J Appl Physiol 117: 492–499, 2014.
First published July 10, 2014; doi:10.1152/japplphysiol.00156.2014.
Acute cardiopulmonary effects induced by the inhalation of concentrated
ambient particles during seasonal variation in the city of São Paulo
Jôse Mára de Brito,1 Mariângela Macchione,1 Kelly Yoshizaki,1 Alessandra Choqueta Toledo-Arruda,2
Beatriz Mangueira Saraiva-Romanholo,2 Maria de Fátima Andrade,3 Thaís Mauad,1
Dolores Helena Rodriguez Ferreira Rivero,1 and Paulo Hilário Nascimento Saldiva1
1
Department of Pathology, Laboratory of Experimental Air Pollution, School of Medicine, University of São Paulo, São
Paulo, Brazil; 2Department of Medicine, Laboratory of Experimental Therapeutics, School of Medicine, University of São
Paulo, São Paulo, Brazil and City of São Paulo University, São Paulo, Brazil; and 3Institute of Astronomy, Geophysics, and
Atmospheric Sciences, University of São Paulo, São Paulo, Brazil
Submitted 18 February 2014; accepted in final form 7 July 2014
particulate matter; climatic change; toxicity; inflammation; responsiveness
THE METROPOLITAN AREA OF SÃO PAULO is one of the largest
megacities in the world, with more than 19 million inhabitants
in an area of 1,523 km2 at an altitude of 824 m, with an
increasing mobile fleet of 7 million vehicles (18). Automobile
sources are, therefore, major contributors to air pollution in São
Paulo (3, 25). Significant detrimental effects have been re-
Address for reprint requests and other correspondence: J. M. de Brito,
Faculdade de Medicina USP, Departamento de Patologia, Av. Dr. Arnaldo
455, 1 andar, sala 1220, Cerqueira César, São Paulo/SP, CEP, 01246-903
Brazil (e-mail: [email protected]).
492
ported in relation to ambient levels of air pollution in São
Paulo, predominantly associated with particles (11, 21, 22).
The tropical and extra-tropical climate defines singular characteristics in São Paulo city. The climate variability could be
defined in two broad categories: 1) cold and dry (mean:
temperature, 18°C; relative humidity, 80%; precipitation, 70
mm), which occurs between April and September and is
characterized by high concentrations of primary pollutants due
to the higher occurrence of thermal inversions, low humidity,
and reduced wind; and 2) warm and humid (mean: temperature, 21°C; relative humidity, 81%; precipitation, 218 mm),
which occurs between October and March and is characterized
by relatively higher concentrations of secondary pollutants,
especially ozone, due to high atmospheric temperatures (10).
Air pollution is characterized as a composite mixture with
variable compositions among different locations. The general
understanding of air pollution composition is complicated by
the direct effects that regional characteristics, climatic variability, and fuel types have on particle composition. Particles are
grossly classified as primary and secondary pollutants. The
primary pollutants are directly emitted by several sources, such
as industry, forest fires, and vehicles. The secondary pollutants
are formed in the atmosphere as a consequence of photochemical processes. The relative toxicity of primary and secondary
particles is not fully understood. In several studies, both primary and secondary particles have been shown to have adverse
health effects on biological systems (12, 13, 31), while other
studies have shown that secondary pollutants of particles appear to be a main source of these effects (26, 33).
Experimental models using concentrated ambient particles
(CAPs) can provide useful information to verify the seasonal
variation of particle toxicity. Recently, a study conducted in
California showed that particulate matter ⱕ2.5 ␮m (PM2.5)
generated during the winter season was associated with relatively higher systemic and pro-coagulant effects in mice (35).
The location of São Paulo, given the complexities of the
climate variation there, presents an opportunity to investigate
the influence of weather conditions on particle toxicity because
only a small number of studies have measured the effects of
pollutants at specific locations (19, 35). Therefore, the objectives of the present study were 1) to determine whether acute
exposure to low levels of particles promotes measurable acute
systemic and cardiopulmonary effects; and 2) to assess if the
magnitude of the observed alterations are influenced by season.
For these purposes, controlled exposures to CAPs were conducted in mice.
8750-7587/14 Copyright © 2014 the American Physiological Society
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de Brito JM, Macchione M, Yoshizaki K, Toledo-Arruda AC,
Saraiva-Romanholo BM, Andrade MF, Mauad T, Rivero DHRF,
Saldiva PHN. Acute cardiopulmonary effects induced by the inhalation of concentrated ambient particles during seasonal variation in the
city of São Paulo. J Appl Physiol 117: 492– 499, 2014. First published
July 10, 2014; doi:10.1152/japplphysiol.00156.2014.—Ambient particles may undergo modifications to their chemical composition as a
consequence of climatic variability. The determination of whether
these changes modify the toxicity of the particles is important for the
understanding of the health effects associated with particle exposure.
The objectives were to determine whether low levels of particles
promote cardiopulmonary effects, and to assess if the observed alterations are influenced by season. Mice were exposed to 200 ␮g/m3
concentrated ambient particles (CAPs) and filtered air (FA) in cold/
dry and warm/humid periods. Lung hyperresponsiveness, heart rate,
heart rate variability, and blood pressure were evaluated 30 min after
each exposure. After 24 h, blood and tissue samples were collected.
During both periods (warm/humid and cold/dry), CAPs induced
alterations in red blood cells and lung inflammation. During the
cold/dry period, CAPs reduced the mean corpuscular volume levels
and increased erythrocytes, hemoglobin, mean corpuscular hemoglobin concentration, and red cell distribution width coefficient variation
levels compared with the FA group. Similarly, CAPs during the
warm/humid period decreased mean corpuscular volume levels and
increased erythrocytes, hemoglobin, hematocrit, and red cell distribution width coefficient variation levels compared with the FA group.
CAPs during the cold/dry period increased the influx of neutrophils in
the alveolar parenchyma. Short-term exposure to low concentrations
of CAPs elicited modest but significant pulmonary inflammation and,
to a lesser extent, changes in blood parameters. In addition, our data
support the concept that changes in climate conditions slightly modify
particle toxicity because equivalent doses of CAPs in the cold/dry
period produced a more exacerbated response.
Toxicity by Concentrated Ambient Particles
MATERIALS AND METHODS
493
de Brito JM et al.
to minimize suffering. Four experimental groups were defined as
follows: cold/dry exposed to CAPs (n ⫽ 46) and a corresponding
control group exposed simultaneously to filtered air (FA) (n ⫽ 44),
and warm/humid CAP-exposed (n ⫽46) and control groups (n ⫽ 44).
All animals were exposed for 1 h at the same time each day (11 AM
to 12 PM). During the intervals between the exposure periods, the
animals were maintained in ventilated racks with FA (high efficiency
particulate air), a controlled temperature (22–25°C), and a light-dark
cycle of 12 h.
CAPs exposure protocol. Male Balb/c mice were exposed to
concentrated ambient PM2.5 from the city of São Paulo using a
Harvard ambient particle concentrator located within the main campus
of the School of Medicine of the University of São Paulo. In this
system, a jet of particle-laden air is injected, and a series of impactors
are used to classify particles according to their aerodynamic size. The
PM2.5 was accelerated through a nozzle and concentrated by inertial
forces, while aspirating peripheral airflow and increasing the concentration of particles in the size range of 0.1–2.5 ␮m, while maintaining
their physicochemical characteristics, as previously described (34).
Animals were exposed to the same amount of particles. The
concentration of CAPs was monitored in real time using a particulate
monitor (DataRam4, Thermo Fisher Scientific) to precisely adjust
concentration during the exposure duration. To achieve an accumulated dose (concentration vs. time product) of 200 ␮g/m3, the time of
the exposure was controlled to ensure the same concentration for all
groups. Groups of seven to eight animals for CAPs and seven to eight
animals for controls (FA) were frequently exposed for less than 1 h
inside of the inhalation chambers. For each season (cold/dry or
warm/humid) and for each group, 6 exposures were conducted on
different days to achieve the final number of 46 CAP-exposed mice
and 44 controls for each season (Table 1). Exposure protocols were
conducted on the following days: 1) cold: July 7, July 19, and August
11, 2010; May 10, May 25, and June 7, 2011; and 2) warm: October
20, October 26, and November 3, 2011; February 15, March 1, and
March 8, 2012. Meteorological data corresponding to the period
between 2007 and 2012, as well as to the exposure dates, are
presented in Table 2.
Heart rate, heart rate variability, and blood pressure. Thirty
minutes after the completion of the CAPs or FA exposure, the animals
were anesthetized with a mixture of isoflurane (inhalatory anesthesia)
and O2. A rodent-specific cuff was attached to the base of the tails of
the mice and was coupled to a Powerlab system (digital system for
acquisition data, ADInstruments) for 3 min of recording. The following parameters were obtained as described previously by Brito et al.
(4): heart rate (HR), HR variability (HRV) for the time domain
Table 1. Outline of the experimental protocol, number of animals per group, and average exposure concentrations
Animals/Group
Date
Period
FA
CAPs
Mean CAPs Mass
Concentration, ␮g/m3
Exposure, min
CAPs Accumulate
(Concentration vs. Time)
Temperature, °C
Humidity, %
7/7/10
7/19/10
8/11/10
5/10/11
5/25/11
6/7/11
10/20/11
10/26/11
11/3/11
2/15/12
3/1/13
3/8/12
Mean
Mean
1
1
1
1
1
1
2
2
2
2
2
2
1
2
7
7
8
7
7
8
7
7
8
7
7
8
44
44
8
8
7
8
8
7
8
8
7
8
8
7
46
46
200.19
504.16
488.36
197.37
506.84
452.90
427.17
296.95
408.04
214.46
387.28
347.28
391.63
346.86
60
24
25
60
25
27
28
40
30
58
32
35
36
37
202.19
201.66
203.48
197.37
211.18
203.81
199.34
197.96
204.02
207.31
206.55
202.58
203.28
202.96
24
24
20
29
27
27
26
25
28
25
31
29
25.16
27.33
54
66
66
52
45
45
45
63
38
64
43
47
54.66
50.00
Period 1, cold/dry; period 2, warm/humid; FA, filtered air; CAPs, concentrated ambient particles.
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Ethics statement. This study was approved by the Ethics Committee of the School of Medicine of the University of São Paulo (permit
no.: 149/10).
Sampling and PM2.5 elementary characterization. Air pollution in
São Paulo has been characterized as mainly vehicular in origin (22).
To characterize PM2.5 in the two periods (cold/dry and warm/humid),
we used data from previously collected PM2.5 samples at the School
of Medicine of the University of São Paulo between June 2007 and
2008. The climatic conditions were similar to those of the study
period. The PM2.5 samples generated during the cold/dry season had
a higher PM2.5, black carbon, and elemental silicon, potassium,
calcium, titanium, vanadium (V), iron (Fe), nickel, zinc, bromine (Br)
(P ⫽ 0.001) content, and lead (Pb) (P ⫽ 0.031) level compared with
the samples generated during the warm/humid season.
The identification of absolute principal component analysis was
based on eigenvalues ⬎ 1 before rotation analysis. This method was
used to estimate the contribution of each factor to the mass concentration variation. The absolute principal component analysis identified
four factors: factor 1 was related to fossil fuel combustion and
secondary pollutants, which have high levels of phosphorus, sulfur, V,
sulfate, and ammonium, explaining their contribution of 12% of the
PM2.5 mass (3.4 ␮g/m3); factor 2 was associated with the burning of
heavy-duty diesel and biomass due to the high loading for PM2.5,
black carbon, Br, and potassium, accounting for 48% of the PM2.5
mass (13.5 ␮g/m3); factor 3 was related to crustal emissions (soil and
construction) due to the high loadings for silicon, calcium, titanium,
and Fe and constituted 11% of the PM2.5 mass (3.2 ␮g/m3); and factor
4 was associated with light-duty vehicle emissions due to the presence
of zinc, Pb, and other metals, accounting for 5% of the PM2.5 mass
(1.4 ␮g/m3). A regression analysis of the four “absolute factor scores”
showed that 21% of the PM2.5 mass in São Paulo was not explained
by any factor.
The polycyclic aromatic hydrocarbon (PAH) concentrations have
been characterized by Vasconcellos et al. (38). Total PAH concentrations were 10.6 ng/m3 in the warm/humid season and 25.9 ng/m3 in
the cold/dry season.
Experimental groups. Adult male Balb/C mice (6 – 8 wk of age)
weighing ⬃25 g were obtained from the animal house at the School
of Medicine of the University of São Paulo. The animals were
maintained at 22–23°C with controlled humidity and a 12:12-h lightdark cycle. Food and water were available ad libitum. All animals
received care in compliance with the “Principles of Laboratory Animal Care” published by the National Institutes of Health. All surgery
procedures were performed under anesthesia, and efforts were made
•
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Toxicity by Concentrated Ambient Particles
•
de Brito JM et al.
Table 2. Mean values (temperature and relative humidity) and sum (precipitation index) of the meteorological parameters
obtained from 2007 to 2012 in São Paulo, Brazil
Parameters
Temperature, °C
Relative humidity, %
Precipitation, mm
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
2007
2008
2009
2010
2011
2012
2007
2008
2009
2010
2011
2012
2007
2008
2009
2010
2011
2012
22
21
22
23
23
21
83
82
82
84
83
82
126
263
234
653
466
333
23
22
23
24
23
23
80
82
82
77
81
79
274
219
219
394
328
255
23
21
22
22
21
22
77
80
82
81
87
79
205
75
247
148
91
150
21
20
20
20
21
20
82
82
83
82
82
84
78
122
52
130
102
177
17
17
18
18
17
17
81
78
81
82
83
82
54
57
56
81
24
49
17
16
15
16
15
17
76
82
81
79
81
86
26
57
42
13
65
191
15
16
16
17
16
16
79
74
87
80
79
78
161
0
200
90
12
88
17
18
17
16
17
18
75
78
77
75
77
74
1
89
51
4
65
2
19
17
19
19
17
19
74
79
84
77
77
75
4
42
203
97
4
22
20
20
19
18
19
21
79
82
86
83
80
77
96
146
138
77
176
92
20
20
23
20
19
20
80
82
80
80
79
81
123
113
234
159
111
163
22
20
22
22
21
24
80
80
84
84
79
81
198
256
209
281
236
345
Source: Institute of Astronomy, Geophysics and Atmospheric Science, University of São Paulo. Values in bold are the exposure months of the mice in the
CAP groups.
(standard deviation of normal beats and root mean square of successive differences in the heart beat interval), frequency domain (low
frequency, high frequency, and low-frequency-to-high-frequency ratio), and blood pressure (BP).
Lung hyperresponsiveness. Lung hyperresponsiveness of conscious
mice was measured during a dose-response curve to aerosolized
methacholine (MCh) through a whole body plethysmography system
(BUXCO, Winchester, UK) after HR and BP acquisition. Briefly,
each mouse was placed in a chamber, and continuous measurements
of the box pressure-time wave were made using a transducer that was
connected to a computer data-acquisition system. The main indicator
of air bronchoconstriction was enhanced pause (Penh), which shows
correlation with airway resistance, as measured according to standard
evaluation methods in Balb/c mice (1). After the measurement of
baseline Penh, either aerosolized saline or MCh in increasing concentrations (6.25, 12.5, 25, and 50 mg/ml) was nebulized through an inlet
of the chamber for 3 min. Penh values for each dose were collected for
3 min and then averaged. The maximum Penh and area under the
curve were calculated.
Blood analysis. After 24 h, the animals were anesthetized with a
mixture of ketamine/xylazine (50 mg/kg by intraperitoneal injection).
Blood samples were collected by heart puncture and then stored in
ethylenediaminetetraacetic acid K3 tubes for complete blood and
reticulocyte counts. The parameters evaluated were as follows: erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), MCH concentration (MCHC),
coefficient variation of the red cell distribution width (RDW-CV),
standard deviation of the red cell distribution width (RDW-SD),
platelets, reticulocytes, leukocytes, polymorphonuclear neutrophils,
lymphocytes, and monocytes. Complete blood counts of red blood
cells (RBCs), platelets, and white blood cells were performed using a
hematological analyzer (Bayer).
Lung histology, immunohistochemistry, and morphometric analysis.
The lungs were removed and fixed by intratracheal instillation of a
10% buffered formalin solution for 24 h and then embedded in
paraffin. Five-micrometer-thick sections of lung tissues were deparaffinized, rehydrated, and stained with hematoxylin and eosin for the
quantification of neutrophil density.
For immunohistochemical staining analysis, the lung sections were
deparaffinized, rehydrated, and blocked for endogenous peroxidase followed by antigen retrieval performed with high-temperature citrate buffer
(pH ⫽ 6.0). The following primary antibodies were used: anti-endothelin
A receptor (anti-ET-Ar) (1:250, Santa Cruz Biotechnology), anti-endothelin B receptor (anti-ET-Br) (1:250, Santa Cruz Biotechnology), and
anti-vascular cell adhesion molecule (VCAM)-1A (1:200, Santa Cruz
Biotechnology). Anti-goat, -rat, or -rabbit antibodies (Vectastain Abc
Kit, Vector Laboratories) were used as secondary antibodies. Sections
were counterstained with hematoxylin. Bovine serum albumin was
used in substitution for primary antibodies for controls.
Table 3. Median (IQR: 25–75) values of the HR variability, HR, and BP for groups exposed to FA and CAPs during cold/
dry and warm/humid periods
FA
Cold/dry (n ⫽ 34)
2
SDNN, ms
RMSSD, ms2
LF, ms
HF, ms
LF/HF
HR, beats/min
BP, mmHg
Warm/humid (n ⫽ 43)
CAPs
All (n ⫽ 77)
Cold/dry (n ⫽ 34)
Warm/humid (n ⫽ 44)
All (n ⫽ 78)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
15.79
20.16
14.44
10.65
1.67
400.75
89.05
12.88–20.77
16.50–28.09
8.76–30.29
5.07–20.73
1.39–2.18
366.86–427.45
69.67–129.83
14.40
18.58
7.66
4.80
1.40
401.27
91.25
11.88–19.53
14.45–24.77
4.04–14.85
2.72–11.96
1.10–2.43
381.36–448.98
74.87–117.13
15.09
19.38
11.14
7.91
1.50
400.81
91.25
12.45–20.17
14.90–26.60
4.58–24.19
3.81–13.71
1.14–2.21
379.77–436.54
72.37–120.55
17.66
21.49
10.41
7.61
1.29
404.14
94.13
14.40–20.87
16.87–28.36
6.53–17.16
5.33–12.98
1.02–2.36
362.90–433.55
58.74–134.44
14.36
17.43
10.55
6.94
1.46
409.34
85.03
11.86–18.06
14.76–22.99
4.18–15.46
3.07–13.46
1.00–1.93
396.79–431.08
76.53–121.01
15.43
18.55
10.41
7.29
1.35
409.16
86.88
12.55–20.07
15.39–25.47
5.81–15.95
4.80–13.43
1.01–2.01
384.43–431.94
71.33–125.02
n, No. of animals. IQR, interquartile range; SDNN, standard deviation of normal beats; RMSSD, root mean square of successive differences in the heartbeat
interval; LF, low frequency; HF, high frequency; LF/HF, ratio of low to high frequency; HR, heart rate; BP, blood pressure. The groups were subdivided and
were exposed to the following conditions: cold/dry-FA, warm/humid-FA, all-FA, cold/dry-CAPs, warm/humid-CAPs, and all-CAPs.
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Fig. 1. Mean (⫾SE) values of enhanced pause (Penh) obtained by a dose-response curve to methacholine. A: cold. B: warm. C: all. The open circles represent
filtered air (FA), and the solid circles represent concentrated ambient particles (CAPs). In A, CAPs ⫽ FA: *P ⫽ 0.012, #P ⫽ 0.038.
RESULTS
HR, HRV, and BP. There were no effects on HR, BP, and all
parameters of HRV (Table 3).
Lung hyperresponsiveness. The CAPs produced during the
cold/dry period elevated the Penh in the baseline dose group
(P ⫽ 0.012) as well as in the MCh 6.25 mg/ml dose group (P ⫽
0.038) compared with the FA group (Fig. 1). There were no
effects observed for the available parameters as evaluated by
area under the curve and maximum Penh (Fig. 2).
Blood. Independent of the exposure period, CAPs induced a
reduction of MCV (P ⫽ 0.001) and an increase in hematocrit
(P ⫽ 0.019), MCHC (P ⫽ 0.004), erythrocytes, hemoglobin,
and RDW-CV (P ⫽ 0.001) compared with the FA group
(Table 4).
Exposure to CAPs generated during the cold/dry period
promoted a reduction in MCV (P ⫽ 0.010) and an increase in
erythrocytes (P ⫽ 0.018), hemoglobin (P ⫽ 0.022), MCHC
(P ⫽ 0.016), and RDW-CV (P ⫽ 0.021) compared with that of
the FA group. Similarly, exposure to CAPs generated during
the warm/humid period induced a decrease in MCV (P ⫽
0.001) and an increase in the number of erythrocytes, hemoglobin (P ⫽ 0.001), hematocrit (P ⫽ 0.024), and RDW-CV
(P ⫽ 0.006) compared with the FA group (Table 4).
The increase in RDW-CV (P ⫽ 0.015) was higher for CAPs
generated during the cold/dry period than those generated
during the warm/humid period, whereas hemoglobin levels
(P ⫽ 0.034) were increased after exposure to CAPs generated
in warm/humid periods (Table 4).
Fig. 2. Mean (⫾SE) values of maximum Penh (Penhmax;
A) and area under the curve (AUC; B).
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An eyepiece with a coherent system of 50 lines and 100 points with
a known area attached to the ocular lens of the microscope was used
for conventional morphometry. The density of neutrophils and macrophages was assessed by point counting. Using a 100-point grid with
a known area (67.500 ␮m2 at ⫻1,000 magnification) attached to the
microscope ocular lens, we counted the number of points that contacted alveolar tissue in each field. Alveolar tissue area in each field
was calculated as the number of points that contacted alveolar tissue
as a proportion of the total grid area. Data are expressed as neutrophil
cell density or macrophage cell density per alveolar area (cells/mm2)
(5, 36). Counting was performed in 20 fields of alveolar parenchyma
for each animal at a magnification of ⫻1,000.
Image analysis. Positively immunostained areas for the ET-Ar,
ET-Br, and VCAM 1 in peribronchiolar vessels were determined
using image analysis. Analyses were performed using Image-Pro Plus
4.1 software for Windows (Media Cybernetics, Silver Spring, MD)
using a light microscope-coupled digital camera (Olympus Q-Color 5,
Tokyo, Japan) connected to a personal computer. We also assessed
ET-Ar, ET-Br, and VCAM in five peribronchiolar vessels at ⫻200
magnification. The results were expressed as positively immunostained area per perimeter of the outer muscular layer of the vessel
(␮m2/␮m) (5). Coded slides were used to ensure that the analysis was
blinded. All measurements were performed by the same observer.
Statistical analysis. The distribution of the biological effects data
was checked for all variables by Kolmogorov-Smirnov analyses.
Normally distributed variables are reported as means ⫾ SD, and the
significance was determined using the independent-samples T-test.
The nonnormally distributed variables were reported as median and
interquartile range (25–75%), and the significance was determined
using the Mann-Whitney U-test. The significance of the lung mechanics was determined using the general linear model for repeated
measures. The level of significance was set at 5%. Statistical analyses
were performed using SPSS 15.0.
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Table 4. Descriptive values of hematological parameters for groups exposed to FA and CAPs during cold/dry and warm/
humid periods
FA
Parameters
1)
2)
3)
4)
5)
6)
7)
8)
a) Cold/dry
(n ⫽ 34)
b) Warm/humid
(n ⫽ 39)
c) All (n ⫽ 73)
d) Cold/dry
(n ⫽ 39)
e) Warm/humid
(n ⫽ 40)
f) All (n ⫽ 79)
10.18 ⫾ 0.47
15.13 ⫾ 0.60
44.76 ⫾ 1.74
43.99 ⫾ 1.05
14.86 ⫾ 0.29
10.28 ⫾ 0.46
15.30 ⫾ 0.74
45.64 ⫾ 1.91
44.41 ⫾ 1.03
14.89 ⫾ 0.54
10.23 ⫾ 0.47
15.22 ⫾ 0.68
45.23 ⫾ 1.87
44.21 ⫾ 1.05
14.88 ⫾ 0.44
10.50 ⫾ 0.63
15.53 ⫾ 0.82
45.29 ⫾ 1.91
43.22 ⫾ 1.37
14.81 ⫾ 0.27
10.75 ⫾ 0.53
15.95 ⫾ 0.89
46.67 ⫾ 2.01
43.45 ⫾ 1.20
14.83 ⫾ 0.54
10.62 ⫾ 0.59
15.74 ⫾ 0.87
45.99 ⫾ 2.07
43.33 ⫾ 1.29
14.82 ⫾ 0.43
33.81 ⫾ 0.71
33.54 ⫾ 1.29
33.67 ⫾ 1.06
34.28 ⫾ 0.87
34.18 ⫾ 1.49
34.23 ⫾ 1.21
22.89 ⫾ 0.77
21.97 ⫾ 1.10
22.40 ⫾ 1.06
23.47 ⫾ 1.25
22.75 ⫾ 1.33
23.10 ⫾ 1.33
28.95 ⫾ 1.49
890.68 ⫾ 220.85
3.19 ⫾ 0.83
2.89 ⫾ 1.33
0.31 ⫾ 0.21
2.45 ⫾ 1.16
0.10 ⫾ 0.16
28.13 ⫾ 1.67
786.29 ⫾ 229.20
3.46 ⫾ 0.77
3.92 ⫾ 1.54
0.58 ⫾ 0.35
3.17 ⫾ 1.29
0.17 ⫾ 0.12
28.52 ⫾ 1.63
835.58 ⫾ 229.78
3.34 ⫾ 0.80
3.43 ⫾ 1.52
0.45 ⫾ 0.32
2.83 ⫾ 1.28
0.14 ⫾ 0.14
29.17 ⫾ 1.54
979.18 ⫾ 226.92
3.03 ⫾ 0.86
3.19 ⫾ 1.38
0.37 ⫾ 0.32
2.71 ⫾ 1.22
0.11 ⫾ 0.12
28.23 ⫾ 1.56
817.63 ⫾ 201.47
3.17 ⫾ 0.83
4.55 ⫾ 1.69
0.70 ⫾ 0.35
3.71 ⫾ 1.49
0.17 ⫾ 0.12
28.69 ⫾ 1.61
897.38 ⫾ 228.01
3.10 ⫾ 0.84
3.88 ⫾ 1.68
0.54 ⫾ 0.37
3.22 ⫾ 1.44
0.14 ⫾ 0.12
Values are means ⫾ SD; n, no. of animals. PMN, polymorphonuclear neutrophils. The groups were subdivided and were exposed to the following conditions:
a) cold/dry-AF; b) warm/humid-AF, c) all-AF; d) cold/dry-CAPs; e) warm/humid-CAPs; f) all-CAPs. Group c ⫽ group f (parameters 1, 2, 4, and 7: P ⫽ 0.001;
3: P ⫽ 0.019; and 6: P ⫽ 0.004). Group d ⫽ group e (parameters 3: P ⫽ 0.003; and 7: P ⫽ 0.015). Group a ⫽ group d (parameters 1: P ⫽ 0.018; 2: P ⫽
0.022; 4: P ⫽ 0.010; 6: P ⫽ 0.016; and 7: P ⫽ 0.021). Group b ⫽ group e (parameters 1, 2, and 4: P ⫽ 0.001; 3: P ⫽ 0.024; and 7: P ⫽ 0.006).
Lung histology. Both CAPs produced during the cold/dry
and the warm/humid periods elevated the number of neutrophils and macrophages (P ⫽ 0.001) compared with the FA
group. However, the increase of neutrophils (P ⫽ 0.001) was
higher in the exposure conducted during the cold/dry period
(Table 5).
Immunohistochemistry. There were no differences between
groups for VCAM, ET-Ar, and ET-Br expression in lung
vessels (Table 6).
Summary of effects. Table 7 summarizes the statistically
significant (P ⬍ 0.05) effects of all parameters studied in mice
exposed to CAPs compared with mice exposed to FA, such as
the electric activity of the heart through HRV, lung hyperresponsiveness during a dose-response curve to aerosolized
MCh, systemic inflammation by blood parameters, lung inflammation by lung histology, and immunohistochemical
markers.
DISCUSSION
We observed that a single exposure to a low concentration of
CAPs derived from a São Paulo urban region evoked lung
inflammation and alterations in RBC count in mice. Neutrophilic lung inflammation was more prominent in mice exposed
to CAPs generated during the cold/dry period compared with
mice exposed to CAPs generated in the warm/humid period.
To our knowledge, this is the first study to address the seasonal
effects of PM in São Paulo on biological outcomes in mice.
Principal component analyses of São Paulo PM2.5 elements
showed that, during the cold/dry seasons, there was a predominance of factors related to the burning of heavy-duty diesel as
well as due to biomass and crustal emissions (soil and construction). It is believed that the weather conditions during the
cold/dry seasons are unfavorable for the dispersion of pollutants, thus worsening the air quality in major urban centers. In
contrast, warm/humid weather conditions perpetuate the formation of secondary aerosols in the atmosphere due the action
of solar radiation on PM2.5.
Tablin et al. (35) demonstrated an association between
increased concentrations of PM0.1 metals and PAHs during
winter periods in Fresno, CA and a greater systemic proinflammatory and procoagulant response. Little is known about the
seasonal effects on particle composition in São Paulo city,
where differences in temperature and humidity are milder
compared with other regions of the globe.
The effect of climate alterations on particle composition and
size in the city of São Paulo has been well documented by
Albuquerque et al. (2), who showed that meteorological conditions have a major influence on the suspended aerosol con-
Table 5. Descriptive values of the lung neutrophils and macrophages for groups exposed to FA and CAPs during cold/dry
and warm/humid periods
FA
CAPs
Parameters
(105 cells/mm2)
a) Cold/dry (n ⫽ 44)
b) Warm/humidy (n ⫽ 44)
c) All (n ⫽ 88)
d) Cold/dry (n ⫽ 46)
e) Warm/humid (n ⫽ 45)
f) All (n ⫽ 91)
1) Neutrophils
2) Macrophages
5.36 ⫾ 1.68
6.99 ⫾ 1.11
4.99 ⫾ 1.92
6.82 ⫾ 1.01
5.18 ⫾ 1.80
6.91 ⫾ 1.06
8.67 ⫾ 2.10
11.38 ⫾ 1.45
7.40 ⫾ 1.25
10.79 ⫾ 1.43
8.04 ⫾ 1.84
11.08 ⫾ 1.47
Values are means ⫾ SD; n, no. of animals. The groups were subdivided and were exposed to the following conditions: a) cold/dry-AF; b) warm/humid-AF,
c) all-AF; d) cold/dry-CAPs; e) warm/humid-CAPs; f) all-CAPs. Group c ⫽ group f (parameters 1 and 2: P ⫽ 0.001). Group d ⫽ group e (parameter 1: P ⫽
0.001). Group a ⫽ group d (parameters 1 and 2: P ⫽ 0.001). Group b ⫽ group e (parameters 1 and 2: P ⫽ 0.001).
J Appl Physiol • doi:10.1152/japplphysiol.00156.2014 • www.jappl.org
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9)
10)
11)
12)
13)
14)
Erytrocytes, ml/mm3
Hemoglobin, g/dl
Hematocrit, %
Mean corpuscular volume, fl
Mean corpuscular hemoglobin, pg
Mean corpuscular hemoglobin
concentration, g/dl
Red distribution with variation
coefficient, %
Red distribution with size
distribution, fl
Platelets, ml/mm3
Reticulocytes, %
Leucocytes, ml/mm3
PMN, ml/mm3
Lymphocytes, ml/mm3
Monocytes, ml/mm3
CAPs
Toxicity by Concentrated Ambient Particles
•
497
de Brito JM et al.
Table 6. Median (IQR: 25–75) values of the lung immunohistochemistry for groups exposed to FA and CAPs during cold/
dry and warm/humid periods
FA
Cold/dry (n ⫽ 39)
CAPs
Warm/humid (n ⫽ 44)
All (n ⫽ 83)
Cold/dry (n ⫽ 41)
Warm/humid (n ⫽ 44)
All (n ⫽ 84)
Parameters
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Median
IQR (25–75%)
Endothelin-A
Endothelin-B
VCAM-1
0.28
0.63
0.09
0.14–0.42
0.37–1.03
0.05–0.13
0.16
0.08
0.16
0.06–0.34
0.03–0.27
0.11–0.23
0.23
0.34
0.11
0.10–0.36
0.08–0.69
0.06–0.20
0.26
0.56
0.07
0.13–0.49
0.41–0.94
0.03–0.17
0.14
0.11
0.16
0.04–0.28
0.04–0.23
0.11–0.27
0.21
0.29
0.12
0.07–0.36
0.09–0.59
0.05–0.21
The groups were subdivided and were exposed to the following conditions: cold/dry-AF, warm/humid-AF, all-AF, cold/dry-CAPs, warm/humid-CAPs, and
all-CAPs.
Our results show that acute, short exposure to ambient
particles induced lung inflammation in both cold/dry and
warm/humid periods, with increases in neutrophils and macrophages in the lung tissue. Previous studies have demonstrated that exposure to CAPs caused an increase in lung
neutrophils in rats, which correlated with the V and bromide
Table 7. Summary of the effects
FA
System/Parameters
HR variability
SDNN
RMSSD
LF
HF
LF/HF
HR
BP
Lung hyperresponsiveness
Baseline Penh
Penh saline
Penh 6.25 MCh
Penh 12.5 MCh
Penh 25 MCh
Penh 50 MCh
Penhmax
AUC
Blood
MCV
Erythrocytes
Hemoglobin
Hematocrit
MCHC
RDW-CV
RDW-SD
Leucocytes totals
Monocytes
Lymphocytes
PMN
Platelets
Lung histology
Neutrophils
Macrophages
Vessels peribronchiolar
Endothelin-A receptor
Endothelin-B receptor
VCAM-1
CAPs
CAPs
Cold
Warm
Cold
Warm
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
—
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2
1
1
1
1
1
—
—
—
—
—
—
2
1
1
—
1
1
—
—
—
—
—
—
2
1
1
1
—
1
—
—
—
—
—
—
—
—
—
—
—
1
—
—
—
—
—
—
—
—
1
—
—
—
—
—
—
—
—
—
1
1
1
1
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
The CAPs, cold, and warm columns under FA compare CAPs group in relation group FA. The cold and warm columns under CAPs compare CAPs cold vs.
CAPs warm. Penh, enhanced pause; Penh saline, Penh with 6.25, 12.5, 25, and 50 mg/ml of methacholine, respectively; Penhmax, maximum Penh; AUC, area
under the curve; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; RDW-CV, coefficient variation of the red cell
distribution width; RDW-SD, standard deviation of the red cell distribution width; 1, increase; 2, decrease; —, unchanged. Significance: P ⬍ 0.05.
J Appl Physiol • doi:10.1152/japplphysiol.00156.2014 • www.jappl.org
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centrations in the atmosphere. Epidemiological studies have
shown that interactions between air pollution and weather
conditions are associated with increases in mortality and morbidity of respiratory and cardiovascular diseases (14, 15, 24).
Few studies have addressed biological outcomes in animals
exposed to particles generated in different climates (28, 35).
498
Toxicity by Concentrated Ambient Particles
de Brito JM et al.
mation, with an increase in the RDW level in canines. The
mechanisms that promote the release of circulating RBCs from
the bone marrow by CAPs are unclear, and there is a lack of
literature focusing on this physiopathological process.
CAPs exposure induced no alterations in HR, HRV, and BP,
parameters that are predictive of cardiovascular disease, as
described in previous studies of exposure to CAPs (17), PM2.5
(29), diesel exhaust particles (4), and interactions between
temperature and ozone levels (29, 39). Similarly, no alterations
in lung adhesion molecules and endothelial receptor values
were observed. Exposure to 14 days of air pollution caused
vascular constriction with increased ET-Ar expression (21). It
is possible that the lack of changes related to endothelial
dysfunction in our model is related to the acute, short exposure
that we employed.
It is important to note the limitations of the present study.
We used historical data from 2007–2008 to analyze PM elements. In the present study, the climatic conditions were
similar to the climatic conditions during the period 2007–2012.
Considering that the quality of the fuel and vehicular engines
did not change during that period, we considered it valid to use
historical PM2.5 composition data. The collection of ambient
particles does not capture potential pollutants that are in gaseous or vapor forms; therefore, this exposure was not simulated
through these experiments. The tail-cuff system used in this
study is a system of limited value to detect small differences
for HR, BP, and HRV and could explain the lack of significant
results in the present study.
In summary, short-term exposure to low concentrations of
CAPs elicited modest but significant pulmonary inflammation
and, to a lesser extent, changes in blood parameters. In addition, our data support the concept that changes in climate
conditions slightly modify particle toxicity because equivalent
doses of CAPs from the cold/dry period produced a more
exacerbated response.
GRANTS
This work was supported by grants from Fundação de Amparo a Pesquisa
do Estado de São Paolo (São Paulo, Brazil, 2010/50841–3). T. Mauad is
funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.M.B. and P.H.S. conception and design of research;
J.M.B., K.Y., and B.M.S.-R. performed experiments; J.M.B., M.M., K.Y.,
A.C.T.-A., M.d.F.A., and T.M. analyzed data; J.M.B., M.M., A.C.T.-A.,
M.d.F.A., and T.M. interpreted results of experiments; J.M.B. prepared figures;
J.M.B. and M.M. drafted manuscript; J.M.B., M.M., A.C.T.-A., T.M., and
D.H.R.F.R. edited and revised manuscript; J.M.B. and P.H.S. approved final
version of manuscript.
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