title: Nephron Toxicity Profiling via Untargeted Metabolome Analysis Employing a High Performance Liquid Chromatography-Mass Spectrometry-based Experimental and Computational Pipeline.
abstract: Untargeted metabolomics has the potential to improve the predictivity of in vitro toxicity models and therefore may aid the replacement of expensive and laborious animal models. Here we describe a long term repeat dose nephrotoxicity study conducted on the human renal proximal tubular epithelial cell line, RPTEC/TERT1, treated with 10 and 35 μmol·liter(-1) of chloroacetaldehyde, a metabolite of the anti-cancer drug ifosfamide. Our study outlines the establishment of an automated and easy to use untargeted metabolomics workflow for HPLC-high resolution mass spectrometry data. Automated data analysis workflows based on open source software (OpenMS, KNIME) enabled a comprehensive and reproducible analysis of the complex and voluminous metabolomics data produced by the profiling approach. Time- and concentration-dependent responses were clearly evident in the metabolomic profiles. To obtain a more comprehensive picture of the mode of action, transcriptomics and proteomics data were also integrated. For toxicity profiling of chloroacetaldehyde, 428 and 317 metabolite features were detectable in positive and negative modes, respectively, after stringent removal of chemical noise and unstable signals. Changes upon treatment were explored using principal component analysis, and statistically significant differences were identified using linear models for microarray assays. The analysis revealed toxic effects only for the treatment with 35 μmol·liter(-1) for 3 and 14 days. The most regulated metabolites were glutathione and metabolites related to the oxidative stress response of the cells. These findings are corroborated by proteomics and transcriptomics data, which show, among other things, an activation of the Nrf2 and ATF4 pathways.
authors.affiliation: From the Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Ranninger
authors.affiliation: the Applied Bioinformatics Group, Center for Bioinformatics, Quantitative Biology Center and Department of Computer Science, University of Tübingen, Tübingen 72076, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Rurik
authors.affiliation: the Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck, Innsbruck 6020, Austria.
authors.forename: Alice
authors.initials: A
authors.lastname: Limonciel
authors.affiliation: From the Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Silke
authors.initials: S
authors.lastname: Ruzek
authors.affiliation: From the Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Reischl
authors.affiliation: the Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck, Innsbruck 6020, Austria.
authors.forename: Anja
authors.initials: A
authors.lastname: Wilmes
authors.affiliation: the Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck, Innsbruck 6020, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Jennings
authors.affiliation: Merck KGaA, Merck Serono, Nonclinical Safety, Darmstadt 64293, Germany, and.
authors.forename: Philip
authors.initials: P
authors.lastname: Hewitt
authors.affiliation: the Department of Toxicology, University of Würzburg, Würzburg 97078, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Dekant
authors.affiliation: the Applied Bioinformatics Group, Center for Bioinformatics, Quantitative Biology Center and Department of Computer Science, University of Tübingen, Tübingen 72076, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kohlbacher
authors.affiliation: From the Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, 5020 Salzburg, Austria, c.huber@sbg.ac.at.
authors.forename: Christian G
authors.initials: CG
authors.lastname: Huber
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000633:toxicity; D000970:Antineoplastic Agents / Q000633:toxicity*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D055442:Metabolome; D009399:Nephrons / Q000187:drug effects / Q000378:metabolism*; D012984:Software; D053719:Tandem Mass Spectrometry
chemical_list: D000970:Antineoplastic Agents; C004656:chloroacetaldehyde; D000079:Acetaldehyde
keywords: KNIME; OpenMS; bioinformatics; chloroacetaldehyde; high performance liquid chromatography (HPLC); mass spectrometry (MS); metabolomics; oxidative stress; toxicity profiling