Biomarker discovery for mitochondrial toxicants using metabolic footprinting

摘要 许多环境应激源通过各种机制对线粒体功能产生有害影响， 时间线有不同的长度。线粒体功能障碍有多种临床表现，通常 从细胞器损伤开始就延迟了。目前，报告线粒体功能的生物标记物 使人口能够研究环境暴露及其后果，是缺乏的。我们建议 使用代谢组学方法识别候选生物标记物，比以前更深入 应用于毒物学调查。 新陈代谢分析提供了一个了解细胞和机体功能的窗口，更接近实际情况 生理学比基因组或转录组阵列更重要。使用多个平台进行分离和质量 复杂混合物的光谱分辨，包括有机酸在内的一套全面的代谢物， 氨基酸、类固醇、复合脂质、能量电荷和线粒体运输代谢物可以成为靶点。 我们将利用这项技术来开发线粒体功能障碍的生物标记物，这将填补 当前的环境毒理学研究。 我们将重点研究一种多溴联苯醚，BDE-47，它是正在出现的 美国的主要持久性有机污染物。我们的合作者卡瓦纳博士和我们的 初步数据表明，BDE-47在体外会损害细胞系的线粒体功能。体内的代谢物 细胞外介质(代谢足迹)将与原代小鼠肝细胞分析，主要之一 BDE-47毒性的目标，作为暴露剂量和时间的函数。我们还将检验这一假设 脂肪酸超载将通过对分离的小鼠进行代谢组学分析来发现微小的线粒体缺陷 线粒体。这些研究将提供BDE-47毒性的代谢特征，下一步将扩展到 BDE-47处理的小鼠的血浆和尿液的体内研究。淋巴细胞可能是一种 替代组织对BDE-47的线粒体毒性将使用脂肪酸超负荷试验进行检测。 遗传背景和环境对BDE-47毒性的影响知之甚少。我们会 测试两个潜在的修饰物：1)低和高谷胱甘肽水平的基因工程小鼠，以及2)脂肪 肝脏因维生素A缺乏。这些实验将提供有关潜在高风险的新信息 暴露于BDE-47的人群。这些研究的一个关键问题是候选生物标记物是否具有 具有毒性(除暴露剂量外)。 除了发现BDE-47毒性的代谢特征外，我们还将检查最近的两个 描述了线粒体通过代谢足迹对应激的反应：1)线粒体蛋白毒性 未折叠/未组装蛋白的聚集，以及2)富马酸呼吸对低氧的响应 和电子传递链的远端阻断。通过选择明确的线粒体反应，一个不利的和 一种适应性，我们开始对线粒体功能障碍进行分类，并寻找与 特定类型。在富马酸呼吸的情况下，分泌物中高水平的琥珀酸的标志 代谢物已为人所知。 这项工作是与代谢组学筛选和数据专家奥利弗·费恩密切合作的 以及氧化应激和线粒体毒理学专家Terry Kavanagh。

Abstract 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. We propose 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. We will use this technology to develop biomarkers of mitochondrial dysfunction that will fill an important gap in current studies of environmental toxicology. We will focus our studies 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 our collaborator, Dr. Kavanagh, and our 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. We will also test the hypothesis 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. We will test two potential modifiers: 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 for these studies will be whether candidate biomarkers scale with toxicity (in addition to exposure dose). In addition to discovering metabolic signatures of BDE-47 toxicity, we will examine two recently described mitochondrial responses to stress 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 electron transport chain. By selecting defined mitochondrial responses, one adverse and one adaptive, we 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.