Predictive Toxicological Paradigms to Establish Inhalation Toxicology Models

描述（由申请人提供）：加州大学洛杉矶分校的一个多学科小组（Andre Nel, Jeffrey Zink, Tian Xia, Ning Li）与NIOSH的Vince Castranova博士和不来梅大学（德国）的Lutz Mddler博士合作，旨在建立小鼠吸入毒理学模型，以筛选与组织培养细胞机械损伤途径相关的组合纳米材料库。这项研究将解决缺乏可重复的筛选方案，以评估纳米材料（NM）的安全性。我们假设两个组合文库(i)通过铁掺杂改变Zn++的释放来调节ZnO纳米颗粒的毒性作用，（ii）通过降低表面阳离子密度来调节阳离子介孔二氧化硅纳米颗粒（MSNP）的细胞毒性，这将有助于建立人支气管上皮（NHBE）和骨髓树突状细胞（DC）的体外毒理学与小鼠非过敏性和过敏性肺部炎症之间的联系。我们认为，非过敏性气道炎症和细胞毒性损伤之间的联系可以通过纳米颗粒的特性来解释，纳米颗粒会导致氧化损伤、有毒金属离子的脱落和触发线粒体损伤的能力。相反，材料特性与变应性气道炎症的联系可能会影响DC“危险信号”的产生，从而使DC启动促进变应性炎症的免疫刺激途径。为了实现我们开发预测吸入毒理学模型的长期目标，我们在Aim 1中提出表征纳米文库的生物物理化学特性，这些特性有望在NHBE和骨髓源性DC中诱导不同的细胞毒性和促炎作用。体外毒性筛选将由加州大学洛杉矶分校加利福尼亚纳米系统研究所的高含量筛选（HCS）设施进行，并通过ELISA进行细胞因子测量。我们也会评估活性氧的产生。将评估的纳米性能包括粒度、粒度分布、分散性、ζ电位、TEM、XRD和BET分析。HCS是用混合染料进行的，这些染料可以显示细胞膜渗漏、DNA损伤、线粒体去极化和细胞内Ca2+通量。颗粒表征和体外筛选将在第一年开始和新批次合成时进行。目的2将利用这些文库通过吸入-抽吸方法建立小鼠过敏性和非过敏性肺部炎症的标准化方案。非嗜酸性粒细胞炎症（Castranova）方案将在暴露后1天至2个月期间测量BAL中的炎症、损伤和氧化应激标志物以及肺组织学。该过敏致敏模型依赖于麻醉动物鼻子的颗粒和OVA吸入，将与BAL差异细胞计数和肺组织学同时评估OVA特异性IgE水平。这些研究将在第1-2年分阶段进行。我们希望开发一种新的和可预测的吸入毒理学范式，作为联盟活动的一部分，以建立NM安全性筛选的体外和体内研究方案。

DESCRIPTION (provided by applicant): A multidisciplinary group at UCLA (Andre Nel, Jeffrey Zink, Tian Xia, Ning Li) in collaboration with Dr. Vince Castranova at NIOSH and Dr. Lutz Mddler at Bremen University (Germany), aim to establish a mouse inhalation toxicology model to screen combinatorial nanomaterials libraries that are linked to mechanistic injury pathways in tissue culture cells. This research will address the lack of reproducible screening protocols for assessment of nanomaterial (NM) safety. We hypothesize that two combinatorial libraries, which have been designed to (i) adjust the toxic effects of ZnO nanoparticles by changing Zn++ release through iron doping, (ii) adjust the cytotoxicity of cationic mesoporous silica nanoparticles (MSNP) by scaling back the surface cationic density, will be useful to establish a link between in vitro toxicology in human bronchial epithelium (NHBE) and myeloid dendritic cells (DC) and non-allergic and allergic pulmonary inflammation in mice. We posit that the link between non-allergic airway inflammation and cytotoxic injury will be explicable by nanoparticle properties that lead to oxidant injury, shedding of toxic metal ions, and ability to trigger mitochondrial injury. In contrast, the linkage of the material properties to allergic airway inflammation is likely to impact the generation of "danger signals" to DC, which allow them to initiate an immunostimulatory pathway that promotes allergic inflammation. To achieve our long-term goal of developing a predictive inhalation toxicology model, we propose in Aim 1 to characterize the bio-physicochemical properties of NM libraries that are expected to induce differential cytotoxic and pro-inflammatory effects in NHBE and bone marrow-derived DC. In vitro toxicity screening will be carried out by the high content screening (HCS) facility in the California Nano Systems Institute at UCLA as well as cytokine measurements by ELISA. We will also assess ROS production. The NM properties that will be assessed include particle size, size distribution, dispersibility, zeta-potential, TEM, XRD and BET analysis. HCS is carried out with a cocktail of dyes that reveal cellular membrane leakage, DNA damage, mitochondrial depolarization and intracellular Ca2+ flux. The particle characterization and in vitro screening will be carried out at the beginning of year 1 and when new batches are synthesized. Aim 2 will use the libraries to establish standardized protocols for allergic and non-allergic pulmonary inflammation in mice by an inhalation-aspiration approach. The protocol for non-eosinophilic inflammation (Castranova) will measure markers of inflammation, damage, and oxidant stress in the BAL along with lung histology for period of 1 day up to 2 months post-exposure. The allergic sensitization model, which relies on particle and OVA aspiration from the nose of anesthesized animals, will assess OVA-specific IgE levels in parallel with BAL differential cell counts and lung histology. These studies will be carried out in phases through years 1-2. We expect to develop a novel and predictive inhalation toxicology paradigm as a component of the consortium activities to establish in vitro and in vivo study protocols for NM safety screening. PUBLIC HEALTH RELEVANCE: This project addresses the design of cellular and animal screening procedures that can be used to assess the potential danger of engineered nanomaterials that are currently being introduced into the marketplace. We delineate a unique approach in which we use in-house synthesized nanoparticles to develop a predictive toxicological model, meaning that we will develop test protocols in tissue culture cells to obtain basic information about the nanoparticle properties that may render them dangerous and then test those ideas out in a mouse model that may reflect what could happen in the lung when those particles are inhaled. The goal of this project is to develop reliable and reproducible protocols that can be used by academic centers, industry and government agencies as a screen for nanomaterial safety.