Real time quantitative assessment of oxidative stress as a marker for differential nanoparticle toxicity

了解纳米颗粒（NPs）与生物系统的相互作用，并评估暴露于NPs如何影响生命系统中的生物和化学机制是至关重要的。其中一个关键的纳米毒性机制是通过产生活性氧（ROS）和氮（RNS）来诱导氧化应激的潜力。然而，评估NPs诱导氧化应激的程度一直是一个挑战，因为大多数ROS和RNS具有高活性且寿命短，因此难以检测到。本研究旨在解决这一基本问题，通过开发直接实时评估活体水生系统、斑马鱼胚胎和2周幼鱼NPs积累位点的ROS和RNS物种的方法，并将观察到的氧化效应与组织和器官水平的细胞毒性联系起来，包括氧化/抗氧化生物机制的改变、细胞损伤、炎症诱导和凋亡。智力优势：生命系统暴露于NPs可能会改变细胞、组织和器官的抗氧化防御系统和氧化还原机制。本提案的目标是获得氧化应激反应机制的基本理解，由于暴露于工程NPs在一个完整的活生物体：斑马鱼。研究工作将集中在直接检测ROS/RNS的新型纳米毒性探针的工程设计，以及它们的应用，以确定NPs是否产生自由基或由于毒性而改变积累NPs的器官的生理氧化状态。结果将提供暴露于不同剂量NPs后斑马鱼肠道中ROS/RNS的局部浓度谱。研究将确定调节与ROS/RNS物种相互作用的NPs的特性，并确定表面反应活性和反应动力学的作用。将研究斑马鱼细胞和组织的损伤、畸形和生存能力与ROS/RNS产生的关系。通过微电极对单NP碰撞研究的体外电化学测量，对颗粒的物理化学和表面性质在与活性ROS/RNS物种接触时如何变化的基本了解，也将用于建立NPs诱导氧化应激的预测模型。该技术可广泛用于测量各种其他器官、细胞培养、组织和其他环境条件下的氧化状态。这些数据将用于NPs的风险评估，这将有助于开发一种使用相对简单、廉价和快速筛选方法预测NPs诱导氧化应激的新范式。该方法的成功将使NPs的高通量筛选更快，作为动物实验的替代方案。更广泛的影响：该项目将为纳米毒性评估创造新的工具和方法，并产生基础知识，以解决理解NPs暴露于环境和生命系统的影响的关键问题，特别是与氧化应激有关的问题。拟议研究的结果将提供生物系统中NPs的行为和运输的“氧化概况”，从材料特性到器官和组织反应。它将促进新型纳米毒性探针的开发，用于直接评估NPs诱导的氧化应激，并允许鉴定NPs表面性质和反应性的关键因素，这些因素可用于预测毒性，允许靶向筛选，并允许基于结构-毒性信息控制生成新的，更安全的NPs。了解这些机制可以为修改NPs提供指导，以防止这种类型的损害。此外，这项工作将使本科生和研究生，特别是少数民族和妇女，在纳米毒理学和可持续材料开发与生物系统界面领域的教育和培训成为可能，并使他们意识到新开发的工程材料在环境、生态系统和生物系统中的潜在影响和风险。氧化应激和环境与健康影响的概念将在纽约州北部的当地高中介绍。将为克拉克森大学跨学科的生物技术、材料科学和环境科学与工程专业开设一门关于纳米技术对环境健康和安全影响的课程，并广泛传播到附近的四年制大学，并在克拉克森网站上开放。

CBET - 1336493 Understanding the interaction of nanoparticles (NPs) with biological systems and assessing how exposure to NPs affects biological and chemical mechanisms in living systems is of critical importance. One of the key nanotoxicity mechanisms is the potential for induction of oxidative stress by generating reactive oxygen (ROS) and nitrogen (RNS) species. However, assessing the extent of NPs induced oxidative stress has been a challenge as most ROS and RNS are highly reactive and short lived and therefore difficult to detect. This proposal addresses this fundamental problem by developing methodologies for direct real-time assessment of ROS and RNS species at the NPs accumulation site in a living aquatic system, zebrafish embryos and 2 week juveniles,and correlating the observed oxidative effects with their cytotoxicity at the tissue and organ level including alteration of the oxidative/anti-oxidative biological mechanism, cellular damage, inflammation induction, and apoptosis.Intellectual Merit :Exposure of living systems to NPs may alter the antioxidant-defense system and redox mechanisms in cells, tissues and organs. The goal of this proposal is to gain fundamental understanding of the mechanism of oxidative stress response due to exposure to engineered NPs in an intact live organism: zebrafish. Research work will focus on the engineering design of new nanotoxicity probes for direct detection of ROS/RNS and their use to establish whether NPs generate free radicals or change the physiological oxidative status of organs that accumulate NPs due to toxicity. Results will provide local concentration profiles of ROS/RNS in the zebrafish intestine following exposure to various doses of NPs. Research will identify properties of the NPs that regulate interactions with ROS/RNS species and determine the role of surface reactivity and reaction kinetics. Cell and tissue damage, malformations and viability will be studied and related with ROS/RNS production in zebrafish. Fundamental understanding on how the physicochemical and surface properties of the particles change in contact with reactive ROS/RNS species, quantified by in vitro electrochemical measurements of single NP collision studies with microelectrodes, will also be used to establish predictive models of NPs induced oxidative stress. The technology can be widely used to measure oxidative status in a variety of other organs, cell cultures, tissues, and other environmental conditions. The data will be used for risk assessment of NPs which will facilitate development of a new paradigm for predicting NPs induced oxidative stress using a relatively simple, inexpensive and rapid screening method. The success of this method would enable faster high throughput screening of NPs, as an alternative to animal experimentation. Broader Impacts :The project will create new tools and methodologies for nanotoxicity assessment and generate fundamental knowledge that can solve key questions in understanding the effect of NPs exposure to the environment and living systems, specifically those related to oxidative stress. The results of the proposed studies will provide an "oxidative profile" of the behavior and transport of NPs in biological systems starting from the material characteristics to organ and tissue response. It will enable development of novel nanotoxicity probes for direct assessment of NPs induced oxidative stress and permit identification of the key factors in the NPs surface properties and reactivity that can be used to predict toxicity, permit targeted screening, and allow controlled generation of new, safer NPs based on structure-toxicity information. Understanding these mechanisms may provide guidelines for modifications to NPs to prevent this type of damage. Moreover, this effort will enable the education and training of undergraduate and graduate students, especially minorities and women, in the field of nanotoxicology and sustainable material development at the interface with biological systems, and make them aware of the potential implications and risks of the newly developed engineered materials in the environment, ecosystem and biological systems. Concepts of oxidative stress and environmental and health impacts will be introduced in local high schools in upstate New York. A course on environmental health and safety implications of nanotechnology will be created for the interdisciplinary Biotechnology, Materials Science and the Environmental Science and Engineering programs at Clarkson University, and broadly disseminated to nearby 4-year colleges and open access on the Clarkson website.