Trophic Transfer of Nanomaterials and TheirInfluences with Functional Proteins

随着纳米技术的高速发展，纳米产品已得到越来越多的应用，其向环境的无 意释放引发了新的环境污染问题，从而可能对生态体系和人类健康构成潜在的威 胁，因此评价纳米材料的环境行为与生物安全性至关重要。本论文围绕几种典型 的纳米材料，分别从它们在环境食物链中的分布富集行为及与功能性蛋白的相互 作用两个方面展开了研究。 与其它环境污染物类似，水生态系统也是纳米材料的重要环境归宿,然而由 于真实环境的复杂性，纳米材料的环境行为研究较为困难。本论文在实验室内构 建了一个包括水、底泥、大薸、水绵、亚洲蛤、螺、水蚤、日本青鳉和大和沼虾 的淡水生态系统，研究了纳米二氧化铈（CeO2 NPs）长期暴露后在这一模拟水生 态系统中的分布，生物富集和潜在影响。结果表明CeO2 NPs 经过三十多天后达 到水相-泥相分配平衡，大多数纳米材料沉积在底泥中（88.7%）。六个物种中的 铈浓度与其营养级呈负相关关系，表明CeO2 NPs 在此食物链中无生物富集现象。 另外，值得注意的是，CeO2 NPs 可以引起水绵和大薸叶绿素含量降低、水绵淀 粉粒数量体积减少。CeO2 NPs 还可以通过诱导羟基自由基生成使大薸根部细胞 壁变薄，根系明显增长。以上模拟水生态体系中CeO2 NPs 的环境行为研究为纳 米材料的环境风险评估提供了重要的参考依据。 在水生态体系研究的基础上，本论文进一步在实验室内构建了丰年虫-日本 青鳉的水生食物链与胡萝卜-黄粉虫-黑斑蛙的陆生食物链，探讨了包括纳米金颗 粒、纳米银颗粒与纳米金棒在内的几种金属纳米材料随食物链的迁移转化行为。 实验结果显示，带正电荷的棒状金属纳米材料更容易在日本青鳉体内富集且不容 易排出。相对而言，纳米银比纳米金的生物富集效应更强，并且其在食物链的传 递过程中可逐渐发生生物转化，主要表现为在日本青鳉体内消化、吸收与转移过 程中的硫化效应，导致远端鱼脑内的银几乎完全被硫化。针对胡萝卜-黄粉虫-黑 斑蛙的陆生食物链研究显示，纳米金和纳米银沿着食物链传递到高营养级生物中 并发生一定的脏器累积效应。纳米材料在食物链不同级间的累积出现不同现象。 具体而言，在胡萝卜-黄粉虫间，纳米材料的累积系数大于1，而在黄粉虫-黑斑 蛙间则发生了生物稀释现象，这可能与所研究的实验动物的摄食习惯和代谢特征 有关。另外，纳米材料暴露的食物引起了黄粉虫和黑斑蛙摄食速率的降低，从而 影响了其生物累积行为。相对而言，纳米银对摄食速率的影响比纳米金更加显著。 黑斑蛙累积纳米材料的主要靶器官包括胃、肠、脑和肝脏，且被累积的纳米材料不易被机体代谢消除。该研究很好揭示了不同金属纳米材料在不同食物链中的具 体迁移转化特征，为其环境污染评价提供了重要的科学数据。 以上研究显示，纳米材料可进入不同有机体并分布富集在生物各组织器官中， 由此表明这种理化性质特殊的化学物质很可能与相关功能性蛋白相互作用从而 产生潜在的生物学效应。纳米金棒是一类物化性质非常独特的纳米材料，在生物 医药领域有着很好的应用前景，然而关于这种纳米材料的生物安全性也同时受到 了人们的关注。当纳米材料在活体中应用时必然会进入循环系统，因此其血液兼 容性评价至关重要。本论文设计了两种纳米金棒，分别是表面带正电荷的十六烷 基三甲基溴化铵修饰的纳米金棒（C-GNRs）和表面带负电荷的聚苯乙烯磺酸钠 修饰的纳米金棒（P-GNRs）。通过测试血液毒性发现，C-GNRs 接触血红细胞可 以有效引起溶血现象。在暴露剂量相对较低的情况下，可以观察到纳米金棒进入 血红细胞。这些进入血红细胞的纳米金棒可与细胞内丰度最高的血红蛋白相互作 用，通过静电力结合形成纳米金棒-蛋白复合物。血红蛋白中的发色基团如Trp 等可与C-GNRs 相互作用，从而引起蛋白荧光强度增加。纳米金棒的作用还可以 引起血红蛋白空间结构出现明显的改变，主要表现为α-螺旋下降，β-折叠与无序 结构增加。虽然C-GNRs 并不改变血红蛋白的载氧能力，但它阻碍了蛋白释放氧 的过程。血红素中结合氧的铁卟啉环在纳米金棒作用下可从血红蛋白上脱落下来， 而游离的铁卟啉可在一定程度上引起血红细胞的溶血现象。该研究表明，表面带 有正电荷的纳米金棒可引起显著的血液毒性效应，因此在医学的相关应用中需要 对纳米材料的生物安全性进行客观系统的评价。 除循环系统外，消化系统也是机体接触纳米材料的重要途径，因此探讨典型 纳米材料与消化道中关键蛋白酶如α-胰凝乳蛋白酶等的相互作用非常重要。由于 碳纳米管的特殊性质，其应用已广泛扩展至日常生活的诸多领域中，因此对于这 类新型材料的生物安全性也备受关注。碳纳米管的羧基化修饰可以改善其水分散 性，以满足特殊的应用需求，然而这种羧基修饰可能对其生物学效应产生潜在的 影响。本研究通过不同剂量的钴辐射造成未修饰的多壁碳纳米管表面缺损，进而 通过酸化引入羧基修饰，由此获得具有不同羧基基团修饰密度的多壁碳纳米管。 利用荧光光谱、紫外-可见吸收光谱、圆二色光谱、zeta 电位、原子力显微镜和 蛋白质定量分析等手段，研究探讨了α-糜蛋白酶和羧基化多壁碳纳米管在模拟生 理环境中的相互作用。结果表明，碳纳米管通过静电作用与α-糜蛋白酶结合，导 致α-螺旋含量下降、β-折叠含量增加，蛋白质荧光发生静态猝灭。碳纳米管表面 羧基修饰密度增加可提高其对蛋白质的吸附，并同时降低了酶的活性。表面羧基 修饰密度较低的碳纳米管对α-糜蛋白酶活性产生非竞争性抑制，而表面羧基修饰 密度较高的碳纳米管对酶活性产生竞争性抑制。在反应体系中增加离子强度可弱化碳纳米管和α-糜蛋白酶之间的相互作用。以上研究结果表明，碳纳米管表面的 功能化修饰对其生物学效应的调节起着重要的作用。 本论文研究工作通过探讨多种典型纳米材料在不同水生或陆生食物链中的 生物分布、富集与迁移转化效应，揭示其不同于其他污染物的环境行为，为这类 特殊的化学物质的环境风险评价提供了重要的科学数据。研究通过深入探讨纳米 材料与功能性蛋白的相互作用，表明其在进入生物体后可能引起的潜在毒理学效 应。本论文工作为纳米材料的环境效应与生态毒理学研究提供了重要的评价技术 手段与科学思路。The widespread use of engineered nanomaterials (ENMs) in consumer and industrial products has provided lots of benefits for our life, but may also bring new environmental problems due to their unintended release into the ecosystem. Their potential deleterious impacts on the environment and human health are being concerned. It is thus of great importance to study environmental behaviors of ENMs and evaluate their biosafety. In this dissertation, we mainly focused on several representative nanomaterials, including CeO2 nanoparticles (CeO2 NPs), gold nanoparticles, silver nanoparticles, gold nanorods (GNRs) and multi-wall carbon nanotubes (MWCNTs), and studied their environmental behavior in some constructed food chains, and the interactions with some functional proteins. In view of the final destination of nanomaterials, the water system would be the important sink. However, the environmental behavior of nanomaterials is rather confusing due to the complexity of the real environment. In this study, a fresh water ecosystem, including water, sediment, water lettuce, water silk, Asian clam, snail, water flea, the Japanese Medaka, and the Yamato shrimp, was constructed to study the distribution, bioaccumulation and potential impacts of CeO2 NPs via long-term exposure. The results demonstrated most of the CeO2 NPs deposited in the sediment (88.7%) when the partition approached constantly 30 days later. The bioaccumulated Ce in 6 tested biota species was negatively correlated with its trophic level, showing no biomagnification of CeO2 NPs through this food web. CeO2 NP exposure induced visual abnormalities in hydrophytes including chlorophyll loss in water silk and water lettuce, ultrastructural changes in pyrenoids of water silk and root elongation in water lettuce. The generation of hydroxyl radical (OH) and cell wall loosening induced by CeO2 NP exposure might mediate the increased root growth in water lettuce. The findings on the environmental behavior of CeO2 NPs in water system have provided useful information on the risk assessment of nanomaterials. Besides the study on the food web in simulated aquatic system, the environmental behaviors of metallic nanomaterials including gold nanoparticles, silver nanoparticles, and GNRs, were also investigated in two lab-constructed aquatic and terrestrial food chains. In the aquatic food chain including fairy shrimp and medaka, the metallic nanoparticles with positive charge and rod shape were more likely to be accumulated, and the bioaccumulation of silver nanoparticles was relatively higher than that of gold nanoparticles. Sulfidation was observed for silver nanoparticles druing their food transfer and biodistribution from digestive orans to some other tissues like liver and brain in medaka. As for the constructed terrestrial food chain, including carrot, mealworm and frog (Pelophylax nigromaculatus), significant biomagnification of gold nanopartilces occurred between carrot and mealworm, but biodilution was observed between mealworm and frog. This could be related with the feeding behaviors of the tested species. The nanoparticle-exposed carrots caused the decreased food intake of mealworm, which in turn decreased that in the frog as well. Silver nanoparticle exposure induced more severe effect on the feeding behavior than gold nanoparticles. The target organs for nanoparticle bioaccumulation mainly included stomach, intenstine, liver and brain, and the issue nanoparticles had rather slow elimination rates. The findings in this study revealed the interesting characteristics in food chain transfer of metallic nanomaterials. As reported extensively that nanomaterials can enter into diverse biotas and distribute in multiple tissues or organs, how they may interact with functionals proteins would be interesting in the explanation of their potential biological effects or toxicities. GNRs are a unique class of metal nanostructures that have attractive potentials in biomedical applications, and the concern on their biological safety is concomitantly increasing. Hemocompatibility is extremely important as their contact with blood circulation is unavoidable during in-vivo delivery. Herein, two kinds of GNRs coated with hexadecyltrimethylammonium bromide (C-GNRs) or poly(sodium-p-styrenesulfonate) (P-GNRs) were used to test their potential toxicological effects in blood. C-GNRs with positive surface charge were found to efficiently induce hemolysis when encountering erythrocytes. Cellular internalization of C-GNRs was found, and they subsequently bound with hemoglobin, thus forming the bioconjugation agglomerates. The interaction between hemoglobin and C-GNR surface (stoichiometry 32.7:1) was regulated by electrostatic forces (KA = 1.05×106 M-1, ΔH○ = -8.35×106 J·mol-1, ΔS○ = -2.79×104 J·mol-1·K-1, ΔG○ = -3.58×104 J·mol-1 at 298 K). Chromophores like Trp were found to interact with C-GNRs, causing enhancement in fluorescence intensity. The conformation of protein was partially altered, evidenced by decrease in α-helical, increase in β-sheet and random coil of hemoglobin. Although C-GNRs did not essentially decrease oxygen binding capacity of hemoglobin, they hampered oxygen release from the protein. Heme, the oxygen binding unit, was detected to be released from hemoglobin upon C-GNR treatment, which could contribute to C-GNR-induced hemolysis. This study has demonstrated hematological effects of GNRs with positively-charged surface, revealing their potential risk in C-GNR-based medical applications. Besideds the circulation system, the digestive system is also one of the most important routes throug which nanomaterials may enter into biotas. It is thus important to investigate how nanomaterials interact with the functional proteins, like α-chymotrypsin (α-ChT), therein. Considering the special properties of MWCNTs and their wide application in many fields, they were selected as the model compounds, and functionlized by different densities of carboxyl groups. The interaction between α-ChT and these carboxylized MWCNTs in simulated biophysical environment was characterized by the techniques of fluorescence, UV-vis, circular dichroism spectroscopy, ζ potential, atomic force microscopy and bicinchoninic acid analysis. It was demonstrated that CNTs interacted with α-ChT through electrostatic forces, causing decrement in α-helix and increment in β-sheet content of the protein. The protein fluorescence was quenched in a static mode. The increase in surface modification density of CNTs enhanced the protein absorption and decreased the enzymatic activity correspondingly. α-ChT activity inhibition induced by CNTs with low surface modification density exhibited non-competitive characteristics, however, competitive feature was observed when CNTs with high surface modification density interacted with the protein. Increase of ionic strength in the reaction buffer may help to reduce the interaction between CNTs and α-ChT because high ionic strength may favor the release of the protein from binding on CNTs surface modified with functional groups. Accordingly, functionalization density on CNTs surface plays an important role in the regulation of their biological effects and is worthy of being concerned when new modified CNTs are developed. The research in this dissertation revealed the biodistribution and bioaccumulation of some typical nanomaterials in both aquatic and terrestrial food chains, and the interactions of ENMs with functional proteins showed their potential hazardous effects on environmental biotas and human health. The research findings provided important scientific data for risk assessment on environmental occurrence of ENMs