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8种双酚A类化合物对MCF-7细胞的毒性及损伤研究

发布时间:2018-04-17 00:30  文章来源:笔耕文化传播

  本文选题:双酚A类化合物 + 微板毒性分析 ; 参考:《西南大学》2012年硕士论文


【摘要】:双酚A(BPA)是广泛存在的环境内分泌干扰物(EDCs), BPA类化合物作为环氧树脂,聚碳酸酯塑料以及阻燃剂等产品添加剂,在水杯、奶瓶、食品饮料容器、医疗设备等生产中被广泛使用,并被不断开发做新的用途。这些制品在长期使用或在高温、碱性条件下,BPA都会缓慢释放到周围环境中,并且很容易在环境中积累,并通过用水,空气,土壤和食品污染等对周围生物产生影响。 BPA类化合物的雌激效应一直是研究的重点,但是关于其化合物毒性效应的研究较少。到目前为止,研究发现BPA在较窄的浓度范围内对MCF-7的损伤作用主要依赖于类雌激素作用干扰胞内酶活性及细胞代谢活性影响细胞增殖,而微高浓度的BPA会对细胞造成损伤,导致细胞凋亡。因此在细胞水平上研究BPA类化合物的毒性效应具有重要的意义。同时,环境中的污染物并不是单独存在的,人群在生产生活中总是处于各种污染物的混合暴露之下。研究污染物的联合作用比污染物的单独作用更加贴近实际,更有现实意义。本实验主要是通过微板毒性分析法研究8种BPA类化合物对雌激素受体缺失MCF-7细胞的毒性效应,并初步结合DNA损伤以及细胞膜损伤研究为进一步评价BPA类化合物的环境毒性效应奠定基础。 实验选用8种双酚A类化合物,包括2,2-双(4-羟基苯基)丙烷(简称BPA),2,2-二(4-羟基苯基)丁烷(简称BPB),4,4'-(1-甲基亚乙基)双(2-甲基苯甲醚)(简称BPC),4,4'-亚乙基双苯酚(简称BPE),2,2-双(2-羟基-5-联苯基)丙烷(简称BPQ,4,4'-硫代二苯酚(TDP),双酚AF(简称BPAF),4,4'-(1-苯乙基)双酚(简称BPAP),采用四唑盐(MTS/PMS)比色法检测此8种BPA类化合物和12种根据等效应浓度比和均匀浓度比设计的混合物对雌激素受体缺失人乳腺癌细胞(MCF-7(ER-))增殖活性的影响,用非线性最小二乘法拟合得到浓度-效应曲线(CRC),以浓度加和模型(CA)和独立作用模型(IA)为参考模型,研究混合物的相互作用规律。通过2,4一二硝基苯肼法检测8种双酚A类化合物对MCF-7(ER-)细胞乳酸脱氢酶(LDH)露出率的影响,单细胞凝胶电泳(SCGE)检测了5种双酚A类化合物对MCF-7(ER-)细胞DNA损伤。 结果发现所有的剂量-效应曲线(CRC)均能用Weibull或者Logit函数有效表征,以模型估算的半数效应浓度负对数值(pEC50)评估8种化合物的毒性大小依次为:BPQBPAFBPAPBPBBPCTDPBPE BPA;应用剂量加和(CA)模型和独立作用(IA)模型对混合物毒性进行评估与预测,结果8种BPA类化合物各种浓度组合的混合物毒性均可用CA模型有效预测;LDH检测以及SCGE检测BPA类化合物对MCF-7(ER-)的损伤作用,首先是干扰酶活性及微量元素代谢,抑制细胞增殖,然后是对细胞核DNA造成损伤,同时细胞膜也受到损伤,细胞开始出现大量坏死。 实验最后对构建稳定表达Metridia荧光素酶的MCF-7(ER-)细胞毒性检测系统进行探索,旨在通过测定荧光素酶活性间接反映环境污染物的毒性作用,使得毒性检测更加直观易行。
[Abstract]:Bisphenol A (BPA) is a widespread environmental endocrine disruptor, BPA compounds are widely used as additives for epoxy resin, polycarbonate plastics and flame retardants in the production of water cups, milk bottles, food and beverage containers, medical equipment, etc.And has been constantly developed for new uses.These products can be released slowly into the surrounding environment under the condition of long term use or high temperature and alkalinity, and can easily accumulate in the environment, and influence the surrounding organisms through water, air, soil and food pollution.The female excitatory effect of BPA compounds has been the focus of study, but the toxicity of its compounds is less studied.Up to now, it has been found that the damage of BPA to MCF-7 in a narrow concentration range mainly depends on the estrogenic effect on the interference of intracellular enzyme activity and cell metabolic activity, while the slightly high concentration of BPA can damage the cells.Leading to apoptosis.Therefore, it is of great significance to study the toxic effects of BPA compounds at the cellular level.At the same time, environmental pollutants do not exist alone, people are always exposed to a mixture of pollutants in production and life.It is more practical and meaningful to study the combined action of pollutants than the single action of pollutants.In this study, microplate toxicity assay was used to study the toxic effects of eight BPA compounds on estrogen receptor deficient MCF-7 cells.Combined with the study of DNA damage and cell membrane damage, the study laid a foundation for further evaluation of the environmental toxicity of BPA compounds.Eight kinds of bisphenol A compounds were used in the experiment.Including BPA-2-Bis 4- hydroxyphenyl) propane (abbreviated as BPBX 4H 4H 4H 4N) BPCO 4H 4N 4 ethylphenyl bisphenol (abbreviated as BPE2O2O2O2-HYDROXY5HYDIPHENYL) propane (abbreviated as BPCO 4HYDROXYLPHENYL) propane (abbreviated as BPCO 4HYDROXYLPHETHYL) propane (BPCO 4HYDROXYLPHENYL for short for short for BPCO 4HYDROXYLPHENYL)Eight BPA compounds and 12 mixtures designed according to the equal effect concentration ratio and uniform concentration ratio were determined by using the method of tetrazolium salt MTS / PMS-colorimetry to detect the effects of eight BPA compounds and 12 mixtures designed according to the equal effect concentration ratio and the uniform concentration ratio, respectively, and bisphenol A F (abbreviated as BPA PAPP) to determine the estrogenicity of the eight BPA compounds and 12 mixtures designed according to the equal effect concentration ratio and the uniform concentration ratio.The effect of deletion of human breast cancer cell line MCF-7 ER-G) on proliferative activity,The concentration-effect curve was obtained by using the nonlinear least square method. The interaction law of the mixture was studied by using the concentration addition model (CAA) and the independent interaction model (IAA) as reference models.The effects of eight bisphenol A compounds on the exposure rate of lactate dehydrogenase (LDHs) in MCF-7ER-cells were detected by 2O4-dinitrophenylhydrazine method. Single cell gel electrophoresis (SCGE) was used to detect the DNA damage of MCF-7ER-cells induced by five bisphenol A compounds.The results show that all dose-response curves can be effectively characterized by Weibull or Logit functions.The toxicity of the eight compounds was evaluated in order of: BPQBPAFBPAPBPBBPCTDPE BPAs, and the toxicity of the mixture was evaluated and predicted by the dose-add-sum model and the independent action model.Results the toxicity of the mixture of 8 kinds of BPA compounds with different concentrations can be predicted by CA model and SCGE. The first step is to interfere with enzyme activity and trace element metabolism and inhibit cell proliferation.Then the nuclear DNA was damaged, the cell membrane was also damaged, and the cell began to suffer a lot of necrosis.At the end of the experiment, we explored the construction of MCF-7 ER-ER-C cytotoxicity detection system which stably expressed Metridia luciferase, in order to reflect the toxicity of environmental pollutants indirectly by measuring luciferase activity, making the toxicity detection more intuitionistic and practical.
【学位授予单位】:西南大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R114

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