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. 2015 Jun 3;10(6):e0126536.
doi: 10.1371/journal.pone.0126536. eCollection 2015.

Particulate Matter From Both Heavy Fuel Oil and Diesel Fuel Shipping Emissions Show Strong Biological Effects on Human Lung Cells at Realistic and Comparable in Vitro Exposure Conditions

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Particulate Matter From Both Heavy Fuel Oil and Diesel Fuel Shipping Emissions Show Strong Biological Effects on Human Lung Cells at Realistic and Comparable in Vitro Exposure Conditions

Sebastian Oeder et al. PLoS One. .
Free PMC article


Background: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling.

Objectives: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols.

Methods: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses.

Results: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification.

Conclusions: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

Conflict of interest statement

Competing Interests: Tobias Krebs is an employee of Vitrocell GmbH, Tübingen, Germany. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.


Fig 1
Fig 1. Experimental set-up and global omics analyses.
(A) An 80 KW common-rail-ship diesel engine was operated with heavy fuel oil (HFO) or refined diesel fuel (DF). The exhaust aerosols were diluted and cooled with clean air. On-line real-time mass spectrometry, particle-sizing, sensor IR-spectrometry and other techniques were used to characterise the chemical composition and physical properties of the particles and gas phase. Filter sampling of the particulate matter (PM) was performed to further characterise the PM composition. Lung cells were synchronously exposed at the air-liquid-interface (ALI) to aerosol or particle-filtered aerosol as a reference. The cellular responses were characterised in triplicate at the transcriptome (BEAS-2B), proteome and metabolome (A549) levels with stable isotope labelling (SILAC and 13C6-glucose). (B) Heatmap showing the global regulation of the transcriptome, proteome and metabolome.
Fig 2
Fig 2. Chemical and physical aerosol characterisation.
(A) The ship diesel engine was operated for 4 h in accordance with the IMO-test cycle. (B) Approximately 28 ng/cm2 and 56 ng/cm2 were delivered to the cells from DF and HFO, respectively, with different size distributions. The HFO predominantly contained particles <50 nm, and the DF predominantly contained particles >200 nm, both in mass and number. (C) Number of chemical species in the EA particles. (D) Transmission electron microscope (TEM) images and energy-dispersive X-ray (EDX) spectra of DF-EA and HFO-EA; heavy elements (black speckles, arrow); and contributions of the elements V, P, Fe and Ni in the HFO particles using EDX (* = grid-material). (E) Exemplary EA concentrations (right) and concentration ratios (left) for particulate matter-bound species. For all experiments, n = 3.
Fig 3
Fig 3. Effects of shipping particles on lung cells.
The net effects from the particles were referenced against the gaseous phase of the emissions. (A) Number of the regulated components in the transcriptome shows more genes regulated by the DF than the HFO particles (in BEAS-2B cells). Similar results were observed for the proteome (B) and metabolome (C) (in A549 cells). (D) Meta-analyses for the transcriptome and proteome using the combined Gene Ontology (GO) term analysis of the 10% most regulated transcripts and proteins. Individual GO terms are listed in S2 Table; the hierarchical pathways are indicated on the right. (E) Gene regulation of Wiki-pathway bioactivation; (F) gene regulation of Wiki-pathway inflammation; g, secreted metabolites; and h, metabolic flux measurements using 13C-labelled glucose. For all experiments, n = 3.

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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. We 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 specific roles of the authors are articulated in the ‘author contributions’ section.