【作者】 王婧；

【导师】 刘汝涛；

【作者基本信息】 山东大学 ， 环境科学， 2016， 博士

【摘要】 镉是一种典型的重金属污染物,主要通过化石燃料燃烧、磷肥、自然来源、钢铁铸造、生活垃圾焚烧、水泥及有色金属生产和其他工业生产等途径进入环境。随着现代工业的快速发展,环境中镉污染的问题已广泛存在于全球的大部分区域,且人体接触镉暴露的机会呈逐年上升的趋势。由于镉在环境中的普遍存在和其潜在的毒性效应,目前镉污染带来的环境问题和对人体健康的毒性效应引起了广泛的关注和研究。镉是生物体内的一种非必需元素,不可以被机体降解。对于非职业暴露人群,镉主要通过被污染的食物和吸烟进入其体内。镉进入人体后,经血液循环系统分布到全身各器官,引发骨、肝、肾和生殖系统等多系统性、多器官性的机体损伤。研究表明重金属在机体内诱发的氧化应激与多种疾病相关。镉可以在体内和体外通过扰乱氧化还原平衡进而诱发氧化应激。镉诱发活性氧物质(ROS)的大量积累会造成机体内的一系列非特异性损伤,导致细胞死亡乃至组织损伤,被认为是疾病的一般机制。目前,镉暴露诱发的氧化应激相关毒性效应与作用机理尚未完全阐明,相关研究的方法学还需要进一步建立和完善。取决于实验条件和研究对象的不同,镉诱发的氧化应激相关毒性效应与作用机理仍存在争议。针对以上问题,本研究从动物、细胞和分子水平上研究了镉诱发氧化应激相关毒性效应与机理,主要包括以下六个部分：第一章简要介绍了环境中镉污染的特点和现状以及镉毒性效应的研究进展,概述了氧化应激、氧化损伤及其调控和作用机制,归纳了镉暴露诱发机体的氧化应激相关毒性效应与机理。通过文献综述,总结了本领域的研究进展和目前存在的尚待解决的科学问题,并针对这些问题设计了实验方案。第二章以斑马鱼作为实验对象,从实验动物层面上通过检测了镉诱发斑马鱼肝脏内氧化应激过程中生物标记物的变化,包括抗氧化酶过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)、谷胱甘肽还原酶(GR)和谷胱甘肽硫转移酶(GST)的活力变化,还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)以及脂质过氧化产物丙二醛(MDA)的含量变化,研究了镉染毒诱发斑马鱼肝脏内的氧化应激相关毒性效应与机理。研究结果表明镉暴露导致GPx酶活力上升,GR和GST酶活力下降；直接抗氧化酶CAT酶活力下降,而SOD酶活力上升；GSH含量下降而GSSG含量上升,GSH/GSSG比率下降；以及MDA含量升高。这些研究结果表明镉暴露诱发斑马鱼肝脏内产生了大量的ROS,打破了氧化还原平衡态,诱发了氧化应激,对斑马鱼肝脏造成了氧化损伤。第三章以小鼠原代肝细胞为研究对象,经体外染毒,从细胞水平研究镉在小鼠原代肝细胞内诱发的氧化应激相关毒性效应与机理。细胞经6或24小时镉暴露后,原位测定了细胞内的Cd2+含量,并测定了细胞活力、凋亡情况,胞外信号调节激酶ERK信号通路和天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)的活化情况,氧化应激相关的生物标记物ROS,GSH,CAT和SOD的变化,DNA损伤及组蛋白磷酸化的情况。研究发现经6h镉暴露后,细胞内无游离的Cd2+存在且镉对细胞没有明显的毒性。而经过24 h镉暴露后,部分细胞内发生了ROS含量持续升高等氧化应激效应、凋亡比例升高以及DNA氧化损伤等毒性效应。采用抗氧化剂N-乙酰基-L-半胱氨酸(NAC)对细胞进行预孵育可以阻止镉暴露造成的肝细胞活力的下降、凋亡比例的升高以及DNA损伤。上述结果说明镉在肝细胞内诱发了氧化应激并对肝细胞产生了毒性效应。镉通过氧化应激介导的凋亡降低了肝细胞活力。镉暴露后肝细胞内ERK通路被激活,NAC可以降低ERK通路的活化程度；NAC和ERK信号通路的抑制剂PD98059降低了细胞凋亡比例和caspase-3的活化程度。上述结果说明氧化应激调控的下游ERK信号通路的活化参与了镉引起的小鼠原代肝细胞的凋亡。本研究发现并证明了镉引起组蛋白H3的磷酸化与氧化应激和ERK通路的活化紧密相关。第四章以抗氧化酶CAT和SOD为研究对象,采用荧光光谱法、紫外-吸收光谱以及圆二色谱等多种光谱法研究了镉对蛋白结构的影响；采用酶活性检测的方法测定了镉暴露后酶活性的变化；采用等温量热滴定法(ITC)研究了镉与两种酶的结合模式,计算了结合参数和热力学参数；采用分子模拟等技术模拟了蛋白上镉的结合位点,建立两者的作用模型,并结合前述实验得到的结论,解释蛋白结构和功能变化的机理。(1)镉与CAT分子的直接相互作用导致CAT酶活性下降。镉与CAT主要通过静电作用力结合,与CAT有一类结合位点,结合常数K=(2.69±0.243)×103M-1。镉静态猝灭了CAT的内源荧光,两者形成了复合物,改变了芳香环氨基酸残基微环境的疏水性。两者的结合改变了CAT的二级结构,导致酶分子发生错折叠现象。对接的结果表明镉与底物进入CAT活性中心通道上的Gln 167和Trp 185相互作用,阻止底物进入酶活性中心,导致CAT的酶活性下降。(2)镉与SOD分子的直接相互作用导致SOD酶活性上升。镉与SOD主要通过静电作用力结合,与SOD有两类结合位点,结合常数分别为K1=(6.07±1.47)×105M-1和K2=(1.02±0.132)×104M-1。镉暴露改变了SOD的二级结构,导致酶分子发生去折叠现象。并且,镉不优先与SOD的发色团氨基酸残基发生作用,而是在SOD构象发生变化发色团外露后才与发色团发生作用。分子对接的结果表明镉结合到了SOD两亚基的交界面,可能造成酶活性位点附近的口袋增大,进而导致酶活性上升。第五章以具有间接抗氧化作用的蛋白溶菌酶(LYZ)和转铁蛋白(TF)为研究对象,采用荧光光谱法、紫外-吸收光谱以及圆二色谱等多种光谱法研究了镉对蛋白结构的影响；采用酶活性检测的方法测定了镉暴露后酶活性的变化；采用ITC研究了镉与蛋白相互作用过程中的结合模式,计算了结合参数和热力学参数；采用分子模拟等技术模拟了蛋白上镉的结合位点,建立两者的作用模型,并结合前述实验得到的结论,解释蛋白结构和功能变化的机理。(1)镉主要通过疏水作用力与LYZ相互作用,改变了LYZ的二级结构。尽管两者形成了复合物,但在低浓度镉暴露时,镉不优先结合于LYZ的活性位点附近,所以LYZ活性无变化。而在相对高浓度镉暴露时,镉与LYZ活性位点的关键氨基酸残基Glu 35和Asp 52发生作用,导致酶活性受到抑制。(2)TF可以结合镉并且中和镉对肝细胞的毒性。镉静态猝灭了TF的内源荧光,与TF分子形成复合物,使TF发生α螺旋含量降低和蛋白质皱缩等结构和构象上的变化。镉主要通过疏水作用力优先与TF上结合力高的位点结合,对TF的铁结合位点没有影响且不会造成铁的释放。高结合力位点饱和后,镉主要通过静电作用力结合到TF上结合力较弱的位点,与铁结合位点周围的Tyr 517和Tyr 95相互作用,造成铁的释放。第六章对本论文的工作进行了总结,归纳了了本研究中的创新点,分析了研究方法的不足,并展望了该领域的发展方向和下一步的研究计划。本研究结合体内动物实验和体外细胞及分子实验三种毒性评价的方法,评价了镉在肝脏及肝细胞内产生的氧化应激相关毒性效应,研究了镉致肝细胞发生凋亡、遗传和表观遗传毒性的作用机理,建立了镉与氧化应激相关蛋白间的作用模型并完善了污染物造成蛋白结构和功能变化及其作用机制的研究方法。三种暴露途径联合全面地评价并阐明了镉诱发的氧化应激相关毒性效应与机理,为更好地理解镉毒性提供了基础数据；为镉致肝损伤的的致病机理以及镉中毒的治疗方案提供了参考；为评估镉类重金属污染物的危害度、为国家制定有效的干预对策和措施等决策提供了科学的参考依据和技术支持。更多还原

【Abstract】 Cadmium is a typical heavy metal pollutant. Cadmium enters into the environment mainly through fossil fuel combustion, phosphatic fertilizer, natural sources, steel foundry, municipal solid waste incineration, cement and nonferrous metals productions and other industrial productions. With the rapid developments of modern industry, the problem of environmental cadmium pollution currently exists in most areas of the whole world and continues to increase in the future. Due to the common presence of cadmium in the environment and its potential toxic effects, the adverse effects of cadmium on the environment and human health have raised wide attention and obtained numerous on-going researches.As a non-essential element to the organism, cadmium can not be biologically degraded in the organism. For non-occupationally exposed people, cadmium enters into their body mainly through contaminated food and tobacco smoking. After entering into the body, cadmium distributes to other organs through blood circulation system and induces the damage of multisystems and multiorgans including bone, liver, kidney and reproductive system. Metal-induced oxidative stress is closely associated with a variety of diseases. In vitro and in vivo studies have demonstrated that cadmium could disrupt the balance of the celluar redox state and lead to oxidative stress. The overproduction of reactive oxygen species (ROS) induced by cadmium could lead to varieties of nonspecific damages, leading to cell death and tissue damage. These are considered as the general mechanism of common damages and diseases.Currently, cadmium induced oxidative stress-related toxic effects and its underlying mechanisms have not been completely clarified. The methodology of related research still needs to be further established and completed. Depending on the difference of experimental conditions and study objects, it remains controversial for cadmium induced oxidative stress-related toxic effects and its underlying mechanisms. To cure the above problems, this paper investigated cadmium induced oxidative stress-related toxic effects and its underlying mechanisms from the level of animal, cell and molecule. This paper is mainly consisted with the following six parts:In chapter one, the characteristics and current situation of environmental cadmium pollution as well as the research development of cadmium toxicity were simply introduced. The induction mechanism of oxidative stress, oxidative damage and the related regulated mechanism were overviewed. The effects and mechanism of cadmium-induced oxidative stress were also summarized. Through reviewing this knowledge, the research developments and the current scientific problems in the toxicology filed were concluded and experimental protocols were set for these problems.In chapter two, in vivo animal experiments were conducted and zebrafish was chosen as the study object. Cadmium induced oxidative stress-related toxic effects were investigated in the liver of zebrafish through measuring the biomarker changes during this process, including the activity change of antioxidant enzyme, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) as well as the level change of reduced glutathione (GSH), oxidized glutathione (GSSG) and the product of lipid peroxidation malondialdehyde (MDA). Results showed that cadmium exposure led to the increase of GPx activity, the decrease of GR and GST activity. The activity of CAT decreased and the activity of SOD increased. The level of GSH decreased and the level of GSSG increased, resulting in the decrease of GSH/GSSG ratio. The level of MDA increased with the concentration of cadmium. These results suggested that cadmium exposure induced amount of ROS production in the liver of zebrafish, disrupted the balance of redox state, induced oxidative stress in zebrafish livers and resulted oxidative damage.In chapter three, the mouse primary hepatocytes were chosen as the study object and exposed to cadmium in vitro. Cadmium induced oxidative stress-related toxic effects and its underlying mechanisms in mouse primary hepatocytes were investigated at the cellular level. The cellular Cd2+ level were measured in situ. The cell viability, apoptosis percentage, the activation of ERK signaling pathway and caspase-3 were measured after 6 or 24 h cadmium exposure. The response of oxidative stress biomarker ROS, GSH, CAT and SOD, DNA damage and histone phosphorylation were also investigated. After 6 h cadmium exposure, no free Cd2+ exists in the hepatocytes and cadmium exhibits no overt toxicity to the hepatocytes. After 24 h cadmium exposure, ROS level elevated and other oxidative stress-related effects occurred in a part of the hepatocytes. The increase of apoptosis percentage, DNA oxidative damage and other toxic effects were also observed in a part of the hepatocytes. The antioxidant N-acetyl-L-cysteine (NAC) prevented the decrease of cell viability, the increase of apoptosis percentage and DNA oxidative damage, suggesting that cadmium-induced cellular oxidative stress had adverse effects on the hepatocytes. Oxidative stress-mediated apoptosis reduced the cell viability of the hepatocytes. The ERK signaling pathway was activated after exposure to cadmium. NAC prevented the phosphorylation of key protein in ERK pathway. NAC and ERK pathway inhibitor PD98059 prevented apoptosis and the activation of caspase-3. These results suggested that oxidative stress-mediated the activation of downstream ERK signaling pathway participated cadmium-induced mouse primary hepatocytes. This study also demonstrated that cadmium-induced the phosphorylation of histone H3 was closely correlated with oxidative stress and the activation of ERK signaling pathway.In chapter four, the antioxidant enzymes CAT and SOD were selected as study objects. Enzyme activity change after cadmium exposure was determined using enzyme activity determination method. The effects of cadmium on protein structure were investigated through measuring fluorescence spectra, UV-vis absorption and circular dichroism. The binding mode of the direct interaction between cadmium and enzymes was explored by isothermal titration calorimetry (ITC), including the calculation of the binding parameters and thermodynamic constants. The possible sites on protein after cadmium binding were simulated by the molecular modelling methods. Combined with these conclusions, the mechanism of the structure and function change of the enzyme was clarified.(1) The direct interaction between cadmium and CAT molecule resulted in the decreased activity of CAT. Cadmium binds to CAT mainly through electrostatic forces with one type of binding sites and binding constant K= (2.69±0.243)×103 M-1. Cadmium statically quenched the intrinsic fluorescence of CAT and formed complexes with CAT, changing the hydrophobicity around the aromatic amino acids. The interaction changed the secondary structure of CAT, leading to the misfolding of the protein. Molecular docking results suggested that cadmium interacts with Gln 167 and Trp 185 located at the entrance to the active site of CAT, hinders the substrate entering into the active site and subsequently results in the decrease of the activity.(2) The direct interaction between cadmium and SOD molecule resulted in the increased activity of SOD. Cadmium binds to SOD mainly through electrostatic forces with two types of binding sites and binding constants K1=(6.07±1.47)×105 M-1 and K2=(1.02±0.132)× 104 M-1. The interaction changed the secondary structure of SOD, leading to the unfolding of the protein. And, cadmium did not interact with the aromatic amino acids of SOD until the aromatic amino acids became exposing to cadmium due to structure and conformation changes induced by cadmium. Molecular docking results suggested that cadmium binds to the interface of the subunit of SOD, which might result in the enlargement of the pocket around the enzyme active sites and then lead to the increase of enzyme activity.In chapter five, the indirect antioxidant proteins lysozyme (LYZ) and transferrin (TF) were selected as study objects. Enzyme activity change after cadmium exposure was determined using enzyme activity determination method. The effects of cadmium on protein structure were investigated through measuring fluorescence spectra, UV-vis absorption and circular dichroism. The binding mode of the direct interaction between cadmium and enzymes was explored by ITC, including the calculation of the binding parameters and thermodynamic constants. The possible sites on protein after cadmium binding were simulated by the molecular modelling methods. Combined with these conclusions, the mechanism of the structure and function change of the enzyme was clarified.(1) Cadmium binds to LYZ mainly through hydrophobic forces, changing the secondary structure of LYZ and the microenvironment of Trp residues. Although complexes were formed, no effects were observed on the activity of LYZ at low cadmium concentrations because cadmium does not preferentially bind to the active site of LYZ. However, at relatively high cadmium concentrations, a moderate loss of activity was observed due to the interaction with Glu 35 and Asp 52 located at the active site of LYZ.(2) The ability of TF to bind cadmium and the potential role of TF in neutralizing cadmium toxicity on the hepatocytes were demonstrated. Cadmium statically quenched the intrinsic fluorescence of TF and formed complexes with TF, leading to a decrease of the a-helix content, a shrinkage of the protein and other structural and conformational changes and TF. Cadmium preferentially binds to the higher binding affinity sites of TF via hydrophobic forces with no release of Fe and no interface to the Fe binding site. Subsequently, cadmium binds to lower binding affinity sites of TF via electrostatic forces and interacts with Tyr 517 and Tyr 95 around the Fe binding site, resulting in the release of the Fe content.In chapter six, every part of this thesis was concluded. The innovation points were also summarized. Subsequently, the development direction and future research in this filed were outlooked according to the shortage of the research protocols. This paper combined the exposure routes of in vivo animal experiments, in vitro cell and molecule experiments and evaluated the oxidative stress induced by cadmium in the liver and the hepatocytes. The mechanism of the apoptosis, genotoxicity and epigenotoxicty induced by cadmium were also explored. We also established a research strategy to explore the binding mode of cadmium interacting with proteins and the mechanism of protein structure and function change. Cadmium induced oxidative stress-related toxic effects and its underlying mechanisms were comprehensively investigated through combining the three exposure routes. This research could provide basic data for understanding cadmium toxicity. This research could help to provide references to the nosogenesis of cadmium-induced liver damage as well as the therapeutic schedule of acute and chronic cadmium exposure. This research could also provide scientific basis and technical supports for the hazardous evaluation of heavy metal toxicity, the establishment of intervene policy and measurements by the government.更多还原