Mechanisms of Embryo Response to Oxidative Stress

描述(申请人提供)：环境暴露引起的氧化应激与多种人类疾病有关，从化学致畸到心血管和神经退行性疾病。发育中的动物似乎对引起氧化应激的化学物质特别敏感。在这些早期生命阶段，抗氧化防御的表达和诱导是影响对氧化剂敏感性的关键因素，但这些反应在胚胎中的个体发育还不是很清楚。在成年动物中，氧化剂通过激活核因子-E2相关因子2及其相关蛋白来启动抗氧化反应，这些蛋白结合到抗氧化反应元件上，并激活谷胱甘肽S转移酶、NAD(P)H-Q氧化还原酶、谷氨酰半胱氨酸连接酶和超氧化物歧化酶等基因的转录。这项研究的总体目标是阐明脊椎动物胚胎在发育过程中对氧化应激做出反应的机制。我们将检验这一中心假设，即对氧化应激的反应和一组受调控的基因在发育过程中会发生变化。由于这些发育差异，某些阶段可能对氧化应激诱导的损伤更敏感。这些研究将使用斑马鱼(Danio Rerio)的胚胎在体内进行，这是一个有价值的模型，可以用来研究发育中动物的毒性机制，并筛选化学物质的发育毒性。目标1将使用转录图谱和表型锚定来确定构成胚胎氧化应激反应的核心基因集，确定氧化应激反应核心基因集的响应性和组成如何随胚胎的发育阶段而变化，并确定在暴露于结构和机械上不同的NRF2激活剂(TBHQ、敌草枯、萝卜硫素)的胚胎中，氧化应激反应的时间和基因谱有何不同。目的2通过用吗啉寡核苷酸靶向下调NRF蛋白的合成，阐明不同的NRF同源基因在体内胚胎发育过程中对氧化应激的转录反应中的作用。目的通过对氧化应答基因启动子的计算和体内实验分析，建立氧化应答基因在胚胎发育过程中的调控机制，建立稳定表达报告基因(GFP)的转基因斑马鱼对氧化应激的应答。最后，我们将测试一组哺乳动物发育毒物在胚胎中激活转基因的能力。这些研究的结果将建立脊椎动物胚胎对氧化应激的转录反应的组成和个体发育，阐明这种反应的基本机制，产生筛选化学物质作为发育毒物或抗氧化剂的活性的工具，并为深入了解氧化应激在人类疾病中的作用提供依据。公共卫生相关性：氧化应激与各种环境影响的人类疾病有关，从化学致畸到心血管和神经退行性疾病。这项拟议的研究将增强我们对一些化学物质如何通过产生氧化应激来干扰胚胎发育以及胚胎如何减轻这些影响的基本理解。这项研究将1)确定胚胎发育过程中抗氧化防御的基本表达和诱导性，2)阐明脊椎动物胚胎对氧化应激反应的基本机制，3)建立作为发育毒物或抗氧化剂的化学物质筛选活性的模型系统，4)提供机制基础，有助于将斑马鱼获得的结果外推到人类，支持风险评估，并深入了解氧化应激在人类疾病中的作用。

DESCRIPTION (provided by applicant): Oxidative stress resulting from environmental exposures is associated with a variety of human diseases ranging from chemical teratogenesis to cardiovascular and neurodegenerative diseases. Developing animals appear to be especially sensitive to chemicals causing oxidative stress. The expression and inducibility of anti-oxidant defenses are critical factors affecting susceptibility to oxidants at these early life stages, but the ontogenic development of these responses in embryos is not well understood. In adult animals, oxidants initiate an anti-oxidant response by activating NF-E2-related factor 2 (NRF2) and related proteins, which bind to the anti-oxidant response element and activate transcription of genes such as glutathione S-transferases, NAD(P)H-quinone oxidoreductase, glutamyl-cysteine ligase, and superoxide dismutase. The overall objective of the research proposed here is to elucidate the mechanisms by which vertebrate embryos respond to oxidative stress during development. We will test the central hypothesis that responsiveness to oxidative stress and the set of regulated genes vary during development. Because of these developmental differences, some stages may be more sensitive to oxidative stress-induced damage. These studies will be performed in vivo using embryos of the zebrafish (Danio rerio), a valuable model in which to examine mechanisms of toxicity in developing animals and to screen chemicals for developmental toxicity. Aim 1 will use transcriptional profiling and phenotypic anchoring to identify the core set of genes that comprise the oxidative stress response in embryos, establish how the responsiveness and composition of the core set of oxidant-responsive genes vary with developmental stage of embryos, and determine how the timing and gene profile of the oxidative stress response differ in embryos exposed to structurally and mechanistically distinct activators of NRF2 (tBHQ, diquat, sulforaphane). Aim 2 will elucidate the roles of different NRF paralogs in the transcriptional response to oxidative stress during development in embryos in vivo, using targeted knock-down of NRF protein synthesis with morpholino oligonucleotides. Aim 3 will establish the mechanism of regulation of anti-oxidant response genes during embryonic development through computational and in vivo experimental analysis of oxidant-responsive gene promoters, leading to the generation of a stable line of transgenic zebrafish expressing a reporter gene (GFP) in response to oxidative stress. Finally, we will test a set of mammalian developmental toxicants for the ability to activate the transgene in embryos. The results of these studies will establish the composition and ontogeny of the transcriptional response to oxidative stress in vertebrate embryos, elucidate fundamental mechanisms underlying this response, generate tools for screening chemicals for activity as developmental toxicants or antioxidants, and provide insight into the role of oxidative stress in human disease. PUBLIC HEALTH RELEVANCE: Oxidative stress is involved in a variety of environmentally influenced human diseases ranging from chemical teratogenesis to cardiovascular and neurodegenerative diseases. The proposed research will enhance our basic understanding of how some chemicals interfere with embryonic development by generating oxidative stress and how embryos can mitigate these effects. This research will i) determine the basal expression and inducibility of anti-oxidant defenses during embryonic development, ii) elucidate fundamental mechanisms of the response of vertebrate embryos to oxidative stress, iii) establish a model system for screening chemicals for activity as developmental toxicants or antioxidants, and iv) provide a mechanistic foundation that will facilitate the extrapolation of results obtained in zebrafish to humans, supporting risk assessment and providing insight into the role of oxidative stress in human disease.