Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions: Different Effects of Heavy Fuel Oil and Refined Diesel Fuel

PLoS One. 2016 Jun 27;11(6):e0157964. doi: 10.1371/journal.pone.0157964. eCollection 2016.

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 inflammation-relevant 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.

MeSH terms

  • Animals
  • Cell Line
  • Fuel Oils / toxicity*
  • Gasoline / toxicity*
  • Macrophages / drug effects*
  • Macrophages / metabolism
  • Metabolome / drug effects*
  • Mice
  • Proteome / drug effects*
  • Vehicle Emissions / toxicity*

Substances

  • Fuel Oils
  • Gasoline
  • Proteome
  • Vehicle Emissions

Grants and funding

HICE partners received funding from the Impulse and Networking Funds (INF) of the Helmholtz Association (HGF), Berlin, Germany. The support of HICE by the Helmholtz Zentrum München and University of Rostock is gratefully acknowledged. Sebastian Oeder also received funding from CK-CARE Teilbereich A. Sean Sapcariu and Karsten Hiller acknowledge financial support from the Fonds National de la Recherche (FNR), specifically the ATTRACT program Metabolomics Junior Group. Funding from the Academy of Finland (Grant No: 258315 & 259946), Saastamoinen foundation and the strategic funding of the University of Eastern Finland for project “sustainable bioenergy, climate change and health” is acknowledged. Funding from the German Science Foundation (DFG ZI 764/5-1, ZI 764/3-1, INST 264/56-1 and 264/77-1) helped to achieve the presented results. The authors also thank SNSF and DFG for funding for the DACH project WOOSHI. Vitrocell GmbH provided support in the form of a salary for author T. Krebs, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The other funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.