【作者】 刘欣梅；

【导师】 邵健忠；

【作者基本信息】 浙江大学 ， 海洋生物学， 2004， 硕士

【摘要】 环境激素是一类存在于环境中能进入人体内部，具有类似雌性激素的作用，危害人体正常激素分泌的化学物质。环境激素问题是继臭氧层、温室效应之后，又一重大的世界性环境问题。马拉硫磷是一种在农业上广泛使用的有机磷杀虫剂，阿特拉津则是一种常用的三嗪类除草剂。这两者均已被世界野生动物基金会（WWF）划分为环境激素类物质。本文以草鱼（Ctenopharyngodon idellus）培养细胞ZC7901为模型，研究马拉硫磷和阿特拉津这两种环境激素的毒理学效应及从凋亡角度探讨了它们的毒理学机制。 在本实验的第一部分中，我们分析了马拉硫磷和阿特拉津的细胞毒性效应。从显微形态观察的结果，可以看出草鱼ZC7901细胞受到马拉硫磷和阿特拉津的胁迫后，活性受到较大的影响，细胞的贴壁程度大大降低。同时，四唑盐（MTT）比色试验结果显示，马拉硫磷和阿特拉津均会对草鱼ZC7901细胞的生长及活性造成抑制，而且这种抑制作用还有显著的时间及剂量依赖性。说明高浓度的药物残留及长时间药物累积，对于草鱼细胞的生长都是不利的。实验还得出：在12hr～72hr范围内，马拉硫磷对草鱼ZC7901细胞的IC₅₀值位于34.935～3.04mg／L之间；而阿特拉津在12hr～72hr范围内，其IC₅₀值则位于198.980～11.567mg／L之间。从马拉硫磷和阿特拉津二者的IC₅₀比较，可以看出，马拉硫磷的细胞毒性强于阿特拉津。 在论文的第二部分，我们研究了马拉硫磷和阿特拉津胁迫诱导鱼类细胞凋亡的情况。结果显示，这两种环境激素均能诱导草鱼细胞发生凋亡。通过荧光显微镜、共聚焦激光显微镜以及透射电镜观察，发现经阿特拉津胁迫后的细胞，均出现了明显的染色质凝聚、趋边化、形成凋亡小体等凋亡形态特征。当用浓度为37.75 mg／L阿特拉津作用细胞48hr及用浓度为28.31 mg／L阿特拉津作用细胞72hr，核酸电泳显示草鱼细胞的DNA发生特异降解并呈现约180bp～200bp的电泳阶梯。而同样的结果出现在14.25mg／L的马拉硫磷作用细胞72hr和19 mg／L马拉硫磷作用细胞48hr后。进一步探讨马拉硫磷和阿特拉津的浓度及诱导时间与草鱼细胞凋亡之间的关系时发现，草鱼细胞凋亡的程度与药物作用的浓度、药物诱导的时间之间均存在着较好的相关性，即当药物浓度较高时，其诱导细胞凋亡的时间就较短，反之，当药物浓度较低时，诱导凋亡的时间就越长。浙江大学硕士学位论文 在论文的第三部分，我仃]初步探讨了马拉硫磷和阿特拉津诱导细胞凋亡的机制。经过检测两种环境激素胁迫诱导后细胞内的ca2+、线粒体膜电位（△甲）、细胞内ATP的变化，以及02、H202等ROs的动态变化，结果发现:当细胞经马拉硫磷或阿特拉津诱导后，细胞内c扩+迅速升高，在24小时内达到最高峰，而线粒体膜电位（△甲）和细胞内ATP含量都则出现了持续快速的下降，并且细胞内02一、H20:出现快速上升，在细胞作用到36小时后，细胞内02一、H20:上升到最高。提示ca2+、线粒体膜电位（△甲）、氧化应激共同参与了马拉硫磷和阿特拉津这两种环境激素诱导的细胞凋亡，同时也可推断，马拉硫磷和阿特拉津诱导的细胞凋亡中，氧化应激途径是启动其凋亡的可能途径之一。更多还原

【Abstract】 Environmental hormones (EHs), also called environmental endocrine disruption chemicals (EDCs), have been confirmed to be a group of newly environmental contaminants and EHs have raised increasing concern from every walk of life. It is considered that pollution of EHs has become one of three great global environmental problems the same as global climatological change and ozonosphere destruction. They can cause endocrine confusion of the healthy body. Atrazine (2-chloro-4-ethylamino-6-isopropoylamino-s-triazine) is a pre- and postemergence herbicide for the control of annual and perennial grass and annual broad-leaved weeds. Malathion is widely used in many countries to control mosquitos and rice pests. In present study, a grass carp(ctenopharyngodon idellus) cell culture ZC7901 was used as an in vitro model system to study the toxic effects of malathion and atrazine.In part I, we studied the cytotoxic effects of malathion and atrazine. The results in inverted microscope showed that the viability of cells stressed by malathion or atrazine have been affected clearly and the percentage of cell adherence have been decreased. The MTT cleavage assay indicated that malathion or atrazine can inhibite the cells growth and this inhibition are dose- and time-dependent. So we can conclude that high concentration of the residue of malathion or atrazine and long time accumulation of the residue are adverse to the growth of cells of grass carp. Moreover, the results described that the IC50 value in 12hr to 72hr of malathion and atrazine on ZC7901 cells were 34.935 to 3.04mg/L and 198.980-11.567mg/L, indicating that malathion is more toxic than atrazine.In part II, we found that malathion and atrazine can all result in apoptosis of fish cell ZC7901. The results through fluorescence microscope and confocal laser microscope and transmissional electronmicroscope showed that the culture chemically stressed by malathion or atrazine exhibited cell death accompanied by various morphological alterations such as nuclear condensation, chromatin abutted sharply against nuclear membrane and develop into distinct apoptotic-like bodies, which were charateristic features seen in apoptosis. By agarose gel electrophoresis, DNA ladderswas clearly visualized, furthermore, the study of malathion or atrazine treatment showed that there were better relationships between the cell apoptosis and concentration and time, namely that higher concentration malathion or atrazine treatment can result in apoptosis rapidly and contrarily lower concentration malathion or atrazine treatment can induce apoptosis tardily.In part III, the changed of intracellular Ca2+, O2-, H2O2, ATP and the mitochondria membrane potential in the apoptosis induced by malathion or atrazine were investigated. The results showed that the intracellular Ca2+ content increased in 24hr, so did O2, H2O2 in 36hr. then decreased. But mitochondria membrane potential and ATP content kept decreasing quickly. These data indicated that the intracellular Ca2+, O2-, H2O2, ATP and mitochondria membrane potential all played roles in apoptosis of malathion or atrazine inducing, maybe the oxidative stress is the way of apoptosis.更多还原