Differential Sensitivity to Oxidative Stress during Embryonic Development

胚胎发育过程中对氧化应激的不同敏感性

• 批准号: 8097330
• 负责人: Alicia R Timme-Laragy
• 金额: $ 5.3万
• 依托单位: WOODS HOLE OCEANOGRAPHIC INSTITUTION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097330
• 关键词: Address; Affect; Antioxidants; Area; Blood flow; Calcium; Calcium Signaling; Cardiac; Cardiotoxicity; Cell physiology; Cells; Chemicals; Cocaine; Detection; Development; Diquat; Disease; Embryo; Embryonic Development; Enzymes; Ethanol; Exposure to; Extravasation; Fertilization; Fiber Optics; Fluorescent Probes; Foundations; Gene Expression Profile; Generations; Genes; Genetic Techniques; Glutathione; Glutathione Disulfide; Government Agencies; Heart; Heart Rate; Hour; Human; Hydrogen Peroxide; Hydroquinones; Image; Individual; Industry; Learning; Measurement; Measures; Microelectrodes; Modeling; Morphology; Movement; National Institute of Environmental Health Sciences; Outer Mitochondrial Membrane; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Oxygen Consumption; Pattern; Pesticides; Pharmacologic Substance; Physiological; Population Dynamics; Reactive Oxygen Species; Research; Resistance; Resolution; Respiration; Response Elements; Role; Screening procedure; Signal Transduction; Source; Specificity; Staging; Stem cells; Sulforaphane; Superoxides; System; Techniques; Teratogens; Testing; Time; Toxic effect; Translational Research; Translations; United States National Institutes of Health; Vertebrates; Xenobiotics; Zebrafish; biological adaptation to stress; catalase; developmental toxicology; gain of function; hydroquinone; interdisciplinary approach; interest; multidisciplinary; nanoparticle; optical imaging; oxidative damage; research study; transcription factor

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.

描述（由申请人提供）：脊椎动物的胚胎发育非常容易受到暴露于化学物质（例如药物，农药和纳米颗粒）的破坏。异种生物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破坏”（2）。斑马鱼是发育毒理学的强大模型，在筛查和理解可能的人类毒素（包括通过氧化应激作用的人）具有巨大的使用潜力。关于发育过程中氧化应激反应的尚待了解。我们发现，尽管胚胎在受精后18-52小时（HPF）越来越抗性地耐药，但随后是72-96 hpf之间敏感性提高的令人惊讶的窗口。采用协作和多学科的方法，该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。 AIM 1将确定敏感性的关键窗口，抗氧化剂响应元件转录因子NRF2的作用以及促氧化剂暴露对心脏祖细胞命运决策的影响。这些实验将通过化学发光，心脏功能和细胞命运分析，使用钙成像进行ROS检测，以及胚胎生存研究以及通过功能实验的丧失和增益来评估NRF2的作用。 AIM 2将使用定量实时PCR，总谷胱甘肽，GSH：GSSG：GSSG比率和GSH含量的调制来检查谷胱甘肽的作用，谷胱甘肽是抗氧化剂防御的最丰富成分的作用。 AIM 3将通过使用自我引用的氧气感应微电极和减少ROS的内源性来源来测量发育过程中呼吸和ROS对氧化剂敏感性的生理增加和相关的ROS对氧化剂敏感性的贡献。鉴于政府机构和私营企业的发展越来越多地采用斑马鱼来致致造性和发育毒性，因此尤其重要的是要理解对氧化应激和潜在机制敏感性的关键窗口。这项研究将对氧化应激如何影响胚胎发育有详细的机械理解，并在检查化学物质时确定必要的考虑（以及引起氧化应激的化学混合物。这也将使氧化应激如何影响氧化应激如何影响人类胚胎的发展和进展。