Differential Sensitivity to Oxidative Stress during Embryonic Development

描述（由申请人提供）：脊椎动物的胚胎发育极易因暴露于化学品（如药物、农药和纳米颗粒）而受到破坏。外源性物质还可以通过产生活性氧（ROS）和氧化应激（1）引起发育性心脏毒性，定义为“氧化还原信号和控制的中断”（2）。斑马鱼是发育毒理学的一个强大模型，在筛选和了解可能的人类致畸剂方面具有巨大的潜力，包括那些通过氧化应激起作用的致畸剂。关于发育过程中的氧化应激反应还有很多东西有待了解。我们发现，虽然胚胎在受精后18-52小时（hpf）对促氧化剂暴露的抵抗力越来越强，但随后在72-96 hpf之间出现了一个令人惊讶的敏感性提高的窗口。采取合作和多学科的方法，本提案的目的是了解胚胎发生过程中对促氧化剂的差异敏感性所涉及的机制。目的1将确定敏感性的关键窗口，抗氧化反应元件转录因子NRF 2的作用，以及促氧化剂暴露对心脏祖细胞命运决定的影响。这些实验将涉及通过化学发光进行ROS检测，使用钙成像进行心脏功能和细胞命运分析，以及胚胎存活研究和通过功能丧失和获得实验评估NRF 2的作用。目的2将研究谷胱甘肽的作用，最丰富的抗氧化防御的组成部分，使用定量实时PCR，总谷胱甘肽，GSH：GSSG的比例，和GSH含量的调制测量。目的3将检查呼吸和相关的活性氧对氧化剂敏感性的生理增加的贡献，通过测量氧消耗量，使用自参考氧传感微电极和减少内源性来源的活性氧的过表达的抗氧化酶的线粒体外膜。鉴于政府机构和私营企业越来越多地使用斑马鱼进行致畸性和发育毒性研究，了解氧化应激敏感性的关键窗口及其潜在机制尤为重要。这项研究将提供氧化应激如何影响胚胎发育的详细和机械的理解，并确定在检查化学品（和引起氧化应激的化学混合物）时需要考虑的重要因素。它还将导致更好地了解氧化应激如何影响人类胚胎毒性的发展和进展以及由此产生的致畸作用和疾病。

DESCRIPTION (provided by applicant): The embryonic development of vertebrates is highly susceptible to disruption by exposure to chemicals, such as pharmaceuticals, pesticides, and nanoparticles. Xenobiotics can also cause developmental cardiotoxicity through generation of reactive oxygen species (ROS) and oxidative stress (1), defined as "a disruption of redox signaling and control" (2). The zebrafish is a powerful model in developmental toxicology and has great potential for use in screening and understanding possible human teratogens, including those that act via oxidative stress. Much remains to be learned about the oxidative stress response during development. We have found that while embryos grow increasingly resistant to pro-oxidant exposure from 18-52 hours post fertilization (hpf), this is followed by a surprising window of heightened sensitivity between 72-96 hpf. Taking a collaborative and multidisciplinary approach, the objective of this proposal is to understand the mechanisms involved in the differential sensitivity to pro-oxidants during embryogenesis. Aim 1 will identify critical windows of sensitivity, the role of the antioxidant response element transcription factor NRF2, and the impact of pro-oxidant exposure on cardiac progenitor cell fate decisions. These experiments will involve ROS detection by chemiluminescence, cardiac function and cell fate analysis using calcium imaging, as well as embryo survival studies and assessment of the role of NRF2 through loss and gain of function experiments. Aim 2 will examine the role of glutathione, the most abundant component of antioxidant defenses, using quantitative real-time PCR, measurements of total glutathione, GSH:GSSG ratios, and modulation of GSH content. Aim 3 will examine the contribution of a physiologic increase in respiration and associated ROS towards oxidant sensitivity during development by measuring oxygen consumption using self-referencing oxygen sensing microelectrodes and reducing endogenous sources of ROS by over-expressing antioxidant enzymes on the outer mitochondrial membrane. Given the growing movement of government agencies and private industry to employ zebrafish for teratogenicity and developmental toxicity, it is especially important to comprehend the critical windows of sensitivity to oxidative stress and the underlying mechanisms. This research will provide a detailed and mechanistic understanding of how oxidative stress can impact embryonic development, and identify important considerations necessary when examining chemicals (and chemical mixtures that cause oxidative stress. It will also result in a better understanding of how oxidative stress influences the development and progression of human embryotoxicity and resulting teratogenesis and diseases.