ORIGINAL 
RESEARCH 

published:17December2020 doi:10.3389/fchem.2020.581752 
Edited by: 
Loredana Serpe, University of Turin, Italy 
Reviewed by: 
Ankush Prasad, Palacký University, Olomouc, Czechia Angela Di Pietro, University of Messina, Italy 
*Correspondence: 
Olga Mazuryk mazuryk@chemia.uj.edu.pl 

Specialty section: 
This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry 
Received: 09 July 2020 Accepted: 03 November 2020 Published: 17 December 2020 
Citation: 
Mazuryk O, Stochel G and Brindell M (2020) Variations in Reactive Oxygen Species Generation by Urban Airborne Particulate Matter in Lung Epithelial Cells—Impact of Inorganic Fraction. Front. Chem. 8:581752. doi: 
10.3389/fchem.2020.581752 

Variations 
in 
Reactive 
Oxygen 
Species 
Generation 
by 
Urban 
Airborne 
Particulate 
Matter 
in 
Lung 
Epithelial 
Cells—Impact 
of 
Inorganic 
Fraction 

Olga Mazuryk*, Grazyna Stochel and Małgorzata Brindell 
Faculty of Chemistry, Jagiellonian University, Krak, Poland 
Airpollutionisassociatedwithnumerousnegativeeffectsonhumanhealth.Thetoxicityof organic components of airpollutionis well-recognized, whiletheimpact oftheirinorganic counterpartsin the overalltoxicityis still a matter of variousdiscussions.Theinfuence of airborneparticulate matter(PM) and theirinorganic components onbiologicalfunction ofhuman alveolar-like epithelial cells(A549) wasinvestigated in vitro.A novel treatment protocol based on covering culture plates with PM allowed increasing the studied pollutant concentrations and prolonging their incubation time without cell exposure on physical suffocation and mechanical disturbance. PM decreased the viability of A549 cells and disrupted their mitochondrial membrane potential and calcium homeostasis. Forthe frsttime,thedifferenceinthereactiveoxygenspecies(ROS)proflesgenerated by organic and inorganic counterparts of PM was shown. Singlet oxygen generation was observed only after treatment of cells withinorganicfraction ofPM, whilehydrogen peroxide,hydroxyl radical, and superoxide anion radical wereinduced after exposure of A549 cells tobothPM and theirinorganicfraction. 
Keywords: air pollution, particulate matter PM, reactive oxygen species, inorganic fraction of PM, treatment protocol 
INTRODUCTION 

Poor air quality is one of the global health problems, since it aects around 91% of the world’s population(WorldHealthOrganization)(http://www.who.int).Numerous reports confrmthat exposure to particulate matter (PM) increases the risks of respiratory tract, cardiovascular, immunological, andotherdiseases(Araujo,2011;Selmiet al.,2012;Kelly andFussell,2017;Zhang et al., 2018; Shen and Lung, 2020; Yang et al., 2020; Wang et al., 2020a). Despite the fact that the health eects of air pollution have been recognized since the last century, the progress in research is still not satisfed and it might arise from the diyculty to reliably assess the impact of air pollutants on human health. The eects of chronic exposure are usually observed only after several or evendozens ofyears, while acute contact oftendoes not resultindetectablebiological alterations(Bräuner etal.,2007;Han et al.,2020).Thebio-accumulative nature ofpollutions makes it possibletoreach up thethresholdlevel thatisharmful tohumanhealth(Ali etal.,2019)and safelevels of exposure arediycultto establish(Liu etal.,2018).Therefore,small constantly applied disturbancesto cellularhomeostasis canleadto pathologicalchanges andresultinthedevelopment of variousdiseases. 
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Air pollution consists of multi-component and multi-phase chemical systems that include traces of transition and main group metals and compounds(minerals, carbonaceous species, inorganic ions), various gaseous components (O2, O3, NOx, HS-/H2S, CO, CO2, SO2), and organic contaminations [polycyclic aromatic hydrocarbons (PAHs), chlorinated pesticides, quinones, and biological materials such as viruses, endotoxins, cell fragments]. All components are constantly interacting in gas–liquid, liquid–solid, and gas–liquid–solid interfaces. The health problems originating from air pollution are mainly linked to PM of <0.1 and 2.5µm in diameter (denoted as PM0.1 and PM2.5, respectively), which can easily penetrate the respiratory tract and accumulate more eyciently (Gangwar et al., 2020; Leikauf et al., 2020; Manigrasso et al., 2020; Novák et al., 2020). PM is a complex mixture of particles with a wide range of size anddiverse composition,depending on the place and time of sample collection(Shao et al.,2018).The natural sources contributingtohighPM emission are windblown dust, volcano eruptions, wildfres, and sea salt aerosols, while industrial, automobile, and construction/demolition sectors are the major anthropogenic contributors to air pollution (Ali et al., 2019). Numerous worldwide government health regulations aimed at the improvement of air quality have shifted PM emission contribution from dierent sources, for example, from being dominated by coal burning to a mix of vehicle and stationary combustion emissions(Shao et al.,2018). Each PM source generates several potential toxic elements. Such large heterogeneity of the naturally collected PM samples can impede the evaluation of the biological eects of PM and obstruct establishing of underlying toxicological mechanisms. Nowadays, standard reference materials for urban air pollutant are commercially available and widely used as the reference samplesin variousbiological and chemical studies(Park et al., 2013; Courtois et al., 2014; Jiang et al., 2014; Lee et al., 2016; O’Driscollet al.,2019;Dijkho et al.,2020). 
In our work, we are focusing on the evaluation of the impact of the inorganic fraction of PM (in particular redox­active transition metal elements) on cells by using the standard reference material for urban air pollutants deprived of organic components. The toxicity of organic compounds is usually based on specifc interaction with biomolecules and is widely described in the literature (Hanzalova et al., 2010; Oh et al., 2011; Falco et al., 2017; Qi et al., 2020; Wang et al., 2020b). Among the most hazardous chemical compounds are PAHs, which have carcinogenic, mutagenic, and/or teratogenic eects. They are lipophilic and can easily penetrate tissues, interacting withdierent enzymes(such as cytochromeP450), producing reactive toxic metabolites and genotoxic DNA adducts, and activatingdierentbiologicalpathways(Bauliget al.,2003;Chew et al., 2020; Longhin et al., 2020). The activityof inorganic PM is predominantly based on its redox chemistry and ability to induce oxidative stress(Kasprzak,2002; Kawanishi et al.,2002; Visalli et al., 2015; Cui et al., 2019; Haghani et al., 2020; Pardo et al., 2020; Samet et al., 2020). Severalstudies were performed to establish an association between toxic eects of air pollution and transition metal components of PM such as Fe, V, Cr, Cu, andZn(Ali et al.,2019;Valacchi et al.,2020).In our opinion, greater attention should be paid to the inorganic components, since they can catalyze dierent oxidation reactions, whereas organic pollutants can produce reactive oxygen species(ROS) mainlyin a stoichiometric way(Risom et al.,2005;Andersson et al., 2009). Dierent mechanisms of pathological cellular response induced by organic and inorganic fractions of PM are postulated, and serious debate on their roles has been going on in the scientifc community in recent years (Stone et al., 2003; Lu et al., 2014; Huang et al., 2015; Kim et al., 2018; Badran et al., 2020). Severalreports disclosed the predominant role of organic pollutants due to their ability to induce a pro­infammatory response in murine macrophages or bronchial epithelial cells (Baulig et al., 2003, 2009; Billet et al., 2007; 

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Gawda et al., 2018; Wang et al., 2018). Alternatively, there are data that indicate the limited importance of endotoxin and organic fraction of air pollutants on infammatory response to PM, suggesting the involvement of other components of PM, which need to be identifed (Herseth et al., 2017). Furthermore, synergistic and antagonistic interactions between PM components could signifcantly alter their redox properties (Gao etal.,2020).Signifcantdiscrepancyinthe currentliterature state ofknowledge mightbe relatedtothedierent experimental protocols applied to the performed research. The studied air pollutant samples varied by not only chemical/physicochemical characteristics(dierent organicandinorganiccontents, particle size, zeta potential, and others) but also dierent experimental models, exposure time, and concentrations used. Hence, direct comparison of the results receivedfor theinorganic and organic PMfractionsis challenging. 
To improve our knowledge on the direct mechanisms involved in PM-induced toxicity with a particular interest on the role of inorganic components, the infuence of PM on type II human alveolar-like epithelial cell line A549 was analyzed in vitro. Several biological parameters such as cells’ viability and mechanism of cellular death as well as ROS production, mitochondrialpotential, and cytosolic calciumhomeostasis were evaluated after exposuretoPM samples,bothintact(inorganic and organicPMfractions) and plasma treated(mostlyinorganic fraction). Additionally to standard exposure protocol involving cell treatment with a suspension of PM, the new protocol based on covering culture plates with PM and coating them with adhesion proteins was applied(Scheme1). Limited contact of thebiggest particles(microparticles) ofPM with cells allowed the avoidance of mechanical disturbance of cells. This approach enables afocus on the evaluation of the impact ofPM as well as itsdissolvedcompounds on cells without additional contribution from dying physically disturbed cells. Particular attention was paid to use small non-toxic concentrations ofPM to avoidfalse­positive results caused by a fraction of dead cells as well as fuorescent probe oxidation causedbyPM. 
MATERIALS AND METHODS 


Sample Preparation 
UrbanPM sampleSRM1648a(encoded asSRM)was purchased from theNationalInstitute ofStandards andTechnology(USA). This sample is composed of PM collected in the St. Louis, MO, area over a 1-year period (1976–1977). Among organic constituents, SRM contained PAHs, nitro-substituted PAHs, polychlorinated biphenyls (PCBs), and chlorinated pesticides. Analysis of the inorganic fraction revealed, among others, components containing Fe, Mn, Al, Ca, Cr, Ti, Pb, Mg, and others. The sample was fully characterized by numerous analytical techniques, and its specifcation is available in the manufacturer’scertifcate.The particlesizevariesfrom0.2toover 100µm,withthe predominanceofparticleswithahydrodynamic diameter of5–20µm. 
The Plasma Zepto system (Diener Electronic GmbH) was used for the removal of organic compounds present in the SRMsample.Low-temperatureplasmatreatmentisawell-known technique for the elimination of organic contamination, which are oxidized, converted into volatile products, and sucked by pump.Samples(17–20mg) were treated with alow-temperature plasma for 120min at 100W power. Content of carbon was determinedbythe elementaryanalysis(Ewlementar,Vario Micro Cube) and total organic carbon (TOC) analyzer (Schimadzu, TOC-V series). Upon plasma treatment, the carbon content decreased from 14% in the SRM sample to 1.8% in the plasma­treated sample (encoded as C-SRM) (Mikrut et al., 2018). Plasma treatment did not induce signifcant changes in the sample morphology or aggregation(Mikrut et al.,2018). 
PM samples were weighed on ahigh-precision microbalance, and stock suspensions were sonicated for a few minutes before use.Fresh suspensions were preparedbefore each experiment. 

Cell Culture Conditions 
Biological studieswere performed using typeIIhumanalveolar­like epithelialcelllineA549.Cells were maintainedinDulbecco’s modifedEagle’s medium(DMEM) supplemented with10%fetal bovineserum(FBS) and1% antibiotics(penicillin100 units/ml, streptomycin 100µg/ml) and were routinely cultured at 37. C in a humidifed incubator in a 5% CO2 atmosphere. Cells wereseededonunmodifedplates24hbefore performingthe experiments, typically with a density of3 × 104 cells percm2.If not stated otherwise, cells were exposedtoPMfor24h(added as a suspension in medium), followed by washing with phosphate buered saline (PBS) (exposure method 1, Scheme1). After that, an appropriated test was applied. Alternatively, cells were seeded on plates that were pre-prepared by covering them with PM samples(asdescribedinthefollowing paragraph) andkept for24h(exposure method2, Scheme1). Allexperiments were performed at least in triplicate and repeated three times. The meanvalues ± standarderrorofthemean(SEM)werecalculated. 

Covering Plates With Particulate Matter Samples (Exposure Method 2) 
To cover the plates with PM samples, SRM, or C-SRM was suspended in cold methanol and added to the wells to achieve dierent concentrations up to 625 µg/cm2. After methanol evaporation, some plates were additionally coated with fbronectin from human plasma or collagen I from calf skin (Sigma).Fibronectin andcollagen werediluted to25µg/mlwith sterile water, and 50-µl solutions were added to the wells of 96­well-plates.Plates werekept at4.Covernight, andthenthey were directlyusedfor seeding cells. 


Cell Viability 
The evaluation of the A549 cell viability after exposure to PM samples was conducted using 3-[4,5-dimethylthiazol-2-yl]-2,5­diphenyltetrazolium bromide (MTT) or resazurin assays. The MTT assay is based on the reduction of yellow tetrazolium salt (MTT) by dehydrogenases of metabolically active cells into purple water-insoluble formazan dye. The concentration of dye determined spectrophotometrically after dissolution in an organic solvent is proportional to the number of live cells. Cell viability wasquantifed measuring absorbance at565nm using 700nm as a reference wavelength(TecanInfnite200microplate 

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reader). Resazurintestisbased onthe reduction ofblue and non­fuorescent substrate(resazurin)toapink andhighlyfuorescent product(resorufn)by thelive cells.Cell viability wasquantifed at605nm using560-nm excitationlight(TecanInfnite200 microplatereader).Experiments were performedintriplicateand repeated at least three times to get the mean values ± SEM. The viability was calculated with respect to the control seeded on uncoated/coatedplate withoutPM samples.The viability was determined after incubation of cells with PM samples prepared as suspensionin a medium[without orwith2/10%fetalbovine serum (FBS)] at concentrations up to 500µg/ml (that refers to 156.25 µg/cm2) for 24 or 72h. Alternatively, viability was assessed after exposing cellstoPM samples coveringplates atthe concentrations upto625 µg/cm2 for24h. 


Evaluation of Reactive Oxygen Species Generation by A549 Cells 
TotalReactiveOxygen SpeciesProduction 
To evaluate the total ROS production induced in cells after incubation with non-toxic concentrations of PM, the cyto-ID Hypoxia/Oxidativestressdetectionkit(EnzoLifeSciences, USA) was used according to the manufacturer’s protocols. A549 cells were seeded with a density of 2 × 104 cells per cm2. A day later, the SRM or C-SRM samples at dierent concentrations suspendedin medium without serum were added andincubated inthedarkfor24h.Then, cells were washed withPBS,treated with trypsin, and analyzed by BD FACSVerse cytometer. As a positive control, pyocyanin (300µM) was used. The level of the oxidative stress was determined as a percentage of the ROS-positive cells of the whole-cell population determined by pyocyanin control. Alternatively, mean fuorescent intensity of the stainedtreated cells was evaluated. 
To mimic the chronic exposure ofA549 cells toPM samples, cells were cultured for 18 days. Firstly, cells were seeded with a densityof2 × 104 cells per cm2 on a six-well-plate.Nextday,the SRM orC-SRM samples atdierentconcentrations suspended in medium with 2% FBS were added. Cells were incubated with pollutants for 3 days and were reseeded in 1:5 ratio to new plates.Thefreshlyprepared suspensions ofPM were added.Cells were incubated in total for 18 days and, every 3 days, cells were reseeded and new suspensions of PM were added. On the 18th dayofincubation,A549 cells were washed withPBS, stained with cyto-ID Hypoxia/Oxidative stress detection kit according to the manufacturer’s protocols, treated with trypsin, and analyzed by BDFACSVerse cytometer. 
To evaluate the infuence of PM on the oxidative stress induced by other stimuli, A549 cells were pretreated with PM samples. For this purpose, A549 cells were seeded on a six­well-plate with a density of 2 × 104 cells per cm2 a day before.The suspension ofSRM or C-SRM samples atdierent concentrations in medium without serum was added. After 24h ofincubation,cellswerewashed withPBS and pyocyanin (300µM) was usedfor30mintoinduce oxidative stress.A549 cells were then stained with cyto-ID Hypoxia/Oxidative stress detection kit according to the manufacturer’s protocols and analyzedbyBDFACSVerse cytometer. 
2 ' ,7 ' -Dichlorodihydrofuorescein Diacetate 
'' 
2 ,7 -Dichlorodihydrofuorescein diacetate (DCF-DA) (Sigma-Aldrich) was used to assess the general ROS production by cells exposed to PM samples at non-toxic concentrations. Cells were stained withDCF-DA(20µM)for45min at37.C. After the staining, the ROS indicator was washed out with PBS, and fuorescence of cells wasquantifedby aTecanInfnite200 plate reader at 535nm using 485nm as an excitation wavelength. Controlexperimentswithidenticalsettingsbutwithoutcellswere madetodeterminetheinfuence ofPM onthedye. 
QualitativeAnalysis oftheProduced Reactive OxygenSpecies 
Various fuorescent probes, specifc to the selected ROS, were used to determine the type of ROS produced by cells after treatment with SRM or C-SRM samples. Each probe was applied for cells exposed to PM-covered surfaces at two non­toxic concentrations,50 and100 µg/cm2.After the staining, the ROSindicatorswerewashedtwicewithPBS,andfuorescenceof cells wasquantifedby aTecanInfnite200plate reader.Controls without cells were made to avoidfalse-positive results and verify theinfuenceofPM ontheusedfuorescentprobes. 
Singlet oxygen sensor green (SOSG, 5µM, 20min; ThermoFisher Scientifc) was used to assess the production of singlet oxygenincells.Aminophenyl fuorescein(APF,5µM, 30min,ENZO) wasused toevaluatethe productionofhydroxyl radical, hypochlorite, or peroxynitrite. Dihydrorhodamine 123 (DhR123, 10µM,20min,AATBioquest) was used to estimate the production of mainly hydrogen peroxide; however, it can be used to detect additionally hypochlorite, peroxynitrite, or cytochrome c. Hydroethidium (HE, 10µM, 20min, AAT Bioquest) was used to check the production of superoxide anion radical. MitoSox Red (5 µM, 10min, ThermoFisher Scientifc) was applied to evaluate the production of superoxide anion radical in mitochondria. Staining cells with probes was performed at 37.C. Fluorescence intensity of cells was measured at535nm using485nm as an excitation wavelength for SOSG/APF and at 529nm using 507nm as an excitation wavelength for DhR123. For HE evaluation, fuorescence intensity of cells was quantifed at 605nm using 520nm as an excitation wavelength, while 510/595nm were used as excitation/emissionwavelengthsfor MitoSox Redquantifcation. 

Lipid Peroxidation 
Lipid peroxidation was assessed using C11-BODIPY581/591­Lipid Peroxidation Sensor(ThermoFisherScientifc). A549 cells were seeded on PM-covered surfaces at two concentrations, 50 and 100 µg/cm2, 24h before the experiment. After that, cells were stained withC11-BODIPY581/591 (1µM) for 30min in the dark at 37.C. The fuorescence intensity of cells was measured at484/510nm(green) and581/610nm(red)—the excitation/emission wavelengths. 


Mitochondrial Membrane Potential 
Mitochondrial membrane potential(m) was evaluated using JC-1 probe (AAT Bioquest). The JC-1 probe is a lipophilic cationic dye that exhibits potential-dependent accumulation in 

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the mitochondria. At low m, the probe exists as a monomer thatemitsgreen fuorescence.Athigherm,the concentration of dye increases and the probe forms J-aggregates that lead to a shiftin fuorescenceemissionfromgreentored.A changefrom red togreen fuorescencerefectsadecreasein m.A549 cells were exposed to PM-covered plates at two concentrations, 50 and100 µg/cm2.After24h of treatment, cells were stained with JC-1(10µM)for30mininthedark at37.C. The fuorescence intensity of cells was measured at 525 and590nm using 490nm as an excitation wavelength. 


Cytosolic Calcium Homeostasis 
Cytosolic calcium concentration was measuredusingFluo-8AM probe(AATBioquest).A549 cells were exposed toPM-covered plates at two concentrations, 50 and 100 µg/cm2.After24h of treatment, cells were stained withFluo-8AM(4µM)for30min in the dark at 37.C. The fuorescence intensity of cells was measured at525nm using490nm as an excitation wavelength. 

The Mechanism of Cellular Death 
The mechanism of cellular death was evaluated using Annexin V-fuorescein isothiocyanate (FITC)/propidium iodide (PI) (ThermoFisher Scientifc) assay. The phosphatidylserine of the cytoplasmic membrane at the early stage of apoptosis fipped on theouter surface ofcellmembrane andinthe presenceofcalcium ionsisboundwithAnnexinV.PI was used to assess the necrotic population of cells.A549 cells were seeded onPM-coveredplates with adensity of4 × 104 cells per cm2, and24h afterthe seeding, thetest was performed.A549 cellswerestained withAnnexinV­FITCfor10mininthedarkandthen withPI(0.5µM)for5min. Cells were analyzedbyaBDFACSVerse cytometer. 

Caspase Activity 
Activation of caspases 3/7 was examined using CellEvent Caspase-3/7GreenDetection Reagent(ThermoFisherScientifc). A549 cells were seeded on PM-covered plates with a density of 4 × 104 cells per cm2. Twenty-four hours later, cells were stained with CellEvent Caspase-3/7 Green Detection Reagent. The fuorescence intensity of cells was analyzed using a BD FACSVerse cytometer. 

Statistical Analysis 
For in vitro experiments, all data were expressed as the mean ± standard error of the mean (SEM). All the experiments were performed in triplicate and repeated at least three times. Signifcant dierences among groups were determined using t-test or a one-way analysis of variance(ANOVA) usingOriginPro 2018 software. Probabilities of p < 0.05 were considered as statistically signifcant. The following notifcation is used * p < 
** 
0.05,p< 0.001. 

RESULTS AND DISCUSSION 
For the evaluation of biological eect of PM with and without organic components in vitro, A549 cell line was used. This line was chosen as a simple model of lung epithelial cell line as has beenroutinely usedforthistypeof studies(Billetetal.,2007; Mehta et al., 2008; Wang et al., 2013; Huang et al., 2014, 2015; Lee et al., 2014; Lu et al., 2014; Schiliret al., 2015; Kim et al., 2018; Li et al., 2020). SRM 1648a sample (SRM) supplied by the National Institute of Standards and Technology, USA, was used as a standardized sample for urban air pollutants. Sample withoutorganic contaminations(C-SRM) was prepared using low-temperature plasma treatment during which the organic carbon contentdecreasedfrom14to1.8%,asdeterminedbyboth elementary analysis andTOC measurements.Neither signifcant changes in the morphology nor aggregation of particles due to plasma treatment was observed (Mikrut et al., 2018). Two dierent exposure protocols were used to fully assess the infuence of PM on biological parameters of A549 cells: i) suspension ofPMin cell culture medium(exposure method1) andii)PM-coveredplates(exposure method2). 


Cell Viability 
The viability of the A549 cells after exposure to SRM and C­SRM samples was assessed using MTT or resazurin tests. It wasdeterminedthat after24h ofincubation with eitherSRM or C-SRM samples suspended in medium, the viability of A549 cells decreased signifcantly for the concentrations higher than 100µg/ml (SupplementaryFigure1A). These results are in agreement with other reports (Holian et al., 1998; Gawda et al., 2018). No dierence in toxicity between SRM and C­SRM was observedfor 24h ofincubation, while after 72h,SRM sample appearedtobe moretoxic(SupplementaryFigure1B). Prolonging the incubation time in case of C-SRM sample did not induce additional toxicity. Serum proteins may have substantial impact on the toxicity of numerous compounds by either reducing the toxicity due to the lower accumulation of compounds or increasing the toxicity by facilitating the uptake (Mazuryk et al., 2014). Therefore, the infuence of fetal bovine serum(FBS)presenceintheincubationmediumwasstudiedby performingthe viability assay additionallyinthe presence of2 or 10%FBS. It wasdetermined thatFBShas a negligibleimpact on thetoxicity ofSRM sample(results are not shown). 
The decrease in viability of the A549 cells after the incubation with SRM or C-SRM samples may not be solemnly caused by chemical nature of PM interaction with cells. Additional factors related to the physical contact and mechanical disturbance of cells by PM sample should be considered (SupplementaryFigure2). PM applied directly on cells, covering them that can eyciently reduce the amount of nutrients and generate mechanical damage, which in consequence mayinduce celldeath. 
To eliminate this eect, plates were coveredwithPM samples at dierent concentrations and then used for cell seeding (see experimental part). Additionally, some plates coveredwith PM samples were coated with collagen or fbronectin prior to seeding of cells. Plates prepared in such a way gave cells the ability to interact with PM without limiting a profcient collection of nutrients from cell medium. Covering plates with PM samples and coating them with adhesion proteins next signifcantly reduced the toxicity of SRM or C-SRM samples (SupplementaryFigure3). No toxicity of PM samples was observedin cells seeded on uncoatedplates or plates coated with 

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collagen, while slight toxicity(up to80% viability) was observed on the surface coated with fbronectin. PM covering technique allowed the use of 20 times more concentrated PM samples (100µg/ml= 31.25µg/cm2)as a nontoxicdosage. 
Attention should be paid to morphological changes of A549 cells upon contact with PM (SupplementaryFigure4). Flow cytometry results revealed only a slightdecreasein cell size(FSC) while cellgranularity parameter(SSC) increased.Thisindicates thatPM was collectedby cells.Plasma-treated and untreatedPM samplesinduced a similar eect ontheSSCparameter. 


Evaluation of Reactive Oxygen Species 
Thegenerationof ROSinA549 cellsuponinteractionwithPM samples was explored using two dierent methods of contact between cells and PM. The samples were applied either in the form ofsuspensionin medium or as alayer coveringthe plate. 
ROS production was assessed using small non-toxic concentrations of PM to avoid false-positive results caused by cells’ response to toxic eect of PM or PM interference with fuorescent probes. Total ROS production in A549 cells after dierent incubation times was evaluated using cyto-ID Hypoxia/Oxidative stress detection kit. One hour of incubation with SRM suspension was not a suycient period of time to produce a signifcant amount of ROS by cells (SupplementaryFigure5). However, treatment with suspension of inorganic part of pollutants (C-SRM sample) was able to cause a detectable increase in the amount of ROS produced by A549 cells even after 1h of incubation (SupplementaryFigure5). Twenty-four hours ofcell exposure to PM suspensions resulted in an increase in ROS-positive cell population for both applied PM samples, SRM and C-SRM (Figure1A). The SRM sample induced a stronger eect, confrming relevance of organic pollutants in oxidative stress induction. Semi-chronic exposure (18 days) to PM suspensions caused a dierent eect (Figure1B). Incubation of A549 cells with SRM sample did not result in an increase in ROS production, indicating an existence of a compensatory mechanismtoward constant small exposureto pollutants.Some of the organic pollutants have ROS-scavenging properties, and they may act as antioxidants. In contrast, C-SRM induced a signifcant rise in the amount of generated ROS in cells, fairly independent of the used concentration of the sample, suggesting a catalytic mechanism of ROS production. High content of metals such as Fe, Cu, and Mn in C-SRM can participate in catalytic production ofROS. 
To further evaluate the infuence of PM on oxidative stress induction, the production of ROS in cells pretreated with PM suspensions(SRM or C-SRM) and exposed to a known ROS inducer was studied. As a ROS inducer, pyocyanin was used, a toxin produced by Pseudomonas aeruginosa (Ando and Yonamoto, 2015; Winter and Zychlinsky, 2018). Pretreatment of cells with PM suspensions for 24h before exposing them to pyocyanin resulted in a higher response to the used stimuli(Figure2). Pretreatment with SRM resulted in a strong dose-dependent increase in ROS production upon exposure to pyocyanin, while pretreatment with C-SRM induced a similar eect but independent of concentration. The results are in agreement with literature data, which showed that exposure of murine macrophages to suspension containing low concentrations of PM may prime cells to a hyper-infammatory response upon contact withthe second stimulus(Gawda et al., 2018). 
In numerous publications, the production of ROS was evaluated using DCF-DA assay (Wilson et al., 2002; Baulig et al., 2004, 2009; Andersson et al., 2009; Lee et al., 2014, 2016; Rodríguez-Cotto et al., 2015; Ahlberg et al., 2016). DCF-DA is a cellular probe that reacts with numerous ROS to produce fuorescent product and is often used to estimate the general ROS production. The level of oxidative stress in A549 cells was measured by this probe after 24-or 72-h incubation with PM suspension. No signifcant ROS production in A549 cells using DCF-DAprobewasobservedafter24h ofincubationforeach of the samples(SupplementaryFigure6A).Prolongingincubation time up to72h resulted only in a slightincreaseinthelevel of oxidative stress (SupplementaryFigure6B). Such discrepancy 

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in the results between two tests used for the evaluation of ROS production can be explained by dierent sensitivities of the probes, since DCF-DA is mostly sensitive toward H2O2, while cyto-ID Hypoxia/Oxidative stress detection kit detects general ROS production (according to the manufacturer’s protocol). The amount of ROS generated by suspension of PM may not be high enough to be detected by DCF-DA probe. Further increase in PM concentration was not possible due to its toxicity(SupplementaryFigure1). Additionally, PM concentration higher than 20µg/ml (for SRM sample) and 10µg/ml(for C-SRM sample)interfered with DCF-DA assay, causingits oxidation. 
To overcome the mentioned problems for further studies, the PM covering technique was used. Covering plates with PM samples and coating them with adhesion proteins resulted in a signifcant decrease of toxicity of air pollutants (SupplementaryFigure3);therefore, the higher amount of PM samples, still in nontoxic range of concentration, can be used in the evaluation of ROS production in A549 cells by using DCF-DA assay. The production of ROS in A549 cells seeded on the surface covered with PM samples and coated with either fbronectin or collagen is shown in Figure3. Both SRM and C-SRM samplesinduce oxidative stressinA549cells. C-SRM samples were more eycient as ROS inducers. They caused ca. twice higher ROS production than SRM samples when cells weretreated with ahigh concentration ofPM(>200 µg/cm2). SRM samples started toinduce signifcant oxidative stressin the concentration higher than 50 µg/cm2, while C-SRM samples caused asimilarlevel ofROSproductionwith aconcentrationas small as1.6 µg/cm2. 
To thoroughly evaluate the role of inorganic fraction of PM samples in ROS formation in treated cells, the mass reduction of plasma-treated sample was taken into account. During cold plasma treatment, ca. 33% decrease in mass of the sample was observed, so the same mass of C-SRM in comparison to SRM contained much more inorganic particles. To compensatefor this eect, the results ofDCF-DA evaluation of ROS formation were recalculated, and ROS formation eect of SRM was compared to 67% of ROS generation induced by C-SRM (SupplementaryFigure7). At small and moderate concentrations ofPM samples(upto100 µg/cm2),SRMinduced a greater increase in ROS production in A549 cells. However, higher concentrations (>200 µg/cm2) resulted in a similar level of oxidative stress induction by both SRM and C-SRM samples. These results confrmed that at small to moderate concentrations of PM, the organic fraction plays an important rolein an oxidative stressformation;however,itis notexclusively responsible for it. At higher concentrations, catalytic activity of inorganic particles became more evident and C-SRM samples inducedsimilarproductionofROS asSRMsamples. 


Qualitative Analysis of the Produced Reactive Oxygen Species 
To determine a profle of the produced ROS, the selective fuorescent probes, described in detail in the experimental part, wereused.A549 cellsweregrown onplates covered withPM at concentrations 50 and 100 µg/cm2 to avoid possible interaction of PM samples with fuorescent probes. Incubation of A549 cells on SRM-covered plates resulted in increased fuorescent signals from APF, DhR123, HE (only on fbronectin-coated plate),and MitoSox(only oncollagen-coatedplate) indicators (Figure4). This indicated that majority of ROS produced by A549 upon contact withSRM arehydrogen peroxide(H2O2), hydroxyl radical (· OH), and superoxide anion radical (O - ). 
2 Plasma-treated air pollutant (C-SRM) sample upon contact with A549 cells additionally to already mentioned ROS induced the production of singlet oxygen, as was manifested by the increased fuorescent signal from the SOSG probe (Figure4). Thelack of singlet oxygen productionby cellstreated withSRM samples can be a consequence of an antioxidant potential of organic part of PM sample. Singlet oxygen can be quenched by and/or reacts with many organic molecules (DeRosa and Crutchley,2002). 


Lipid Peroxidation 
Lipid peroxidation was assessed using C11-BODIPY581/591­Lipid Peroxidation Sensor (Figure5). This liposoluble probe upon oxidation with lipid peroxyl radicals (ROO· ) alters its fuorescence properties, shifting emission intensity to shorter wavelengths, from red to green fuorescence. Incubation of A549 cells on the PM-covered surfaces resulted in increased green-to-red fuorescence ratio of C11-BODIPY581/591 probe, displaying the occurrence of lipid peroxidation. The observed eect was similar for SRM and C-SRM samples, which can indicate that the organic part of PM has a negligible eect on lipidperoxidation. 

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Mitochondrial Membrane Potential 
To evaluate m, JC-1 probe was used (Figure6). The shift in mitochondrial potential was represented as a change in red/green fuorescenceratio.Both untreated andplasma-treated PM samples caused the decrease in red/green fuorescence intensity ratio, indicating depolarization of the mitochondrial membrane. The eect was concentration dependent and was slightly stronger after exposure cellstoSRM samples. 


Intracellular Calcium Concentration 
Calcium ion is one of the most important signal transducers in cells, involved in numerous physiological and pathological processes including proliferation, dierentiation, and cellular motility. Both calcium spatial localization and magnitude might determinethe cell’sfaith.Itis well-knownthatvarious metals can modify intracellular Ca2+ signaling (MacNee and Donaldson, 2003). Fluo-8 AM probe was used to determine the changes 

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FIGURE 4 | hydroethidine(O- );MitoSoxTM (O- in mitochondria). (D) 
22 
Representativeimages ofROSproductioninducedinA549 cells on PM-coveredplates(100 µg/cm2)measuredbyDhR123.Scalebaris50µm. *p < 0.05, **p < 0.001. 

in intracellular calcium concentration in A549 cells after 24­h incubation on the PM-covered plates uncoated or coated withfbronectin/collagen(SupplementaryFigure8).Incubation of A549 cells on PM samples resulted in the decrease in Fluo­8 AM probe fuorescence, indicating the disruption of calcium homeostasis andthedecreasein cytosolic[Ca2+].The results are more pronounced for cells seeded on uncoated plates than for one coated with adhesion proteins. 


Mechanism of Cellular Death 
To investigate the mechanism of cellular death, the activity of executioner caspases 3/7 was examined. These enzymes are activatedduring regulated cellulardeath(extrinsicorintrinsic apoptosis) and are responsible for many morphological and biochemical changes such as DNA fragmentation, phosphatidylserine exposure, and formation of apoptotic bodies (Galluzzi et al., 2018). The percentage of caspase 3/7­positive cells after24-hincubation withdierent concentrations of PM-covered plates is shown in Figure7A. In A549 cells after exposure to PM samples, the percentage of caspase 3/7-positive cells signifcantly increased. SRM sample caused activation of executioner caspases in the whole population of the treated cells, while C-SRM sample only in around 30%. The results indicate thatapoptosisis a main mechanism of cellulardeathfor SRM-treated cells, while exposure to C-SRM caninduce some additional caspase-independent celldeath mechanisms. 
To further investigate the mechanism of cellular death, morphological signs of apoptosis and necrosis were examinedby Annexin V-FITC/PI assay. This assay allows for determination of a potential loss of cellular integrity (during necrosis) and measures transition of phosphatidylserine (PS) on the outer surfaceof thecell membrane(during apoptosis).The percentage ofliving, necrotic, and apoptoticA549 cells after24-hincubation with dierent concentrations of PM-covered plates is shown in Figure7B.The presenceofSRMand C-SRMsamplesincreased the population of apoptotic and necrotic cells. While some necrotic fraction of the populations can be explained by end­stage apoptosis(Galluzziet al.,2018),inorganicPMsample, C­SRM, undoubtedly induced a stronger eect on A549 cells than the SRM sample, producing a signifcantly higher amount of necrotic cells. The results indicate involvement of necrotic cell deathas cells’failed responses to adapt toinduced stress.Similar results were obtained for collagen-coated surfaces (results are not shown). 


CRITICAL DISCUSSION OF THE RESULTS 
Several studies tried to estimate the relative contribution of inorganic and organic components to PM-induced toxicity (Gao et al., 2020). Some studies try to do this by multiplying 

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the intrinsic oxidative potential of individual compounds of PM with their ambient concentrations and provide evidence of the importance of either transition metals (Charrier and Anastasio,2012) or organic components(Verma et al.,2015). Others compared atmospheric particles with dierent amounts of organic constituents such as PM2.5, carbon black particles, anddiesel exhaust particles(DEPs).Baulig et al. concluded that organic compounds presented in DEP (Baulig et al., 2003) or isolated from PM2.5 (Baulig et al., 2009) mainly contributed to the observed biological eects in human bronchial epithelial cells.Our approach relies on the evaluation ofinorganicfraction contribution onbiological responseto airbornePMby removing organic compounds usingcoldplasmatreatment.Suchtreatment does notinduce changesin particle morphologyor size;however, we are not able togaindetailedinformation on the speciation of thePM components. 
Due to the cumulative nature of pollution, adverse health eectsareoftenresultsof chronicexposuretoharmful particles, so when cells are exposed to urban airborne PM at quantities expected from the known content of PM in air, the observed changesareataverylowlevelornegligiblysmall.Largequantities of PM that have often been used to detect changes in various biological parameters in vitro can induce additional mechanical damage,whichwillfalsifytheobtainedresultsduetotheabilityof adyingcellto expose or release moleculesthat alert other cells.In these studies, special emphasis was placed on the elimination of false-positiveresultsarisingfrommechanical pressureofPM on cell culture.This was achievedthrough aninverting cell exposure protocolby covering cell culture plates withPM.Such alternative way of celltreatment allowed ustoincrease20timesthe usedPM concentration without causing unnecessary cell death from the mechanicaldamage. 
Another serious issue in this type of research concerns the oxidation of the fuorescent probes by using PM due to the oxidative potential of both organic and inorganic components of airborne air pollution. To eliminate such a possibility, several control experiments were carried out and carefully analyzed to avoid artifcial positiveresults.Thecombinationof thesefactors allowedforthe selection ofanappropriatecelltreatmentprotocol andPM concentrationsthat makeit a possibilityto study narrow dierencesbetweenthetestedPMsamples. 
We are aware that such a model system also has some limitations. One of the most relevant is the inability to assess synergistic and antagonistic interactions between dierent PM components that could signifcantly alter their redox properties. This methoddoes notconsiderthe cumulative eect ofpollution and can underestimate the infuence of fraction due to its low atmospheric concentration. Additionally, bulk PM mass measurements do not accurately represent a complex chemical mixture that is air pollution, which additionally varies both spatially and temporally. In vitro studies using lung epithelial cells allowed assessing the eects of dissolved components and components of the studied PM with a size smaller than 2.5µm. Only such particles are capable of reaching the alveolar epithelium, crossing cell membranes, and directly interacting with cellular structures. SRM predominantly contains particles from 5 to 20µm, which can lead to underestimation of the observed eects. The negative eects of air pollution on humanhealthisdiycult toimitate underlaboratory conditions; however, by using thoroughly characterized Standard Reference 

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Materials(availablefromtheNationalInstituteofStandard and Technology), the eect of airborne PM can be studied among dierentlaboratories worldwide. 


CONCLUSIONS 
Air pollution is now recognized as one of the main health risks in the developed world. The presented work is focused on the comparison of biological eects in vitro for the organic and inorganic fractions of PM. To achieve the goal, the infuence of intact standardized urban PM (SRM) and plasma-treated SRMcontaining predominantly inorganicfractionofPM(C­SRM) on various biological parameters of A549 lung epithelial cells was directly compared following the newly elaborated experimentalprotocols. 
The obtained results indicate a signifcant infuence of the inorganic partofPMon oxidative stressgeneration.Bothorganic and inorganic fractions of PM are involved in the induction of ROS production by cells. In the case of small and moderate concentrations ofPM and shorterincubationtime(24h),the organic fraction of PM plays a pivotal role in oxidative stress induction.The signifcance oftheinorganicfractionis enhanced with increasing PM concentrations and prolonging incubation time(up to18days), mimickinginthis way chronic exposure. CoveringplateswithPMallowedtheuseofhigherconcentrations of air pollutants in the studied suspensions without observing thetoxic eectofthe particles.Athigh concentrations,treatment with C-SRMdemonstratedasimilareect on ROS production as SRM, and the catalytic activity of inorganic particles became more apparent. 

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Both air pollutant samples decreased the viability of DATA AVAILABILITY STATEMENT 
A549 cells by decreasing the m and disrupting calcium homeostasis. Despite the SRM sample being slightly more toxic toward A549 cells, the inorganic fraction (C-SRM) appeared to be more dangerous due to its superior ability to induce necrosis in the studied cell line. The biological alterations observed in vitro might contribute to lung infammation and increased probability ofpulmonarydiseases. 
Detected changes in the observed biological eects of SRM and C-SRM might be related to dierences in the ROS profle induced in cells. Singlet oxygen production was observed only after treatment of cells with C-SRM. Hydrogen peroxide, hydroxyl radical, and superoxide anion radical are the most abundant ROS produced after exposure of A549 cells to SRM, while C-SRM preferably induced the production of H2O2, singlet oxygen, andhydroxyl radical. 
The obtained fndings are of importance for our future understandinghowdierent organic andinorganic components of PM contribute to the adverse health eects of PM and could help to develop a strategy to identify the most harmful components. The proposed novel research protocol is crucial for accurate evaluation of the biological eects of PM in vitro without cell exposure to additional stressful factors such as physicaldisturbance. 


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AUTHOR CONTRIBUTIONS 
All authors listed have made substantial, direct and intellectual contributiontothe work, and approveditfor publication. 

FUNDING 
The authors wouldliketothankSYMPHONYproject(GrantNo. 2015/16/W/ST5/00005)forthe fnancialsupport. 

ACKNOWLEDGMENTS 
TheauthorsaregratefultoMagdalena Mikrutfor preparationof the C-SRM sample. 


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Confict of Interest: The authors declare that the research was conducted in the absence of any commercial or fnancial relationships that could be construed as a potential confict ofinterest. 
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FrontiersinChemistry| www.frontiersin.org 15 December2020|Volume8|Article581752