Biomarker discovery for mitochondrial toxicants using metabolic footprinting

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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.