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

Abstract

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.
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.
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.
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.
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.

Full text

RESEARCH ARTICLE
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
1,2,3☯
, Tamara Kanashova
1,4☯
, Olli Sippula
1,5☯
, Sean C. Sapcariu
1,6☯
,
Thorsten Streibel
1,7,8☯
, Jose Manuel Arteaga-Salas
1,8☯
, Johannes Passig
1,7☯
,
Marco Dilger
1,9,10
, Hanns-Rudolf Paur
1,9
, Christoph Schlager
1,9
, Sonja Mülhopt
1,9
,
Silvia Diabaté
1,10
, Carsten Weiss
1,10
, Benjamin Stengel
1,11
, Rom Rabe
1,11
,
Horst Harndorf
1,11
, Tiina Torvela
5
, Jorma K. Jokiniemi
1,5,12
, Maija-Riitta Hirvonen
1,5,13
,
Carsten Schmidt-Weber
2
, Claudia Traidl-Hoffmann
3,14
, Kelly A. BéruBé
1,15
, Anna
J. Wlodarczyk
1,15
, Zoë Prytherch
1,15
, Bernhard Michalke
16
, Tobias Krebs
1,17
, André S.
H. Prévôt
18
, Michael Kelbg
1,19
, Josef Tiggesbäumker
1,19
, Erwin Karg
8
, Gert Jakobi
8
,
Sorana Scholtes
1,8
, Jürgen Schnelle-Kreis
8
, Jutta Lintelmann
8
, Georg Matuschek
8
,
Martin Sklorz
7
, Sophie Klingbeil
1,7
, Jürgen Orasche
8
, Patrick Richthammer
1,8
,
Laarnie Müller
8
, Michael Elsasser
8
, Ahmed Reda
8
, Thomas Gröger
8
, Benedikt Weggler
1,8
,
Theo Schwemer
7
, Hendryk Czech
7
, Christopher P. Rüger
7
, Gülcin Abbaszade
8
,
Christian Radischat
1,7
, Karsten Hiller
1,6
, Jeroen T. M. Buters
1,2,3‡
, Gunnar Dittmar
1,4‡
,
Ralf Zimmermann
1,7,8‡
*
1 HICE—Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health—Aerosols and
Health, www.hice-vi.eu, Neuherberg, Rostock, Munich, Karlsruhe, Berlin, Waldkirch, Germany, Kuopio,
Finland, Cardiff, UK, Esch-Belval, Luxembourg, 2 Center of Allergy and Environment (ZAUM), Helmholtz
Zentrum München and Technische Universität München, Member of the German Center for Lung Research
(DZL), Munich, Germany, 3 CK-CARE, Christine Kühne Center for Allergy Research and Education, Davos,
Switzerland, 4 Mass Spectrometry Core Unit, Max Delbrück Center for Molecular Medicine Berlin-Buch,
Germany, 5 University of Eastern Finland, Department of Environmental Science, P.O. Box 1627, FI-70211
Kuopio, Finland, 6 Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362 Esch-
Belval, Luxembourg, 7 Joint Mass Spectrometry Centre, Chair of Analytical Chemistry, Institute of
Chemistry, University Rostock, Rostock, Germany, 8 Joint Mass Spectrometry Centre, CMA—
Comprehensive Molecular Analytics, Helmholtz Zentrum München, Neuherberg, Germany, 9 Institute for
Technical Chemistry (ITC), Karlsruhe Institute of Technology, Campus North, Karlsruhe, Germany,
10 Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology, Campus North, Karlsruhe,
Germany, 11 Chair of Piston Machines and Internal Combustion Engines, University Rostock, Rostock,
Germany, 12 VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Espoo, Finland,
13 National Institute for Health and Welfare, Department of Environmental Health, P.O. Box 95, FI-70701,
Kuopio, Finland, 14 Institute of environmental medicine, UNIKA-T, Technische Universität, Munich,
Germany, 15 Lung and Particle Research Group, School of Biosciences, Cardiff University, Cardiff, Wales,
United Kingdom, 16 Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum München—German
Research Center for Environmental Health GmbH, Neuherberg, Germany, 17 Vitrocell GmbH, Waldkirch,
Germany, 18 Laboratory of Atmospheric Chemistry, Paul Scherrer Institute (PSI), Villigen, Switzerland,
19 Institute of Physics, University Rostock, Rostock, Germany
☯ These authors contributed equally to this work.
‡ These authors also contributed equally to the manuscript.
* ralf.zimmermann@helmholtz-muenchen.de
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 1/17
OPEN ACCESS
Citation: Oeder S, Kanashova T, Sippula O,
Sapcariu SC, Streibel T, Arteaga-Salas JM, et al.
(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. PLoS
ONE 10(6): e0126536. doi:10.1371/journal.
pone.0126536
Academic Editor: Shama Ahmad, University of
Alabama at Birmingham, UNITED STATES
Received: October 14, 2014
Accepted: April 2, 2015
Published: June 3, 2015
Copyright: © 2015 Oeder et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Additionally, transcriptomics data are available from
Gene Expression Omnibus (accession number
GSE63962). Proteomics data are available from
ProteomicsDB (ID: PRDB004215).
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

Abstract
Background
Ship engine emissions are important with regard to lung and cardiovascular diseases espe-
cially in coastal regions worldwide. Known cellular responses to combustion particles in-
clude oxidative stress and inflammatory signalling.
Objectives
To provide a molecular link between the chemical and physical characteristics of ship emis-
sion particles and the cellular responses they elicit and to identify potentially harmful frac-
tions in shipping emission aerosols.
Methods
Through an air-liquid interface exposure system, we exposed human lung cells under realis-
tic 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 ex-
haust aerosols were combined with transcriptional, proteomic and metabolomic profiling in-
cluding 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, where-
as DF emissions induced generally a broader biological response than HFO emissions and
affected essential cellular pathways such as energy metabolism, protein synth esis, 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.
Introduction
Epidemiological studies provide compelling evidence that pollution by airborne particulate
matter (PM) derived from fossil fuel combustion is an important cause of morbidity and
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 2/17
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.
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.

premature death [1, 2]. Chronic PM exposure can induce short-term (e.g., cardiovascular dis-
eases or asthma) and long-term health effects, most notably cancer. Diesel automobile emis-
sions were recently classified as human carcinogens by the Intern ational Agency for Research
on Cancer [3].
Diesel ship emissions substantially contribute to worldwide anthropogenic PM levels, which
account for up to 50% of the PM-related air pollution in certain coastal areas, rivers and ports
[4–7]. Epidemiological studies attribute up to 60,000 annual deaths from lung and cardiovascu-
lar disease [ 8] to ship engine PM. A variety of new regulations will soon be implemente d to en-
sure cleaner ship emissions [ 9–11]. Low-grade heavy fuel oils (HFOs) contain high levels of
sulphur, toxic polycyclic aromatic hydrocarbons (PAHs) and transition metals. Current regula-
tions target HFO use by limiting their sulphur content. In this context, the maximum sulphur
content in shipping fuel is internationally regulated by the International Maritime Organisa-
tion (IMO) at 3.5%; in most European and US coastal areas, the maximum allowed sulphur
content is 1% (Sulphur Emission Control Areas, SECA) [12, 13]. Furthermore, in 2015, a 0.1%
sulphur fuel limit will be implemented in the Baltic and North Sea SECAs [14]. To comply
with these new sulphur limits, highly refined distillate fuels are necessary (diesel fuel, DF, or
marine gas oil, MGO). Currently, MGO is the most used distillate fuel for marine shipping and
contains up to 1% sulphur. In 2011, 170 million tons of HFO and 43 million tons of MGO and
DF were consumed by ship diesel engines worldwide [15, 16]. This volume corresponds to ap-
proximately 21% of global fuel consumption [17].
The biological and health effects of land-based diesel engine emissions have been extensively
studied using submersed cell cultures subjected to collected diesel exhaust particles [18, 19].
This submersed cell culture approach neglects the effect of airborne particle exposure, which
can result in low sensitivity in measuring biological effects [20]. An alternative is the air-liquid
interface (ALI) cell exposure technology. Current systems are technically mature enough to en-
able reproducible, direct, on-site exposure of lung cell culture to emission aerosols under realis-
tic dilution, flow and humidity conditions [21]. Multiple ALI-exposure studies using car diesel
engines [22–25] highlight the improved sensitivity of ALI systems compared with submerged
toxicological test systems that use collected diesel exhaust particles (DEP) [20].
Up to now three main causes for PM-induced health effects have been identified: genotoxi-
city, inflammation and oxidative stress; other mechanisms have also been described [19]. Thus
far, all information on diesel PM has been inferred from research on car emissions. However,
diesel emissions from ships differ greatly from car or truck diesel emissions due to the fuel
composition (HFO) and combustion characteristics of ship engines [26]. Thus, the practice
currently used to estimate the health impacts of ship diesel emissions based on analogous car
or truck emissions [8, 12, 27] is problematic. The high levels of toxic compounds [6, 28] suggest
that HFO emissions produce more detrimental acute and chronic toxic effects than car or
truck diesel emissions.
This study targets the biological effects of airborne PM from both diesel and HFO ship
emissions based on their chemical compositions. The joint analysis of the biological multi-
omics data with the comprehensive aerosol analysis results provides an extensive overview of
affected biological mechanisms and pathways and further identifies potentially harmful frac-
tions of the shipping aerosols.
Results and Discussion
Experimental setup
The experimental setup is illustrated in Fig 1 (details in S1 and S2 Figs and in S1 Text). Briefly,
we operated a four-stroke, one-cylinder common rail research ship diesel engine (80 kW)
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 3/17

using either HFO (HFO 180) containing 1.6% sulphur or DF containing less than 0.001% sul-
phur and 3.2% plant oil methyl ester in compliance with the 2014 IMO-SECA-legislation (DIN
EN590, see S1 Fig for the engine and fuel properties), which represents the common dual-fuel
use in commercial shipping [10, 29]. The engine was operated according to the test cycle ISO
8178–4 E2 for ship diesel engines with a balance between harbour-manoeuvring and cruising
engine-loads (Fig 2). The combustion aerosol was cooled and diluted with sterile air. Chemical
and physical properties of the HFO and DF aerosol were comprehensively characterised using
state-of-the-art, on-line and real-time techniques as well as off-line filter sample analyses. Re-
sults are summarised in Fig 2 (for details, see SI). In parallel with aerosol characterisation, con-
fluent layers of two human epithelial lung cell lines (the human lung alveolar cancer cell line
A549, purchased from the American Type Culture Collection, ATCC CCL-185; http://www.
lgcstandards-atcc.org/Products/All/CCL-185.aspx, and human SV40-immortalised bronchial
epithelial cells BEAS-2B, purchased from ATCC, CRL-9609; http://www.lgcstandards-atcc.org/
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
13
C
6
-glucose). (B) Heatmap showing the global regulation of the transcriptome, proteome
and metabolome.
doi:10.1371/journal.pone.0126536.g001
Lung Cell Responses to Shipping Emissions
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Products/All/CRL-9609.aspx)) [30] were exposed to the diluted engine exhaust for 4 h at the
ALI (Fig 1). Epithelial lung cells have direct contact to inhaled aerosol particles and gases and
were therefore used as a model of aerosol inhalation. The cell lines A549 and BEAS-2B have
been widely used for testing particles and gases at the air-liquid-interface [31–36]. The BEAS-
2B cells are considered to better resemble the situation in human lung tissue while require-
ments for the cultiva tion of the cancer derived cell line A549 are better suited for labeling with
the L-D
4
-Lysine isotope maker for the quantitative proteomics. The transcriptomics methodol-
ogy is not based on metabolic labelling and thus well suited for the analysis of BEAS-2B cells.
The quantitative comparative proteomics approach requires the labelling of the cells with
D4-Lysine. However the BEAS-2B cells require specialized media and coating of the plates,
which is currently incompatible with the metabol ic labelling. Therefore simultaneuos SILAC-
based proteomic and metabolic analysis was performed with the establis hed A549 cell model.
In summary the cells were analysed using transcriptome (BEAS-B), SILAC-proteome (A549),
metabolome and metabolic flux measurements (A549) as well as cytotoxicity tests (A549). The
omics data are stored in Gene Expression Omnibus (GSE63962) and Proteomics DB
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/cm
2
and 56 ng/cm
2
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.
doi:10.1371/journal.pone.0126536.g002
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 5/17

(PRDB004215), respectively. All experiments were performed in triplicate (3 independent ex-
posures) and referenced to filtered aerosol (for normalising the effects induced by the gas
phase) because particles and particle-related chemicals play an important role in the health rel-
evance of diesel exhaust [ 37] and are therefor in the focus of this study.
The first phase of the experiment was used to find the optimal dose for the large-scale analy-
sis. Cells in the setup were exposed to different concentrations of aerosols. The reaction of the
cells was moni tored using the Alamar Blue viability test. Due to the higher particle concentra-
tion in HFO- exhaust (see belo w) a dilution of 1:100 was required to achieve a non-impaired
cell status while for DF-exhaust a lower dilution of 1:40 was possible without any viability im-
pairment (i.e., a no acute toxicity exhaust dilution; S3A Fig). By applying the different dilution
ratios of 1:40 (DF) and 1:100 (HFO) for the exhaust gases for no acute toxicity at 4 h exposure,
a similar deposition dose (deposited particle mass per confluent cell culture surface area, see
below) was achieved. Based on a gravimetric filter analysis of PM 2.5 and assuming a size-inde-
pendent, constant deposition probability of 1,5% after Comouth et al. [38], the accumulated
particle mass deposited on the lung cell monolayer surface area was roughly estimated as
28 ± 1.5 (DF) and 56 ± 0.7 ng/cm
2
(HFO) per 4 h exposure duration (see S3C Fig) with the var-
iance of the mass measurement expressed by the standard deviation of the filter samples. A
more elaborated model taking into account the particle size distribution from an electric low
pressure impactor (ELPI) and a size dependent deposition probability after Comouth et al.
[38], which was determined using previous measurements from ALI exposure systems , predicts
15.7 (DF) and 41.5 ng/cm
2
(HFO) per 4 h exposure. Even for improved deposition approxima-
tion model, the estimated uncertainties, however, are rather high (about a factor of 2). There-
fore the deposition dose in both cases can be considered being approximately equal for DF and
HFO. We decided to perform the exposure for omics measureme nts at these dilutions, in order
to compare th e specific molecular biological effect strength at an about equal deposition dose.
Note that in the following all aerosol parameters are reported considering the specific emis-
sion-aerosol dilution factors (i.e. the exposure aerosol, EA, as delivered to the cells).
Chemical and physical analysis
Consistent with previous studies [29], only small concentration differences of gaseous com-
pounds were found in the emissions of the ship engine using the two fuels. An exception was
SO
2
(4 mg/m
3
), which was below toxicity threshold after dilution in the HFO-EA. In addition,
the EA concentrations of the further potential ly toxic gases NO, NO
2
and CO were below 16.3,
0.4 and 7 ppm, respectively. These values are below the reported toxicity thresholds for the air-
liquid interface [39, 40] and even below the general NIOHS lifetime workplace 8-hr exposure
limit values of 25, 1 and 35 ppm, respectively [41].
The concentration of particles with an aerodynamic diameter larger than 200 nm was higher
for the DF-EA (particle number and mass concentration), whereas nanoparticles smaller than
50 nm were approximately 100-fold more abundant for the HFO-EA (see the size distributions
in Fig 2). However, note that the mass contribution of these nanoparticles is very small. TEM
images of the particles show that the smaller HFO particles (Fig 2) contained high levels of
amorphous organic material around carbonaceous fractal cores with metal inclusions. The
DF-EA particle analysis reveals a different picture (Fig 2), in which the particles appear larger
and are mostly composed of pure carbonaceous aggregates with spherical soot cores (Ø ~ 20–
30 nm). A layered graphite-like car bon structure became visible at a higher TEM magnification
(S3 Fig). Based on the size-dependent deposition function described by Comouth et al.[38](S3
Fig) and the low specific density of the observed fractal soot aggregates in DF-EA (Fig 2), the
deposited mass for the DF-EA cell exposure experiments is slightly lower than the above
Lung Cell Responses to Shipping Emissions
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estimates. The particles deposited from the HFO-EA were of a higher dose in mass and number
compared to the DF-EA exposure.
Energy-dispersive X-ray spectroscopy (EDX, Fig 2) on TEM showed large differences be-
tween HFO-EA and DF-EA particles with regard to the abundance of heavy elements. High in-
tensities of elements such as vanadium, nickel, sulphur and iron were detected in the HFO
particles, whereas the DF particles primarily contained carbon and oxygen in the EDX spec-
trum. Fig 2 shows an overview of the differences in the inorganic and organic chemical compo-
sition (Fig 2) as well as the absolute concentrations of the respective substances in the DF- and
HFO-exposure aerosol particles (Fig 2 and S4 Fig). Almost all of the measured components, ex-
cept elemental carbon and black carbon, were more abundant in HFO-EA compared with
DF-EA, despite a 2.5-fold higher dilution for HFO-EA.
On-line aerosol mass spectrometry and off-line analyses showed considerably higher mass
concentrations of particle-bound organic material and much more complex organic material
in the HFO-EA (S1 Table). High-resolution mass spectrometry (ESI-FTICR-MS) revealed
3631 different polar organic compounds in the HFO particles compared with only 321 in the
DF particles (Fig 2); 244 compounds were common to both fuel types. The quantification of ar-
omatic and aliphatic compounds (S4 Fig ) revealed that higher molecular weight components
were more abundant in the HFO particles (green text in Fig 2), such as the higher molecular
weight carcinogenic PAH benzo[a]pyrene (Fig 2 and S4 Fig). The sum of PAH toxicity equiva-
lency factor s (Fig 2), which ranks different toxic PAHs weighted by their concentration and rel-
ative toxicity, was more than 10-fold higher in HFO-PM compared with DF-PM (Fig 2). The
only component over-represented in the DF-PM was the elemental carbon fraction (EC) and
the corresponding optically measured “black carbon” factor (BC; Fig 2).
Summarising the chemical and physical characterisations, particles emitted from ship en-
gines differ in concentration, size distribution, morphological appearance and chemical com-
position depending on whether DF or HFO is used. The DF particles in the inhalable size
region were dominated by elemental carbon-rich soot-aggregate particles [29], whereas the
HFO particles were smaller (nanoparticles) and rich in organic material, including known or-
ganic air toxicants (PAHs and their derivatives) and reactive transition metals such as V, Ni, Fe
and Zn (S4 Fig). However, it shall be noted that also DF-PM contains organic compounds in
relatively high concentrations. The HFO-PM just contains much higher concentrations (Fig 2).
Exposure and deposition dose
We exposed human lung cells for 4 h to concentrations which are corresponding to occupation-
al exposure scenarios or 10 times the concentration of an ambient high concentration scenario
(EA ~ 390 μg DF PM2.5/m
2
and ~760 μg HFO PM2.5/m
2
). This concentration corresponds to
an ALI mass deposition dose of about 28 and 56 ng PM/cm
2
for DF and HFO respectively.
These doses can be related to the human respiratory tract using the specific deposition effi-
ciency for different lung regions. From the measured size distribution and an estimated effective
particle density based on the mass-mobility-relation for aggregated diesel particles (between 1.1
and about 0.1 g cm
-3
, derived from [42, 43]), a deposition simulation was performed using a re-
cently updated model [44, 45] for the tracheobronchial lung region. A 4 h exposure of a human
being to the EA concentrations used in our experiments would result to a tracheobronchial de-
position of about 1.5 and 5 ng PM/cm
2
for DF and HFO, respectively. Thus the deposited mass
in an ALI experiment corresponds to about 3 days (DF) or 2 days (HFO) exposure time for an
exposed person (note that for an equal dilution of 1:100 in both EAs the actual deposited tra-
cheobronchial dose for DF would correspond to a 7.5 days exposure of a person). However, one
should keep in mind that the size distribution may change quickly in the ambient atmosphere
Lung Cell Responses to Shipping Emissions
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and in the airways, e.g. by coagulation or water condensation, causing additional uncertainty
thereby. The similarity between the size dependent deposition curve [38] for the ALI-system
and for the lung deposition curve [44] suggests a good transferability of the results, in particular
for the tracheobronchial region. In conclusion, the deposited mass concentration of at an equal
dilution of DF PM mass would be about ¼ of the deposited HFO PM. This however, only holds
true for directly emitted aerosols. In the atmosphere the more polar, sulphate containing HFO
emission particle will quickly grow considerably by water condensation, while the hydrophobic
DF particles size distribution will remain stable for longer time [46, 47]. Therefore, emission
size distributions might, to some extent, equalise soon.
Biological analysis
To relate the extensive chemical and physical characterisation of the exhaust aerosols to biolog-
ical effects, the HFO and DF emission particles were directly deposited on human lung cells
using ALI exposure technology. Transcriptome, proteome, metabolome and metabolic flux
analyses were performed, which yielded parallel and relative quantification of 42205 different
transcripts, 6192 proteins and 400 metabolic molecules. To reduce variability, the proteins and
metabolites were extracted from the same cell material (A549) that was previously metabolical-
ly labelled using D
4
-lysine (SILAC proteomics) and
13
C
6
-glucose (metabolic flux analysis). Ri-
bonucleic acid (RNA) was isolated from BEAS-2B cells exposed through the same ALI
exposure system and was used for the transcriptome analyses [20].
The transcriptome, proteome and metabolome analyses revealed widespread changes in the
cellular system upon exposure to both HFO and DF aerosol particles. Surprisingly, more gene
expression levels were regulated in the DF-particle-exposed cells (i.e., the response was more
widespread compared with the HFO-particle-treated cells on all “omic”-levels; p<0.001, Figs 1
and 3 and S5 Fig). The most significantly regulated genes, proteins and metabolites also dif-
fered between the DF and HFO (S6 Fig), which shows that the response to emissions of each
type of fuel differed quantitatively and qualitatively in both human lung cell lines. A higher reg-
ulation alone only proofs a stronger biological reaction onto the deposited PM at the given ex-
posure conditions (i.e. 4 h exposure at a deposition dose below measurable cytotoxicity) and
does not necessarily indicate a higher toxicity or risk of disease.
Further conclusions can be drawn from a specific biological pathway analysis. Pro-inflamma-
tory signaling, chemical response (such as xenobiotic metabolism) and oxidative stress pathways
were indicated by the regulated genes (Fig 3 and S6 Fig)[19]. The HFO particles specifically in-
duced the transcription of primary and secondary inflammation markers (IL-8, IL-6 and IL-1),
and both fuel types affected the cytokines CSF3, CXCL1, and CXCL2. Considering xenobiotic
metabolism, CYP1A1 (PAH metabolism) was induced by exposure to HFO particles (which cor-
responds to the higher PAH concentrations in HFO PM), whereas the DF particles affected
other cytochromes (CYP3A4 and CYP17A1) and the carbosulphotransferase CHST6 (Fig 3).
In addition to these, in the context of aerosol exposure well-known pathways [19, 48], we
searched for other cellular responses undergoing modulation. A meta-analysis combining the
proteome and transcriptome data was performed to examine the significant enrichment of
gene ontology (GO) terms. The results indicate that the HFO and DF particle effects were dis-
tinct (Fig 3 and, in more detail, S7 Fig). Particles from both fuels induced effects on cell motili-
ty, th e cellular stress response, the response to organic chemicals, proliferation and cell death
(Fig 3 and S7 Fig). Genes and proteins associated with vesicle transport pathways were en-
riched, which might be connected to the endocytosis of diesel particulate matter.
The pathways specifically regulated by DF particle exposure included the general translation
pathway (Fig 3, S7 Fig and S2 Table). The translational elongation, RNA-processing and
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 8/17

ribosome translation pathways were down-regulated, whereas the pathways that affect chroma-
tin organisation and modification were up-regulated. The down-regulated pathways included
histone acetylation, which may result in DF particle-induced epigenetic changes. Other path-
ways modulated by DF particles were involved in processes such as cell junction organisation
and cell adhesion. Pathways such as the energy metabolism, cell junction and cell adhesion
were clearly affected in both cell lines when assessed using transcriptomics and proteomics but
differed in the direction of regulation (Table 1, S2 Table and Fig 3), which indicates a time-de-
layed reaction in the cell. Exposure to DF particles induced mitochondria-associated genes and
proteins, which indicates that mitochondrial stress was induced, whereas the HFO particles did
not yield this response.
Pathways specifically regulated by the HFO particles include the homeostasis, oxidative
stress and inflammatory response pathways, whereas the metabolic and biosynthetic processes
were slightly down-regulated (Fig 3 and S2 Table).
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
13
C-labelled glucose. For all experiments, n = 3.
doi:10.1371/journal.pone.0126536.g003
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Interestingly, the proteomics data reveal a direct induction of cell-cell interaction remodel-
ling, whereas the transcriptomics data show a down-regulation of simila r GO terms. This find-
ing can be explained by assuming an immediate response of the proteome e.g. by stabilizing
the already synthesized proteins, while the transcriptome shows the shut-down of the system
in a time-de layed response. Although 40% of the observed protein regulation can be explained
by the changes in mRNA abundance, most of the changes indicate other modes of regulation.
Protein can be degraded in direct response to PM exposure, and translation or transcription
may be too slow to change the protein concentrations after 4 h of exposure [49].
The metabolome analyses supported the finding that biosynthetic and protein synthesis
processes were down-regulated in the DF particle-treated cells. ATP-binding cassette trans-
porters, which are involved in actively transporting biomolecules across membranes, were also
affected (S2 Table). Further information supporting the inhibition of biosynthetic activities in-
cludes the negatively affected metabolites secreted by the cells (Fig 3). The pathways affected
by HFO particle exposure include glycolysis and pyrimidine metabolism. Glycolysis is a path-
way that is typically altered during inflammation and is generally increased in cells under in-
flammatory conditions [50].
Glucose flux into lactic acid through glycolysis was significantly reduced (p<0.05) in cells
treated with DF particles (Fig 3 and S9 Fig). Mammalian cells oxidise glucose and glutamine in
the TCA cycle to produce NADH/H
+
, which is re-oxid ised in the respiratory chain to produce
ATP. DF exposure strongly decreases the levels of relative glucose oxidation in the TCA cycle
compared with HFO, as reflected by the significantly lower levels of labelled citric acid
(p<0.001; ratio data: Fig 3). Simultaneously, we observed an increase in gl ucose-derived carbon
flux into glycine (Fig 3); enhanced glycine metabolism has previousl y been associated with
tumourigenesis in lung cancer [51]. These observations suggest a lower ATP production and,
hence, lower available energy compared with HFO. Increased carbon flux into glycine is
Table 1. Summary of the main HFO- and DF-particle exposure effects.
Effect HFO DF
Pro-inflammatory signaling " -
Oxidative stress " -
Cell homeostasis " -
Response to chemicals "#"
Cellular stress response ""
Motility ""
Endocytosis ""
Cellular signalling MAPK, TGF beta, PDGF, EGF, GPCR ID, kinase cascade
Energy metabolism - #"
x
Protein synthesis - #
Protein degradation - "
RNA metabolism - #
Chromatin modifications - "
Cell junction and adhesion - #"*
The arrows indicate the direction of regulation for cellular functions derived from the most statistically
significant enriched Gene Ontology terms from the transcriptome, proteome, and metabolome (details in S2
Table).
x
BEAS-2B up, A549 down
* BEAS-2B down, A549 up
doi:10.1371/journal.pone.0126536.t001
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 10 / 17

directly linked to the increased transformation of hydroxymethyl groups through one-carbon
metabolism. The latter is essential for DNA synthesis and repair.
Conclusions
We assessed human lung cell resp onses to ship exhaust particles. A unique combination of ex-
tensive chemical and physical aerosol characterization and multiple omics analysis was used to
generate a broad overview on cellular mechanisms affected by shipping particles and to identify
possibly harmful constituents of two types of ship exhaust aerosols. While not providing a clas-
sical toxicological risk assessment, which would require the testing of multiple doses and time-
points, this study rather gives a comprehensive picture on the cellular responses to ship exhaust
particles after short-term exposure, which should be used as starting point for more mechanis-
tic studies. Although the HFO particles deposited in the ALI system were about equal in mass,
higher in number and contained a large excess of toxic compounds, DF particle exposure in-
duced a broader biological reaction in the human lung cells (BEAS-2B and A549) on all investi-
gated "omic" levels. As discussed, a stronge r affected cell metabolism is not an adverse effect
per se, but it holds a higher risk of disturbance of normal cell functions. Within known path-
ways, such as pro-inflammatory signaling, oxidative stress and xenobiotic metabolism, the lev-
els of certain well-known indicators (e.g., IL-1/6/8 and CYP1A1) surged followin g HFO
particle exposure. In contrast, DF particles strongly affected basic cellular functions (energy
and protein metabolism) and mechanisms little yet known to be affected by aerosol treatment,
such as mRNA proc essing and chromatin modification.
The obtained results also suggest formulating specific hypotheses and are motivating further
experiments to proof or disproof those. In this context the role of freshly formed “elemental
carbon, EC” fractions and the influence of organic compounds on the biological activity should
be investigated. The relatively large EC fractio n in DF exhaust is one of the prominent differ-
ences between the two particle types. The chemical and physical surface propertie s of freshly
formed EC fractions might be of relevance here. Laboratory experiments using e.g. combustion
aerosol standard generator (CAST,[52]), which allows to generate fresh combustion particles
with adjustable EC/OC ratios, are currently under preparation.
There is no doubt that the carcinogenic emissions from HFO-operated vessels need to be
minimized and HFOs should be replaced by refined modern DF (at least if no flue gas cleaning
systems are installed). HFO emissions contain among other constituents high concentrations
of toxic metals (V, Ni etc.) and polycyclic aromatic hydrocarbons. However, also emission of
diesel engines operated with refined DF, are known to be toxic and carcinogenic, although the
toxicant concentrations are much lower [8] than in HFO emissions. Consequently the imple-
mentation of emission reduction measures for land-based diesel engines started decades ago
(e.g. with sulfur-reduced fuels) [18] and current efforts are directed towards the reduction of
particle emissions from diesel automobiles. Due to the substantial contribution of ship emis-
sions to global pollution, ship emissions are the next logical target for improving air quality
worldwide, particularly in coastal regions and harbour cities. In this contex t our findings on
the biological effects of HFO and DF ship diesel emissions can contribute to the current debate
about the reduction measures to be implemented for shipping. The results from this study pro-
vide the information that at comparable lung deposition doses the acute biological activity of
particles of ship emissions from DF fuelled ships is not less relevant than the activity of HFO
emission particles. This supports the suggestion that a general reduction of the PM emissions
(not the SO
2
emission) from shipping in harbours and the vicinity of the coast should be imple-
mented for both, HFO- and DF-opera ted ships. Efficient particle filter technology (e.g., electro-
static precipitation or bag-filtration) is available. From a regulatory perspective, the next step
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 11 / 17

should be the introduction of legal emissions limits for respirable PM (e.g. PM 2.5, in [mg/m
3
])
from ship emissions [29].
Supporting Information
S1 Fig. Sampli ng setup. (A) Simplified scheme of the sampling and measurement setup.
DR = dilution ratio, TC = temperature control, T = temperature measurement, P = pressure
meter. (B) Detailed setup of the used sampling train with porous tube and ejector diluter units.
(C) Properties of the used diesel fuel (DF) and heavy fuel oil (HFO). Most noticeable are the
high viscosity and high sulfur content of HFO compared with distillate fuels like EN 590 diesel.
(D) Experimental engine parameters. The engine is a single cylinder engine with common rail
injection system representing state of the art medium speed marine diesel engines. The dual
fuel system allows operation with both distillate and residual fuels.
(EPS)
S2 Fig. Air-Liquid-Interface (ALI) exposure. HICE exposure system: the left part shows the
data acquisition and control unit for the mass flow controllers, humidity and temperature. The
exposure unit in the right part contains three Vitrocell modules and is thermostated to 37°C.
Each module has six positions for cell exposure to either complete or filtered aerosol for gas
phase referencing. The flow through each of the exposure positions is individually controlle d
by a mass flow controller (lower left) Cell exposure: the aerosol passes through the aerosol inlet
and is streaming directly over the cell cultures.
(EPS)
S3 Fig. Particle dosing and morphology. (A) Cell viability at DF and HFO aerosol particle
dose. A549 cells were exposed for 4h to 1:40 diluted DF or 1:100 diluted HFO. Directly after ex-
posure, cell viability was mea sured by reduction of Alamar Blue and compared to cells exposed
to the filtered aerosols. Reported are the means relative to filtered aerosol ± SD from 3 (HFO)
or 2 (DF) independent experiments. As requested for the further ‘omics study, the viability is
not impaired by the DF or HFO particle exposure. (B) Size dependent deposited dose of DF
and HFO particles (left ordinate) as well as deposition probability (W, right ordinate) calculat-
ed according to Comouth et al. (1) for a size dependent density profile. (C) Mass dose of DF
and HFO particles deposited per cell area. Data are estimated from gravimetric filter samples
(case 1, 2) and from electrical low pressure impactor (ELPI) size distributions (case 3, 4). Cal-
culations are performed assuming a constant deposition probability of W = 1.5% for all particle
diameters (case 1, 2). For comparison, calculations are performed additionally using the size
dependent probability Wρ(D) based on Comouth et al. (31) and a particle density based on a
mass-mobility relationship for DF and HFO (case 3, 4). In all cases the deposited PM dose is
about a factor 2 higher for the HFO case. d-g, TEM images of diesel fuel exposure aerosol parti-
cles. The typical soot agglomerate structure (D,E) and the layered graphitic structure (F,G) is
typical for rather pure, elemental carbon containing soot. (H-L), TEM images of heavy fuel oil
exposure aerosol particles. The often much smaller particles consist of heavier elements (black
speckles) and tarry substance (crusted appearance). The HFO-EA soot particles have a more
amorphous structure than the diesel fuel soot (J).
(EPS)
S4 Fig. Compounds in particulate matter. (A) Exemplary sum-parameters and compound-
class data for exposure aer osol (EA) particu late matter for HFO-EA and DF-EA. Particular
abundance and statistic parameters’ ratios (a), absolute concentrations (b) and statistic param-
eters on the sample complexity (c) reveal a substantial complexity of the organic-chemical
composition of the particulate matter.
1
EC/OC coupled to SPI,
2
EC/OC coupled to REMPI,
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 12 / 17

3
EC/OC-analysis (thermal-optical method),
4
AMS,
5
Filter weighing.
6
Aethalometer,
7
Compre-
hensive two-dimensional gas chromatography/Time-of-flight mass spectrometry,
8
Fourier-
Transform Ion Cyclotron Resonance Mass Spectrometry with atmospheric chemical ioniza-
tion,
9
Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry with electrospray ioni-
zation,
10
Thermal desorption/direct derivatization gas chromatography/Mass spectrometry,
11
GC/MS. (B) Elemental analysis of the particulate matter. Exemplary concentrations-ratios
(HFO-EA- over DF-EA-particles) of elements (left). Absolute concentrations of the species in
the HFO-EA- (red bars) and DF-EA-particles (blue bars) are also shown (right). Method:
ICP-AES. (C) Exemplary concentration-ratios (HFO-EA- over DF-EA-particles) of polycyclic
aromatic hydrocarbons (PAH) (left). Absolute concentrations of the species in the HFO-EA-
(red bars) and DF-EA-particles (blue bars) are also shown (right). The larger the PAH-struc-
ture, the stronger is the prevalence of the compound in the HFO-EA-particles. Methods:
1
Thermal desorption/derivatization gas chromatography/Mass spectrometry,
2
Gas chromatog-
raphy/mass spectrometry,
3
Liquid chromatography/Tandem mass spectrometry. (D) Exempla-
ry concentration-ratios (HFO-EA- over DF-EA-particles) of aliphatic hydrocarbons (left).
Absolute concentrations of the species in the HFO-EA- (red bars) and DF-EA-particles (blue
bars) are also shown (right). The same behaviour as in the PAH compound class is observed:
The larger the aliphatic-structure, the stronger is the prevalence of the compound in the
HFO-EA-particles.
(EPS)
S5 Fig. DF regulates more transcripts, proteins and metabolites than HFO. (A-C) Compari-
son of regula tion magnitude and regulation significance (obtained with a two-tailed t-Student’s
t-test on the replicate measurements). Mean of log2 fold change aerosol/filtered is plotted vs.
-log10 p-value of comp lete datasets of transcriptome in BEAS-2B cells (A), proteome (B) and
metabolome (C) in A549 cells for DF and HFO. (D-F), Comparison of regulation magnitude
and abundance of regulated transcripts, proteins or metabolites. Mean of log
2
fold change aero-
sol/filtered is plotted vs. mean of log
10
fold intensity of complete datasets of transcriptome (D),
proteome (E) and metabolome (F) for DF and HFO.
(EPS)
S6 Fig. Cellular responses to DF and HFO differ qualitatively. (A-C) Distinct patterns of reg-
ulation of DF and HFO. Hierarchical clustering of highest regulated entities of each omic ap-
proach: transcriptomics (A) (BEAS-2B), proteomics (B) and metabolomics (C) (A549). (D,E)
Pathways known to be affected by diesel particle exposure. Transcriptome pathway analysis
was performed using 1.5-fold regulated genes. Typical PM-influenced pathways were selected
and according gene regulation were clustered hierarchically. Apoptosis (D, pro- and anti-apo-
ptotic genes), Oxidative stress (E).
(EPS)
S7 Fig. Meta-analysis of gene ontology-terms in the proteomi c and transcriptomic mea-
surement of DF and HFO particle-treated samples. Significantly regulated proteins in A549
cells were determined using 10% of lowest and 10% of highest log2 fold change in the ratio
Aerosol/Gas and a cut-off of —log10(p-value) >1 for 3 replicates. According to the high identi-
fication number, significantly regulated transcripts in BEAS-2B cells were determined using
5% of lowest and 5% of highest log2 fold change of Aeroso l/Gas and a cut-off of—log10(p-
value) >1 for 3 replicates. GO term analysis was performed using David Tool. The p-values of
GO-terms were z-transformed, hierarchically clustered, and plotted as a heat map.
(EPS)
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 13 / 17

S8 Fig. DF- and HFO-particles disrupt lung epithelial integrity. (A) Histopathology of
HFO-/DF-particle treated NHBE cells. Light microscopy histological analysis of sections of the
NHBE cultures treated with PBS (control), and (B) HFO, (C) DF and (D) CB120 at a dose
150 μg/cm
2
for 24 h. Hematoxylin and eosin staining, scale bar = 50 μm. (E) TEM micrographs
of HFO- and (F) DF-particle treated NHBE cells. Ribosome agglomeration in cells of the
NHBE cultures after 24 h incubation at a dose 150 μg/cm
2
; n = 5. Scale bar = 2 μm.
(EPS)
S9 Fig. Secreted metabolites and metabolomic flux analysis. Met abolism of U-
13
C-Glucose
through central carbon metabolism in A549 cells. Reduced model of central carbon metabo-
lism, with labeled atom transition marked for selected metabolit es. Red circles =
13
C labeled
carbon; Blue circles =
13
C labeled carbon from Malic Enzyme activity; White circles =
12
C unla-
beled carbon. Selected Secreted Metabolite Ratios. Selected metabolites were measured through
GC/MS analysis of cellular medium post exposure. Values shown are the ratios of unfiltered
treatments to filtered treatments for each fuel type during three replicates. Metabolic flux mea-
surements based on
13
C-labeled glucose. Filtered and unfiltered aerosol samples were
analyzed separately.
(EPS)
S10 Fig. Exemplary light microscopic image of a confluent A459 cell layer. 4x10
5
A549 cells
were seeded into a 24mm trans-well insert. After 24h and just before ALI-exposure, confluence
was checked by light microscopy.
(TIF)
S1 Table. Chemical Analytics of Ship Exhaust Particles.
(XLSX)
S2 Table. Biological Responses to Ship Exhaust Particles.
(XLSX)
S1 Text. Mater ials and Methods.
(DOCX)
Acknowledgments
The technical efforts of Anita Wüst, Evelyn Hübner, Renate Effner, Jenny Ghelfi, Thekla
Cordes, and Christian Jäger are greatly appreciated. We thank Patrick Beaudette for carefully
reading the manuscript.
Author Contributions
Conceived and designed the experiments: SO T. Kanashova OS SCS T. Streibel JP MD HRP
SM SD CW HH JKJ MRH KAB MK EK GJ MS JO LM ME AR TG CR KH JB GD RZ. Per-
formed the experiments: SO T. Kanashova OS SCS JP MD CS BS RR TT AJW ZP BM AP MK
EK GJ JL GM MS JO PR LM ME AR BW T. Schwemer HC CPR GA CR. Analyzed the data:
SO T. Kanashova OS SCS T. Streibel JMAS JP MD SD CW BS RR TT AJW ZP BM AP MK JT
EK GJ JSK JL GM MS SK JO PR LM ME AR TG BW T. Schwemer HC CPR GA CR KH JB GD
RZ. Contributed reagents/materials/analysis tools: SO T. Kanashova OS SCS T. Streibel JMAS
JP MD HRP CS SM SD CW BS RR HH TT JKJ MRH CSW CTH KAB AJW ZP BM T. Krebs
AP MK JT EK GJ SS JSK JL GM MS SK JO PR LM ME AR TG BW T. Schwemer HC CPR GA
CR KH JB GD RZ. Wrote the paper: SO T. Kanashova OS SCS T. Streibel JMAS JP MD SM BS
KAB AJW ZP JT EK GJ SS JSK JL GM MS SK JO LM ME AR TG BW T. Schwemer HC CPR
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 14 / 17

GA CR KH JB GD RZ. Initially conceived and designed the study: T. Streibel HRP CW HH JKJ
MRH KAB T. Krebs JT EK JSK JL KM MS TG KH JB GD RZ.
References
1. Pope CA 3rd, Dockery DW. Air pollution and life expectancy in China and beyond. Proc Natl Acad Sci
USA. 2013; 110(32):12861–2. doi: 10.1073/pnas.1310925110 PMID: 23847200
2. Chen Y, Ebenstein A, Greenstone M, Li H. Evidence on the impact of sustained exposure to air pollu-
tion on life expectancy from China's Huai River policy. Proc Natl Acad Sci USA. 2013; 110(32):12936–
41. doi: 10.1073/pnas.1300018110 PMID: 23836630
3. Benbrahim-Tallaa L, Baan RA, Grosse Y, Lauby-Secretan B, El Ghissassi F, Bouvard V, et al. Carcino-
genicity of diesel-engine and gasoline-engine exhausts and some nitroarenes. Lancet oncol. 2012; 13
(7):663–4. PMID: 22946126
4. Dalsoren SB, Eide MS, Endresen O, Mjelde A, Gravir G, Isaksen ISA. Update on emissions and envi-
ronmental impacts from the international fleet of ships: the contribution from major ship types and ports.
Atmos Chem Phys. 2009; 9:2171–94.
5. Matthias V, Bewersdorff I, Aulinger A, Quante M. The contribution of ship emissions to air pollution in
the North Sea regions. Environ pollut. 2010; 158(6):2241–50. doi: 10.1016/j.envpol.2010.02.013 PMID:
20226578
6. Ault AP, Moore MJ, Furutani H, Prather KA. Impact of emissions from the Los Angeles port region on
San Diego air quality during regional transport events. Environ Sci Technol. 2009; 43(10):3500–6.
PMID: 19544846
7. Poplawski K, Setton E, McEwen B, Hrebenyk D, Graham M, Keller P. Estimation and assesment of
cruise ship emissions in Victoria, BC, Canada. Atmos Environ. 2011; 45:824–33.
8. Corbett JJ, Winebrake JJ, Green EH, Kasibhatla P, Eyring V, Lauer A. Mortality from ship emissions: a
global assessment. Environ Sci Technol. 2007; 41(24):8512–8. PMID: 18200887
9. EPA. Diesel Boats and Ships. Available: http://www.epa.gov/otaq/marine.htm. Accessed March 2014)
2014.
10. Lack DA, Cappa CD, Langridge J, Bahreini R, Buffaloe G, Brock C, et al. Impact of fuel quality regula-
tion and speed reductions on shipping emissions: implications for climate and air quality. Environ Sci
Technol. 2011; 45(20):9052–60. doi: 10.1021/es2013424 PMID: 21910443
11. Blatcher DJ, Eames I. Compliance of Royal Naval ships with nitrogen oxide emissions legislation. Ma-
rine pollution bulletin. 2013; 74:10–8. doi: 10.1016/j.marpolbul.2013.07.010 PMID: 23906471
12. Winebrake JJ, Corbett JJ, Green EH, Lauer A, Eyring V. Mitigating the health impacts of pollution from
oceangoing shipping: an assessment of low-sulfur fuel mandates. Environ Sci Technol. 2009; 43
(13):4776–82. PMID: 19673264
13. Borrell Fontelles J, Straw J. Directive 2005/33/EC of the Europen Parliament and the council. OJEU.
2005; L191/59(22.7.2005).
14. Khan MY, Giordano M, Gutierrez J, Welch WA, Asa-Awuku A, Miller JW, et al. Benefits of two mitigation
strategies for container vessels: cleaner engines and cleaner fuels. Environ Sci Technol. 2012; 46
(9):5049–56. doi: 10.1021/es2043646 PMID: 22468877
15. Gaetjens. http://smm-hamburg.com/fileadmin/img/content/programme/downloads/programmpunkte_
de/491_7351_gaetjens.pdf 2012.
16.
Eyring V, Isaksen ISA, Berntsen T, Collins WJ, Corbett JJ, Endresen O, et al. Transport impacts on at-
mosphere and climate: Shipping. Atmospheric Environment. 2010; 44(37):4735–71. doi: 10.1016/j.
atmosenv.2009.04.059
17. Eyring V, Köhler HW, van Aardenne J, Lauer A. Emissions from international shipping. J Geophys Tes.
2005; 110:D17305.
18. Lloyd AC, Cackette TA. Diesel engines: environmental impact and control. J Air & Waste Manag
Assoc. 2001; 51(6):809–47.
19. Schwarze PE, Totlandsdal AI, Lag M, Refsnes M, Holme JA, Ovrevik J. Inflammation-related effects of
diesel engine exhaust particles: studies on lung cells in vitro. BioMed research international. 2013;
(685142: ):1–13. doi: 10.1155/2013/685142 PMID: 23509760
20. Holder AL, Lucas D, Goth-Goldstein R, Koshland CP. Cellular response to diesel exhaust particles
strongly depends on the exposure method. Toxicol Sci: an official journal of the Society of Toxicology.
2008; 103(1):108–15. doi: 10.1093/toxsci/kfn014
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 15 / 17

21. Paur H-R, Cassee F, Teeguarden J, Fissan H, Diabate S, Aufderheide M, et al. In-vitro cell exposure
studies for the assessment of nanoparticle toxicity in the lung—A dialog between aerosol science and
biology. J Aerosol Sci. 2011; 42(10):668–92. doi: 10.1016/j.jaerosci.2011.06.005
22. Tsukue N, Okumura H, Ito T, Sugiyama G, Nakajima T. Toxicological evaluation of diesel emissions on
A549 cells. Toxicol in vitro. 2010; 24(2):363–9. doi: 10.1016/j.tiv.2009.11.004 PMID: 19900534
23. Cooney DJ, Hickey AJ. Cellular response to the deposition of diesel exhaust particle aerosols onto
human lung cells grown at the air-liquid interface by inertial impaction. Toxicol in vitro. 2011; 25
(8):1953–65. doi: 10.1016/j.tiv.2011.06.019 PMID: 21756993
24. Oostingh GJ, Papaioannou E, Chasapidis L, Akritidis T, Konstandopoulos AG, Duschl A. Development
of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular
effects. Toxicol in vitro. 2013; 27(6):1746–52. doi: 10.1016/j.tiv.2013.04.016 PMID: 23684770
25. Kooter IM, Alblas MJ, Jedynska AD, Steenhof M, Houtzager MM, Ras M. Alveolar epithelial cells
(A549) exposed at the air-liquid interface to diesel exhaust: First study in TNO's powertrain test center.
Toxicol in vitro. 2013; 27(8):2342–9. doi: 10.1016/j.tiv.2013.10.007 PMID: 24161370
26. Adam TW, Chirico R, Clairotte M, Elsasser M, Manfredi U, Martini G, et al. Application of modern online
instrumentation for chemical analysis of gas and particulate phases of exhaust at the European Com-
mission heavy-duty vehicle emission laboratory. Anal Chem. 2011; 83(1):67–76. Epub 2010/12/04. doi:
10.1021/ac101859u PMID: 21126058
27. Mueller D, Uibel S, Takemura M, Klingelhoefer D, Groneberg DA. Ships, ports and particulate air pollu-
tion—an analysis of recent studies. J Occup Med Toxicol. 2011; 6:31. doi: 10.1186/1745-6673-6-31
PMID: 22141925
28. Cooper J. Exhaust emissions from ships at berth. Atmos Environ. 2003; 37:3817–30.
29. Winnes H, Fridell E. Particle emissions from ships: dependence on fuel type. J Air & Waste Manag
Assoc. 2009; 59(12):1391–8.
30. Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, et al. Transformation of human
bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via
strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res.
1988; 48(7):1904–9. PMID: 2450641
31. Kooter IM, Alblas MJ, Jedynska AD, Steenhof M, Houtzager MM, van Ras M. Alveolar epithelial cells
(A549) exposed at the air-liquid interface to diesel exhaust: First study in TNO's powertrain test center.
Toxicol in vitro. 2013; 27(8):2342–9. doi: 10.1016/j.tiv.2013.10.007 PMID: 24161370
32. Steinritz D, Mohle N, Pohl C, Papritz M, Stenger B, Schmidt A, et al. Use of the Cultex(R) Radial Flow
System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparti-
cles with special focus on the intra- and inter-laboratory reproducibility. Chemico-biol Int. 2013; 206
(3):479–90. doi: 10.1016/j.cbi.2013.05.001 PMID: 23669118
33. Herzog F, Clift MJ, Piccapietra F, Behra R, Schmid O, Petri-Fink A, et al. Exposure of silver-nanoparti-
cles and silver-ions to lung cells in vitro at the air-liquid interface. Part Fibre Toxicol. 2013; 10:11. doi:
10.1186/1743-8977-10-11
PMID: 23557437
34. Persoz C, Achard S, Momas I, Seta N. Inflammatory response modulation of airway epithelial cells ex-
posed to formaldehyde. Toxicol Lett. 2012; 211(2):159–63. doi: 10.1016/j.toxlet.2012.03.799 PMID:
22484645
35. Baber O, Jang M, Barber D, Powers K. Amorphous silica coatings on magnetic nanoparticles enhance
stability and reduce toxicity to in vitro BEAS-2B cells. Inhal Toxicol 2011; 23(9):532–43. doi: 10.3109/
08958378.2011.592869 PMID: 21819260
36. Diabate S, Mulhopt S, Paur HR, Krug HF. The response of a co-culture lung model to fine and ultrafine
particles of incinerator fly ash at the air-liquid interface. Atla-Altern Lab Anim. 2008; 36(3):285–98.
PMID: 18662093
37. Patel MM, Chillrud SN, Deepti KC, Ross JM, Kinney PL. Traffic-related air pollutants and exhaled mark-
ers of airway inflammation and oxidative stress in New York City adolescents. Environ Res. 2013;
121:71–8. doi: 10.1016/j.envres.2012.10.012 PMID: 23177171
38. Comouth A, Saathoff H, Naumann K-H, Muelhopt S, Paur H-R, Leisner T. Modelling and measurement
of particle deposition for cell exposure at the air—liquid interface. J Aerosol Sci. 2013; 63(0):103–14.
doi: 10.1016/j.jaerosci.2013.04.009
39. Karthikeyan S, Thomson EM, Kumarathasan P, Guenette J, Rosenblatt D, Chan T, et al. Nitrogen diox-
ide and ultrafine particles dominate the biological effects of inhaled diesel exhaust treated by a cata-
lyzed diesel particulate filter. Toxicol Sci. 2013; 135(2):437–50. doi: 10.1093/toxsci/kft162 PMID:
23897985
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 16 / 17

40. Ghio AJ, Dailey LA, Soukup JM, Stonehuerner J, Richards JH, Devlin RB. Growth of human bronchial
epithelial cells at an air-liquid interface alters the response to particle exposure. Part Fibre Toxicol.
2013; 10(1):25. doi: 10.1186/1743-8977-10-25
41. Dept. of Health and Human Services. NIOHS pocket guide to chemical hazards. Barsan M, editor. Cin-
cinnati Ohio: NIOHS publications; 2007.
42. Pagels J, Khalizov AF, McMurry PH, Zhang RY. Processing of Soot by Controlled Sulphuric Acid and
Water Condensation: Mass and Mobility Relationship. Aerosol Sci Technol. 2009; 43(7):629–40. doi:
10.1080/02786820902810685
43. Park K, Cao F, Kittelson DB, McMurry PH. Relationship between particle mass and mobility for diesel
exhaust particles. Environ Sci Technol. 2003; 37(3):577–83. doi: 10.1021/es025960v PMID: 12630475
44. Ferron GA, Upadhyay S, Zimmermann R, Karg E. Model of the Deposition of Aerosol Particles in the
Respiratory Tract of the Rat. II. Hygroscopic Particle Deposition. J Aerosol Med Pulm Drug Deliv. 2013;
26(2):101–19. doi: 10.1089/jamp.2011.0965 PMID: 23550602
45. Karg E, Ferron GA. The hygroscopic particle lung deposition model Neuherberg / Munich: Helmholtz
Zentrum München; 2014 [cited 2014]. Available: http://www.helmholtz-muenchen.de/en/neu-cma/
research/facilities/lung-deposition-model/index.html.
46. Wehner B, Birmili W, Gnauk T, Wiedensohler A. Particle number size distributions in a street canyon
and their transformation into the urban-air background: measurements and a simple model study.
Atmos Environ. 2002; 36(13):2215–23.
47. Vignati E, Berkowicz R, Palmgren F, Lyck E, Hummelshoj P. Transformation of size distributions of
emitted particles in streets. Sci Total Environ. 1999; 235(1–3):37–49. PMID: 10535125
48. Oeder S, Jorres RA, Weichenmeier I, Pusch G, Schober W, Pfab F, et al. Airborne indoor particles from
schools are more toxic than outdoor particles. Am J Respir Cell Mol Biol. 2012; 47(5):575–82. doi: 10.
1165/rcmb.2012-0139OC PMID: 22904196
49. Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, et al. Global quantification of mam-
malian gene expression control. Nature. 2011; 473(7347):337–42. doi: 10.1038/nature10098 PMID:
21593866
50. Palsson-McDermott EM, O'Neill LA. The Warburg effect then and now: from cancer to inflammatory dis-
eases. BioEssays. 2013; 35(11):965–73. doi: 10.1002/bies.201300084 PMID: 24115022
51. Zhang WC, Shyh-Chang N, Yang H, Rai A, Umashankar S, Ma S, et al. Glycine decarboxylase activity
drives non-small cell lung cancer tumor-initiating cells and tumorigenesis. Cell. 2012; 148(1–2):259–
72. doi: 10.1016/j.cell.2011.11.050 PMID: 22424234
52. Mueller L, Jakobi G, Orasche J, Karg E, Sklorz M, Abbaszade G, et al. Online determination of polycy-
clic aromatic hydrocarbon formation from a flame soot generator. Anal Bioanal Chem. 2015. doi: 10.
1007/s00216-015-8549-x
Lung Cell Responses to Shipping Emissions
PLOS ONE | DOI:10.1371/journal.pone.0126536 June 3, 2015 17 / 17