Full Length Article
Cellular responses of human astrocytoma cells to dust from the Acheson process: An in vitro study
Yke Jildouw Arnoldussen
a, Torunn Kringlen Ervik
a, Balazs Berlinger
a, Ida Kero
b, Sergey Shaposhnikov
c, Shanbeh Zienolddiny
a,*
aDepartmentofBiologicalandChemicalWorkEnvironment,NationalInstituteofOccupationalHealth,Pb8149Dep.,N-0033,Oslo,Norway
bDepartmentofIndustrialProcess,TechnologySINTEFMaterialsandChemistry,PB4760,N-7465,Trondheim,Norway
cNorgenotechAS&CometBiotechAS,N-1290Oslo,Norway
ARTICLE INFO
Articlehistory:
Received8August2017
Receivedinrevisedform2November2017 Accepted2November2017
Availableonlinexxx
Keywords:
Achesonprocess Neurotoxicity Astrocytes Siliconcarbide
ABSTRACT
Siliconcarbide(SiC)islargelyusedinvariousproductssuchasdieselparticulatefiltersandsolarpanels.It isproducedthroughtheAchesonprocesswhereaerosolizedfractionsofSiCandotherby-productsare generatedintheworkenvironmentandmaypotentiallyaffecttheworkers’health.Inthisstudy,dustwas collected directly on afilter ina furnacehall overatime periodof24h. The collected dustwas characterizedbyscanningelectronmicroscopyandfoundtocontainahighcontentofgraphiteparticles, andcarbonandsiliconcontainingparticles.Only6%wasclassifiedasSiC,whereofonly10%hadafibrous structure.Tostudyeffectsofexposurebeyondtherespiratorysystem,neurotoxiceffectsonhuman astrocyticcells,wereinvestigated.Bothlow,occupationallyrelevant,andhighdosesfrom9E-6mg/cm2 upto4.5mg/cm2wereused,respectively.Cytotoxicityassayindicatednoeffectsoflowdosesbutaneffect ofthehigherdosesafter24h.Furthermore,investigationofintracellularreactiveoxygenspecies(ROS) indicatednoeffectswithlowdoses,whereasahigherdoseof0.9mg/cm2inducedasignificantincreasein ROSandDNAdamage.Insummary,lowdosesofdustfromtheAchesonprocessmayexertnoorlittle toxiceffects,atleastexperimentallyinthelaboratoryonhumanastrocytes.However,higherdoseshave implicationsandarelikelyaresultofthecomplexcompositionofthedust.
©2017TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.Introduction
Siliconcarbide(SiC)producedbytheAchesonprocessisawell- knownceramicmaterialknownforitspropertiessuchaschemical inertness, elevated thermal stability and excellent mechanical properties.Itisusedinawidevarietyofindustrialpurposesbothin theceramicandcompositematerialfields(Oliverosetal.,2013).
Furthermore,thereisanincreasedinterestintheuseofmicroand nanoscaleSiCmaterialsintheareasofceramics,electronicsand catalysis.Thus,asSiClikelywillcontinuetobeusedinavarious numberofproducts, researchonpotentialhealth effectsdue to occupationalexposurewillbeofimportance.
Most toxicological effects related to SiC from the Acheson processhavefocusedontherespiratoryeffects andmostof the knowledge is on SiCwhiskers. Analyses on the effects of dust exposureofworkersintheSiCindustryhaveindicatedincreased loss of lung function, increased mortality from non-malignant
respiratorydiseasesandincreasedincidenceoflungcancer(Bugge etal.,2011,2012;Johnsenetal.,2013).Duetotheirabilitytocause lungcancer,occupationalexposureassociatedwiththeAcheson process has been classified as carcinogenic to humans by the InternationalAgencyforResearchonCancer(Grosseetal.,2014).
SubchronicinhalationandintrapleuralinjectionofSiCwhiskers in rats inducedinflammatorylesions, thickeningof the pleural wall, pleural fibrosis and mesotheliomas (Lapin et al., 1991;
Johnsonand Hahn,1996).StudiesonSiCmicroparticlesshowed thattheparticlestriggeredlunginflammation(Cullenetal.,1997), granulomas (Vaughanetal.,1993),fibroticchangesinthelungs (Akiyama et al., 2007), exerted cytotoxic and genotoxiceffects (Vaughan et al., 1993), induced reactive oxygen species (ROS) (Svenssonetal.,1997)andincreasedtheexpressionofinflamma- torycytokines(Cullenetal.,1997).Furthermore,accumulationof nanoscaleSiCparticleswasobservedinlungepithelialcells and induced ROSand DNA damage(Fan etal., 2008; Barillet et al., 2010).
Thereislittleknowledgeonpotentialneurotoxiceffectsofdust fromtheAchesonprocess.DustemittedfromtheAchesonprocess hasseveralcomponents,includingsilicaparticlesthatmayhavean
* Correspondingauthor.
E-mailaddress:shan.zienolddiny@stami.no(S.Zienolddiny).
https://doi.org/10.1016/j.neuro.2017.11.001
0161-813X/©2017TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/
).
xxx–xxx ContentslistsavailableatScienceDirect
NeuroToxicology
impactonthecentralnervoussystem.Nanoscalesilicaparticles showedincreasedoxidativedamageandinflammatoryresponses inthebrainafterintranasalinstillationinexperimentalanimals (Wu et al., 2011). Furthermore, uptake of silica nanoparticles decreased neuron cell viability, induced ROS, apoptosis, and increasedAlzheimer-likepathology(Yangetal.,2014).Supporting this,arecentstudyshowedthatsilicananoparticlescancrossthe blood-brainbarrierandinduceneuronalcelldamage(Zhouetal., 2016).Otherelements,suchascarbon,aluminumandvanadium, thatcanbepartofoccupationalexposureintheAchesonfurnace hallhave allbeen showntohave neurotoxic effects (Campbell, 2002;Garciaetal.,2005;Onodaetal.,2017).
We soughttoinvestigatetheeffectsof dustcollectedinthe furnacehallofaSiCfacilityonahumanastrocyticcelllinecultured inthe laboratory.Thehumancells were exposedtoa range of doses,includinglowoccupationallyrelevant,andhighdoses.The resultsshowedminimaldose-dependenttoxicity,ROSproduction andDNAdamage.
2.Materialsandmethods
2.1.CollectionoftheAchesondust
A 142mm stainless steel filter holder, YY3014236 (Merck Millipore,Massachusetts, USA) wasplaced inthemiddle ofan Acheson furnace hall close to one of the furnaces. A 142mm diameterpolycarbonatefilter withporesize10
m
mwasplacedontopofthefilterholderandthen connectedtoa highoutput vacuum pump, WP6222050 (Merck Milllipore, Massachusetts, USA), withadjustable output.This was left on for a period of 24hwheredustwascollectedfromthefilterthreetimesduring this period. The dust can beconsidered as an impure powder with no industrial application and is representative of the airbornedustthatcanbeinhaledintheworkenvironmentofa furnacehall.
2.2.PreparationoftheAchesondustforcharacterizationandcell cultureexperiments
Thedustwas weighedand fordispersiona slightlymodified versionoftheNANOGENOTOXprotocolwas used(Jensenetal., 2011;Phuyaletal.,2017).Briefly,toobtainwell-dispersedparticles asolutionofsterile-filtered0.05%BovineSerumAlbumin (BSA) (dilutedinH2O,m/v)wasaddedtoobtainastocksolutionof1mg/
ml.Afterabriefvortexing,thesolutionwassonicatedusingaprobe sonicatorat 10%amplitude (Sonifier450S, BransonUltrasonics, Danbury,USA)for 15min.Foreach single experimenta freshly preparedstockwasused.Inthecellcultureexperiments,controls wereexposedtothehighestvolumeof0.05%BSAthatwasusedto preparethehighestdoseof Achesondustfor exposure.Forthe highestdoseofAchesondust(4.5
m
g/cm2)thiscorrespondedtoa finalBSAconcentrationof0.000675%inthecellculturemedia.2.3.Achesondustcharacterization 2.3.1.SEM
The Acheson dust was prepared as follows: a volume correspondingto100
m
gwastakenfroma1mg/mlstockdispersedin0.05%BSAwhichwassonicatedasdescribedabovefollowedby filteringona47mmWhatmanNucleporepolycarbonatefilterwith 50nm pore size. Thereafter the filter was coated with a thin platinum film in a sputter coater (Cressington 208HR sputter coater,UK).Specimensof1010mmwerecutfromthefilterand gently fixed on aluminum specimen stubs with double-sided
carbonadhesivediscs.ThespecimenswereanalyzedwithaHitachi SU 6600 (Ibaraki-ken, Japan) field emission scanning electron microscope(FE-SEM)equippedwithaBrukerenergy-dispersiveX- raydetector. Theinstrument was operatedunderthefollowing conditions: accelerating voltage 15keV and working distance 10mm.Highresolutionimagesoftheparticleswereobtainedby acquiring at slow scanning speed. Initially, specimens were examined in theSEM todeterminetheirmorphology and size.
The chemical composition of the Acheson dust particles was obtainedbyenergydispersivex-rayspectroscopy(EDX).
2.3.2.Dynamiclightscattering
To obtain information on the dusts’ hydrodynamic size distributionafterdispersion,ZetaSizerNanoZS(MalvernInstru- mentsLtd,UK)wasused.Immediatelyaftersonication1mlofthe sonicatedsolutionwaspipettedintoacuvette,leftonthebenchfor 5minandwasthereafterleftfor5minintheZetaSizerapparatus before measuring over 10 cycles. ZetaSizer software (Malvern InstrumentsLtd, UK)wasused toanalyzethedata.Theresults shownarefromthreeindependentmeasurements.
2.4.Cellsandcellculture
Thehumanastrocytoma1321N1celllinewaspurchasedfrom Sigma-Aldrich(catalogueno.86030402).Theseareglialcellsfrom ahumanbrainastrocytomathatwasinitiallyisolatedin1972asa subcloneofthecellline1181N1(Macintyreetal.,1972).Cellswere routinelykeptinahumidified5%CO2and95%air incubatorat 37C in Dulbecco’s Modified Eagle’s Medium (DMEM, Fisher Scientific) containing 10% fetal bovine serum (FBS, Biochrom), 50U/mlpenicillinand50
m
g/mlstreptomycin(ThermoScientific).Thepassagenumberofthecellswaskeptbelow30.
2.5.Estimationofdustdosesusedforcellcultureexperiments The doses usedfor cell cultureexposures werekeptlow to mimic occupational exposure and were calculated following a mathematicalcalculationmodifiedfromAntoniniandcoworkers (Antoninietal.,2010,2013)todeterminethedailylungburdenofa worker working 8h per day. Incorporated factors were the occupational exposure limit for respirable dust in the silicon carbideindustry(0.5mg/m3),humanminuteventilationvolume (20.000ml/minE-6m3/ml),theexposureduration(8h/day),the deposition efficiency (set to 20%; (Oberdorster et al., 2005a, 2005b)).
Thedailydepositeddosewas:
0.5mg/m3(20.000ml/min106m3/ml)(8h60min/h) 0.20=0.96mg
Whenusingthesurfaceareaofthealveolarepithelium(human 102m2(Stoneetal.,1992))thisleadstoadepositeddoseof9E- 4
m
g/cm2 (0.96mg/1.020.000cm2=0.9E-6mg/cm2!0.0009m
g/cm2).Thisdosewas setas 1x andthe otherdoses(0,0.01, 0.1,10,100,1000,5000)usedinthisstudywerecalculated accordingly.Thuscellswereexposedto0,9E-6,9E-5,9E-4,9E-3, 0.09,0.9and4.5
m
g/cm2takingintoaccountthesurfaceareaofthecellculturedish,respectively.
2.6.Cytotoxicityassay
For eachtoxicity experiment 5000 cells/wellwere seededin triplicate in black 96-well plates with a transparent bottom (Nunclon,ThermoScientific).Cellswereallowedtoattachfor24h priortoadditionofdispersedAchesondustattheindicateddoses.
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Thereafter themedium was removed and thecellswere washed once withphosphate-buffered saline (PBS) toremoveexcessparticles.The Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich) was used to measurecytotoxicitylevelsbydilutingitinthecellculturemedia withoutsupplementsaccordingtothemanufacturer’sinstructions.
Afterincubationat37Cfor1h,absorbanceasopticaldensity(OD) wasmeasuredat450nmusingaSpectraMaxi3(MolecularDevices, California, USA).In addition, ODwas measured at 750nm asa referencewavelengthforbackgrounddetectionandwassubtracted
Fig. 1.CharacterizationofthecollectedAchesondust.A)Avolumecorrespondingto100mgdustwastakenfroma1mg/mlstockdispersedin0.05%BSAandfilteredthrougha 47mmWhatmanNucleporepolycarbonatefilterwith50nmporesize.ThedustwasinvestigatedbySEMandrepresentativeimagesweretakenfromthedifferentparticles thatwerefound.RepresentativeimagesofparticlescontainingSiC,C>80%,80%>C>50%and25%>Si,Ti-Alareshown.B)Particlesweredividedintogroupsaccordingto theirmorphologyandelementalcontents.PercentagesandrepresentativeSEMimagesofeachparticletypeareshown.n=308.SiC:siliconcarbide,C:carbon,Si:silicon,Ti:
titanium,Al:aluminum.C)Overviewofparticlesizemeasuredbydynamiclightscattering.AftersonicationoftheAchesondust1mlofsolution(1mg/ml)wasimmediately transferredtoatransposablecuvetteandleftonthebenchfor5minandthereafterinsidethemachinefor5minbeforemeasuring.10cycleswererun.TheZ-averagefrom threeindependentdispersedbatchesisshownstandarddeviation(SD).
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fromsampleODat450nm.Astandardcurvewithaknownnumber ofcellswasestablishedtocalculatethenumberofcellsineachwell.
2.7.MeasurementofintracellularROS
IntracellularROSlevelsweremeasuredusingdichlorodihydro- fluorescein (DCF) fluorescence. 2070-dichlorodihydrofluorescein diacetate (DCFH/DA) (Sigma-Aldrich) is a cell-permeable com- poundthatyieldsa fluorescent productwhenoxidizedbyROS.
1321N1cellswereseededinsixwellplates,allowedtoattachfor twodaysandexposedtothedispersedAchesondustfor24,48and 72h.TheprotocolforROSmeasurementismodifiedfrom(Liand Ellis,2014).Briefly,followingexposurethemediumwasremoved andmediumcontaining100
m
MDCFH/DAwasaddedtothecellswhichwerethenincubatedat37Cfor1h.Positiveandnegative controls were included in each experiment. Cells were then washedwith1xPBSandincubatedwith2%TritonX-100inPBSon icefor5min.CellswerecollectedinEppendorftubesbyscraping and then sonicated on a VialTweeter (UIS250V, Hielscher Ultrasonics GmbH, Teltow, Germany) for 25s at maximum amplitude.Thereaftertubeswerecentrifugedat15.000RPMfor 10minat4CandsupernatantwastransferredtonewEppendorf tubes.Eachsamplewasaddedintriplicatetoa96-wellplateand fluorescence was measured using SpectraMax i3 (Molecular Devices,California,USA)upon excitationof488nm.Finally,the protein concentration of each sample was measured and DCF fluorescencewascalculatedrelativetotheproteincontentofeach sample.
2.8.AssessmentofDNAdamagewiththecometassay
ToexamineDNAdamagingeffectsafterAchesondustexposure, themodifiedcometassaywasperformedfollowingapreviously describedprotocol(Azquetaetal.,2014).Briefly,cellswereseeded onsixwellPlates24hbeforeexposure.Cellswerethenexposedto theAchesondustfor24,48and72h.Attheendofexposure,cells wereharvestedbytrypsinization,mixedwithlowmeltingpoint (LMP)agaroseandtwodropsfromthis mixturewereplaced on glassslidesthatwerepre-coatedwith0.5%standardmeltingpoint agarose.Afterlysis incoldlysis buffer(2.5MNaCl,0.1MEDTA, 10mMTris,1%TritonX-100,pH10),incubationincold0.3MNaOH, 1mMEDTAfor20minandelectrophoresisinthesamebufferat 0.8V/cmfor20min,slideswereneutralizedwithPBSandstained withSybrGold1(Invitrogen)dilutedinTEbuffer.Slideswerethen examined under a Nicon fluorescent microscope using Comet assayIVsoftware(PerceptiveInstruments,UK),wheremean%DNA incomettailsfrom100comets/gelwasusedasameasureofDNA
strand breaks. To detect oxidative DNA damage, post-lysis incubation with the formamidopyrimidine DNA glycosylase (Fpg) was also carried out. Fpg is a multifunctional DNA base excisionrepairenzymethatremovesawiderangeofoxidatively- damaged bases (N-glycosylase activity) creating a so called apurinic/apyrimidinic site (AP site); and its AP lyase activity cleavesboththe30-and50-phosphodiesterbondsoftheresulting APsite.TheAPlyaseactivityintroducesnicksintheDNAstrand, cleavingtheDNAbackbonetogenerateasingle-strandbreakatthe siteoftheremovedbasewithboth30-and50-phosphates.Fpghasa preferenceforoxidizedpurines,excisingoxidizedpurinessuchas 7,8-dihydro-8-oxoguanine(8-oxoG).Since Fpgconvertsoxidized purinestostrandbreaks,thisallowsindirectreadoutofoxidative baselesions.
2.9.Statistics
StatisticalanalyseswereperformedinSigmaPlot(Systat,USA).
ThedatawereanalyzedusingonewayANOVAfollowedbyTukey’s posthoctest.IncaseoflackofnormaldistributiontheKruskal- WallistestfollowedbyTukey’sposthoctestwasused.Statistical significancewasassessedatP<0.05.
3.Results
3.1.CharacteristicsoftheAchesondust
The dust collectedin the furnace hall of a SiC factory was characterizedbyscanningelectronmicroscopy(SEM)andenergy dispersive x-ray spectroscopy (EDX). Representative images of someof the particlestructuresare shown in Fig.1A. Thetotal numberofparticlesinvestigated(n=308)weredividedintogroups taking into account their morphology and elemental content (Fig.1B).Thelargestgroup(34%)ofparticleswerecharacterizedby ahighpercentageofcarbon(>80%C)andflake-likemorphology.
The next particlegroup represents 29% of thetotal number of particlesexaminedandhadacarboncontentbetween50and80%, combinedwithasiliconcontentbelow25%,inadditiontooxygen.
Theseparticleshaddifferentstructures,includingdroplet-likeand more irregularly shaped particles. As seen from Fig. 1A, the particlesappeartobeagglomeratesofsmallerparticleswhichmay explain the somewhat large variations in carbon and silicon content.TheagglomeratesmayconsistofsmallerSiCparticles.The interactionvolumeofx-raysislargeandgeneratesEDXsignalfrom an effective volume much larger than the actual particle investigatedand quantitativeEDXanalysisis thereforedifficult.
Furthermore,22%of theexaminedparticles containedtitanium
0 20 40 60 80 100
Cell viability %
0 1 2 3 4 5 6
Fold change
*
* *
* *
*
*
*
*
*
*
*
*
(24 h)
- 9E-6 9E-5 9E-4 9E-3 0.09 0.9 4.5
dust
*
Fig.2. Achesondust-inducedcytotoxicityandROSisdose-andtime-dependent.A)Human1321N1astrocytomacellsweregrownandexposedtoshamortoAchesondustat theindicatedconcentrationsfor24,48and72hbeforemeasurementofcellularcytotoxicity.Cellviabilityofsham-treatedcellswassetto100%.B)Theindicateddoseswere usedtomeasurethelevelsofintracellularROSafter24,48and72h.Sham-treatedcontrolsweresetto1andthemeanfoldchangefromthreeindependentexperimentsin triplicateisshown.Bars:SE.*:P<0.05indicatesasignificantdifferencebetweenexposedcellsandthecorrespondingsham-treatedcontrol.
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(25% Ti) and aluminum (2.7% Al), and 8% of the particles containedtitanium(38%Ti),aluminum(3%Al)andvanadium (2.7%V).Atthesiteofcollection,theairborneparticulatematter seemtohavecomefromrawmaterialsandsourcesunrelatedto theactivefurnace heat zone. The smallfractionof SiCmay be attributedtothefactthatnofurnaceswerebeingdismantledinthe proximityofthesamplingequipmentduringsampling.Moreover, onlytwoofthe308(approx.0.65%)investigatedparticleshada fibrous shape. Measurements of the hydrodynamic size by dynamic light scattering indicated that the majority of the particleshad a Z-averageof 42657nmfollowed bya peakof 14430nm that are higher than the suggested 100nm to be considerednanoscaleparticles(Fig.1C).Thesesizescorrespondto the sizes of the smallest particles, including titanium and aluminum(22%)andtitanium,aluminumandvanadiumcontain- ingparticles(8%)foundbySEM.Inaddition,apeakof537478nm wasdetected andlikely consistsof thelargerSiC-,carbon- and silicon-containingparticles.
3.2.TheeffectofAchesondustoncellviability,ROSgenerationand DNAdamage
Exposure of the cells to the increasing concentrations of Achesondustindicateda dose-dependenteffectoncellviability whichwasreversibleatlatertimepoints(Fig.2A).Alldosesexcept for9E-6and9E-5
m
g/cm2induceda significantreductionincell viabilityafter24h.Forboth9E-4and9E-3m
g/cm2thisresponsewasbiphasicascellviabilityincreased.After72hthehighestand notoccupationalrelevantdosesof0.09,0.9and4.5
m
g/cm2hadasignificantimpactonthecellviability.Thus,thecytotoxiceffectof the Acheson dust on the 1321N1 cells is dose-dependent but transientwheretheinitialreductionincellviabilityobservedafter 24hisreversibleafter48and72hwithlowoccupationalrelevant doses(Fig.2A).Fourdoses,including9E-5,9E-4,9E-3and0.9
m
g/cm2werechosenforfurtherexperiments.Thelowdoseswouldbe comparable of occupational exposure scenarios, whereas the higher doses were included to look for extreme and overload exposurescenarios.
InvestigationofthelevelsofintracellularROSshowedthatthe low doses up to 9E-3
m
g/cm2 did not induce ROS (Fig. 2B).Moreover,a significantreductionwasobservedforthe9E-5
m
g/cm2 dose after 24h. The highest dose, 0.9
m
g/cm2, gave asignificant increase in ROS levelsat all the time points tested andpeakedat48h.Thus,lowandoccupationallymorerelevant dosesoftheAchesondustdonotinduceROS.
TofurtherinvestigatethemoleculareffectsoftheAchesondust onthe1321N1cells,cometassaywasperformedtodetectpossible damagetotheDNA.TheresultsshowedanincreaseinDNAdamage after24hwhichwassignificantforthehighestdose,0.9
m
g/cm2(Fig. 3A). This response was reversible as no changes were observedafter48and72h,exceptforasignificantdecreaseinDNA damage for the 0.9
m
g/cm2 dose after 48h. Furthermore, a significant, although small, increase was shown with 9E-4and 9E-3m
g/cm2 after 72h. To investigate possible oxidative DNA damagefollowingexposuretoAchesondust,post-lysisincubation withFpgwas alsoperformed.Theindirectreadoutof oxidative baselesionsshowedthattherewerenosignificantchangesforall thedosesandtimepointstested(Fig.3B).4.Discussion
Exposure to emitted dust in industrial processes poses a potential human health problem. There may be a correlation betweenhealtheffectsandexposurestoknownaircontaminants inSiCplants.Littleresearchhasbeenperformedontheeffectsof dustcollecteddirectlyfromthefurnacehallintheSiCindustryon
humanbraincells.However,inothercelltypessuchasRAW264.7 macrophages that were exposed to various manufactured SiC powderscollectedfromtheAchesonprocessandtoaSiCpowder representativeofairbornedustthisdidnotinducecytotoxicityin dosesrangingfrom20to120
m
g/106cellsafter24h(Boudardetal.,2014).However,theairbornedustrepresentativeinducedtumor necrosisfactor
a
(TNFa
)possiblyduetothepresenceofcrystalline silicaandironimpurities(Boudardetal.,2014).Toxicityofparticlesiscloselyrelatedtotheirphysico-chemical properties, including size, shape, surface area, and chemical composition. The raw materials for SiC production consist of quartzsandandpetroleumcoke,inadditiontobothunreactedand partlyreactedmaterialfrompreviousfurnacecycles.Graphiteis usedasanelectricconductorandthereforetracesofgraphitemay alsobepresent.MostconcernondustfromtheAchesonprocessis on SiC fibers and most studies focus on this. In ourcollected material only 6% consisted of SiC and among the particles detected asSiC,only10%had a fibrous structure.Otherstudies frombothNorwayandCanadahavereportedonairbornefibers and presence of carbonaceous matter, quartz, cristobalite, SiC, respirabledust,benzene solublematterand commonpolycyclic aromatichydrocarbons(Byeetal., 1985;Dufresneetal.,1987, 1995;
Dionetal.,2005).Asthefurnacehallcontainsamixtureofdusts andgasestheexposurepatternmaybedifferentatvariousplaces inthefurnacehall.Moreover,thelevelsofimpuritieswilllikelybe differentforthevariousAchesonprocessworkplaceswheredustis collected.Thesefactorswouldcomplicatecomparisonofdifferent studies.
0 0.5 1 1.5 2
Fold change
24 h 48 h 72 h
*
*
0 0.5 1 1.5 2
Fold change
- 9E-5 9E-4 9E-3 0.9
dust µg/cm2
* *
Fig.3.DNAdamageinthe1321N1astrocytomacellsafterexposuretotheAcheson dust.A)CellswereexposedtotheindicateddosesofAchesondustfortheindicated times.ThereafterdamagetotheDNAwasinvestigatedusingthecometassayas describedinMaterialsandMethods.Foranalysisthepercentageoftailintensitywas calculated.B)OxidativedamagetotheDNAwasalsoinvestigatedbypost-lysis incubationwithFpg.Foranalysisthepercentageoftailintensitywascalculated.
Foldchangeforeachexposurefromthreeindependentexperimentsintriplicateis shownwith sham-treated controls set to 1. Bars: SE. *: P<0.05 indicates a significantdifferencebetweenexposedcellsandthecorrespondingsham-treated control.
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Thedosesinthepresentstudywerekeptverylowtomimic occupationalexposurescenariosandwereestimatedtakinginto considerationtheoccupationalexposuresituationsforparticulate matterfromtheAchesonprocess.TheAchesondusthadadose- andtime-dependenteffectonthecells.Withthelowestdosesthe initialdecreaseincellviabilityasaresponsetoacutetoxicitywas biphasicas cellviabilitywas restored atlater time points.The highestdose,0.9
m
g/cm2,inducedasignificantincreaseinROSand correspondedtoacontinuingdecreaseincellviability.Thus,for thelowerdosesdetoxificationprocessesmighthaverestoredcell viabilityprobablyduetoincreasedcellproliferation,whereasthis wasnotobtainedforthehigherdoses.Itisknownthatparticle- inducedROScanoccurinvariousways,includingthepresenceof pro-oxidantgroupsonthesurfaceoftheparticles,redoxreactions onthesurfaceofmetalsandparticle-cellinteractions(Mankeetal., 2013).Furthermore,astheinductionof ROSwastransientwith 0.9m
g/cm2, peaking after 48h, this may indicate activation ofdetoxificationprocessestoreducetheselevels.Transientincreases inROSlevelshavealsobeenreportedbyothers(Barilletetal.,2010;
Mankeetal.,2013;Carrascoetal.,2016).However,atthistimecell viability was not completely restored and indicates that other cellularprocessesareactivatedthatcontributetothereducedcell viability.Asthereweresmallandtransienteffectsoncellviability and ROS production it was of interest to investigate possible damagetotheDNA.Also,ROSlevelsmayincreaseduetodamageto theDNA(Roweetal.,2008;Kangetal.,2012)andhasbeenshown tobeimportantfortheregulationofcellsurvivaland apoptosis (Simon et al., 2000; Hamanaka and Chandel, 2010). The data indicatedDNAdamageafter24hwiththehighestdosetested,and after72hwith9E-4and9E-3
m
g/cm2.However,thelevelsareverylowandforthehighestdosereversible,indicatingthatDNArepair mechanisms possibly are activated. Furthermore, testing for oxidative damage by including Fpg in the assay indicated no significantdifferences,butothertypesofDNAdamagecannotbe ruledout.
Exactly what fraction of the Acheson dust that causes the increaseincytotoxicity,ROSandDNAdamageisnotknown.Metal impuritieshaveanimpactontheseendpoints andtheAcheson dustusedherecontainsavarietyofelementsthatmaycausethe observedeffects.Severalstudieshaveinvestigatedtheeffectsof differenttypesofparticlesonprimaryastrocytes.Astudyfocusing oncarbonshowedthatastrocytesgrownoncarbondecreasedtheir function(Mckenzieetal.,2004).Workperformedoncarbonblack nanoparticlesshowedthatmiceexposedtothismaterialinduceda long-termactivationofastrocytesresultinginreactiveastrogliosis inthebrains oftheiroffspring,probablyduetothepresenceof carbon(Onodaetal.,2017).Moreover,severalneurotoxicological studieshavebeenperformedontheeffectsofaluminumexposure.
It has beenassociated with a variety of neurological disorders linkedtoanincreaseinoxidativeandinflammatoryeventsleading totissuedamage(Campbell,2002).Culturedastrocytesexposedto aluminum had an intracellular accumulation and reduced cell viabilitywascharacterizedbyDNAfragmentationandapoptosis (Suarez-Fernandez et al., 1999). Vanadium, another element presentinthedust,haspreviouslybeenshowntobeneurotoxic to the central nervous system in rats where it increased the presenceofenlargedastrocytesinthecerebellumandhippocam- pus(Garcia etal.,2005).AsmoststudiesondustfromtheSiC industrymainlyfocusonSiC,inparticularSiCfibers,onemustnot forgetthepossibleeffectscausedbyotherelementalcontents.
Inthecurrentstudy,anastrocytomacelllinewasemployed.The 1321N1 cell lineis well-established and used in many studies investigatingneurotoxiceffectsofvariouscompounds,andcould bea suitable cellmodeltogain insightin possibleeffectsafter exposuretoAchesondust.However,thecharacteristicsofthese cellsmaydifferfromprimarycellsandtherefore,asimilarstudy
using primary astrocytes would be more relevant. During the experiments serum was used in the cell culture media as recommended by the manufacturer. As serum may vary from batchtobatchthismayhavehadasmallpotentialimpactonthe results. Theoccupationallyrelevantdoses wehaveused inthis study do not reflect the actual amount of particles that are translocatedintothecirculationthroughuptakebythebloodinthe lungsorbytheolfactorybulb.Differencesinbreathing,deposition inupperairways,andclearanceoftheparticlesfromtheairways andparticlecharacteristicswillinfluencetheactualdepositionin the alveolar region. Studies on the actual amount of particles passingfromthelungstothebloodarefewandinconclusive.A studybyGeiserandKreylingshowedforexamplethataverylow percentagemaytranslocatefromlungsandcrossthebloodbrain barrierandenterthebrain(GeiserandKreyling,2010).Therefore, thedosesusedinthepresentstudywouldstillbehighcomparedto thedosethat mayultimatelyenter thebrainafteroccupational exposure.
Conclusively, the main idea of this study was to look for cytotoxiceffectsofdustfromtheAchesonprocess,andthentolook if the toxicity could be related to increased ROS production followedbyifROSproductionwasduetodamagetoDNAbases, specificallypurines,orwhetheritwasnotrelatedtoDNAdamage.
Atthispoint,ourmechanisticdatasuggestthattheAchesondust maynotbehighlytoxicatleastatlowdosesortheremightbea transienttoxicitywhichmaynotberelatedtopermanentdamage toDNAbases.
Conflictofinterest
None oftheauthorshasany potentialconflict ofinterest or financialintereststodisclose.
Acknowledgements
This work was supported by a postdoctoral grant from the ResearchCouncilofNorwaytoYJA(RCNgrantno.245216O70).We wouldliketothankVainetaVebraiteforexcellenthelpwiththe cometassays.
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