I ett samarbete mellan franska och israeliska forskare har dessa igår 10/8 fått sina studier publicerade i det vetenskapliga organet Atmosphere,en division till det mer kända organet MDPI ,“Molecular Diversity Preservation International”.Det handlar om en miljörapport som i allra högsta grad har aktualitet i dagens allmänna miljökonsekvensresonemang.Forskaren
The Toxic Effect of Water-Soluble Particulate Pollutants from Biomass Burning on Alveolar Lung Cells
Abstract
In 2018, 3.8 million premature deaths were attributed to exposure to biomass burning nanoparticles from wood combustion. The objective of this study was to investigate and compare the toxic effect of wood-combustion-related biomass burning nanoparticles from three different combustion stages (i.e., flaming, smoldering, and pyrolysis) on alveolar lung cells, by studying cell proliferation, and structural and behavioral parameters. A549 lung epithelial cells were treated with 31, 62, 125, 250, and 500 µg/mL of water-soluble particulate pollutants from wood burning, and measured by means of real-time cell analysis, cell imaging, and phase imaging microscopy. At low concentrations (31 and 62 µg/mL), all three types of wood burning samples exhibited no toxicity. At 125 µg/mL, they caused decreased cell proliferation compared to the control. Exposure to higher concentrations (250 and 500 µg/mL) killed the cells. Cell physical parameters (area, optical volume, eccentricity, perimeter, and optical thickness) and behavioral parameters (migration, motility, and motility speed) did not change in response to exposure to wood burning materials up to a concentration of 125 µg/mL. Exposure to higher concentrations (250 and 500 µg/mL) changed cell perimeter, optical thickness for smoldering and flaming particles, and led to decreased migration, motility, and motility speed of cells. In conclusion, all three of the combustion water-soluble organic pollutants were identified as equally toxic by real-time cell analysis (RTCA) results. The parameters describing cell structure suggest that pyrolysis particles were slightly less toxic than others.1. Introduction
The
World Health Organization (2018) estimates that 3.8 million premature
deaths per year are due to exposure to environmental pollution,
especially particulate matter (PM).
These nanoparticles (NPs) arise from household air pollution,
industrial and traffic sources, and wildfires. In fact, around 3 billion
people still cook or heat their houses using solid fuels due to
economic conditions, and wood is the main fuel used for domestic biomass
combustion.
While wood is often considered as a renewable fuel, its combustion
produces primary nanoparticles more efficiently than oil or natural gas
burning systems. As of 2005, domestic biomass combustion was responsible for more than 45% of PM2.5 over Europe.
Such particulate pollution causes not only lung-related diseases, such
as pneumonia, chronic obstructive pulmonary disease, stroke, lung
cancer, and cardiovascular diseases, but also negatively affects
cognitive functioning among the elderly.
Under
the changing climate and land use change, the frequency of wildfires
gradually increased worldwide. Large fires are frequently reported in
countries such as Canada and the USA, Australia, Brazil, and Indonesia.
These fires can exert impacts ranging from regional to global on
climate and air quality, in contrast to household pollution. Global
exposure to wood-related aromatic compounds is inevitable. These
compounds have a short lifetime in the atmosphere (several hours up to a
few days) and they are ubiquitously present in fog and precipitation.
In addition, two different subfractions (water-soluble vs.
organic-soluble) of wood fuel pyrolysis brown carbon (BrC) were tested
and shown to be toxic to epithelial lung cells.
Wood
burning can be described by three identified combustion stages:
flaming, smoldering, and pyrolysis. Flaming is defined by the burning of
wood with flames or complete combustion, while smoldering is the slow
and incomplete burning of wood, with low-temperatures, and is flameless.
Pyrolysis is an intricate process that is not yet fully understood, but
it is the genesis for both flaming and smoldering conditions.
In
vitro studies have been performed with lung cell lines, demonstrating
that wood-burning biomass NP is toxic, due to the presence of phenolic
compounds, organic peroxides, and polycyclic aromatic hydrocarbons
(PAH). PAH are considered as human carcinogens, with well-documented
mechanisms of action that involve the production of reactive oxygen
species (ROS).
In addition, in vivo studies were performed to establish which burning
condition produces the most toxic outcome, but the results were
inconclusive, with different studies highlighting different combustion
conditions.
Forest
fires are unexpected, and smoke can be transported over large
distances. Hence, it is almost impossible not to be exposed to smoke
particles and gaseous species. It is critical to understand what the
most toxic mechanisms of action of biomass burning particles are, in
order to prevent their adverse health effects. Three different but
complementary methods were used to determine the cytotoxicity of
particles. All three methods are without labelling, and are conducted in
real-time. First, real-time cell analysis (RTCA) allows for the
monitoring of cell proliferation and viability. It analyzes cell
viability using electrical impedance. Second, the quantitative phase
microscope Holomonitor produces 3D reconstructive images of cells, and
measures the structural and behavioral parameters of cells, such as
their area and migration. Third, the multi-mode brightfield microscope
Cytation 3 provides images of cells, allowing the visualization of cell
division and death.
All three, flaming, smoldering and pyrolysis stages, can occur during
wood fires. In this study, we focused on their effects on cell
proliferation, which is an important feature of the maintenance of
epithelia, and on the effects on the cell structure and cell behavior of
epithelial lung cells.
2. Materials and Methods (urval)
2.3.2. Quantitative Phase Imaging
Quantitative
phase imaging was performed using the Holomonitor M4 digital
holographic cytometer (DHC) from Phase Holographic Imaging (PHI, Lund,
Sweden). The microscope was placed in a standard 37 °C cell culture
incubator with 5% CO2. The microscope measured eight
parameters obtained from 3D reconstructed images, every 10 min for 10 h:
area, perimeter length, optical thickness, optical volume,
eccentricity, migration, motility, and motility speed.
2.4. Statistical Analysis
The
figures presented here depict three experiments performed
independently. Data are presented as mean values ± SEM for RTCA and
Holomonitor (each value represents the average of eight wells).
Statistical analysis was performed with Stat View 4.5 software (Abacus
Corporation, Baltimore, MD, USA) for Windows; the data were analyzed
using one-way ANOVA followed by Fisher’s protected least significance
difference (PLSD), post hoc test. Differences were considered to be
significant at a probability level of p ≤ 0.05.
3.2. Effects of Water-Soluble Particulate Pollutants from Biomass Burning on A549 Behavioral and Structural Parameters
Cells
were monitored with a Holomonitor, a quantitative phase contrast
microscopy for non-invasive analysis of cellular events by long-term
digital phase imaging, allowing for the quantification of behavioral and
structural parameters. The analyses were performed for 10 h, and
results are presented as the mean of the last two hours.
4. Discussion
The present study is also an opportunity to compare three different
methods for cell analysis: RTCA, phase imaging microscopy, and
time-lapse microscopy. The three methods provide real-time monitoring of
the cells without labelling. Each process has its own advantages and
drawbacks. RTCA is based on impedance measurements, which are affected
by the surface of electrodes covered by the cells and the adhesion
strength of the specific cell line. Cell size is rather constant and
homogeneous during cell proliferation. Adhesion strength is a
characteristic of the cell line. Under normal conditions, it is not
expected to change; however, the possibility that adhesion may be
altered by various chemicals cannot be excluded. For this reason, the
results obtained from RTCA were cross-checked using phase imaging
microscopy (Holomonitor), which measures numerous cell parameters, such
as cell volume and mobility, which are normally difficult to measure.
5. Conclusions
Materials
extracted from biomass burning particles from three combustion
processes did not exhibit differences in toxicity with respect to the
cell index of A549 cells, exerting toxic effects on cells at 125, 250,
and 500 µg/mL. All three types of water-soluble particulate pollutants
from biomass burning caused a decrease in the migration, motility, and
motility speed at 250 and 500 µg/mL, with the exception of PWS, which
did not cause a decrease in motility for 250 µg/mL, as measured by a
Holomonitor for 10 h. Overall, the particles from FWS and SWS were the
only ones that affected cell structure by increasing the cell optical
thickness (FWS and SWS 500 µg/mL,
and decreasing the perimeter length (FWS and SWS 500 µg/mL).
Furthermore, the different real-time and label-free methods, used here
to study the toxicity of these particles, shared an essential
complementarity to properly assert toxicity studies of atmospheric
pollutants. Our results demonstrate that a short but intense exposure to
water-soluble particulate pollutants from biomass burning may exert
long-term, persistent, deleterious effects.
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