【作者】 刘慧刚；

【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】 三丁基锡(Tributyltin，TBT)是一种影响范围较广的有机锡污染物。TBT最初是作为聚氯乙烯(PVC)的稳定剂开发使用的，后来作为杀虫剂主要用作船舶油漆中的防污剂，用来防止微生物、植物、动物等附着生长于船体和海洋建筑及钻井平台等。TBT对海洋的污染及其对海洋生物的毒性研究已成为各国关注的热点问题。由于有机锡在海洋环境中的长期残留，即使在限制使用近十年后，海水和底泥中的有机锡含量仍无明显下降。我国沿海和内陆水域地区也广泛存在着TBT污染。TBT对海洋生物的污染，会通过食物链的作用，不可避免地对人体健康构成威胁。TBT对生物的毒性作用是多方面的，机理比较复杂，主要包括免疫毒性、生殖毒性和神经毒性等。国内外学者对TBT的危害性、作用机理以及污染治理问题进行了广泛深入的研究，但是如何采用有效、可行的方法来减少TBT对人类健康的危害方面的研究还不多见。茶多酚(Green Tea Polyphenols，简称GTPs)是一种从绿茶中提取的天然复合物，其组成除酚酸外，主要是类黄酮化合物，其中羟基取代基作为质子的供体，使其具有特殊的生理功能，这些功能与茶多酚清除自由基的抗氧化效果密切相关。茶多酚中最重要的成分是黄烷醇类的多种儿茶素(catechins)，儿茶素类主要由epicatechin(EC)、epicatechin gailate(ECG)、epigallocatechin gallate(EGCG)等几种单体组成。在GTPs中，EGCG是研究得最多的抗氧化剂。已经研究证实的茶多酚的主要生物学活性有：(1)作用于与自由基生成与清除相关的酶。(2)直接作用于自由基。(3)与诱导氧化的过渡金属离子络合。大蒜(Animasatik)为百合科植物，世界上将其鳞茎用于防病治病已有悠久的历史。大蒜素(Allicin)为大蒜鳞茎中提取的多硫化合物，研究发现大蒜素中含有的多种有机硫化合物和大蒜的保健、抗病作用有关，其主要作用机理是抗氧化和清除自由基。已有研究表明，大蒜素的抗氧化活性可有效地对抗脂质过氧化物对膜结构的损伤，可以拮抗化学物质导致的毒性。实验证明大蒜素不但能有效清除白由基、抑制过氧化物对细胞的伤害、避免DNA受损，而且具有抑制解毒系统Ⅰ相酶和诱导解毒系统Ⅱ相酶的作用。茶多酚和大蒜素都具有抗氧化、抑制脂质过氧化反应和清除自由基作用，被认为是天然的植物来源的抗氧化剂。本研究针对国内外对环境化学污染物对人体危害预防措施方面的研究尚欠缺的情况，从主动采取保护性措施的角度入手，着重研究是否可用人们日常接触到的食用植物来抵抗环境化学污染物对人体的危害。本研究体外实验与体内实验相结合，以TBT引起的氧化损伤、DNA损伤和细胞凋亡干扰三个方面为主要内容，研究天然植物有效成份茶多酚和大蒜素对TBT所导致这三种损伤的拮抗效果，同时对拮抗作用的机理进行初步探讨。主要结果：1、茶多酚和大蒜素能有效清除TBT诱导产生的ROS。本研究从体内和体外实验证实茶多酚和大蒜素均可以有效抑制TBT诱导的细胞ROS水平升高，并呈明显的剂量-效应关系，证明茶多酚和大蒜素能有效清除TBT诱导产生的ROS。2、茶多酚和大蒜素对TBT引起的小鼠肝脏MDA含量升高有明显的拮抗作用。小鼠肝脏MDA检测结果证明茶多酚和大蒜素对TBT引起的MDA含量升高有明显的抑制作用，证明茶多酚和大蒜素能有效防止脂质过氧化的发生。3、茶多酚和大蒜素对TBT引起的DNA损伤有明显保护作用。从小鼠外周血彗星实验结果分析，无论是尾长还是尾相，均提示茶多酚和大蒜素对TBT引起的DNA断裂有明显的保护作用。4、茶多酚和大蒜素对TBT引起的胸腺细胞损伤有明显保护作用。小鼠胸腺电镜观察结果证明茶多酚和大蒜素对TBT引起的细胞结构损伤有明显的保护作用，儿茶素(EGCG)对TBT引起的小鼠胸腺细胞损伤的保护作用与茶多酚的保护作用相似。5、茶多酚和大蒜素对TBT引起的FL细胞活性下降有明显拮抗作用。随着TBT浓度升高FL细胞活性明显下降，而FL细胞预先与一定浓度的茶多酚或大蒜素作用后再进行TBT染毒，其细胞活性与单纯TBT染毒细胞相比明显改善。6、EGCG和大蒜素对TBT引起的小鼠肝细胞凋亡率升高有明显拮抗作用。TUNEL实验可以看出，无论是高剂量保护组还是低剂量保护组，EGCG和大蒜素对TBT引起的小鼠肝细胞凋亡率升高都有明显的拮抗作用。7、EGCG和大蒜素对TBT诱导的凋亡相关蛋白Bax和Bcl-2水平改变有明显影响。本研究中各组Bcl-2和Bax水平的变化提示EGCG和大蒜素对TBT诱导的促凋亡蛋白(Bax)的升高、抑凋亡蛋白的降低有明显拮抗作用。8、EGCG对TBT引起的FL细胞骨架改变有明显的保护作用。正常FL细胞骨架F-actin结构清晰可见，纹理规则。而在TBT染毒后细胞骨架F-actin结构紊乱，纹理不清晰。在EGCG保护组，细胞骨架F-actin的改变明显好转，高剂量EGCG保护效果更加明显。主要结论：1、茶多酚和大蒜素对TBT引起的氧化损伤具有明显的保护作用。体内和体外实验结果都表明茶多酚和大蒜素均可以有效地拮抗TBT导致的细胞ROS升高，证明茶多酚和大蒜素能有效清除ROS；小鼠肝脏MDA检测结果证明茶多酚和大蒜素能阻止脂质过氧化的发生。2、茶多酚和大蒜素对TBT引起的DNA损伤和胸腺细胞损伤具有保护作用。小鼠外周血彗星实验结果提示，无论是从尾长还是尾相结果分析，茶多酚和大蒜素对TBT引起的小鼠外周血淋巴细胞的DNA断裂有明显的保护作用。电镜观察结果证明，茶多酚和大蒜素对TBT引起的胸腺细胞核和线粒体损伤都有明显的保护作用，儿茶素(EGCG)的保护作用与茶多酚的保护作用相似。综合茶多酚和大蒜素对TBT引起的氧化损伤和DNA损伤的保护结果，可以推测茶多酚和大蒜素对胸腺细胞的保护作用的机制可能是阻止了氧化损伤，从而保护了胸腺细胞。3、茶多酚及大蒜素能明显抑制TBT诱导的细胞凋亡。TUNEL实验表明EGCG和大蒜素能抑制TBT引起的细胞凋亡，Bcl-2和Bax的变化证明EGCG和大蒜素能使TBT引起的凋亡调节相关蛋白表达水平的改变还原到接近正常值，FL细胞骨架F-actin的改变也证明了EGCG对TBT引起的细胞凋亡具明显的拮抗作用。4、茶多酚和大蒜素对TBT引起的氧化损伤、DNA损伤及凋亡的拮抗作用有密切的内在关系。TBT引起的毒性效应是从氧化损伤开始，茶多酚和大蒜素对TBT引起的氧化损伤的保护作用可能是对TBT毒性拮抗作用的关键环节。由于TBT引起的氧化损伤、DNA损伤及凋亡有密切的内在关系，所以可以推测，茶多酚和大蒜素对TBT引起的氧化损伤、DNA损伤及凋亡的拮抗作用也可能是多位点、多层面、多阶段的。5、本研究提示，日常摄入一定量的茶多酚和大蒜素，有可能减少TBT污染的食品给人们带来的危害。茶多酚和大蒜素拮抗TBT毒性作用的确切机理，以及是否对其它化学污染物的毒性作用具有类似的解毒作用，需要进一步研究。更多还原

【Abstract】 The organotin compound tributyltin (TBT), has been extensively used in boat paints because of its excellent and long-lasting antifouling properties. Since the early 1980s, the harmful effects of the TBT on marine organisms have been determined. In some selected lakes, rivers and coastal environments from China, concentrations of TBT were higher than the acute and chronic toxicity threshold of fresh water and marine organisms. The toxic effects of TBT have been observed in invertebrates, micro- and macro-algae. It was found that exposure of TBT could promote both oxidative damage and DNA damage in mammals. TBT-related toxicity, especially immunotoxicity, reproductive toxicity and neurotoxicity have been well documented.Recently, restrictions on the application of TBT compounds as vessel antifouling agents have been introduced. However, TBT pollution in fish and shellfish, marine water and sediment are still being reported. The persistent presence of TBT in coastal ecosystems poses an ecotoxicological threat to humans through the food chain because TBT could be transported to the human body by contaminated seafood. Thepossible health hazard caused by human dietary exposure to TBT through ingesting contaminated fish and shellfish is a growing concern. Currently, there is no known effective strategy to eliminate TBT’s toxic effects from contaminated food.Green tea polyphenols (GTPs) have gained great interest due to their potent anti-oxidative activities combined without side effects in humans. A number of green tea polyphenolic compounds have been found to be good antioxidants against lipid peroxidation in biological systems, and they also possess anti-cancer properties. The main components of GTPs are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA), and EGCG is the most reported compound among these GTPs. The main biologic activities of GTPs include: (1) Affect enzyme relating with free radical generation and scavenging. (2) Act on free radical directely. (3) Combin with the metallic ion which inducing oxidation.Garlic (Allium sativum) is a widely consumed herb in foodstuffs and has been traditionally used in the treatment of fungal and bacterial infections. Numerous studies have shown that garlic exhibits diverse biological and pharmacological properties, including antitumorigenesis, antiatherosclerosis and detoxifying, and has also been shown to protect against chemical-induced toxicity in animals. The organosulfur compounds are believed to play key roles in these biological effects. Among the organosulfur compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are the three major constituents. These compounds vary in their efficacy depending on the number of sulphur atoms in their molecules. The detoxifying effects of organosulfur compounds were mostly thought to be related to not only their ability to prevent oxidative damage and DNA damage, but also their ability to inhibit phase I enzymes, induce phase II enzymes, or bind to exogenous toxins through sulfhydryl groups.Base on the conclusion of previous study that both green tea polyphenols (GTPs) and garlic oil (GO) are natural phytochemicals with powerful antioxidant capacity. This study was conducted to investigate the protective effect of GTPs and GO on toxicities induced by TBT in vivo as well as in vitro. The present study aimed to determine if GTPs and garlic oil, the two wildly consumed foodstuffs in the world, are able to reduce TBT-induced toxicities, and leads to find more convenient strategies to reduce the potential damage from chemical pollutants.Main results:1. GTPs and garlic oil can scavenge ROS induced by TBT effectively. The elevation of ROS production was inhibited by GTPs or garlic oil in mice liver and FL cell in a dose-depend manner in this study. It is indicated that GTPs or garlic oil can prevent ROS increasing both in vivo and in vitro.2. GTPs and garlic oil can prevent TBT-induced MDA contents increase. The mice liver MDA content increased significantly in the TBT treated group but was almost unchanged in the GTPs or garlic oil pre-treated groups compared to control.3.GTPs and garlic oil can prevent TBT induced DNA damage. Both tail length and tail moment data (comet assay) demonstrated that the number of cells with damaged DNA in un-protected mice was found to be significantly higher compared to GTPs or garlic oil protected mice.4. GTPs and garlic oil can prevent thymocytes from TBT-induced damage. Damage to the nuclei and mitochondria in un-protected group was apparent under electron microscope observation, while in GTPs, EGCG or garlic oil protected group, the damage of thymocytes induced by TBT observably alleviated.5. TBT-induced reduction of cell viability was prevented by GTPs and garlic oil in human FL cells. Mice in GTPs or garlic oil protected group exhibited similar cell viability to control. The cell viability decreased in a dose-dependent in TBT treated cells, and exhibited almost no change in GTPs or garlic oil protected cells.6.Increased apoptosis ratio induced by TBT was inhibited by GTPs or garlic oil in mice liver. As detected by TUNEL assay in mice liver, both high and low concentrations of GTPs or garlic oil exhibite obvious protective effects on the increasing of apoptosis ratio induce by TBT.7. EGCG and garlic oil can affect the change of apoptosis related protein (Bax and Bcl-2) level induced by TBT. The data indicated that both EGCG and garlic oil can affect the increasing of Bax and the decreasing of Bcl-2 protein level induced by TBT. The antagonizing effects of EGCG or garlic oil on TBT-induced apoptosis protein change were obvious.8.EGCG can protect FL cells cytoskeleton (F-actin) from TBT induced alteration. Data from FL cells cytoskeleton (F-actin) observation showed that EGCG could avoid TBT induced cells cytoskeleton (F-actin) change in a dose-depend manner. The variety of cytoskeleton (F-actin) induced by TBT was alleviated after FL cells pretreated with EGCG.Main conclusion:1.GTPs and garlic oil can prevent TBT-induced oxidative damage. Data of ROS level and MDA contents in this study indicated that ROS induced by TBT can be efficiently scavenged by GTPs and garlic oil, and TBT-induced lipid peroxidation in mice was also prevented. Therefore it can be concluded that GTPs and garlic oil can prevent oxidative damage induced by TBT.2. GTPs and garlic oil can prevent TBT-induced DNA damage and thymocytes damage. The number of cells with damaged DNA in the lymphocyte of peripheral blood of the un-protected group differed significantly from both GTPs and garlic oil protected group. The damage of nucleolus and mitochondria in thymus, the main target orgain of TBT, was prevented by GTPs, EGCG and garlic oil. It is confirmed that GTPs, EGCG and garlic oil can prevent TBT-induce DNA breakage and thymocytes damage. Taking together the data of GTPs and garlic oilpreventing oxidative damage and DNA damge, it could be speculated that GTPs and garlic oil prevent TBT-induced thymocytes damage by inhibiting oxidative damage.3. GTPs and garlic oil can antagonize TBT-induced apoptosis. Aapoptosis ratios (TUNEL assay) confirm the fact that EGCG and garlic oil can reduce TBT-induced apoptotic rate. Data from western-blot indicated that both EGCG and garlic oil can affect Bax and Bcl-2, the two apoptosis related protein level. And data of F-actin showed that EGCG could prevent TBT induced cytoskeleton (F-actin)alteration, which is related to apoptosis.4. The preventive effects of GTPs and garlic oil on TBT-induced oxidative damage, DNA damage, nucleus damage and apoptosis are close related. Data obtained from this study implied that TBT-induced toxicities initiated at oxidative damage, and the preventing of oxidative damage may be the key step due to the powerful ability of GTPs and garlic oil to scavenge ROS and prevent DNA breaks. So it could be speculated that the the preventive effects of GTPs and garlic oil on TBT-induced toxicities are compositive and extensive.5. Daily taking in green tea polyphenols or garlic oil can reduce the harm from TBT- contaminated sea food. This study suggested that GTPs and garlic oil can be applied as chemopreventive agents against TBT-induced toxicity. The exact mechanism of how green tea polyphenols and garlic oil reducing the toxicity of TBT, or if the protective effects also work in preventing the toxicities of other chemical pollutants needs further investigation.更多还原