黄绿青霉毒素对HepG2细胞的DNA损伤毒性及氧化应激机制的研究
发布时间：2018-11-19 18:10
【摘要】：前言：黄绿青霉毒素（citreoviridin, CIT）是主要由黄绿青霉菌（Penicilliumcitreoviridin）产生的具有毒性的次级代谢产物。CIT广泛存在于霉变作物中，例如霉变的谷物，大米，玉米等。研究发现黄绿青霉菌在大米中生长速度最快，产毒数量也最高。温度与环境中的PH值对黄绿青霉菌的生长及产毒有着密切影响。 CIT最早引起人们关注是在100多年前的日本。1891年，日本学者Sakaki发现用发霉的大米喂养实验动物会导致其出现中枢神经系统麻痹的症状。1920年，日本学者Miyake和Takada首次报道了来自霉变食物的青霉菌属（Penicilliumcommune）引起实验动物兔和老鼠的病变。随后Miyake等人从日本本土大米和台湾大米中提取出了一种真菌并命名为黄绿青霉菌（P. citreonigrum）。该菌株可产生一种毒性很强的代谢产物即黄绿青霉毒素。 CIT可通过干扰受试老鼠脑部的糖代谢进而导致中枢神经系统功能障碍。研究认为CIT的毒性效应源于其导致的呼吸系统及心血管系统的损伤，如呼吸暂停、脑电图异常、窦性心律失常、低血压等，从而出现由于主动脉组织缺氧而导致的中枢神经系统症状。CIT可致大鼠心肌原发性坏死，心肌细胞呈颗粒状变性，心肌纤维凝集，溶解。CIT可结合并抑制线粒体中的三磷酸腺苷合成酶。研究认为CIT与“黄变米中毒”，“心源性脚气病”以及克山病有着密切关系。 CIT可致心肌细胞，人胃癌细胞(SGC-790)DNA损伤。但具体机制尚不清楚。本研究选用HepG2细胞，探讨CIT的DNA损伤毒性及其可能机制。HepG2细胞来源于人类肝胚细胞瘤，分化程度较高，不仅保留了人类正常肝实质细胞的许多功能和特点，还保留了较完整的生物转化代谢Ⅰ相酶和Ⅱ相酶。被认为是用来检测外来化合物DNA损伤毒性的理想细胞系。CIT为亲脂性毒素，吸收后在肝脏中浓度最高。这也就是我们采用HepG2细胞作为该试验系统的原因之一。 本研究选用HepG2细胞，探讨CIT的DNA损伤毒性及其可能机制，旨在为评估CIT对人类健康的潜在危害提供实验室依据。 方法：本研究选用HepG2细胞作为试验系统。通过单细胞凝胶电泳（SCGE）试验检测细胞DNA损伤情况，评价CIT的DNA损伤毒性。为探讨其可能的DNA损伤毒性机制，以2＇，7＇—二氢二氯荧光素(DCFH)法测定细胞内ROS水平，以苯二醛（OPT）测定细胞内还原型谷胱甘肽（GSH）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，用吖啶橙（Acridine orange）和罗丹明123（Rhodamine123）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。实验结果用SPSS v11.5统计软件包进行统计分析。 结果：2.5-5μM的CIT作用于HepG2细胞1h后，引起细胞DNA链断裂，细胞形成彗星样拖尾，其尾DNA%明显大于未用CIT处理的细胞。2.5-5μM的CIT染毒60min引起细胞内ROS水平明显增加，GSH水平显著降低，与对照组相比差异具有统计学意义（P 0.01）；10mM的N-乙酰半胱氨酸(NAC)预处理60min后可降低CIT引起的HepG2细胞内ROS水平增加，对CIT所致HepG2细胞DNA损伤具有保护作用。 1.25-5μM CIT作用于HepG2细胞3h后，可致8-OHdG的表达升高；1.25-5μM的CIT作用于HepG2细胞60min后，可引起细胞内溶酶体膜稳定性明显改变；5μM的CIT作用60min引起细胞内线粒体膜电位的改变，，与对照组比较，其差异有统计学意义（P＜0.05）。 结论：CIT可引起HepG2细胞DNA损伤，提示CIT对HepG2细胞具有DNA损伤毒性。CIT可诱发细胞内ROS水平增高，GSH水平降低，提示CIT对HepG2细胞产生的DNA损伤毒性可能与氧化应激机制有关。N-乙酰半胱氨酸(NAC)预处理60min后可降低CIT引起的HepG2细胞内ROS水平增加，对CIT所致HepG2细胞DNA损伤具有保护作用。CIT作用于细胞后导致氧化性DNA损伤的标志性产物8-OHdG形成增强，进一步表明CIT引起HepG2细胞的DNA损伤为氧化性损伤。此外，CIT对HepG2细胞溶酶体膜稳定性有显著影响，并使得HepG2细胞线粒体膜电位显著降低。
[Abstract]:Citroviridin (CIT) is a toxic secondary metabolite, which is mainly produced by Penicillium citriviridin. CIT is widely used in mildewed crops, such as mildewed grains, rice, corn, etc. The results showed that the growth rate of the fungus in the rice was the fastest in the rice, and the number of the production was the highest. The pH value in the temperature and the environment has a close effect on the growth and the production of the yellow-green-green mould. CIT first raised concerns over the 100-year-old Japan. In 1891, Japanese scholar Sakaki found that feeding experimental animals with musty rice could lead to the symptoms of central nervous system paralysis. In 0, the Japanese scholar, Miyake and Takada, reported for the first time that the Penicillium communis from mildewed food caused experimental animal rabbits and mice The lesion. Then Miyake et al. extracted a fungus from Japanese native rice and Taiwan rice and named P.citregoniru. m) the strain can produce a metabolite with strong toxicity, namely, the yellow-green-green mildew The toxin. CIT can result in the central nervous system by interfering with sugar metabolism in the brain of the tested mouse The study found that the toxic effect of CIT is due to the damage of the respiratory system and the cardiovascular system, such as apnea, abnormal electroencephalogram, ventricular arrhythmia, hypotension, and so on, so that the central God caused by the hypoxia of the aorta Through systemic symptoms, CIT can induce primary necrosis of the myocardium of the rat, and the cardiac muscle cells are in the form of granular degeneration, cardiac muscle fiber, Agglutination and dissolution. CIT can bind and inhibit the triphosphoric acid in the mitochondria Adenoid synthase. It is considered that CIT is associated with 鈥測ellow rice poisoning鈥
本文编号：2343033
[Abstract]:Citroviridin (CIT) is a toxic secondary metabolite, which is mainly produced by Penicillium citriviridin. CIT is widely used in mildewed crops, such as mildewed grains, rice, corn, etc. The results showed that the growth rate of the fungus in the rice was the fastest in the rice, and the number of the production was the highest. The pH value in the temperature and the environment has a close effect on the growth and the production of the yellow-green-green mould. CIT first raised concerns over the 100-year-old Japan. In 1891, Japanese scholar Sakaki found that feeding experimental animals with musty rice could lead to the symptoms of central nervous system paralysis. In 0, the Japanese scholar, Miyake and Takada, reported for the first time that the Penicillium communis from mildewed food caused experimental animal rabbits and mice The lesion. Then Miyake et al. extracted a fungus from Japanese native rice and Taiwan rice and named P.citregoniru. m) the strain can produce a metabolite with strong toxicity, namely, the yellow-green-green mildew The toxin. CIT can result in the central nervous system by interfering with sugar metabolism in the brain of the tested mouse The study found that the toxic effect of CIT is due to the damage of the respiratory system and the cardiovascular system, such as apnea, abnormal electroencephalogram, ventricular arrhythmia, hypotension, and so on, so that the central God caused by the hypoxia of the aorta Through systemic symptoms, CIT can induce primary necrosis of the myocardium of the rat, and the cardiac muscle cells are in the form of granular degeneration, cardiac muscle fiber, Agglutination and dissolution. CIT can bind and inhibit the triphosphoric acid in the mitochondria Adenoid synthase. It is considered that CIT is associated with 鈥測ellow rice poisoning鈥
本文编号：2343033
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