Toxicology Letters 202 (2011) 209–217

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

The acute toxic effects of particulate matter in mouse lung are related to size and
season of collection
Francesca Farina a , Giulio Sancini a,∗ , Paride Mantecca b , Daniele Gallinotti b , Marina Camatini b ,
Paola Palestini a
a
Department of Experimental Medicine, POLARIS Research Center, University of Milano-Bicocca, 48 via Cadore, Monza 20052, Italy
b
Department of Environmental Science, POLARIS Research Center, University of Milano-Bicocca, 1 piazza della Scienza, Milano 20126, Italy

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

Article history: The toxicity of size-fractionated particulate matter (PM10 and PM2.5) collected in Milano during two dif-
Received 24 September 2010 ferent seasons (summer and winter) has been evaluated in vivo. The focus is on time related (3 h, 24 h and
Received in revised form 27 January 2011 1 week) lung response following a single intratracheal aerosolization in BALB/c mice. The bronchoalveo-
Accepted 31 January 2011
lar lavage fluid (BALf) and the lung parenchyma were screened for different markers of inflammation and
Available online 1 March 2011
cytotoxicity. Histology and immunohistochemistry were performed on excised fixed lungs to assess the
effects produced by the different PM fractions. All the analyzed inflammatory markers (PMNs percentage,
Keywords:
TNF-␣, Hsp70 in the BALf, HO-1 in lung parenchyma), increased after summer PM10 administration; on
Particulate matter
Acute lung inflammation
the contrary winter PM10 and PM2.5 specifically increased the amount of the Cyp1B1, a protein puta-
Broncho-alveolar lavage tively involved in the induction of pro-carcinogenic effect. Moreover, we detected an intensification of
BALB/c mice LDH activity in the BALf after the administration of winter PM10 and PM2.5, potentially related to an
in progress necrotic process while after summer PM10 and PM2.5 administration, the initiation of the
caspase cascade suggested a cytotoxic effect sustained by apoptosis. Our results evidenced the toxicity
mechanisms elicited by size fractionated PM samples, collected in winter and summer seasons, which dif-
fers for dimensions, chemical and microbiological composition. PM10 has been indicated to elicit above
all a pro-inflammatory response, linked to its specific biological components, while PM2.5 is supposed to
be more harmful due to its smaller dimension and the ability to distribute into the lung alveolar districts.
We hypothesized that adverse health effects observed after a single dose of winter PM2.5 is at least partly
caused by specific winter PM components, i.e. PAH and transitional metals.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Particulate matter (PM) is a mixture of airborne particles derived
from the combustion of fossil fuels or from the breakdown of crustal
components. Currently, PM standards are based on total mass and
size, which can range from few nanometers to tens of micrometers.
Particles below 10 ␮m in diameter have the potential to reach
and be deposited in the alveoli, while those greater than 10 ␮m are
likely to land in proximal airways and be removed by the ciliary
activity (Kim et al., 1994). Particles smaller than 2 ␮m in diame-
ter show a clear tendency to achieve greater peripheral deposition
than those greater than 2 ␮m (Conway et al., 1998). Numerous epi-
demiological studies (Calderón-Garcidueñas et al., 2007; Pope et al.,
2004) presented convincing evidence of the association between
increase in respiratory disease morbidity and increased levels of
coarse PM, especially among susceptible populations (Pope et al.,
∗ Corresponding author. Tel.: +39 02 64488310; fax: +39 02 64488068.
E-mail address: giulio.sancini@unimib.it (G. Sancini).
1995). Some researches suggest that this fraction may contribute
predominantly to possible toxic and pro-inflammatory effects by
its constituents, which include biological agents (Camatini et al.,
2010), metals (Gerlofs-Nijland et al., 2009) and organic compounds
(Li et al., 2002).
Recently, attention has been focused on the fine fraction, con-
stituted of particles with an aerodynamic diameter below 2.5 ␮m
(PM2.5). These particles, which are usually present in high number
in PM samples, may be more harmful than larger ones, as they are
more efficiently retained in the alveolar lung portion. As a general
rule, the primary biological targets of inhaled particles are cells of
the pulmonary epithelium as well as resident macrophages (Cohen
and Pope, 1995). Among these cells, alveolar epithelial cells are
affected in a number of respiratory PM induced diseases (Floreani
et al., 2003; Pourazar et al., 2004). The fine fraction is frequently
suggested to be responsible of cardiac disorders and acute car-
diovascular events, such as myocardial infarction (Dockery and
Stone, 2007; Pope et al., 2006; Zanobetti and Schwartz, 2005),
as well as inflammatory pathologies (Scapellato and Lotti, 2007).
Increased plasma viscosity and other changes in blood-related

0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2011.01.031
210 F. Farina et al. / Toxicology Letters 202 (2011) 209–217
parameters have been detected after inhalation of fine PM (Peters
et al., 1997). However, the biological mechanisms of such events
are still unclear; even the pulmonary endothelium appears to be
the initial target site for ultrafine particles, which can translo-
cate into the blood vessels, and reach other organs (Peters et al.,
2006).
LPS, a constituent of the outer membrane of Gram-negative bac-
teria associated with PM, and polycyclic aromatic hydrocarbons
(PAHs), an organic class of PM components, differ in PM con-
centration between summer and winter seasons (Camatini et al.,
2010; Lonati et al., 2005; Marcazzan et al., 2001). PM composi-
tion seasonal variation has been related to the different biological
responses, and cytokine release is one of the parameters affected
(Hetland et al., 2005).
In vivo studies have been performed by means of various pollu-
tant exposure methods (e.g. inhalation, intratracheal or intranasal
instillation). Inhalation mimics the most natural route of exposure,
even though it presents some experimental limitations (Costa et al.,
2006). One of these is the estimation of the real dose delivered
into the lungs, which is one of the most powerful advantages of
intratracheal aerosolization. This method is very useful for the anal-
ysis of dose–response effects in comparative studies of exposure
to various PM collected in different areas (Gerlofs-Nijland et al.,
2009; Wegesser and Last, 2008). Recently, we used intratracheal
aerosolization to assess the lung toxicity of a single dose of tire
debris and PM (10 and 2.5) and we found that the effects pro-
duced were related both to the particle dimension and composition
(Mantecca et al., 2009, 2010).
To analyze if the dimension and the seasonality of PM may
elicit different lung responses, we examined, in intratracheally
aerosolized mice, the acute effect of PM10 and PM2.5 collected in
Milano during summer 2008 and winter 2009.
Bronchoalveolar lavage fluid (BALf) was screened to evaluate
total and differential cell counts, inflammatory markers (tumor
necrosis factor ␣, TNF-␣; heat shock protein 70, Hsp70; secretory
phospholipase A2 , sPLA2 ) and markers of cell damage (total protein
content; lactate dehydrogenase, LDH; alkaline phosphatase, ALP).
Lung pathologic effects were assessed by immunoblotting
analyses and histological observations. In lung parenchyma we
evaluated the levels of Caspase8-p18, involved in the programmed
cell death, of heme oxygenase-1 (HO-1), an enzyme induced by
stimuli such as oxidative stress, cytokines and hypoxia, nuclear fac-
tor kappa-light-chain-enhancer of activated B cells (NF-KB-p50),
involved in inflammatory pathways, cytochrome 1B1 (Cyp1B1), a
member of cytochrome P450 superfamily involved in pro-oxidant
action and heat shock protein (Hsp70), an heat shock protein
involved in cytokines release.
2. Materials and methods
2.1. Animals
Male BALB/c mice (7–8 weeks old) were purchased from Harlan; food and water
were administered ad libitum. The mice were housed in plastic cages under con-
trolled environmental conditions (temperature 19–21 ◦ C, humidity 40–70%, lights
on 7 a.m. to 7 p.m.). The established rules of animal care approved by Italian Ministry
of health (DL 116/92) were followed.
2.2. PM sources and characterization
Atmospheric PM10 and PM2.5 were collected during summer 2008 (PMsum)
and during winter 2009 (PMwin) in a Milano urban area as described in previous
papers (Gualtieri et al., 2009, 2010; Perrone et al., 2010). The chemical characteri-
zations of PM collected during summer 2008 and winter 2009, do not differ from
those of PM collected in years 2006, 2007 and 2008 (Camatini et al., 2010; Perrone
et al., 2010; Bolzacchini et al., personal communication).
Briefly, size fractionated particulate samples were collected onto Teflon filters
using a low volume gravimetric sampler (EU system 38.33 l/min, FAI Instruments,
Rome, Italy). For in vivo experiments, particles were recovered from filters by
sequential sonications (four cycles of 20 min each) in sterile water. Detached parti-
cles were dried into a desiccator, weighed, re-suspended in sterile water at a final
concentration of 4 ␮g/␮l and stored at −20 ◦ C until use.
2.3. Experimental design
Mice were aerosolized with 100 ␮l of saline solution (Mantecca et al., 2009,
2010; Shwe et al., 2005) containing 100 ␮g of PM10 (winter or summer) or PM2.5
(winter or summer) and euthanized at different times (3 h, 24 h and 1 week). Control
mice, running parallel to PM treated ones, were aerosolized with 100 ␮l of saline
solution. BALf was collected and analyzed for inflammatory markers evaluation.
Lungs were excised and the right lobes were submitted to biochemical analysis,
while the left lobes were fixed and processed for histological analyses. Animal test-
ing was carried out by instilling three mice for each experimental group and the
experiment was replicated two times.
2.4. Intratracheal PM aerosolization
Male BALB/c mice were briefly exposed to a mixture of 2.5% isoflurane
(Flurane), 70% O2 and 30% N2 O anesthetic gas and then anesthetized with
Tiletamine/Zolazepam-Xylazine (TZX 40 + 5 mg/kg) i.m. administered. Once a deep
stage of anesthesia was reached, mice were intratracheally aerosolized by means
of MicroSprayer® Aerosolizer system (MicroSprayer® Aerosolizer-Model IA-1 C and
FMJ-250 High Pressure Syringe, Penn Century, USA) with 100 ␮g of PM2.5 or PM10 in
100 ␮l of isotonic saline solution, or 100 ␮l of isotonic saline solution (control). Each
mouse was placed in a supine position, the mouth was opened, and the tongue was
gently moved aside using a pince to better incannulate the trachea (Mantecca et al.,
2009). The particles were suspended in the appropriate solution immediately prior
to aerosolization, according to the previously described procedure. Immediately fol-
lowing aerosolization, treated and control mice were allowed to recover under visual
control before placing them back in plastic cages under controlled environmental
conditions.
Mice were aerosolized with the dose of 100 ␮g of different PM samples; Gavett
et al. (2003) estimated PM concentration required to produces human dose equiva-
lent to mouse. During winter 2009 in Milan the highest peak of PM10 was estimated
about in 164 ␮g/m3 . We calculated that the 24 h exposure PM equivalent dose is
about 100 ␮g/24 h for mice of 20–25 g body weight.
After 3 h, 24 h or 1 week, mice from each experimental group were euthanized
with an anesthetic mixture overdose (tiletamine/zolazepam-xylazine and isoflu-
rane), the trachea was exposed, cannulated and secured with suture thread, and
three in-and-out washes with 0.6 ml of isotonic saline solution were performed
(Henderson, 2005). The efficacy of BALf collection ranged from 50% to 100% of
the total solution injected. The BALf was centrifuged at 1500 × g for 15 min at 4 ◦ C
(Mantecca et al., 2009, 2010) and pellets collected for cell counts. Supernatants were
divided into aliquots and appropriately stored for subsequent biochemical analyses.
2.5. Bronchoalveolar lavage fluid analysis
The BALf analysis provided significant informations to predict the pulmonary
response to PM, both in terms of early biochemical changes and time–response
parameters, as well as of comprehensive screening of PM-induced inflammation or
lesions (Gerlofs-Nijland et al., 2009; Mantecca et al., 2009, 2010).
Cell counts. After centrifugation, the BALf pellets were resuspended in 500 ␮l
of DMEM (10% FBS, 1% penicillin–streptomycin and 1% glutamine), and total cells
count performed with a Burker chamber, using the Trypan Blue exclusion method. A
cell aliquot (240,000 cells, 800 cells/␮l) was smeared in duplicate onto slides using
Cytofuge 2 (StatSpin, USA) at 40 × g for 7 min at room temperature. Subsequently,
the smears were stained with Diff Quik (Medion Diagnostic) for cell differential
count, according to manufacturer instructions. Macrophages, polymorphonuclear
leukocytes (PMNs) and lymphocytes were identified by their characteristic shapes
(Fig. 1A–E).
Biochemical analyses. The following biochemical analyses were performed on
cell-free BALf supernatants.
Total protein content was measured spectrophotometrically at 570 nm accord-
ing to BCA method (Sigma Aldrich, USA). The commercially available kits for alkaline
phosphatase (DALP-250 QuantiChrom Alkaline Phosphatase Assay Kit, Gentaur
Molecular) and lactate dehydrogenase (DLDH-100 QuantiChrom Lactate Dehydro-
genase Kit, Gentaur Molecular) were employed according to the manufacturers’
instructions.
Cytokine analyses. The analyses of pro-inflammatory cytokine released in the
BALf were performed by DuoSet ELISA kits for tumor necrosis factor-␣ (TNF-␣; R&D
Systems, Minneapolis, MN) according to the manufacturer’s protocols.
Other proteins. 30 ␮g of BALf proteins of control and treated mice were loaded
on SDS–PAGE (12% polyacrylamide) and submitted to electrophoresis, followed by
Western blot, and tested for Hsp70 and sPLA2 (anti-Hsp70 1:200, anti sPLA2 1:200,
all from Santa Cruz), according to the procedures described in the next section.
2.6. Lung parenchyma analysis
The lungs of control and treated mice exposed to PM10 sum and win, and to
PM2.5 sum and win, at the end of BAL procedure, were quickly excised from the
 F. Farina et al. / Toxicology Letters 202 (2011) 209–217
Fig. 1. Histograms showing total and differential cell counts in bronchoalveolar lavage fluid (BALf) collected 3 h, 24 h or 1 week post-aerosolization from controls and
PM10sum-, PM10win-, PM2.5sum-, PM2.5win-treated mice. The data are mean ± SE from 6 controls and 6 treated mice per group. *Significantly different from control
(p < 0.05).
chest and washed in ice cold isotonic saline solution. Lungs were finely minced at
4 ◦ C, briefly homogenized for 30 s at 11,000 rpm with Ultra-Turrax T25 basic (IKA
WERKE), sonicated for other 30 s and then resuspended in Buffer A (Tris–HCl 20 mM
pH–7.4, EDTA 2 mM, EGTA 0.5 mM and NaF 10 mM) and centrifuged at 100,000 × g
for 1 h at 4 ◦ C. The supernatant was the fraction enriched in cytosol. The pellets
were resuspended in Buffer A added with 1% of Triton-X 100, sonicated for 30 s and
then centrifuged at 15,000 × g for 20 min at 4 ◦ C. The supernatant was the fraction
enriched in membrane (Giordano et al., 2005).
Then samples were submitted to trichloroacetic acid (TCA) precipitation. An
equal volume of 30% TCA was added, samples were incubated on ice for 1 h and
then centrifuged at 18,400 × g for 30 min at 4 ◦ C. After one wash of the pellets with
cold acetone, samples were centrifuged at 18,400 × g for 20 min at 4 ◦ C. The wash
with cold acetone was replicated twice. The pellets were suspended in water and
protein quantity determined by BCA method (Sigma Aldrich, USA).
Thereafter, 30 ␮g of membrane and cytosol fractions of control and PM-treated
mice were loaded on SDS–PAGE (12% polyacrylamide) and submitted to elec-
trophoresis, followed by Western blot. The membranes were stained with Ponceau
S and the protein loading was assessed by densitometry (BIORAD Densitometry 710,
program Quantity one) as described (Daffara et al., 2004). After blocking, blots were
incubated for 2 h with the primary antibody diluted in PBS-Tween20/milk. Mem-
brane enriched fractions were tested for Cyp1B1 and HO-1, while cytosol fractions
were examinated for Caspase8-p18, NF-kB-p50, Hsp70 (anti-Cyp1B1 1:200, anti-
HO-1 1:200, anti-Casp8-p18 1:200, anti-NF-kB-p50 1:200, anti-Hsp70 1:200, all
from Santa Cruz). Then, blots were incubated for 1.5 h with horseradish peroxidase-
conjugated anti-rabbit IgG (1:5000) or anti-goat IgG (1:2000) diluted in PBS-T/milk.
Proteins were detected by ECL using the SuperSignal detection kit (Pierce, Rockford,
IL). Immunoblot bands were analyzed and the optical density (OD) quantified by
KODAK (Kodak Image Station 2000R).
2.7. Histological analyses
At the end of BAL procedure, the left lungs of control and PM treated mice were
excised and immediately formalin fixed and processed for routine histology. Briefly,
after being preserved for 24 h in the fixative, lung fragments were rinsed in distilled
water, dehydrated in an ethanol series from 70% to 100% and embedded in Bio-
plast tissue embedding medium. For each control and PM exposed sample, 7 ␮m
serial sections were cut by a rotary microtome, mounted on slides and stained with
Mayer’s haemalaun and alcoholic eosin.
The immunohistochemical localization of Cyp1B1 in lung tissues was performed
by an indirect immunochemical method using a rabbit anti-Cyp1B1 polyclonal anti-
body (Santa Cruz Biotechnology, Inc.), and the peroxidase-based Vectastain Elite
ABC Kit (Vectastain Laboratories, Burlingame, CA) to visualize the immunochemical
reaction, following the protocol reported in Mantecca et al. (2010).
Histological samples were qualitatively screened by using a Zeiss Axioplan 40
light microscope and images taken with a Zeiss AxioCam MRc5 digital camera inter-
faced with the Axiovision Real 4.6 software.
211
2.8. Statistical analyses
For each cytological and biochemical parameter measured in control and PM
experimental groups, the means (±standard error, SE) were calculated. Statistical
differences were tested by t-test. To test the tendency toward increase or decrease
in the different cytological and biochemical parameters, linear regressions were
carried out. Statistical differences were considered to be significant at the 95% level
(p < 0.05).
3. Results
3.1. Bronchoalveolar lavage fluid analysis
Cellular inflammatory response. BALf was analyzed for cellular
indicators of inflammation and differential count was investigated
as an inflammatory marker.
Total cell number never provided a clear indication of
lung inflammatory status, at any of the considered times
(Fig. 1A).
However, differential count confirmed a significant increase in
PMNs percentage 3 h after the aerosolization of PM2.5sum, and
24 h after aerosolization of all PM samples (Fig. 1B). Furthermore,
a decrease in AMs percentage was observed, though at 3 h it was
significant only for PM2.5sum, while it became significant at 24 h
for all the PM samples, except for PM2.5win (Fig. 1C). At 1 week,
there were no differences in PMNs and AMs percentage between
control and treated mice.
No statistical difference in lymphocyte percentage was observed
between control and PM treated groups (Fig. 1D).
Cytotoxicity. Total protein content, lactate dehydrogenase (LDH)
and alkaline phosphatase (ALP) activities were analyzed as markers
of cytotoxicity.
Concerning total protein content, a decrease was evident 24 h
after aerosolization of PM2.5sum; on the contrary, statistical
increases were noticed 1 week after treatments with summer PM
samples and PM10win (Fig. 2A).
LDH and ALP activities did not show modifications between con-
trol and treated mice, except a significant increase in LDH activity
24 h after aerosolization of PM2.5win and PM10win (Fig. 2B; for
212 F. Farina et al. / Toxicology Letters 202 (2011) 209–217

Fig. 2. Histograms showing cytotoxic markers in bronchoalveolar lavage fluid (BALf) collected 3 h, 24 h or 1 week post-aerosolization from controls (Co) and PM10sum-
, PM10win-, PM2.5sum-, PM2.5win-treated mice. The data are mean ± SE from 6 controls and 6 treated mice per group. *Significantly different from control (p < 0.05);
#
significantly different from corresponding time of PM2.5win or PM2.5sum (p < 0.05).

3.2. Lung parenchyma analysis

HO-1, one of the defense protein against oxidative and inflam-

matory insults, was largely increased 3 h after summer PM samples
aerosolization (Fig. 5A and Table 2). PM10sum caused the highest
levels of HO-1, with OD values about ten times higher than those
of control mice, while PM2.5sum caused an increase of about six
times, compared to control. No modifications were identified at

Fig. 3. Histograms showing TNF-␣ concentrations in bronchoalveolar lavage fluid

(BALf) collected 3 h, 24 h or 1 week post-aerosolization from controls (Co) and
PM10sum-, PM10win-, PM2.5sum-, PM2.5win-treated mice. The data are mean ± SE
from 6 controls and 6 treated mice per group. *Significantly different from control
(p < 0.05).

ALP: data not shown). PM2.5win treated mice, at 24 h showed a

significant increase in LDH activity, compared to PM2.5sum group
(Fig. 2B).
Cytokine release. A significant increase in TNF-␣ was evident at
3 h in BALf of both PM10sum and PM10win treated mice, while the
aerosolization of both PM2.5 caused no significant increase in TNF-
␣ release (Fig. 3). At 24 h and 1 week, TNF-␣ levels were notably
lower, tending to be similar to control levels.
Other proteins. Hsp70, a protein present in the extracellu-
lar milieu in response to a wide variety of stressful stimuli,
was sensibly increased only 24 h after PM10sum and PM2.5sum
aerosolization (Fig. 4A and Table 1).
A slight increment in sPLA2 , enzyme that hydrolyze the ester
bond at the sn-2 position of phospholipids, was detected 24 h after
PM10sum treatments, while PM2.5sum induced a large expression
of sPLA2 (Fig. 4B).

Fig. 4. (A) Western blottings (WB) showing Hsp70 in bronchoalveolar lavage
fluid (BALf) collected 24 h post-instillation from controls (Co), PM10sum and
PM2.5sum aerosolized mice. (B) WB showing sPLA2 levels in broncho-alveolar
lavage fluid (BALf) collected 24 h post-aerolization from controls (Co) and PM10sum-
, PM2.5sum-treated mice.
Fig. 5. (A) Western blottings (WB) showing HO-1 in lung parenchyma 3 h post-
aerosolization from controls (Co) and PM10sum- and PM2.5sum- treated mice.
(B) WB showing NF-kB-p50 levels in lung parenchyma 1 week post-aerosolization
from controls (Co) and PM10sum-, PM2.5sum-treated mice. (C) WB showing Casp8-
p18 levels in lung parenchyma 3 h and 24 h post-aerosolization from controls (Co)
and PM10sum-, PM2.5sum-treated mice. (D) WB showing Hsp70 levels in lung
parenchyma 3 h and 24 h post-aerosolization from controls (Co) and PM10win-
, PM2.5win-treated mice, and from PM10sum-, PM2.5sum- treated mice. (E) WB
showing Cyp1B1 levels in lung parenchyma 24 h and 1 week post-aerosolization
from controls (Co) and PM10win-, PM2.5win-treated mice, and from PM10 sum-,
PM2.5sum-treated mice.
 F. Farina et al. / Toxicology Letters 202 (2011) 209–217 213

Table 1
Statistical analyses of Hsp70 in bronchoalveolar lavage FLUID (BALf): all the data have been normalized to control, and in the table mean ± SE are reported for each experimental
group at any time; sPLA2 was completely lacking in control. *Significantly different from control (p < 0.05).

Hsp70 Co PM10sum PM2.5sum PM10 win PM2.5 win

Mean Standard Mean Standard Mean Standard Mean Standard Mean Standard
error error error error error

3h 1 0.15 0.53 0.05 0.69 0.34 1.23 0.24 0.53 0.1

24 h 1 0.15 3.31* 0.19 4* 0.2 1.21 0.5 1.76 0.4
1w 1 0.15 0.17 0.08 0.33 0.03 0.74 0.14 1.2 0.34
*
p < 0.05.

24 h or 1 week after the aerosolization of both summer and winter
PM samples.
The increased level of TNF-␣ evaluated in the BALf of all treated
mice 3 h after aerosolization, suggested the activation of NF-kB in
the lung tissue. However, no increment of NF-kB-p50 was found in
PM10sum treated mice at any time, but a large increase in NF-kB-
p50 level was estimated 1 week after the treatment with PM2.5sum
(Fig. 5B and Table 2).
Caspase8-p18, a pro-apoptotic marker, at 3 h was increased in
the lungs as a consequence of summer PM samples aerosolization
(Fig. 5C and Table 2). In exposed lungs, winter PM samples were
indeed unable to induce modifications in Caspase8-p18 levels.
Intracellular Hsp70, an indicator of cell stress, increased 3 h after
the aerosolization of PM2.5win, and 24 h after aerosolization of
PM10sum, PM2.5sum and PM2.5win. On the contrary, it resulted
decreased 24 h after the aerosolization of PM10 win (Fig. 5D and
Table 2).
Cyp1B1, a cytochrome of the P450 superfamily, involved in the
activation of many xenobiotics and in PAHs metabolism, increased
24 h after the aerosolization of PM2.5win, and 1 week after treat-
ments with both PM2.5win and PM2.5sum as well as with PM10win
(Fig. 5E and Table 2).
Immunohistochemical detection of Cyp1B1 expression in lung
tissues confirmed the biochemical analyses, with PMwin displaying
the most evident positive reaction when compared to both control
and PMsum treated mice. Cyp1B1 induction appeared to be dis-
tributed along both airways and respiratory tract, but particularly
distinguishable at the level of the epithelium of distal bronchioles
(Fig. 6A–D).
As evidenced by the histological analysis of the lungs, PMsum
was able to induce inflammatory cells recruitment in the connec-
tive surrounding terminal bronchioles and the proximal alveolar
sacs. With PM2.5sum these morphological pictures were evi-
dent even at 3 h (Fig. 7B), while PM10sum elicited similar effects
especially at 24 h (Fig. 7C). At 24 h, PM2.5sum-treated mice also
showed intra-alveolar infiltration of inflammatory cells (Fig. 7D).
Lungs aerosolized with PMwin showed less extended inflam-
matory tissues when compared to those treated with PMsum,
but evidenced cell lyses at the level of the alveolar walls,
and alveolar macrophages engulfed with particles undergoing
necrotic changes (Fig. 7E–F). These histological evaluations sup-
ported the cytological and biochemical measurements on BALf,
which ultimately showed increased PMNs lung infiltration in
mice aerosolized with all PM samples, but particularly with
summer samples. Moreover histology of PMwin exposed mice
seemed to reflect the augmented alveolar cell lyses, as indicated
by increased LDH activity found in BALf. At 1 week, lung his-
tology indicated an almost complete tissue recovery (data not
shown).
4. Discussion
The focus of this study is on time related (3 h, 24 h and 1 week)
lung response following a single intratracheal aerosolization of size
fractioned summer and winter PM in BALB/c mice.
The toxic effects elicited by the aerosolization of PM could be
related not only to physical characteristics, such as the different
dimensions of the particles, but also to their specific chemical com-

Table 2
Statistical analyses of lung parenchyma proteins; all the data have been normalized to control, and in the table mean ± SE are reported for each experimental group at any
time. *Significantly different from control (p < 0.05).

Co PM10 sum PM2.5sum PM10 win PM2.5 win

Mean Standard Mean Standard Mean Standard Mean Standard Mean Standard
error error error error error

HO-1
3h 1 0.15 11.68 4.37 6.56* 1.67 1.08 0.1 1.1 0.17
24 h 1 0.07 1.21 0.25 1.55 0.19 1.82 0.24 1.61 0.27
1w 1 0.11 1.22 0.6 1.47 0.16 1.38 0.29 1.01 0.07
NF-kB-p50
3h 1 0.24 0.68 0.34 1.43 0.72 1.2 0.15 1.47 0.08
24 h 1 0.24 0.9 0.26 0.87 0.34 1.4 0.55 1.71 0.22
1w 1 0.14 0.71 0.37 2.37* 0.15 1.03 0.23 1.11 0.04
Casp8-p18
3h 1 0.04 2.17* 0.2 2.12* 0.06 0 0 0 0
24 h 1 0.28 3.75 0.87 2.6 0.84 0.44 0.23 0.62 0.42
1w 1 0.18 1.02 0.29 1.2 0.36 0.37 0.35 0.39 0.2
Hsp70
3h 1 0.24 1.09 0.26 1.66 0.08 0.9 0.06 3.04* 0.15
24 h 1 0.11 4.2* 0.32 5.09* 0.63 0.49 0.14 3.3* 0.37
1w 1 0.13 0.7 0.26 0.67 0.07 0.33* 0.06 0.97 0.17
Cyp1B1
3h 1 0.23 1.78 0.08 1.23 0.1 1.66 0.19 2.04 0.14
24 h 1 0.11 0.72 0.09 0.57 0.09 1.22 0.09 2.24* 0.18
1w 1 0.11 1.77 0.41 2.5* 0.31 2.85* 0.34 3.83* 0.36
*
p < 0.05.
214 F. Farina et al. / Toxicology Letters 202 (2011) 209–217

Fig. 6. Immunohistochemical analyses of Cyp1B1 expression in lung tissue 24 h post-aerosolization. The brown color reflects the site and intensity of Cyp1B1 expression. (A)
Control lung aerosolized with NaCl 0.9%; in the box, a control of the immunohistochemical reaction specificity, where the primary antibody has been avoided, in a PM2.5win
aerosolized mice. (B) PM10sum treated lung. (C) PM10win treated lung. (D) PM2.5win treated lung. Bars = 50 ␮m. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)

ponents. These last aspects depend both on particles dimensions
and on the season of collection.
The experiments were performed with a single seasonal batch
of PM (PM10sum, PM2.5sum, PM10win, PM2.5win) collected in an
urban site of Milano (Torre Sarca) characterized by intense vehic-
ular traffic, during summer 2008 and winter 2009. All PM samples
have been previously chemically and microbiologically character-
ized (Camatini et al., 2010; Perrone et al., 2010; Franzetti et al.,
2010).
4.1. Pro-inflammatory effect
As already demonstrated by the previous literature, coarse PM
collected in summer is able to induce strong inflammatory response
possibly due to the presence of endotoxins (Alfaro-Moreno et al.,
2007). Endotoxins are components of the outer membrane in
Gram-negative bacteria associated to the fine and coarse PM frac-
tions (Schins et al., 2004). The microbiological analyses of our PM
samples confirmed a larger amount of bacteria in PM10 than in
PM2.5; moreover, in PM10sum were mainly found Gram-negative
bacteria, while in PM10win Gram-positive bacteria were predom-
inant (Franzetti et al., 2010), thus the LPS content was greater in
PM10sum (60,5 EU/mg) than in PM10win (20,7 EU/mg) (Camatini
et al., 2010).
Consistent with the literature, a huge inflammatory response
was detected after the aerosolization of all the PM samples,
with highest effects elicited by the summer PM. Indeed, the
influx of PMNs (almost 100% neutrophils), a specific marker of
a pro-inflammatory response induced by particulate matter, was
significantly increased after the aerosolization of PM2.5sum, prob-
ably because of the smallest dimensions of the particles, which
allows a deeper penetration in alveoli, but 24 h after the aerosoliza-
tion a significant neutrophils influx was also evident in all the
PM treated mice, in agreement with previous studies (Gerlofs-
Nijland et al., 2005; Mantecca et al., 2009, 2010). A concomitant
decrease in AMs percentage suggested that macrophage mediated
PM particles clearance occurred during the first 24 h. Probably, the
unchanged total cell number may depend on a compensatory effect
between decrease in AMs percentage and a contemporary increase
in PMNs.
There are several known macrophages and epithelial cells
derived chemoattractans that might be directly or indirectly
responsible for the neutrophils recruitment to the lung. TNF-␣ is the
principal inflammatory mediator upstream of MIP-2 (Henderson,
2005; Tessier et al., 1997) and it has been suggested to be a
key-player in particles-induced lung inflammation (Driscoll, 2000).
Each different PM sample elicited specific response in terms of
TNF-␣ release (PM10sum > PM10win > PM2.5sum > PM2.5win), 3 h
after the aerosolization. Consistent with previously reported data
(Mantecca et al., 2009), 24 h after the aerosolization of all PM sam-
ples only slight deviations in TNF-␣ concentration between control
and treated mice were detected, confirming that TNF-␣ release is
an acute response to an inflammatory event.
We investigated other pro-inflammatory markers in order to
deal with a larger panel of putative indicators of early lung dam-
age consequent to PM administration. Secretory phospholipase
A2 (sPLA2 ) belongs to a family of enzymes that hydrolyze phos-
pholipids generating arachidonic acid (Lambeau and Gelb, 2008);
sPLA2 increased in the airways of patients with acute inflamma-
tory disease (Granata et al., 2005) and when alveolar inflammation
occurs (Attalah et al., 2003). Hsp70 released in the extracellular
milieu can act as a cytokine to human monocytes, stimulating
pro-inflammatory signal transduction cascade that results in a fur-
ther up-regulation of cytokines (Asea et al., 2000). The increased
sPLA2 and Hsp70 levels in the BALf of PM10sum- and mainly in
PM2.5sum-treated mice, 24 h after the aerosolization, seems to cor-
roborate the situation previously suggested, indicating a stronger
inflammatory effects sustained by summer PM.
 F. Farina et al. / Toxicology Letters 202 (2011) 209–217 215

Fig. 7. Histology of PM aerosolized lungs. (A) Control lung parenchyma showing bronchiolar and alveolar epithelia. (B) PM2.5sum treated lung parenchyma 3 h post-instillation
and (C) PM10sum treated lung parenchyma 24 h post-aerosolization: inflammatory cells recruitment involve the connective surrounding terminal bronchioles and proximal
alveolar sacs; (D) PM2.5 sum treated lung parenchyma 24 h post-aerosolization: infiltration of inflammatory cells in intra-alveolar spaces; (E and F) PM2.5win treated lung
parenchyma 24 h post-aerosolization: inflammatory tissue is less extended, but cell lyses is evident and alveolar macrophages showed necrotic changes. Bars = 50 ␮m.

Histological analyses also confirmed this situation: PMsum
induced inflammatory cells recruitment surrounding terminal
bronchioles and proximal alveoli, while PMwin induced less
extended inflammation. So, we can conclude that the main mecha-
nisms of PM collected in summer, rich in endotoxin, are prevalently
pro-inflammatory (Osorio-Vargas et al., 2003; Wegesser and Last,
2008).
In conclusion and in agreement with Gualtieri et al. (2010), the
pro-inflammatory potential of PM10 was strictly related to summer
season.
4.2. Protection against inflammation
The inflammatory status induced by the aerosolization of PM
samples could be controlled and even reduced by the expression of
some protection proteins, such as Hsp70 and HO-1.
Intracellular Hsp70 can be induced by PM exposure (Watterson
et al., 2009), coke oven emissions (Xiao et al., 2002) and its syn-
thesis can be stimulated in mammalian cells by transition metals
(Levinson et al., 1980). Hsp70 accumulates in the cytoplasm of
alveolar cells and attenuated the expression of pro-inflammatory
cytokines (Singleton and Wischmeyer, 2006). So, the higher PAHs
and transition metals content in PM2.5win (Gualtieri et al., 2010;
Perrone et al., 2010) could explain the large increase in Hsp70 intra-
cellular levels that we found after the aerosolization of PM2.5win.
Thus we can speculate that the low TNF-␣ release found in the BALf
after the treatment with PM2.5win could be a consequence of the
increase of Hsp70 intracellular levels.
On the contrary, the intracellular increase of Hsp70 observed
24 h after the aerosolization of PM10sum and PM2.5sum could be
imputable to endotoxin-mediated delayed effect (Kang et al., 2005).
HO-1 activity and its enzymatic products have been shown to
down-regulate the inflammatory response (Wagener et al., 2001);
consistent with the previous observations, the large increase of HO-
1 in lung parenchyma 3 h after the aerosolization of PMsum could
be due to a protective response of the lungs, in order to control and
then stop the inflammatory status. Indeed, one of the products of
HO-1 is CO, which decreases the TNF-␣ release: so, the reduction
in TNF-␣ concentration in the BALf observed at 24 h in PMsum-
treated mice could be due to the specific anti-inflammatory action
of CO. We cannot exclude that part of this HO-1 was expressed not
only by alveolar cells, but also by the inflammatory cells infiltrated
216 F. Farina et al. / Toxicology Letters 202 (2011) 209–217
in the lung parenchyma, especially to control the cytokine release
after the PM2.5 treatment (Morse et al., 2003).
4.3. Cytotoxic effect
Three different markers were considered to asses the cytotoxic
effects of PM: LDH, ALP and caspase8-p18.
Significant increments in LDH activities in the BALf were
observed 24 h after the aerosolization of winter PM, and we argue
that the necrotic effect is probably related to the major PAHs and
transition metals content (Gerlofs-Nijland et al., 2009). However,
this increase in LDH activity is not supported by any change in
ALP activity. ALP is a specific marker of damage to type I pneu-
mocytes and of proliferation of type II pneumocytes (Fehrenbach,
2001), thus we can hypothesize that the cytotoxic damage induced
by the aerosolization of winter PM mainly involves AMs, and only
marginally pneumocytes. Histological analyses confirmed the pres-
ence of cell lyses close the alveolar walls and AMs engulfed with
particles undergoing necrotic changes, after PMwin aerosolization.
In the lungs of mice aerosolized with PMsum, caspase8-p18
increased, thus suggesting an early onset of pro-apoptotic events.
Indeed, caspase8 is an initiating caspase and this pathway could be
started by TNF-␣ binding to its receptor (Chen and Goedel, 2002),
being related to the high pro-inflammatory potential of PMsum due
to their high endotoxin content. Once more, we cannot exclude that
the cells involved in the apoptotic mechanisms are the inflamma-
tory cells infiltrating in the lung parenchyma, as so far supported
by the histological analyses mainly for PM2.5.
4.4. Cytochrome induction
Cytochrome P450 1B1 is a recently identified member of the
CYP gene family. This enzyme is particularly known to catalyse
a number of PAHs to carcinogenic epoxides in several organs,
causing genotoxicity and mutation, and leading to tumor initi-
ation (Nebert and Dalton, 2006). Cyp1b1−/− mice demonstrated
increased protection against many different PAHs-induced malig-
nances, compared with wild type mice (Nebert and Dalton, 2006).
The higher PAHs content of PM2.5win (Perrone et al., 2010) may be
responsible for the increase in Cyp1B1 levels that we found in lung
parenchyma of treated mice, as confirmed by immunohistochem-
istry analyses.
Nebert and Dalton (2006) suggested that the over-expression of
cytochrome of CYP family could promote pro-carcinogenic events;
further investigations may clarify if a chronic exposure to winter
urban PM could be linked to carcinogenic mechanisms.
4.5. Delayed effects
One week after all PM aerosolization, almost all the above con-
sidered markers went back to control values, indicating that the
lung inflammatory and cytotoxic effects were in attenuation or
nearly completely reverted, as confirmed by histological analyses
(data not shown). However, a still ongoing lung damage were sup-
ported by the outlined high concentration of total proteins in the
BALf, which may be related to increased alveolar/capillary barrier
permeability (Henderson, 2005); similarly, a late PM2.5 effect was
suggested by the increased levels of NF-kB-p50 and Cyp1B1. We can
speculate that after 1 week from PM aerosolization the main dis-
trict still involved in lungs damage is the alveolar capillary barrier
which disclose an increased permeability.
In conclusion, the results here presented evidenced the toxicity
mechanisms elicited by size fractionated PM sample, collected in
winter and summer seasons, which differs for dimensions, chemi-
cal composition.
These data corroborates the already hypothesized role of par-
ticle diameter and surface area in eliciting lung inflammatory
responses (Stoeger et al., 2006) and the importance of the differ-
ent PM composition: for winter PM the relevant presence of PAHs
and transition metals (Perrone et al., 2010) and for summer PM the
endotoxin component (Alfaro-Moreno et al., 2007; Camatini et al.,
2010; Wegesser and Last, 2009).
Here, we examined the lung pathologic events induced by a
single and acute aerosolization of particulate matter, while the
realistic situation implies the continuous exposure of lungs to pol-
lutants. Indeed, it must be considered that the inflammatory status
could be protracted, and that the repeated and persevering produc-
tion of pro-inflammatory mediators could lead in the long run to
chronic inflammation. The in vivo analyses of the outlined markers
after a chronic exposure to different PM samples could elucidate
the role of pollutants in inducing chronic inflammation.
Funding
The authors are indebted to Fondazione Cariplo for research
support (TOSCA Project).
Conflict of interest
None.
Acknowledgements
Authors thank the researchers of the Atmospheric Chemistry
group directed by Prof. E. Bolzacchini, operating in the POLARIS
Research Center, Department of Environmental Science, University
of Milano Bicocca, for their precious contribution in PM sampling
and chemical characterization. Thanks to Prof. A. Pesci, Azienda
Ospedaliera San Gerardo, Monza, for his precious contribution in
microscope cells analyses in the BALf.
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