ISSN 1806-3713
© 2016 Sociedade Brasileira de Pneumologia e Tisiologia
http://dx.doi.org/10.1590/S1806-37562015000000342
ABSTRACT
Objective: To evaluate the effects of passive inhalation of cigarette smoke on the
respiratory system of guinea pigs. Methods: Male guinea pigs were divided into two
groups: control and passive smoking, the latter being exposed to the smoke of ten
cigarettes for 20 min in the morning, afternoon and evening (30 cigarettes/day) for ve
days. After that period, inammatory parameters were studied by quantifying mesenteric
mast cell degranulation, as well as oxidative stress, in BAL uid. In addition, we
determined MIP, MEP, and mucociliary transport (in vivo), as well as tracheal contractility
response (in vitro). Results: In comparison with the control group, the passive smoking
group showed a signicant increase in mast cell degranulation (19.75 ± 3.77% vs. 42.53
± 0.42%; p < 0.001) and in the levels of reduced glutathione (293.9 ± 19.21 vs. 723.7 ±
67.43 nM/g of tissue; p < 0.05); as well as a signicant reduction in mucociliary clearance
(p < 0.05), which caused signicant changes in pulmonary function (in MIP and MEP; p
< 0.05 for both) and airway hyperreactivity. Conclusions: Passive inhalation of cigarette
smoke caused signicant increases in mast cell degranulation and oxidative stress. This
inammatory process seems to inuence the decrease in mucociliary transport and to
cause changes in pulmonary function, leading to tracheal hyperreactivity.
Keywords: Inammation; Inhalation exposure; Tobacco smoke pollution.
Effects of passive inhalation of cigarette
smoke on structural and functional
parameters in the respiratory system of
guinea pigs
Thiago Brasileiro de Vasconcelos
1
, Fernanda Yvelize Ramos de Araújo
1
,
João Paulo Melo de Pinho
2
, Pedro Marcos Gomes Soares
1
,
Vasco Pinheiro Diógenes Bastos
3
Correspondence to:
Thiago Brasileiro de Vasconcelos. Rua Aveledo, 501. apto. 201, Torre 2, Messejana, CEP 60871-210, Fortaleza, CE, Brasil.
Tel.: 55 85 3231-5125. E-mail: thiagobvasconcelos@hotmail.com
Financial support: This study received nancial support from the Scientic Initiation Program of the
Centro Universitário Estácio do Ceará
(Estácio University Center of
Ceará).
INTRODUCTION
Smoking is a risk factor for the leading causes of death
worldwide, including cardiac and pulmonary diseases.
(1,2)
There are no safe levels of exposure to cigarettes.
Passive or active smoking is directly related to irritation,
inammation, and changes in lung function within the
rst few hours of exposure.
(3,4)
Airway contact with cigarette smoke induces changes
in the respiratory system, such as mucus hypersecre-
tion, decit in mucociliary transport, tracheobronchial
tree defects, small airway restriction accompanied by
increased closing capacity, and a trend toward changes
in the ventilation-perfusion ratio.
(5)
A passive smoker is an individual who inhales envi-
ronmental cigarette smoke. The immediate harmful
effects of such exposure include irritation of the eyes,
nose and throat, as well as increased respiratory and
heart rates.
(6)
There is also an increase in inammatory
cytokine levels within the rst few hours of inhalation,
especially in men.
(3)
The concern regarding passive inhalation of cigarette
smoke is recent, having begun in the early 1980s.
According to the Brazilian National Health Oversight
Agency, approximately two thirds of the smoke produced
by cigarettes (passive smoking, secondary exposure,
passive inhalation, or involuntary smoking) is released
into the ambient air through the lit end of the product.
(7)
In the USA, 3,000 lung cancer deaths per year are
estimated to have been caused by passive inhalation of
cigarette smoke.
(8)
In Brazil, the National Health Survey
conducted in 2013 points out that the proportion of
people aged 18 years or older who were exposed to
passive smoking was 10.7% in the home and 13.5% in
the indoor workplace. Regarding gender, that proportion
is higher among women in the home (11.7%) and men
in the workplace (16.9%).
(9)
Some studies have pointed out that the harmful effects
of passive inhalation of cigarette smoke may begin in
childhood, causing cough,
(10)
endothelial dysfunction,
(11)
and prolonged expiratory apnea.
(12)
Currently, the incidence of death and disease caused
by cigarette smoking remains high. However, there have
been few studies reporting the possible structural and
functional changes in the respiratory system in passive
smokers. This aroused our interest in developing this
study. We hope to use the study ndings to alert the
population to and raise its awareness about the prevention
of diseases caused by smoking, in order to contribute to
improving public health in general.
1. Universidade Federal do Ceará.
Fortaleza (CE) Brasil.
2. Hospital Dr. Carlos Alberto Studart
Gomes, Fortaleza (CE) Brasil.
3. Centro Universitário Estácio do Ceará,
Fortaleza (CE), Brasil.
Submitted: 16 January 2016.
Accepted: 31 July 2016.
Study carried out in the Laboratório de
Biofísica, Fisiologia e Farmacologia,
Centro Universitário Estácio do Ceará,
Fortaleza (CE) Brasil.
J Bras Pneumol. 2016;42(5):333-340
333
ORIGINAL ARTICLE
Effects of passive inhalation of cigarette smoke on structural and functional parameters in the respiratory system of guinea pigs
Therefore, the present study aimed at evaluating
structural and functional aspects of the respiratory
system of guinea pigs after passive inhalation of
cigarette smoke.
METHODS
This was an experimental longitudinal exploratory
study with quantitative analysis of results. We used male
guinea pigs (Cavia porcellus; 5-8 per group) obtained
from the animal facilities of the Estácio University Center
of Ceará, located in the city of Fortaleza, Brazil. The
mean body weight of the animals was 321.00 ± 6.72 g
at the beginning of the experiments. All animals were
handled in accordance with the Brazilian College for
Animal Experimentation’s guidelines for animal care
and welfare. The study was approved by the Animal
Research Ethics Committee of the Federal University
of Ceará (Protocol no. 052/10).
The experiments were performed in the Laboratory
of Biophysics, Physiology and Pharmacology of the
Centro Universitário Estácio do Ceará (Estácio University
Center of Ceará), in cooperation with the Department of
Physiology and Pharmacology of the Federal University
of Ceará, also located in the city of Fortaleza.
Cigarette smoke inhalation
The protocol consisted of inhalation of smoke from ten
commercial lter cigarettes (Derby Autêntico; Souza
Cruz S.A., Rio de Janeiro, Brazil), each containing 8 mg
of tar, 0.7 mg of nicotine, and 7 mg of carbon monoxide.
Those cigarettes were lit concomitantly for 20 min in
the morning, afternoon, and evening, adding up to 30
cigarettes/day, for 5 days.
(13,14)
During inhalation, the
animals were placed into acrylic boxes (whole body;
n = 3 per box) measuring 30.0 × 16.6 × 19.8 cm.
(15)
Each box had a removable upper lid for introduction of
the animals and two holes on its side walls: one for air
drainage, allowing the smoke to escape; and one for
placing the cigarettes (cigarette smoke was introduced
into the acrylic box passively). Control animals did
not inhale any type of toxic substance, and they were
placed into control group acrylic boxes (whole body; n
= 3 per box) to simulate the same conditions as those
of the cigarette smoke inhalation groups.
For the purposes of this study, two groups were
formed: control, consisting of guinea pigs that did not
inhale any toxic substance and were killed after 25
days; and passive smoking, consisting of guinea pigs
that were killed 25 days after completing the process
of passive inhalation of cigarette smoke (Figure 1).
Analysis of the inammatory process on the
basis of mast cell degranulation counts
After anesthesia (urethane, 1.2g/kg, i.p.), the
mesentery was removed and a wire was carefully
inserted into the small intestine, which was then given
a circular shape. Subsequently, the small intestine was
transferred to a Petri dish containing a toluidine blue
solution (toluidine blue dissolved in 70% alcohol at a
concentration of 0.1g/L and diluted with 1% NaCl at a
ratio of 1:10, resulting in a concentration of 0.01 g/L)
for staining at room temperature. The preparation was
then washed with Tyrode’s solution (136 mM of NaCl;
5 mM of KCI; 0.98 mM of MgCl
2
; 0.36 mM of NaH
2
PO
4
;
11.9 mM of NaHCO
3
; 2 mM of CaCl
2
; and 5.5 mM of
glucose) and left to dry on a glass slide (for 10 min)
at room temperature. After drying and excess tissue
removal, 100 cells were counted in different elds by
optical microscopy (magnication, ×200), and, on
that basis, the percentage of mast cell degranulation
was determined.
Assessment of oxidative stress
Oxidative stress was measured indirectly by quanti-
cation of nitrite and reduced glutathione (GSH) in BAL
uid. To that end, the lungs were initially lled with
5 mL of saline solution at 37ºC, which was instilled
into the tracheal tube using a syringe. After a 3-min
period, the instilled uid was slowly recovered by
aspiration. That procedure was repeated with another
5 mL of saline solution. The material was stored in a
freezer (−70ºC).
(16)
Determination of nitrite
Assays were prepared with 100 µL of Griess rea-
gent—0.1% N-(1-naphthyl)ethylenediamine in water;
and 1% sulfanilamide in 5% phosphoric acid—and
100 µL of the (centrifuged) supernatant of the 20%
BAL uid homogenate from the guinea pigs or 100
µl of various concentrations of the standards. Blanks
were prepared with 100 µL of Griess reagent and 100
µL of saline solution (0.9% NaCl). Absorbance was
measured at 560 nm using a plate reader.
(17)
Results
are expressed as µM/g of tissue.
M A E
25 days after
inhalation
Euthanasia
Inhalation
(30 cigarettes/day)
5 days
Figure 1. Model of passive inhalation of cigarette smoke. In the center, a photograph of the guinea pigs during the
inhalation process. M: morning; A: afternoon; and E: evening.
334
J Bras Pneumol. 2016;42(5):333-340
Vasconcelos TB, Araújo FYR, Pinho JPM, Soares PMG, Bastos VPD
Determination of GSH
The reagent was prepared using 0.02 M of EDTA
and 50% trichloroacetic acid. After this process,
centrifugation was performed (5,000 rpm for 15 min
at 4°C). Subsequently, the supernatant was collected
and homogenized. The samples were mixed with 0.4
M Tris-HCl buffer (pH = 8) and 0.01 M of 5.5-dithio-
bis-2-nitrobenzoic acid. The material was kept cooled
throughout the assay. The GSH activity was measured
at 412 nm using a plate reader. Results are expressed
as ng/g of tissue.
Measurement of mucociliary transport
Initially, the guinea pigs were anesthetized with
urethane (1.2 g/kg, i.p.) and xed horizontally in
the supine position. Subsequently, 2 µL of a 0.3 g/
mL gelatin solution containing 0.5% Evans blue dye
was injected into their tracheas with a microsyringe.
Two minutes later, the tracheas were opened, and
mucociliary transport was measured, from the injection
point, by using a caliper.
(15,18)
Measurement of pulmonary pressures
The animals were anesthetized (urethane, 1.2 g/kg,
i.p.). Subsequently, the trachea was cannulated and
connected in a closed system to a pressure transducer,
at the end of inhalation, not allowing airow leaks.
The connection was maintained until the animal had
intercostal retraction (approximately 20 seconds).
Three MIP and MEP measurements were taken in each
animal, and the time interval between measurements
depended on normalization of the respiratory pattern
and rhythm.
(19)
The ventilatory parameters were
recorded by PowerLab/8sp (ADInstruments Pty Ltd.,
Bella Vista, Australia) data acquisition system.
Isometric recordings of the tracheal rings
After anesthesia (urethane, 1.2 g/kg, i.p.) and
subsequent euthanasia by exsanguination (through
the left carotid artery), a ventral midline incision was
made and the trachea was rapidly excised in one single
segment of approximately 10-12 mm.
(19)
The excised
segment was then rapidly transported to a Petri dish
containing Krebs-Henseleit solution (in nmol/L: 118
NaCl; 4.7 KCI; 2.5 CaCl
2
; 1.2 MgSO
4
; 25.0 NaHCO
3
;
1.2 KH
2
PO
4
; and 10 glucose). After removal of adjacent
tissues, the trachea was cut into four ring-shaped
segments, which were transferred to individual organ
bath chambers containing 5 mL of Krebs-Henseleit
solution that was continuously aerated with carbogen
((95% O
2
and 5% CO
2
); the pH of the solution was
manually adjusted to 7.4 at a temperature of 37°C,
which was kept constant through water circulation from
a pumped water bath. The lumen of the tracheal rings
was crossed with two pieces made of thin stainless
steel, which were tied to two points: one xed in the
chamber; and one connected to a force transducer
(ML870B60/C-V; ADInstruments) suitable for recording
isometric contractions. The signals generated by the
force transducer were recorded by a computerized data
acquisition system (PowerLab™ 8/30; ADInstruments).
The tension applied to each tracheal segment was
set at 1 gf. The equilibration period was 1 h, and the
incubation liquid was changed every 15 min.
(20)
After
the stabilization period, concentration-effect curves
were constructed for potassium (10-120 mM) and
for carbachol (0.001-10 µM), with a 5 min-interval at
each concentration. After completion of the protocol,
the tracheas were removed from the incubation liquid
and left to dry at room temperature (approximately
25°C) for 2 h. Subsequently, each tissue was weighed.
Data analysis
Results are expressed as mean ± standard error of
the mean, and the number of experimental observations
is noted in parentheses (n). For group comparison,
we used one-way or two-way ANOVA, as well as the
Student’s t-test, Holm-Sidak’s test, the Kruskal-Wallis
test, and the Mann-Whitney test, according to the
normality test. For all tests, values of p < 0.05 were
considered statistically signicant.
RESULTS
Mast cell degranulation
Mesenteric mast cell degranulation in the guinea pigs
was 19.75 ± 3.77% (n = 5). However, in the guinea
pigs that inhaled cigarette smoke over a short period
of time (25 days), mast cell degranulation was 42.53
± 0.42% (n = 5), being signicantly higher than that
observed in the control group (p < 0.001; Student’s
t-test; Figure 2).
Nitrite and GSH levels
Determination of nitrite (control group = 0.073
± 0.007 µM/g of tissue vs. passive smoking group
= 0.065 ± 0.004 µM/g of tissue) and GSH (control
group = 293.9 ± 19.21 nM/g of tissue vs. passive
smoking group = 723.7 ± 67.43 nM/g of tissue) in the
BAL uid from the guinea pigs revealed a signicant
difference (p < 0.05; Student’s t-test) only in the GSH
levels (Figure 3).
Mucociliary transport and pulmonary
pressures
Analysis of mucociliary transport revealed that, in the
control group, the distance traveled was 0.65 ± 0.08
cm; however, that distance was signicantly reduced
(p < 0.05; ANOVA followed by the Kruskal-Wallis
post-test) to 0.30 ± 0.03 cm in the passive smoking
group (n = 6; Figure 4A).
The mean MIP was −9.93 ± 0.94 cmH
2
O in the
control group (n = 5) and −40.44 ± 9.26 cmH
2
O in the
passive smoking group (n = 6); therefore, the latter
group showed a signicant decrease (p < 0.05; ANOVA
followed by Holm-Sidak’s post-test) as compared with
the former (Figure 4B).
The mean MEP was 0.58 ± 0.05 cmH
2
O in the
control group (n = 6) and 3.60 ± 0.60 cmH
2
O in the
335
J Bras Pneumol. 2016;42(5):333-340
Effects of passive inhalation of cigarette smoke on structural and functional parameters in the respiratory system of guinea pigs
passive smoking group (n = 6); therefore, the latter
group showed a signicant increase (p < 0.05; ANOVA
followed by Holm-Sidak’s post-test) as compared with
the former (Figure 4C).
Tracheal contractility
Adding increasing cumulative concentrations of K
+
(10-120 mM) in the control group (n = 6) produced a
contractile response with an amplitude of 0.05 ± 0.00
gf/mg of tissue. Comparatively, the passive smoking
group (n = 8) showed a signicantly increased (p
< 0.05; two-way ANOVA followed by Holm-Sidak’s
post-test) contractile response, with a mean value of
0.10 ± 0.01 gf/mg of tissue (Figure 5A).
Concentration-effect curves were also constructed
to determine the contractile response to carbachol
(0.001-10 µM): the animals in the control group (n
= 6) showed a mean value of 0.09 ± 0.02 gf/mg
of tissue; comparatively, the animals in the passive
smoking group (n = 8) showed a signicantly increased
(p < 0.05; two-way ANOVA followed by Holm-Sidak’s
post-test) contractile response (0.23 ± 0.03 gf/mg of
tissue; Figure 5B).
DISCUSSION
The ndings of the present study reveal that passive
inhalation of cigarette smoke over a short period of
time produces structural and functional changes in the
respiratory system. Such effects were proven by evidence
of a decit in lung tissue; an increase in inammatory
cells, leading to mucus accumulation; and pulmonary
function impairment caused by tracheal hyperreactivity.
Although some inhalation models
(13,14,21,22)
have reported
the damages caused by constant and prolonged use
of cigarettes, the relevance of the present study lies
in reporting the changes caused by passive inhalation
of cigarette smoke over a short period of time (5
days), with a change in the amount and duration of
exposure to cigarette smoke as compared with previous
studies.
(23-25)
In their study, Hernandez et al.
(23)
also
opted to analyze the effects of passive inhalation of
cigarette smoke over a short period of time. To that end,
the animals were submitted to a protocol consisting of
three 10-min exposures to smoke from one cigarette,
separated by 30-min intervals, each day, for 4 days, and
it was found that exposure of guinea pigs to cigarette
smoke produced airway hyperreactivity to histamine
and recruitment of inammatory cells.
It is of note that, in recent years, the understanding
of the pathophysiological mechanisms related to passive
inhalation of cigarette smoke has enabled increasingly
specic approaches, with the use of concise and
reproducible methods to investigate the contractile and
inammatory repercussions of this inhalation process.
The animals that inhaled cigarette smoke exhibited
destruction of lung tissue architecture, accompanied by
migration of proinammatory cells (data not shown),
which corroborates other experimental models of
cigarette smoke inhalation.
(23-27)
Such changes were
pointed out by the study of Banerjee et al.,
(28)
in
which the guinea pigs that inhaled cigarette smoke
exhibited inammation and apoptosis, which leads
to destruction of alveolar membranes and septal
cells, causing pulmonary airspace enlargement, and
such enlargement may have inuenced the changes
in pulmonary pressures in the present study. Page
PSControl
% of mast cell degranulation
*
50
40
30
20
10
0
Control PS
Control PS
*
1000
800
600
400
200
0
GSH
(ng/g of tissue)
0.10
0.08
0.06
0.04
0.02
0.00
A B
Nitrite
(µM/g of tissue)
Figure 2. Comparison of the proportion of mast cell
degranulation between the control group and the passive
smoking (PS) group. Data expressed as mean ± standard
error. *p < 0.001 in relation to the control group.
Figure 3. Comparison between the control group and the passive smoking (PS) group regarding tissue levels of nitrite
(in A) and reduced glutathione (GSH; in B). Data expressed as mean ± standard error. *p < 0.05; Student’s t-test.
336
J Bras Pneumol. 2016;42(5):333-340
Vasconcelos TB, Araújo FYR, Pinho JPM, Soares PMG, Bastos VPD
et al.
(29)
add that, after exposure of airway sensory
nerves to cigarette smoke, there is damage caused
by cytotoxic mediators to the ciliary epithelium layer,
increased mucus secretion, hyperresponsiveness, and
vasodilatation, ndings that are in agreement with
those of the present study.
In this sense, Valença & Porto
(21)
highlighted the
participation of macrophages in the destruction of
extracellular lung matrix in animals exposed to cigarette
smoke over different periods of time; in contrast, in
animals exposed to room air (control group), the
alveoli were preserved and there were few alveolar
macrophages. The study by Zhong et al.,
(30)
in which
guinea pigs were exposed to inhalation of smoke
from 10 cigarettes for 20 min, twice a day, for 14
days, reported that the animals exposed to cigarette
smoke showed increased proinammatory cytokine
levels (TNF-α and IL-8), as well as an increase in
inammatory cells and tracheal thickness.
Another important harmful effect observed in the
present study was the decrease in mucociliary transport
in the guinea pigs submitted to passive inhalation of
cigarette smoke, a nding conrmed by Furtado
(5)
when he states that smoking leads to a decrease
in mucociliary transport because of the many toxic
substances found in the cigarette that exhibit ciliostatic
and ciliotoxic properties. Vasconcelos et al.
(15)
also
observed a decit in mucociliary transport in guinea
pigs that mimicked asthma symptoms, corroborating
the present study when they state that inammatory
diseases cause changes in ciliary function and in the
amount of mucus secreted, which directly inuences
the protection of the respiratory system.
Therefore, to protect itself, the organism has an
intracellular defense system that can act in two ways:
either by reducing the toxic substances in the organism
before they cause injury, through the activity of antiox-
idants, such as GSH, superoxide dismutase, catalase,
and vitamin E; or by inhibiting the injury with ascorbic
acid, glutathione reductase, glutathione peroxidase,
etc.
(31)
Indirectly, measurement of antioxidant enzyme
activity and determination of the concentration of
tripeptides are the most widely used methods for the
assessment of oxidative stress.
(32)
That same group
of authors
(32)
points out the important antioxidant
effect of GSH on the respiratory tract, an effect that
can reduce oxidative stress.
The levels of GSH, one of the major antioxidant
enzymes in the respiratory tract, were signicantly higher
in the passive smoking group than in the control group,
demonstrating an adaptive response of the organism
to an injury, but there were no changes in the nitrite
Figure 4. Comparison between the control group and the passive smoking (PS) group regarding mucociliary transport
(in A), MIP (in B), and MEP (in C).Data presented as mean ± standard error.
*
p < 0.05 for all.
*
*
0
-10
-20
-30
-40
-50
cmH
2
O
cmH
2
O
Distance traveled (cm)
*
1.0
0.8
0.6
0.4
0.2
0.0
4
3
2
1
0
A
B
C
Control
Control
Control
PS
PS
PS
337
J Bras Pneumol. 2016;42(5):333-340