AaliyahWilliams

TheEffectofCadmiumonthe
CardiacDevelopmentof
EmbryonicZebrafish

AaliyahWilliams
NilesNorthHighSchool
2015

AaliyahWilliams

TableofContents

Acknowledgements3

Purpose/Hypothesis4

ReviewofLiterature510

Procedure11

Materials12

Variables13

Results1418

Conclusion19

References20

AaliyahWilliams

Acknowledgements

Iwouldliketoacknowledgemyteacher,Ms.Camel,whoguidedmethroughoutmyprojectand
keptmeontherighttrack.

AaliyahWilliams

Purpose/Hypothesis

Hypothesis:
Ifzebrafishembryosareexposedtovaryingconcentrationsofcadmiumthentheirheartswill
exhibitdevelopmentaldefects.Zebrafishtreatedwithhigherconcentrationsofcadmiumwill
haveahigherheartrateandalargerratioofdistancebetweensinusvenosusandbulbus
arteriosus(heartchambers)tobodylengthofthezebrafishembryo.

Purpose:

Toinvestigateiftherearemorphologicalandfunctionaldefectsofthezebrafishheartdueto
varyingconcentrationsofexposuretotheenvironmentaltoxicantcadmium,intheearlystagesof
development.

AaliyahWilliams

ReviewofLiterature

By:AaliyahWilliams
Camel
Period9

Therearenumerouschemicalsinourenvironmentthatcanbeconsideredtoxic,butare
hardtocompletelyavoid,suchasthechemicalcadmium.Cadmiumisanenvironmentaltoxicant
thathasbeenshowntohavemanyhealthrelatedsideeffects.Cadmium,likemanyothertoxic
metals,hasbeensuspectedofcausingahighamountofreactiveoxygenspecies(ROS),thus
leadingtooxidativestressdamageassociatedwiththedevelopmentofmanydiseasesandaging.
TheoverproductionofROScanleadtodamageinproteins,DNA,lipidfunctionandevencell
death.ThisoverproductionofROScanleadtodiseases,suchascardiovasculardisease.Sinceit
isverydifficulttoobservetheeffectofcadmiumonactualorgans,suchastheheart,many
scientistshavebegantousezebrafishasamodelbecauseofitssimilaritytothehumanheart.
Cadmiumisatoxicchemicalthatisnonessentialtobiologicalprocessesandthereis
usuallylowlevelsofexposureinourenvironment,buttherearestillmultiplewaysoneis
exposedtocadmium.Humansmaybeexposedtocadmiumthroughcigarettesmokeinhalation
duetotobaccoplantshighabsorptionofcadmium.Ithasbeenreportedthatonecigarette
containsabout12gofcadmiumandthatabout10%ofthecadmiumcontentisinhaledwhen
thecigaretteissmoked(ICDAn.d).Also,workersintheindustrysectorarehighlysusceptible
tocadmiumexposure,workersusuallyinthemanufacturingandconstructionfield.Workersare
exposedthroughthemakingofbatteries,plastics,coatings,andsolarpanels,andsmeltingof

AaliyahWilliams

metals.landfilloperations,recyclingofelectronicpartsorplastics(OSHAn.d).Another
commonsourceofcadmiumexposureisexposurethroughingestionoffood.Ingeneral,leafy
vegetablessuchaslettuceandspinach,potatoesandgrains,peanuts,soybeans,andsunflower
seedscontainhighlevelsofcadmium,approximately0.050.12mgcadmium/kg.Exposureto
cadmiumisalsopossiblethroughitspresenceinwatersourcesnearindustriesemittingcadmium.
Theexposureofwatertocadmiumputsaquaticlifeatrisk,possiblycontaminatingfood
supply(WHO2010).
Whencadmiumentersthehumanbody,itaccumulatesinthebodythroughoutapersons
lifetimemakingitverydangerous.Itshalflifeisactually1730yearsinahumanbeing.Once
cadmiumisuptakenfromthelungorgastrointestinaltract,isistransportedbyproteins,suchas
Albumin.Usually,thecadmiumthatisattachedtotheAlbuministakenupbytheliver,thus
inducingthesynthesisofmetallothionein.Thenthemetallothioneinboundcadmiumappearsin
thebloodplasma.Sincecadmiumhasalowmolecularweight,thecadmiummetallothioneinis
efficientlyfilteredthroughtheglomeruliandtakenupbythetubules(Haschek2013).
CadmiumexposuremayresultinoverproductionofROS(reactiveoxidativespecies).
ROSisgeneratedbyprooxidants,thusinducingoxidativestress.ROSandconsistoffree
radicalsandreactivespeciesandbreakdownproductsoflipidsproteins,nucleicacidsand
carbohydratesmadebythem.Freeradicalscanbepositively,negativelyorneutrallychargedand
containunpairedelectrons.Themostcommonandmostimportantfreeradicalsaresuperoxide
anion,freehydroxylradicalandnitricoxidethatareabletoproduceotherfreeradicalsfrom
unsaturatedfattyacidsmostly.Thesefreeradicalshaveaggressivecharacteristicsthatallow
themtotakeelectronsfromothermoleculesorgiveawaytheirunpairedelectroninorderto

AaliyahWilliams

maintainstability.Theyarecommonlyknownforthisabilitytofragmentothermoleculesand
areproducedfromthecellularmembranemitochondria,lysosomes,nucleus,cytoplasm,
peroxisomes,andendoplasmicreticulum.Thesefreeradicalscanplayanimportantrole
physiologically,butcanalsoleadtothedevelopmentofchronicinflammatorydiseaseswhenin
excess(Singh).Oxidativestressisdefinedasacontinuousimbalancebetweenantioxidantsand
prooxidants,thatcanresultindamagetocells.ROSaspreviouslydiscussedbefore,isan
oxidantthatmaybeoverproducedbythemitochondrialrespiratorychain.TheROScreates
oxidativestressdamagethatcanbevisuallyrepresentedinFigure1.Oxidativestressiscaused
byROSduetoitbeingoverlyreactivecausingbreaksandmutationinDNA,byinactivating
proteinsandenzymes,byoxidizingsugars,andbyinducinglipidperoxidationamongthe

polyunsaturatedfattyacidsoflipoproteinsorofthecellmembrane(Probiox).

Figure1.Mechanismsofoxidativestressinducedcelldamage(Agarwal2005).
TheeffectsofCadmiumhavebeenresearchedthroughoutmanystudies,andrecentlya
connectionbetweencardiovasculardiseaseandcadmiumexposurehasbeenhypothesizeddueto

AaliyahWilliams

increasedROSproduction.Researchershaveperformedasystematicreviewofepidemiologic
studiesandhaveconcludedthatthereisreasontosuspectthatcadmiummaybeariskfactorof
cardiovasculardisease.Theirexperimentalevidencesuggeststhatthisconnectionisdueto
cadmiuminitiatingandprogressingatherosclerosisduetoitsabilitytoraisethebloodpressure.
Cadmiummayincreasereactiveoxygenspeciesformationandinterferewithantioxidative
stressresponsesbybindingmetallothionein,alowmolecularweightproteinthatregulateszinc
homeostasisandactsasafreeradicalscavenger.Thestudyalsoexplainsacorrelationbetween
cadmiumandbloodpressure,asideeffectofCardiovascularDisease.Thissystematicreview
supportsthetheorythatCadmiumisresponsibleforROSproduction,leadingtooxidativestress,
potentiallycreatingacausalrelationshipbetweencadmiumandcardiovasculardisease
(TellezPlaza).

Manyresearchersuseazebrafishmodelinordertoexaminethecardiovascularsystem.
Figure2showsavisualrepresentationoftheheartdevelopmentofzebrafishduringthe5hour
postfertilizationphasethroughthe48hourpostfertilizationphase.

AaliyahWilliams

Figure2.Heartdevelopmentofzebrafish(Stainier2001).
Theventricularendofthehearttubeassemblesfirst,thenthetheatrialenddevelops.At24hpf,
thetubeliesalongtheanterioposterioraxiswiththeatrialendtotheleftofthemidline.At30
hpf,visiblydistinctventricularandatrialchambersform.At36hpf,theheartundergoeslooping
morphogenesisand,by48hpf,functionalvalvesareformed(Stainier2001).During
embryogenesisofzebrafish,theheartisthefirstorgantofullydevelopandfunction.Even
thoughtheheartisabletofunctionandisdevelopeditwillnotbeessentialtotheembryosability
toliveuntilafterseveraldays.Thisisbecausetheconnectionofcirculationhasnotdeveloped
yetpreventfullbloodcirculation.Sincetheheartisnotessentialduringtheearlystagesof
development,zebrafishembryosaregreatmodelsbecausetheyareabletosurvivewithout
cardiacfunctionbecausetheyreoxygenisdeliveredbydiffusionthanthecardiovascularsystem.
Thehearthas4chambers,sinusvenosus,atrium,ventricle,andbulbusarteriosus,in
9

AaliyahWilliams

zebrafish(Hu2011).
Theembryoniczebrafishhearthasthesameelectricalexcitationasthe
humanheart.Ithaspulsesgeneratedinthesinoatrialnode,propagatedthroughtheatrium,
pausingintheatrioventricularnode,andthensenttotheventricle.Aregularrestingheartbeatis
usuallybetween6090beatsperminuteforhumans,butforzebrafishaheartrateisusually
between120180beatsperminute,whichisrelativelysimilartohumans.(DeLuca2014).
Thezebrafishheartisagreatmodelbecauseitconsistsofmammaliantraitsallowingittoserve
asamodelforhumanheartdevelopment.Thezebrafishandhumansbothhaveaclosed
cardiovascularsystem,sotheirdevelopmentsareextremelysimilar.Itisanidealmodeltobe
studiedbecauseduringitsstageasanembryoitistransparenteasilyallowingobservationunder
adissectingmicroscope
(Hu2011).
Thetoxiceffectsofcadmiumanditsroleinthedevelopmentofdiseasesisvery
importanttoinvestigatebecauseofthehighchancesofexposuretocadmium.Itisknownthat
cadmiumistoxic,butresearchersarenotawareofthedirecteffectsrelatingtodiseasessuchas
cardiovasculardisease.Exposuretocadmiumcouldexplainthedevelopmentofvariousdiseases
thatmanyarediagnosedwithunexpectedly.Asuddenhighrateofcertaindiseasesthatmaybe
relatedtocadmiumexposuremayhelptodetectwhentheirisahigherthannormalcadmium
exposureincertainareas.Ifcadmiumisapotentialcauseforsomediseases,thenthemethodof
treatmentcouldbealteredinordertoensureabetterresultandhaltanyfurthercadmium
exposure.

10

AaliyahWilliams

Procedure:

PartI
1. Labelthepetridishesdependingontheconcentrationofcadmium
chloride(0mg/L,0.25mg/L,0.50mg/L,1.0mg/L)thattheembryoswillbe
exposedto.
2. Separatethealiveembryosfromthedeadonesusingforceps.Thenrinse
thealiveones.
3. Transferembryosinto20miniaturepetridishes(6embryosperpetridish).
Placepetridishesincoolerandkeepat28degreescelsius.
4. Usingthebalancemassout0.25mg,0.50mg,1mgofcadmiumchloride
andmixinto1Lofwatertomakecadmiumsolutions.
5. Distribute120hatchedzebrafish(24hpf)embryosinto10sixwell
microplatesin3mLoffishwaterwithdifferingcadmiumconcentrations
and30embryosperconcentrationgroup(0mg/L,.25mg/L,.5mg/L,1
mg/L)for18hours.30zebrafishembryoswillonlycontain.1%DMSO
andfishwater(0mg/L).
6. Transferzebrafishfrommicroplatestodepressionglassslidesandexamine
underdissectingmicroscope.
7. Usingatimer,countthebeatsperminuteofeachofthezebrafishembryos
heartfor30seconds(multiplytogetbeatsperminute).
8. Returnzebrafishembryostopetridishesfilledwithfishwater.

PartII(At72hpfofthezebrafishembryos):

1. Usingapipetteplaceadropof2%methylcelluloseinsideofthewells.
2. Examineunderadissectingmicroscopeunderroomtemperature.
3. Countthenumberofembryosthathavediedandthosethatarestillalive.
4. Takepicturesofthetheembryosunderthedissectingmicroscope.

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AaliyahWilliams

a.Examinemorphologicalchanges.MeasurethedistancebetweenSABA,
andthebodylengthoftheembryo.

Materials:

106wellMicroplates
120ZebrafishEmbryos
25gCadmiumChlorideSolution
DissectingMicroscope
Methylcellulose
Stopwatch
3mLPipette(AutomaticPipette)
DMSO(1%),Depressionslides
3MicroDissectionForceps
30Petridishes
Computer
Motic2.0
FaceMask
Gloves
Goggles
Balance

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AaliyahWilliams

Variables

IndependentVariables:
CadmiumConcentrations(0mg/L,0.25mg/L,0.50mg/L,1mg/L)

DependentVariables:
HeartRate(BeatsPerMinute)
DistanceBetweenSABA/BodyLength

Control:
0mg/LCdCl2

ControlledVariables:
Temperature
AmountofCadmiumSolution
Numberofembryoswithineachexperimentalgroup
AmountofHoursPostFertilization(hpf)datawasrecorded
Hoursofcadmiumexposure
AmountofHoursPostFertilizationcadmiumwasintroducedtoembryos

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Results:
Data:

Figure1.

Figure1.visuallycomparestheheartbeatsperminuteofzebrafishembryos48hours
postfertilizationafterexposuretocadmium.

Figure5.ZebrafishHeartRateAfterExposuretoVaryingConcentrationsof
CadmiumChloride
0mg/L
CdCl2

Embryos

0.25mg/L
CdCl2

0.50mg/L
CdCl2

1mg/LCdCl2

128

152

178

204

138

153

166

198

142

168

182

194

134

158

178

202
14

AaliyahWilliams

146

168

174

192

130

170

168

190

126

160

176

204

138

164

182

196

124

168

174

188

10

154

154

176

198

11

132

172

166

190

12

136

162

182

208

13

143

156

174

197

14

128

166

178

15

138

162

16

132

168

17

144

162

18

136

19

142

20

140

21

134

22

132

23

128

24

136

Figure5.givestheheartbeatsperminuteforthezebrafishembryosintheexperimental
groups.

Figure6.
AverageHeartRate(BeatsPerMinute)ofZebrafish
EmbryosTreatedwithVaryingConcentrationsofCadmium

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AaliyahWilliams

0mg/LCdCl2
135.875

0.25mg/L
CdCl2

0.50mg/L
CdCl2

162.5294118

1.0mg/L
CdCl2

175.2857143

197

Figure6.showstheaverageoftheratioofSABA/BodyLength.

Figure3.MortalityRates(%)ofZebrafishEmbryosafterCadmiumChloride
ExposureatVaryingHoursofPostfertilization
Concentration
ofCadmium
Chloride
0mg/L

24hpf

48hpf

72hpf

6.67%

23.33%

43.33%

.25mg/L

13.33%

53.33%

60.00%

.50mg/L

10.00%

56.67%

66.67%

1mg/L

6.67%

63.33%

73.33%

Figure3.hasthepercentagesofzebrafishembryosthatdiedduringeachintervalofhours
postfertilization.Zebrafishweretreatedwithcadmiumafter24hourspostfertilization.

Figure4.TheDistanceBetweentheSinusVenosusand
BulbusArteriosusoftheZebrafishEmbryoHeart
0mg/LCdCl2

0.25mg/LCdCl2 0.5mg/LCdCl2

1mg/LCdCl2

0.09

0.14

0.16

0.24

0.14

0.15

0.19

0.26

0.1

0.16

0.2

0.22

0.12

0.17

0.22

0.24

0.11

0.18

0.16

0.22

0.16

0.17

0.21

0.24

0.14

0.15

0.19

0.21

0.13

0.19

0.18

0.25

0.09

0.14

0.17

0.12

0.15

0.23

0.14

0.18

0.08

0.12

0.15

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0.13

0.11

0.14

0.17

Figure4.givestheratiosoftheheartchambersSABA.

Figure2.

Figure2.showstheratioofthedistancebetweentheheartchambers,sinusvenosus
andbulbusarteriosus,ofthezebrafishembryohearttothebodylengthoftheembryo.

Figure7.TheAverageRatiosoftheDistanceBetweentheSinus
Venosusand
BulbusArteriosusoftheZebrafishEmbryoHeart:
Concentration 0mg/LCdCl2
Ratios

0.1247058824

0.25CdCl2
0.1583333333

0.50mg/L
CdCl2
0.191

1.0mg/L
0.235

Figure7.showstheaverageratiosofSABA/bodylengthofzebrafishembryos.

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AaliyahWilliams

DataAnalysis:

Themortalityrateofthezebrafishembryoswererecordedinfigure3.Thepercentagedeadat
eachintervalofhourpostfertilization(24,48,72)ispresented.Thezebrafishembryoswere
treatedwithcadmiumat24hpf,sothe48hpfand72hpfshowtheeffectsofthecadmiumon
mortality.Bytheendoftheexperimentthemortalityratesofthezebrafishwere43.33%for0
mg/LCdCl2,60.00%for0.25mg/LCdCl2,66.67%for0.50mg/LCdCl2,73.33%for1.0mg/L
CdCl2.Thehighestpercentmortalityratewasthe1.0mg/LCdCl2treatmentgroupandthe
lowestwasthecontroltreatmentgroup.Figure1.visuallyshowstheheartbeatsperminute
recordedat48hourspostfertilizationforthedifferenttreatmentgroups.Theaverageheart
beatforthecontrolwas136bpm,for0.25mg/LCdCl2theaverageis163bpm,for0.50mg/L
CdCl2theaverage175bpm,for1.0mg/LCdCl2theaverageis197bpm.Figure2visually
showstheratiosofthedistancebetweensinusvenosusandbulbusarteriosusoftheheart,to
itsbody.Thegraphshowsatendencyofahigherratioforthezebrafishembryostreatedwith
higherconcentrationsofcadmiumchloride.

ExperimentalError:

Althoughtheconcentrationsofcadmiumandamountofsolutionwerepreciselymeasured,a
differenceincadmiumconcentrationanddistributionmayhavetakenplacethroughoutthe
trials.Thismayhavecausedvastdifferenceswithinthecardiacdevelopmentofzebrafish
embryosinthesameexperimentalgroup.Also,errormayhaveoccurredthroughthecounting
ofzebrafishheartbeatsduetohumaneyescountingthebeatsperminute.

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AaliyahWilliams

Conclusion:

Ifzebrafishembryosaretreatedwithvaryingconcentrationsofcadmiumchloride
solutionthentheirheartswillabnormallydevelop.Theratioofthedistancebetweensinus
venosusandbulbusarteriosusoftheheartandbodylengthwillbehigherinembryostreated
withhighamountsofcadmiumchloridecomparedwiththecontrolgroupof0mg/LCdCl2.It
wasalsopredictedthattheheartrateoftheembryoswouldbeconcentrationdependent,
resultinginhigherheartratesforlargerconcentrationtreatmentofCdCl2.Basedon
experimentationresults,theoriginalhypothesiswassupportedbecauseasthecadmium
chlorideconcentrationincreased,sodidtheheartrateofthezebrafishembryos.Theratioof
SABA/bodylengthalsoincreasedasconcentrationsincreasedshowingapossible
morphologicaldefect.Theimplicationsofthisexperimentinvolveexposingspecificeffects
toxicheavymetalssuchascadmium.cannotonlyhaveonzebrafish,butpossiblyonhumans
duetooursimilarities.Discoveringacausualrelationshipbetweencardiovasculardisease
andcadmiumexposureisveryusefulbecauseitmayexplainsuddenonsetsofcardiovascular
diseasecasesanditcanbeusedtoevenfurtherunderstandthedrasticeffectsofcadmium.
Inordertoimprovethisexperiment,moretrialswithmorezebrafishembryoswouldhavetobe
usedduetothelackofquantityofzebrafishduetotheirhighdeathratewithinthis
experiment,causingredesignoftheexperimentthroughouttheprocess.Also,further
researchcouldincludemeasuringtheheartdevelopmentofanactualfullydeveloped
zebrafishanditsheartfunction,sothefulleffectsofcadmiumcanbeobserved.

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References:

Chang,K.,Hsu,C.,&Liu,S.(2013).CadmiumInducesApoptosisinPancreaticCells
throughaMitochondriaDependentPathway:TheRoleofOxidativeStressMediatedcJun
NTerminalKinaseActivation.
PlosOne,

8(2)
(Doi:10.1371/journal.pone.0054374).Retrieved
October26,2014.

DeLuca,Elisa.(2014).
ZebraBeat:
aflexibleplatformfortheanalysisofthecardiacratein
zebrafishembryos.
ScientificReports.
4898.
Haschek
,WandaM.(2013).
HandbookofToxicologicPathology:Elsevier

Hu,Norman.(2000)StructureandFunctionoftheDevelopingZebrafishHeart.
THE
ANATOMICALRECORD
.260:148157.

ICDA(Ed.).(n.d.).CadmiumExposureandHumanHealth.RetrievedOctober30,2014.

OSHA.(n.d.).SafetyandHealthTopics|Cadmium.RetrievedNovember2,2014.

WHO.(2010,January1).ExposuretoCadmium:AMajorPublicHealthConcern.Retrieved
November4,2014.

ProBiox.(n.d.).WhatisOxidativeStress.RetrievedNovember1,2014.

Agarwal
etal.

ReproductiveBiologyandEndocrinology
2005
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:28

Stainier,DidierY.R.(January2011).Zebrafishheartdevelopment.
NatureReviewsGenetics
2.3948.doi:10.1038/35047564

TellezPlaza,M.,Jones,M.,DominguezLucas,A.,Guallar,E.,&NavasAcien,A.(2013).
CadmiumExposureandClinicalCardiovascularDisease:ASystematicReview.
Current
AtherosclerosisReports,

15
(10).

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