【作者】 王晓峰；

【导师】 徐立红；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2007， 博士

【摘要】 铬是自然界中广泛存在的一种元素，主要以三价铬（Cr（Ⅲ））和六价铬（Cr（Ⅵ））的形式存在。受环境中物理、化学和生物等因素的影响，Cr（Ⅲ）与Cr（Ⅵ）之间可以相互转化，但两者对人类健康有着截然不同的影响。Cr（Ⅲ）是一种人体必需的微量元素，它是葡萄糖耐量因子的组成部分，对调节糖代谢、维持体内正常的耐量起重要作用。而Cr（Ⅵ）则被列为对人体危害最大的化学物质之一，也是国际公认的重金属致癌物。随着铬化合物在工业上的广泛使用，这些生产工业排放的废水、废气和废渣造成的铬污染问题已受到全世界的普遍关注。散布在环境中的Cr（Ⅵ）以离子状态随水循环，它不能被微生物分解，但可被动植物吸收向人体转移，并沿食物链出现生物放大效应，进而影响整个生态系统的平衡，危害人类健康。Cr（Ⅵ）可通过皮肤接触、呼吸吸入和经口摄入三种方式进入人体。其中，皮肤接触和呼吸吸入是职业人群接触Cr（Ⅵ）的主要途径。研究证实，长期暴露于Cr（Ⅵ）环境中的作业工人易患过敏性皮炎和湿疹，还能出现鼻粘膜穿孔或溃疡，慢性鼻炎和支气管炎，甚至导致肺气肿，从而影响肺功能。呼吸吸入的Cr（Ⅵ）对人体造成的最严重的危害在于增加了罹患呼吸道肿瘤的危险性。经口摄入包括饮水和摄入食物，是非职业人群接触Cr（Ⅵ）的最主要方式，国内外学者先后报道了多起因食入Cr（Ⅵ）污染的食物影响人群健康的事件。大量体外实验证实Cr（Ⅵ）对多种细胞均具有毒性效应，包括肝细胞、肾细胞、成纤维细胞以及淋巴细胞等。此外，皮下注射和腹腔内注射Cr（Ⅵ）的动物实验研究也证实Cr（Ⅵ）能引起机体多个脏器的损伤，其中以肝脏和肾脏的损伤较为明显。但是，由于Cr（Ⅵ）特殊的致毒机制，以皮下注射和腹腔内注射方式建立的动物模型实验并不能正确反映经口摄入的Cr（Ⅵ）对机体产生的毒性效应。而在为数不多的以灌胃或自由饮水方式摄入Cr（Ⅵ）的动物实验中，对Cr（Ⅵ）引起机体毒性效应的研究至今未有定论。为进一步阐明经口摄入的Cr（Ⅵ）在体内的毒性效应，有必要从分子和细胞水平上研究其在体内可能的致毒机理。综合对Cr（Ⅵ）的一系列体外和体内的研究可以得出，Cr（Ⅵ）引起毒性的最为重要的一个方面就是大量活性活性氧（ROS）的生成并由此引起的氧化应激。近年来，大量研究表明，ROS与细胞凋亡存在着十分密切的关系，它可通过多个信号途径干扰正常的细胞凋亡。因此，通过研究Cr（Ⅵ）对细胞凋亡的诱导有可能阐明Cr（Ⅵ）在体内的致毒机理。本研究对受试动物以灌胃方式给予Cr（Ⅵ），通过比较其对不同组织器官造成的损伤，进一步研究经口摄入的Cr（Ⅵ）在体内引起的毒性效应；同时，通过研究Cr（Ⅵ）对细胞凋亡的诱导，以凋亡重要相关蛋白的改变为研究中心探讨其在体内的毒作用机理。按实验设计，将健康雄性ICR小鼠随机分为4组，每组均为10只；对照组用蒸馏水灌胃，处理组以K₂Cr₂O₇溶液灌胃，剂量设计分别为25、50、100mg/kg体重，灌胃量以小鼠体重计为0.1mL/10g体重。每天灌胃1次，分别给予灌胃1d或5d。染毒结束后继续饲养24h，处死前对小鼠进行摘眼球取血，并立即分离血淋巴细胞进行彗星实验。处死小鼠后迅速切取部分新鲜肝脏和肾脏组织，用于电镜实验和TUNEL实验；剩余组织用液氮速冻后用Western blot进行p53、Bcl-2、Bax、细胞色素c蛋白水平测定以及caspase-3酶活化水平测定，并进行ROS水平、脂质过氧化产物丙二醛（MDA）含量以及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性的检测。实验结果如下：1．K₂Cr₂O₇染毒小鼠外周血淋巴细胞彗星实验结果显示，1d或5d染毒后随剂量的增加各染毒组尾长也随之升高，并具有显著性差异；除5d染毒的最低剂量组外，其余两个时间段的各染毒组尾相均显著高于对照组；但5d染毒与1d染毒的尾长及尾相数值相比，升高趋势有所降低。2．K₂Cr₂O₇染毒1d或5d后小鼠肝脏ROS水平均高于对照组，并且，除1d染毒的最低剂量组外，其余各剂量组与对照组比均具有显著性差异。3．小鼠经K₂Cr₂O₇染毒1d后最低剂量组肝脏SOD活性稍高于对照组，但并无显著性差异，其余两个剂量组SOD活性与对照组相比均无明显变化；5d染毒后各剂量组的SOD活性均显著低于对照组。4．K₂Cr₂O₇染毒小鼠肝脏CAT活性除1d和5d染毒的最低剂量组与对照组无明显差异外其余各组均低于对照组且具有显著性差异。5．小鼠经K₂Cr₂O₇染毒1d或5d后，随剂量的升高肝脏MDA含量有上升趋势，但与对照组相比未见统计学差异。6．经K₂Cr₂O₇染毒1d或5d后，各剂量组小鼠肾脏ROS、MDA、SOD和CAT与对照组相比均没有显著性差异。7．透射电镜显示，与对照相比，经K₂Cr₂O₇染毒1d或5d后小鼠肝脏细胞超微结构均出现凋亡特征性改变：细胞核皱缩，染色质浓缩并发生边聚化；线粒体变的致密，嵴消失，并且线粒体发生空泡化改变。8．本研究用TUNEL实验检测K₂Cr₂O₇染毒后小鼠肝脏细胞DNA降解情况，结果所示：与对照组相比，K₂Cr₂O₇染毒1d后小鼠肝细胞核呈棕色的细胞即凋亡细胞数明显增多；5d染毒小鼠肝细胞内也发生相似改变。在1d和5d的两个染毒时间段，随剂量的升高调亡率也随之上升，并显著高于对照组，呈现出明显的剂量反应关系。9．小鼠经0、25、50、100mg/kg K₂Cr₂O₇染毒1d或5d后，肝组织细胞内caspase-3酶原裂解产生的caspase-3活性片段（17kD）蛋白条带逐渐变粗。与对照组相比，随染毒剂量的增加，caspase-3酶活化逐渐增加，并在最高剂量组100mg/kg差异具有显著性。10．小鼠染毒后肝组织细胞内p53蛋白水平情况显示，与对照组相比，1d或5d染毒的各剂量组小鼠肝组织细胞内p53蛋白水平均有升高，并在最高剂量组100mg/kg时差异具有显著性。11．经K₂Cr₂O₇染毒1d或5d后小鼠肝组织细胞内Bcl-2蛋白水平随染毒剂量的增加均呈下降趋势，并且，1d染毒的100mg/kg以及5d染毒的50mg/kg和100mg/kg剂量组均显著低于对照组。Bax蛋白水平随染毒剂量的增加均有升高，但仅在5d染毒的100mg/kg剂量组蛋白水平显著高于对照组。12．1d或5d染毒小鼠肝组织细胞胞浆内细胞色素c蛋白水平随染毒剂量的增加均呈升高趋势，并且，1d染毒的50mg/kg和100mg/kg剂量组以及5d染毒的100mg/kg剂量组均显著高于对照组。主要结论：1．Cr（Ⅵ）灌胃引起小鼠肝脏ROS的显著升高，同时抗氧化酶SOD和CAT活性也明显降低，表明经口摄入的Cr（Ⅵ）破坏体内抗氧化系统的平衡并使机体处于氧化应激状态是Cr（Ⅵ）引起毒性效应的一个重要原因。2．与肝脏相比，本研究未发现Cr（Ⅵ）灌胃后小鼠肾脏出现明显的氧化应激，表明经口摄入的Cr（Ⅵ）经过体内氧化还原代谢后对机体各个组织器官造成的毒性效应会有较大差异。3．Cr（Ⅵ）染毒后，小鼠淋巴细胞DNA出现损伤，表明经口摄入的Cr（Ⅵ）在体内产生的ROS和/或还原过程中产生的低价态Cr所引起的DNA损伤是Cr（Ⅵ）的毒性作用之一。4．Cr（Ⅵ）染毒导致肝细胞发生凋亡，同时肝脏组织中凋亡相关蛋白p53、Bcl-2、Bax、细胞色素c蛋白水平以及caspase-3酶活化水平也发生明显改变，表明p53、Bcl-2、Bax、细胞色素c以及caspase-3参与了Cr（Ⅵ）诱导的细胞凋亡。该结果提示：Cr（Ⅵ）引起机体毒性效应的一个重要原因很有可能就是干扰了机体正常的细胞凋亡过程。更多还原

【Abstract】 Chromium is an ubiquitous element in nature that exists in primarily two valences states, trivalent （Cr（III）） and hexavalent （Cr（VI））. The two valence states can be transformed mutually under the influence of physical, chemical and biological factors in the environment. However, the effects of the two states on human health are quite different. Cr（III） is an essential micronutrient for human. It is a part of the glucose tolerance factor, which plays an important role in regulating glucose metabolism for the maintenance of normal glucose levels. In contrast, Cr（VI） is regarded as one of the most toxic chemical substances, as well as an international recognized heavy metal carcinogen.With wide use of the chromium compounds in industries, the waste water, exhaust gas and waste residue emitted from these industries resulted in severe chromium pollution. Great attention has been payed to this increasing severe pollution by the world. Cr（VI） scattered in the environment cycles with the water in ion state. It can not be decomposed by microorganism, but can be absorbed by animals and plantsand then affect the balance of the whole ecosystem. Cr（VI） is toxic to humans due to the effect of biological amplification of Cr（VI） along food chain. Cr（VI） may enter into the human body via dermal contact, inhalation and ingestion. Dermal contact and inhalation are the two major routes of occupational Cr（VI） exposure. Studies have demonstrated that the workers exposed to Cr（VI） chronically were vulnerable to dermatitis and eczema, as well as perforation, ulcer, chronic rhinitis and bronchitis. Cr（VI） may also result in emphysema, and consequently affect lung function. The most toxic effect on humans via inhalation is that it increases the risk of suffering from respiratory tract cancer. Water and food intake is a common route of Cr（VI） exposure for nonoccupational people. Several cases about which ingestion of food contaminated by Cr（VI） caused adverse effects on humans have been reported by researchers at home and abroad.A plenty of in vitro experiments have demonstrated that Cr（VI） could induce toxic effects on many types of cells including hepatocytes, renal cells, fibroblasts and lymphocytes. Animal experiments also confirmed that Cr（VI） exposed by subcutaneous or intraperitoneal injection could cause several organs damage, especially for liver and kidney. However, due to the special toxic mechanisms of Cr（VI）, animal experiments established by subcutaneous or intraperitoneal injection could not properly reflected the toxicity induced by oral administration of Cr（VI） in vivo. Up to now, only a few animal experiments administrated with Cr（VI） by gavage or drinking at liberty have been reported, moreover, conclusions on toxicity induced by oral administration of Cr（VI） remains uncertain.To illustrate the toxicity of Cr（VI） exposed by oral administration in vivo, it is necessary to study the potential toxic mechanisms at molecular and cellular level. Overview on a series of in vitro and in vivo researches, it can be concluded that an important aspect of Cr（VI） toxicity is the formation of plentiful reactive oxygenspecies （ROS） and the consequent oxidative stress. Recently, a large number of studies have showed a close relationship between ROS and apoptosis because ROS could disturb normal apoptosis through many cell signaling pathways. Therefore, investigating the apoptosis induced by Cr（VI） may contribute to illustrating the toxic mechanisms of Cr（VI） in vivo.The present study was designed to give Cr（VI） to the experimental animals via oral administration （gavage）. It was planed to study the toxic effects of Cr（VI） in vivo by comparing the damage in different organs. Furthermore, evaluating the changes of apoptotic related proteins in the process of cell apoptosis was also planed to investigate the toxic mechanisms of Cr（VI） in vivo.According to the experimental design, the animals were randomly distributed into four groups and were orally administrated （gavage） with potassium dichromate （K₂Cr₂O₇） at a dose of 0, 25, 50, 100 mg/kg body weight once daily for 1 day or consecutive 5 days. The mice were continued breeding for 24 h after the last Cr（VI） treatment. The blood was collected by extirpating the eyeballs, and the blood lymphocytes were separated out for comet assay immediately. The tissues of liver and kidney were quickly dissected off after the mice were sacrificed. One part of the tissues were used for TUNEL assay and transmission electron microscope, the remains were frozen in liquid nitrogen for p53, Bcl-2, Bax, cytochrome c protein level and caspase-3 activation determined by Western blot, and also for ROS level, malondialdehyde （MDA） content, superoxide dismutase （SOD） activity and catalase （CAT） activity evaluation.Main results:1. The comet assay of peripheral blood lymphocytes showed that the tail length in treatment groups significantly increased in a dose-dependent manner after 1 day or 5days of K₂Cr₂O₇ exposure. Except for the lowest dose group at 1 day of exposure, the tail moment at the other treatment groups were obviously higher than that of the control groups. However, comparing with 1 day of exposure, the increasing tendency of the value on the tail length and the tail moment declined after the mice exposed to K₂Cr₂O₇ for 5 days.2. After the mice were exposed to K₂Cr₂O₇ for 1 day or 5 days, ROS levels in mice liver in treatment groups were all higher than those in control groups. Except for the lowest dose group at 1 day of exposure, there was a significant difference between the control group and the other treatment groups.3. SOD activity in mice liver was not modified by the administration of K₂Cr₂O₇ at 50 and 100 mg/kg dose for 1 day. However, the value showed a non-significant increase at 25 mg/kg dose compared with the controls. SOD activity declined significantly at the three doses after 5 days of exposure as compared to the controls.4. CAT activity in mice liver showed a dose-dependent decrease in all K₂Cr₂O₇ treated groups and the difference was significant at 50 and 100 mg/kg doses as compared to the controls.5. The MDA content in mice liver increased in a dose-dependent manner after administration of K₂Cr₂O₇ to mice for 1 day or 5 days, but no statistical differences were observed between the control and treatment groups.6. No significant changes were observed on ROS, MDA, SOD and CAT in kidney after administration of K₂Cr₂O₇ to mice between the control and treatment groups.7. Transmission electron microscopy revealed the ultrastructural alterations of hepatocytes undergoing apoptosis in mouse liver treated with K₂Cr₂O₇ for 1 and 5 days. The typical apoptosis alterations were described as follows: the nucleus of mouse hepatocytes become shrunken. The chromatin was condensed to form dense compact masses and arranged along the crinkly nuclear membrane. In addition, themitochondria became a compact and vacuolar structure with loss of cristae.8. TUNEL assay was used to evaluate DNA degradation in liver after the mice were treated with K₂Cr₂O₇ . The number of cells stained with brown significantly increased in K₂Cr₂O₇ -treated mice compared with the control animals after 1 day of exposure. Such phenomenon also occurred in liver cells after administration of K₂Cr₂O₇ for 5 days. Dose- and time-dependent increase was observed after 1 and 5 days of K₂Cr₂O₇ exposure to mice and the increase was significant between the control and treatment groups.9. After the mice were exposed to K₂Cr₂O₇ at 0, 25, 50, 100 mg/kg doses for 1 day or 5 days, it could be seen that the protein band of caspase-3 active segment （17 kD） hydrolyzed from caspase-3 zymogen in liver cells gradually became thick. Caspase-3 activation enhanced in accordance with the increase of K₂Cr₂O₇ , and the difference compared with the control animals was significant at the dose of 100 mg/kg.10. p53 protein level in liver showed that the administration of K₂Cr₂O₇ to mice for 1 or 5 days enhanced the p53 protein level, and the difference compared with the control animals was significant at the dose of 100 mg/kg.11. Bcl-2 protein level in liver showed a declining tendency with the increase of K₂Cr₂O₇ at 1 or 5 days. Furthermore, the Bcl-2 protein level at 100 mg/kg dose for 1 day as well as at 50 mg/kg and 100 mg/kg doses for 5 days decreased significantly compared with the control groups. Bax protein level increased in a dose-dependent manner, and the difference was significant only at 100 mg/kg dose for 5 days.12. Cytochrome c protein level in hepatocytes cytoplasm enhanced in accordance with the increase of K₂Cr₂O₇ after 1 day or 5 days of exposure, and the values at 50 mg/kg and 100 mg/kg doses for 1 day as well as at 100 mg/kg dose for 5 days showed a statistical significance compared with the controls.Main conclusions:1. ROS level increased and SOD and CAT activity decreased in mice liver after the animals were exposed with oral administration of Cr（VI）. The results indicated that one important reason for the toxicity induced by Cr（VI） is that Cr（VI） exposed by oral administration could destroy the balance of antioxidative system and cause the following oxidative stress in vivo.2. Compared with liver, no significant oxidative stress was observed in kidney after oral administration of Cr（VI） to mice, indicating that the toxic effects on different organs may be different after Cr（VI） underwent redox metabolism in vivo.3. Oral administration of Cr（VI） induced DNA damage in mice lymphocytes, indicating that the DNA damage induced by ROS and/or Cr intermediates which formed in the Cr（VI） metabolism was one of the Cr（VI） toxic effects.4. Cr（VI） could induce hepatocytes apoptosis and alter the p53, Bcle-2, Bax, cytochrome c protein levels and caspase-3 activation, indicating p53, Bcle-2, Bax, cytochrome c and caspase-3 take part in the cell apoptosis induced by Cr（VI）. These results suggests that an important mechanism on the Cr（VI） toxicity is to disturbe the normal process of cell apoptosis.更多还原