characterization of particulate matter in urban environments...

_____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected]; E-mail: [email protected];

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

1Department of Studies in Environmental Sciences, University of Mysore, Manasagangotri,

Mysore-570006, Karnataka, India. 2School of Forestry and Environmental Studies, Yale University, New Haven, Connecticut, USA.

3Environment 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

Received 14th

July 2017 Accepted 21st September 2017 Published 16

th 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

Original Research Article

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

237

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

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].

In India, PM generated mainly by traffic emission 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 15C and the maximum temperature in summer is 35C. Mysore gets most of its rain during the monsoon


238

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


239

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

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.


240

2.4 Bronchoalveolar Lavage Fluid Analysis

Bronchoalveolar lavage (BAL) of lungs was performed on half of the mice from each study group. Within 2 h following the end of particle exposure of 5, 15, 21, 30 and 90 days mice were anesthetized using chloroform. Preparation of BAL was done following Harrod’s protocol with some modifications [14]. Immediately after respiratory mechanics assessment, BAL was performed by introducing 1 ml sterile phosphate-buffered saline (PBS) into the lungs via a tracheal cannula, and the recovered fluid was kept in a test tube on ice. This procedure was repeated three times. The fluid collected was centrifuged at 1000 rpm for 10 minutes at 5°C to separate cells from the supernatant. The cell pellet was resuspended in 300 μl PBS. A volume of 100 μl resuspended pellet was removed and stored in an Eppendorf tube with 100μl PBS. A minimum of 106 cells was used to prepare slides in duplicates. Cells were stained with Giemsa to determine the proportion of macrophages, monocytes, lymphocytes, and neutrophils. Total and differential cell counts and viability were determined using an improved Neubauer hemocytometer chamber and an optical microscope with a 400 zoom.

2.5 Histological Analysis The left lungs were fixed by intratracheal instillation of formalin (10% formaldehyde in 2% buffer) at a pressure of 20 cmH2O for 24 hours. Longitudinal lung sections were paraffin-embedded and 5 mm thick histological sections were stained with Hematoxylin and Eosin for qualitative and morphometric analyses. Histological slides were coded for blind analysis. Morphometric measurements were also performed by the same observer.

2.6 Statistical Analysis Data are presented as mean ± S.E.M., unless otherwise specified. Comparison between numerical parameters was performed using Student’s t test. The comparison degree of alveolization among the eight animal groups was performed using general linear models and the level of significance was set to 5%. 3. RESULTS

3.1 PM Concentrations and Meteoro-logical Factors

Gravimetric analysis of the sampled particles found that ambient air in Site 1 located in commercial areas has the highest PM concentration than any other areas (Table 1). Ambient air in industrial and residential areas (Site 3, 4, and 5) was found to have quite similar concentrations of particulate matter. The meteorological data are also presented in the same table. The trend data of PM air quality in Mysore are obtained from the Karnataka State Pollution Control Board (KSPCB) Mysore as presented in Figs. 2 and 3. It is noted that using statistical data analysis, almost no significant correlation was found between any weather condition variable and PM concentration for this study, except for outdoor temperature. The PM concentrations were considerably correlated with outdoor temperatures (r = 0.9, p < 0.05; 4 d.f). This may be due to commercial area has higher traffic volume that generates heat from vehicles’ exhaust fumes. Annual average of PM

concentrations in Mysore city from 2012 to 2015 shows values reaching the national ambient air quality standards and at some points exceeding the permissible limits [15].

Fig. 2. PM10 concentrations (μg/m3) at Mysore commercial and industrial area

Fig. 3. PM10 and PM2.5 concentrations (μg/m

3) at Mysore industrial area

0

10

20

30

40

50

60

70

80

2012 2013 2014 2015

PM

co

nce

ntr

atio

ns

(mg/

m3)

Commercial area Industrial area

PM10 standard

0

10

20

30

40

50

60

70

80

2012 2013 2014

PM

co

nce

ntr

ati

on

s (m

g/m

3)

PM10 PM2.5

PM10 standard

PM2.5 standard


241

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

Site Concentration μg/m

3

Temperature C

Humidity %

Pressure kPa

Heat index C

Wind speed 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

The EDX instrument obtained results in the form of spectra showing a number of peaks that correspond to the specific elements presented in the sample. The analysis shows that roadside particulate matter consists of carbon (C) 56.38%, oxygen (O) 33.66%, and other metal and metalloid elements in smaller fraction (Fig. 4). In nature, these elements may present as single elements like carbon, iron, and aluminium, or as chemical compounds in combination of different elements, such as silicon presents as silica, iron as rust, aluminium as alumina, calcium as lime or gypsum, sodium and potassium present as marine salts, or other possible forms of compounds like oxides, hydroxides, chlorides, carbonates, sulfates, phosphates, etc.

3.3 Particle Size Distribution The dynamic light scattering method was used to identify the size variation of particles taken from the sampling site. The samples were suspended in a specific liquid before taken into the instrument. The scattered light signal collected by the DLS detectors was analyzed by the system and presented in particle size distribution graph (Fig. 5). The result shows that ambient particulates entrapped in the sampler were largely fine particles (PM2.5) with size less than 2.5 μm (Table 2). The larger the size, the less percentage of particles was identified. The relative weight of SPM collected in different places of Mysore historical city at different intervals is presented in Table 3.

Fig. 4. Weight percentage of elements present in particulate matter

Fig. 5. Particle size distribution obtained 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

56.38

33.66

0.99 1.593.39

0.56 1.46 1.97

0

10

20

30

40

50

60

70

C O Na Al Si K Ca Fe

We

igh

t %


242

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

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.

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) Lung weight (mg) Lung/body weight ratio (mg/g)*

21 05 days 08.17 210.09 25.71 36 15 days 09.54 220.18 23.07 57 21 days 10.90 243.24 22.31 87 30 days 13.46 245.34 18.22 177 90 days 22.37 286.60 12.81

* There was no statistical significance in the lung/body weight ratios among all groups


243

Fig. 6. Size distribution of roadway PM analyzed from SEM digital image

Fig. 7. Classification data for the 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 Lungs

The morphologic lesions in lungs of mice were an allergic bronchiolitis and alveolitis. Inflammatory and epithelial lesions were usually more severe in the proximal bronchioles (Fig. 9B) compared to those in the distal preterminal and terminal bronchioles. Induced bronchiolitis was characterized by peribronchiolar edema associated with a mixed inflammatory cell influx of eosinophils and mast cells. Peribronchiolar inflammation was located in the subepithelial interstitial tissues (e.g. lamina propria and submucosa) on the surface epithelium (Fig. 9C). In 30 days exposure mice had a mucous cell metaplasia with increased amounts of mucus substances (Fig. 9D) in the surface epithelium (i.e. intraepithelial mucus substances) including the proximal and distal axial airways in

comparison to the control mice. There was no significant difference in the amounts of intraepithelial mucus substances between control (Fig. 9A) and 5 days exposed mice. The lung parenchyma of mice exposed to airborne PM for 90 days was characterized by accumulations of large numbers of alveolar macrophages, monocytes, and eosinophils, with less numbers of lymphocytes and plasma cells in the alveolar airspace (Table 5). The alveolar septa in these areas of alveolitis were thickened due to type II pneumocyte hyperplasia and hypertrophy, intracapillary accumulation of inflammatory cells, and capillary congestion (Fig. 10A and B). In contrast to the exposure, the longer duration brings relatively more changes in alveolar regions since there are few major emission sources in that direction.

0

5

10

15

20

25

30

35

<0.5 <1 <1.5 <2 <2.5 <3 <3.5 <4 <4.5 <5 <5.5 <6 <6.5 <7

% D

istr

ibu

tio

n

Diameter (mm)

0

5

10

15

20

25

30

35

<0.5 <0.55 <0.6 <0.65 <0.7 <0.75 <0.8 <0.85 <0.9 <0.95 <1

% D

istr

ibu

tio

n

Circularity value


244

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 parameters

Control 5 days 15 days 21 days 30 days 90 days

Cells/ml, 106 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 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

observed in an extensive bronchial-associated 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

A B

C D


245

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

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.


246

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.

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 parameters

Control 5 days 15 days 21 days 30 days 90 days

Cells/ml, 106 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)

Ab

B

C D


247

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 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. Several studies have demonstrated that cigarette 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


248

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

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.


249

COMPETING INTERESTS Authors have declared that no competing interests exist.

REFERENCES 1. Larsson BM, Sehlstedt M, Grunewald J,

Sköld CM, Lundin A, Blomberg A, Sandstro T, Eklund A, Svartengren M. Road tunnel air pollution induces bronchoalveolar inflammation in healthy subjects. J Eur Respir. 2007;29:699-705.

2. Pereira FAC, Lemos M, Mauad T, Saldiva N. Urban, traffic-related particles and lung tumors in urethane treated mice. J Clinics. 2011;66:1051-4.

3. Aaron CP, Chervona Y, Kawut SW, Diez RAV, Shen M, Bluemke DA, Van Hee VC, Kaufman JD, Barr RG. Particulate matter exposure and cardiopulmonary differences in the multi-ethnic study of atherosclerosis. Environ Health Perspect. 2016;124:1166-73.

4. Watkinson WP, Campen MJ, Nolam JP, Costa DL. Cardiovascular and systemic responses to inhaled pollutant in rodents: Effect of ozone and particulate matter. Environ Health Prospec. 2001;4:539-46.

5. Bagate K, Meiring JJ, Gerlofs-Nijland ME, Vincent R, Cassee FR, Borm PJA. Vascular effects of ambient particulate matter instillation in spontaneously hypertensive rats. Toxicol Appl Pharmacol. 2004;197:1-11.

6. Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem. 2001;276:7614-20.

7. Fortoul TI, Rojas-Lemus M, Avila-Casado MC, Rodriguez-Lar V, Montaño LF, Muñoz-Comonfort A. Endogenous antioxidant and nasal human epithelium response to air pollutants: Genotoxic and inmmunocytochemical evaluation. J Appl Toxicol. 2010;30:661-5.

8. James GW, Morishita M, Gerald JK, Harkema JR. Divergent effects of urban particulate air pollution on allergic airway responses in experimental asthma: A comparison of field exposure studies. J Environ Health. 2012;11:2-13.

9. Andrew J, Candice B, Smith CB, Madden MC. Diesel exhausts particles and airway inflammation. Curr Opin Pulm Med. 2012; 18(2):144-50.

10. Ying Z, Xie X, Bai Y, Chen M, Wang X, Zhang X, Morishita QMS, Rajagopalan S. Exposure to concentrated ambient particulate matter induces reversible increase of heart weight in spontaneously hypertensive rats. J Particle Fibre Toxicol. 2015;12(15):2-10.

11. Central Ground Water Board. Ground Water Information Booklet, Mysore District, Karnataka. Ministry of Water Resources, Government of India; 2012.

12. Harish M. Urban transport and traffic management - For sustainable transport development in Mysore city. Int J IT Engg Appl Sci Res. 2013;2:86-92.

13. Ap Gwynn I, Wilson C. Characterizing fretting particles by analysis of SEM images. Eur Cells Mater. 2001;1:1-11.

14. Harrod KS, Mounday AD, Stripp BR, Whitsett JA. Clara cell secretory protein decreases lung inflammation after acute virus infection. Am J Physiol Lung Cell Mol Physiol. 1998;275:924-30.

15. Fauzie AK, Venkataramana GV. Vehicular particulate emissions in Mysore city. Asian J Environ Sci. 2016;11(1):78-86. DOI: 10.15740/HAS/AJES/11.1/78-86

16. Sielicki P, Janik H, Guzman A, Namiésnik J. The progress in electron microscopy studies of Particulate Matters to be used as a standard monitoring method for air dust pollution. Crit Rev Anal Chem. 2011; 41:314-34. DOI: 10.1080/10408347.2011. 607076

17. Ulrich RS, Simons RF, Losito BD, Fiorito E, Miles MA, Zelson M. Stress recovery during exposure to natural and urban environments. J Environ Psychol. 1991;11: 201-30.

18. Kodavanti UP, Mette CJ, Schladweiler AD, Ledbetter RH, David CC, James MS, McGee J, Judy HR, Daniel LC. Pulmonary and systemic effects of zinc-containing emission particles in three rat strains: Multiple exposure scenarios. Toxicol Sci. 2002;70:73-85.

19. Kevin R, Smith JMV, Kodavanti UP, Ann EA, Kent EP. Acute pulmonary and systemic effects of inhaled coal fly ash in rats: comparison to ambient environmental particles. Toxicol Sci. 2006;93:390-9.

20. Saldiva PH, Clarke RW, Coull BA, Stearns RC, Lawrence J, Murthy GG, Diaz E,


250

Koutrakis P, Suh H, Tsuda A, Godleski JJ. Lung inflammation induced by concentrated ambient air particles is related to particle composition. Am J Respir Crit Care Med. 2002;165:1610-7.

21. Kim I, Moon SO, Park SK, Chae SW, Koh GY. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1 and E-selectin expression. Circ Res. 2001;89:477-9.

22. Wellenius GA, Coull BA, Godleski JJ, Koutrakis P, Okabe K, Savage ST, Lawrence JE, Murthy GG, Verrier RL. Inhalation of concentrated ambient air particles exacerbates myocardial ischemia in conscious dogs. Environ Health Perspect. 2003;111:402-8.

23. Campen MJ, Nolan JP, Schladweiler MC, Kodavanti UP, Costa DL, Watkinson WP. Cardiac and thermoregulatory effects of instilled particulate matter-associated transition metals in healthy and cardiopulmonary-compromised rats. J Toxicol Environ Health. 2002;65:1615-31.

24. Kodavanti UP, Schladweiler MC, Ledbetter AD, Watkinson WP, Campen MJ, Winsett DW, Richards JR, Crissman KM, Hatch GE, Costa DL. The spontaneously hypertensive rat as a model of human cardiovascular disease: Evidence of exacerbated cardiopulmonary injury and oxidative stress from inhaled emission particulate matter. Toxicol Appl Pharmacol. 2000;164:250-63.

25. Suwa T, Hogg JC, Quinlan KB. Particulate air pollution induces progression of atheroscrosis. J Am Coll Cardiol. 2002;39: 935-42.

26. Gavett SH, Madison SL, Dreher KL, Winsett DW, McGee JK, Costa DL. Metal and sulfate composition of residual oil fly ash determines airway hyperreactivity and lung injury in rats. Environ Res. 1997;72: 162-72.

27. Kodavanti UP, Jaskot RH, Costa DL, Dreher KL. Pulmonary proinflammatory gene induction following acute exposure to residual oil fly ash: Roles of particle-associated metals. Inhal Toxicol. 1997;9: 679-701.

28. Kodavanti UP, Costa DL, Bromberg PA. Rodent models of cardiopulmonary disease: Their potential applicability in studies of air pollutant susceptibility. Environ Health Perspect. 1998;106:111-30.

29. Nadadur SS, Kodavanti UP. Altered gene expression profi les of rat lung in response to an emission particulate and its metal constituents. J Toxicol Environ Health A. 2002;65:1333-50.

30. Dye JA, Lehmann JR, McGee JK, Winsett DW, Ledbetter AD, Everitt JI, Ghio AJ, Costa DL. Acute pulmonary toxicity of particulate matter filter extracts in rats: Coherence with epidemiological studies in Utah Valley residents. Environ Health Perspect. 2001;3:395-403.

31. Soukup JM, Ghio AJ, Becker S. Soluble components of Utah valley particulate pollution alter alveolar macrophage function in vivo and in vitro. Inhal Toxicol. 2000;12:401-14.

32. Adamson IYR, Prieditis H, Hedgecock C, Vincent R. Zinc is the toxic factor in the lung response to an atmospheric particulate sample. Toxicol Appl Pharmacol. 2000;166:111-9.

33. Vincent R, Bjarnason SG, Adamson IY, Hedgecock C, Kumarathasan P, Guenette J, Potvin M, Goegan P, Bouthillier L. Acute pulmonary toxicity of urban particulate matter and ozone. Am J Pathol. 1997;151: 1563-70.

34. Henderson RE, Benson JM, Hahn FF, Hobbs CH, Jones RK, Mauderly JL, McClellan RO, Pickrell JA. New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fundam Appl Toxicol. 1985; 5:451-8.

35. Lehnert BE, Valdez YE, Stewart CC. Translocation of particulates to the tracheobronchial lymph nodes after lung deposition: Kinetics and particle–cell relationships. Exp Lung Res. 1986;10:245-66.

36. Schmid-Schonbein GW, Seiffge D, DeLano FA, Shen K, Zweifach BW. Leukocyte counts and activation in spontaneously hypertensive and normotensive rats. Hypertension. 1991;17:323-30.

37. Brooke L, Heidenfelder DM, Reif JR, Harkema EA, Hubal C, Edward E, Hudgens LA, Bramble JG, Wagner MM, Gerald JK, Stephen WE, Jane EG. Comparative microarray analysis and pulmonary changes in brown Norway rats exposed to ovalbumin and concentrated air particulates. Toxicol Sci. 2009;108:207-21.

38. Zhong CY, Zhou YM, Kevin R, Smith IMK, Chao-Yin C, Ann EA, Kent EP. Oxidative injury in the lungs of neonatal rats following


251

short-term exposure to ultrafine iron and soot particles. J Toxicol Environ Health. 2010;73:837-47.

39. Deng X, Zang F, Rui W, Long F, Wang L, Feng Z, Chen D, Ding W. PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells. Toxicol In vitro. 2013;27(6):1762-70.

40. Mauad T, Olivera RC, Coimbra AJF, Guimara ET, Andre PA, Kashara DL, Siqueira HM, Saldiva BPHN. Chronic exposure to ambient levels of urban particles affects mouse lung development.

Am J Respir Crit Care Med. 2008;178:721-8.

41. Kotecha S. Lung growth for beginners. Paediatr Respir Rev. 2000;1:308-13.

42. Burri PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: Mcdonald JA, editor. Lung growth and development: Lung biology in health and disease. New York: Marcel Dekker, Inc. 1997;1-32.

43. Schwartz J. Air pollution and children’s health. Pediatrics. 2004;113:1037-43.

© 2017 Venkataramana et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

characterization of particulate matter in urban environments...

Documents