Toxicology 262 (2009) 265–270

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Wood dusts induce the production of reactive oxygen species and caspase-3
activity in human bronchial epithelial cells
Lea Pylkkänen, Helene Stockmann-Juvala, Harri Alenius, Kirsti Husgafvel-Pursiainen, Kai Savolainen ∗
Finnish Institute of Occupational Health, Topeliuksenkatu 41 a, 00280 Helsinki, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Wood dusts are associated with several respiratory symptoms, e.g. impaired lung function and asthma,
Received 5 December 2008 in exposed workers. However, despite the evidence from epidemiological studies, the underlying mech-
Received in revised form 18 June 2009 anisms are not well understood. In the present study, we investigated different wood dusts for their
Accepted 21 June 2009
capacity to induce cytotoxicity and production of radical oxygen species (ROS) as well as activation of the
Available online 30 June 2009
apoptotic caspase-3 enzyme in human bronchial epithelial cells (BEAS-2B). Dusts from three different
tree species widely used in wood industry were studied; birch and oak represented hardwood species,
Keywords:
and pine a common softwood species. All the experiments were carried out in three different concentra-
Wood dust
Reactive oxygen species
tions (10, 50, and 500 ␮g/ml) and the analysis was performed after 0.5, 2, 6, and 24 h exposure. All wood
Caspase-3 dusts studied were cytotoxic to human bronchial epithelial cells in a dose-dependent manner after 2 and
Apoptosis 6 h treatment. Exposure to pine, birch, or oak dust had a significant stimulating effect on the production
Bronchial epithelial cell of ROS. Also an induction in caspase-3 protease activity, one of the central components of the apoptotic
BEAS-2B cascade, was seen in BEAS-2B cells after 2 and 6 h exposure to each of the wood dusts studied. In sum-
mary, we demonstrate that dusts from pine, birch and oak are cytotoxic, able to increase the production of
ROS and the apoptotic response in human broncho-epithelial cells in vitro. Thus, our current data suggest
oxidative stress by ROS as an important mechanism likely to function in wood dust related pulmonary
toxicity although details of the cellular targets and cell–particle interactions remain to be solved. It is
though tempting to speculate that redox-regulated transcription factors such as NF␬B or AP-1 may play
a role in this wood dust-evoked process leading to apparently induced apoptosis of target cells.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Background nose and para-nasal sinuses particularly among workers exposed to

Exposure to wood dust is an important health hazard in the
work environment although the underlying mechanisms are not
well understood. It is estimated that in the European Union approx-
imately 3.6 million workers are exposed to inhalable wood dust
(Kauppinen et al., 2006). Occupational exposure to dust from var-
ious tree species has been shown to be associated with a variety
of health effects, including multiple respiratory effects such as
asthma, chronic bronchitis, and emphysema (SCOEL, 2003; ACGHI,
2005). Wood dust exposure is also strongly associated with nasal
cancer (Demers et al., 1995; Barcenas et al., 2005; Delclos et al.,
2005). Furthermore, in 1995, wood dust was classified by the Inter-
national Agency for Research on Cancer (IARC) as a known human
carcinogen (Group 1) based on increased risk for cancer of the
∗ Corresponding author.
E-mail addresses: lea.pylkkanen@ttl.fi (L. Pylkkänen),
helene.stockmann-juvala@ttl.fi (H. Stockmann-Juvala), harri.alenius@ttl.fi
(H. Alenius), kirsti.husgafvel-pursiainen@ttl.fi (K. Husgafvel-Pursiainen),
kai.savolainen@ttl.fi (K. Savolainen).
hardwood dusts (IARC, 1995). In addition, there are less conclusive
epidemiological data suggesting increased risk of other respira-
tory cancers, including lung cancer, in association with wood dust
exposure (Demers et al., 1995; Barcenas et al., 2005; Delclos et al.,
2005).
In industrial processing of wood, airborne wood dust parti-
cles are generated in large quantities. In woodworkers, asthma
and other respiratory symptoms have been attributed to dust
from many different tree species, including those from common
softwood species such as pine, based on epidemiological stud-
ies (SCOEL, 2003; ACGHI, 2005; Douwes et al., 2001), and there
is increasing evidence that these symptoms occur already at low
wood dust levels (Demers et al., 1997; Douwes et al., 2001; Schlun-
ssen et al., 2002). Moreover, inhalation of pinewood dust has been
shown to lead to the recruitment of inflammatory cells to the
airways of healthy subjects exposed to wood dust, suggesting a
contribution of inflammatory mechanisms to the development
of respiratory disorders among woodworkers (Gripenback et al.,
2005).
Morphological analysis on human lung epithelial cells has
shown that fine particles interact with the cell surface, where

0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2009.06.019
266 L. Pylkkänen et al. / Toxicology 262 (2009) 265–270
they induce alterations and, subsequently, are internalized into the
cytoplasm (Calcabrini et al., 2004). Cellular exposure to fine par-
ticulate matter may lead to increased production of radical oxygen
species (ROS) which can play a central role in signal transduction
pathway during apoptosis (Kannan and Jain, 2000), with the activa-
tion of a cascade of caspases preceding the morphological changes
occurring during apoptosis (Turk and Stoka, 2007). In particular,
caspase-3 is essential in this process as it is responsible for the
proteolytic cleavage of many of the key proteins (Villa et al., 1997;
Abu-Qare and Abou-Donia, 2001; Lakhani et al., 2006). Increased
ROS production may also increase the expression of genes encod-
ing redox-regulated transcription factors such as NF␬B and AP-1
that are known to be associated with the induction of apoptosis
(Gwinn and Vallvathan, 2006).
The present study was conducted as a continuation of a series
of studies on inflammatory effects of wood dusts in murine cells in
vitro and in allergic asthma murine model in vivo (Long et al., 2004;
Määttä et al., 2005, 2006a,b, 2007; Bornholdt et al., 2007). Focus-
ing on cells of human airway origin, we investigated the capacity
of wood dusts to induce ROS production and to activate the apop-
totic caspase-3 enzyme in cultured human bronchial epithelial cells
(BEAS-2B), a cell line successfully applied for studying toxic effects
in human respiratory cells (Steerenberg et al., 1998; Han et al., 2007;
Schmid et al., 2007; Veranth et al., 2007). Dusts from three different
wood species widely used in wood industry in Finland and else-
where were studied. From those, birch and oak represent hardwood
species, and pine a common softwood species.
2. Materials and methods
2.1. Wood dusts
Dusts from pine, birch, and oak were obtained from the Kuopio Regional Institute
of Occupational Health (Kuopio, Finland). Good-quality dry raw wood not impreg-
nated with wood preservatives was carefully selected for production of dusts; dusts
were generated with a dust collecting face-grinding machine with 400-grit sand-
ing paper. To separate coarse particles from the total dust, the dust was aerosolized
with a Pitt number 3 generator after which the large particles entered into a sam-
pling chamber. For particle size distribution analyses, wood dust specimens were
gold-coated for 170 s with BAL-TEC SCD 005 Sputter Coater (BAL-TEC AG, Liechten-
stein) and thereafter examined on a JEOL JSM-6400 scanning electron microscope
(JEOL Inc., Peabody, MA) at an acceleration voltage of 20 kV. At least 1300 particles
for each dust were analysed from electron micrographs for particle size distribution
and more than 90% of particles of all the used wood dusts had a geometric diameter
less than 5 ␮m. Preparation, physicochemical characterization, and the size distri-
bution monitoring of wood dusts used have been described in detail earlier (Naarala
et al., 2003; Long et al., 2004; Määttä et al., 2005).
To assure the absence of contamination of bacterial origin, endotoxin levels in the
wood dusts were measured with a kinetic Limulus amebocyte lysate (LAL) test. The
endotoxin concentrations of the wood dusts were 50 pg/mg for pine dust, 220 pg/mg
for birch dust, and 70 pg/mg for oak dust (Määttä et al., 2006b). In addition, volatile
organic compounds were released at 70 ◦ C (Naarala et al., 2003). For experiments,
wood dusts were suspended in BEGM-medium and vortexed before adding on the
cells. The size distribution of the wood dusts used in these experiments is shown
in Fig. 1 (modified from the figures published earlier in Määttä et al., 2005 and
2006b).
2.2. Cell culture and exposure
Human bronchial epithelial cells (BEAS-2B) were obtained from the American
Type Culture Collection (ATCC; Rockville, USA). BEAS-2B is an immortalised cell-line,
derived from normal human bronchial epithelium transformed by SV-40 adenovirus
(Reddel et al., 1988).
The cells were cultured in 25 cm2 tissue culture flasks (5 × 106 cell/flask) in
serum free Bronchial Epithelial Cell Basal Medium (BEBM) (Clonetics, San Diego,
CA) supplemented with epidermal growth factor (0.5 ng/ml), insulin (5 ␮g/ml),
hydrocortisone (0.5 ␮g/ml), transferrin (10 ␮g/ml), epinephrine (0.5 ␮g/ml), tri-
iodothyronine (6.5 ng/ml), bovine pituitary extract (BPE), retinoic acid (0.1 ng/ml),
gentamicin (50 ␮g/ml), and amphotericin-B (50 ␮g/ml). The cells were incubated in
a humidified atmosphere at 37 ◦ C with 5% CO2 , detached from the flasks with 0.25%
trypsin-EDTA (Sigma–Aldrich, Inc.) solution and sub-cultured after neutralisation
with soybean inhibitor (Sigma–Aldrich, Inc.). After two days incubation the culture
medium was changed and wood dusts suspended in the medium were added to the
cells. The final concentrations of the wood dusts were 10, 50, and 500 ␮g/ml, and
Fig. 1. Size distribution of the wood dusts used in the current experiments as based
on electron microscopy analysis of the wood dust material used in these studies.
the durations of the incubations were 0.5, 2, 6, 12, and 24 h. Untreated cells, serving
as control cells, were incubated for the same time periods.
2.3. Cell viability
Cell survival was determined by bright-field microscopy using the 0.4% trypan
blue dye exclusion method. The cells were trypsinized after 2, 6, and 12 h exposure
and cell survival was determined by counting viable cells in a randomly selected
four non-overlapping fields using a hemocytometer.
2.4. Measurement of reactive oxygen species (ROS)
BEAS-2B cells were cultured on 48 well plates and after exposure to
wood dusts at the final concentrations of 10, 50, or 500 ␮g/ml the cells were
loaded with 40 ␮M 2 ,7 -diclorodihydrofluorescein diacetate (2-[2,7-dichloro-3,6-
diacetyloxy-9H-xanthen-9-yl]-benzoic acid by Cayman Chemical, MI, USA) for
20 min at room temperature. The formation of a fluorescent compound, 2 ,7 -
diclorodihydrofluorescein, was monitored at an excitation wavelength of 535 nm
and emission wavelength of 485 nm using a Wallac Victor II Multilabel Counter
(Wallac, Turku, Finland). Background fluorescence was obtained from cell-free wells
containing same volumes 2 ,7 -diclorodihydrofluorescein diacetate as the samples,
and it was subtracted from the fluorescence values of the cell samples. The ROS pro-
duction was calculated as percent of the untreated control cells at each time point
(0.5, 2, 6, and 12 h). In addition, wells with two concentrations of wood dusts (50
and 500 ␮g/ml) were included in each experiment to control the background.
2.5. Measurement of caspase-3 protease activity
Caspase-3 protease activity was measured using a fluorogenic caspase-3 sub-
strate (Ac-DEVD-AMC). Cells were cultured in 25 cm2 tissue culture flasks, exposed
to 50 or 500 ␮g/ml of different wood dusts for 2, 6, and 24 h, washed with PBS,
and collected into tubes. The tubes were centrifuged and the supernatants were
removed. The cell pellets were suspended into lysis buffer (10 mM Tris, 1% Triton X-
100 in PBS, pH 7.5), incubated on ice for 20 min and centrifuged (30 min, 12,000 × g,
4 ◦ C). The supernatants containing cytosolic fractions were collected and the total
protein concentration was determined. Aliquots of this fraction (25 ␮g of protein)
were dissolved in protease assay buffer (20 mM HEPES, pH 7.5, 2 mM dithiotreitol,
10% glycerol; final volume 175 ␮l), and 15 ␮M Ac-DEVD-AMC in 25 ␮l assay buffer
was added to start the reaction. After 1 h of incubation at 37 ◦ C at dark, the substrate
cleavage was measured fluorometrically on black 96-well plates at an excitation
wavelength of 355 nm and an emission wavelength of 460 nm using a Wallac Victor
II Multilabel counter. Fluorescence values of cell-free wells containing 15 ␮M Ac-
DEVD-AMC in assay buffer were subtracted from the fluorescence values of the cell
samples.
2.6. Statistical analysis
The software package GraphPad Prism (GraphPad Software, Inc., San Diego,
CA, USA) was used for the statistical analyses. Results are given as mean ± S.E.M.,
*P < 0.05, **P < 0.01 and ***P < 0.001 was considered as statistically significant. For
the data of cell viability, ROS production and caspase-3 activity were analysed using
two-way analysis of variance (ANOVA) followed by Bonferroni’s post-comparison
tests.
 L. Pylkkänen et al. / Toxicology 262 (2009) 265–270 267

Fig. 2. Cell viability of BEAS-2B cells after 2 and 6 h’ exposure to three different concentrations (10, 50, and 500 ␮g/ml) of pine, birch, and oak dust. The columns indicate the
quantity of live cells and the results are shown as % of decrease compared to controls, and represent mean ± S.E.M. of 3–6 independent experiments. *P < 0.05, **P < 0.01, and
***P < 0.001 significantly different from control; using two-way analysis of variance (ANOVA) followed by Bonferroni’s post-comparison tests.

3. Results
3.1. Cell viability
All wood dusts induced cytotoxicity in human bronchial epithe-
lial cells in a dose-dependent manner at 2 and 6 h the decrease
being statistically significant when compared to the control level.
The cytotoxicity of birch dust was somewhat less pronounced at 2 h
as compared to the other two species (Fig. 2). The maximal decrease
(14% with pine dust, 11% with birch dust and 16% with oak dust) in
cell viability was reached with the highest wood dust concentra-
tion (500 ␮g/ml) at 2 h with pine dust and at 6 h with birch and
oak dust. At 12 h, cells were recovered and the number of viable
cells was close to that detected in the control cultures (data not
shown).
3.2. Production of ROS
Exposure to all of the three wood dusts had a marked stimulat-
ing effect on the production of ROS in human BEAS-2B cells (Fig. 3).
The maximal induction of ROS production was seen already after
30 min of exposure to pine dust. With birch and oak dust the max-
imal induction was observed after 2 h exposure. Compared to the
control level, the mean increase was 3.3-fold for pine dust at the
concentration of 50 ␮g/ml after 30 min, 2.1-fold for birch dust at
500 ␮g/ml after 2 h, and 2.3-fold for oak dust at 50 ␮g/ml after 2 h
(Fig. 3).
For pine dust, production of ROS was significantly induced at
0.5, 2, and 6 h at the smallest concentrations (10 and 50 ␮g/ml),
while no increase was seen after exposure to the highest concen-
tration (500 ␮g/ml). For birch dust, the induction of ROS production
occurred in a dose-dependent manner after 2 h of exposure, show-
ing a statistically significant effect with the highest concentrations.
After 6 h exposure to birch dust the increase of ROS production was
only marginal and after 12 h exposure ROS levels returned to the
same levels as in untreated control cells (data not shown). The cells
exposed to 50 ␮g/ml of oak dust for 2 h showed statistically signifi-
cant increased ROS levels, but this effect declined at longer exposure
time (Fig. 3).
3.3. Caspase-3 activity
Caspase-3 protease activity was increased in BEAS-2B cells after
2 and 6 h exposure to each of the wood dusts studied. Two-hour
exposure to the highest concentration (500 ␮g/ml) of pine dust
resulted in dramatic, maximally 8.9-fold increase in enzyme activ-
ity compared to the control level, and after 6 h, exposure to higher
concentration of pine dust still produced a 3.6-fold increase (Fig. 4).
The lower concentration (50 ␮g/ml) of pine dust produced a minor
induction (1.5–2.1-fold) at 6 and 24 h. For birch dust, the increase of
caspase-3 activity was in general less marked than those induced by
pine or oak dusts. Birch dust induced caspase-3 activity after 2 and
6 h of exposure at the lowest (50 ␮g/ml) and highest (500 ␮g/ml)
concentrations (2.7-fold and 3.8-fold, respectively) (Fig. 4). Oak dust
induced a markedly elevated (16.4-fold) caspase-3 activity at the
highest dose after 2-h exposure. At 24 h, the induction levelled off
(Fig. 4), probably, at least in part, due to cytotoxicity. Stimulation
of caspase-3 activity seemed to be associated with the increased
ROS production even though the relationship may not have been a
totally straight one.

Fig. 3. Effects of wood dust on the production of reactive oxygen species (ROS) in BEAS-2B cells exposed for 0.5, 2, and 6 h to pine, birch, and oak dust in final concentrations
of 10, 50, and 500 ␮g/ml. The results are shown as % of control (dashed line) and represent mean ± S.E.M. of 3–6 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
significantly different from control; using two-way analysis of variance (ANOVA) followed by Bonferroni’s post-comparison tests.
268 L. Pylkkänen et al. / Toxicology 262 (2009) 265–270
Fig. 4. Caspase-3 protease activity of BEAS-2B cells exposed to pine, birch, and oak dust for 2, 6, and 24 h in final concentrations of 50 and 500 ␮g/ml. Data are expressed as
% of control cell cultures, mean ± S.E.M. of two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 significantly different from control; using two-way analysis of
variance (ANOVA) followed by Bonferroni’s post-comparison tests.
4. Discussion
In the present study, we showed the capacity of dust from
three wood species, pine, birch and oak, to induce ROS produc-
tion and to evoke apoptosis (caspase-3 activity) in cultured human
bronchial epithelial cells (BEAS-2B), a widely used airway epithe-
lial cell model. Epithelial cells lining the airways form an important
physical barrier, being the primary targets for inhaled particles.
Exposure to external particles may induce cellular apoptosis and
necrosis of epithelial cells which again may lead to shedding of the
bronchial mucosa, to bronchial hyperreactivity, and exacerbation
of asthma (Nel et al., 2001).
The present study showed that short-term exposure to pine,
birch and oak dusts induces a significant increase in the produc-
tion of ROS in human bronchial epithelial cells. The formation of
ROS occurred rapidly, with the most remarkable induction of ROS
measured after 30-min exposure to each of the wood dusts. ROS are
known to be molecules with a strong cytotoxic capacity, and there-
fore a lack of response after a longer treatment time, as observed
here, may be related to up-regulation of the intracellular compen-
satory redox buffering mechanisms (Ferret et al., 2002). Our current
findings match well with data from our previous investigations
where wood dust was found to increase the expression of TNF-␣ and
MIP-2 in primary rat alveolar macrophages by a mechanism that is,
at least in part, mediated by ROS (Long et al., 2004). Dusts from
several wood species (birch, beech, teak and pine) have also been
shown to generate in human lung cell line A549 a dose-dependent
induction of DNA single-strand breaks (Bornholdt et al., 2007), sug-
gested as an indicator of ROS-mediated carcinogenic mechanism
(Risom et al., 2005). Consistent with these collective experimen-
tal data as discussed above, buccal cells from wood dust exposed
workers were found to exhibit increased levels of toxicity and geno-
toxicity, with suggestion that such biomarker responses may be
related to the increased cancer risk among wood workers (Celik
and Kanik, 2006).
Several hypotheses have been put forward to explain the effects
of wood dust in the airways, among them cellular production of
ROS. Current knowledge on particle toxicology indicates that many
particles as such, especially fine to ultrafine particles, are able
to generate ROS (Fubini and Hubbard, 2003; Stone et al., 2007).
Particles can also activate inflammatory cells to produce ROS, or
recruit other mediators of inflammation, such as TNF-␣, that oper-
ate through ROS-related mechanisms (Churg, 2003). In the present
study, the particle size of the wood dusts used was less than 5 ␮m,
i.e. in the size range a capacity to induce harmful biological effects.
Morphological analysis on human lung epithelial cells has shown
that fine particles interact with the cell surface, where they induce
alterations and, subsequently, are internalized into the cytoplasm
(Blank et al., 2006; Singh et al., 2007; Kemp et al., 2008). It is
of interest, that an increase in the production of ROS is an early
cellular response to the fine particulate matter, that may account
for cytoskeletal changes and the production of proinflammatory
cytokines as observed by earlier studies (Calcabrini et al., 2004). A
survey of size-fractionated dust generation in 10 wood processing
plants across the United States demonstrated that the respirable
fraction (particle size ≤5 ␮m) accounted for 16.7% of the inhalable
dust mass. This longitudinal 5-year study emphasized the relevance
of ROS production to human respiratory health effects (Kalliny et
al., 2008).
In the present study, caspase-3 activity was also increased in
human bronchial epithelial cells subsequent to exposure to all wood
dusts following increased ROS production. Oxidants can cause cel-
lular injury and death by modifying and disturbing the structure
and function of cellular components (Haddad, 2004; Genestra,
2007). Induction of the apoptosis cascade, i.e. mitochondrial mem-
brane depolarization, lipid peroxidation, caspase-3 activation and
GSH depletion, is believed to be closely related to increased ROS
production (Cervia et al., 2007; Pathak and Khandelwal, 2007). Fur-
thermore, excessive apoptosis is proposed to be involved in the
mechanisms leading to cellular damage due to oxidants; in par-
ticular this has been proposed to occur in airway epithelial cells
(Truong-Tran et al., 2003). Also, increased ROS production associ-
ated with induction of apoptosis may evoke a cascade of events
in which the stimulation of redox-regulated transcription factors,
notable NFкB and AP-1, is evoked, and also expression of the genes
encoding these transcription factors may be induced. Hence, the
role of altered redox state of the target cells and subsequently a role
of these transcription factors in the wood dust-induce apoptosis
cannot be excluded (Gwinn and Vallvathan, 2006).
Adding to the complexity of cellular mechanism likely to play
a role in oxidant-related cellular toxicity, antioxidants, known
components of various wood species (IARC, 1995), are important
components of cellular detoxification pathways. Antioxidants have
a wide range of biochemical activities, including inhibition of ROS
generation, and inhibition or delay of the onset of apoptosis caused
by blocking caspase-3 activation (Ju et al., 2004). The antioxidative
capacity of BEAS-2B cells is significant, and biochemical analy-
ses have shown remarkably similar antioxidant enzyme activities
between primary cultured human epithelial cells and BEAS-2B cells
(Kinnula et al., 1994), thus rendering BEAS-2B cells as a well-suited
in vitro model.
Finally, a multitude of epidemiological studies have reported
that adverse effects of the upper and lower respiratory tract, such
as irritation and other nasal effects, asthmatic symptoms, asthma,
lung function changes, commonly occur among workers exposed
to wood dust (SCOEL, 2003; ACGHI, 2005). Importantly, the studies
indicate that such effects are related not only to dusts from hard-
woods but similarly to dusts from softwood species (Demers et al.,
 L. Pylkkänen et al. / Toxicology 262 (2009) 265–270
1995; Hessel et al., 1995; Douwes et al., 2001). In agreement with
these human data, we did not detect significant differences in ROS
production and caspase-3 expression between the cells exposed to
softwood or hardwood dusts, but dusts from both hardwood and
softwood species were capable of inducing such effects in vitro.
Also, the results from our earlier studies were well in agreement
with these findings (Long et al., 2004).
In summary, cellular oxidative stress interacts with many
vital intracellular macromolecules, such as glutathione, DNA,
RNA, proteins, enzymes, lipids, and ATP, as well as induction of
redox-regulated transcription factors (Schmid et al., 2007). Since
concentration gradients of such components are very delicately bal-
anced for normal cellular functioning, a gross perturbation leads to
cell injury and cell death. Compatible with experimental data from
rodent macrophages and lung cells, oxidative stress induced by ROS
appears a likely fundamental mechanism involved in wood dust
toxicity in human airway epithelial cells. In future work, however,
many questions about specific components and details of the cel-
lular targets and cell–particle interactions remain to be addressed.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This research was supported by The Academy of Finland (no.
53631) and by the EU 5th Framework Programme, Key Action 4,
Environment and Health, Quality of Life and Management of Living
Resources, Project No. QLK4-2000-00573. The views and opinions
expressed in this paper do not necessarily reflect the views of the
European Commission.
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