British Journal of Environment & Climate Change

7(4): 236-251, 2017; Article no.BJECC.2017.019

ISSN: 2231–4784

Characterization of Particulate Matter in Urban

Environments and Its Effects on the Respiratory
System of Mice
Venkatareddy Venkataramana1,2*, Azis Kemal Fauzie1,3 and Sreenivasa1
1
Department of Studies in Environmental Sciences, University of Mysore, Manasagangotri,
Mysore-570006, Karnataka, India.
2
School of Forestry and Environmental Studies, Yale University, New Haven, Connecticut, USA.
3
Environment Management Board, Government of Karawang Regency, Karawang-41316,
West Java, Indonesia.

Authors’ contributions

This work was carried out in collaboration between all authors. Author VV conceived and designed the
experiments, managed the laboratory analyses, analyzed the data and wrote the paper. Author AKF
performed the field-work experiments and data collection, analyzed the data, and wrote the paper.
Author S performed the field-work experiments and wrote the paper. All authors read and approved
the final manuscript.

Article Information

DOI: 10.9734/BJECC/2017/36547

st
Received 31 August 2017
Accepted 21st September 2017
Original Research Article th
Published 16 December 2017

ABSTRACT

Aims: To investigate the characteristics of ambient particulate matter (PM) and its impacts on
animal respiratory system.
Place and Duration of Study: The study was conducted in urban area of Mysore city from 2014 to
2017.
Methodology: The elemental composition, image interpretation, and size distribution of particles
was analysed using energy dispersive X-ray spectroscopy, scanning electron microscopy, and
dynamic light scattering methods, respectively. Bronchoalveolar lavage analysis was performed to
determine the differential cell counts of leucocytes and lymphocytes in the mice lungs. Histological
and histopathological studies have been demonstrated to observe the effect of PM exposure on
the lungs tissue of mice.
Results: The particle characterization analysis found that roadside PM was dominated by 56%
black carbon and trace amount of metal elements. The analysis also shows that almost 90% of
ambient particulate matter collected in the urban traffic roads was fine particles (PM2.5). By using
bronchoalveolar lavage fluid, bronchial biopsies studies have found the compositional changes in
_____________________________________________________________________________________________________

*Corresponding author: E-mail: venkataraman_1970@yahoo.co.in;

E-mail: aziskemalfauzie@gmail.com;
 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019

neutrophils, eosinophils, mast cells, monocytes and lymphocytes after exposure to PM. Elevated
expression and concentrations of inflammatory mediators have similarly been observed in the
respiratory tract of mice. The pathological change like degeneration of alveolar region, pycnotic
nuclei, and intercellular spaces with prominent vacuolization in epithelial cells followed by
parenchyma and accumulation of particle laden macrophages was evident.
Conclusion: Exposure to PM induces pathological changes, differential cell counts, and
inflammatory response in the mice lungs in a dose and duration dependent pattern.

Keywords: Particulate matter; particle characterization; alveolization; respiratory system; differential

leucocytes.

1. INTRODUCTION In India, PM generated mainly by traffic emission

Air pollution is an emerging biggest public health
threat. Several studies worldwide have reported
that air pollution causes significant illnesses and
mortality contributed largely by cardiopulmonary
diseases [1-3]. Recent reports from Western
world also suggest causal role of air pollution in
obesity, metabolic syndrome and diabetes, which
is growing to an epidemic proportion in India.
Another alarming fact is that children or adults
living close to high traffic areas were associated
with poor cognitive function suggesting that air
pollution exposure could impact cognitive
function. Air pollution levels in Indian major cities
are 2 to 3 times higher than western world. With
Indian economy projected to attain 10% growth
in next decade, the levels of air pollution will
further rise dramatically as a result of increase in
urbanization and industrialization. Despite these
concerns, large proportion of public in India is
still unaware of adverse health effects of air
pollution.
In epidemiologic studies, the impact of long-term
exposure to air pollution on lung function is
probably the result of exposure to both gases
and particles, making it difficult to ascribe a
single agent as the cause of the observed
changes. The use of animal models allows the
role of selected pollutants on developmental
parameters to be studied. For instance,
alterations of the development of distal airways
and pulmonary parenchyma have been elegantly
demonstrated after prolonged exposure to
particulate matter and ozone in rodents [4].
Among the components of air pollution,
particulate matter (PM) has been consistently
associated with chronic respiratory symptoms
and pulmonary function alterations in mammals
[5]. In addition, data provided by autopsy studies
reveal that young adults living in areas with high
ambient levels of inhalable particles have
pathologic alterations of airways and distal
pulmonary parenchyma [6].
is major threat to urban public health contributing
to various diseases such as asthma, chronic
obstructive pulmonary disease (COPD),
cardiovascular disease, metabolic syndrome,
and respiratory infections [7,8]. It is well
documented that oxidative stress plays an
important role in PM-induced adverse respiratory
and systemic health effects [9,10]. Our research
interest is in the area of exposure assessment
with objective of estimating the exposure–
response relationship between air pollutants and
cardiopulmonary diseases. The data can be
useful for designing effective interventional
strategies to mitigate adverse health effects of air
pollutants in population and providing the policy
makers a hard scientific evidence to implement
effective policy to improve air quality. The study
is aimed to characterize PM in urban traffic roads
and to analyze its effects on the respiratory
system of mice after exposure in different
intervals, focusing on pathophysiological
consequences of the inflammatory response. We
hypothesize that in addition to levels, oxidative
potential of PM is an important exposure metric
to predict adverse respiratory and systemic
effects.
2. MATERIALS AND METHODS
2.1 The Study Area
Mysore is one of the largest districts in the state
of Karnataka, India. It is located at 135 km south
of Bangalore metropolitan city and lies at 12° 18'
25" N latitude and 76° 38' 58" E longitude. Mean
sea level or altitude of the city is 765 m. The city
is well connected to the neighbouring states of
Kerala and Tamil Nadu through road transport
and rail network. Mysore city has a warm, cool
and salubrious climate throughout the year; the
minimum temperature in winter is 15C and the
maximum temperature in summer is 35C.
Mysore gets most of its rain during the monsoon

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season between June and September with an
annual average of 782 mm [11].
Mysore city has witness tremendous population
growth after 2001 due to establishment of new
industries, information technology, parks, new
educational institutions, etc. It has 1,119,031
populations as per 2011 census and is projected
to have 2,834,000 populations in 2041. As the
city is being connected to the state capital of
Bangalore through a new dedicated express
highway, it is estimated that the city will grow at a
much faster rate due to large scale population
migration. The population is booming,
demanding increase in public amenities like
transportation, and leading to increase in traffic
density and thus the vehicular emissions. Like
many other Indian cities, Mysore city has also
high vehicular growth and emissions problem. It
has over 523 thousand vehicles registered in
2015 and is projected to expand about 120% in
2020 [12]. At the intersection near the monitoring
station of the Karnataka State Pollution Control
Board (KSPCB) Mysore, it was estimated that
approximately 13,221 cars, 6,712 diesel vehicles,
and 7,656 motorcycles circulate daily on the
main street, and 15,590 cars, 8,234 diesel
vehicles, and 18,567 motorcycles circulate on the
lateral street of the crossing. There are no
significant biomass burning sources near the
surroundings.
The locations of sampling points are distributed
around the city (Fig. 1). Irwin road (Site 1) is the
highly narrow and congested traffic lane in
Mysore city which is near KSRTC bus stand (Site
2); both have been selected as study sites,
followed by moderate traffic area of Metagalli
extension (Site 3) and Siddarthanagar (Site 4),
and low traffic area of Mysore University campus
(Site 5) and Mysore Sandalwood oil factory
(Site 6).

Fig. 1. Location of sampling points (arrows) in Mysore urban area. Inset: Location of Mysore
city in Karnataka, India

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2.2 Particle Collection and Characteriz-
ation
Air pollution in most of the sampling sites has
been characterized as mainly vehicular. Previous
characterization of PM2.5 mass (size < 2.5 μm)
collected at the monitoring station and from the
roof of the school has shown that approximately
90% of the PM2.5 mass is derived from vehicular
sources, with a black carbon/organic carbon ratio
ranging between 40% and 70% throughout the
day. We have further performed elemental
analyses of PM2.5 collected at the exposure site
confirming that vehicular emissions and crust
resuspension are the major PM2.5 components at
this site.
The particulate material was collected by a
particle retainer that consists of filters and
capable of retaining PM emitted from the exhaust
of vehicles. The particle collection sampling was
carried out on the roadside in the daytime when
traffic density was relatively high, usually about
five to six hours per day in the daytime.
Particulate matter entrapped in the filters were
then taken out thoroughly and stored to the
laboratory for further analytical studies. At the
same time, the outdoor temperature, humidity,
heat index, barometric pressure and wind speed
were also measured at the same sampling points
using a specific weather station.
The characteristics of suspended particulate
matter were analyzed according to the
concentrations of their elements, which were
determined by energy-dispersive X-ray
spectrometry (EDX). The data generated by EDX
instrument consists of spectra showing peaks
corresponding to the elements. The count
number of emitted X-ray versus their energy is
evaluated to determine the true elemental
composition of the sampled volume being
analyzed. The X-ray energy was converted to
voltage pulse and recorded in voltage units. The
height of the peaks represents the relative
abundance of X-rays emitted by the elements
and the concentration of each element in the
sample.
Particle size distribution can be determined by
measuring the random changes in the intensity
of light scattered from a suspension. The
technique is commonly known as Dynamic Light
Scattering (DLS) method. DLS works by
measuring the dynamic thermal motion of the
particles dispersed in the liquid suspension,
known as the Brownian motion. Combining AC
electric field with Brownian motion enables the
239
system to measure the velocity distribution of
charged particle movement The variations in the
signal arise due to random Brownian motion will
be used to extract particle size.
A scanning electron microscope (SEM) was used
to capture an image of particle samples by
scanning the samples with a focused electron
beam over their surface. The electrons in the
beam interact with the sample, producing various
signals that can be used to obtain information
about its surface topography and composition.
SEM can produce very high resolution images of
a sample surface to about 250 times the
magnification limit of the best light microscopes.
The digital images were then processed in a
computer to quantify their size (length or
diameter), area, perimeter, and circularity value
by employing specific image processing
software. The circularity, also known as degree
of roundness, that represents the shape of
particles is dimensionless [13].
2.3 Animal Exposure
Exposure was performed using inhalation
chambers installed on the traffic roads in the
sampling site. The exposure chamber consisted
of a cuboidal plastic structure. Air entered the
chamber at the top, exited at the bottom, and
was uniformly distributed throughout the
chamber. The average air flow was maintained
at 25 L/m using a flowmeter gauge. It was a
normobaric system; the pressure inside the
chambers did not exceed 30 mmH2O. In the
filtered system, two stages of filters were in line
(the screen and the bag filters) that can eliminate
large particles and trap fine particles.
Three weeks old mice were exposed via whole-
body inhalation to suspended particulate matter
in different intervals of six hours per day and
five days per week for 5, 15, 21, 30 and 90
days respectively. Animals were fed ad libitum
with water and commercial pelleted food. Eight
animals were included in each group and two
sets of exposure for each group were
performed. Control animals were kept in the
laboratory. Exposure to particles was repeated
with the same group of mice under identical
conditions done at 21 days age. Following
exposure, samples of bronchoalveolar lavage
(BAL) and lung tissues were collected for
analysis. At the termination day, the lungs of
mice were feasible for conducting BAL
procedure. Different sets of exposure were
done to allow BAL and lung tissue analyses
separately.
 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019

2.4 Bronchoalveolar Lavage Fluid 2.6 Statistical Analysis

Analysis
Data are presented as mean ± S.E.M., unless
Bronchoalveolar lavage (BAL) of lungs was otherwise specified. Comparison between
performed on half of the mice from each study numerical parameters was performed using
group. Within 2 h following the end of particle Student’s t test. The comparison degree of
exposure of 5, 15, 21, 30 and 90 days mice were alveolization among the eight animal groups was
anesthetized using chloroform. Preparation of performed using general linear models and the
BAL was done following Harrod’s protocol with level of significance was set to 5%.
some modifications [14]. Immediately after
respiratory mechanics assessment, BAL was 3. RESULTS
performed by introducing 1 ml sterile phosphate-
buffered saline (PBS) into the lungs via a 3.1 PM Concentrations and Meteoro-
tracheal cannula, and the recovered fluid was logical Factors
kept in a test tube on ice. This procedure was
repeated three times. The fluid collected was Gravimetric analysis of the sampled particles
centrifuged at 1000 rpm for 10 minutes at 5°C to found that ambient air in Site 1 located in
separate cells from the supernatant. The cell commercial areas has the highest PM
pellet was resuspended in 300 μl PBS. A volume concentration than any other areas (Table 1).
of 100 μl resuspended pellet was removed and Ambient air in industrial and residential areas
stored in an Eppendorf tube with 100μl PBS. A (Site 3, 4, and 5) was found to have quite similar
minimum of 106 cells was used to prepare slides concentrations of particulate matter. The
in duplicates. Cells were stained with Giemsa to meteorological data are also presented in the
determine the proportion of macrophages, same table. The trend data of PM air quality in
monocytes, lymphocytes, and neutrophils. Total Mysore are obtained from the Karnataka State
and differential cell counts and viability were Pollution Control Board (KSPCB) Mysore as
determined using an improved Neubauer presented in Figs. 2 and 3.
hemocytometer chamber and an optical
microscope with a 400 zoom. It is noted that using statistical data analysis,
almost no significant correlation was found
2.5 Histological Analysis between any weather condition variable and PM
concentration for this study, except for outdoor
The left lungs were fixed by intratracheal temperature. The PM concentrations were
instillation of formalin (10% formaldehyde in 2% considerably correlated with outdoor
buffer) at a pressure of 20 cmH2O for 24 hours. temperatures (r = 0.9, p < 0.05; 4 d.f). This may
Longitudinal lung sections were paraffin- be due to commercial area has higher traffic
embedded and 5 mm thick histological sections volume that generates heat from vehicles’
were stained with Hematoxylin and Eosin for exhaust fumes. Annual average of PM
qualitative and morphometric analyses. concentrations in Mysore city from 2012 to 2015
Histological slides were coded for blind analysis. shows values reaching the national ambient air
Morphometric measurements were also quality standards and at some points exceeding
performed by the same observer. the permissible limits [15].

80 80
PM concentrations (mg/m3)
PM concentrations (mg/m3)

70 70
PM10 standard PM10 standard
60 60
50 50
PM2.5 standard
40 40
30 30
20 20
10 10
0 0
2012 2013 2014 2015 2012 2013 2014
Commercial area Industrial area PM10 PM2.5

Fig. 2. PM10 concentrations (μg/m3) at Fig. 3. PM10 and PM2.5 concentrations

3
Mysore commercial and industrial area (μg/m ) at Mysore industrial area

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Table 1. Descriptive statistics of PM concentration and weather condition*

Site Concentration Temperature Humidity Pressure Heat index Wind speed

3
μg/m C % kPa C kph
1 6464 32.09  0.58 23.82  1.11 1.0110  0.0003 30.55  0.45 3.73  0.52
2 2954 30.82  0.35 18.73  0.97 1.0107  0.0003 29.18  0.30 5.91  0.44
3 1808 29.73  0.51 22.09  0.94 1.0103  0.0004 28.36  0.36 10.27  0.80
4 1322 29.22  1.10 31.11  4.24 1.0114  0.0005 29.57  0.37 3.56  0.84
5 1674 30.62  0.40 42.00  0.95 1.0117  0.0002 30.92  0.35 5.08  0.46
* All weather values are mean  S.E.M.

3.2 Elemental Composition of PM 3.3 Particle Size Distribution

The EDX instrument obtained results in the form The dynamic light scattering method was used to
of spectra showing a number of peaks that identify the size variation of particles taken from
correspond to the specific elements presented in the sampling site. The samples were suspended
the sample. The analysis shows that roadside in a specific liquid before taken into the
particulate matter consists of carbon (C) 56.38%, instrument. The scattered light signal collected
oxygen (O) 33.66%, and other metal and by the DLS detectors was analyzed by the
metalloid elements in smaller fraction (Fig. 4). In system and presented in particle size distribution
nature, these elements may present as single graph (Fig. 5). The result shows that ambient
elements like carbon, iron, and aluminium, or as particulates entrapped in the sampler were
chemical compounds in combination of different largely fine particles (PM2.5) with size less than
elements, such as silicon presents as silica, iron 2.5 μm (Table 2). The larger the size, the less
as rust, aluminium as alumina, calcium as lime or percentage of particles was identified. The
gypsum, sodium and potassium present as relative weight of SPM collected in different
marine salts, or other possible forms of places of Mysore historical city at different
compounds like oxides, hydroxides, chlorides, intervals is presented in Table 3.
carbonates, sulfates, phosphates, etc.
70

60 56.38

50
Weight %

40
33.66
30

20

10
3.39 1.97
0.99 1.59 0.56 1.46
0
C O Na Al Si K Ca Fe

Fig. 4. Weight percentage of elements Fig. 5. Particle size distribution obtained

present in particulate matter from DLS analysis

Table 2. Percentile of particles measured by DLS according to their size

Percentile 10 20 30 40 50 60 70 80 90 95
Size above (nm) 377.9 335.7 311.9 293.6 277.7 263.1 248.6 232.5 211.8 197.8

Table 3. Relative weight (mg) of SPM collected in different places at Mysore city

Sampling locations 5 days 15 days 21 days 30 days 90 days

1-Irwin road 5.00 11.00 12.00 15.00 26.00
2-KSRTC bus stand 4.80 09.40 11.00 14.08 24.83
3-Metagalli extention 3.40 04.00 07.22 12.30 20.20
4-Siddharthanagar 2.37 02.89 05.30 08.60 10.23
5-University campus 1.87 02.04 03.80 05.13 09.31
6-Sandalwood oil factory 2.21 02.89 04.45 06.50 11.21

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3.4 Particle Morphology
Analysis of SEM digital images resulted in
graphs of particle size distribution (Fig. 6) and
circularity values of the particles (Fig. 7). It was
found that under electron microscope the mean
diameter of particles collected in the sampling
site was 1.66 μm. In details, they were
distributed in a variety of size, mainly from less
than 1 μm to less than 2.5 μm. It suggested that
majority of airborne PM in urban traffic road falls
to PM2.5 category.
The shape of urban roadway particles was found
close to circular; even they were not exactly
spherical. Majority (31%, 27%, and 13%) of the
sampled particles have circularity values or
degree of roundness up to 0.85, 0.8, and 0.9,
respectively. A perfectly rounded particle sphere
should have circularity value of 1. Liquid droplets
or aerosols are almost nearly spherical. Particle
objects with less circularity values are classified
as irregular shape particles. Particles generated
from unburned fuel tend to have circular shape
[16].
3.5 Anatomy of Mice Lungs
The bronchi branch repeatedly after entering the
lung, diminishing in size with each division. The
smaller bronchi are lined by simple columnar
ciliated cells and the bronchioles by low
columnar epithelium lacking both cilia and goblet
cells. As the tubes become smaller their wall
become thinner and consists of less connective
tissue and smooth muscle. The terminal
bronchioles give rise to respiratory bronchioles,
each branching into several alveolar ducts and
alveolar ducts lead into alveolar sacs, each
composed of several alveoli. Respiratory
bronchioles are lined with cuboidal epithelial cells
surrounded by thin connective tissue sheets
containing scattered smooth muscle cells. The
cuboidal epithelium ends abruptly at the junctions
of respiratory bronchioles and alveolar ducts
where it is replaced by squamous epithelium.
Alveolar ducts, alveolar sacs, and alveoli have
Table 4. Body weights, lung weights, and lung/body weight ratio of mice in the different study
groups at the time of dissection
Age (days) Exposure group Body weight (g)
21 05 days 08.17
36 15 days 09.54
57 21 days 10.90
87 30 days 13.46
177 90 days 22.37
* There was no statistical significance in the lung/body weight ratios among all groups
242
very thin walls invested with fine close-meshed
networks of large thin-walled capillaries (Fig. 8a
and b).
3.6 Effects of PM on Lung/Body Weight
Our animals were exposed in chambers in close
vicinity to a road with high traffic density and
without nearby industries. The exposure site was
located near an environmental monitoring
station, which performed continuous monitoring
of gases and particles. Emissions at this site
have been characterized as typically vehicular,
as demonstrated by PM elemental analysis. Both
control and PM-exposed mice were weighed
before and after experiment. Body weight (Bw,
g), lung weight (Lw, mg), and lung/body weight
(Lw/Bw, mg/g) ratio of mice were examined after
each exposure to airborne PM for 5, 15, 21, 30
and 90 days (Table 4). There were slight
differences in Lw/Bw ratios among the groups.
3.7 Effects of PM on BALF
Biochemical parameters of bronchoalveolar
lavage fluid of mice after PM exposure are
presented in Table 5. Variation was calculated in
median range number of cells in six visual fields
for the biological indices in both control and PM-
exposed animals. Some parameters showed
substantial differences that were attributed to the
assays and need to be taken into account as a
confounding factor in the overall statistical
analysis. There was a significant reduction of the
bronchoalveolar lavage lymphocytes (9.0, 8.1,
7.9 and 8.7) after 15, 21, 30 and 90 days
exposure, respectively. This inflammation has
elevated the expression of neutrophils (3.7),
monocytes (30.2), and eosinophils (1.12)
compared to control. Percentage viability of
leucocytes has decreased and differential cell of
macrophages (98.10±1.37) and neutrophils
(1.34±0.17) were increased in BALF following
mice exposure to PM2.5 in 90 days. The total cell
concentration in the BALF increased after the
exposure to particulate matter as compared to
control mice.
Lung weight (mg) Lung/body weight ratio (mg/g)*
210.09 25.71
220.18 23.07
243.24 22.31
245.34 18.22
286.60 12.81
 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019

35 35

30 30

% Distribution 25 25

% Distribution
20 20

15 15

10 10

5 5

0 0
<0.5 <1 <1.5 <2 <2.5 <3 <3.5 <4 <4.5 <5 <5.5 <6 <6.5 <7 <0.5 <0.55 <0.6 <0.65 <0.7 <0.75 <0.8 <0.85 <0.9 <0.95 <1

Diameter (mm) Circularity value

Fig. 6. Size distribution of roadway PM Fig. 7. Classification data for the

analyzed from SEM digital image circularity values of particulate matter

(a) (b)

1. Larynx 3. Trachea 5. Azygous Lobe 7. Diaphragmatic Lobe

2. Thyroids 4. Apical Lobe 6. Cardiac Lobe 8. Left Lobe

Fig. 8. (a) Anatomical lobe structure and (b) specimen of control mice lungs

3.8 Effects of PM on Histopathology of comparison to the control mice. There was no

Lungs significant difference in the amounts of
intraepithelial mucus substances between control
The morphologic lesions in lungs of mice were (Fig. 9A) and 5 days exposed mice.
an allergic bronchiolitis and alveolitis.
Inflammatory and epithelial lesions were usually The lung parenchyma of mice exposed to
more severe in the proximal bronchioles (Fig. 9B) airborne PM for 90 days was characterized by
compared to those in the distal preterminal and accumulations of large numbers of alveolar
terminal bronchioles. Induced bronchiolitis was macrophages, monocytes, and eosinophils, with
characterized by peribronchiolar edema less numbers of lymphocytes and plasma cells in
associated with a mixed inflammatory cell influx the alveolar airspace (Table 5). The alveolar
of eosinophils and mast cells. Peribronchiolar septa in these areas of alveolitis were thickened
inflammation was located in the subepithelial due to type II pneumocyte hyperplasia and
interstitial tissues (e.g. lamina propria and hypertrophy, intracapillary accumulation of
submucosa) on the surface epithelium (Fig. 9C). inflammatory cells, and capillary congestion (Fig.
In 30 days exposure mice had a mucous cell 10A and B). In contrast to the exposure, the
metaplasia with increased amounts of mucus longer duration brings relatively more changes in
substances (Fig. 9D) in the surface epithelium alveolar regions since there are few major
(i.e. intraepithelial mucus substances) including emission sources in that direction.
the proximal and distal axial airways in

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A B

C D

Fig. 9. The photomicrograph of the respiratory epithelium (B) lining the proximal axial airways
in the left lobe of control mice lung. Significant morphologic changes are present in mice after
21 and 30 days exposure (C and D). The most prominent histological changes due to
particulate matter exposure included a thickened, hypertrophic, respiratory epithelium with
increased numbers of mucous goblet cells, and a mixed inflammatory cell infiltrate (arrows)
consisting of scattered eosinophils (double arrows) in the interstitium of the airway followed
by vacuolization compared to the normal airway in the control mice (A). All tissues are stained
with Hematoxylin and Eosin. 400

Table 5. Bronchoalveolar lavage cell numbers* in control mice and after exposure to PM at
different intervals
Biochemical Control 5 days 15 days 21 days 30 days 90 days
parameters
6
Cells/ml, 10 91 (51-97) 118 (76-125) 110 (68-130) 128 (74-140) 130 (78-148) 143 (68-154)
Macrophages, % 66 (55-78) 93 (62-103) 72 (60-111) 83 (62-123) 85 (68-110) 96 (82-113)
Lymphocytes, % 11.6 (6.1-12) 10.0 (7.6-11) 9.0 (6.6-10) 8.1 (9.6-13) 7.9 (6-11) 8.7 (6-10)
Monocytes, % 8 (0.5-8.8) 11 (6.7-12.3) 23 (6.7-25.3) 27 (6.7-28.3) 30 (7-43) 27 (7-30)
Neutrophils, % 1.2 (0.8-1.7) 2.3 (0.7-4.0) 1.3 (0.5-3.0) 3.7 (0.6-4.7) 2.8 (1.6-3.0) 3.3 (0.6-4.2)
Eosinophils, % 0.04 (0-0.43) 0.08 (0-0.94) 0.07 (0-0.88) 0.19 (0-1.21) 1.08 (0-1.38) 1.12 (0-1.94)
Mast cells, % 3.5 (0.4-4.7) 2.1 (1.0-3.7) 2.8 (1.0-3.6) 3.3 (2-5.8) 3.9 (2-6.9) 2.8 (1.2-6.8)
* Data are presented as median range number of cells in six visual fields with 400 magnifications

An increase in the number of alveolar observed in an extensive bronchial-associated

macrophages and simultaneously an increase in
the cellularity of centriacinar septa were noticed
after SPM exposure in all mice. Focal
hypertrophy of the bronchiolar epithelium
occurred in few animals, as well as a minimal
perivascular influx of polymorphonuclear (PMN)
granulocytes. The pathological pulmonary
characteristics of the exposed mice were
lymphoid tissue (BALT) at many bifurcations of
the airways, and a minimal to moderate
perivascular infiltrate of lymphocytes. Small to
large alveolar foci were seen in septa with
increased cellularity and an influx of alveolar
macrophages. It is noticeable in many
experimental animals that small hemorrhages
were present in alveolar spaces. In about half of

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the animals, it shows a minimal to moderate
inflammatory foci with interstitial pneumonia and
alveolitis consisting of macrophages and some
neutrophilic granulocytes (Fig. 10C and D). In 5
and 15 days exposed mice, relatively less
histopathological changes were observed as
compared to the control.
4. DISCUSSION
The study was designed to investigate the
adverse health effects of fine particulate matter
(PM2.5). It was hypothesized that SPM could
affect the health status of mice (mild pulmonary
inflammation) by inducing or worsening
inflammation. This may lead to disturbed
differential leucocyte count [17,18]. For this
reason, but also due to the inherent variability of
the ambient PM composition, the experiments
were repeated more than five times. We
therefore applied the strategy to combine a
series of experiments to establish a duration
concentration–effect relationship and to increase
the statistical power of our experiments. The
present study confirms the findings of pulmonary
inflammation due to exposure to SPM in mice.
Although the results presented in Tables 5 and 6
tend to show a somewhat stronger effect on PM,
the differences in duration reflect the
substantially higher exposure-concentration to
the present study, but may also reflect the
differences in the procedure to retrieve the cell
by using lung lavage techniques [19].
The BALF parameters were statistically and
significantly different in the overall analysis of the
experiments. In addition, visible inclusions of PM
in macrophages as well as increased neutrophils
suggest that PM were deposited in the lungs of
mice. Again, this confirms the findings of Saldiva
[20]. Similar health parameters as described in
the present article were measured at 5, 15, 21,
30 and 90 days after PM exposure. It was
demonstrated that by using SPM inducement
statistically significant pulmonary cytotoxicity was
found as indicated by differential count variations
in BAL fluid, and these continued for days after
exposure. Based on available evidence of
studies using PM exposure in either animals or
human subjects, it seems that PM mass is not
the optimal metric to be associated with adverse
health effects [20]. This suggests that other
metrics may be more appropriate like chemical
composition or physical properties. For example,
an emerging study using ultrafine PM that
became possible through the development of
ultrafine aerosol concentrators [6,21] indicates
that these particles appear to be far more toxic
245
than those of particle accumulation as
demonstrated in the present study.
The evidence that PM produces systemic effects
or induces effects that go beyond the lung as
target organ is growing. A common feature in all
of these studies seems to be an increase in the
number of PMNs in the lungs, as well as adverse
effects on heart function, which could often not
be related to the PM mass concentration or
duration of PM exposures. People with ischemic
heart disease are likely to be disposed to
develop life-threatening cardiac effects, as
suggested by the results from the studies in dogs
[22]. These results from studies in which animals
are exposed by intratracheal instillation [4,23,24]
support the hypothesis that PM affects the
vascular system – for instance, by inducing
endothelial damage [5,25]. Such effects can
explain the changes in heart rate and heart-rate
variability. Although changes in biological
endpoints were occasionally statistically
significant, evidence is provided that ambient PM
can alter the homeostasis.
The marked increases in total lavageable cells
due primarily to macrophages and neutrophils
occurred in a duration-dependent manner.
However, the neutrophilic influx was largely
reversed by day 30 and 90, which was also
reflected in the reversal in total cell numbers
(Tables 5 and 6). Accumulation of particle laden
macrophages (Fig. 10C and D) was the only
prominent and consistent effect of PM inhalation
in all groups of mice in comparison to control.
These increases in macrophages were largely
concentration-duration-dependent and reflected
the increases in total lavageable cell counts. The
scenario of five days per week exposures for 5,
15, 21, 30, and 90 days revealed a significant
increase in macrophages in 30 and 90 days.
Industrially generated combustion and ambient
PM differ significantly in the type and quantity of
bioavailable elements. Because of these
differences, the mechanisms or targets of toxicity
may also vary [26-29]. Recently it has been
shown that the toxicity of ambient-derived PM
from Utah Valley [30,31] and Ottawa, Canada
[32,33] could in part be related to the level of
bioavailable zinc when rats were exposed to the
aqueous extracts of these particles by
intratracheal instillation. However, the
mechanism by which SPM may induce cell
signaling, nuclear translocation of nuclear factors
and down-stream transcription of inflammatory
genes is not clear.
 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019

The effect of PM inhalation in mice was a dose
and a time dependent accumulation of particle-
laden alveolar macrophages with apparent
neutrophil increase. The macrophage activation
[34] was also elevated in BALF and followed a
similar pattern of increase as macrophages with
acute exposure scenarios, suggesting that
phagocytosis of particles may have triggered
macrophage activation. The differences in BALF
may serve as an important marker for acute
pulmonary changes without apparent neutrophilic
inflammation. In the present study it is suggested
that in any mice strain the duration and
concentration of PM used were sufficient to
trigger a significant neutrophilic inflammation and
matrix alterations. This finding may have an
impact on the health risk involving this outcome
associated with air pollution PM exposure.

A B b

C D

Fig. 10. Histological distal lung samples (A) of 30 days exposed mice showing sparse, mild
foci of macrophages (arrows) represents alveolar parenchyma. Lung parenchyma (B) of 90
days exposed mice showing the alveolar spaces that are enlarged, hypertrophy and irregular
(a and b) when compared with control as result of incomplete alveolization (arrow). The mild
foci of macrophage accumulations occurred followed by predominant vacuoles (arrow head)
in the alveolar areas (C). Accumulation of particles within alveolar macrophages was readily
apparent in lung parenchyma and in lymph nodes (C and D). It appeared that there were more
particle laden macrophages (double arrows) found in lymph nodes (D) of 90 days exposed
mice in comparison to control. All tissues are stained with Hematoxylin and Eosin. 400 and
1,000

Table 6. Changes in cell number, viability and differential cell count* in BAL fluid of mice
following exposure to PM at different intervals

Biochemical Control 5 days 15 days 21 days 30 days 90 days

parameters
6
Cells/ml, 10 2.24±0.28 3.12±0.31 2.51±0.11 3.14±0.23 4.70±0.43 7.56±2.47
Cell viability, % 82.24±11.10 80.74±20.32 79.43±23.01 88.23±39.22 70.25±72.98 65.54±28.22
Macrophages, % 96.46±2.25 95.88±3.23 98.73±1.23 95.46±3.21 97.12±4.25 98.10±1.37
Lymphocytes, % 2.34±0.41 1.28±0.27 0.62±0.24 0.34±0.21 0.64±0.16 0.61±3.10
Neutrophils, % 0.33±0.23 1.09±0.26 1.02±0.17 1.97±0.23 1.03±0.10 1.34±0.17
Eosinophils, % 0.03±0.13 0.18±0.17 0.33±0.13 0.93±0.42 0.79±0.33 0.23±0.14
Mast cells, % 0.20±0.10 0.14±0.13 0.03±0.13 1.73±0.77 0.43±0.22 0.03±0.13
* Values are mean ± S.E.M. (n=8 per group)

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 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019

Particle clearance by alveolar macrophage
phagocytosis and migration to the bronchus-
associated lymph tissue is one of the many
different mechanisms of host defense [35].
Histological examination of bronchus-associated
lymph nodes in mice exposed for 30 and 90 days
(five days per week) revealed the presence of
particle clusters, possibly associated with
mononuclear cells, suggesting that PM may be
cleared by this route, in addition to ciliary
clearance and interstitial uptake. In general,
larger and more pronounced bronchus-
associated lymph nodes exist in compared to
control may be due to chronic systemic and
baseline pulmonary inflammation as a Several studies have demonstrated that cigarette
consequence of their genetic predisposition [36].
In this study, we have demonstrated that chronic
exposure to PM2.5 particles trigger alterations in
lung structure of alveolar parenchyma associated
with cell inflammation. Many studies showed
associations between air pollution and
exacerbations of pre-existing COPD, but the role
of air pollution in the development and
progression of COPD is still uncertain [9].
Particle retention in lung tissue (Fig. 11b, c, and
d) resulted in a chronic, low-grade inflammatory
response that may be pathogenetically important
in the progression of lung disease. It is possible
that longer exposures could have more
significant impact on lung mechanics or
remodeling. In addition, it is possible that in pre-
injured lungs, like the smoker’s lungs, chronic
exposure to air pollution could have a synergic
effect on the development of emphysema
[37,38].
Another possibility is the induction of air
pollution-induced autophagy in lung cells.
smoke induces autophagy in lung cells and this
autophagic process appears to play a critical role
in the pathogenesis of emphysema [1,10]. Deng
et al. [39] found that PM2.5 can elicit oxidative
stress, resulting in accumulation of intracellular
reactive oxygen species and autophagic cell
death in human epithelial lung A549 cells. It is
possible that longer exposures would detect
more pronounced inflammatory or extracellular
matrix changes.

(a) (b)

(c) (d)
Fig. 11. (a) Images captured by SEM showing no particulate in control lung sample. (b) The
lung sample after 21 days exposure showing significant amount of PM concentrated in the
inner region. (c) The image showing the sign of particle accumulation in the tissues and their
persistence in the parenchyma. (d) The image of higher magnification showing different
morphological structure of PM in the lung tissues. 5,000 and 10,000

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 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019
In mice, alveoli are absent at birth and the gas
exchanging units are the primary saccules.
Secondary crests appear within the first days of
life to form the alveoli, and the process is
believed to be largely complete by 14 days of
age [40]. In humans, alveolization begins in
utero, by week 36. Since it is estimated that 85%
of alveoli are formed after birth, alveolization in
humans is also considered a postnatal event.
Controversy exists regarding the time needed to
complete alveolization in humans. It is accepted
to occur by 2 to 3 years of age, but development
of full functionality does not occur until 6 years of
age [41-43]. Despite differences in postnatal
alveolization, we believe that studies in mice can
provide useful insights into the potential effects of
urban air pollution. Adequate lung growth during
the pre- and early-postnatal periods is highly
dependent on developmental, genetic, and
environmental factors [43].
Increasing experimental and epidemiological
evidence shows that ambient air pollution alters
structures involved in lung development. The
present study shows that chronic exposure of
mice to airborne particles can affect lung
structure in the absence of overt inflammation.
Future studies should be conducted to elucidate
the pathways related to alveolar damage caused
by air pollution. Given that the majority of PM2.5
reacts within the epithelial lining fluids via free
radical mechanisms, the antioxidant composition
of this fluid may be critically important in
determining the individual's sensitivity to air
pollution.
5. CONCLUSION
Study of particle characterization has generated
important findings that majority of urban roadway
particles are circular PM2.5 particles consist
mostly of elemental black carbon generated
mainly from unburned fossil-fuel of motor
vehicles. It therefore indicates that the sources of
particulate pollution in the urban sites are mainly
from the emissions of motor vehicles. The SPM
exposure in mice for a particular period has
found the effects on the development and
pathophysiological consequences of the
inflammatory response. The results of the study
confirmed the hypothesis that chronic exposure
of mice model to suspended particulate matter
has resulted in a significant airway and lung
parenchymal inflammation and changes in the
alveolar structure.
The only prominent effect of PM inhalation was
duration and time-dependant particle uptake with
248
a concomitant increase in alveolar macrophages
in mice exhibited marked accumulation of particle
laden macrophages. These findings suggest that
exposure to the mixture of suspended particulate
matter induces pathological changes, differential
cell counts and inflammatory response in the
mice lungs in a dose and duration dependent
pattern. The responses observed from the
present study are associated with the
bioavailability of inhaled particulate mixtures.
Therefore, the current observations for the
inhibition of eosinophil and mucus responses by
PM runs counter to epidemiological findings for
traffic-associated asthma symptoms.
However, there is still a need for further studies
in characterization of particulate matter, using
pilot data as a demand surface for allocation of
further sampling sites. While this represents one
of the first known systematic study of the
composition and distribution of air pollution
sampling for exposure assessment, the low
sample size of the pilot study represents a
limitation. Future studies may be needed to
compare multiple sampling technologies as well
as to conduct sensitivity analyses through
comparison with on-going long-term air pollution,
its composition and impacts on biological
systems.
ETHICAL APPROVAL
All experiments have been examined and
approved by the appropriate ethics committee.
Animals received with care in compliance with
the “Laboratory Animal Care” formulated by the
Ethical Committee of the University of Mysore
and the “Guiding Principles in the Care and Use
of Animals” approved by our Institutional Animal
Care and Ethical Committee at the number of
UOM/IAEC/05/2013.
ACKNOWLEDGEMENTS
We are grateful to the University Grants
Commission Raman Fellowship for Post Doctoral
Research in USA, Science and Engineering
Research Board (SERB) Department of Science
and Technology, and Department of Studies in
Environmental Sciences, University of Mysore for
financial support; and to the Imaging Facility and
Materials Science and Technology Laboratory,
Institute of Excellence (IOE), University of
Mysore, School of Forestry and Environmental
Studies, Yale University, and Yale Tropical
Research Institute, New Haven, Connecticut,
USA for the laboratory and technical support.
 Venkataramana et al.; BJECC, 7(4): 236-251, 2017; Article no.BJECC.2017.019
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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