Mechanisms of Embryo Response to Oxidative Stress

项目概要/摘要 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。发展中的动物 似乎对引起氧化应激的化学物质特别敏感。抗- 氧化剂防御是影响这些早期生命阶段对氧化剂敏感性的关键因素， 这些反应在胚胎中的个体发育还没有很好的理解。在成年动物中， 通过激活NF-E2相关因子2（NRF 2）和相关蛋白质来启动抗氧化反应， 抗氧化反应元件并激活基因如谷胱甘肽S-转移酶的转录， NAD（P）H-醌氧化还原酶、谷氨酰-半胱氨酸连接酶和超氧化物歧化酶。总体目标 本文提出的研究的目的是阐明脊椎动物胚胎对 发育过程中的氧化应激。我们将测试的核心假设，反应氧化应激 而受调控的基因组在发育过程中也会发生变化。由于这些发展差异，一些 阶段可能对氧化应激诱导的损伤更敏感。这些研究将在体内进行 使用斑马鱼（Danio rerio）的胚胎，这是一种有价值的模型，用于研究 发展中的动物和筛选化学品的发育毒性。 目的1将使用转录谱和表型锚定来确定核心基因组， 包括胚胎中的氧化应激反应，建立如何反应和组成的 一组核心的氧化剂反应基因随胚胎发育阶段而变化，并决定了胚胎发育的时机。 和基因谱的氧化应激反应不同的胚胎暴露于结构和机制 NRF 2的不同活化剂（tBHQ、敌草快、萝卜硫素）。目标2将阐明不同的NRF的作用 旁系同源物在体内胚胎发育过程中对氧化应激的转录反应中，使用 用吗啉代寡核苷酸靶向敲低NRF蛋白合成。目标3将建立 胚胎发育过程中抗氧化反应基因的调控机制 和氧化剂响应基因启动子的体内实验分析，导致产生稳定的 转基因斑马鱼品系表达报告基因（GFP），对氧化应激作出反应。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转录反应的组成和个体发生， 脊椎动物胚胎中的氧化应激，阐明这种反应的基本机制，产生 用于筛选化学品作为发育毒物或抗氧化剂的活性的工具，并提供深入了解 氧化应激在人类疾病中的作用。

Project Summary/Abstract 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.