环境化学与生态毒理学国家重点实验室知识库

      本论文旨在探讨环境与生物因素对 碳纳米材料 毒性 效应的影响机制 。 以典型纳米材料氧化石墨烯为研究对象，分别从肺暴露、环境转化和体内代谢三个方面进行了研究。论文 共 分 为 四 个 部分：一、 建立 基于 评 价 肺表面活性剂的物理化学性质变化来评价纳米材料肺毒性 的方法 ；二、 探索 碳黑 颗粒 氧 含 量对 其 肺毒性和肺巨噬细胞 毒性效应 的 影响 机制 ；三、 考察 还原 对 氧化石墨烯 对 原代巨噬细胞毒性 和细胞系巨噬细胞 毒性的影响及 其 对两种细胞 的 毒性差异 ；四、 探索 氧化石墨烯 暴露 对小鼠 肝脏毒性和对 肝实质细胞 的表观毒性效应 。
一、建立 通过评价肺表面活性剂的物理化学性质变化来评价纳米材料肺毒性的方法
      作为天然表面活性物质，肺表面活性剂（pulmonary s urfactant PS ）是肺部抵御纳米材料入侵 和 与纳米材料相互作用的初始屏障。我们 采用 约束液 体表面 测量 c onstrained d rop S urfactometry CDS 的方 法 实现 了 对由 工程纳米材料engineering nanomaterial ENM 引起的肺表面活性剂表面张力变化的 实时观测 和定量评估。结果表明，在非常低的浓度下，包括 氧化石墨烯 g raphene o xide GO 碳纳米管 c arbon nanotube CNT ）），氧化锌 z inc oxide ZnO 纳米颗粒和纳米 银 颗粒 s ilver nanoparticles AgNP s 在内的四种有代表性的 E NM 均提高了来源 于 小牛肺的表面活性剂的表面张力。这些体外实验 的 结果 与气管滴注模型中 观察到的 小鼠 体内肺泡 大面积 塌陷 和炎症 反应 具有很好的一致性 。因此，ENM 引起 的 肺表面活性剂体外表面张力增加与 其 体内肺毒性之间 具有 可能存在直接 的 相关性。与常用动物模型相比， CDS 法 在开发用于快速和低成本预测吸入性 ENM 的急性肺毒性的测定方法方面具有很大的应用潜能。
      二、探索 氧 含 量对碳黑颗粒 肺毒性和肺巨噬细胞 毒性效应 的 影响 机制

尽管已经有 大量 的研究探索了 细颗粒 （如 PM 2.5 对 环境健康和安全environmental health an d safety EHS 的 影响，但仍然存在重大的知识空白。关于不同 的 物理 结构和 化学 性质如何决定细颗粒物的毒性效应尚没有 取得 确切的理解。我们比较了四种 碳黑 c arbon Blacks, CB ）颗粒的细胞毒性 这四种颗粒物 除了它们的氧含量（ C824455 C1864> Printex U> SB4A 。在这些CB 颗粒中， C82 4455 和 C1864 表现出 最显着的肺损伤（例如 肺泡 塌陷和 肺局部 炎症）和巨噬细胞活化，特别是 C824455 。所有这些毒性 效应 的差异，包括体外和体内细胞毒性 ，促炎症效应和对肺上皮的直接损伤，应该（至少部分）归因于这些 CB 颗粒中的氧含量 的差异 而含氧量的差异 又决定了 碳黑颗粒的生物转化 的不同 ，即不同的聚合状态。 同时， PM 2.5 同样对肺细胞和巨噬细胞造成严重的体内和体外毒性。 综上所述 ，这项研究 为了解颗粒物的 结构 活性关系 structure and reactivity SAR 提供了 更多 的 见解，并为阐明细 空气颗粒引发肺损 伤的物理化学决定因素开辟了新的途径 。
三、考察还原对氧化石墨烯对原代 巨噬细胞毒性和细胞系巨噬细胞毒性的影响及其对两种细胞的毒性差异
      我们使用骨髓来源的 原代 巨噬细胞（ b one marrow derived macrophagesBMDMs ）和 J774A.1 巨噬细胞系 比较了 硫化钠（ Na 2 S 还原前和还原后 的 GO对巨噬细胞生物相容性（例如细胞毒性，促炎症效应和细胞形态 等 损伤的差异 。我们 使用 Na 2 S 作为 还原剂制备 了 两种相对于亲本 GO 具有不同 还原 水平的RGO RGO1 和 RGO2 ）。有趣的是 还原 导致 GO 发生了 显着形态改变，从 还原前 GO 更多 的片状结构到 还原后 RGO 的 多边形边缘卷曲 结构 且短时间内几种材料 在生物培养基中没有 显著 的 聚集 现象 。细胞毒性评估 结果 显示 RGO 比GO 对 两种细胞 的 毒性 更强 ，并且 J774A.1 细胞更容易受到这些材料诱导的毒性的影响。 RGO 比 GO 诱导 更 显著 的促 炎症 反应，尤其是在 BMDM 暴露 中。此外， GO 和 RGO 对 BMDM 的 细胞 形态变化产生 了 不同的影响， GO 处理的BMDM 细胞的突触更长而 RGO 暴露后的 BM DM 细胞的突触缩短且更加扁平 。机制研究表明 RGO 的细胞相容性受损可能是其氧含量及官能团变化和边缘形态变化共同作用的结果 ，从而造成 RGO 在巨噬细胞中 激发 更 强 氧化应激 反应 。 综上所述 ，这项研究揭示了 GO 和 RGO 之间 由还原 介导的有害细胞效应对巨噬细胞 毒性影响 的重要贡献。
四、揭示 纳米氧化石墨烯 暴露 对小鼠肝脏 和肝实质细胞 的表观 毒性 效应
      如前所述，氧化石墨烯在纳米医学当中表现出优越的性能 。如超大的比表面
积，光热转化效应等，为其在载药和肿瘤光热治疗等领域提供了广阔的应用潜能。然而，氧化石墨烯的生物安全性，特别是其在体内的代谢机制和表观毒性机制仍然知之甚少。本文选取生物医学中 推荐 使用的 粒径为 几十纳米的纳米氧化石墨烯为研究对象，通过尾静脉暴露小鼠，分析纳米氧化石墨烯在小鼠体内，特别是肝脏内的分布和代谢情况。通过对肝脏实质细胞的分离和提纯，进一步研究纳米氧化 石墨烯暴露对小鼠肝脏实质细胞的转录组和全基因组 D NA 甲基化的影响。结果表明，纳米氧化石墨烯在肝脏 当中会发生累积，并伴随着暴露时间 延长 而发生代谢去除。此过程中，实验动物并未表现出明显的病理学变化。 但， 肝脏实质细胞的全基因组 D NA 甲基化 ，特别是 羟甲基 化水平 发生了显著的变化，代谢相关基因的表达水平也发生了相应的变化。综上所述，本文为揭示纳米氧化石墨烯的体内代谢通路和机制及其在生物医学中的应用提供了新的见解和 实验 依据。

      This dissertation aims to explore the effects of environmental and biological factors on the biological effects of carbon nanomaterials. Part 1: Biophysical assessment of pulmonary surfactant PS predicts the lung toxicity of nanomaterials; Part 2: Oxygen content determines the bio-reactivity and toxicity profiles of carbon black (CB) particles; Part 3: Reduction of graphene oxide (GO) by Na2S differentially alters its cyto-compatibility towards primary macrophages and cell line macrophages; Part 4: Epigenetic toxicity of nano-GO on mouse hepatic parenchymal cells.
      Part 1: Biophysical assessment of pulmonary surfactant predicts the lung toxicity of nanomaterials
      As a natural surface-active substance, PS is the initial barrier in the lungs against the invasion of nanomaterials and the interaction with nanomaterials. Together with our team at the University of Hawaii, we have implemented an in vitro experimental method called the Constrained Drop Surfactometry (CDS) method by which the effect of ENM on the surface tension of the lung surfactant could real-time observation and quantitative assessment of tension changes. At very low concentrations, four kinds of representative ENMs, including carbon nanotubes (CNTs), GO, zinc oxide (ZnO), all increase the surface tension of surfactant extract from calf lungs. The results of these in vitro experiments correlate well with the extensive alveolar collapse and inflammatory responses observed in mice exposed to these ENMs in the tracheal instillation model. Therefore, there may be a direct correlation between increased surface tension of PS in vitro and pulmonary toxicity of ENM in vivo due to inhibition of ENM. Compared with the general use of animal models, the CDS method has great potential in the development of a method for the determination of acute pulmonary toxicity for rapid and low-cost predictive inhalation of ENMs.
      Part 2: Oxygen content determines the bio-reactivity and toxicity profiles of CB particles
      Although a large number of studies have explored the impact of fine particles (such as PM2.5) on environmental health and safety (EHS), significant gaps still exists. There is no exact understanding of how different physical structures and chemical properties determine the toxic effects of fine particulate matter. In this article, we compared the cytotoxicity of four CB particles that had similar physicochemical properties except for their oxygen content (C824455 C1864> Printex U> SB4A. Among these CB particles, C824455 and C1864 showed the most prominent lung injury (such as alveolar collapse and local lung inflammation) and macrophage activation, especially C824455. All of these differences in toxic effects, including in vitro and in vivo cytotoxicity, proinflammatory effects, and direct damage to the lung epithelium should be attributed, at least in part, to differences in oxygen content in these CB particles, which determine the different biological transformation of them, that is, different polymerization state. At the same time, PM2.5 also causes severe in vivo and in vitro toxicity to lung cells and macrophages. Taken together, this study provides additional insight into understanding the structure and reactivity (SAR) of particulates and opens up new avenues for elucidation of the physico-chemical determinants of air-lung particle-induced lung injury.
      Part 3: Reduction of GO by Na2S differentially alters its cyto-compatibility towards Primary macrophages and cell line macrophages
      We compared the differences in biocompatibility (eg, cytotoxicity, proinflammatory effects, and cell morphology, etc.) of sodium sulphite (Na2S) pre-reduction and post-reduction of GO on bone marrow-derived macrophages (BMDMs) and J774A.1 macrophage cell line. We used Na2S as a reducing agent to prepare two RGO (RGO1 and RGO2) with different reduction levels relative to the parent GO. Interestingly, the reduction of Na2S resulted in significant morphological changes of GO, from a flatter GO structure before reduction to the bilayer concave RGO structure after reduction, and the aggregation status did not change significantly. Cytotoxicity assessment showed that RGO was more toxic than GO to both cell types and that J774A.1 cell was more susceptible to the toxicity induced by these materials. RGO induced more pronounced pro-inflammatory responses than GO, especially in BMDM. In addition, GO and RGO exerted different effects on the morphological changes of BMDM cells. The synapses of GO-treated BMDM cells were longer and the synapses of BMDM cells exposed to RGO were shortened and flatter. Mechanistic studies showed that the impaired cell compatibility of RGO may be the result of a combination of oxygen content and changes in functional groups and edge morphology, resulting in RGO stimulating a stronger oxidative stress response in macrophages. Taken together, this study reveals an important contribution of GO and RGO to macrophage cytotoxicity mediated by reductive-mediated deleterious cellular effects.
      Part 4: Epigenetic toxicity of nano-GO on mouse hepatic parenchymal cells
      As mentioned above, GO shows superior properties in nanomedicine. Such as large specific surface area, photothermal conversion effect, etc., which provide broad potential for application in the fields of drug-carrying and photothermal therapy of tumors. However, the biosafety of GO, especially its in vivo metabolism and apparent toxicity mechanisms, remains poorly understood. In this paper, nano-sized GO, which is used in the recommended biomedicals in current literatures, was selected as the research object. The mice were exposed through the tail vein to analyze the distribution and metabolic conditions of nano-GO in mice, especially in the liver. Through the isolation and purification of liver parenchymal cells, we further studied the effects of nano-GO exposure on the transcriptome and genome-wide methylation of mouse hepatic parenchymal cells. The results show that nano-GO accumulates in the liver parenchyma cells, accompanied by metabolic elimination during the exposure time. During this process, the experimental animals did not show obvious pathological changes. However, whole-genome DNA methylation and methylation levels of liver parenchymal cells have undergone significant changes, and metabolic-related gene expression levels have also changed correspondingly. In summary, this article provides new insights and evidence for revealing the in vivo metabolic pathways and mechanisms of nano-GO and its application in biomedicine.