Characterization of Urban Traffic Particulate Matter

and Its Pulmonary Effects in Mouse Model

G.V. 1,2
Venkataramana , 1
Sreenivasa , Azis, K. 1
Fauzie , Amruta 2
Nori-Sarma , and Michelle Bell2

1 Department of Studies in Environmental Sciences, University of Mysore, Mysore, Karnataka, INDIA

2 School of Forestry and Environmental Studies, Yale University, New Haven, Connecticut, USA

ABSTRACT
Introduction: Traffic is a major contributor to air pollution in Indian cities, and traffic-related
exposure has been shown to induce acute inflammation in humans, both in chamber studies
using diesel exhaust and in real-life environments such as road traffics. In urban centers,
diesel exhaust particles are considered to be the hazardous pollutants released from
automotive engines due to their aerodynamic and chemical characteristics.
Methods: Elemental composition and size distribution of particulate matter (PM) were
analyzed using Energy Dispersive X-Ray (EDX) and Dynamic Light Scattering (DLS) method,
respectively. Preparation of BAL was done following Harrod’s protocol with some
modifications (Harrod et al., 1998). The mice were exposed to particulate pollution for
5,15,21,30 and 90 days (each 6 hours/day) in inhalation chambers placed 2.5 m from a street
with heavy traffic in Mysore city. Total and differential cell count and viability were determined
using an improved Nebur’s hemocytometer and optical microscope with a 400x zoom.
Results: DLS and EDX analysis found that roadside PM was mainly fine particles (PM2.5) and
consists of 56% black carbon that generated mostly from vehicular emissions. The samples
also contain trace metal elements like Si, Fe, Ca, Al, Na and K. There was a significant
reduction of the bronchoalveolar lavage lymphocyte cell numbers (8.75) after 30 and 90 days
exposure. This inflammation has elevated expression of neutrophils, monocytes and
eosinophils compare to control. Percentage viability of leucocytes has decreased and
differential cell of macrophages and neutrophils were increased in BAL fluid following mice
exposure to PM2.5 in different intervals.
Conclusions: The exposure to the mixture of suspended particulate matter particles induces
pathological changes, differential cell counts and inflammatory response in the lungs of mice
in a dose and duration dependent pattern. The responses observed from the present study
are associated with the bioavailability of inhaled particulate mixtures.

INTRODUCTION
• Air pollution is an emerging biggest public health threat in developing countries especially in India and China.
• As per recent estimates around 4·2 million deaths globally were attributed to long-term air pollution exposures, which represent 7·6% of total global mortality.
• Air pollutants have direct effect on cardiopulmonary health. Several studies worldwide have reported that exposures to ambient air pollution accelerates lung function decline in adults and
increase risk for COPD; impairs lung development in children; significantly increases risk for cardiovascular diseases. Additionally, recent reports also suggest causal role of air pollution in
obesity, metabolic syndrome and diabetes, which is growing to an epidemic proportion in India.
• Air pollution levels in major Indian cities are 2-5 times higher than Western world. In urban settings, automobile emission followed by industrial emission are the major contributors of PM.
• Although air pollution exposures are linked with adverse respiratory health, the key components of PM and underlying cellular and molecular mechanism are not well understood. Our long-
term goal is to identify the key components in particulate matter that elicits toxic responses and eventually leads to lung injury.
• This study was conducted in Mysore city located 135 Km south of Bangalore, Karnataka, India. Mysore is a moderate size city with 128 sq Km area and approximately 1 million people. As
per the Government of India, Mysore city was awarded as cleanest city in India in 2014-2015. Despite being tagged as a clean city, the levels of PM2.5 ranged from 25-30 ug/m3. We carried
this study to characterize the composition of Mysore PM and its effect on pulmonary inflammation in mouse models.

MATERIALS AND METHODS

• The study area is located at Mysore city that lies at 12°18’25“ N and 76°38’58” E. Like many
other Indian cities, Mysore has also high vehicular growth and emissions problem. Six sites
were selected as the sampling points comprising high, moderate and low traffic area.
• Elemental composition of PM was analyzed using Energy Dispersive X-Ray (EDX) method
by measuring X-rays emitted from the sample during bombardment by an electron beam.
• Particle size distribution was determined using Dynamic Light Scattering (DLS) method by
measuring random changes in the intensity of light scattered from a liquid suspension.
• The mice exposures were performed from Jul to Oct 2015, using two inhalation chambers
installed on the traffic roads. Mice were exposed to 6 h/day for 5, 15, 21, 30 and 90 days.
This study was approved by the Ethics Committee for Research, University of Mysore at
the number of UOM/IAEC/05/2013. Following exposure, mice (n=4-5) were sacrificed using
chloroform and blood was collected for serum preparation. Bronchoalveolar lavage (BAL)
assay and differential cell counts were performed to quantify lung inflammation. The lungs
were inflated and fixed with 10% formalin for histopathological analysis.

RESULTS
EDX analysis found that ambient particulate matter collected from roadside sampling site
consists of 56.38% carbon, 33.66% oxygen, and other elements in smaller fraction such as
silicon, iron, aluminium, calcium, sodium, and potassium (Fig. 1). Maximum number of
particles suspended in the DLS sample container has aerodynamic diameter of 318 nm (Fig.
2). Peak summary report from DLS analysis also indicates that 97.4% of the total sample
volume contains particles with an average diameter of 275.8 nm.
Fig. 3 shows sparse, mild foci of macrophages (A) represents alveolar parenchyma in 30 days
exposed mice. Alveolar spaces (B) are enlarged, hypertrophy and irregular (a and b) in lung
parenchyma of 90 days exposed mice. Accumulation of particles within alveolar macrophages
was readily apparent in lung parenchyma (C) and in lymph nodes (D) of 90 days exposed
mice. There was a significant variation of BAL cell counts after 30 and 90 days exposure; this
inflammation has elevated the expression of neutrophils and macrophages (Table 1).

A B b Table 1

C D

Fig. 1 Fig. 2 Fig. 3

Conclusions and Limitations

• Study in PM elemental composition using energy dispersive X-ray analysis has generated important findings that majority (56%) of urban roadway particles are carbon-rich particles – widely
known as black carbon – emitted mostly by fossil-fueled vehicles.
• PM concentrations were found 2 to 4 times higher in commercial areas compared to industrial and residential areas, and are considerably correlated with number of urban vehicles and
atmospheric temperature.
• The effect of SPM2.5 exposure for a period (5,15,21,30 and 90 days) in mice, focusing on the development and pathophysiological consequences of the inflammatory response and
hypothesized that chronic exposure of mice to SPM would result in significant airway and lung parenchymal inflammation and changes in the alveolar structure.
• Future studies will seek to further study in the characterization and composition of particulate matter, using the pilot data as a demand surface for allocation of further sampling sites. While this
represents one of the first known systematic study of composition and distributions of air pollution sampling for exposure assessment in urban India, the low sample size of the pilot study
represents a limitation.
• Future studies will continue to compare multiple sampling technologies, as well as conducting sensitivity analyses through comparison with ongoing long-term air pollution, its composition and
impacts on biological systems.

Acknowledgements
We thank University Grants Commission (UGC) for awarding Raman Postdoctoral Fellowship to VGV and acknowledge funding from Science and Engineering Research Board (SERB),
Department of Science and Technology.