Differential Sensitivity to Oxidative Stress during Embryonic Development

描述(申请人提供)：脊椎动物的胚胎发育非常容易受到药物、杀虫剂和纳米颗粒等化学品的干扰。外源生物还可通过产生活性氧簇(ROS)和氧化应激(1)造成发育中的心脏毒性，其定义为“氧化还原信号和控制的中断”(2)。斑马鱼是发育毒理学中的一个强大的模型，在筛选和了解可能的人类致畸物质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物质。关于发育过程中的氧化应激反应，还有很多需要了解的地方。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，但紧随其后的是72-96 HPF之间的一段令人惊讶的高敏感性窗口。采用协作和多学科的方法，这项建议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将确定敏感的关键窗口，抗氧化反应元件转录因子NRF2的作用，以及促氧化剂暴露对心脏祖细胞命运决定的影响。这些实验将包括通过化学发光检测ROS，使用钙成像技术分析心脏功能和细胞命运，以及胚胎存活研究和通过功能丧失和获得实验评估NRF2的作用。目的2将利用实时定量聚合酶链式反应、测定总谷胱甘肽、GSH/GSSG比率以及调节GSH含量来研究谷胱甘肽的作用，谷胱甘肽是抗氧化防御中含量最丰富的成分。目的3将通过使用自参照氧传感微电极测量氧气消耗，并通过在线粒体膜外过度表达抗氧化酶来减少内源性ROS来源，来研究在发育过程中呼吸和相关ROS的生理性增加对氧化敏感性的贡献。鉴于政府机构和私营企业越来越多地将斑马鱼用于致畸和发育毒性，了解对氧化应激敏感的关键窗口及其潜在机制尤为重要。这项研究将提供对氧化应激如何影响胚胎发育的详细和机理的理解，并确定在检查导致氧化应激的化学品(和化学混合物)时所需的重要考虑因素。它还将有助于更好地理解氧化应激如何影响人类胚胎毒性的发展和进展，以及由此导致的畸形和疾病。

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.