Toxicology Letters 214 (2012) 218225

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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Cerium dioxide nanoparticles can interfere with the associated cellular

mechanistic response to diesel exhaust exposure
Sandro Steiner a, , Loretta Mueller b , Olga B. Popovicheva c , David O. Raemy a , Jan Czerwinski d ,
Pierre Comte d , Andreas Mayer e , Peter Gehr f , Barbara Rothen-Rutishauser a,g , Martin J.D. Clift a,
a

University of Fribourg, Adolphe Merkle Institute, Route de lAncienne Papeterie, 1723 Fribourg, Switzerland
University of North Carolina at Chapel Hill, Center for Environmental Medicine, Asthma and Lung Biology, 104 Mason Farm Road, Chapel Hill, NC 27510-7310, USA
c
Institute of Nuclear Physics, Moscow State University, 119991 Moscow, Russia
d
AFHB, Bern University of Applied Sciences, Gwerdtstrasse 5, 2560 NIdau, Switzerland
e
Technik Thermische Maschinen (TTM), Fohrhlzlistrasse 14 b, 5443 Niederrohrdorf, Switzerland
f
University of Bern, Institute for Anatomy, Baltzerstrasse 2, 3000 Bern, Switzerland
g
Respiratory Medicine, Bern University Hospital, Inselspital, Freiburgstrasse, 3010 Bern, Switzerland
b

h i g h l i g h t s
 First study to assess dual effects of diesel exhaust and cerium dioxide NPs.
 Cerium dioxide NPs interfere with the secondary toxicity of diesel exhaust.
 Cerium dioxide NPs could be advantageous fuel borne catalysts.

a r t i c l e

i n f o

Article history:
Received 11 June 2012
Received in revised form 27 August 2012
Accepted 28 August 2012
Available online 6 September 2012
Keywords:
Cerium dioxide nanoparticles
Diesel exhaust
Co-exposure study
Fuel additives
Respiratory epithelium

a b s t r a c t
The aim of this study was to compare the biological response of a sophisticated in vitro 3D co-culture
model of the epithelial airway barrier to a co-exposure of CeO2 NPs and diesel exhaust using a realistic
airliquid exposure system. Independent of the individual effects of either diesel exhaust or CeO2 NPs
investigation observed that a combined exposure of CeO2 NPs and diesel exhaust did not cause a significant cytotoxic effect or alter cellular morphology after exposure to diesel exhaust for 2 h at 20 g/ml
(low dose) or for 6 h at 60 g/ml (high dose), and a subsequent 6 h exposure to an aerosolized solution of
CeO2 NPs at the same doses. A signicant loss in the reduced intracellular glutathione level was recorded,
although a signicant increase in the oxidative marker HMOX-1 was found after exposure to a low and
high dose respectively. Both the gene expression and protein release of tumour necrosis factor- were
signicantly elevated after a high dose exposure only. In conclusion, CeO2 NPs, in combination with diesel
exhaust, can signicantly interfere with the cell machinery, indicating a specic, potentially adverse role
of CeO2 NPs in regards to the biological response of diesel exhaust exposure.
2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Corresponding author at: Adolphe Merkle Institute, University of Fribourg,

Route de lAncienne Papeterie, P.O. Box 209, CH 1723 Fribourg, Switzerland.
Tel.: +41 026 300 9515; fax: +41 026 300 9624.
Corresponding author at: Adolphe Merkle Institute, University of Fribourg,
Route de lAncienne Papeterie, P.O. Box 209, CH 1723 Fribourg, Switzerland.
Tel.: +41 026 300 9517; fax: +41 026 300 9624.
E-mail addresses: sandro.steiner@bfh.ch (S. Steiner),
loretta mueller@med.unc.edu (L. Mueller), david.raemy@unifr.ch (D.O. Raemy),
jan.czerwinski@bfh.ch (J. Czerwinski), pierre.comte@bfh.ch (P. Comte),
ttm.a.mayer@bluewin.ch (A. Mayer), peter.gehr@ana.unibe.ch (P. Gehr),
barbara.rothen@unifr.ch (B. Rothen-Rutishauser), martin.clift@unifr.ch
(M.J.D. Clift).
0378-4274/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxlet.2012.08.026

Diesel engines are known to emit high amounts of diverse toxic

compounds, such as nitrogen oxides (NOx ), polyaromatic hydrocarbons (PAHs) and diesel exhaust particles (DEPs) (Westerholm and
Egeback, 1994). Between 1994 and 2010 the European automobile
manufacturers association (www.acea.be) reported an increase in
the percentage of passenger cars containing a diesel engine from
23% to 51% within the European Union. This observed trend has
been suggested to contribute to a higher tropospheric load of these
compounds. The consequence of which therefore, is a continuously
higher exposure to these volatile compounds towards a large fraction of the population, with a heightened risk of detrimental effects
towards human health.

S. Steiner et al. / Toxicology Letters 214 (2012) 218225

Of this increase in volatile compounds within the atmospheric

environment, DEPs are of particular concern due to their specic
context regarding (diesel) exhaust toxicity. DEPs are known to be
readily inhaled and retained in the central airways for prolonged
periods (Moller et al., 2008) and have been reported to deposit on
the lung surface and interact with the local cellular environment
(Muhlfeld et al., 2008). The association between DEP inhalation,
particularly the nanoparticle (NP) content retained within the DEP
sample, and adverse human health effects is well documented.
This is most prominent in the epidemiological study by Peters
et al. (1997) in which twenty-seven, non-smoking asthmatics, who
resided within a highly polluted city in central Europe were examined over a six month period. Analysis of the peak expiratory ow
(PEF) of the lungs for each individual volunteer demonstrated the
number of atmospheric NPs to have a signicant mean decrease in
PEF, compared to the mass of larger particulates in the atmosphere.
It was concluded therefore, that the adverse health effects observed
could be related to the size distribution of environmental air pollution particles, and thus suggesting that DEP associated NPs could
drive lung associated toxicity.
DEPs also contain a wide variation of particulate materials,
including carbon, a complex mixture of metals ranging from 2.5 m
to <100 nm in size (Wilson et al., 2002). It is postulated therefore that DEPs may also mediate the generation of reactive oxygen
species (ROS) and reactive nitrogen species (RNS), which via the
subsequent induction of inammatory responses, have been suggested as being responsible for DEP toxicity (Donaldson et al., 2005;
Pope and Dockery, 2006). Due to these observed effects, increased
efforts have been undertaken in order to reduce the level of DEP
emissions released into the atmosphere and exposed to the human
population (Mayer et al., 2010).
Fuel borne catalysts (FBCs) are a commonly used strategy to
reduce DEP emissions (Mayer et al., 2010). FBCs have been shown to
increases fuel combustion efciency and thereby reduce the particle mass formation in the combustion chamber (Mayer et al., 2010).
In addition, FBCs can assist in diesel particle lter (DPF) regeneration since they are emitted into the exhaust as part of the particulate
phase, where they are able to considerably lower the ignition temperature of soot on the DPF (Setiabudi et al., 2004). A commonly
used metal in FBCs is cerium, specically in the form of cerium
dioxide (CeO2 ). Although DPFs are efcient in retaining particles
originating from FBCs (Mayer et al., 2010), due to the widespread
use of fuel additives, a high emission of CeO2 (nano-) particles cannot be excluded, especially as their use is expected in the absence
of DPFs (e.g. in countries where exhaust emission regulations do
not limit particle emissions sufciently).
It has recently been reported that the biological response to
an individual exposure of NPs can be inuenced by the presence of other NPs or chemicals (Ahmad et al., 2012; Salnikow and
Zhitkovich, 2008; Thomson et al., 2006). In this context, metals
appear to play an important role, for example as co-carcinogens
when applied in combination with engine exhaust (Anttila et al.,
1995; Guo et al., 2009). Based on these observations therefore, it is
reasonable to assume that diesel exhaust toxicity may also be inuenced by the presence of CeO2 NPs. The associated health effects
of CeO2 NPs in general, or as a particular component of fuel additives however, are of great interest. Despite this, few toxicological
studies have been conducted (Cassee et al., 2011). Of the limited
investigations performed however, one particular study compared
the biological response to the exposure of CeO2 NPs alone, diesel
exhaust generated from Ce-based FBC additized fuel and exposure
to diesel exhaust generated from non-additized fuel (Fall et al.,
2007). It was reported that a Ce-based FBC (EnviroxTM ) was not
found to increase the toxicity of diesel exhaust, suggesting that
CeO2 NPs may, benecially, interfere with the biological impact
following diesel exhaust exposure. FBCs however, change the

219

composition of diesel exhaust substantially, and thus studies

comparing the toxicity of exhaust generated from additized and
non-additized fuel are not able to answer the question as to
whether or not a certain metal is advantageous as catalytic component of fuel additives. The aim of this study therefore, was to
compare the toxicity of diesel exhaust in the absence and presence of CeO2 NPs using a sophisticated 3D in vitro co-culture of the
epithelial airway barrier in combination with a novel, established
airliquid interface exposure system. CeO2 NPs were not mixed
with the diesel fuel, but instead applied after diesel exhaust exposure in order to co-expose the cell cultures to DEPs and CeO2 NPs.
In this way, we were able to determine how cellular responses to
diesel exhaust exposure are inuenced by CeO2 NPs independently
to changes in diesel exhaust composition. It was hypothesized that
a decrease in exhaust toxicity brought by the presence of and FBC
(i.e. CeO2 ) may be compensated by the intrinsic toxicity of the FBC
on its own (or that an increase in exhaust toxicity may be compensated by benecial effects of the FBC). As per the knowledge of
the authors, this research study is the rst of its kind, the results of
which will shed a new light upon the suitability of a metal for use
in FBCs.
2. Materials and methods
2.1. Experimental procedure
The experimental procedure is graphically described in Fig. S1. Briey, six sets
of cell cultures were used for each experimental replication. The six cultures were
split into three groups: (i) incubator control, (ii) ltered air exposure and (iii) exhaust
exposure. For each group, two sets of cell cultures were treated (either with (i) no
treatment, (ii) ltered air exposure or (iii) exhaust exposure) for 2 h or 6 h (low
and high dose), followed by 6 h post-incubation at 37 C, 5%CO2 . For all CeO2 NP
exposures, 50 l of 20 or 60 g/ml (low or the high dose) CeO2 NP solution was
aerosolized over the cell cultures prior to the post-incubation period. CeO2 NPs were
therefore exposed to cultures for only 6 h in total. Following all exposures, cell cultures were processed for biological analysis. Cellular morphology was assessed by
uorescent microscopy. Cytotoxicity was estimated by quantication of extracellular lactate dehydrogenase (LDH) activity. The induction of cell death was determined
by assessment of the gene expression levels of two pro-apoptotic genes; caspase7
(CASP7) and FAS. The onset of oxidative stress was estimated by quantication
of total reduced glutathione (GSH) and by measurement of the gene expression
levels of two oxidative stress-responsive genes; heme oxygenase 1 (HMOX1) and
superoxide dismutase 1 (SOD1). (Pro)-inammatory responses were assessed via
measurement of the gene expression levels of tumour necrosis factor (TNF) and
interleukin-8 (IL-8), as well as by quantication of their gene products (protein)
TNF- and IL-8 secreted in the culture medium.
2.2. Cell cultures
A sophisticated 3D in vitro co-culture model of the epithelial airway barrier was
used. The properties and the production of this cell culture model are described in
Blank et al. (2007) and Rothen-Rutishauser et al. (2005) with the following adaptations: human whole blood monocytes were isolated from buffy coats (provided
by the Blutspendendienst SRK Bern AG, 3008 Bern) using CD14+ MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturers
manual.
2.3. Test vehicle type and settings
The test vehicle was an Opel Astra X20DTL 2.0L without exhaust after treatment system. The vehicle ran upon a dynamometer at a constant velocity of
35 km/h (engine speed 2180 rpm) with a low sulfur diesel (Greenergy SA, Steinhausen, Switzerland) and normal lubrication oil (V10.237, Motorex, Langenthal,
Switzerland).
2.4. Exhaust characterization and particle deposition on the cell cultures
To determine the size-number distribution and elemental carbon (EC) content
of the particulate exhaust fraction a differential mobility analyzer (TSI 3071), a condensation particle counter (TSI 3025 A) and a photoelectric aerosol sensor (EcoChem
PAS 2000) were employed The concentration of carbon monoxide (CO), total gaseous
hydrocarbons (HC), NOx and nitrogen monoxide (NO) was measured using the
Horiba MEXA-9400H exhaust gas measuring system. All measurements were conducted in parallel to the exposure experiments with samples of the same (ten-fold
diluted) exhaust. During diesel exhaust exposures, DEP samples (from undiluted
exhaust) were collected on PallFlex lters and analysed for their surface chemistry

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S. Steiner et al. / Toxicology Letters 214 (2012) 218225

by Fourier Transform Infrared spectroscopy (FTIR, IRPrestige-21, Shimazdu). DEPs

and CeO2 NPs deposition inside the exposure chambers (on the cell cultures) was
Deposited DEPs and CeO2 NPs were subsequently quantied via transmission electron microscopy, as previously described in Muller et al. (2010). Briey, for each
exposure setting two TEM grids were placed in the exposure system (described
below) for each previously stated exposure period. The TEM grids were then analysed, with a total of 1520 pictures per grid over an area of 46.2 m2 assessed and
the number of deposited particles counted by hand.

control (absolute values as well as all normalized values are shown in Appendix
Table B). In addition, in light of these variations, statistical analysis was performed
on the average of the difference between the experimental groups rather than on
the measured average cellular response. The procedure is described in Cumming
and Finch (2005) and corresponds to a t-test on the observed differences between
responses to different exposures. All raw data values were calculated using Microsoft
Excel 2010. All statistical analysis was performed using IBM SPSS statistics19. All
data is presented as the mean the standard error of the mean (SEM). Results were
considered signicant if the p < 0.05.

2.5. Exhaust exposure system and exposure conditions

The exposure system used has previously been described in detail by Muller
et al. (2010). Briey, the cell cultures were exposed to a ten-fold diluted (in ltered
air) exhaust sample at the airliquid interface for 2 h or 6 h (low and high dose)
followed by 6 h post-incubation period at 37 C, 5%CO2 , 80% relative humidity for
6 h.
2.6. Preparation of CeO2 NPs and CeO2 NP exposure
A CeO2 NP aerosol was prepared by ame spray synthesis as described in Raemy
et al. (2011). Directly after the exposure to diesel exhaust or ltered air or for the
CeO2 NP only exposure, 50 l of particle suspension (20 g/ml after 2 h, 60 g/ml
after 6 h exhaust/ltered air exposure) was applied in aerosolized form to the cells
using a microsprayer (Model FMJ-250 high pressure syringe and model IA-1C 1
long microsprayer tip; Penn-Century Inc., Philadelphia, USA).
2.7. Microscopy
Samples were stained with phalloidin-rhodamine to label the F-actin cytoskeleton, as well as with 4 ,6-diamidino-2-phenylindole (DAPI) to highlight the cell nuclei
as previously described by Muller et al. (2010) and subsequently imaged by uorescent microscopy (Leica DMI 4000B, Leica Application Suite Advanced Fluorescence
2.3.5 software).
2.8. Cytotoxicity
Cytotoxicity was assessed using the cytotoxicity Detection Kit (LDH), Roche
Applied Science, Rotkreuz, Switzerland) according to the manufacturers protocol.
2.9. Quantication of total reduced glutathione
The total amount of reduced glutathione (GSH) in the cell cultures was quantied by using a glutathione assay kit (Cayman Chemical Company, Ann Arbor,
USA) according to the manufacturers instructions. The detected concentrations of
reduced GSH are reported relative to the concentrations of total protein in the samples (determined the Pierce BCA Protein Assay kit (Pierce Protein research Products,
Thermo Scientic, Rockford, USA) according to the manufacturers guidelines).
2.10. Real-time reverse-transcriptase polymerase chain reaction (real-time
RT-PCR)
Immediately after post incubation, cells were transferred into RNA protect buffer
(Qiagen AG, Hombrechtikon, Switzerland) and stored at 4 C until further use. RNA
isolation was achieved by using an RNeasy plus kit (Qiagen AG, Hombrechtikon,
Switzerland) according to the manufacturers guidelines. Reverse transcriptase reactions were performed in 10 l volumes with an RNA concentration of 25 ng/l
(Omniscript RT, Qiagen AG) and Oligo dT primers (Qiagen). To induce a reverse
transcriptase reaction, 0.25 l RNasin Plus RNase Inhibitor (Promega AG, Dbendorf, Switzerland) was added. A total of 2 l of the ten-fold diluted cDNA was used
for real-time PCR in reaction volumes of 10 l with SYBR Green as reporter dye
(Fast SYBR Green master mix, 7500 fast real-time PCR system, Applied Biosystems
International Inc., Rotkreuz, Switzerland). Relative expression levels were calculated
using the Ct method with glyceraldehyde-3-phosphate reductase as an internal
standard. The genes assessed, their GenBank (GenBank ID: BA123456) accession
numbers and the sequences of the primers used are listed in Appendix Table A.
2.11. Quantication of (pro-)inammatory cytokines/chemokines
The quantity of both the TNF- and IL-8 protein released into the culture
medium was assessed by enzyme linked immunosorbent-assay (ELISA) using the
human TNF-alpha DuoSet and the human CXCL8/IL-8 DuoSet (R&D Systems, Abingdon, UK) as per the manufacturers instructions.
2.12. Data analysis and statistics
For each day of exposure, human blood derived monocytes were isolated from a
different buffy-coat and hence from a different blood donor. Therefore, variations
in the background (negative control) between different sets of cell cultures were
expected and observed. Due to the presence of such variations, instead of using
the measured absolute values, all measurements were normalized to the untreated

3. Results and discussion

In the present study, the ability for CeO2 NPs to affect the toxicity of diesel exhaust in vitro was assessed using a sophisticated
3D model of the human epithelial barrier. Due to the complexity of
the used cellular model, the present study goes beyond considering
the respiratory epithelium as a simple mechanical barrier, but takes
an important set of immunological functions and cell-cell interactions into consideration (Blank et al., 2007; Rothen-Rutishauser
et al., 2005). Furthermore, exposure of diesel exhaust and CeO2 NPs
were conducted at the air liquid interface, which reects the situation in vivo and reduces the interaction between exhaust and
culture medium (Holder et al., 2008; Muller et al., 2011). In addition,
the used exhaust exposure system allows application of freshly
produced and whole exhaust samples under controlled conditions
(Muller et al., 2010). Aging effects or biased sample generation can
therefore be ruled out. Since whole diesel exhaust was used for
exposures, the dose applied could only be adjusted by variation
in exposure time and exhaust dilution. As previously described by
Muller et al. (2010), diesel exhaust exposure periods of 2 h and 6 h
are short in comparison to other studies. Since exposure at the
airliquid interface may pose a certain level of stress to the cell
cultures however, we decided to keep the exposure duration short
in order not to provoke the risk of biological artefacts occurring. To
compensate for the short exposure period, it was decided to use
ten-fold diluted exhaust, which is ten-fold the concentration experienced from a distance of two to three meters from a busy road
(Gautam et al., 2003).
As previously reported (Schnelle-Kreis et al., 2005), the analysis
of the DEP surface chemistry by FTIR revealed that the particle surfaces are dominated by alkenes and alkanes (distinct bands at 1375,
1465 and 28003000 cm1 ). Aromatic compounds and carbonyl
groups appeared to be present only in low amounts, as indicated
by very weak bands at 1595 cm1 and 1717 cm1 respectively. The
characterization of the diluted exhaust as well as the numbers of
DEPs and CeO2 NPs deposited on the cell cultures are listed in
Appendix Table C and Fig. S2. The decision for the deposition of
a higher number of CeO2 NPs than DEPs per area was based on the
fact that during combustion of diesel containing CeO2 NPs, each
DEP will adsorb the CeO2 NPs, but that the occurrence of free CeO2
NPs cannot be excluded (Mayer et al., 2010), which will result in
an overall higher interaction between cells and CeO2 than between
cells and carbonaceous DEP surfaces.
Independent of the exposure setting and the dose, uorescent
microscopy revealed no rupture in the layer of epithelial cells and
the cellular morphology also appeared to not be affected (Fig. 1A).
In addition, the quantication of extracellular LDH showed no statistically signicant cytotoxic effects for any exposure or dose used
(Fig. 1B). This implies that the measured biological responses can
be considered a reaction of properly functioning cellular signalling
networks. Considerably elevated average LDH release was only
observable upon exposure to high doses of ltered air + CeO2 NPs
and diesel exhaust + CeO2 NPs. These elevated values however, can
be attributed to an outlier within the data set.
For CASP7 expression (Fig. 1C), low dose exposures did not
reveal a consistent pattern of responses. High doses of CeO2 NPs
however clearly showed a repressive effect despite the lack of a

S. Steiner et al. / Toxicology Letters 214 (2012) 218225

221

Fig. 1. Cellular morphology, cytotoxicity and pro-apoptotic responses. (A) Fluorescent microscopic pictures of uorescently labelled cell cultures. Green colour shows F-actin
(cytoskeleton), yellow colour shows nucleic acids (cell nuclei). The size bar corresponds to 25 m. Only cultures exposed to high dose (60 g/ml for 6 h and 6 h post-incubation)
are shown. (B) Cytotoxicity as estimated by quantication of extracellular LDH release (shown relative to the untreated control). (C and D) Transcriptional activity of the
pro-apoptotic genes CASP7 and FAS. Data is presented as the mean standard error of the mean (SEM) (n = 3). *A statistical signicance compared to the negative control and
as indicated (p < 0.05). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of the article.)

statistically signicant outcome: CeO2 NPs resulted in a 0.71 0.14

fold gene expression relative to the untreated control, ltered
air + CeO2 NPs resulted in a 0.29 0.12 and diesel exhaust + CeO2 in
a 0.28 0.10 fold expression compared to the ltered air exposure
whereas diesel exhaust alone caused no change in gene expression (1.13 0.46 fold relative to ltered air). Diesel exhaust + CeO2
NPs therefore resulted in a 0.25 0.09 fold expression when
compared to diesel exhaust only. This indicates that CeO2 NPs
reduce pro-apoptotic responses and that this effect is enhanced by
diesel exhaust exposure. FAS expression (Fig. 1D) was found to be
repressed by all treatments, but a consistent pattern of CeO2 NPs
and diesel exhaust acting individually or in combination could not
be observed.
All exposures resulted in lower concentrations of reduced GSH
compared to the untreated control, except for the high dose exposure to CeO2 NPs (116 25%) (Fig. 2A). Effects of CeO2 NPs alone,
ltered air and ltered air + CeO2 NPs were small. The strongest
effect was a decrease to 69 6% of the untreated control upon 2 h
exposure to ltered air. It is noteworthy however, that exposure
to low doses of ltered air + CeO2 NPs resulted in higher levels
of reduced GSH than exposure to ltered air alone (131 6% of
the untreated control). Relative to ltered air, exposure to diesel
exhaust resulted in a statistically signicant decrease in reduced
GSH levels to 4.5 2.9% (low dose) and 1.7 1.7% (high dose). Similarly, diesel exhaust + CeO2 NPs caused a decrease to 7.5 4.7%

(low dose) and 16 10%, only the low dose effect being statistically
signicant. The nding that diesel exhaust exposure drastically
decreases the cellular pool of antioxidant molecules was expected,
since the high oxidative potential of diesel exhaust is well described
(Pan et al., 2004). Also, the slight antioxidant effect of CeO2 NPs
observed was not surprising since similar observations are also
reported (Xia et al., 2008). However, the decrease in GSH oxidation
brought by CeO2 NPs is small and may not be considered biologically relevant.
The consequence of the depleted cellular pool of antioxidant
molecules is the induction of oxidative stress-responsive genes
(Motterlini et al., 2000). However, the results of the present study
clearly show that the levels of reduced GSH cannot be directly
correlated with the transcriptional activation of HMOX1 (Fig. 2B),
a fact that has to be attributed to the more complex regulation
of the expression of the gene (Ryter and Choi, 2010). In particular, the presence of additional stressful stimuli appears to play
a role. In the absence of such stimuli (untreated control) CeO2
NPs act weakly inducing at low doses (2.6 2.2 fold expression)
and strongly inducing at high doses (40 4 fold expression). As
soon as ltered air is included (causing a certain degree of stress
due to the continuous air ow), a suppressive effect of CeO2 NPs
is seen at low doses (0.14 0.10) but an inducing effect at high
doses (18 2) (both expressed relative to ltered air alone). The
picture is similar when diesel exhaust exposures are compared

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S. Steiner et al. / Toxicology Letters 214 (2012) 218225

Fig. 2. Cellular oxidative stress response. (A) Total reduced GSH/total protein in the
cell culture expressed relative to the untreated control. (B and C) Transcriptional
activity of the oxidative stress-responsive genes HMOX1 and SOD1. Data is presented
as the mean standard error of the mean (SEM) (n = 3). * A statistical signicance
compared to the negative control and as indicated (p < 0.05).

to diesel exhaust + CeO2 NP exposure; at low doses, the transcriptional induction caused by diesel exhaust (21 2) is completely
abolished as soon as CeO2 NPs are included (diesel exhaust + CeO2
NPs: 1.1 0.8). In contrast, when looking at the high dose exposures, the induction caused by diesel exhaust (34 13) is strongly
enhanced by CeO2 NPs (diesel exhaust + CeO2 NPs: 293 45). This
corresponds to a 0.06 0.09 (low dose) and an 8.6 1.3 (high dose)
fold expression upon diesel exhaust + CeO2 NP exposure compared
to diesel exhaust only, both being statistically signicant. Independently of whether or not the cells had been exposed to diesel
exhaust, the effect of CeO2 NPs on HMOX1 expression is therefore
qualitatively the same but the amplitude of the response is more
than two times as high (low dose) or only half as high (high dose)
as soon as the cells are exposed to diesel exhaust + CeO2 NPs. This
observation implies that how CeO2 NPs affect biological systems
depends strongly on what additional stimuli the system experience
and on the dose in which they are applied. At very low stress levels (untreated control) low doses of CeO2 NPs do not affect HMOX1
gene expression, whereas at high doses they act in an inducing
manner. Moderate stress (ltered air exposure) results in downregulation of the gene when low doses of CeO2 NPs are applied
but in up-regulation in the presence of high doses of CeO2 NPs.
High stress levels (diesel exhaust exposure) cause low doses of
CeO2 NPs to down-regulate HMOX1 expression strongly, whereas
high doses cause an up-regulation, resulting in very high gene
expression.
As it is the case for HMOX1, SOD1 expression could not be correlated with the levels of reduced GSH, which we as well assume
to be due to the complex regulation of the gene (Zelko et al.,
2002). The transcriptional responses of SOD1 were small in all cases
(Fig. 2C), but it is noteworthy that upon high dose exposures CeO2
NPs elicited a limited cellular response, as relative to the untreated
control, CeO2 NPs led to a 0.7 0.09 fold gene expression. Relative to ltered air, ltered air + CeO2 NPs resulted in a 0.7 0.08
fold, diesel exhaust + CeO2 NPs in 1.4 0.6 fold gene expression. It
is worth noting though that the result obtained for the high dose
diesel exhaust + CeO2 NP exposure is strongly inuenced by one
value considerably higher than the other two. Omission of this value
yields a 0.8 0.1 fold expression relative to ltered air, which relative to diesel exhaust exposure corresponds to a 0.7 0.08 fold
expression). We do not assume this result to be due to the slightly
higher levels of reduced GSH observed upon exposures in which
CeO2 NPs were applied since those differences are small compared
to the total cellular pool of the molecule. It also cannot be assumed
that the small observed differences in SOD1 expression are biologically relevant.
One of the consequences of oxidative stress is the induction of
and inammatory cascade (Donaldson et al., 2005; Xiao et al., 2003).
Considering the drastically decreased levels of reduced GSH upon
exposure to diesel exhaust and diesel exhaust + CeO2 NPs, it was
therefore expected that transcriptional activation of the genes TNF
and IL-8 and/or an increase in the secretion of their gene products
TNF- and IL-8 could occur. Indeed, relative to ltered air, diesel
exhaust exposure resulted in a 1.3 0.2 (low dose) and a 2.1 0.3
(high dose), diesel exhaust + CeO2 NPs in a 1.2 0.1 (low dose) and
a 1.9 0.6 (high dose) fold expression of TNF (Fig. 3A). For IL-8,
the according values are 1.4 0.5 (low dose) and a 3.7 1.9 (high
dose) upon exposure to diesel exhaust and 1.3 0.6 (low dose)
and 2.5 1.1 (high dose) upon exposure to diesel exhaust CeO2
NPs (Fig. 3B). Even though these values are lower than expected,
they clearly demonstrate an inammatory response, at least for
high dose exposures. CeO2 NPs appeared to act suppressive on TNF
expression since low and high doses of CeO2 NPs alone resulted
in 0.8 0.1 fold gene expression relative to the untreated control
and exposure to ltered air + CeO2 NPs in a 0.8 0.1 (low dose) and
0.6 0.14 (high dose) fold gene expression relative to ltered air

S. Steiner et al. / Toxicology Letters 214 (2012) 218225

223

Fig. 3. Inammatory response. (A and B) Transcriptional activity of the gene TNF and IL-8. (C and D) Extracellular concentration of the (pro-)inammatory proteins TNF-
and IL-8. Data is presented as the mean standard error of the mean (SEM) (n = 3). *A statistical signicance compared to the negative control and as indicated (p < 0.05).

exposure. With diesel exhaust + CeO2 NPs resulting in a 1.2 0.15

(low dose) and a 1.9 6 (high dose) fold TNF expression relative
to ltered air exposure, CeO2 NPs reduced the expression of the
gene to 0.9 0.1 (low dose) and 0.9 0.3 (high dose) fold relative to
diesel exhaust. These effects were not statistically signicant and
since they are small are not assumed to be biologically relevant.
A conclusive effect of CeO2 NPs on IL-8 expression could not be
deduced.
The protein quantication data for TNF- and IL-8 (Fig. 3C
and D) do not reect the patterns observed at the transcriptional level, which can be explained by the time shift that has
to be expected between transcription of a gene, the presence
of its gene product and by regulatory events between transcription and protein secretion. The relative to ltered air exposure
increased TNF- secretion upon exposure to diesel exhaust and
diesel exhaust + CeO2 NPs expected based on the gene expression data is observable (diesel exhaust: 1.3 0.1 (low dose) and
1.4 0.2 (high dose), diesel exhaust + CeO2 NPs: 1.4 0.2 (low dose)
and 1.4 0.14 (high dose)), but the suppressive effect of CeO2 NPs
observed for TNF expression could not be detected at the protein
level (e.g. diesel exhaust + CeO2 NPs relative to diesel exhaust alone:
1.1 0.2 (low dose) and 1.0 0.1 (high dose) fold TNF- secretion).
Rather, low doses of CeO2 NPs alone resulted in a (small) increase
in TNF- secretion (1.3 0.1). For IL-8, upon low dose exposures no
changes were measured at all. High doses of ltered air + CeO2 NPs,
diesel exhaust and diesel exhaust + CeO2 NPs resulted in slightly
increased IL-8 secretion relative to ltered air (1.5 0.4, 1.2 0.2
and 1.4 0.4).

Taken together, the results of the present study show that following a short exposure period to a sophisticated in vitro model
system of the epithelial airway barrier, CeO2 NPs do not increase
the cytotoxicity of diesel exhaust at the doses used. Rather they act
in a cytoprotective manner by reducing the expression of the proapoptotic gene CASP7. Considering the fact that diesel exhaust is
known to have mutagenic properties (Bao et al., 2007) (Human 1A
carcinogen) however, it is questionable whether or not the suppression of apoptotic responses can be considered benecial. In
accordance with other studies (Schubert et al., 2006), a certain
antioxidant effect of CeO2 NPs was observed, although in the context of the strongly oxidative diesel exhaust this cannot be assumed
to signicantly reduce the level of oxidative stress. Hence, it was
not possible to detect the antioxidant effect of CeO2 NPs in the
transcriptional response to oxidative stress. Even though CeO2 NPs
appeared to result in a slightly decreased activation of SOD1 expression, this effect is too small to be considered biologically relevant.
HMOX1 expression is affected by both diesel exhaust and CeO2 NPs
and the combination of the two leads to responses that besides the
dose applied appear to depend upon the level of stress to which the
cells are subjected. Besides being involved in the defence against
oxidative stress, the gene product of HMOX1 is known to have a regulatory function within the inammatory and apoptotic processes
(Prawan et al., 2005). Even though the effects on inammatory and
apoptotic responses observed in this study cannot be correlated
with the observed expression patterns of HMOX1 and despite the
fact that the observed effects on inammatory responses are not
likely to be biologically relevant, it is reasonable to assume that over

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S. Steiner et al. / Toxicology Letters 214 (2012) 218225

longer periods of exposure CeO2 NPs will have effects on these two
key players in the processes involved in the onset of air pollution
related respiratory diseases.
The present study provides insight into the biological effects
caused by the complete exhaust (i.e. gas-phase components, particles and semivolatile compounds adsorbed to the particles), in
the presence or absence of CeO2 NPs. In order to obtain a detailed
picture on which component(s) of the exhaust is/are responsible
for any observed biological response, additional studies using ltered exhaust, organic and inorganic particle extracts and denuded
particles, each individually and in co-exposure with CeO2 NPs, are
required. For such experiments, new protocols for cell exposures
at the airliquid interface with particles that have been denuded
from adsorbed surface chemicals and the according extracts need to
be established, since suspension exposure models will wash away
any non-soluble compounds from the core particle, and thus do not
represent a realistic exposure scenario (Holder et al., 2008; Muller
et al., 2011).
In conclusion, it appears that CeO2 NPs do not have adverse
effects on acute cellular responses to diesel exhaust exposure. This
suggests that with regard to human respiratory health, CeO2 NPs
are in fact applicable as an efcient FBC. Since however, it cannot
be deduced from the present study whether the observed changes
in the regulation of pro-apoptotic and oxidative stress-responsive
genes will result in undesirable long-term effects, additional work
employing longer exposure periods and more detailed investigation of the mentioned cellular responses is needed in order to derive
a proper understanding of the potential risk posed by such an exposure.
Conict of interest
The authors declare that Dr. Andreas Mayer is the owner and
general manager of TTM Andreas Mayer, Switzerland, an emission consulting company. All other authors declare no conicts of
interest. All authors are responsible for the writing and content of
the manuscript.
Acknowledgements
The authors would like to acknowledge the nancial support of
the European Respiratory Society, Fellowship LTRF-MC1572-2010
to Dr. MJD CLIFT, the Swiss Federal Ofce for the Environment,
Erdlvereinigung EV and VSS lubes, as well as and the Adolphe
Merkle Foundation. Furthermore, the authors would like to thank
the Bern University of Applied Sciences, the Institute of Aerosol and
Sensor Technology, Northwestern Switzerland and the University
of Rouen for their invaluable technical assistance. The authors also
acknowledge the input of E. Kireeva (associated with afliation c in
the authors list) in respect to the FTIR measurements and the kind
donation of the CeO2 NPs from W. Stark (ETH Zurich, Switzerland).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.toxlet.2012.08.026.
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