Respiratory Physiology & Neurobiology 186 (2013) 53–60

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Respiratory Physiology & Neurobiology

journal homepage:www.elsevier.com/locate/resphysiol

Regular exercise training attenuates pulmonary inflammatory responses to inhaled

alumina refinery dust in mice
a b b
Valéria Marques Ferreira Normando , Flavia Mazzoli-Rocha , Dayse Kelly Molina Moreira , Bárbara
b c b,∗
Chaves Barcellos , Domingos Wanderley Picanc¸ o-Diniz , Walter Araújo Zin
a Laboratory of Neuroendocrinology, Federal University of Pará, Belém, Brazil
b Laboratory of Respiration Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
cInstitute of Biological Science, Postgraduate Program of Neuroscience and Cell Biology, Laboratory of Neuroendocrinology, Federal University of Pará, Belém, Brazil

article info abstract

Article history: Exposure to alumina dust has been recently associated with impaired lung mechanics and inflammation. We aimed at
Accepted 3 December 2012 evaluating if moderate exercise training prevents these outcomes. Twenty-three female BALB/c mice (25–30 g) were
randomly divided in two main groups: control (C) and exercise (E), which were submitted, or not, to 15 min of swimming, 5
Keywords: days/week during 4 weeks. Then, the animals were exposed for 1 h to either saline solution (CS or ES) or to a suspension of 8
Alumina 3
mg/m of alumina dust (CA or EA). Twenty-four hours later pulmonary mechanics was determined by the end-inflation
Particulate matter
occlusion method. Left lungs were prepared for histology and right lungs for TGF- determination. Static elastance increased
Air pollution
Lung mechanics
after alumina dust exposure independently of swimming. In CA group the viscoelastic component of elastance, the
Inflammation viscoelastic/inhomogeneous pressure, the polymorphonuclear amount, the fraction area of alveolar collapse and TGF-
increased. Thus, exercise training may mitigate the pro-inflammatory response to inhaled aluminum refinery dust.

© 2013 Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction increases the activity of anti-oxidant enzymes in mice exposed to cigarette

Aluminum refinery workers are constantly exposed to alu-minum oxide
(Al2 O3 ) obtained from bauxite (Musk et al., 2000), reporting respiratory
symptoms. Decreased lung function and lung inflammation have been
observed in epidemiologic (Kraus et al., 2000; Fritschi et al., 2003) and
experimental (Halatek et al., 2005; Ichinose et al., 2008; Mazzoli-Rocha et al.,
2010) studies. Neigh-borhoods of the aluminum oxide industry are also
exposed to concentrations of alumina dust, making these communities sus-
ceptible to develop respiratory alterations (Chattopadhyay et al., 2007).
smoke (Menegali et al., 2009). Recently, Toledo et al. (2012) demonstrated
that physical training minimized the reduc-tion in lung elastance and reduced
oxidative stress in mice exposed to cigarette smoke. Hence, the aim of this
study was to evalu-ate whether regular exercising prevents pulmonary
alterations induced in a murine model of acute exposure to alumina dust.
2. Materials and methods

2.1. Animal preparation

High intensity exercise practiced under stressed conditions triggers a
transitory state of low immunity (Brenner et al., 1994). On the other hand,
while regular exercise can be beneficial to health, a sedentary style of life is
detrimental to it (Brines et al., 1996). Daily physical activity may be able to
modulate the immune system (Brines et al., 1996), increasing the resistance to
respiratory infections (Oliveira et al., 2007; Malm, 2006). Regular exercise
improves histology, decreases free radical production and
∗ whose lids were perforated; the containers rested side by side inside the
Corresponding author at: Universidade Federal do Rio de Janeiro, Instituto de Biofísica
Carlos Chagas Filho – C.C.S., Av. Carlos Chagas Filho 373, Ilha do Fundão, 21941-902, Rio de
Janeiro, RJ, Brazil. Tel.: +55 21 2562 6557; fax: +55 21 2280 8193.
E-mail addresses: walter zin@hotmail.com, wazin@biof.ufrj.br (W.A. Zin).
Twenty-three female BALB/c mice (20–25 g) were randomly divided into
2 groups: control (C, n = 10) and exercise (E, n = 13) that swam for 15
min/day, 5 days per week during 4 consecutive weeks (E), or remained
sedentary (C). After a 4-week training, all animals were exposed for 1 h in a
whole-body chamber to either sterile saline (CS, n = 6 or ES, n = 4) or to a
3
suspension of 8 mg/m of alumina dust (CA, n = 6 or EA, n = 7) collected in
an aluminum refinery, both delivered by an ultrasonic nebulizer. Each animal
rested in a container, which was made of high clarity polypropy-lene falcon
tubes whose conical tips were cut off and replaced by metal meshes and
exposure chamber (Mazzoli-Rocha et al., 2010). All animals were analyzed 24
h after saline or alumina

1569-9048 © 2013 Elsevier B.V. Open access under the Elsevier OA license.
http://dx.doi.org/10.1016/j.resp.2012.12.010
54 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60
dust exposure. They received humane care in compliance with the “Principles
of Laboratory Animal Care” formulated by the National Society for Medical
Research and the “Guiding Principles in the Care and Use of Animals”
approved by the Council of the American Physiological Society. The study
was approved by the Institutional Committee for Animal Care and Use,
Health Sciences Center, Federal University of Rio de Janeiro (Protocol no.
IBCCF 046).
2.2. Particle sampling and preparation
3 is reached. This plateau corresponds to the elastic recoil pressure of the lung
A suspension of 8 mg of particles/m of air was obtained by
ultrasonicating 5 mg of the collected dust in 83.3 mL of sterile saline solution
(NaCl 0.9%). The dose was calculated based on the body chamber volume (7
L) and on the airflow of the nebulizer (1 mL/min), taking into consideration
the high dose reported by Fritschi et al. (2001).
2.3. Particle analysis
The particulate matter was digested in a HNO3 –HClO4 mixture and after
dissolution was brought to a final volume of 15 mL of HCl 0.1 M. The extract
was analyzed by flame atomic absorption spectroscopy (VARIAN AA1475,
Varian, Inc., Palo Alto, CA, USA) following recommended standard operating
procedures (Varian, 1981) and previous reports (Trindade et al., 1981; Azcue
et al.,
1988). Trace elements, nickel (Ni), manganese (Mn), aluminum (Al), iron
(Fe), lead (Pb), chromium (Cr), cadmium (Cd), copper (Cu), zinc (Zn) and
mercury (Hg), were measured and the results expressed as g/g of particles.
Three independent samples of the particulate matter were analyzed for this
purpose.
The distribution of particle sizes, as measured by their volume and
surface, and the diameters encompassing 90%, 50% and 10% of the
particulate matter were determined by laser diffraction (Long Bench
Mastersizer S, Malvern Instruments Ltd., Malvern, Worces-tershire, United
Kingdom). The particulate matter was visualized by scanning electron
microscopy (JEOL 5310, Tokyo, Japan).
2.4. Pulmonary mechanics
Twenty-four hours after exposure to either aerosolized sterile saline
3
solution (CS and ES) or to 8 mg/m of aluminum dust (CA and EA) in a
whole-body chamber during 1 h (1 mL/min), the animals were sedated with
diazepam (1 mg i.p.), anesthetized with pentobarbital sodium (20 mg/kg body
weight i.p.), placed in the supine position on a surgical table, tracheotomized,
and a snugly fitting cannula (0.8 mm ID) was introduced into the trachea. The
animals were then paralyzed with pancuronium bromide (0.1 mg/kg) and their
anterior chest wall was surgically removed. A pneumotachograph (1.5 mm ID,
length = 4.2 cm, distance between side ports = 2.1 cm) (Mortola and Novoraj,
1983) was connected to the tracheal cannula for the measurements of airflow
(V ). Tidal volume (VT ) was determined by digital integration of the flow
signal. The pressure gradient across the pneumotachograph was determined
by a Validyne MP45-2 differential pressure transducer (Engineering Corp,
Northridge, CA, USA). The flow resistance of the equipment (R eq ), tracheal
cannula included, was constant up to flow rates of 26 mL/s and amounted to
0.12 cmH2 O/mL/s. Equip-ment resistive pressure (= R eq V ) was subtracted
from pulmonary resistive pressure so that the present results represent
intrinsic values. Transpulmonary pressure was measured with a Vali-dyne
MP-45 differential pressure transducer (Engineering Corp, Northridge, CA,
USA). All signals were conditioned and amplified in a Beckman type R
Dynograph (Schiller Park, IL, USA). Flow and pressure signals were then
passed through 8-pole Bessel low-pass filters (902LPF, Frequency Devices,
Haverhill, MA, USA) with the corner frequency set at 100 Hz, sampled at 200
Hz with a 12-bit
analog-to-digital converter (DT2801A, Data Translation, Marlboro, MA,
USA), and stored on a microcomputer. All data were collected using
LABDAT software (RHT-InfoData Inc., Montreal, QC, Canada).
Lung resistive ( P1 ) and viscoelastic/inhomogeneous ( P2 ) pressures,
static elastance (Est ), and viscoelastic component of elas-tance ( E) were
computed by the end-inflation occlusion method (Bates et al., 1985, 1988).
Briefly, after end-inspiratory occlusion, there is an initial fast drop in
transpulmonary pressure ( P1 ) from the pre-occlusion value down to an
inflection point (Pi ) followed by a slow pressure decay ( P 2 ), until a plateau
(Pel ). P1 selectively reflects airway resistance in normal animals and humans
and P2 reflects stress relaxation, or viscoelastic proper-ties of the lung,
together with a small contribution of time constant of alveoli (Bates et al.,
1988; Saldiva et al., 1992). Lung static and dynamic elastances (E st and Edyn ,
respectively) were calculated by dividing P el and Pi by tidal volume,
respectively. E was calculated as E st − Edyn , and reflects the viscoelastic
component of elastance (Bates et al., 1985, 1988).
2.5. Histological study
Heparin (1000 IU) was intravenously injected immediately after the
determination of pulmonary mechanics. The trachea was clamped at end-
expiration and the animals were euthanized by exsanguinations via sectioning
of the abdominal aorta and the vena cava. The lungs were removed and
weighed. Functional residual capacity (FRC) was determined by volume
displacement (Scherle, 1970). Left lungs were then fixed with Millonig
formaldehyde (100 ml HCHO, 900 ml H2 O, 18.6 g NaH2 PO4 , 4.2 g NaOH),
routinely prepared for histology, embedded in paraffin, and two 3- m-thick
longitudinal slides from the left lung were cut and stained with hematoxylin–
eosin.
Morphometric analysis was performed with an integrating eye-piece with
a coherent system made of a 100-point and 50-line (1250- m-long each) grid
coupled to a conventional light micro-scope (Axioplan, Zeiss, Oberkochen,
Germany). The fraction areas of collapsed and normal alveoli were
determined by the point-counting technique at a magnification of ×200 across
10 random non-coincident microscopic fields per animal. Points falling on
nor-mal or collapsed alveoli were expressed as percentage of points hitting
those alveoli (Weibel, 1990). Polymorphonuclear (PMN) and pulmonary
tissue were evaluated at ×1000 magnification across 10 random non-
coincident microscopic fields in each animal. Points falling on PMN was
counted, and divided by the total number of points falling on tissue area in
each microscopic field (Weibel, 1990).
In all animals exposed to alumina dust the presence of alumina crystals in
the lung (alveolar spaces and airways) was qualita-tively evaluated under
polarized light (Axioplan, Zeiss, Oberkochen, Germany) at 1000×
magnification.
2.6. Preparation of lung homogenates for determination of
inflammatory cytokines
The right lungs were homogenized in 1 mL of PBS with protease
inhibitors (1 g/mL leupeptin and 1 g/mL pepstatin). Homogenates were
centrifuged (Centrifuge 5415R, Hamburg, Germany, 4 ◦ C, 6700 × g, 15 min)
and then, the supernatant was collected for transforming growth factor beta
(TGF- ) and interleukin-1beta (IL-1 ) assays by ELISA (R&D Systems Inc.,
Min-neapolis, MN, USA), according to the manufacturer’s protocol. Total
protein concentration in lung homogenates was determined by Bradford’s
Concentration of cytokines in lung
method (Bradford, 1976).
homogenates was further normalized to protein concentra-
tion in the samples and expressed as picograms per milligram
of
 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60 55

Table 1
Metals in aluminum dust particulate matter.

Elements Mean ( g/g) SD

Ni 24.71 12.92
Mn 102.65 112.42
Al 97,451.16 49,725.90
Fe 19,204.09 10,547.38
Pb 158.16 87.46
Cr 56.54 29.82
Cd 1.08 1.37
Cu 255.04 139.52
Zn 392.38 431.36
Hg 7211.48 3949.32

Concentration of metals were measured three times and the results expressed as mean and SD.

protein. Optical density was measured at 450 nm by a microplate reader Fig. 2. Absolute values of body weight during 4 weeks in control (C) and exercising mice (E).
(SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). Values are mean ± SEM of 4–7 animals in each group. *Significantly dif-ferent from day 28; **
#
significantly different from day 21; significantly different from E.

2.7. Statistical analysis

The normality of the data and the homogeneity of variances were tested training, but thereafter the values did not differ from those in con-trol mice
(Fig. 2).
by Kolmogorov–Smirnov test with Lilliefors’ correc-tion and Levene median
test, respectively. In all instances both conditions were satisfied and All mechanical parameters ( P2 , E and Est ) but P1 were higher after
parametric tests were run. One-way ANOVA was used to compare the values alumina dust exposure in animals not submitted to physical exercise.
of body weight measured every 7 days, throughout 4 weeks in each group. Additionally, exercise training before particle exposure caused no changes in
Weight differences between control and exercise groups at every 7 days were resistive and viscoelastic compo-nents, but Est increased in this group (Fig. 3).
evaluated by Student’s t-test. Two-way ANOVA was applied to the remaining
parameters (factors: exercise and alumina). For all ANOVAs, the Student–
Newman–Keuls was used as a post hoc test. The morpho-metric data,
originally expressed as percent, underwent an arcsine transformation, in order
to generate a normal distribution. The sta-tistical analyses were carried out by
the SigmaStat 9.0 software (SYSTAT, Point Richmond, CA, USA). In all
instances p < 0.05 was considered a statistically significant difference.

3. Results

Metal composition of alumina dust is presented in Table 1. A high

concentration of the element Al, followed by Fe and Hg was found.

Scanning electron micrographs of particles are shown in Fig. 1,

demonstrating the frequency distribution of diameters of particle sample. 90%
of particles diameter are under 150 m, being 50% below 100 m and 10%
smaller than 57 m.
A progressive increase in body weight was observed along time in animals
not submitted to physical exercise. In exercising group, a decrease in body
weight occurred during the first week of aquatic

Fig. 3. Lung static elastance (Est ), elastic component of viscoelasticity ( E) (upper

Fig. 1. Histogram of the frequency distribution of particle diameters. Accumulated
frequency is represented by the solid line.
panel), and pressures spent against resistive components of lung mechanics ( P 1
and P2 : pressures spent to overcome resistive and viscoelastic/inhomogeneous
mechanical components, respectively, lower panel) in mice that had undergone (E
group), or not (C group), exercise training and were then exposed for 1 h to either
3
sterile saline (CS and ES) or to a suspension (in saline) of 8 mg/m of particulate
matter (CA and EA) with a high content of aluminum, both delivered by an ultra-
sonic nebulizer. Measurements were performed 24 h after saline or alumina dust
exposure. Bars are mean ± SEM of 4–7 animals in each group (10–15 determina-
tions per animal). * Significant difference between alumina and saline exposed mice
(p < 0.05); ** significant difference (p < 0.001).
56 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60

Crystals of alumina were observed in alveolar spaces of all ani-mals

exposed to alumina. A representative image is shown in Fig. 4. In one
instance a crystal was detected in an airway.
Fig. 5 depicts photomicrographs of lung parenchyma showing pulmonary
inflammation after inhalation of alumina dust, as sug-gested by the more
important influx of PMN cells (upper right-hand corners inserts), and
increased alveolar collapse (evidenced by #). Exposure to particulate matter
increased the fraction area of alve-olar collapse in CA and EA, being more
pronounced in the former (Table 2). Also in Table 2, one can see that physical
exercise pre-vented PMN cell influx into the lung parenchyma in EA group.

Functional residual capacity decreased in animals exposed to alumina dust

(CA = 0.12 ± 0.07 mL and EA = 0.12 ± 0.04 mL, mean ± SD) in relation to
their controls (CS = 0.25 ± 0.16 mL and ES = 0.23 ± 0.10 mL), independently
of previous exercise.
An increase in TGF- was observed after exposure to alumina dust. This
alteration was minimized by exercise (Fig. 6, upper panel). IL-1 expression
Fig. 4. Photomicrograph of lung parenchyma under polarized light showing two crystals of
did not differ among the groups (Fig. 6, lower panel).
alumina in the alveolar space and on the alveolar epithelium. Sample was obtained 24 h after
exposure. Hematoxylin–eosin (1000×). Bar: 50 m.
The survival rate was 100% in all groups throughout the experiment.

Fig. 5. Photomicrographs of lung parenchyma from mice that had undergone (E group), or not (C group), exercise training and were then exposed for 1 h to either sterile saline (CS and ES) or to a
suspension (in saline) of 8 mg/m 3 of particulate matter (CA and EA) with a high content of aluminum, both delivered by an ultrasonic nebulizer. Samples were obtained 24 h after inhalation and
stained with hematoxylin–eosin (200×). Upper right-hand corners inserts were obtained at 1000× magnification; note that cell influx is more prominent in CA lung.
# indicates alveolar collapse. Bar: 100 m.
 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60
Table 2
Morphometrical parameters and cellularity in lung parenchyma.
CS
Normal area (%) 88.8 ± 1.8a
Alveolar collapse (%) 11.2 ± 1.8a
−3 2 2.8 ± 0.4a
PMN (cells ×10 / m )
Values are mean ± SD of 4–7 animals/group. Data were gathered from ten random, non-coincident fields per mouse in mice that had undergone (E group), or not (C group), exercise training and were
3
then exposed for 1 h to either sterile saline (CS and ES) or to a suspension (in saline) of 8 mg/m of particulate matter (CA and EA) with a high content of aluminum, both delivered by an ultrasonic
nebulizer. Measurements were performed 24 h after exposure. PMN, polymorphonuclear cells. Different letters represent significantly different values (p < 0.05).
Fig. 6. Concentrations of TGF- and IL-1 in lung homogenates of mice that had undergone (E
group), or not (C group), exercise training and were then exposed for 1 h to either sterile saline
3
(CS and ES) or to a suspension (in saline) of 8 mg/m of particulate matter (CA and EA) with a
high content of aluminum, both delivered by an ultrasonic nebulizer. Measurements were
performed 24 h after saline or alumina dust exposure. Bars are mean ± SEM of 4 animals in
each group (in each mice two analyses were run for each sample). * Significantly different
groups are linked by horizontal solid lines (p < 0.05).
4. Discussion
In the present study in mice we demonstrated that lung mechan-ical and
histological alterations after a single aerosolization of dust containing a high
concentration of aluminum were in part mini-mized by previous aquatic
exercise training.
3 3
As previously mentioned, 4 mg/m breathable dust content and 1.5 mg/m
respirable dust content may not affect the healthy of aluminum refinery
workers (Deutsche Forschungsgemeinschaft, 2006). Additionally, the
3 to the lung periphery. Although an increase in resistive pressure was not found
concentration should not exceed 10 mg/m for up to a 10-h workday (NIOSH,
2005). Accordingly, the admin-istered dose was identical to that used in a
previous study (Mazzoli-Rocha et al., 2010), in which the animals were
3 histology analy-ses. This phenomenon could be explained by the fact that in
exposed to a suspension (in saline) of 8 mg/m of alumina dust, a dose
smaller than that recommended for human exposure (NIOSH, 2005). An
3
exposure to 8 mg/m of particulate matter corresponds to 0.48 mg/kg in mice
(body weight: 20 g, breathing frequency:
57
CA ES EA
66.5 ± 2.1b 84.5 ± 4.0a 75.5 ± 3.6a,b
33.5 ± 2.1b 15.6 ± 4.0a 24.5 ± 3.6a,b
8.8 ± 3.0b 4.9 ± 0.4a,b 4.9 ± 0.7a,b
100 bpm, and tidal volume: 0.2 mL), a dose smaller than that used in rats and
mice: 75 mg/kg (Halatek et al., 2005) and 4 mg/kg (Ichinose et al., 2008),
respectively, and that approximates an 8-fold value in relation to the highest
dose reported by Fritschi et al. (2001) in human beings.
Decreased lung function after alumina dust exposure has been observed
even in small doses (Schlesinger et al., 2000; Abbate et al., 2003; Barnard et
al., 2004; Chattopadhyay et al., 2007; Mazzoli-Rocha et al., 2010).
Additionally, accumulated exposure seems to be one of the most important
factors related to the pulmonary tox-icity and may be involved in alveolar
macrophage influx (Pauluhn, 2009a, 2009b); we avoided such design because
our aim was to investigate the role of exercise in mice acutely exposed to low
doses of aluminum dust.
In the present study, size frequency distribution and composi-tion of the
dust particles were analyzed. Since 10% of the particulate matter had a
diameter smaller than 57 m (Fig. 1), some of them reached alveolar spaces, as
illustrated in the photomicrograph under polarized light (Fig. 4). As depicted
in Table 1, particulate matter showed a high concentration of the element
aluminum. The second most frequently element, iron, has been described as
the main culprit in triggering oxidative stress (Park et al., 2006) and producing
reactive oxygen species (ROS) (Smith and Aust, 1997). Some authors suggest
that other metals act as coadjutants in the genesis of pulmonary injury
(Prahalad et al., 2000, 2001 ). The ini-tial phase of the pulmonary reaction to
particle exposure seems to be influenced by individual metals, whereas the
persistence of the response would reflect the complexity of the interaction
among dif-ferent metals (Dreher et al., 1997; Antonini et al., 2004). However,
it is not possible to exclude the contribution of other non-determined
constituents of the particle composition.
We measured elastic, resistive and viscoelastic parameters by the end-
inflation occlusion method, allowing the identification of elastic, resistive, and
viscoelastic and/or inhomogeneous lung mechanical components (Bates et al.,
1985, 1988). In line with previous results (Mazzoli-Rocha et al., 2010),
viscoelastic pres-sure, static elastance and viscoelastic component of elastance
were higher in CA than in CS (Fig. 3), which implies that lung parenchyma
was compromised, whereas large airways were not. Additionally, an influx of
polymorphonuclear cells and an increase in alveo-lar collapse were more
important in group CA than in CS (Fig. 5 and Table 2). The cell influx into the
alveolar walls, as well as the decreased lung function reported in this study
was previously observed in hamsters (Drew et al., 1974), mice (Mazzoli-
Rocha et al., 2010), and rats (Halatek et al., 2005) after aluminum exposure.
According to Donaldson et al. (2001), the coarse particles may be mostly
restrained in the superior airways and cause local irritation unchaining
symptoms as cough. On the other hand, ultrafine parti-cles can cause damage
(in accordance with previous results), decreased lung function and
parenchymal inflammation could be observed by pulmonary mechanics and
general coarse particles are comprised of up to 50% by mass of ultrafine
58 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60
particles (Donaldson and Stone, 2003) and these small aggregated particles
may be the active component of the coarse ones (Anderson et al., 2001).
Particulate inhalation from environmental (Liu et al., 2007) and
occupational (Trupin et al., 2003) air pollutants has been identified as being
among the primary causes and exacerbations of pulmonary diseases. The
inflammation injury is followed by an unbalanced repair process
characterized by an inappropriate production or degradation of the organic
matrix, leading to abnormal lung archi-tecture and impairment of lung
function (Wolff and Crystal, 1997; Van den Brûle et al., 2005). The secretion
of growth factors, such as TGF- , contributes to the increased production of
matrix com-ponents by fibroblasts, yielding to lung remodeling (Wolff and
Crystal, 1997; Wang et al., 2009). In order to verify a possible remodeling
process in mice exposed to alumina dust, two cytokines were determined in
lung homogenate (TGF- and IL1- ). TGF-signaling controls cell proliferation,
recognition and differentiation (Shi and Massagué, 2003), and represents a
potent fibrogenic agent that stimulates fibroblast chemotaxis, and enhances
the production of collagen, fibronectin, and proteoglycans (Leask and
Abraham, 2004). In animal model of bleomycin-induced pulmonary fibrosis,
TGF- production is increased before collagen synthesis, mainly by alveolar
macrophages (Khalil et al., 1989). In a human fibrotic lung disease (idiopathic
pulmonary fibrosis), increased TGF- pro-duction can be detected by
immunohistochemical staining, in epithelial cells and macrophages in areas of
lung regeneration and remodeling (Khalil et al., 1991). In the present study,
Fig. 6 shows an increase in the production of TGF- in CA group in relation to
CS. Accordingly, Wistar rats intratracheally exposed to a unique instillation of
silica had an increase of TGF- in bronchoalveolar lavage fluid (BALF) after 7
days of exposure (Wang et al., 2009). Van den Brûle et al. (2005)
demonstrated an increase in TGF- in lung homogenate of C57BL/6, but not
BALB/c mice, one month after silica intratracheal instillation. The difference
between our results and those of Van den Brûle et al. (2005) could be due to
the dura-tion between the end of exposure and the experiments and/or to the
different particulate matter used. In this connection the patho-genesis of
silicosis involves alveolar cell injury, cytokine signaling and cell recruitment
in the areas of silica dust deposition (Brown et al., 2007; Kühlmann et al.,
2009). This finding suggests that lung fibrosis could take place in CA mice
after the completion of lung remodeling.
Lung fibrosis is dependent on the influx and activation of inflam-matory
cells that release key pro-inflammatory cytokines such as TNF- and IL-1 that
directly stimulate fibroblast functions and pulmonary deposition of matrix
proteins (Lundblad et al., 2005; Di Giuseppe et al., 2009). IL-1 has been
shown to be among the most biologically active cytokines in the lungs early
after the onset of lung injury (Olman et al., 2002; Ganter et al., 2008). In
addition, this cytokine is a potent inducer of TGF- , and part of its profibrotic
effects is probably mediated through this growth factor (Kolb et al., 2001). In
the present study, we observed a non-significant trend to higher IL-1
concentration in lung homogenate of CA group in relation to CS (Fig. 6).
Interestingly, qualitatively TGF- and IL-1 depicted the same group-wise
behavior and indeed an increase in IL-1 expression could have preceded TGF-
induction (Kolb et al., 2001). Taken together, the results of TGF- and IL-1
suggest that lung fibrosis could take place in CA mice after the completion of
lung remodeling.
Exercise modifies homeostasis leading to a reorganization of systems
responses, including the immune system (Brenner et al., 1994). In general,
regular and moderate exercise improves the reac-tion capacity of the immune
system (Woods et al., 2009; Beavers et al., 2010), whereas high intensity
exercise practiced under stressed conditions yields to a transitory state of low
immunity (Brenner et al., 1994). Chronic practice of regular exercise exerts a
marked anti-inflammatory effect in different lung disease, such as asthma
(Pastva et al., 2004; Vieira et al., 2007, 2008) and chronic obstructive
pulmonary disease (Menegali et al., 2009; Toledo et al., 2012). In this way, we
decided to expose mice to alumina dust after a 4-week exercising routine in
order to evaluate its putative pro-tective action against the particulate matter
aggression. For such purpose we studied trained animals exposed (EA) or not
(ES) to alumina dust. Fig. 3 discloses that exercise training prevented the
increase in E and P2 in EA in relation to ES, although the increase in E st could
not be avoided (Fig. 3). P2 normalization could be attributed to attenuation of
lung tissue stiffness owing to reduced alveolar collapse (Fig. 5 and Table 2).
However, although exercise resulted in less heterogeneous lungs, it was not
enough to keep all alveoli open and restore FRC (lower in EA than in ES) and
Est (higher in EA than in ES). We could find only one study relating exer-cise
and lung mechanics. It reports that moderate exercise training (60 min/day, 5
days/week, during 24 weeks) did not modify tissue damping and prevented the
reduction of tissue elastance in mice (C57BL/6) exposed to cigarette smoke
(Toledo et al., 2012). Air-way resistance behaved similarly in both studies.
The difference between their and our results could be due to a species differ-
ence, exposure to different pollutants, method used to determine mechanical
parameters, and duration and/or intensity of training.
In this study alveolar collapse and cell influx to lung parenchyma were
also minimized by previous exercise (Fig. 5 and Table 2). Accordingly,
exercise training alleviates lung inflamma-tion, demonstrated by the reduction
in total cell count in BALF of mice exposed to cigarette smoke (Yu et al.,
2012). This reduction was attributed to decreased number of lymphocytes,
macrophages and neutrophils (Yu et al., 2012). Vieira et al. (2012) also
observed a beneficial effect of aerobic exercise (5 times/week, during 5
weeks) in reducing neutrophils and lymphocytes influx caused by diesel
exhaust particles (DEP) exposure.
According to Nemet et al. (2003), exercise modifies the concentration of
circulating cytokines involved in the immune responses. Physical exercise can
induce the sequential release of pro-inflammatory cytokines (TNF- and IL-1 ),
anti-inflammatory cytokines (IL-10) and also IL-6 (classified as both pro- and
anti-inflammatory cytokine) (Petersen and Pedersen, 2005). In the present
study, exercise training maintained TGF- at the same lev-els as in groups CS
and ES and smaller than in CA. Physical exercise did no alter IL-1 expression
(Fig. 6). Exercise training prevents the increase of nitric oxide in BALF of
mice exposed to DEP and reduced lung parenchymal remodeling by inhibiting
collagen accumulation in lung parenchyma (Vieira et al., 2012).
It is important to note that exercise alone (ES group) did not modify lung
function and histology as well as cytokine release (val-ues similar to CS).
Our study presents limitations: we did not measure levels of different
markers of inflammation and oxidative stress after/before inhalation with/out
exercise, as well as damage to epithelial cells, mucociliary transportation and
the surfactant system that could have been modified by exposure to particulate
matter.
In this study, we demonstrated for the first time in mice exposed to
alumina dust that regular exercise partially prevented lung mechanical
impairment and the triggering of TGF- . Addition-ally, the recruitment of
PMN cells and the increase of alveolar collapse observed in CA were
minimized in EA group. To our knowl-edge no animal studies on pulmonary
mechanics, lung histology and cytokine concentration in lung homogenate
after aluminum exposure and pretreated with exercise could be found in the
literature.
In conclusion, we demonstrated that regular exercise could
partially prevent lung inflammation induced by a single
aerosoliza-tion of small amounts of particulate matter
containing mostly aluminum.
 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60
Acknowledgments
The authors are grateful to Joao Luiz Coelho Rosas Alves and Antonio
Carlos de Souza Quaresma (Laboratory of Respiration Physiology) for their
skillful technical assistance and to Fabianno Ferreira Dutra and Marcelo
Torres Bozza (Laboratory of Inflam-mation and Immunity) for their
assistance in the determination of cytokines.
This study was supported by: PRONEX/FAPERJ, Brazilian Council for
Scientific and Technological Development (CNPq), and Carlos Chagas Filho
Rio de Janeiro State Research Supporting Foundation (FAPERJ).
References
Abbate, C., Giorgianni, C., Brecciaroli, R., Tringali, M.A., D’Arrigo, G., 2003. Spirometric
function in non-smoking workers exposed to aluminum. American Journal of Industrial
Medicine 44, 400–404.
Anderson, H.R., Bermmer, S.A., Atkinson, R.W., Harrison, R.M., Walters, S., 2001. Par-ticulate
matter and daily mortality and hospital admissions in the west midlands conurbation of the
United Kingdom: associations with fine and coarse parti-cles, black smoke and sulphate.
Occupational and Environmental Medicine 58, 504–510.
Antonini, J.M., Taylor, M.D., Leonard, S.S., Lawryk, N.J., Shi, X., Clarke, R.W., Roberts, J.R.,
2004. Metal composition and solubility determine lung toxicity induced by residual oil fly
ash collected from different sites within a power plant. Molecular and Cellular
Biochemistry 255, 257–265.
Azcue, J.M.P., Pfeiffer, W.C., Fiszman, M., Malm, O., 1988. Heavy metal removal from
different water treatment plants, in Rio de Janeiro state, Brazil. Environmental Technology
Letters 9, 429–436.
Barnard, C.G., McBride, D.I., Firth, H.M., Herbison, G.P., 2004. Assessing individual employee
risk factors for occupational asthma in primary aluminum smelting. Occupational and
Environmental Medicine 61, 604–608.
Bates, J.H.T., Ludwig, M.S., Sly, P.D., Brown, K.A., Martin, J.G., Fredberg, J.J., 1988.
Interrupter resistance elucidated by alveolar pressure measurements in open-chest normal
dogs. Journal of Applied Physiology 65, 408–414.
Bates, J.H.T., Rossi, A., Milic-Emili, J., 1985. Analysis of the behavior of the respira-tory
system with constant inspiratory flow. Journal of Applied Physiology 58, 1840–1848.
Beavers, K.M., Brinkley, T.E., Nicklas, B.J., 2010. Effect of exercise training on chronic
inflammation. Clinica Chimica Acta 411, 785–793.
Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry
72, 248–254.
Brenner, K.M., Sherk, P.N., Shepard, R.J., 1994. Infection in athletes. Sports Medicine 17, 86–
107.
Brines, R., Hoffman-Gotez, L., Pedersen, B.K., 1996. Can you exercise to make your immune
system fitter? Immunology Today 17, 252–254.
Brown, J., Swindle, E.J., Kushnir-Sukhov, N.M., Holian, A., Metcalfe, D.D., 2007. Silica-
directed mast cell activation is enhanced by scavenger receptors. American Journal of
Respiratory Cell and Molecular Biology 36, 43–52.
Chattopadhyay, B.P., Saiyed, H.N., Roychowdhury, A., Alam, J., 2007. Pulmonary func-tion in
aluminum smelter and surrounding community – a case study. Journal of Environmental
Science and Engineering 49, 309–316.
Deutsche Forschungsgemeinschaft’s Senate Commission on the Investigation of Health
Hazards of Chemical Compounds in the Work Area – list of maximum concentration at the
workplace (MAK value) and biological tol-erance value at the workplace (BAT value),
press release no. 34, 2006. www.dfg.de/aktuelles presse/reden
stellungnahmen/download/mak2006.pdf
Di Giuseppe, M., Gambelli, F., Hoyle, G.W., Lungarella, G., Studer, S.M., Richards, T.,
Yousem, S., McCurry, K., Dauber, J., Kaminski, N., Leikauf, G., Ortiz, L.A., 2009.
Systemic inhibition of NF-kappaB activation protects from silicosis. PLoS ONE 4, e5689.
Donaldson, K., Stone, V., 2003. Current hypotheses on the mechanisms of toxicity of ultrafine
particles. Annali dell Istituto Superiore di Sanita 39, 405–410.
Donaldson, K., Stone, V., Clouter, A., Renwick, L., Macnee, W., 2001. Ultrafine parti-cles.
Occupational and Environmental Medicine 58, 211–216.
Dreher, K.L., Jaskot, R.H., Lehmann, J.R., Richards, J.H., McGee, J.K., Ghio, A.J., Costa, D.L.,
1997. Soluble transition metals mediate residual oil fly ash induced acute lung injury.
Journal of Toxicology and Environment Health 50, 285–305.
Drew, R.T., Gupta, B.N., Bend, J.R., Hook, G.E., 1974. Inhalation studies with a glycol
complex of aluminum chloride-hydroxide. Archives of Environment Health 28, 321–326.
Fritschi, L., Klerk, N., Sim, M., Benke, G., Musk, A.W., 2001. Respiratory morbidity and
exposure to bauxite, alumina and caustic mist in alumina refineries. Journal of
Occupational Health 43, 231–237.
Fritschi, L., Sim, M.R., Abramson, A.F.M.J., Benke, G., de Klerke, A.W.M.N.H., 2003.
Respiratory symptoms and lung-function changes with exposure to five sub-stances in
aluminum smelters. International Archives of Occupational and Environmental Health 76,
103–110.
59
Ganter, M.T., Roux, J., Miyazawa, B., Howard, M., Frank, J.A., Su, G., Sheppard, D., Violette,
S.M., Weinreb, P.H., Horan, G.S., Matthay, M.A., Pittet, J-F., 2008. Interleukin-1 causes
acute lung injury via v 5 and v 6 integrin-dependent mechanisms. Circulation Research 102,
804–812.
Halatek, T., Opalska, B., Lao, I., Stetkiewicz, J., Rydzynski, K., 2005. Pneumotoxicity of dust
from aluminum foundry and pure alumina: a comparative study of mor-phology and
biomarkers in rats. International Journal of Occupational Medicine and Environmental
Health 18, 59–70.
Ichinose, T., Yoshida, S., Sadakane, K., Takano, H., Yanagisawa, R., Inoue, K., Nishikawa, M.,
Mori, I., Kawazato, H., Yasuda, A., Shibamoto, T., 2008. Effects of Asian sand dust,
Arizona sand dust, amorphous silica and aluminum oxide on allergic inflammation in the
murine lung. Inhalation Toxicology 20, 685–694.
Khalil, N., Bereznayj, O., Sporn, M., Greenberg, A.H., 1989. Macrophage production of
transforming growth factor and fibroblast collagen synthesis in chronic pulmonary
inflammation. Journal of Experimental Medicine 170, 727–737.
Khalil, N., O‘Connor, R.N., Unruh, H.W., Warren, P.W., Flanders, K.C., Kemp, A., Bereznay,
O.H., Greenberg, A.H., 1991. Increased production and immuno-histochemical localization
of transforming growth factor-beta in idiopathic pulmonary fibrosis. American Journal of
Respiratory Cell and Molecular Biology 5, 155–162.
Kolb, M., Margetts, P.J., Anthony, D.C., Pitossi, F., Gauldie, J., 2001. Transient expression of
IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. Journal
of Clinical Investigation 107, 1529–1536.
Kraus, T., Schaller, K.H., Angerer, J., Letzel, S., 2000. Aluminum dust-induced disease in the
pyro-powder-producing industry: detectin by high-resolution computed tomography.
International Archives of Occupational and Environmental Health 73, 61–64.
Kühlmann, U.C., Chwieralski, C.E., van den Brule, S., Röcken, C., Reinhold, D., Welte, T.,
Bühling, F., 2009. Modulation of cytokine production and silica-induced lung fibrosis by
inhibitors of aminopeptidase N and of dipeptidyl peptidase-IV-related proteases. Life
Sciences 84, 1–11.
Leask, A., Abraham, D.J., 2004. TGF- signaling and the fibrotic response. FASEB Journal 18,
816–827.
Liu, S., Zhou, Y., Wang, X., Wang, D., Lu, J., Zheng, J., Zhong, N., Ran, P., 2007. Biomass fuels
are the probable risk factor for chronic obstructive pulmonary disease in rural South China.
Thorax 62, 889–897.
Lundblad, L.K., Thompson-Figueroa, J., Leclair, T., Sullivan, M.J., Poynter, M.E., Irvin, C.G.,
Bates, J.H., 2005. Tumor necrosis factor-alpha overexpression in lung disease: a single
cause behind a complex phenotype. American Journal of Respi-ratory and Critical Care
Medicine 171, 1363–1370.
Malm, C., 2006. Susceptibility to infections in elite athletes: the S-curve. Scandina-vian Journal
of Medicine and Science in Sports 16, 4–6.
Mazzoli-Rocha, F., Santos, A.N., Fernandes, S., Normando, V.M.F., Malm, O., Saldiva, P.H.N.,
Picanc¸ o-Diniz, W., Faffe, D.S., Zin, W.A., 2010. Pulmonary function and his-tological
impairment in mice after acute exposure to aluminum dust. Inhalation Toxicology 22, 861–
867.
Menegali, B.T., Nesi, R.T., Souza, P.S., Silva, L.A., Silveira, P.C.L., Valenc¸ a, S.S., Pinho, R.A.,
2009. The effects of physical exercise on the cigarette smoke-induced pulmonary oxidative
response. Pulmonary Pharmacology & Therapeutics 22, 567–573.
Mortola, J.P., Novoraj, A., 1983. Two-sidearm tracheal cannula for respiratory airflow
measurements in small animals. Journal of Applied Physiology 55, 250–253.
Musk, A.W., de Klerke, N.H., Beach, J.R., Fritschi, L., Sim, M.R., Benke, G., Abramson, M.,
McNeil, J.J., 2000. Respiratory symptoms and lung function in alumina refinery employees.
Occupational and Environmental Medicine 57, 279–283.
Nemet, D., Rose-Gottron, C.M., Mills, P.J., Cooper, D.M., 2003. Effect of water polo practice
on cytokines, growth mediators and leukocytes in girls. Medicine and Science in Sports and
Exercise 35, 356–363.
NIOSH, National Institute for Occupational Safety and Health, 2005. http://www.
atsdr.cdc.gov/toxprofiles/tp22-c8.pdf
Oliveira, C.A.M., Luciano, E., Marcondes, M.C.C.G., Mello, M.A.R., 2007. Effects of
swimming training at the intensity equivalent to aerobic/anaerobic metabolic transition in
alloxan diabetic rats. Journal of Diabetes and Its Complications 21, 258–264.
Olman, M.A., White, K.E., Ware, L.B., Cross, M.T., Zhu, S., Matthay, M.A., 2002. Microar-ray
analysis indicates that pulmonary edema fluid from patients with acute lung injury mediates
inflammation, mitogen gene expression, and fibroblast proliferation through bioactive
interleukin-1. Chest 121, 69S–70S.
Park, S., Nam, H., Chung, N., Park, J.D., Lim, Y., 2006. The role of iron in reactive oxygen
species generation from diesel exhaust particles. Toxicology in Vitro 20, 851–857.
Pastva, A., Estell, K., Schoeb, T.R., Atkinson, T.P., Schwiebert, L.M., 2004. The Journal of
Immunology 172, 4520–4526.
Pauluhn, J., 2009a. Pulmonary toxicity and fate of agglomerated 10 and 40 nm alu-minum
pxyhydroxides following 4-week inhalation exposure of rats: toxics effects are determined
by agglomerated, not primary particle size. Toxicological Sciences 109, 152–167.
Pauluhn, J., 2009b. Retrospective analysis of 4-week inhalation studies in rats with focus on fate
and pulmonary toxicity of two nanosized aluminum oxyhydroxides (boehmite) and
pigment-grade iron oxide (magnetite): the key metric of dose is particle mass and not
particle surface area. Toxicology 259, 140–148.
Petersen, A.M.W., Pedersen, B.K., 2005. The anti-inflammatory effect of exercise.
Journal of Applied Physiology 98, 1154–1162.
Prahalad, A.K., Inmon, J., Dailey, L.A., Madden, M.C., Ghio, A.J., Gallagher, J.E., 2001. Air
pollution particles mediated oxidative DNA base damage in a cell free system
60 V.M.F. Normando et al. / Respiratory Physiology & Neurobiology 186 (2013) 53–60
and in human airway epithelial cells in relation to particulate metal content and
bioreactivity. Chemical Research in Toxicology 14, 879–887.
Prahalad, A.K., Inmon, J., Ghio, A.J., Gallagherj, E., 2000. Enhancement of 2 - deoxyguanosine
hydroxylation and DNA damage by coal oil fly ash in relation to particulate metal content
and availability. Chemical Research in Toxicology 13, 1001–1019.
Saldiva, P.H., Zin, W.A., Santos, R.L., Eidelman, D.H., Milic-Emili, J., 1992. Alveolar pressure
measurement in open-chest rats. Journal of Applied Physiology 72, 302–306.
Scherle, W., 1970. A simple method for volumetry of organs in quantitative stereol-ogy.
Mikroskopie 26, 57–60.
Schlesinger, R.B., Snyder, C.A., Chen, L.C., Gorczynski, J.E., 2000. Clearance and
translocation of aluminum oxide (alumina) from the lungs. Inhalation Toxico-logy 12, 927–
939.
Shi, Y., Massagué, J., 2003. Mechanisms of TGF- signaling from cell membrane to the nucleus.
Cell 113, 685–700.
Smith, K.R., Aust, A.N., 1997. Mobilization of iron from urban particles leads to gen-eration of
reactive oxygen species in vitro and induction of ferritin synthesis in human lung epithelial
cells. Chemical Research in Toxicology 10, 828–834.
Toledo, A.C., Magalhaes, R.M., Hizume, D.C., Vieira, R.P., Biselli, P.J.C., Moriya, H.T.,
Mauad, T., Lopes, F.D.T.Q.S., Martins, M.A., 2012. Aerobic exercise attenuates pul-
monary injury induced by exposure to cigarette smoke. European Respiratory Journal 39,
254–264.
Trindade, H.A., Pfeiffer, W.C., Londres, H., Costa-Ribeiro, C.L., 1981. Atmospheric
concentration of metals and total suspended particulates in Rio de Janeiro. Envi-ronmental
Science and Technology 15, 84–89.
Trupin, L., Earnest, G., San Pedro, M., Balmes, J.R., Eisner, M.D., Yelin, E., Katz, P., Blanc,
P.D., 2003. The occupational burden of chronic obstructive pulmonary disease. European
Respiratory Journal 22, 462–469.
Van den Brûle, S., Misson, P., Bühling, F., Lison, D., Huaux, F., 2005. Overexpression of
cathepsin K during silica-induced lung fibrosis and control by TGF- . Respiratory Research
6, 84.
Varian, A.A., 1981. 1275-1475 series Spectrophotometers Service Manual. Varian Techtron Pty.
Limited Australia.
Vieira, R.D.P., Toledo, A.C., Silva, L.B., Almeida, F.M., Damaceno-Rodrigues, N.R., Caldini,
E.G., Santos, A.B.G., Rivero, D.H., Hizume, D.C., Lopes, F.D.T.Q.S., Olivo, C.R., Castro-
Faria-Neto, H.C., Martins, M.A., Saldiva, P.H.N., Dolhnikoff, M., 2012. Anti-inflammatory
effects of aerobic exercise in mice exposed to air pollution. Medicine and Science in Sports
and Exercise 44, 1227–1234.
Vieira, R.P., Andrade, V.F., Duarte, A.C.S., Santos, A.B.G., Mauad, T., Martins, M.A., Dol-
hnikoff, M., Carvalho, C.R.F., 2008. Aerobic conditioning and allergic pulmonary
inflammation in mice. II. Effects on lung vascular and parenchymal inflammation and
remodeling. American Journal of Physiology – Lung Cellular and Molecular Physiology
295, L670–L679.
Vieira, R.P., Claudino, R.C., Duarte, A.C.S., Santos, A.B.G., Perini, A., Neto, H.C.C.F., Mauad,
T., Martins, M.A., Dolhnikoff, M., Carvalho, C.R.F., 2007. Aerobic exercise decreases
chronic allergic lung inflammation and airway remodeling in mice. American Journal of
Respiratory and Critical Care Medicine 176, 871–877.
Wang, X., Chen, Y., Lv, L., Chen, J., 2009. Silencing CD36 gene expression results in the
inhibition of latent-TGF- 1 activation and suppression of silica-induced lung fibrosis in the
rat. Respiratory Research 10, 36.
Weibel, E.R., 1990. Morphometry: stereological theory and practical methods. In: Gil, J. (Ed.),
Models of Lung Disease – Microscopy and Structural Methods. Marcel Decker, New York,
pp. 199–247.
Wolff, G., Crystal, R.G., 1997. Biology of pulmonary fibrosis. In: Crystal, R.G., West, J.B.
(Eds.), The Lung: Scientific Foundations. , second edition. Lippincott/Raven Publishers,
Philadelphia, pp. 2509–2524.
Woods, J.A., Vieira, V.J., Keylock, K.T., 2009. Exercise, inflammation, and innate immunity.
Immunology and Allergy Clinics of North America 29, 381–393.
Yu, Y.-B., Liao, Y.-W., Su, K.-H., Chang, T.-M., Shyue, S.-K., Kou, Y.R., Lee, T.-S., 2012. Prior
exercise training alleviates the lung inflammation induced by sub-sequent exposure to
environmental cigarette smoke. Acta Physiologica 205, 532–540.