Advanced in vitro exposure systems.

Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions

27. Jun. 2016

Different Effects of Heavy Fuel Oil and Refined Diesel Fuel

DOI:10.1371/journal.pone.0157964

Sean C. Sapcariu1,12, Tamara Kanashova2,12, Marco Dilger3,4,12, Silvia Diabaté3,12, Sebastian Oeder5,6,12,13, Johannes Passig7,12, Christian Radischat7,12, Jeroen Buters5,6,12,13, Olli Sippula8,12, Thorsten Streibel7,9,12, Hanns-Rudolf Paur4,12, Christoph Schlager4,12, Sonja Mülhopt4,12, Benjamin Stengel10,12, Rom Rabe10,12, Horst Harndorf10,12, Tobias Krebs11,12, Erwin Karg9, Thomas Gröger9, Carsten Weiss3,12, Gunnar Dittmar2,12, Karsten Hiller1,12, Ralf Zimmermann7,9,12

1 Luxembourg Centre for Systems Biomedicine 6, avenue du Swing, L-4362 Esch-sur-Alzette, Luxembourg,
2 Mass Spectrometry Core Unit, Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany,
3 Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology, Campus North, Karlsruhe, Germany,
4 Institute for Technical Chemistry (ITC), Karlsruhe Institute of Technology, Campus North, Karlsruhe, Germany,
5 Center of Allergy and Environment (ZAUM), Helmholtz Zentrum München and Technische Universität München, Munich, Germany,
6 CK-CARE, Christine Kühne Center for Allergy Research and Education, Davos, Switzerland,
7 Joint Mass Spectrometry Centre, Division of Analytical and Technical Chemistry, Institute of Chemistry, University Rostock, Rostock, Germany,
8 University of Eastern Finland, Department of Environmental Science, P.O. Box 1627, FI-70211 Kuopio, Finland,
9 Joint Mass Spectrometry Centre, CMA – Comprehensive Molecular Analytics, Helmholtz Zentrum München, Neuherberg, Germany,
10 Chair of Piston Machines and Internal Combustion Engines, University Rostock, Rostock, Germany,
11 Vitrocell GmbH, Waldkirch, Germany,
12 HICE – Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health – Aerosols and Health, Neuherberg, Rostock, Munich, Karlsruhe, Berlin, Waldkirch, Germany; Kuopio, Finland; Cardiff, United Kingdom; Esch-Belval, Luxembourg,
13 German Center for Lung Research (DZL), Munich, Germany

An automated ALI exposure system station with 18 exposure positions was used as the interface for mouse macrophage RAW 264.7 cell line exposures of the diesel engine exhaust. The evaluation is devided in LDH release assay, Metabolite extraction and GC-MS processing, Stable isotope labeling by amino acids in cell culture (SILAC), Proteome extraction and LC-MS/MS analysis of peptides and Proteomics Data An

Abstract

Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammationrelevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols.

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