MackenzieNalepa,BrettSmith,SamanthaWargo

11/24/15
CellBiologyLabBL248

EffectofPhenolontheExpressionofBAXinMouseLungFibroblasts

Introduction

Muchresearchhasbeenconductedoncigarettesmoke,itscomponentcompounds,and

theirharmfuleffectsonhumans.Althoughthepublicknowledgeofcigarettesmokehasbeen

increasing,recentstudiesreportthatcigarettesmokeisstillresponsiblefor480,000American

deathseachyear.Cigarettesmokingislinkedtonumerousdiseases,includingbutnotlimitedto

varioustypesofcancers,pneumonia,stroke,coronaryheartdisease,andchronicobstructive

pulmonarydisease(U.S.DepartmentofHealthandHumanServices,2014).Statisticsdisplay

thatsmokingistheleadingcauseofpreventabledeathintheUnitedStates.Notonlyiscigarette

smokeresponsiblefordestructionwithinthepopulation,butstudiesalsoshowthatcigarettesare

responsiblefor$289billioninhealthcarespendingeachyear(Jamaletal.,2014).

Notonlyiscigarettesmokingdirectlyrelatedtohealthcomplications,butinhaling

secondhandsmokecanalsoproducethesameeffects.Eachyearthereareapproximately7,000

deathsfromlungcancerand33,950deathsfromheartdiseaseinadultsthatresultedfrom

exposuretosecondhandsmoke(AmericanLungAssociation,2014).Thesedeathsarearesultof

theexposureofmanytoxicchemicalsfoundincigarettes.Whenisolated,tobaccosmokeis

comprisedofover7,000chemicals,70ofwhichareknowntobecancercausingagents.Someof

thesecompoundsincludeformaldehyde,benzene,hydrogencyanide,andarsenic.These

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compoundsarealsofoundinresins,gasoline,chemicalweapons,andpesticides,respectively

(CentersforDiseaseControlandPrevention,2011).

Ourcolleaguesinthebiochemistrylaboratoryextractedvariouschemicalsfromcigarette

smoke,oneofwhichisphenol.Phenoliscomposedofonehydroxylgroupattachedtoan

aromaticbenzeneringandisoneofthemostcommonrepresentativesoftoxicorganic

compounds.Studieshavealreadydisplayedtheharmfuleffectsofphenolinmiceembryos

injectedthroughtheplacenta.Theseharmfuleffectsweredisplayedthroughdecreased

embryonicweight,increasedplacentalthickness,andhigherconcentrationsandsizesof

spongiotrophoblastsandtrophoblasticgiantcells(Monfaredetal.,2013).Somesubstituted

phenolderivativesareknowntohaveapoptosisinducingeffectsonleukemiacellsinmice

(Tsygankova,I.M.&Zhenodarova,S.M.,2008).

Apoptosisisamechanismofprogrammedcelldeathinwhichthecontentsofthecellare

compartmentalizedintoapoptoticbodies.Thereleaseoftheseapoptoticbodiesdestroysthecell

inawaythatdoesnotcauseinflammationinthesurroundingtissue(Reed,2000).Inthisrespect,

apoptosisoccursasamechanismofhomeostasisorimmunologicdefenseforcolonieswithin

tissue.Thispathwaycanoccurwhencellsaregeneticallyprogrammedtoundergoapoptosis

continuallyorwhenthecellisstimulatedbyligandbinding.Twocommoncharacteristicsof

apoptosisareoverallcellularshrinkageandpyknosis.Pyknosisinvolvesthecondensationof

chromatininthenucleus(Elmore,2007).Caspasesareproteasesthatareinvolvedinthese

processesandtheoverallbreakingapartofthecellularcomponentswithATPhydrolysis(Reed,

200).

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 Anotherformofcellulardeathisnecrosis,whichdoesnotrequireenergyandismore

toxicandinflammatorytothesurroundingtissue.Inthenecroticpathway,thecellswellsand

bursts,breakingdownthechromaticmaterialthroughkaryolysisinsteadofcondensingit.

NecrosisisthereforeinducedwhenthecellhaslowlevelsofcaspasesorATP,bothofwhich

mustbepresentforapoptosistooccur(Elmore,2014).Whenthecellbursts,intracellularleakage

fromthecelloccursbecausetheplasmamembraneisbrokendown.Itwaspreviouslythought

thatnecrosiswasaccidental,butrecentstudieshaveshownthatnecrosiscanalsobeplanned,

likeapoptosis(Chanetal.,2013).

TheproteinofinterestforthisstudyisBAX,whichisintheBcl2familyofproteins.In

thisfamily,someoftheproteinsinhibitapoptosis,whilesomeofthemactivateapoptosis

(Albertsetal.,2014).WhenBAXisactivated,itactstomaketheoutermembraneof

mitochondriamorepermeable,whichreleasescytochromecintothecytosol.Thisprocesstakes

under10minutestooccur(Green,2005).Cytochromecthenbindstoaproteinthatformsan

apoptosome,whichhassevenarmsinapinwheelshape(Albertsetal.,2014).These

apoptosomesthenbecomedispersedthroughphagocytoticmechanisms,aprocessthattakes

hoursordays(Green,2005).SomemembersoftheBcl2familyareapoptoticinhibitors.For

instance,BclxLisaninhibitorandregulatorforBAX(Edlichetal.,2011).Someresearch

showstherelationshipbetweenphenolandtriazolesmoietesandtheirabilitytobreakthe

interactionsofBAXandBclxLinawaythatpromotescelldeath(Vo,D.D.,etal.,2014).

Inthisstudy,thegoaloftheexperimentwastotestthetoxicityofphenolandthelevelof

BAXexpressiononnormalfibroblastmouselungcells.Thecelllinesusedforthisstudywere

CCL206fibroblastmouselungcellsandCCL196epithelialadenomamouselungcells,studied

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byapartnergroup,purchasedfromATCC(ATCC,2014).Thefibroblastlungcellsarereferred

toasnormalmouselungcellswithinthecontextoftheexperiment.Thenormallungcells

wereculturedanddosedwithvariouslevelsofphenolinordertoperformthenecessary

experimentsneededforanalysis.

Trypanblueassayisananalysisperformedtodeterminecellsurvivalafterchemical

treatment.Whencellsaredead,oronthevergeofdying,theirmembranebecomesdamaged

therefore,themembraneismorepermeable.Thischaracteristicallowsthetrypanbluetodiffuse

acrossthemembranesofdeadcells,givingthecelladarkbluetint.Thecellsthatappearblueare

classifiedasdead,andthecellsthatarestilltransparentareclassifiedasliving.

ALIVE/DEADassayisperformedusingaLIVE/DEADViability/CytotoxicityKit.The

chemicallytreatedcellsarestainedwithchemicalcompoundsknownasfluorochromesthat

absorbandemitdifferentwavelengths.Cellsthatarealiveandmaintainanintactmembraneemit

greenfluorescentcalceinAM.Cellsthatarepresumeddeadduetodysfunctionalandpermeable

membranesemitredfluorescentethidiumhomodimer1(EthD1).Fluorescentmicroscopyis

usedtoexaminethecells.

qRTPCRisusedtoanalyzetheBAXgeneexpressioninisolatedmRNAfromthetreated

cells.ThisassayusestheAmbionTaqManPreAmpCellstoCTKitfromLifeTechnologiesfor

theanalysis.ThiskitallowsforquickRNApurificationwithouttheriskofcontaminatingor

losinganyofthesample.Thecellislysed,themRNAisisolated,anygenomicDNAis

degraded,andRNaseswithinthecellaredeactivatedthroughthisprocess.Onlythepurified

mRNAremains,whichisthenconvertedtocDNAthroughreversetranscription.ThecDNAis

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thenamplifiedforthetargetproteinexpressiontobeassessedthroughquantitativerealtime

PCR.

TheWesternblottechniquewasthefinalanalysisusedinthisstudy.Sodiumdodecyl

sulfatepolyacrylamidegelelectrophoresis(SDSPAGE)isusedtoconfirmthelevelsofgene

expressionperformedbyqRTPCR.Electrophoresisallowsproteinstobeseparatedbasedon

molecularweight,asproteinfoldingandchargeareeliminated.Theseparationmeansthatthe

largerproteinswilllocatenearthebottomandthesmalleratthetop.LumiGLOallowsthe

presenceofprobedantibodiestocapturelightonanXrayfilmandresultintheformationof

blackbandsthatcorrespondtotheproteinofinterest.StainssuchasPonceauandCoomassie

bluearealsousedinthistypeofanalysistointensifythebandsshownintheWesternblot.

Wehypothesizedthatthatthetreatmentofnormalmousefibroblastlungcellswith

variousdosesofphenolwilldecreasethesurvivalcomparedtoadenomamouseepitheliallung

cellsandincreasetheexpressionofBAXcomparedtoadenomamouseepitheliallungcells.

MaterialsandMethods

CellCulture

Tomaintainthecelllineofnormalmouselungcells(catalognumberCCL206

purchasedfromATCC),thecellswereperiodicallyculturedandpassagedtoamoreoptimal
in

vitro
environment.Thispassagingensuredthatthesamplesdidnotbecomeconfluentandperish.

Thecellswerekeptinamediacontainingnutritionalfetalbovineserum,antibiotics,andapH

indicator.Foreachpassage,thismediawasaspirated,andthecellswerethenwashedwith5mL

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of1XPBSbuffersolution.Followingthebufferwash,thecellsweretreatedwith1mLof

trypsinEDTAandplacedina37Cincubatorfor5minutes.Thecellswereexaminedundera

microscopetoconfirmdetachmentfromthebottomoftheplate.Thedetachedcellswerethen

mixedwith3mLofmediaandtransferredtoa15mLstoragecontainer.10uLofsamplewere

pipettedtocounttheconcentrationofcellsinthemedia,usingahemocytometer.The

concentrationofcellsneededforanoptimalconcentrationofcells/mLinanewplatewas
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calculated.Thisconcentrationwasdeterminedtobe7x10cells/mL.Thecalculatedvolumeof

mediasolutiontoobtainthisoptimalconcentrationwasaddedtoanewplate,andmediawas

addedsothetotalvolumeofthesolutionwasbetween57mL.

TrypanBlue&Hemocytometry

Followingthetrypsinizationoftheplates,3mlofmediawasaddedtothecellplateand

mixedwiththetrypsinEDTA.Thismixturewasplacedinatesttube,fromwhich100ulwas

transferredtoaneppendorf.Tocountthecellsstainedwithtrypanblue,10ulofthecellsand10

uloftrypanbluewereplacedinaneweppendorf.Fromthismixture,10ulwereplacedona

hemacytometerandcountedunderthemicroscope.

SeedingandDosing

Thecelllineusedforchemicaltreatmentwithphenolwasmaintainedbycellpassaging

andculturing.Afterthecellswerepassagedintotwoplates,thoseplateswereeachpassagedinto

twoplatesforatotaloffourplates.Thecellsweredosedwithphenol16hoursbeforeanalysis.

Thefourplatesaccountedforeachofthefourconcentrationsofphenolused:0.0mM,0.12mM,

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0.3mM,and0.6mMofa60Mstocksolution.Thecellswerecountedusingahemocytometerand

theappropriateamountofphenolwascalculatedtocorrespondtothecorrectconcentrationfor

eachplate.Inplatewiththe0.0mMconcentration,0.0ulofphenolwasadded.Forthe0.12mM

concentration,8.3ulofphenolwasadded.Intheplateintendedforthe0.3mMofphenol,25ul

wasadded.Forthe0.6mMconcentration,10ulwasaddedtoeachplate.Inorderforeachplateto

contain5000uloftotalvolume,thesameamountofmediaastheamountofphenoladdedwas

pipettedoutoftheplatebeforechemicaldosing.

Forthepreparationofthefluorescentmicroscopyanalysis,thecellswereseededintoa

fourchamberwellratherthanfourindividualplates.Thecellswereseededintothewellstwo

daysbeforethemicroscopywascompleted.Theamountofcellsusedforthewellswasatenfold

differencelessthantheamountusedfortheplates.Theamountofphenoladdedtothewellswas

0.0ul,1.6ul,5ul,and10ulrespectivetothe0.0mM,.12mM,0.3mM,and0.6mMphenol

concentrationsofeachwell.Thefinalvolumeofthewellswas1000ul.Inorderforeachwellto

contain1000uloftotalvolume,thesameamountofmediaastheamountofphenoladdedwas

pipettedoutofthewellbeforechemicaldosing.

FluorescentMicroscopy

Topreparethewellsformicroscopy,themediawasfirstaspirated.Ineachwell,150ulof

EthD1andcalceinAMsolutionwereadded.Thecellswereincubatedwiththisfor45minutes.

Thechamberwasremovedfromtheslideandtheedgesaroundeachblockwerescraped.The

cellswereobservedwithtwodifferentfilterstoseetheliveanddeadcellsthatemittedthe

fluorochromes.

RNAExtraction

Thecellswerecountedwiththehemocytometerandcentrifugedforfiveminutes.The

PBSwasaspiratedfromthetube.ThecellswereresuspendedinPBStomake1x10^6cellsfor

every500ulofPBS.50ulofcellsweretransferredtoamicrocentrifugetube.Thesupernatant

wasremovedandthecellsofeachconcentrationwereresuspendedin50ullysissolution.Each

samplewasmixedfivetimeswiththemicropipette.Thesampleswereincubatedatroom

temperaturefortwominutes,afterwhich6ulofstopsolutionwasaddedandmixedfivetimes

withthemicropipette.Thesampleswereincubatedatroomtemperaturefortwominutesagain,

andthentheywereplacedinthefreezer.

qRTPCR

ThetubewiththecDNAwasfirstcentrifuged,verybriefly,tobringthecondensation

down.ThemastermixesfortheBAXandGAPDHwerepreparedinmicrocentrifugetubesas

follows:10uloftheprobe(specifictoeitherBAXorGAPDH),100ulTaqmanmastermix,and

50ulPCRgradewater.Thewellsofa96wellreactionplatewerethenprepared.Each

concentrationhad2wellsofBAXand2wellsofGAPDH.Eachwellhad16ulofthemastermix

(BAXorGAPDH)and4ulofthecDNAcorrespondingtothatconcentration.Theplatewas

brieflycentrifugedandthenloaded.

ProteinSamplePreparation

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 A3XReducingSDSLoadingBufferwasdilutedtoa1XReducingSDSLoadingBuffer.

Afterthemediawasaspiratedandthesampleswerewashedwith1XPBSsolution,175mLof

the1XReducingSDSLoadingBufferwasaddedtotheplate.Arubberpolicemanwasusedto

scrapethesamplesfromthebottomoftheplateandintoamicrocentrifugetube.Thesamples

werekeptonicethroughoutthispreparation.Thesamplesweremixedbybeingpassedthrougha

26Gneedle10timesandthenheatedto95100forfiveminutes.Thesampleswerecooled

andcentrifugedforfiveminutes.Theseproteinsampleswerethenputinthefreezeruntilthe

Westernblottingwasperformed.

WesternBlotting

Theglassplatesofthegelmoldwerecleanedwithwaterandthenethanol,thenplaced

together.Thelargerrectanglewasplacedwiththespacersfacingupandthenthesmallerplate

wasplacedontop,withthebottomedgesliningup.Theplateswereplacedinthegreencasting

framewiththeshortplatefacingtheclampside.Thiswasplacedintothecastingstandwiththe

clampsfacingoutward.Theresolvinggelwaspreparedbyadding3.1mlofAcryl:bis(30%),3

mlof1.0MTrisCLpH8.8,38ulof20%SDS,and1.3mldHO.Afterinvertingthetube
2

slowly3times,theother2ingredientswereadded:36ul10%APSand5ulTEMED.Thetube

wasinverted2additionaltimesandthenpipettedimmediatelybetweentheplates.Theplates

werefilledapproximatelythreefourthsfullandthen12mlofwaterwasaddedontopofthegel.

Afterpolymerizationoccurred,indicatedbyasharplinebetweenthegelandwater,thewater

waspouredoutandblottedwithfilterpaper.Thestackinggelwaspreparedbyadding1.1mlof

Acryl:bis(30%),630ulof1.0MTrisCLpH6.8,25ulof20%SDS,and3.5mlofdHO.The
2

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tubewasinverted3timesslowly,then25ulof10%APSand5ulofTEMEDwasadded.The

tubewasinvertedagain2timesandimmediatelyaddedtotheplatesontopoftheresolvinggel.

Thecombwasplacedintotheresolvinggel,gently,sobubbleswerenotintroduced.Afterthe

stackinggelwassolidified,thecombwasgentlyremovedandthewellswererinsedwithwater.

Thepreviouslypreparedproteinsampleswereboiledwithparafilmwrappedaroundthetop,for

10minutes.Whilethesampleswereboiling,theelectrodeassemblywaspreparedbyplacingthe

plateswiththegelinside.Whentheproteinsampleswereready,theywereplacedintothewells.

Thegelwasrunforapproximately1houruntilthebromophenolbluedyehadmigratedtothe

bottom.

Afterthegelwasrun,ithadtobeblotted.Twopiecesoffilterpaperandonepieceof

membrane,withacornercutoff,weresoakedintransferbufferfor15minutes.Thegelwas

scrapedofftheglassandintotransferbuffertosoakfor5minutes.Intheclamp,thesetupwas

inthisorder:sponge,filterpaper,nitrocellulosemembrane,gel,filterpaper,sponge.Theclamp

wasplacedinthechamberalongwithanicepackandtransferbuffer.Transfertookplaceforone

hourafterwhichthemembranewastakenoutandwashedin1XTBSTforfiveminutes.At

roomtemperature,themembranewasplacedinblockingbufferforonehour.Themembranewas

thenstoredin1XTBSTuntilreadytoproceedtoimmunodetectionthefollowingweek.

ThemembranewascutintotwodifferentpieceswithactinononepieceandBAXonthe

otherpiece,inrespecttowherethebandsforeachwouldshowup.TBSTwasusedtowashthe

membranethreetimeswithjustenoughtocoverthemembrane.Whenwashing,thecontainers

wereshookforfiveminuteseachtime.Themembraneswereplacedin10mloffreshblocking

solutionthatcontainedtheprimaryantibodyatadilutionof1:1000.Theprimaryantibodiesused

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wereactinandBAXrespectively.Thecontainerswereplacedontheshakerforonehour.The

antibodysolutionwasdiscardedandthemembraneswerewashed3times.Fivemlofthemilk

solutionandfiveulofthesecondaryantibodywereaddedtothemembranes.Thesecondary

antibodyusedwasrabbitmAb,forboth.Again,theywereshookforonehour.Themembranes

werewashedthreemoretimes.Thesubstratesolution,0.5mlLumiGLO,0.5mlperoxide,and

9.0mlwater,wasaddedtothemembraneswhichwerelaidonplasticwrap.Thesubstrateand

membraneswereincubatedforoneminute.Theexcesssolutionwasremovedandthe

membraneswerewrappedinplastic.TheXrayfilmwasexposedtothemembraneforone

minuteandonehourandthenthefilmwasdeveloped.

Results

Thetrypanblueanalysisshowsthatcellsurvivalremainedrelativelyhigh,between

95100%survival,forthenormalmouselungcells.Thenumbersfluctuateandshownogeneral

trend.Incomparison,thesurvivaloftheadenomacellsshowedaslightincreasebetween0.0mM

and0.12mM,andthenadecreasebetween0.12mMandthehigherconcentrations.Overall,the

normalmousecellsshowedmoresurvivalthantheadenomacells.ThedatainFigure1shows

theaveragedatafromtwotrypanblueanalyses.

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Figure1.Quantitativeanalysisofpercentsurvivalofnormalandadenomamouse

lungcellstreatedwithconcentrationsof0.0mM,0.12mM,0.3mM,and0.6mM

phenol.
Thepercentsurvivalwastestedforcellsthatremainedattachedtothepetridish

followingaspirationofmediaaftertreatment.Cellsthatdiedanddetachedfromthepetri

dishwouldnothavefactoredintothiscalculationforsurvivalrate.Forthenormalcells

(showninblue),thecellsweretreatedwithphenolfor16hours.Fortheadenomacells

(showninred),thecellsweretreatedwithphenolfor24hours.Theerrorbarsdenote

standarddeviationofthesamplesaftertwoindependentruns.

Totakeaccountforanydeadcellsthatmayhavecomedetachedfromtheplateand

aspiratedduringcellculture,percentattachmentofcellstothepetridishwasanalyzed.The

percentattachmentisbasedonthecontrolplate(0.0mM)beingthenormalizedamount.The

normalcellsshowageneraldecreasingtrendinattachment.Thisisinexceptionofthehighest

concentration,0.6mMthatshowsaslightincreasefromthe0.3mMconcentration.In

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comparison,theadenomacellsshowamuchhigherpercentattachment.Theirtrendisthe

opposite,wherethepercentattachmenthasageneralincreasewiththeexceptionofthehighest

dosage.Thenormalcellswereexposedtothephenolfor16hourswhiletheadenomacellswere

exposedfor24hours.

Figure2.
Quantitative analysis of percent attached of normal and adenoma

mouse lungs cells treated with concentrations of 0.0 mM, 0.12 mM, 0.3 mM,

and 0.6 mM phenol.

Thepercentattachmentwastestedforbothsamplesofcellsat

varyingconcentrationsofphenol.Forthenormalcells(showninblue),thecellswere

treatedwithphenolfor16hours.Fortheadenomacells(showninred),thecellswere

treatedwithphenolfor24hours.Theadenomacellsdisplayedamuchhigherpercent

attachmentandrange.Theerrorbarsdenotestandarddeviationofthesamplesaftertwo

independentruns.

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 ThequalitativeeffectsofpercentsurvivalwereanalyzedusingaLIVE/DEADassay

shownthroughfluorescentmicroscopy.Survivalanddeatharerepresentedbythereflectionof

fluorochromes.ThecellsthatappeargreenrepresentthelivecellsthatreflectthecalceinAM

andthecellsthatappearredrepresentthedeadcellsthatreflecttheEthHD1.Thephotographs

(Figure3)showthatastheconcentrationofphenolincreases,theamountofsurvivingcellsalso

decreases,shownbyalackofgreenfluorescence.Inthehighestconcentration,itappearsthatthe

majorityofthecellsaredead.Figure4showsamagnified,1000x,imageofthecellsdosedin

0.6mMphenoltoshowinmoredetailtheeffectsthechemicalhadoncellsurvival.

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Figure3.FluorescentmicroscopyandLIVE/DEADassayqualitativeanalysisofnormal

fibroblastmouselungcellsdosedinconcentrationsof0.0mM,0.12mM,0.3mM,and

0.6mMofphenolovera16hourtimeperiod.
Inthefigureabove,livingcellsarerepresented

byagreencolorobtainedbythereflectionoffluorescentgreencalceinAMdeadcellsare

representedbyaredcolorobtainedbythereflectionofredfluorescentethidiumhomodimer1.

Thephotosweretakenthroughtheeyepieceofafluorescentmicroscopezoomedinto400x

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magnificationonaniphone6camera.(A)representsthecontrolgroupandshowsmostlyliving

cells.(B)representsthecellsdosedwith0.12mMphenol.Mostofthecellsshownareliving,

despitethefewdeadcellsmarkedwithaglowingredcolor.(C)showsslightlylesssurvivalwith

theappearanceofmostlydeadcellsinthe0.3mMconcentration.(D)showssignificantdeathat

the0.6mMconcentration.Thereislittletonoliferepresentedbythelackofgreencellspresent

inthisphotograph.

Figure4.Fluorescentmicroscopyqualitativeanalysisofcellsdosedin0.6mMphenolover

a16hourtimeperiod.
Thisisazoomedinphotoat1000xmagnificationtoshowinmoredetail

theeffectsofthephenol.

TheresultsfromtheqRTPCRshowtheexpressionlevelofBAXinnormalcells

exposedtophenolfor16hours.OurresultsoftheqRTPCRarecloselyrelatedtothesecond

analysisofthepercentsurvivalgraph,whichshowsmasteryofthetechnique.Thegraphbelowis

16
anaverageofthefirstandsecondtrialforbothcelltypes.Therewasanoveralldecreasein

expressionofBAXexceptfortheconcentration,0.3mM,thatshowedahigherexpressionthan

therestoftheconcentrations.Relativetothecontrol,the0.12mMconcentrationhada0.482

folddifference,the0.3mMconcentrationhada0.78folddifference,andthe0.6mMhada

0.198folddifference.

Figure5.RelativequantificationofBAXexpressionincellschemicaltreatedwith0.0mM,

0.12mM,0.3mM,and0.6mM.
WeperformedaqRTPCRanalysistodeterminethe

expressionofBAXofuntreatednormalmouselungcellsincomparisontonormalfibroblast

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mouselungcellstreatedwith0.12mM,0.3mM,and0.6mMphenol.TheBAXexpressionwas

quantifiedthroughcalculationstofindthefolddifferenceinreferencetotheGAPDHcontrol.

Theerrorbarssignifystandarddeviationsbetweentwoseparaterunsofeachconcentration.Note

thatthebarsinthegraphdonotgoinincreasingorderofphenoldosage.

TheWesternblotanalysisshowedthequalitativeresultsoftheqRTPCR.Theresultsof

theWesternblotareinconclusiveduetolackofvisibleactinbandmarkers.Inthepositionof

0.12mMconcentrationwell,thereisabandlocatedat21kDaindicatingBAXexpression.There

isalsoafaintbandlocatedat21kDainthepositionofthe0.3mMconcentration.Themarker

wellislabeledinthefigurebelow.

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Figure9.WesternblotqualitativeanalysisofBAXandactinexpression.
(A)showsthe

membraneafteritwasstainedwithponceaustaintoindicatetheBAXexpression.(B)showsthe

membraneafteritwasstainedwithcoomassiebluestain.Thearrowsindicatetherowwhere

BAXexpressionbandsappear.

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Discussion

ThehypothesiswasrejectedbaseduponthegeneraldecreaseinBAXexpressionwith

increasingdosageofphenol.Notably,theresultsfromtheadenomasamplesshowedopposite

effects,withincreasingBAXexpressionwithmorephenoliceffects.Celldeathincreasedwith

higherphenoldosages.Thistrendwasmorestronglyevidentthroughthepercentattachment

results(Figure2)thanthepercentsurvivalresults(Figure1).Thenormalcellsdisplayedhigher

survivabilitythanadenomacellswithincreasingdosages.Thepercentsurvivalresultsfornormal

cellsshowedhighnumbersofsurvivalthroughouttheincreasingdosages,althoughmuchless

cellswerereported.Webelievethiswasbecausethecellsthatdiedduringdosingweredetached

fromthebottomoftheplate,andwerethusaspiratedwiththemediabeforetrypsinizationand

hemocytometry.Withthisinmind,thepercentattachmentresultsdisplayedamoreaccurate

insightintocelldeath,asthedetachedcellswerepresumeddead.Thegeneraldecreasingtrendin

theamountofcellsattachedisindicativeofincreasinglevelsofcelldeath.Celldeathwasalso

reportedsimilarlythroughqualitativemeansinfluorescentmicroscopy(Figures34).

TheqRTPCRresults(Figure5)showedageneraldecreasingtrendinBAXexpression

throughhigherdosagesaswell.OneinterestingaspectofthisanalysiswastheincreaseinBAX

expressionfromthe0.12mMdosedgrouptothe0.3mMdosedgroup,whichisagainstthe

generaltrendofthedata.Thisslightincreaseisbelievedtobetheresultofperformingonlyone

runofourdata.IftheqRTPCRwasperformedmultipletimes,itwouldbeexpectedthatthe

increasewouldbelessenedornonexistent,andthegeneraldownwardtrendofBAXexpression

wouldbeconsistent.

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 ActinwasusedasthecontrolmarkerfortheWesternblotbecauseitispresentinall

eukaryoticcellsinabundance.ActinbandsshouldhavebeenpresentintheWesternblot,but

theywerenot.Onereasonthatthiscouldhavehappenedwasthattheactindimerizedwithitself.

Iftheactindimerized,thenitwouldnotformabandat45kDa.Thebandswouldinsteadappear

atadifferentmolecularweightmarkerbecausethedimersareheavierthanthemonomers.This

dimerizationwouldhavehappenedonlyiftheactinwasdefective(Fornsaglio,2015).

Anotherreasonthatthebandsforactinmightnothaveshownupwasbecausethe

membranewasdroppedfacedownonapapertowel.Thecompoundsonthenitrocellulose

membranecanbeeasilyremovedforreprobingpurposes(20XLumiGLOReagentand20X

Peroxide#7003).Whenthemembranewasdropped,thiscouldhaveaffectedtheLumiGLO

andhydrogenperoxidesolution.Ifsomeofthesolutionwaswipedoff,thenthelightemission

wouldhavebeenlowerthanitshouldhaveornonexistent.WiththelumiGLOandhydrogen

peroxide,themaximumemittancehappensrightafterthemembranesareexposedtoit,solength

oftimepriortodevelopmentmayhavealsobeenafactorintheminimalexpression.Thelight

emissiontypicallycontinuesfor30minutestoonehour(20XLumiGLOReagentand20X

Peroxide#7003).Ifwehadnotdonetheprocedurequickenoughatthisstep,thenitispossible

wewouldnothavegottenthemaximumeffectoflightemittance.

ThedecreaseinBAXexpressionwasmostlikelyduetodifferencesbetweenapoptosis

andnecrosis.Previousstudieshaveshownthatphenolshaveinhibitoryeffectsonmitochondrial

ATPproductionthroughtheinhibitionofdehydrogenases,phosphokinases,andATPases.These

enzymesallhaveimportantfunctionsintheATPproductionpathway,andsotheirinhibition

wouldgreatlyreduceATPproductionandhydrolysiswithinthecell(Stockdale&Selwyn,

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1971).WithreducedATPproduction,andaninabilitytohydrolyzeATPmoleculestouse

energy,thecellsmayhaveinsufficientenergyforthecaspasestoformapoptosomes.BAX

wouldnotbeexpressedwithoutapoptoticorganizationandinsteadthecellswouldundergoa

necroticpathway.Throughthisreasoning,astheconcentrationofphenolincreased,lessATP

wouldbeproducedandthecellswouldbeunabletoundergoapoptosis,resultinginlessBAX

expression.Researchalsoshowsthatapoptosisandnecrosismaynotbemutuallyexclusive,but

existonacontinuuminwhichmorphologicalevidenceforbothpathwaysexistsimultaneously

(Zeiss,2003).Thisevidencesupportsourdata,inthatthelowerconcentrationsofphenolcould

haveallowedforslightapoptoticactionandBAXexpression,whichwouldhavedissipatedwith

higherconcentrationsasthespectrumshiftedmoretowardnecrosis.

AnotherproposalfordecreasingBAXexpressionisthatthenormalcellsweredosedwith

phenolforonly16hours,whiletheadenomacellsweredosedfor24hours.Wepreviously

believedthatthecellsdidnothaveenoughtimeforBAXtobeexpressedinhigherquantities

withthe16hourdosing.Thisbelief,however,isrefutedwithevidencefromotherstudies

showingthatthemitochondrialaspectofapoptosis,whichisassociatedwithBAXexpression,

onlytakesabouttenminutestooccur(Green,2005).Withthisshorttimeframe,themajorityof

apoptosisoccursafterBAXhasreleasedcytochromecintothecytosol,andthereforetheshorter

dosingtimewouldnotaffecttheBAXexpression.

Theresearchconductedinthisstudyonlycontributestothepresentknowledgeonhow

chemicalsisolatedfromcigarettesmokecanhavedetrimentaleffectsonthehealthoforganisms

thatareexposed.Eventually,incontinuationofresearch,thegoalistounderstandhowthese

isolatedchemicalsaffecthumans.Onewaytoclarifydoubtinafutureexperimentcouldbeto

22
studynecrosisincellsafterbeingdosedwithphenol.Ifapoptosisisnotthepathwaythecellsare

taking,thenitcouldbenecrosisthatisoccurring.Thenumberofassaystohelpstudynecrosisin

cellsarelimited.Onewaytodosoistomeasurelactatedehydrogenase.Lactatedehydrogenase

isanenzymethatleaksfromcellsafternecrosistakesplace(Chanetal.,2013).Anotherfurther

experimentthatcouldbeperformedisusingconcentrationsofphenollowerthan0.12mM.Ifthe

concentrationsaretoohighforthenormalcells,thentheymighttakeadifferentpathwayinstead

ofapoptosis.Lessconcentrateddosagesmaybemoreconducivetoinducingapoptosisinthe

cells.Moreresearchcanbedonetodeterminehowmuchphenolistoomuchforthecelltohave

apoptotictendencies.Inthecontinuationofresearchpertainingtophenolinducedcelldeath,the

hopeistomorecompletelyunderstandtheharmfulcellulareffectsoftoxinsfoundincigarette

smoke.

23
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