Development of methods and models for nanoparticle toxicity screening: Applicatio

纳米颗粒毒性筛选方法和模型的开发：应用

• 批准号: 7455201
• 负责人: Andre Elias Nel
• 金额: $ 12.7万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7455201
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DESCRIPTION (provided by applicant): Data for performing a preliminary risk assessment of manufactured nanomaterials are just beginning to emerge. However, early studies of nanomaterial toxicity in aqueous media have tended to be more observational than mechanistic, and have often focused on a single, advanced stage of toxicity that could yield contradictory results. Moreover, the ability to generalize findings to other nanomaterials is limited by the lack of a rational basis for categorizing nanomaterials. Elucidating the mechanisms of toxicity for a given nanomaterial will provide a basis for classifying materials for regulatory purposes, postulating dose-response curves, screening potential risks, and prescribing strategies for risk management. The primary objective of this work is to elucidate the mechanism(s) by which manufactured nanoparticles may induce toxicity in vitro and in vivo. Specifically, this study will consider fullerene-based materials, comparing them with, (i) reference standards (TiO2 and carbon black); (ii) ultrafine particles obtained from an urban airshed (well characterized by in vitro toxicology studies); We will explore a methodology for rapidly screening potentially toxic nanoparticles based on their propensity to generate ROS. The principal hypothesis is that certain classes of nanoparticles such as fullerenes induce ROS production, cellular oxidative stress and cytotoxicity. Fullerenes are selected based on the relatively novel properties (e.g. strength.arid electron affinity) that make them attractive for commercialization. The investigators propose that oxidative stress induced by fullerene derivatives occurs in several stages (tiers), beginning with the induction of phase II antioxidant defenses at the lowest tier of oxidative stress (tier 1), followed by pro-inflammatory (tier 2) and mitochondrion-mediated cytotoxic effects (tier 3) as the level of oxidative stress increases. Particle size, shape, surface area, charge, and chemical composition are important physical variables that could determine their ROS-generating or scavenging properties. Rapid physicochemical determination of ROS production might provide a paradigm to assess the possible toxicity of nanomaterials that act via these mechanisms. Specific Aim 1 will characterize commercial nanoparticles and their derivatives in terms of particle size, shape, surface area, charge, aqueous solubility, propensity to aggregate, and their ability to catalyze or quench ROS production in vitro. Materials will also be characterized in model solutions containing naturally occurring organic matter, proteins and ions at levels similar to those present in natural waters. Aim 2 will determine whether various fullerenes can generate a hierarchical oxidative stress response in macrophages, bronchial epithelial cells, endothelial cells, neural cells and hepatocytes. This will be accomplished by comparing the effects of fullerenes and reference nanoparticles on, (i) phase II enzyme expression and activation of the heme oxygenase 1 (HO-1) promoter (tier 1); (ii) cytokine and chernokine expression as well as assays for MAP kinase activation (tier 2); (iii) mitochondria! perturbation and induction of cellular apoptosis (tier 3). These biological responses will be compared to the physicochemieal properties of nanomaterials elucidated in Aim 1. Aim 3 will perform in vivo imaging of the oxidative stress-sensitive HO-1 promoter linked to a luciferase reporter in transgenic mice. Organs and tissues showing increased luciferase activity will be investigated for histological evidence of inflammation and cytotoxicity. Aim 4 will compare the biologic responses elicited by each of the nano-scale particles with their ability to generate ROS abiotically, and test the hypothesis that ROS generation can be used to screen toxicity. By focusing on mechanisms of toxicity rather than outcomes alone, this work will provide the basis for classifying nanomaterials for regulatory purposes. Based on preliminary results presented in this proposal, we anticipate that ROS generation in solution and under UV radiation will be good predictors of nanoparticle toxicity and that ROS measurements can be adapted to screen nanomaterials. A broader assessment of nanomaterial toxicity in the context of the hierarchical oxidative stress response is likely to yield a more sensitive paradigm for toxicity testing, perhaps resolving inconsistencies reported in the literature.

描述（由申请人提供）： 用于对人造纳米材料进行初步风险评估的数据刚刚开始出现。然而，早期对水性介质中纳米材料毒性的研究往往更多地是观察性的，而不是机理性的，并且往往集中在一个单一的、晚期的毒性阶段，这可能会产生相互矛盾的结果。此外，由于缺乏合理的纳米材料分类基础，将研究结果推广到其他纳米材料的能力受到限制。阐明给定纳米材料的毒性机制将为监管目的的材料分类、假定剂量-反应曲线、筛选潜在风险和制定风险管理策略提供基础。这项工作的主要目的是阐明机制（S），其中制造的纳米粒子可能会在体外和体内诱导毒性。具体而言，本研究将考虑富勒烯基材料，将其与以下各项进行比较：（i）参考标准（TiO 2和炭黑）;（ii）从城市空气中获得的超细颗粒（通过体外毒理学研究进行了很好的表征）;我们将探索一种基于其产生ROS的倾向快速筛选潜在毒性纳米颗粒的方法。主要假设是某些类别的纳米颗粒如富勒烯诱导ROS产生、细胞氧化应激和细胞毒性。富勒烯的选择是基于相对新颖的性质（例如强度和电子亲和力），使它们具有商业化的吸引力。研究人员提出，由富勒烯衍生物诱导的氧化应激发生在几个阶段（层），从氧化应激最低层（层1）的第二阶段抗氧化防御的诱导开始，随着氧化应激水平的增加，随后是促炎（层2）和炎症介导的细胞毒性作用（层3）。颗粒大小、形状、表面积、电荷和化学组成是重要的物理变量，可以决定其ROS生成或清除特性。ROS产生的快速物理化学测定可能提供一个范例，以评估通过这些机制发挥作用的纳米材料的可能毒性。 具体目标1将表征商业纳米颗粒及其衍生物的粒度，形状，表面积，电荷，水溶性，聚集倾向，以及它们在体外催化或淬灭ROS产生的能力。还将在含有天然存在的有机物、蛋白质和离子的模型溶液中对材料进行表征，这些物质的水平与天然沃茨中存在的水平相似。目的2将确定各种富勒烯是否可以在巨噬细胞、支气管上皮细胞、内皮细胞、神经细胞和肝细胞中产生分级氧化应激反应。这将通过比较富勒烯和参考纳米颗粒对以下各项的影响来实现：（i）II相酶表达和血红素加氧酶1（HO-1）启动子的活化（第1层）;（ii）细胞因子和切诺因子表达以及MAP激酶活化的测定（第2层）;（iii）线粒体！干扰和诱导细胞凋亡（第3层）。这些生物学反应将与目标1中阐明的纳米材料的物理化学性质进行比较。 目的3将在转基因小鼠中对与荧光素酶报告基因连接的氧化应激敏感的HO-1启动子进行体内成像。将研究荧光素酶活性增加的器官和组织是否存在炎症和细胞毒性的组织学证据。目标4将比较由每种纳米级颗粒引起的生物反应与它们非生物产生ROS的能力，并测试ROS产生可用于筛选毒性的假设。 通过关注毒性机制而不仅仅是结果，这项工作将为监管目的的纳米材料分类提供基础。基于本提案中提出的初步结果，我们预计溶液中和UV辐射下的ROS生成将是纳米颗粒毒性的良好预测因子，并且ROS测量可用于筛选纳米材料。在分级氧化应激反应的背景下，对纳米材料毒性进行更广泛的评估可能会产生更敏感的毒性测试范例，或许可以解决文献中报告的不一致性。