞系。本研究选用HepG2细胞,研究PB的遗传毒性及氧化性DNA损伤的机制,为进一步评估PB对人类的危害提供实验资料方法：以HepG2细胞作为试验系统。通过单细胞凝胶电泳(SCGE)试验及微核试验(MNT)检测细胞DNA和染色体损伤情况,评价PB的遗传毒性。为探讨其可能的遗传毒性机制,以2’,7’—二氢二氯荧光素(DCFH)法和苯二醛(OPT)分别测定细胞内ROS以及GSH水平,采用DL——甲硫氨酸磺酰亚胺(BSO)和羟基酪醇(HT)干预法观察细胞内GSH水平对PB所致的DNA与染色体损伤效应的影响,用吖啶橙(Acridine orange)和罗丹明123 (Rhodamine 123)分别测定细胞内溶酶体膜稳定性和线粒体膜电位的改变水平,以免疫组化方法检测细胞内8-羟基脱氧鸟苷(8-OHdG)的表达水平。实验结果用SPSS v 11.5统计软件包进行统计分析结果：1.56 mM—12.5 mM的PB作用于HepG2细胞1 h后,引起细胞DNA链断裂,形成彗星样脱尾,尾长、尾DNA含量及尾距均明显大于未用PB处理的细胞。而相同剂量梯度下的PB接触细胞40min,与未用PB处理细胞比较,未见有统计学意义的差别。12.5μM—50μM的HT预处理后,1 2.5 mM的PB接触HepG2细胞1 h,不同剂量HT干预组形成的彗星样脱尾,尾长、尾DNA含量及尾距均明显小于未用HT处理的细胞。0.12 mM—1 mM的PB作用于HepG2细胞24 h可引起细胞微核率显著升高。采用150μM的BSO预处理HepG2细胞20 h,以耗竭细胞内GSH,可明显增强PB对HepG2细胞DNA及染色体的损伤,且呈剂量依赖关系。采用50μM HT可提高细胞内GSH水平,并可明显地降低PB对HepG2细胞的染色体损伤效应。PB作用于HepG2细胞可引起细胞内ROS表达水平的明显增加以及细胞内GSH的耗竭,PB的作用剂量分别12.5 mM和1.56 mM—12.5 mM。6.25 mM—12.5 mM的PB作用于HepG2细胞3 h后,细胞内8-OHdG水平的表达增强,与对照组比较,有明显差别。6.25 mM—12.5 mM的PB作用于HepG2细胞可引起细胞内溶酶体膜稳定性明显改变。而1.56 mM—12.5 mM的PB作用细胞后检测细胞内线粒体膜电位的变化,则未见明显改变。结论：PB可引起HepG2细胞DNA损伤和染色体损伤,提示PB具有遗传毒性。PB引起8-OHdG形成增强,表明PB引起的DNA损伤为氧化性损伤。PB引起细胞内ROS增高,GSH耗竭,8-OHdG的形成增强以及溶酶体膜稳定性改变,表明PB引起遗传毒性的机制可能与氧化性DNA损伤有关。此外,HT可预防PB引起的HepG2细胞DNA损伤。更多还原

【Abstract】 Introduction:Potassium bromate (PB) is a by-product of the use of ozone in drinking water disinfection, and also as a widely used food additive that is employed to improve the quality of flour, by acting as a bleaching agent. Considering the genotoxicity and caicinogenesis of it, PB was banned to be the flour additive in many countries. In 2007, The Chinese government proposed the provisional guideline value of PB 0.01 mg/1 in drinking water what was in accord with the one proposed by WHO in 2004.In 1987 the International Agency for Research on Cancer (IARC) assessed PB as a Group 2B carcinogen. It proved that PB was a kind of compound which could increase the risk of human cancer. PB has been evaluated for its genotoxic effects. However, the mechanisms of the effects are not well understood. PB was able to induce the generation of reactive oxygen species (ROS) and a significant reduction of intracellular GSH. In addition, it reported that the changes of lysosomal membrane stability and mitochondrial membrane potential may be involved in the mechanisms of PB-induced genotoxicity. Therefore, the aim of this study was to assess the genotoxic effects of PB in vitro and to elucidate the mechanism of PB-induced genotoxicity. Since oral exposure is the route of contamination by PB and the liver is the initial site of PB metabolism, HepG2 cell line is considered to be a more suitable system in our study. The HepG2 cell line retains many of the functions of normal liver cells and expresses the activities of several phases I and II xenobiotic metabolizing enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.Methods:HepG2 cells were selected as test system. We used the single cell gel electrophoresis assay (SCGE) and the micronucleus test (MNT) to study the genotoxic effects of PB. To elucidate the oxidative DNA damage mechanism in HepG2 cells, we used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of ROS and glutathione (GSH). DL-buthionine sulphoximine (BSO) and hydroxytyrosol (HT) were used to modulate the level of GSH in HepG2 cells, and the effects of GSH on PB-induced genotoxicity were determined by the SCGE and MNT. To further investigate the mechanism of genotoxicity of PB in HepG2 cells, we used Acridine orange (AO) and Rhodamine 123 to measure the changes of lysosomal membrane stability and mitochondrial membrane potential. In addition,8-hydroxyderoxyguanosine (8-OHdG), which is a reliable marker for oxidative DNA damage, was also measured by immunocytochemistry staining analysis. The data were statistically analyzed by SPSS v 11.5 software.Results:In the SCGE and MNT, PB increased the DNA migration and the MN frequencies at the concentrations of 1.56 mM—12.5 mM and 0.12 mM—1 mM, respectively. A statistical significance was not observed in HepG2 cells when the cells exposed with PB (0 mM—12.5 mM) for 40 min. DNA damage induced by PB was significantly reduced a dose-dependent manner in cells pre-treated with HT (12.5μM—50μM) for 30 min in the SCGE assay. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the PB-induced genotoxic effects and that when the intracellular GSH content was elevated by HT, the chromosome damage induced by PB was significantly prevented in the concentrations (0.12 mM—1 mM). Moreover, PB significantly caused 8-OHdG formation and the change of lysosomal membrane stability in HepG2 cells at concentrations from 6.25 mM to 12.5 mM. In addition, the change of mitochondrial membrane potential induced by PB (0 mM—12.5 mM) in the assay.Conclusion:The results suggest that PB caused DNA strand breaks and chromosome damage, which indicate that PB induced genotoxic effects in HepG2 cells. PB- induced increase of 8-OHdG formation was considered as oxidative damage of DNA. PB exerts genotoxic effects in HepG2 cells, probably through the formation of ROS, depletion of GSH, increase of 8-OHdG formation and the change of lysosomal membrane stability, which cause oxidative DNA damage.更多还原