2. 534 R. M. Damiani et al.
Toxicology Mechanisms and Methods
These authors also found a tissue-dependent increase in
some antioxidant enzymes such as superoxide dismutase
(SOD) and catalase (CAT). Furthermore, the authors
demonstratedthat24hafterCAPsexposure,lungsrestored
their oxidant balance (Gurgueira et al. 2002).
Based in the facts that the absolute number of deaths
attributable to PM is higher for cardiac toxicity than the
pulmonar adverse effects (Dockery, 2001; Frampton,
2001), we decided to investigate whether oxidants play a
role in the cardiac PM toxicity induced by chronic treat-
ment with different doses of ROFA, including its involve-
ment in the lung inflammation.
Methods
Animals
Male Wistar rats, aged 45 days, from the Animal Facility
of Universidade Federal de Ciências da Saúde de Porto
Alegre were used. The animals were kept in plastic cages
(47 cm × 34 cm × 18 cm) under controlled humidity
(75–85%), temperature (22 ± 2°C), with a 12 h light-dark
period. They had free access to water and to a standard
laboratory diet (Supra-lab, Alisul Alimentos S/A, Brazil).
All animals used in the research were treated humanely,
with due consideration to the alleviation of distress and
discomfort. All experimental procedures were approved
by the Universidade Federal de Ciências da Saúde Ethical
Committee for Research (370/07).
Characterization of particles
Residual oil fly ash was obtained from a steel industry
placedinSaoPaulo,Brazil.Theparticleelementswereana-
lyzed by neutron activation analysis and presented the fol-
lowed composition: Br, 1.4 ± 19 μg g-1
; Ce, 16.3 ± 0.3 μg g-1
;
Co, 9.9 ± 0.25 μg g-1
; Cr, 107.7 ± 1.4 μg g-1
; Fe, 1058.9 ± 2.37
μg g-1
; La, 10.3 ± 0.1 μg g-1
; Mn, 3.8 ± 24 μg g-1
; Rb, 719.7 ±
1.0 μg g-1
; Sb, 2.2 ± 0.9 μg g-1
; As, 154.4 ± 0.8 μg g-1
; V, 35 ±
4 μg g-1
; Zn, 491.9 ± 3.1 μg g-1
. The values are expressed as
the means ± standard deviation. The mean aerodynamic
diameter was 1.2 ± 2.24 μm (Medeiros et al., 2004).
Experimental design
Rats were divided into four treatment groups: ROFA 500
µg/10 µL (n = 20), ROFA 250 µg/10 µL (n = 20), ROFA 50
µg/10 µL (n = 20) and Saline 10 µL (n = 20). The animals
wereexposedtoROFAbyintranasalinstillation,onceaday,
during 90 days. Twenty-four hours after the last instillation,
10 rats from each group were used to obtain the bron-
choalveolar lavage (BAL). The remainder were euthanized
by decapitation and lung and heart were removed and
immediately frozen (−80°C) to perform TBARS, superoxide
dismutase and catalase determinations.
Inflammation parameters
Bronchoalveolar lavage
The rats were anesthetized with sodium pentobarbital
(50 mg/Kg body weight) and their lungs were washed
through the trachea using three aliquots of 7 mL of sterile
saline. Each aliquot represents one in-and-out recovery of
fluid. The obtained fluid was centrifuged at 400 × g at 4°C.
Total cell counts were determined after trypan blue stain-
ing using a Neubauer chamber. Total protein levels, as a
measure of vascular permeability, were measured in the
first lavage from each sample using the Bradford method
(Bradford, 1976). As a marker of toxicity, lactate dehy-
drogenase (LDH) activity was analyzed by a colorimetric
method (Labtest, Brazil). These measurements were car-
ried out in a Perkin Elmer Lambda 35 spectrophotometer
(Perkin Elmer Life and Analytical Sciences, Shelton, USA).
Oxidative stress parameters
Tissue preparation
Tissue samples were homogenized in 5 volumes (lung)
and 7 volumes (heart) of 120 mM KCl and 30 mM sodium
phosphate buffer, pH 7.4, containing 0.5 mM phenylmeth-
anesulfonyl fluoride as a protease inhibitor, at 0–4°C. The
suspensionswerecentrifugedat600×gfor10minat0–4°C
to remove nuclei and cell debris. The pellets were dis-
carded and the supernatant were used as homogenates.
Determination of lipid peroxidation
Lung and heart tissue homogenates were precipitated
with 10% TCA, centrifuged and incubated with thiobar-
bituric acid (0.67%) (Sigma Chem. Co., St Louis, MO) for
60 min at 100°C. Malondialdeyde (MDA) were extracted
using butanol (1:1;v/v) and measured at 535 nm. The
concentration of MDA was expressed in nM MDA/mg of
protein. Tissue protein was quantified using the Bradford
assay (Bradford, 1976).
Catalase activity
The CAT tissue activity was performed according to Aebi
(1984) at 240 nm, during 120 s. Data are expressed in
pmol/mg protein.
Superoxide dismutase activity
The SOD tissue activity was measured as described by
Maklund (1985). This method is based on capacity of
pyrogallol to autoxidize. The pyrogallol autoxidation is
inhibited in presence of SOD, whose activity can be mea-
sured using a double-beam spectrophotometer at 420 nm.
One unit of SOD is represented as units per milligram
protein.
Statistical analysis
Data are given as mean ± standard deviation. One-Way
Analysis of Variance (ANOVA) followed by Tukey’s HSD
test was used to compare data among the different
groups. The level of significance was set at 5%. All
statistical analyses were carried out using Sigma-Stat 2.0
Software (Jandel Corporation, 1992–1995). The sample
size were based in previous studies from our laboratory
which demonstrated that this number of animals is fully
sufficient for a statistical analysis (Pereira et al. 2007;
Zanchi, 2008).
4. 536 R. M. Damiani et al.
Toxicology Mechanisms and Methods
detected, in the same group, an increase in the total cell
counts from bronchoalveolar lavage fluid. This is the first
report that links oxidative cardiopulmonary changes and
long-term exposure to ROFA.
The doses of pollutant used in our study were based in
a previous work which demonstrated that a single intra-
tracheal instillation of 500 µg PM was capable of inducing
functional cardiopulmonary changes in rats (Rivero et al.
2005). We chose the above mentioned concentration and
two lower doses (250 and 50 µg) trying to determinate a
dose-response influence. ROFA has been useful as sur-
rogate for ambient air PM in many biological studies
because of its composition, especially rich in metals.
Data suggests that ambient air and other particles emis-
sion sources follow a comparable mechanism of action
as ROFA including phosphorylation reactions, transcrip-
tion factor activation, mediators release and inflamma-
tory injury (Ghio et al., 2002).
In terms of pulmonary inflammatory parameters, we
observedanincreaseinthecountoftotalcellsinthegroup
of animals which received the highest dose of ROFA,
when compared to Saline group. In addition, we did not
detect any difference in protein and LDH concentration
in BAL when compared to all treatment groups. ROFA
exposure triggers an inflammatory process that includes
leukocyte recruitment, activation and increased alveolar
macrophages count (Becker, 2002) Alveolar macrophages
are the most important cells involved in lung inflamma-
tion response caused by particle inhalation. (Lohmann-
Matthes et al. 1994). Oberdörster et al. have demonstrated
that the alveolar macrophage recruitment and the over-
flow of plasmatic proteins in alveoli, after inhalation of
particles with less than 2.5 µm, are triggered by differ-
ent events and could occur separately. Because higher
doses resulted in an increased interstialized fraction of
particles, those authors suggested that inflammatory
Figure 2. Oxidative stress in lung of rats exposed for ninety days to three different concentrations of Residual Oil Fly Ash (ROFA) or Saline
(n = 10 per group). Data are demonstrated as mean ± standard deviation of the mean. A: malondialdehyde (MDA) concentration in lung. B:
Superoxide Dismutase (SOD) activity in lung. C: Catalase activity in lung. *Statistical difference when compared with all others treatment
groups. Tukey's HSD test, p = 0.013.
6. 538 R. M. Damiani et al.
Toxicology Mechanisms and Methods
a direct contact with the environment with respect to
both injury and treatment. The alveoli are in a unique
position in the body, where exogenous air encounters
a thin cellular layer consisting of only about two cells
beyond which immediate contact occurs with a refined
organ with particular tasks, definitely requiring the
structural integrity of the organ (Lohmann-Matthes,
1994).
On the other hand, the increased level of CAT
observed in heart tissue of rats which received 500
µg/10 µL (ROFA) was not greater enough to protect tis-
sue against ROS. This is demonstrated by the high level
of MDA in heart tissue of animals that were submitted
to high concentration of ROFA during ninety consecu-
tive days. Similarly to muscles and brain, heart has high
endogenous levels of hydrogen peroxide because of
its poor concentration of CAT in physiological status
(Scandalios, 2005). A large number of reports have
suggested that H2
O2
is an important mediator in the
vasculature inducing vascular constriction (Matoba
et al. 2000; Jones Morice 2000). Suvorava and Kojda
(2009) described that a reduction of steady-state con-
centrations of vascular hydrogen peroxide induced by
an endothelial-specific overexpression of human CAT
resulted in a marked reduction of systolic blood pressure
in mice, demonstrating the importance of maintenance
of basal levels of hydrogen peroxide in the circulatory
system. Exogenous H2
O2
also evokes airway reflexes
involving lung vagal afferents that results in changes in
autonomic tonus in heart (Ruan et al. 2003) Pulmonary
exposure to ROFA causes oxidative stress in heart first
by autonomic stimulation (Ghelfi et al. 2008, Rhoden
et al. 2005), production of ROS, release of inflammatory
mediators in lung and heart (Rhoden et al. 2004; 2005)
and by PM fractions that gains access to the systemic cir-
culation and also by a direct interaction with the heart
(Oberdörster et al. 2002). All of those observations have
a common sense: lead to an increased production of
H2
O2
causing an oxidative misbalance in heart. Several
new epidemiological studies have demonstrated that
living in locations with higher long-term average PM
concentrations increases the risk for cardiovascular
morbidity and mortality vastly exceeding the risk noted
with short-term exposure (Miller et al. 2007; Puett et al.
2008). Also, PM air pollution has been linked with endo-
thelial dysfunction, systemic oxidative and inflamma-
tory responses and the progression of atherosclerosis
(Mills et al. 2007; Sun 2005).
Conclusion
The present study demonstrated cardiopulmonary oxi-
dative changes after a chronic ROFA exposure. More spe-
cifically, the heart tissue seems to be more susceptible to
oxidative effects of long-term exposure to ROFA than the
lung. These results suggest that oxidative damage medi-
ated by H2
O2
, may be one of the mechanisms involved in
cardiac toxicity related to PM exposure.
Declaration of interest
This work was supported by Universidade Federal de
Ciências da Saúde de Porto Alegre, Brazil; Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior –
CAPES and Conselho Nacional de Desenvolvimento
Científico e Tecnológico – CNPq.
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