Development of methods and models for nanoparticle toxicity screening: Applicatio

描述(由申请人提供)： 对人造纳米材料进行初步风险评估的数据才刚刚开始出现。然而，早期对纳米材料在水介质中的毒性的研究往往更多的是观察性的，而不是机械性的，而且往往集中在单一的高级毒性阶段，可能会产生相互矛盾的结果。此外，由于缺乏对纳米材料进行分类的合理基础，将研究结果推广到其他纳米材料的能力受到限制。阐明特定纳米材料的毒性机制将为出于监管目的对材料进行分类、推定剂量-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制成的纳米粒在体外和体内可能产生毒性的机制(S)。具体地说，这项研究将考虑基于富勒烯的材料，将它们与(I)参考标准(二氧化钛和碳黑)；(Ii)从城市机场获得的超细颗粒(体外毒理学研究很好地表征)进行比较；我们将探索一种基于其产生ROS的倾向来快速筛选潜在有毒纳米颗粒的方法。主要的假设是某些类型的纳米粒子，如富勒烯，会诱导ROS的产生，细胞氧化应激和细胞毒性。富勒烯是基于相对新颖的性质(例如，强度和电子亲和力)而选择的，这些性质使它们具有商业化吸引力。研究人员提出，富勒烯衍生物诱导的氧化应激发生在几个阶段(TILES)，首先是在氧化应激的最低层诱导第二阶段的抗氧化防御(TIER 1)，然后是随着氧化应激水平的增加而产生促炎(TIER 2)和线粒体介导的细胞毒效应(TIER 3)。颗粒大小、形状、比表面积、电荷和化学成分是决定其产生或清除ROS特性的重要物理变量。ROS产生的快速物理化学测定可能为评估通过这些机制作用的纳米材料的可能毒性提供一个范例。 具体目标1将从颗粒大小、形状、比表面积、电荷、水溶解性、聚集倾向以及它们在体外催化或抑制ROS产生的能力等方面表征商业纳米颗粒及其衍生物。材料还将在模型溶液中进行表征，其中包含自然产生的有机物、蛋白质和离子，其水平与自然水中存在的水平相似。目的2将确定不同富勒烯是否能在巨噬细胞、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粒对以下方面的影响来实现：(I)第二相酶表达和激活血红素加氧酶1(HO-1)启动子(TIER 1)；(Ii)细胞因子和Chernokine的表达以及MAP激酶激活的分析(TIER 2)；(Iii)线粒体！细胞凋亡的扰动和诱导(第3层)。这些生物反应将与AIM 1中阐明的纳米材料的物理化学性质进行比较。AIM 3将在转基因小鼠中对氧化应激敏感的HO-1启动子进行体内成像，该启动子与荧光素酶报告相连。显示荧光素酶活性增加的器官和组织将被调查以寻找炎症和细胞毒性的组织学证据。目的4将比较每一种纳米粒子引起的生物反应与它们非生物产生ROS的能力，并检验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，这项工作将为出于监管目的对纳米材料进行分类提供基础。根据这项建议中提出的初步结果，我们预计在溶液中和在紫外线辐射下产生的ROS将是纳米颗粒毒性的良好预测指标，ROS测量可以用于筛选纳米材料。在分级氧化应激反应的背景下对纳米材料的毒性进行更广泛的评估，可能会为毒性测试产生更敏感的范例，或许可以解决文献中报告的不一致之处。

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.