Biomarker discovery for mitochondrial toxicants using metabolic footprinting

使用代谢足迹发现线粒体毒物的生物标志物

• 批准号: 8218308
• 负责人: DAVID M. HOCKENBERY
• 金额: $ 39.56万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-21 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218308
• 关键词: Affect; Amino Acids; Biochemical; Biochemistry; Bioenergetics; Biological Assay; Biological Markers; Biology; Body Fluids; Cell Line; Cell physiology; Cells; Characteristics; Charge; Classification; Clinical; Collaborations; Complex; Complex Mixtures; Data; Data Analyses; Defect; Disease; Distal; Dose; Environment; Environmental Exposure; Exhibits; Exposure to; Fatty Acids; Fatty Liver; Fumarates; Functional disorder; Generations; Genetic; Genetically Engineered Mouse; Genomics; Glutathione; Harvest; Hepatocyte; Histocompatibility Testing; Hypoxia; In Vitro; Investigation; Length; Lipids; Liquid substance; Lymphocyte; Machine Learning; Membrane Potentials; Metabolic; Metabolic syndrome; Methods; Mitochondria; Mitochondrial Diseases; Modeling; Mus; Organelles; Oxidative Stress; Physiology; Plasma; Population; Population Study; Pre-Clinical Model; Proteins; Publishing; Reactive Oxygen Species; Reporting; Resolution; Respiration; Screening procedure; Staging; Steroids; Stress; Succinates; Technology; Testing; Time; TimeLine; Tissues; Toxic Environmental Substances; Toxic effect; Toxicology; Urine; Vitamin A; Vitamin A Deficiency; Work; base; environmental stressor; environmental toxicology; extracellular; high risk; human population study; in vivo; insight; metabolomics; mitochondrial dysfunction; nonalcoholic steatohepatitis; novel; nucleotide metabolism; organic acid; phenyl ether; pollutant; protein misfolding; public health relevance; research study; response; statistics; toxicant; transcriptomics

DESCRIPTION (provided by applicant): Many environmental stressors have deleterious effects on mitochondrial functions, by a variety of mechanisms, and with timelines of different lengths. Mitochondrial dysfunction has multiple clinical presentations, often delayed from the onset of organelle damage. At present, biomarkers that report on mitochondrial function, to enable population studies of environmental exposures and their consequences, are lacking. This proposal seeks to identify candidate biomarkers using a metabolomics approach, in greater depth than has previously been applied to toxicologic investigations. Metabolomic analysis provides a window on cellular and organismal functions, closer to the actual physiology than genomic or transcriptomic arrays. Using multiple platforms for separation and mass spectrometric resolution of complex mixtures, a comprehensive set of metabolites including organic acids, amino acids, steroids, complex lipids, energy charge and mitochondrial transport metabolites can be targeted. This technology will be employed to develop biomarkers of mitochondrial dysfunction that will fill an important gap in current studies of environmental toxicology. Studies here will focus on a polybrominated diphenyl ether, BDE-47, that is emerging as one of the major persistent organic pollutants in the U.S. Published data from a collaborator, Dr. Kavanagh, and preliminary data indicate that BDE-47 impairs mitochondrial function in cell lines in vitro. Metabolites in extracellular media (metabolic footprinting) will be analyzed with primary mouse hepatocytes, one of the main targets of BDE-47 toxicity, as a function of dose and time of exposure. The hypothesis to be tested is that fatty acid overload will uncover subtle mitochondrial defects by performing metabolomic analysis in isolated mitochondria. These studies will provide metabolic signatures of BDE-47 toxicity that will next be extended to in vivo studies of plasma and urine from BDE-47 treated mice. The possibility that lymphocytes may be a surrogate tissue for the mitochondrial toxicities of BDE-47 will be examined using the fatty acid overload assay. The effects of genetic background and environment on BDE-47 toxicity are poorly understood. Two potential modifiers will be examined: 1) genetically engineered mice with low and high glutathione levels, and 2) fatty liver due to vitamin A deficiency. These experiments will provide novel information on potential high risk populations for BDE-47 exposure. A key question will be whether candidate biomarkers scale with toxicity (in addition to exposure dose). In addition to discovering metabolic signatures of BDE-47 toxicity, two recently described mitochondrial responses to stress will be tested by metabolic footprinting: 1) mitochondrial proteotoxicity due to aggregation of unfolded/unassembled proteins, and 2) alternative fumarate respiration in response to hypoxia and distal block of the ETC. By selecting defined mitochondrial responses, one adverse and one adaptive, once can begin to categorize mitochondrial dysfunction and look for signatures that associate with specific types. In the case of fumarate respiration, a signature of high levels of succinate in secreted metabolites is already known. This work is a close collaboration with Oliver Fiehn, expert in metabolomics screening and data analysis, and Terry Kavanagh, an expert in oxidative stress and mitochondrial toxicology. PUBLIC HEALTH RELEVANCE: Public Health Relevance/Project Narrative Many environmental toxins inhibit mitochondria, the powerhouses of the cell, as a mechanism of toxicity. Current research in this area is hampered by the lack of convenient tests to measure early mitochondrial damage, which would promote appropriate preventive/treatment efforts for individuals and at risk populations. The goal of this research is to identify chemical changes in blood or urine that are indicators of mitochondrial damage in the body, using mass spectrometry tools to survey thousands of compounds.

描述（由申请人提供）：许多环境压力源通过各种机制和不同时间长度对线粒体功能产生有害影响。线粒体功能障碍具有多种临床表现，通常延迟于细胞器损伤的发作。目前，缺乏报告线粒体功能的生物标志物，以使环境暴露及其后果的人口研究成为可能。本提案旨在使用代谢组学方法识别候选生物标志物，比以前应用于毒理学研究的方法更深入。代谢组学分析提供了细胞和有机体功能的窗口，比基因组或转录组学阵列更接近实际生理学。使用多种平台进行复杂混合物的分离和质谱分析，可以针对包括有机酸、氨基酸、类固醇、复杂脂质、能量电荷和线粒体运输代谢物在内的综合代谢物。这项技术将用于开发线粒体功能障碍的生物标志物，这将填补当前环境毒理学研究中的一个重要空白。这里的研究将集中在多溴联苯醚，BDE-47，这是美国主要的持久性有机污染物之一，合作者Kavanagh博士发表的数据和初步数据表明，BDE-47在体外会损害细胞系的线粒体功能。细胞外介质中的代谢物（代谢足迹）将与原代小鼠肝细胞（BDE-47毒性的主要靶点之一）进行分析，作为剂量和暴露时间的函数。要测试的假设是脂肪酸过载将通过对分离的线粒体进行代谢组学分析来发现细微的线粒体缺陷。这些研究将提供BDE-47毒性的代谢特征，接下来将扩展到BDE-47治疗小鼠的血浆和尿液的体内研究。淋巴细胞可能是BDE-47线粒体毒性的替代组织的可能性将使用脂肪酸过载试验进行检查。遗传背景和环境对BDE-47毒性的影响尚不清楚。将研究两种可能的修饰剂：1)谷胱甘肽水平高低的基因工程小鼠，以及2)维生素A缺乏导致的脂肪肝。这些实验将提供有关BDE-47潜在高危人群的新信息。一个关键问题将是候选生物标志物是否与毒性（除了暴露剂量）有关。除了发现BDE-47毒性的代谢特征外，还将通过代谢足迹测试最近描述的两种线粒体应激反应：1)未折叠/未组装蛋白质聚集引起的线粒体蛋白质毒性，以及2)缺氧和ETC远端阻断时富马酸呼吸的替代反应。通过选择明确的线粒体反应，一种是不利的，一种是适应的，一旦可以开始对线粒体功能障碍进行分类，并寻找与特定类型相关的特征。在富马酸呼吸的情况下，高水平的琥珀酸分泌代谢物的特征是已知的。这项工作是与代谢组学筛选和数据分析专家Oliver Fiehn和氧化应激和线粒体毒理学专家Terry Kavanagh的密切合作。