Forskningsrapport från Frankrike

Oscar mejlar över en nyuppgrävd studie från Frankrike, en gedigen doktorsavhandling kommen från en av Dr Alain Géloën´s adepter,Yuri Lima de Albuquerque. Ämnet för avhandlingen är då såklart luftföroreningar och dess påverkan på människa.

Which atmospheric nanoparticles trigger healthdiseases?

Abstract 

In 2017 around 5 million people had premature deaths due to air pollution. Our objectives were to identify which atmospheric nanoparticles cause negative effects on human health, their mechanisms of action and how to prevent them. 

Two types of particles were tested, naphthalene (anthropogenic origin) and particles from biomass burning (from both anthropogenic and natural origin). 

Three different methodologies were used: real time cell analysis, a measure of cell proliferation and adhesion; quantitative phase contrast microscope (Holomonitor, PHI), that gives behavioral and structural parameters of cells (e.g. area, motility and migration) and bright field real time microscopy (Cytation, Biotek), for real time cell imaging. 

Fresh naphthalene nanoparticles were more toxic than aged. Glutathione at 1 mM partially restored cell proliferation. Four by-products of naphthalene nanoparticles were tested on lung cell model A549: 1.4-naphthoquinone (1.4-NQ); 2-hydroxy-1.4-naphthoquinone (2-OHNQ), phthalic acid (PA) and phthaldialdehyde (OPA). 

According to their EC50, 1.4-NQ showed a toxicity 100 times higher than other compounds. 1.4-NQ and OPA decreased the optical volume, migration, motility and motility speed of cells. 1.4-NQ increased the oxidative stress (DHR staining). Addition of glutathione protected the cells against 1,4- NQ. 

The toxicity of particles from different mode of burning wood (flaming (FWS), smoldering (SWS) and pyrolysis (PWS)) were similarly toxic on A549 cell proliferation. In conclusion, toxicity of naphthalene nanoparticles depends on 1,4 naphthoquinone which is mediated through oxidative stress. 

Key-words: RTCA, holomonitor, naphthalene, 1,4-naphthoquinone, glutathione, biomass burning.

Den observante noterar att holomonitor finns med i nyckelordsangivelsen.Det visar sig att forskaren har hela 59 hänvisningar till instrumentet i sin studie.Jag kommer inte att radda upp samtliga av utrymmesmässiga skäl.Värt att notera i samma observation är att HoloMonitors kompis finns med, Agilentas RTCA, Real-Time Cell Analysis.

1 Introduction 

In 2017 around 5 million people had premature deaths due to air pollution, according to the Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD, 2017), an epidemiological study comparing results of 145 countries led by the researches at the World Health Organization and based at the Institute for Health Metrics and Evaluation (IHME)/University of Washington. 

This number previously cited for air pollution deaths is higher in 2017, for example, than child malnutrition (3.1 million of deaths) or alcohol related deaths (2.8 million of deaths), or even illegal drugs abuse (585 thousand of deaths). 

That is a growing factor for health problems according to this epidemiological study, when showing that in the leading causes of premature deaths, Ambient Particulate Matter (particulates in suspension in air) occupied the 17th position on 1990, then the 13th in 2007 and the 10th on 2017 (GBD, 2017). 

In addition, the Global Burden of Diseases, Injuries, and Risk Factors Study 2017 adds a list of the 10 leading risks for premature death, where air pollution is the only environmental, the others being behavioral or metabolic.

 

In the 10 leading risks for premature death in 2007, unsafe water source was the 9th leading cause, being lowered to 14th by 2017, showing that the world’s population does not share the same preoccupations about the quality of water and air. 

Human activities are the main determinant for intensifying a range of environmental issues (Wang et al., 2013a). An increasing concentration of people and economic activities in emerging megacities has been accompanied by a growing in motor vehicle and factories emissions (Atash, 2007; Zhang et al., 2015a), a development that has led to rapid increases in energy consumption, exhaust emissions, and consequently air pollution, especially in developing countries (Tanaka, 2015). 

 

There is no doubt that ambient air pollution, which constitutes an increasing environmental concern, poses a huge range of negative health implications at local, regional, and global scales (Grimm et al., 2008), including, but not limited to, higher instances of cardiorespiratory diseases (lower respiratory infections), cancer (tracheal,bronchus and lung cancer), ischemic heart disease, ischemic stroke, intracerebral hemorrhage, type 2 diabetes mellitus, among other diseases in urban populations (Vella et al., 2015; Eze et al., 2015; GBD, 2017; Boudrel et al., 2017; Khilnani & Tiwari, 2018). 

These impacts incur real costs for individuals, medical systems, and economies (Matus et al., 2012).

 

Air pollution is a worldwide recognized threat to health, since it has been associated with many diseases, including increased mortality and morbidity rates but also with ecosystems damage, impacts on the built environment and the climate (EEA, 2017; Landrigan et al., 2018). 

Some air pollutants can persist for long periods of time and accumulate in the environment and food chain, thus affecting humans and animals via multi-route of exposure (inhalation, dermal, ingestion). According to the European Environment Agency (EEA), transports, industries, energy, power plants, agriculture, households and waste management are the economic sectors that contribute the most to air pollution (EEA, 2017). 

So far, several epidemiological studies had affirmed that air pollutants cause health diseases, diseases which are growing in number every year and becoming more and more problematic in a world scale. But it still rests unknown: what are these pollutants and why are they toxic, with the mechanisms of toxicology still to be studied. To start understanding these questions we first have to identify what are the toxic compounds produced by pollution.

3 Methods (urval)

3.1.5.1 Single cell lines 

The cells were seeded at the amount of 2500 cells per well of 96-wells e-plates for cell proliferation assay (xCELLigence, RTCA, Agilent, Santa Clara, CA, USA) and 96-wells plates for imaging in Holomonitor (PHI, Boston, MA, USA) and Cytation 3 (Biotek Instruments, Winooski, VT, USA), and 150.000 cells per 0.4µm PET membrane inserts (Thincert cell culture insert for 12-wells plate, sterile, translucent membrane (pet) (Greiner Bio-One, Courtaboeuf, France) for Vitrocell experiments, all under conditions of 5% CO2 and 37°C.

3.3.2 Microscopy

3.3.2.1 Holomonitor 

Holomonitor M4 (PHI, Boston, MA, USA) is a label-free imaging microscope that uses a low power laser to analyze cells for extended periods of time. 

From 3D images collected with the low power laser, Holomonitor allows the study of dose response assays and kinetic morphology, given by behavioral and structural parameters, such as area, optical volume, perimeter length, migration, motility, and motility speed (Zhang & Judson, 2018; Lajkó et al., 2018; Berlin et al., 2021). 

Cells were plated in 96-wells plates and were able to proliferate for 48h under control conditions. 

After the addition of treatment (naphthalene by-products or biomass burning mass wood nanoparticles dissolved in 150 µL culture medium), the plate was placed in the microscope to initiate the analysis. The plates were covered with six HoloLids (PHI, Boston, MA, USA), lids specially designed to eliminate image disturbances caused by condensation inside the cell culture vessel. 

For every experiment, 3 to 5 wells were analyzed for each treatment. Time-lapse changed according to the experiment: 5 min intervals during 10 h of experiment for naphthalene by-products and 10 min interval for 10 h for the experiments on burning mass wood nanoparticles. 

After the experiment ended, the data were collected in Excel files for analysis.

 

Toxicity will be determined by changes on the cell index, parameters of cell monitored by Holomonitor and results of fluorescence of zombie red (viability of cell membrane) or DHR (oxidative stress production) when statistically different from control. 

 

4.2.2. 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 index, 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. 

As for the cell index assay, the effect of DMSO was also tested with Holomonitor to observe if the toxic effect induced by the chemicals was not induced by DMSO 0.1% alone. 

Results show that DMSO 0.1% did not affect any of the studied parameters (area, optical volume, perimeter length, migration, motility, and motility speed).

The results for the toxicity of naphthalene NPs and its by-products were obtained with real time cell analysis (RTCA). This system uses the cell index, a unit given by the occupied area of gold-microelectrodes present inside each well of the E-plates. 

Cell index is given by the occupied area of the microelectrodes, which, in its turn, depends on the number of cells, their size or their adhesion strength. RTCA is proven to be a good method to study the toxicological effects of nanoparticles and/or chemicals because cell number is directly related to cell division, thus, critical for epithelium renewal (Altmann & Enesco, 1967). 

However, since RTCA depends on three different cell characteristics (number, size and adhesion) to establish the cell index, the determination of which parameter affected the cell index is unknown. 

The system does not show which of the three previously cited characteristics affected the cell index.

Using RTCA alone, it is not possible to determine if the cell number, size or adhesion were changed. 

To complement the results of RTCA and go even further in our research, we used the Holomonitor microscope.

The four naphthalene NPs by-products, compounds were tested with Holomonitor at concentrations corresponding to their EC50. 

Holomonitor is a quantitative phase contrast microscope that uses a lower-power laser to analyze cells.

 It produces 3D pictures that are later utilized to obtain the quantification of several parameters, such as area, perimeter and optical volume (structural parameters) and migration, motility and motility speed (behavioral parameters), in real time and without any labelling. 

Regarding the structural parameters, the area and perimeter of cells were not affected by 1,4-NQ, but the optical volume was decreased (Figure 35). This indicates a change in the shape of the cell, where the optical volume would be able to change without interfering in the area. 

The change in the size of the cell can be related to a loss of local adhesions. Mechanisms linking 1.4-NQ with a decrease in cell adhesion have never been reported, however one study with furano-1,2-naphthoquinone (Su et al., 2010) and one study with a naphthoquinone-coumarin hybrid (NPQ-C6) (MartínRodríguez et al., 2019) stated that both quinones suppressed the activation of BCRABL1/STAT5, two proteins connected to cell adhesion. 

RTCA or Holomonitor do not precise the effect on the adhesion of cells, even if a change in it may interfere in the cell index. Although studies relating 1,4NQ and cell adhesion have not been performed yet, Wang et al. (2013b) studied the effect of shikonin, another naphthoquinone, on the cell adhesion of A549 cells. They have found that shikonin at 1.5 µM have decreased A549 cell adhesion to extracellular matrix by inhibiting integrin β1 expression.

Migration, motility and motility speed are closely related as parameters of Holomonitor.

The effect on cells of the three other chemicals: 2-OH-NQ, PA and OPA, were also measured using Holomonitor.

6 Conclusions and perspectives 

My thesis brings light to the study of the toxicity of atmospheric nanoparticles and its effects on the human health. 

In conclusion, our study showed that anthropogenic naphthalene nanoparticles are toxic to in vitro cell lines, using RTCA. 

This toxicity is mainly caused by 1,4-naphthoquinone (EC50 31.8 µM), the most toxic by-product of naphthalene. 

Both naphthalene nanoparticles and 1,4-naphthoquinone have their toxic effects counteracted by the antioxidant glutathione (at 1 mM for naphthalene nanoparticles and 2.5 mM for 1,4-naphthoquinone). The effect of glutathione was tested under two different conditions, either was mixed with naphthoquinone during one hour before addition on cells, or cells were exposed to glutathione for 15hours and then removed before the addition of 1,4NQ on cells.

 

In both case glutathione antagonized the effect of 1,4NQ arguing for an action through oxidative stress. 

Further analysis with Holomonitor showed that 1,4-naphthoquinone and phthaldialdehyde also altered in the cells’ behavioral (migration, motility and motility speed) and structural (optical volume) parameters. 

Furthermore, dual anthropogenic-natural particles of water -soluble biomass burning were also toxic to cells, with EC50 of 188.3 ± 43.3 µg/mL for flaming; 188.7 ± 24.3 µg/mL for smoldering; and 202 ± 20.9 µg/mL for pyrolysis particles, also affecting the cell’s behavior and structure. 

Moreover, the present study also cleared the way for more toxicological studies, showing that three real time and label free methods (RTCA, Holomonitor and Cytation) share a perfect complementarity when assessing the toxicity of chemicals. 

 

Min kommentar

Doktorsavhandlingen avslutas med en referens till Dr Alain Géloën`s studie från april 2021. 

Bloggen uppmärksammade den naturligtvis när den offentliggjordes. 

Frankrike levererar miljörapport byggd på HoloMonitor. 

Samma rubrik skulle man kunna använda på denna doktorsavhandling.

Tack till Oscar som mejlade över denna doktorsavhandling.

Man sänder även en tacksamhetens tanke till Dr Alain Géloën och hans team för att de studerar ett så osexigt men ack så viktigt område som luftföroreningar och vilken negativ effekt de har på människa.

Jag avslutar som vanligt med Dr Alain Géloën´s väl valda ord.

 

 

 

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