双酚A遗传毒性研究

发布时间：2018-03-22 08:23

本文选题：双酚A　切入点：遗传毒性　出处：《苏州大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：双酚A（bisphenol A, BPA），是一种广泛使用的化工原料，添加BPA可使塑料制品具有轻巧耐用、无色透明以及加强抗冲击性等特点，并有效防止水果和蔬菜从内部侵蚀金属容器，所以BPA成为制造聚碳酸酯、环氧树脂、食品包装、牙科填充剂、食品罐头涂料和医疗器械等产品的重要原料。BPA具有毒作用剂量小、潜伏时间长等特点，对健康造成多种潜在危害，国内外许多学者对BPA的毒性作用进行了研究报道。目前对BPA的毒性研究多集中在内分泌系统、生殖系统、神经系统等方面，其遗传毒性还有待进一步研究。本实验通过体外染毒检测BPA对中国仓鼠卵巢细胞（CHO细胞）的细胞存活率、DNA的直接损伤作用、微核率以及染色体畸变率，并通过Ames试验检测BPA的致突变性，通过将上述检测不同遗传学终点的试验相结合探讨BPA的遗传毒性。方法：(1)以仓鼠卵巢细胞（CHO细胞）作为实验系统，选用四甲基偶氮唑盐法（即MTT比色法）检测不同浓度双酚A对CHO细胞作用不同时间后，对细胞存活率的影响；（2）采用单细胞凝胶电泳试验（即SCGE彗星试验），了解不同浓度及不同作用时间下双酚A对CHO细胞DNA单链断裂的损伤程度；（3）采用微核试验研究不同浓度双酚A对CHO细胞微核率的影响；（4）采用染色体畸变分析检测不同浓度双酚A对CHO细胞染色体畸变率及诱导的主要畸变类型；（5）通过Ames试验了解不同浓度双酚A的致突变效应。结果：（1）MTT试验结果显示，分别经12h及24h染毒处理后，当双酚A终浓度为40μmol/L时，CHO细胞的存活率显著高于阴性对照组。当浓度高于80μmol/L时，，细胞存活率呈现下降趋势，各染毒组吸光度值与阴性对照组相比均具有统计学差异。（2）单细胞凝胶电泳实验结果显示，经12h及24h处理后，双酚A浓度高于80μmol/L时，可引起CHO细胞的拖尾率、尾部DNA百分含量及尾长增加。（3）微核试验和染色体畸变试验表明，在双酚A处理24h后，当浓度高于100μmol/L时，CHO细胞的微核率和染色体畸变率显著增加。其中染色体畸变的类型以断裂、无着丝粒断片和裂隙为主。（4）Ames试验检测的BPA染毒浓度为10μg/皿～5mg/皿共九个剂量组，其结果为阴性。在活化和非活化条件下，与阴性对照组比较，可见部分菌株的回变菌落数有所增加，但并无统计学差异。结论:（1）外培养条件下，双酚A在染毒浓度为40μmol/L作用12h后，可显著刺激细胞的生长，表现出一定的刺激效应。从80μmol/L开始，染毒12h对CHO细胞的增殖有明显的抑制作用，表现出细胞毒性作用。（2）双酚A可引起CHO细胞的拖尾率、尾部DNA百分含量、尾长及Olive尾矩增加。提示双酚A可引起体外培养的细胞产生DNA的单链损伤。（3）双酚A能显著增加CHO细胞的微核率和染色体畸变率。其中染色体畸变的类型以断裂、无着丝粒断片和裂隙为主。（4）Ames试验未检测到双酚A的致基因突变作用。在活化和非活化条件下，与阴性对照组相比，可见部分菌株的回变菌落数有增加趋势，但并无统计学意义。提示双酚A可能并不存在致基因突变的作用。
[Abstract]:Objective: bisphenol A (bisphenol A BPA), is a widely used chemical raw materials, adding BPA can make plastic products is lightweight and durable, colorless and transparent and strengthening the impact resistance, and effectively prevent the erosion of metal containers of fruit and vegetables from the inside, so BPA is manufacturing polycarbonate, epoxy resin, food packaging. Dental filler,.BPA important raw material of canned food coatings and medical equipment and other products with the toxic effect of small dosage, the characteristics of the potential for a long time, causing a variety of potential health hazards and toxic effects of many domestic and foreign scholars have conducted the research to the BPA report. The present study toxicity of BPA mainly focus on the endocrine system, reproductive system. The neural system, the genetic toxicity remains to be further studied. The experiments of in vitro detection of BPA China hamster ovary cells (CHO cells) cell viability, direct injury DNA The micronucleus rate and chromosome aberration rate were detected. The mutagenicity of BPA was detected by Ames test. The genotoxicity of BPA was investigated by combining the above test of different genetic endpoints.
Methods: (1) in Chinese hamster ovary cells (CHO cells) as an experimental system, using four methyl thiazolyl tetrazolium method (MTT assay) to detect different concentrations of bisphenol A on CHO cells for different time, the rate of cell survival; (2) using single cell gel electrophoresis assay (comet SCGE to understand the extent of damage test), different concentrations and different action time of bisphenol A on CHO cells of DNA single strand breaks; (3) the experimental study on Micronucleus of different concentrations of bisphenol A on the effects of micronucleus rate of CHO cells; (4) by chromosome aberration detection and analysis of different concentration of bisphenol A on the main CHO cell chromosome aberration and distortion induction type; (5) to understand the mutagenic effects of different concentrations of bisphenol A by Ames test.
Results: (1) MTT test results showed that 12h and 24h respectively after exposure after treatment, when the final concentration of bisphenol A is 40 mol/L, the survival rate of CHO cells was significantly higher than that of the negative control group. When the concentration is higher than 80 mol/L, the cell survival rate decreased, the absorbance values were compared with the exposure group significant difference with the negative control group. (2) experimental results of single cell gel electrophoresis showed that the 12h and 24h after treatment, the concentration of bisphenol A is higher than 80 mol/L, can cause CHO cell comet frequency, tail DNA% and tail length increased. (3) showed that micronucleus test and chromosome aberration test in bisphenol A after 24h treatment, when the concentration is higher than 100 mol/L, CHO cell micronucleus rate and chromosome aberration rate increased significantly. The type of chromosome aberration to fracture, acentric fragment and fissures. (4) the exposure concentration of BPA Ames test for 10 g/ ~ nine 5mg/ dish dish In the dose group, the results were negative. When activated and non activated, compared with the negative control group, the number of the colony of some strains increased, but there was no statistical difference.
Conclusion: (1) the culture conditions, bisphenol A exposure concentrations in 40 mol/L after 12h treatment can significantly stimulate cell growth, showed some stimulation effect. From the beginning of the 80 mol/L 12h exposure on the proliferation of CHO cells was significantly inhibited and showed cytotoxicity (2.) bisphenol A can cause CHO cell comet frequency, tail DNA%, tail length and Olive tail moment increased. Single strand damage that bisphenol A can cause the cells to produce DNA. (3) bisphenol A could significantly increase CHO cell micronucleus rate and chromosome aberration rate of chromosome aberration in the type. Fracture, acentric fragment and fissures. (4) Ames test did not detect mutagenic effects of bisphenol A. In the activation and non activation conditions, compared with the negative control group, visible strain change counts increased, but there was no statistically significant. Bisphenol A may prompt There is no effect of gene mutation.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】