【作者】 熊丽林；

【导师】 唐萌；

【作者基本信息】 东南大学 ， 卫生毒理学， 2006， 硕士

【摘要】 微囊藻毒素-LR(Microcystin-LR, MCLR)是有毒蓝藻细胞合成的二级代谢产物,它是一种胞内毒素,只有在细胞死亡分解时才被释放进入水中。近年来,由于工农业的发展和人口的增长,大量富含氮磷的污水被排放进入河流湖泊,水体的富营养化使蓝藻水华频繁暴发。大量的蓝藻繁殖不仅使水质恶化,鱼类因缺氧而死亡,而且藻细胞腐败后释放出的微囊藻毒素也给人类的健康和其它生物的生长带来很大危害。饮用含毒素的水或食用被毒素污染的食物是人群MCLR的主要暴露途径。MCLR具有明显的肝毒性,它不仅会引起动物和人类的肝脏功能损害,长期低剂量的摄入MCLR还与肝癌等消化道肿瘤的高发有关。MCLR是一种促癌剂,它可以引起细胞内活性氧的积聚,通过诱导脂质过氧化反应损伤细胞,但是目前对MCLR诱导的氧化应激后果,如膜毒性、对蛋白质的氧化损伤及对DNA的氧化损伤等方面的研究仍不够。此外,虽然大多数研究认为MCLR是促癌剂,没有遗传毒性,但是仍有一些学者认为它可以引起受试对象遗传物质的损伤。为了进一步加深对微囊藻毒素毒性和毒作用机制的认识,本课题以MCLR为代表对培养细胞进行体外染毒,主要探讨MCLR和黄曲霉毒素B1、伏马菌素B1的联合毒作用,MCLR和四氯化碳联合染毒对DNA断裂的影响,以及MCLR单独作用引起的细胞毒性、氧化应激、膜毒性和DNA损伤,为微囊藻毒素毒作用的评价提供一些依据。上述的试验结果表明:1、在本试验条件下,MCLR可以引起体外培养Hep G2细胞的氧化应激反应,细胞产生羟自由基的能力变强,脂质过氧化产物MDA含量增高,细胞膜流动性下降,说明其对生物膜有一定的损伤作用。但是MCLR在本试验剂量范围内,没有引起细胞膜通透性和Na+,K+-ATP酶活性的改变,这可能是因为不同试验对象对MCLR的敏感性不同所致。此外,MCLR引起的DNA损伤方面的体外试验研究提示,MCLR的作用诱发了DNA的断裂,但是不能引起小牛胸腺DNA和Hep G2细胞DNA-DNA交联。2、由MCLR与其它有害物质的联合毒性作用研究结果来看, MCLR和黄曲霉毒素B1、伏马菌素B1的联合毒作用类型为相加作用,这三种毒素同时存在,对Hep G2细胞的细胞毒性增强;另外,MCLR和氯化消毒副产物四氯化碳共同染毒致Hep G2细胞DNA损伤的析因分析试验结果提示两毒素单独作用均可以诱导DNA断裂,但两者间没有交互影响,联合毒作用类型为相加作用。综上所述,我们可以初步认为:MCLR可以干扰细胞中自由基的正常代谢,通过诱导脂质过氧化反应、膜流动性改变和DNA断裂等途径造成细胞的损伤。MCLR是一个促癌剂,一般将它列为非遗传毒性致癌物一类,但它却能引起一定程度的DNA损伤,至于它是否有可能为遗传毒性致癌物,还有待进一步研究。此外,在日常生活中,人们通过饮食途径同时摄入MCLR、黄曲霉毒素B1和伏马菌素B1,或者饮用含有氯化消毒副产物和MCLR的水将加大对人类健康的威胁。更多还原

【Abstract】 Microcystin-LR is a secondary metabolize production of poisonous cyanobacteria. It is a kind of toxin in cells which only can be released into water with the death and decomposing of these cells. In the recent, with the increasing of human population and the consequent intensification of agricultural and industrial activities, a great deal of sewage riched in nitrogen and phosphorus streams into freshwater bodies. The enhancement of eutrophication leads to the frequent formation of surface planktonic cyanobacteria. The occurrence of blue-green algae blooms contributes to deterioration of water which cause the death of fish. And the biotoxins released by decayed cyanobacteria do great harm to the health of human beings and other creatures.People contact these toxins mainly by drinking water or eating foods contaminated by them. MCLR is liver specific toxins, which can lead to the damage of liver. The development of enteron cancer, such as primary liver cancer, has been linked to long-term chronic exposure to MCLR. MCLR, being a tumour promoter, may induce the overproduction of free radicals. Oxidative stress can lead to severe adverse effects on cells by causing lipid peroxidation. But we still need more research on injury caused by oxidative stress, such as membranous toxicity, protein and DNA damage. On the other hand, according to most researches MCLR is a tumour promotor, but not a carcinogen with genotoxicity. However, there are still some scholars who believe that MCLR results in the damage of genetic material. To strengthen the perception of people on the toxicity and toxicological mechanism of Microcystin, We treat human hepatoma cells with MCLR in vitro. In the article, we mainly explore the combinative toxicity of MCLR, Aflatoxin B1 and Fumonisin B1, the combinative action of MCLR and Carbon Tetrachloride on DNA Damage. Moreover, we also investigate cytotoxicity, oxidative stress, membrane toxicity and DNA damage. We can conclude that:1, MCLR may enhance oxidative stress in Hep G2 cells. The excessive production of reactive oxygen species(ROS) and the lipid peroxidation products, malondialdehyde(MDA), was significantly increased in microcystin-treated cells. And after exposure to MCLR, membrane fluidity of these cells decreases. MCLR can lead to the injury of biomembrane. But in the experimental dosage scope, MCLR fails to influence membrane permeability and Na+,K+-ATPase activity. This possibly is because different cells have different sensitivity to MCLR. In addition, according to the results of MCLR-induced DNA damage in vitro, MCLR can result in DNA strand break, but can not cause DNA Cross-links of the Calf Thymus DNA and Hep G2 Cells DNA.2, According to the research on combinative toxicity of MCLR and other hazardous substance, combinative toxicity of MCLR, Aflatoxin B1 and Fumonisin B1 falls into the category of additive effect. So after treatment by the three biotoxins, cytotoxicity of human hepatoma cells is enhanced. Moreover, we explore the effect of DNA Damage of MCLR and Carbon Tetrachloride, which is the by-product of chlorination disinfection with factorial analysis. According to the result, we can see that each of the two更多还原