DanielKwak

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TheEffectOfFreeRadicals
ontheLongevityofCells

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TableofContents

Acknowledgements.........................................3
Purpose&Hypothesis....................................4
ReviewofLiterature.......................................5
Materials.........................................................10
Procedure........................................................12
Variables.........................................................14
Data.................................................................15
DataAnalysis..................................................21
ExperimentalError.......................................22
Conclusion......................................................23
ReferenceList.................................................25

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Acknowledgements

First,Iwouldliketoacknowledgemyteacher,Mr.RichardJ.Thielsen.Hehashelpedmein
innumerableways,andIwouldnothavefinishedthisprojectwithouthim.Iwouldalsoliketorecognize
Ms.ChristineCamelforhelpingmewithvariouschemistryconcerns.Withouther,Iwouldnothave
beenabletoconductmyexperiment.
Iwouldalsoliketoacknowledgemyparents,TonyKwakandSaraMaeng,foralways
supportingme.WhenIwasforcedtoredesignmyexperiment,theirsupportwasextremelyhelpful.
IwouldliketothankMaryDelaCruzandGitaDevaforhelpingmecutmyboardandpasteit
down.Withouttheirhelp,myboardwouldnotlooknearlyasgoodasitisnow.Iwouldalsoliketo
thankRaisaSalikforallherhelp.TheirencouragementhashelpedmerealizethatIamcapableofdoing
this,andthatIcanfinishmyproject.
Inaddition,IwouldliketoacknowledgeAmbriaBeneschandElizabethPoozhikunnel.They
havehelpedmeagainandagainwithmyexperiment.Theyarebothpositivepeoplewhohavehelped
mestaycalmandmotivated.

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Purpose
ThepurposeofthisexperimentistotestthecredibilityoftheFreeRadicalTheoryofAging.Totestthis,
hydrogenperoxidewasbeusedtoaccumulatefreeradicalsinCanineBLymphomacells.

Hypothesis:
IfCanineBLymphomacellsareexposedtohigherconcentrationsofhydrogenperoxidethatincrease
freeradicalproduction,thentheywilllivelongerthanthecellsnotexposedtolowerconcentrationsof
hydrogenperoxide.
TheFreeRadicalTheoryofAgingstatesthattheaccumulationoffreeradicalscausesprematureaging.
However,insomecases,thishasbeenprovenwrong.SigfriedHakimisexperimentwithworms
showedthatmutatedwormsexposedtofreeradicalinducingtoxinsactuallylivedlongerthanthose
whohadanormalgeneticpattern.Therefore,cancercellsshouldreactinasimilarwayastheworm
cells.

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ReviewofLiterature

Tryingtounderstand,slow,andevenhalttheprocessofaginghaslongbeenadreamandgoal
forscientistsallovertheworld.Eachofthetrillionsofcellsinourbodyages,contributingtotheoverall
effectthatsomanyhumansfear.Since1956thefreeradicaltheory,introducedbyDenhamHarman,
haspresentedalogicalandwidelyacceptedexplanationfortheagingofcells(Harman,1956).
However,in2010,newexperimentssuggestedaradicaldeparturefromtheoldacceptedtheory.Dr.
SiegfriedHekimiandhisstudentDr.WenYangintroducedtheideathatfreeradicals,seeninFigure1
(AquaHealthProducts(n.d.)),contrarytoHarmanstheory,arenotdetrimentaltoacellslifespan
thatthey,infact,mightincreaseacellslongevity(Hekimi,2013).Ifthisholdtruewithhumancells,it
willcreateacompletelynewunderstandingandusherinanageofintenseresearchaboutthetrue
processofagingandwhatwecandotocounteractitseffects.
Figure1:Exampleofafreeradicalandhowanantioxidantdonatesitselectrontofillthefreeradicals
unstablevalenceshell.
DefinitionandFunctionofFreeRadicals
Tounderstandhowthiscouldoccur,onemustfirstknowwhatfreeradicalsare,andtheir
functions.Atomsconsistsofthreesubatomicparticles:protons,electrons,andneutrons.Electronsplay
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alargepartinatomsinteractionswithoneanother.Theyarearrangedinshells,oraseriesof
differentlysizedorbitsaroundthenucleusoftheatomwhereprotonsandneutronssit.Theoutermost
orbitiscalledthevalenceshell.Therearedifferentnumbersofelectronsthatareabletofilleachorbit,
butoftentimesinthenaturalworldalloftheseorbitsarenotalwaysfilled,asinFigure2(TheValence
Shell,2012).

Figure2:Simplediagramofanatomwithnucleusandorbitalshells,withtheoutermostshell,orvalence
shell,holdingonlyoneelectron.
Atomsaremoststablewhentheiroutermostvalenceshelliscompletelyfilled(FreeRadical
Introduction,2013).Thismeansthattwoatomswithincompletevalenceshellscreateachemical
connectioncalledacovalentbondwheretheysharethepowersoftheelectronandsohavefilled
valenceshells.
Whencovalentbondsbetweenatoms,molecules,andionsarebroken,onlyoneelectron
remainswitheachparticle.Thisishowfreeradicalsareformedtheyareessentiallyanyatom,molecule,
orionthathasatleastoneunpairedvalenceelectron(FreeRadicalIntroduction,2013).Freeradicals
canalsobecreatedfromessentialmetabolicprocessesinthehumanbodyandexternalsourcessuchas
exposuretoXrays,cigarettesmoking,ozone,industrialchemicals,airpollutants,andradiation.The
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formationoffreeradicalscontinuouslyoccursinthecellsasaresultofbothenzymaticandnonenzymatic
reactions.Theycaneithergiveawayanelectronoracceptanelectronfromothermolecules,thusthey
canbehaveasoxidantsorreductants.Oxidantsoccurasaresultofbodilyfunctions,butcanbeharmful
asfreeradicals.Theyaresimplyaformofoxygenthattendstoacquireelectronsinchemicalreactions.
Reductantsaretheoppositetheylose,ordonate,electronsinchemicalreactions(Sigust,n.d.).
Themostpotentiallyharmfulfreeradicalsarehydroxylradical,superoxideanionradical,
hydrogenperoxide,oxygensinglet,hypochlorite,nitricoxideradical,andtheperoxynitriteradical(Lobo
etal.,2013).Formyproject,Iwillbeusinghydrogenperoxidetoproducefreeradicals.Inaddition,
freeradicalsarehighlyreactivetheunpairedelectronisalwaysdriventocreateabond,sotheywill
stealelectronsfromotheratoms.Thiscreatesanotherfreeradicalbecauseofthesubsequentlossof
electrons,creatingacycle(FreeRadicalIntroduction,2013).Thisprocessoffreeradicalcreation
continuesinachainreaction,inflictingdamageonnearbycells,macromolecules(moleculescontaining
largenumbersofatomssuchasproteins,nucleicacids,andlipids),andcellularmembranes.Theyare
abletodamagetheseparticlesbydrasticallyalteringtheirbiologicallyrelevantmoleculessuchasDNA,
proteins,carbohydrates,andlipids(Antioxidants,2013).Thisleadstocelldamageandhomeostatic
disruption(Lobo,2013).
Freeradicalscandamagetheparticlesinanumberofways,includingchangingthecoding
instructionsinastrandofDNA,creatinglowdensitylipoprotein(cholesterol)thatoftenclogsarteries,
changingacellsmembrane(calledlipidperoxidation),allowingharmfulparticlestoenterthecell
(Antioxidants,2013),andalteringthefunctioningofreceptorsinthecellmembrane(Bowen,2013).
However,freeradicalsarentnecessarilyalwaysharmful.Theyarecreatedthroughan
assortmentofreactionscrucialtolife,...includingsynthesiswithverydiluteorrarefiedreagents,
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reactionsatverylowtemperatures,orbreakupoflargermolecules.Thelattercanbeaffectedbyany
processthatputsenoughenergyintotheparentmolecule,suchasionizingradiation,heat,electrical
discharges,electrolysis,andchemicalreactions(FreeRadical,2013).Inaddition,therehasbeen
researchconductedthatdemonstratesthatradicalsareinvolvedinbothintercellularandintracellular
signalling(Bowen,2013).Inaddition,freeradicalsarevitaltotheimmunesystem.Freeradicalsare
usedbytheimmunesystemtomarkforeigninvadersanddamagedtissue(FreeRadicalIntroduction,
2013).Themarkingsareanindicationofwhichtissueandinvadersneedtoberemovedfromthebody.
Oxidantsandreductants,twotypesoffreeradicals,arenaturallyoccurringasaresultofessential,
everydaybodilyfunctions(Sigust,n.d.).Theseareclearexamplesofhow,inmanysituations,free
radicalsarecrucialtomaintainingourhealthandwellbeingandarekeytomanyaspectsofthenatural
world.

FreeRadicalTheoryofAging
ThefreeradicaltheoryofagingwasintroducedbyDenhamHarmanin1956.Itstatesthat,
agingchangesarecausedbyfreeradicalreactions.Theaccumulationoffreeradicalsisresponsible
forthesequentialchangesthataccompanyagingandtheincreasesinthepossibilityofillnessordeath
(Hardman,2013).However,researchdoneongeneticallymodifiedwormsseemstodisprovethis
theory.Dr.HekimiandhisstudentDr.YangatMcGillUniversitygeneticallymodifiedwormsto
acceleratetheirproductionoffreeradicals.Theytreatedthewormswithatoxicherbicidecalled
paraquatinordertoincreasetheirproductionoffreeradicals.Theresultsoftheexperimentwere
astoundingthewormstreatedwiththeparaquatlived60percentlongerthanthewormsthatwerenot
treatedwiththeparaquat.Dr.Hekimihadthistosay,Ifyouaskmostpeopleonthestreetwhatcauses
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aging,manywouldsayfreeradicals,butitsacomplexstory("MutantWormsDefyAgingTheory.",
2013).
Therehavealsobeenresearchconductedonbatsthatappeartogoagainstthefreeradical
theoryofaging.Dr.BrunetRossinnifromtheUniversityofMinnesotameasuredtheamountofhydrogen
peroxideproducedinadultandjuvenilebats.Shehadexpectedtheadultbatstohaveproducedmore
hydrogenperoxide,fortheyareolderandhavemorefreeradicals.However,contrarytoherbelief,the
juvenilebatshadproducedmorehydrogenperoxide,thusproducingmorefreeradicals(Rossinni,
2013).Herexperimentdirectlygoesagainstthetheorytheyoungerbat,whohadagedlessthanthe
olderbat,hadmorefreeradicalsthantheolderbat.

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Materials
CanineBLymphomaCells
PipetteTips
Micropipettes
AutomaticPipetteIncubator
70%Ethanol
BioHood
Autoclave
WaterBath
CompleteMedia
VentedCellFlasks
CellWellPlates
CultureFlasks
Scissors
NonLatexGloves
SharpieMarker
Hemocytometer
InvertedMicroscope
TrypanBlue
30%HydrogenPeroxide
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DistilledWater
GraduatedCylinder

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MethodsofProcedure
SplittingandCulturingCells:
1.Placethecompletemediaintothewaterbathat37C.
2.Place8mLofcompletemediainto2newcultureflasks.
3.Add2mLofCanineBLymphomacells(ormediafromtheoldculture)intothe2newflasks.
4.Placethenewculturesintotheincubatorat37C.
5.Cleanthebiohoodworkingsurfacewith70%ethanol.
HydrogenPeroxideSolution
1.Fromtheformula:concentration1(volume1)equalsconcentration2(volume2),
(30/100)V1=(1/100)(10mL),V1(volumeof30%HydrogenPeroxideneeded)=0.333mLor333L
isaddedtoagraduatedcylindertoproduce1%hydrogenperoxide.
2.Adddistilledwatertothesolutionuntil10mLoftotalsolutionisreached.
3.Toproduce0.5%hydrogenperoxide,3mLofthe1%solutionshouldbeextractedandaddedto3
mLdistilledwater.
4.Add10Lof1%hydrogenperoxideinto9cellwellplateslabeledtrials1,2,and3,and24hours,
48hours,and72hours.Repeatwith0.5%hydrogenperoxideand0percenthydrogenperoxide
5.Add50Lofmediafromthecultureplatesintoeachindividualwellplate.
6.Placethecellwellplatesintotheincubatorat37C.
7.Cleanthebiohoodworkingsurfacewith70%ethanol.
HemocytometerCellCount
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1.Afterlettingthecellsincubatefor24hours,add0.5mLoftrypanblueintothewellplateslabeled24
hours.
2.Theliquidwiththecellsisthenpipettedtoahemocytometerslide.
3.Thetopleft,topright,bottomleft,bottomright,andcentersquaredgridcontainingthecellswere
recorded.
4.Usingtheformula:totalcellinsample=totalcellscounted/5*10,000*dilutionfactortofindthe
totalcellsinthesolution.10,000isthevolumeinM/mmofthehemocytometer.

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Variables
IndependentVariable:
ConcentrationofHydrogenPeroxideexposedtoCanineBLynchcancercells.
0.5%,1%,at24,48,and72hours.

DependentVariable:
CellapoptosisofCanineBLynchcancercells

Control:
Thetrialwith0percentconcentrationofHydrogenPeroxideat24,48,and72hours.

Constants:
AmountofHydrogenperoxideadded(10L),temperaturethecellswerekeptat(37C),amountof
trypanblueaddedtothecellwellplates(0.5mL),
no

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Data
Control
TimePassed Alive Dead
24 11 4
24 8 2
24 8 3
48 10 4
48 8 5
48 6 2
72 5 3
72 10 4
72 9 6

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0.5%
TimePassed Alive Dead
24 7 3
24 6 1
24 3 0
48 4 3
48 8 2
48 6 2
72 8 1
72 10 2
72 9 3

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1%
TimePassed Alive Dead
24 6 1
24 5 1
24 8 4
48 7 2
48 9 2
48 9 4
72 5 3
72 5 1
72 7 1

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DataAnalysis:
Inthefirsttrial,at24hours,thecontrolwithnohydrogenperoxideaddedhadthelowestratio
ofalivetodeadcells.Thesolutionwith0.5%hydrogenperoxidehadthehighestratioofalivetodead
cells.Theratioofalivetodeadcellsat1%concentrationwasinbetweentheratioat0%and0.5%
concentrations.Inthesecondtrial,at48hours,thecontrolsratiodroppedby0.82,the0.5%
concentrationby0.4,andthe1%concentrationdroppedtheleastby0.045.Likethefirsttrial,the
controlhadthelowestratio,thenthe1%,andthe0.5%concentrationhadthehighestratioofaliveto
deadcells.Inthelasttrial,at72hours,thehighestratiowasat0.5%,asitwasinthefirsttwotrials.The
controlsratiodroppedevenmoreto1.85,butthe0.5%remainedthesameastrial2.However,the
1%concentrationactuallywentupto3.4.Aftergraphingthedata,itisshownthattheratiosgoupan
downastheconcentrationincreases.Theratiowasthehighestat24hourswiththe0.5%concentration.
Thelowestratiowasat72hourswiththecontrol.Therefore,sincethecontrolwithnofreeradical
causingagentaddedhadthelowestratio,itcanbeconcludedthatthefreeradicaltheoryisinvalid.
However,itappearsthattoohighofaconcentrationisalsonotbeneficiallytothecell.

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ExperimentalError:
Thereweresomeerrorsthatweremadeduringtheexperiment.Theexperimenterhadto
performtheexperimenttwice,fortheydidnotknowtheyhadtousetheHemocytometertocountthe
cells.Also,whencountingthecells,manycellswerecounted,butitisnotthefullsamplethathadbeen
exposedtotheHydrogenPeroxide.

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Conclusion:
ThisexperimentwasconductedinordertotestthevalidityoftheFreeRadicalTheoryofAging.
ThepurposeofthisexperimentwastoexposehydrogenperoxidetoCanineBLymphomacellsto
accumulatefreeradicals,andseeiftheywouldlivelongerthanthecellsthatwerenotexposedto
hydrogenperoxide.Hydrogenperoxidewasusedbecauseitisanoxidantthatcanreactwithcertain
moleculesandconvertthemintofreeradicals.Experimentationbeganwiththegrowingofthecancer
cellsinculture.Theyweresplitandaddedto0.5%and1%concentrationsofhydrogenperoxide.The
cellswerethencountedusingahemocytometeranddyedwithtrypanblue.Thedatawasrecordedat
24,48,and72hoursforthreetrials.Aratioofalivetodeadcellswasmadeusingtheaveragenumber
ofcellsaliveanddeadatthedifferentconcentrationsandtimes.Accordingtotheresults,thesolution
withthe0.5%concentrationofhydrogenperoxidehadthehighestratioofalivetodeadcells.Therefore,
myhypothesiswasnotsupportedbythedatamyoriginalhypothesiswasifcancercellsareexposedto
higherconcentrationsofhydrogenperoxide,thentheywilllivelongerthanthecellsexposedtolower
concentrationsofhydrogenperoxide.Thehighestconcentrationat1%hadalowerratioofalivetodead
cellsthanthelowerconcentrationat0.5%.However,thisdoesdisprovethevalidityofthefreeradical
theoryofagingthetheorystatesthattheaccumulationoffreeradicalscausesprematureaging,butthe
controlwithnofreeradicalcausingagenthadthelowestalivetodeadratio.Itcanalsobeconcluded
thattoomuchfreeradicalsisalsonotnecessarilybeneficialforthecell.
Knowingthatfreeradicalsarenotalwaysnecessarilyharmfulcanbebeneficialinmanyways.
Asshownintheexperiment,cellsthatwereexposedtofreeradicalshadahigheralivetodeadratio.
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Thus,withfurtherresearchandexperimentation,freeradicalscouldhaveahugeimpactonawayto
prolongtheagingprocess.

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State,n.d.Web.18Nov.2013.
Rossinni,Brunet."TestingtheFreeRadicalTheoryofAginginBats."NCBI.U.S.NationalLibraryof
Medicine,n.d.Web.18Nov.2013.
Sigust,A.(n.d.).WhatAreOxidants?.IneHow.RetrievedJanuary17,2014,from
http://www.ehow.com/info_8324835_oxidants.html
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TheValenceShell(2012).InNDTResourceCenter.RetrievedFebruary2,2014,from
http://www.ndted.org/EducationResources/HighSchool/Electricity/valenceshell.htm
AquaHealthProducts(n.d.).Freeradicalsandantioxidants.RetrievedJanuary12,2014,from
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