Biomarker discovery for mitochondrial toxicants using metabolic footprinting

摘要 许多环境应激源通过多种机制对线粒体功能产生有害影响， 时间线长短不一线粒体功能障碍有多种临床表现，通常 延迟了细胞器损伤的发生目前，报告线粒体功能的生物标志物， 缺乏对环境暴露及其后果的人口研究。我们建议 使用代谢组学方法更深入地鉴定候选生物标志物， 用于毒理学研究。 代谢组学分析为细胞和生物体功能提供了一个窗口，更接近于实际 比基因组或转录组阵列更好。使用多个平台进行分离和质量 复杂混合物的光谱解析，包括有机酸在内的一整套代谢物， 可以靶向氨基酸、类固醇、复合脂质、能量电荷和线粒体转运代谢物。 我们将利用这项技术开发线粒体功能障碍的生物标志物，这将填补 环境毒理学研究现状。 我们将把研究重点放在多溴二苯醚BDE-47上， 主要持久性有机污染物在美国公布的数据，从我们的合作者，博士卡瓦纳，和我们的 初步数据表明，BDE-47会损害体外细胞系的线粒体功能。代谢产物 细胞外介质（代谢足迹）将用原代小鼠肝细胞进行分析， BDE-47毒性的目标，作为剂量和接触时间的函数。我们还将检验以下假设： 脂肪酸超载将通过对分离的线粒体进行代谢组学分析， 线粒体这些研究将提供BDE-47毒性的代谢特征，下一步将扩大到 对BDE-47处理小鼠的血浆和尿液进行的体内研究。淋巴细胞可能是一种 将使用脂肪酸过载试验对BDE-47线粒体毒性的替代组织进行检查。 遗传背景和环境对BDE-47毒性的影响知之甚少。我们将 测试两种潜在的调节剂：1）低和高谷胱甘肽水平的基因工程小鼠，2）脂肪 肝脏由于维生素A缺乏。这些实验将为潜在的高风险提供新的信息 接触BDE-47的人群。这些研究的一个关键问题是，候选生物标志物是否 毒性（除暴露剂量外）。 除了发现BDE-47毒性的代谢特征外，我们最近还将研究两种 通过代谢足迹描述了线粒体对应激的反应：1）由于线粒体蛋白质毒性， 未折叠/未组装蛋白质的聚集，和2）响应缺氧的交替富马酸呼吸 和电子传递链的末端阻断。通过选择确定的线粒体反应，一种不利的， 我们开始对线粒体功能障碍进行分类，并寻找与 具体类型。在富马酸呼吸的情况下，分泌的高水平琥珀酸的特征 代谢物是已知的。 这项工作是与代谢组学筛选和数据专家奥利弗菲恩的密切合作 Terry Kavanagh是一位氧化应激和线粒体毒理学专家。

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.