Sent igårkväll publicerades en studie på det vetenskapliga organet Life Sciences som handlar om luftföroreningar. 5 franska forskare från bland annat Universitetet i Lyon (HoloMonitoranvändare) har studerat ämnet Polycykliska Aromatiska Kolväten / Polycyclic Aromatic Hydrocarbons (PAHs) och dess inverkan på människa.Redan i abstractet får vi skrämmande info att 2018 dog 7 miljoner människor av luftföroreningar.
Vad är då PAH?
Wiki berättar :
Polycykliska aromatiska kolväten, även polyaromatiska kolväten eller polyaromater, ofta förkortat PAH av engelska Polycyclic Aromatic Hydrocarbons, är en grupp ämnen som finns i stenkol och petroleum samt bildas vid förbränning av organiskt material. De är aromatiska kolväten med minst två sammankopplade aromatiska ringar som ofta avger en stark lukt. Exponering för material som innehåller PAH kan medföra en ökad risk för cancer.
Eftersom PAH:er finns i petroleum och kol så förekommer de i
stadsmiljöer. De bildas även vid grillning av mat. Olika
råoljeraffineringsprocesser bidrar till att koncentrationen PAH ökar i
vissa slutprodukter. Det är således mycket PAH i bl.a. tung
eldningsolja. Användning av ångkrackers för framställning av eten och
propen ur petroleum(nafta) ger PAH-olja som biprodukt. PAH-oljan används
sedan som tillsats till gummidäck för bil och cykel. Småskalig vedeldning, trafik
inklusive fartygstrafik och rökning är de huvudsakliga källorna till
utsläpp av PAH i luft i Sverige. Spill av eldningsolja, till exempel
bunkerolja till fartyg, ger utsläpp av PAH till mark och vatten.
De 5 forskarna med Dr. Alain Geloen i spetsen har utfört en omfattande studie som berättar hur förödande luftförorening via ämnena i PAH i speciellt stadsmiljö är på människa.
Dr. Alain Geloen är f.ö. en varm förespråkare av HoloMonitor och finns med på PHI`s hemsida.
I studien lyfter forskarna fram HoloMonitor inte mindre än 16 gånger vilket visar på deras syn på detta excellenta instrument.
Men till studien då.
by
Yuri Lima de Albuquerque ,
Emmanuelle Berger
, Sophie Tomaz
, Christian George
and Alain Géloën
Published: 6 April 2021 Abstract
In 2018, seven million people died prematurely due to exposure to
pollution. Polycyclic aromatic hydrocarbons (PAHs) are a significant
source of secondary organic aerosol (SOA) in urban areas. We
investigated the toxic effects of by-products of naphthalene SOA on lung
cells. These by-products were 1,4-naphthoquinone (1,4-NQ),
2-hydroxy-1,4-naphthoquinone (2-OH-NQ), phthalic acid (PA) and
phthaldialdehyde (OPA). Two different assessment methodologies were used
to monitor the toxic effects: real-time cell analysis (RTCA) and the
Holomonitor, a quantitative phase contrast microscope. The chemicals
were tested in concentrations of 12.5 to 100 µM for 1,4-NQ and 1 to 10
mM for 2-OH-NQ, PA and OPA. We found that 1,4-NQ is toxic to cells from
25 to 100 µM (EC50: 38.7 µM ± 5.2); 2-OH-NQ is toxic from 1 to 10mM
(EC50: 5.3 mM ± 0.6); PA is toxic from 5 to 10 mM (EC50: 5.2 mM ± 0.3)
and OPA is toxic from 2.5 to 10 mM (EC50: 4.2 mM ± 0.5). Only 1,4-NQ and
OPA affected cell parameters (migration, motility, motility speed and
optical volume). Furthermore, 1,4-NQ is the most toxic by-product of
naphthalene, with an EC50 value that was one hundred times higher than
those of the other compounds. RTCA and Holomonitor analysis showed a
complementarity when studying the toxicity induced by chemicals.
Keywords:
PAHs; secondary organic aerosols; RTCA; Holomonitor; naphthoquinone; A549
1. Introduction
In 2010, the World Health Organization (WHO) expressed concerns regarding the effects of several air pollutants on health.
In 2018, the WHO estimated that 7 million people suffered premature
death caused by exposure to fine particles in polluted air. These
particles can penetrate deep into the lungs and cardiovascular systems,
causing diseases including stroke, heart disease, lung cancer, chronic
obstructive pulmonary diseases and respiratory infections, including
pneumonia.
Naphthalene,
a ubiquitous polycyclic aromatic hydrocarbon (PAH), is one of the
several air pollutants that affect the human health. Naphthalene is a semi-volatile organic compound (SVOC) according to the U.S. Environmental Protection Agency classification,
and it has a series of anthropogenic sources, such as chemical
industries, gasoline evaporation or oil burning, leading locally to
acute exposures.
In urban areas, vehicle emissions represent the most important source,
but naphthalene is also present in the smoke of cigarettes.
Once in the air, naphthalene undergoes atmospheric oxidation, mainly by
reacting hydroxyl radicals (•OH), and thereby acts as a precursor of
secondary organic aerosols (SOA). These ultrafine particles have a
complex chemical composition and therefore possess chemical complexity
and yield products such as 1,4-naphthoquinone (1,4-NQ), 2-hydroxy-1,4
naphthoquinone (2-OH-NQ), phthalic acid (PA), phthaldialdehyde (OPA).
Studies on lung cell lines showed that 1,4-naphthoquinone (1,4-NQ) is toxic.
Quinones are a class of organic compounds with chemical properties
allowing them to interact with biological targets by forming covalent
bonds and transferring electrons in oxidation–reduction reactions. Among
all quinones, 1,4-NQ and its secondary products are of particular
interest because of their occurrence as natural chemicals and/or
products. These compounds have diverse pharmacological properties, such as antimicrobial, antiviral, antiprotozoal and anthelmintic, also presenting cytotoxic effects on cancer cell lines.
Moreover, 2-hydroxy-1,4-naphthoquinone (lawsone) (2-OH-NQ), a
photochemical reaction product of 1,4-naphthoquinones, can also be
extracted from the leaves of Lawsonia inermis.
It is one of the most widely used skin dyes, commonly known as Henna.
It has been intensively utilized for many years in the biology,
medicine, agriculture and industries as a skin dye and antifungal.
Regarding its toxicity, Sauriasari et al. concluded that lawsone is not
mutagenic, but it is toxic to cells in a dose-dependent manner.
Phthalic acid (PA) is a benzene dicarboxylic acid with no reports on its toxic effects on the respiratory system.
Phthaldialdehyde (OPA) is a dialdehyde used mostly as a fluorescent
marker or a disinfectant, but in vivo experiments proved OPA to be toxic
to the respiratory systems of rats.
To
study the cytotoxic effects of chemicals, the colorimetric MTT
(tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide)) assay, is widely used. However, this technique only shows the
end-points and not the stage of cell proliferation.
Nowadays, more sophisticated approaches are available. Real-time cell
analysis (RTCA) allows the label-free and real-time monitoring of cell
proliferation and viability. It monitors the viability of cultured cells
using electrical impedance. The continuous monitoring of cell
proliferation makes it possible to recognize different perturbations of
cell viability, such as toxicity (cell death) and reduced proliferation
(cell cycle arrest). Technical improvements also allow the creation of
3D reconstructive images of cells and the measurement of the structural
and behavioral parameters of cells.
Similarly, the Holomonitor microscope performs label-free and
non-invasive measurements with the use of a low-power laser. Cell
structural (area, optical volume and perimeter) and behavioral
(motility, motility speed and migration) parameters can be measured in
real time with this microscope.
Bearing in mind
the importance of ultrafine particles derived from PAH photo-oxidation
in urban areas, we studied here the toxicity of the main by-products of
naphthalene SOA using the abovementioned new methodologies to determine
their impacts on lung cells.
Materials and Methods (urval)
2.3. Measurements of Cytotoxicity
2.3.1. Effects on Cell Proliferation
Real-time
cell analysis system (RTCA) (Agilent, Santa Clara, CA, USA) measures
cell indexes resulting from cell surface occupancy, taking into
consideration cell number, cell size and adhesion force. In the case of
cytotoxicity, a decrease in cell index can result from a decreased cell
number (=cell death), a decrease in cell adhesion or a decrease in cell
surface. Cell surface and number can be measured by the Holomonitor.
RTCA results are represented as delta cell indexes (differences in final
− initial cell index = time of treatment). In order to measure the
cytotoxic response of A549 cells in real time, cells were seeded on gold
microelectrodes embedded at the bottom of 96-well microplates
(E-plates; Roche Diagnostics, Basel, Switzerland) at a density of 2500
cells/well. The impedance was recorded at 15-min intervals for 24 h in a
standard 37 °C cell culture incubator with 5% CO2. The EC50 of each compound was calculated using the RTCA data with Excel.
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 platform was housed in a standard 37 °C cell culture
incubator with 5% CO2. The behavioral (motility, motility
speed and migration) and structural (area, optical volume and perimeter)
parameters were measured from 3D reconstructed images obtained for 10 h
with intervals of 5 min between measurements.
2.4. Statistical Analysis
Data
presented are representative of experiments performed at least in
triplicate, as mean values ± SEM for RTCA and Holomonitor (n = 3 for 96-
and n = 8 for E96 wells). Statistical analysis was performed with
StatView 4.5 software (Abacus Corporation, Baltimore, MD, USA) for
Windows. Data were analyzed using one-way ANOVA followed by Fisher’s
protected least significance difference (PLSD) post hoc test.
Significance level was accepted at p < 0.05.
3.2. Effects of Naphthalene SOA By-Products on A549 Behavioral and Structural Parameters
To
determine whether the by-products of naphthalene SOA alter the
structure and behavior of cells, A549 cells were exposed to
concentrations corresponding to the EC50 observed for cell
proliferation, i.e., 1,4-NQ (50 µM), 2-OH-NQ, PA and OPA (5 mM). Cells
were monitored by means of the Holomonitor for 10 hours post-exposure to
the chemicals.
Firstly, 1,4-NQ impacted the
optical volume, migration, motility and motility speed of the cells,
while their area and perimeter did not differ from the control. Optical
volume showed a significant decrease when compared to the control in the
last 2 h of the experiment (Figure 3A–C).
Migration of cells treated with 1,4-NQ showed a significant decrease
when compared to the control after 5 h, as did motility and motility
speed after 3 h (Figure 3D–F).
The curve for migration, differing from the control, did not evolve
during the experiment. Motility, however, slightly increased throughout
the experiment but remained much lower than the control.
To better determine the effects of the different
chemicals on cell structure and behavior, we used the Holomonitor
microscope. To identify their cellular effects, compounds were tested at
concentrations corresponding to their EC50. The cell index produced by
RTCA depends on the surface occupied by the cell and their adhesion
strength. The surface occupied by cells results from cell number and
cell size. The Holomonitor measures different cell parameters, such as
surface, perimeter and volume. The fact that the cell index was
significantly decreased at the EC50 for 1,4-NQ and OPA, but the area and
perimeter of cells were not changed, points out an effect on cell
adhesion; both chemicals decreased the optical volume of cells without
affecting cell perimeter and area.
The
Holomonitor also produces effects on cell behavior, such as motility,
motility speed and migration. Firstly, 1,4-NQ and OPA significantly
decreased all these parameters while 2-OH-NQ and PA did not alter them.
Cell migration is intrinsically connected to the upregulation of the
signal transducer and activator of transcription 3 (STAT-3). Quinones derived from 1,4-NQ are recognized for decreasing the expression levels of this protein,
which could explain the decrease in all behavioral parameters
associated with 1,4-NQ. It is also interesting to note that both 1,4-NQ
and OPA induced a decrease in the migration, motility and motility
speed.
When comparing RTCA and the Holomonitor, both methods are real-time,
without labelling, and useful for assessing the toxicity of all
components. However, as RTCA produces an impedance value reflecting cell
proliferation, it can be affected by cell surface and adhesion
strength. In this manner, the Holomonitor brings a complementary method
of determining the impact of a chemical on cells.
5. Conclusions
In
conclusion, 1,4-NQ is the most toxic by-product of naphthalene, with an
EC50 value that is one hundred times higher than those of other
compounds, inducing structural and behavioral alterations in A549 cells.
Despite their similar EC50 values on RTCA, 2-OH-NQ and PA showed no
alteration in cell parameters, while OPA significantly altered optical
volume, motility, motility speed and migration.
The present study
highlights the perfect complementarity of RTCA and the Holomonitor
microscope to study alterations induced by chemicals.
Min kommentar
HoloMonitor ånyo i miljöns tjänst kan vi notera.Utöver de ruggiga konstateranden och resultat ämnet PHA har på människa har den gode Dr Alain Geloen smugit in en brasklapp (mitt nya favoritord) jag själv tidigare konstaterat. Nämligen att HoloMonitor trivs förträffligt tillsammans med Agilents instrument xCELLigence. I studien använder man förkortningen RTCA istället för xCELLigence Real-Time Cell Analysis som är dess fullständiga namn.
Här kan man läsa mer om RTCA hos Agilent.
Dr. Geloens slutkläm jag skriver under på :
" The present study
highlights the perfect complementarity of RTCA and the Holomonitor
microscope to study alterations induced by chemicals. "
Bjässen Agilent Technologies kanske borde slå en pling till VD Egelberg innan nån annan aktör skaffar sig första tjing på HoloMonitor.
Å oss phi,ares vägnar till Dr. Geloen et al avslutar jag med : Vive la France !
Mvh the99
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