环境化学与生态毒理学国家重点实验室知识库

      纳米银作为一种广谱的抗菌试剂，已被广泛地应用于个人护理产品、保健品以及医疗用品等诸多领域。然而，纳米银在这些产品的生产、使用和处置过程中，会不可避免地释放到环境当中，对环境生物和人体健康造成潜在的威胁。因此，纳米银所引发的健康效应正引起人们的关注。纳米银在释放进入环境以及生物体之后，会发生不同的物理、化学和生物转化，其高度动态性导致不同形态的银共存于环境以及生物体内。尽管不同形态银所诱导的毒性与其形态密切相关，但对于不同形态银的毒性效应与机制研究依旧面临挑战，主要原因是缺乏合适的分析方法来定量甄别生物基质中不同形态银。因此，发展在复杂生物基质中不同形态银的分离定量方法对于研究其在体内的生物分布及代谢转化行为具有十分重要的意义。此外，结合传统的毒理学分析手段，将银的转化行为与相应的毒性联系起来，可为深入探究并阐释不同形态银所诱导的毒性效应机制以及科学地评价其生物安全性提供可靠的数据和技术支持。
      本文主要研究不同形态银在环境生物体以及高等动物体内的分离定量、生物分布、代谢转化以及毒性效应。建立了基于四甲基氢氧化铵（TMAH）的生物基质消解的不同形态银提取的方法，并将其与尺寸排阻色谱-电感耦合等离子体质谱（SEC-ICP-MS）在线联用的粒径分离和定量测定技术相结合，发展了细菌微生物和大鼠体内不同形态银的分离、浓度定量和形态表征方法；基于所建立的分析方法，研究了不同形态银的生物分布及代谢转化过程；结合传统的毒理学分析手段，将不同形态银的分布、转化行为与其毒性相结合，探究了不同形态银所诱导的毒性效应机制。本文主要包括以下几部分研究内容：
      首先，基于SEC-ICP-MS技术，建立了大肠杆菌细胞内颗粒态银、络合态银离子以及自由态银离子等不同形态银的分离和定量方法。为了完全排除细胞壁所吸附的银对内化进入细胞的银的测定的干扰，使用溶菌酶去除细胞壁以得到大肠杆菌的原生质体细胞；之后在破碎的细胞中立即加入表面活性剂FL-70以稳定纳米银的形态，再加入四甲基氢氧化铵（TMAH）进行细菌细胞的消解以及不同形态银的提取；最后利用SEC-ICP-MS实现大肠杆菌中不同形态银的分离和定量。本方法对于银离子以及不同粒径纳米银（10 nm, 20 nm和30 nm）均有较低的检出限（3 ng/mL）；通过大肠杆菌的三个水平加标实验表明，对于不同形态银的方法加标回收率为82.19%-115.8%，SEC分离保留时间的相对标准偏差< 0.9%，说明本方法适用于大肠杆菌内部不同形态银的分离定量。此外，研究发现银离子在大肠杆菌内以银离子-生物分子络合物的形式存在，但在一定量（107 CFU/mL）的大肠杆菌中，有限的生物分子数量使得络合态银离子的浓度存在上限（2 μg/mL），超过此上限的银离子则以自由态形式存在。本方法为细菌中银的形态分布和代谢转化研究提供了有效的分析方法。
      其次，本文研究了不同形态银在细菌中的转化行为及毒性效应。利用菌落计数法测得10 nm、30 nm纳米银和银离子对大肠杆菌的EC10、EC50和EC90值；在此暴露剂量下，基于前期所建立的细菌中不同形态银的分离、定量方法测定了大肠杆菌内部的银形态和含量；同时，基于SEC-ICP-MS能够测定细胞外培养基中不同形态银的含量；因此，利用差减法可以得到结合在细菌细胞壁表面的银含量，进而得出不同形态银的生物分布。研究发现，不同形态的银在大肠杆菌中的生物分布规律基本类似，少量的银（占总暴露量5.98%-15.21%）被大肠杆菌内化，而25.13%-64.43%的银锚定在细菌细胞壁，说明不同形态银同时存在细胞壁的锚定以及内化进入细胞内部的过程。此外，通过暴露不同粒径纳米银，发现了纳米银被细菌内化并发生溶解的单向过程，并且粒径越小，氧化溶解程度越高。与银离子暴露组相比，尽管纳米银暴露组（10 nm、30 nm）中细胞内部的溶解态银离子含量均更高，但细菌致死量却相同，说明由纳米银溶解释放出的银离子对于细菌的毒性作用低于最初暴露的自由态银离子。这可能是由于纳米银溶解释放的银离子与生物分子络合而导致的解毒效应。研究还发现，在实际环境条件下，大肠杆菌体内不容易出现自由态银离子。
      论文的第三部分建立了动物体内不同器官中不同形态银的分离、质量浓度定量和粒径表征方法。首先利用TMAH对猪肝进行消解及不同形态银的提取，先后优化了消解时间和消解浓度，将组织器官中的颗粒态和离子态银以其原始形态同时提取出来，最后基于SEC-ICP-MS进行分离测定。研究发现，进入生物基质中的纳米银表面会包裹一层生物分子冠（biomolecule corona, BCs），形成核壳结构AgNP@BCs。用ICP-MS进行银浓度定量时的检出限为0.1 μg/g，基于SEC色谱分离的保留时间可以测定组织中AgNP@BCs的有效粒径，且粒径分辨率为0.6-3.3 nm。更为重要的是，研究发现AgNP@BCs表面的BCs厚度与纳米银核粒径大小无关，且在组织样品中AgNP@BCs的有效粒径与核粒径呈线性关系，据此可以校正生物组织中纳米银的实际核粒径（标准偏差为0.2-1.1 nm）。本方法也可扩展到大鼠的其他器官（心、脾、肺、肾、脑）的分析，实现了大鼠不同器官中不同形态银的分离定量以及有效粒径/核粒径的测定。
      论文的第四部分研究了不同形态银在大鼠体内的生物分布、转化和毒性的联合效应。通过给大鼠尾静脉注射银离子（5 mg Ag/kg）、纳米银（5 mg Ag/kg, 20 nm）或银离子与纳米银的混合物（各5 mg Ag/kg），借助于已建立的生物组织中不同形态银的分离、浓度和粒径的定量表征方法，测定大鼠体内不同形态银的浓度和粒径分布，并将银的生物分布与组织病理学分析、血清生化指标与免疫组织化学分析相结合来描述颗粒态与离子态银的联合效应。研究发现，在大鼠器官中累积的银大多数都是以离子的形式存在，只有一小部分纳米银在肝脏和肺中检出，被器官摄取的纳米银一旦开始溶解，则会在短时间内完全溶解；尚未开始溶解的部分则在暴露后的3天内维持其原始粒径，到第5天时所有纳米银已经完全转化为银离子。尽管不同形态银在生物摄取和再分配方面的联合效应不明显，但在转化行为方面，生物体内共存的银离子能够加速所暴露的纳米银的溶解，可能由于金属硫蛋白的显著过量表达所致；此外，共存的纳米银在一定程度上能够缓解银离子所诱导的急性毒性。

      Silver nanoparticles (AgNPs), as a broad-spectrum antimicrobial agent, have been widely applied in numerous sectors, including personal care products, dietary supplements, and medical supplies. Inevitably, AgNPs can be released into the environment during the manufacturing, usage, and disposal of AgNP-based products. These arouse emerging concerns regarding to the risks to the environmental organisms and human health. After entering into the environment and organisms, AgNPs may undergo different morphology and chemical transformations as well as biotransformations, and their highly dynamic property can lead to the co-existence of different Ag species. While the toxicities induced by different silver species are closely related to their morphology, the clarification of the toxicity by silver species is still remaining a challenge, due to the lack of a versatile analytical technique to quantitatively discriminate different Ag species. Thus, developing a robust method to separate and quantify different Ag species in complex biological matrices is of paramount importance for understanding of the biodistribution and transformation behavior of Ag species. In addition, based on the toxicology methods, the silver transformation behaviors can hopefully be linked to their corresponding toxicity, which opens new opportunities for shedding new light on the toxic mechanism and safety assessments of Ag species.
      This dissertation focuses on the characterization and quantification of Ag species in environmental organism and mammals, as well as clarifing their biodistribution, transformation and toxic effects. Coupling the tissue digestion and Ag species extraction by tetramethylammonium hydroxide (TMAH) with size exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICP-MS), the methods for separation, quantification and characterization of different Ag species in microorganisms and rats were developed for the investigation of the biodistribution and transformation of Ag species in vivo. Furthermore, combining conventional toxicity tests, we discussed the toxic effects from different Ag species based on their biodistribution and transformation behavior to study the toxic mechanisms of different species in vivo.
      Firstly, a method based on the SEC-ICP-MS system was proposed for simultaneous separation and quantification of various Ag species including particulate Ag, complexed Ag(I) and free Ag+ in Escherichia coli (E.coli). To completely eliminate the interferences of cell wall-adsorbed Ag to the determination of the internalized Ag in E.coli, the lysozyme was employed to remove the cell wall from E.coli and then obtain its protoplasmic cell. After that, into the cell lysates was added the surfactant FL-70 to stabilize AgNPs, and then TMAH for the cell matrices digestion and various Ag species extraction. Finally, SEC-ICP-MS was utilized to realize the separation and quantification of different Ag species in E.coli. The proposed method provided low detection limites with 3 ng/mL for ionic Ag and AgNPs with nominal sizes of 10 nm, 20 nm and 30 nm. The determined recoveries and precisions of different Ag species in E.coli cells at three spiked levels were 82.19%-115.8% and < 0.9%, respectively, suggesting the practicality of this proposed method for the separation and quantification of Ag species in E.coli. In addition, it was found that the ionic Ag in E.coli existed in the form of Ag(I)-biomolecule complexes. However, within a certain amount of E.coli (107 CFU/mL), the limited amount of biomolecules available for complexing with Ag+ set up an upper limit for the amount of complexed Ag(I) (2 μg/mL), and the Ag+ at a spiked level above this limit would exist in the form of free Ag+. This method provided an efficient strategy for the investigation of Ag biodistribution and transformation in E.coli.
      Secondly, the transformation behavior and the related toxicity of different Ag species in E.coli were studied. The E.coli mortalities (EC10, EC50 and EC90) in treatment with Ag+ and AgNPs with nominal sizes of 10 nm and 30 nm were determined by viable plate counts, and then the quantification and speciation of Ag species within E.coli under these three doses were performed based on the proposed method described above. More importantly, since SEC-ICP-MS system is applicable for the quantification and speciation of Ag species in the extracellular matrix, the contents of the cell wall-adsorbed Ag could be obtained by subtracting the contents of the intracellular and extracellular Ag from the total exposure dose of Ag, and therefore the biodistribution of Ag species could be profiled. The results revealed that different doses of Ag+ and different sized AgNPs exposed to E.coli showed similar amplitude of heterogeneity, i.e, the intracellular uptake Ag varied from 5.98% to 15.21% of the dose and the cell wall-bound Ag from 25.13% to 64.43% of the dose, demonstrating that both of the cell wall-adsorption and cellular internalization of Ag species existed during the exposure. Furthermore, after exposure to different sized AgNPs, it was found that AgNP could be internalized by E.coli and further release Ag+; and the released Ag+ increased with the decrease of the AgNP particule size. Although the dissolved Ag(I) concentrations in E.coli exposed to different sized AgNPs were higher than that after Ag+ exposure, they showed almost the same E.coli mortality, demonstrating that the toxicity induced by the Ag+ released from AgNP was lower than that by the initial exposed Ag+ to E.coli. The results might be attributed to the complexation of the released Ag+ from AgNP with the biomolecules, which led to the detoxification. Besides, due to the dose-response of different Ag species, the free Ag+ was not likely to be existed in the living E.coli cells unless the total ionic Ag is > 2 μg/mL.
      In the third part, another method for simultaneous separation, mass quantification and size characterization of different Ag species in various organs of mammals was established. The tissue digestion and Ag species extraction by TMAH were performed by spiking Ag species into swine liver. The digestion conditions (digestion time and TMAH concentration) were optimized, and then both particulate and ionic silver were extracted in their original forms from the organs and analyzed by SEC-ICP-MS. It was reported that when AgNPs enter into physiological fluids, biomolecules tend to adsorb onto the particle surface to form a biomolecule corona (BC) and this AgNP would exist in the form of AgNP@BCs. While the silver mass concentrations were quantified by ICP-MS with a detection limit of 0.1 μg/g, the effective diameter of AgNP@BCs was determined based on the retention time in SEC separation with size discrimination of 0.6–3.3 nm. More importantly, we found that the BC thickness of AgNP@BCs is core size independent, and a linear correlation was found between the effective diameter and core diameter of AgNP@BCs in extracted tissues, which was used to calibrate the core diameter with standard deviations in the range of 0.2–1.1 nm. The utility of this strategy was demonstrated through validation to various rat organs including liver, heart, spleen, lung, kidney and brain, providing an efficient approach for separation, mass quantification and size characterization of the core-biocorona structure and dissolved species of AgNPs in different organs in rats.
      While the highly dynamic properties of AgNPs lead to coexposure to particulate and ionic silver, their combined effects on biodistribution, transformation and toxicity in vivo are still unclear. Herein, after intravenous exposure to either Ag+, AgNPs, or a mixture of Ag+ and AgNPs, the distribution of different silver species in rat organs was quantitatively determined based the proposed method as described above, and the corresponding toxicities were profiled by combining the Ag species biodistribution with histopathological, biochemical and immunohistochemical analyses to elucidate the combined effects of particulate and ionic Ag. Most of the accumulated silver existed in ionic form in all organs, only a small fraction of AgNPs was detected in liver and lung, and the AgNPs taken up in organs were either dissolved completely in a short time or maintained in their original core size in 3 days postadministration, and completely transformed into Ag(I) at day 5. While there was a negligible combined effect on the uptake or redistribution of different silver species, the coexistence of Ag+ accelerated the dissolution of administered AgNPs by the remarkable overexpression of metallothioneins, and the presence of AgNPs could alleviate the Ag+-induced toxicity.