Deferasirox-induced cytogenetic responses

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environmental toxicology and pharmacology 39 (2015)787–793

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Deferasirox-induced

cytogenetic

responses

Mehmet

Arslan

a,∗

,

Hasan

Basri

Ila

b

a_Ardahan_University,_School_of_Health_Sciences,_Department_of_Nursing,₇₅₀₀₀_Ardahan,_Turkey b_Cukurova_University,_Faculty_of_Science_and_Letters,_Department_of_Biology,₀₁₃₃₀_Adana,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4November2014

Receivedinrevisedform

4February2015

Accepted5February2015

Availableonline16February2015

Keywords: Deferasirox Ironchelator Genotoxicity Cytotoxicity Invitro Invivo

a

b

s

t

r

a

c

t

Deferasirox(commerciallyformulatedasExjade®₎_is_one_of_the_effective_iron_chelators_used

intreatmentofironoverloaddiseases.Inthisstudytheeffectofthissubstancefor

chromo-someaberration,sisterchromatidexchangeandmitoticindexwasstudiedbyinvitro(by

usinghumanperipherallymphocytes)andinvivo(byusingrat)analysis.

Deferasiroxincreasedthesisterchromatidexchangefrequencyinalltested

concentra-tionsandperiodsinvitro.Also,inthepresenceofmetabolicactivator,thesubstanceledto

astatisticallysigniﬁcantincreaseinthesisterchromatidexchangefrequenciesonlyathigh

concentration.Whileininvitroanalysisthesubstancesigniﬁcantlyincreasedabnormal

cellpercentagesinallconcentrations,ininvivostudythesubstanceincreased

chromo-someaberrationsonlyintwoconcentrationsat12htreatment.Intheculturedlymphocytes,

deferasiroxshowedcytotoxicitybysigniﬁcantlyreducingproliferationindexandmitotic

indexvalues.Whileinthepresenceofmetabolicactivationitdidnotaffectthe

prolifera-tionindexfrequency,ithadastimulanteffectonthemitoticindexfrequency.Deferasirox

reducedsigniﬁcantlythemitoticindexvalueinthebonemarrowcellsespeciallyinhigh

concentrationandshorttreatmentperiod(12h).

1. Introduction

Theironoverloadinhumantissuesemergesdueto

hered-itary reasons or chronic blood transfusions (transfusional

hemosiderosis).Asaconsequence,lossofcellviabilityand/or

metabolicdysfunctionsoccur(Pietrangelo,2003).Ifthis

con-ditionisnotdiagnosedandtreatedtimely,certainliver,heart

andendocrineglandsdiseasesmaydevelop(Andrews,1999).

Thereductionofironloadinthetissueprotectsindividual

against having heart diseases, diabetes mellitus, or

pre-ventsprematuredeathinthalassemia patients(Brıttenham

etal.,1994).Thephlebotomymethod(AssiandBaz,2014)or

ironchelating agentsare usedfor removingexcessive iron

∗ _{Corresponding}_author._Tel.:₊₉₀_{4782112687/5136;}_fax:₊₉₀_4782112973.

E-mailaddresses:mehmetarslan@ardahan.edu.tr,marslan76@gmail.com(M.Arslan).

accumulatinginthetissuesofhemochromatosispatients.The

ﬁrstoneofthreetypesofchelatorsfrequentlyusedfor

remov-ingironisdeferoxamine(DFO)thatisappliedintravenously.

Deferiprone,whichisanotheragentusedforsamepurpose,

istakenorally.Deferasirox,whichisaneworalchelator,is

thethirdtypetakenorallyandusedeasilyandeffectivelyfor

controllingtheironload.Deferasiroxhashighspeciﬁc

afﬁn-ityforiron.Allpatientsabove2yearssufferingfromsevere

chronicironoverloadcanusedeferasiroxfortherapy(Wang

etal.,2010).

Despiteoftheworthyadvantagesofchelators,their

muta-genic potentials must not be ignored because someother

chelatorssuchasDFOhasbeen showntohaveheavytoxic

andmutagenicpotential(Whittakeretal.,2001).Also,itwas

http://dx.doi.org/10.1016/j.etap.2015.02.001

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determinedthatDFOwasnotclastogenicbyitself,butalong

withgamma raysincreasedacentricchromosomefragment

and ring chromosome formation frequency (Juckett et al.,

1998).Inanotherstudy,itwasfoundthatdeferipronecause

dchromosomefragment(Marshall etal.,2003).Tothe

con-trary of the above-mentioned ﬁndings, it was shown that

deferiproneconsiderablydecreasedtheDNAdamage

result-ingfromironaccumulation(Andersonetal.,2000).

Althoughdeferasiroxhasmanybeneﬁt–costadvantagesin

treatment,thereisonlyourpreviousinvestigationaboutits

genotoxicnature.Deferasiroxhasshowedgenotoxicand

cyto-toxiceffectsinsomecellsystemsaccordingtoresultsofthat

study(Ilaetal.,2014).Therefore,inthecurrentstudy,human

peripherallymphocyteswere exposedtothe testsubstance

indifferentconcentrations inthe absenceand presenceof

metabolicactivator.Itwasattemptedtoinvestigategenotoxic

andcytotoxiceffectofthesubstancebyinvitrosister

chro-matidexchange(SCE)tests,chromosomeaberration(CA)tests,

alongwithinvivochromosomeaberration(CA)testsonrat

bonemarrowcells.

2. Materials

and

methods

The test substance of the present study was deferasirox

(Exjade®_,_obtained_from_a_pharmacy_in_{Adana/Turkey)}_(CAS

No.:201530-41-8).Nonmedical ingredientsinExjade®_tablet

are:lactose, crospovidone,povidone,sodium laurylsulfate,

microcrystallinecellulose,silica-colloidalanhydrous,

magne-siumstearate(Exjade®_–_Consumer_Medicine_Information_)._In

thestudy,short-termgenotoxicitytestswereperformedon

culturedhumanperipherallymphocytesunderinvitroand

invivoconditionswithrat.Sincethetestsubstancewasnot

absolutelywater-soluble,dimethylsulfoxide(DMSO)(CASNo.:

67-68-5)wasusedasasolvent.Thecellsusedfor

quantita-tiveanalyseswereexposedtothetestsubstanceinvarious

concentrationsindifferentperiods,andtheresultswere

com-paredwiththeirowncontrols.OECDguidelinewasconsidered

todoseselectionforinvitrostudies(OECDTG473,paragraph

21,2012).Deferasiroxconcentrationsforinvivo studywere

selectedbasedonitsoralLD50forrats(≥1000mg/kg).

2.1. Material

This study was approved by the Ethics Committee of

the Cukurova University Medical Sciences, Experimental

Research,andApplicationCentre(no.:11,date:2July2010).In

thepresentstudy,Exjade®_(its_active_substance_is_{deferasirox),}

whichisan ironchelatingdrug, wasused asthetest

sub-stance,and peripheralblood takenfrom twonon-smoking,

healthy,andvoluntarywomenandtwonon-smoking,healthy,

and voluntary men ofthe same age (23) was used as the

material for in vitro tests. Four healthy 10 to 12 week

rats (two females+two males) were used in in vivo tests.

Colchicine (SigmaC9754), mitomycin-C MMC(CAS No.:

50-07-7),colchicine(3mg/kgb.w.,SigmaC9754),Urethane(ethyl

carbamate;EC;CASNo.:51-79-6)andchromosomemedium

(Pb-Max,Gibco,cat.no.:12557-013)wereusedinthisstudy.

2.2. Method

2.2.1. InvitroCAandSCEassay

HumanperipherallymphocytesweresubjectedtoCAandSCE

teststoinvestigatethegenotoxiceffectsofdeferasirox(aniron

chelator)anddeferasiroxmetabolite.

Wholeblood(0.2mL)fromfourhealthydonors(twofemale

andtwomale,non-smokers,aged23years,bloodsamplesnot

pooled) wasadded to2.5mLchromosomemedium

supple-mentedwith10␮g/mLsterile5-bromo-2-deoxyuridine(BrdU).

Themediumcontainedphytohaemagglutinin(PHA)for

stim-ulatingofthecellproliferation.Thecultureswereincubated

totaloftime72hat37◦Casfollowing:

The blood culture was treated by 10, 20 and 40mg/kg

concentrationsofdeferasiroxdissolvedindimethylsulfoxide

(DMSO),for24h(deferasiroxwasaddedat48hafterinitiation

timeofincubation)and 48h(deferasiroxwasaddedat24h

afterinitiationtimeofincubation).

Colchicine (0.06␮g/mL) wasadded to the cultureat the

last 2h (70thh) of incubation time for arrest cell cycle at

metaphase. Thenthe cultures were centrifuged (2000rpm,

5min)andtreatedwithhypotonicsolution(0.4%KCl)for5min

at37◦C,andﬁxedincoldmethanol:glacialaceticacid(3:1)

for15minatroomtemperature.Treatmentwithﬁxativewas

repeatedthreetimes,andthenthecellswerespreadonglass

slidesandair-dried.

InordertoCAanalysisthepreparationsweresubjectedto

stainingwith5%Giemsafor24h.Also,forstudyofSCEthe

Flu-orescencePlusGiemsa(FPG)methoddevelopedbySpeit(1984)

andSpeitandHauper(1985)wasused.DMSO(10␮L/2.7mL)

andMitomycin-C(0.25␮g/mL)wasconsideredasanegative

controlandapositivecontrol,respectively.DMSOisagood

solventthatdidnotinduceCA.

InordertostudyofdeferasiroxmetaboliteonCAandSCE,

thecellculturetreatedwithdeferasiroxalongwithS9mix

(3-methylcolanthrene-inducedratliversubcellularenzymes).To

mimicinvivoconditions,exogenousmetabolizingsystemwas

usedforemployingcellswhichhaveinadequateendogenous

metaboliccapacity.Inthisway,thepossiblecontributionof

deferasiroxmetabolismonitsgenotoxicitycanbedisplayed

(MaronandAmes,1983).Forthispurpose,thethree

concen-trationsoftestsubstance(10,20and40mg/kg)togetherwith

S9mix(0.5mL)wereaddedthecultureat48thhofthe

incuba-tiontime.After3htreatment,theculturewaswashedtwice

foreliminatingoftheS9mixfromculture.Followingthelast

washing,BrdU-supplementedfreshmediumwasaddedinto

the cells inthetube, andthe cellswere incubated for19h

at37◦C (untilthe70thhofincubationtime).Afterthatthe

cells were treatedbycolchicine,centrifuged and continued

theprocessmentionedabovetostudyofCAandSCE.

2.2.2. Invivoassay

TheinvivotestwasperformedaccordingtoTopaktasetal.

(1996). Deferasirox concentrations for in vivo study were

selectedbasedonitsoralLD50forrats(≥1000mg/kg).Forthis

purposetwogroupsofrats(twofemales+twomales;fourrats

intotal)were treatedbythreeconcentrationofdeferasirox

(250,500,and1000mg/kg)viaoralgavage.Bonemarrowcells

were taken out from one group after 12h treatment and

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789

colchicine (3mg/kg) was injected intraperitoneally for one

groupat10thtreatmenttimeandforanotherat22nd

treat-menttime(2hbeforetheanimalsweresacriﬁced).

Afterthat,theanimalsweresacriﬁcedbycervical

disloca-tion,andthefemurswerestrippedproximally,andthebone

marrowcellswereaspiratedin0.9%NaCl(37◦C).Theobtained

suspensionwascentrifuged for5minat2000rpm,and the

bonemarrow pelletwas resuspended in0.4% KCl at 37◦C

for10min,thenthecellswereﬁxedincoldmethanol:glacial

aceticacid(3:1)for20minatroomtemperature.Thetreatment

withﬁxativewasrepeatedtwice.Thenthecellswerespread

onglass slidesand air-dried.The slideswere stained with

Giemsa(5%inSorensenbuffer)for15minand100well-spread

metaphasesperanimal(atotalof400metaphasespergroup)

weresubjectedtostudyofstructuralandnumericalCAsby

1000×magniﬁcation.MI(mitoticindex)wasalsodetermined

byscoring3000cellsfromeachanimal.

Untreatedanimalgroup(asanegativecontrol),treatedby

DMSO(asasolventgroup),andtreatedbyethylcarbamate(as

positivecontrol)werestudiedinthisresearch.

2.3. Microscopicinvestigationandstatisticalmethod

TheCAwasclassiﬁedaccordingtotheinternationalsystem

forhumancytogeneticnomenclature(ISCN)(Paz-y-Minoetal.,

2002).Ahundredwell-spreadmetaphasesperdonor(atotalof

400metaphasesperconcentrations)wereexaminedat1000×

magniﬁcationfortheoccurrencesoftheCA.Gapswere not

countedasCA,accordingtoMaceetal.(1978).SCE scoring

wascarried out accordingtoAlbertiniet al.(2000). For the

numberofSCEs,atotalof100cells(25cellsfromeachdonor)

undersecondmetaphaseswereexamined.Theresultswere

usedtodetermine the mean numberofSCEs (SCE/cell).In

addition,atotalof400cells(100cellsfromeachdonor)were

scoredforthedeterminationofthereplicationindex(RI).The

mitoticindex(MI)wasalsodeterminedbyscoring3000cells

fromeachdonor.TheMIexplainstheeffectsofthe

chemi-calsontheG2stageofthecellcycle,andtheRIreﬂectsthe

effectsofthechemicalsontheSandG2stagesofthecycle.

TheRI was calculated according tothe followingformula:

RI=[(M1×1)+(M2×2)+(M3×3)]/totalscoredcells,whereM1,

M2,and M3are the fractionofcells undergoingtheir ﬁrst,

second,andthirdmitosisduringthe72-hcellcultureperiod.

Thesigniﬁcanceofdifferencesbetweenthe mean SCEs,

RI, MI, structural, numerical, and total CAsin treated

cul-tures and their controls were determined using the t-test.

Dose–responserelationshipsweredeterminedfromthe

cor-relationandregressioncoefﬁcientsforthemeanSCEs,RIand

MI,structural,numerical,andtotalCAs.

3. Results

3.1. Invitrosisterchromatidexchange(SCE)

AlthoughallconcentrationsincreasedtheSCEfrequencyin

the24htreatmentofthedeferasirox,theSCEfrequencywas

foundtobesigniﬁcantlyhigherincomparisontothecontrol

(P<0.05),onlyinthelowestconcentration(10␮g/mL).

In the48h treatment, allthe usedconcentrations (even

thelowestconcentration)signiﬁcantlyincreasedtheSCE

fre-quencyincomparisontothecontrol(Table1).

Itwasdeterminedthatwhentheculturedlymphocytecells

wereexposedtothedeferasiroxinthepresenceofmetabolic

activator,onlythehighestconcentrationledtoSCEincreases,

andtheincreasesweresigniﬁcantincomparisontothe

nega-tivecontrolandthesolventcontrol(P<0.05)(Table2).

3.2. Invitrochromosomeaberration(CA)

Inthe24happlication,deferasiroxincreasedCAsinallthree

concentrations,buttheabnormalityincreasedeterminedin

highconcentrations(20and40␮g/mL)wasstatistically

signif-icant(Table1).Inthe48happlication,theCAsformationsinall

dosesinvestigatedwerefoundsigniﬁcantlyhigherin

compar-isontothenegativecontrol.Inthisapplication,twoincreases

inhighdosewerefoundsigniﬁcantlyhigherincomparisonto

thesolventcontrol(P<0.05)(Table1).Thetestsubstanceinthe

presenceofexogenousmetabolicactivator(S9mix)

demon-stratedimportantgenotoxiceffectsinallconcentrations.The

observedCAﬁndingsweresigniﬁcantlyhigherincomparison

tobothcontrols(P<0.01orP<0.001).Theratesof

abnormal-itypercellshowedsimilaritieswiththeabnormalcellrates

(Table2).

3.3. Invivochromosomeaberration(CA)

In in vivo study, two high deferasirox concentrations (500

and 1000mg/kg) used in treatment of rats caused

signiﬁ-cantchromosomeaberrationsat12hoftreatment(Table3),

Whilethelowestconcentrationofthesubstancedidnotshow

anysigniﬁcanteffectonchromosomeaberration.The

high-estconcentrationofdeferasiroxcausedtopartialincreaseof

chromosomeaberrationat24htreatment,buttheeffectwas

notstatisticallysigniﬁcant.

3.4. EffectofdeferasiroxonDNAreplicationand mitosis

IninvitroteststhedeferasiroxshowedasigniﬁcantPI

(pro-liferationindex) reductioninallconcentrations inthe 48h

treatment, though in the 24htreatment, all concentration

exceptthelowestonedecreasedthereplicationrate

signiﬁ-cantlyincomparisontothenegativecontrolandthesolvent

control(P<0.05)(Table1).However,deferasiroxdidnotshow

anysigniﬁcantincreaseinthePIvalueswhenitwasusedalong

withmetabolicactivator(S9mix)(Table2).

Thedeferasiroxhadaninhibiting effectonmitotic

divi-sion(MI).In24htreatmentofcellculture,themitoticindexes

decrease obviously and signiﬁcantly. Thebiggestreduction

occurredinthehighestconcentration.AlthoughalloftheMI

reductionsoccurringinthatperiodweresigniﬁcantin

com-parison tothe negativecontrol, the MI falling inthe high

concentrationwassigniﬁcantincomparisontoboththe

con-trol and the solventcontrol.In the48happlicationin this

test,signiﬁcantdecreasesoccurredinMIvaluesinallofthe

appliedconcentrationsincomparisontothecontrolandthe

solventcontrol(Table1).Asdistinctfromtheprevioustests,

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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 787–793

Table1–Theinvitrosisterchromatidexchange,abnormalcellpercentage,CA/cellratio,proliferationindex(PI),andmitoticindex(MI)valuescausedbythedeferasirox inthehumanperipherallymphocytesintheabsenceofmetabolicactivator(S9mix).

Testsubstance Treatment SCE/Cell±SE Chromosomeaberration Cytotoxicity

Period(h) Conc.(␮g/mL) Abnormalcell±SE% CA/cell±SE PI±SE MI±SE

Control – – 5.06±0.23 3.75±0.47 0.037±0.002 2.28±0.10 3.69±0.26 DMSO 24 3.7␮L/mL 5.46±0.36 9.50±0.64 0.095±0.003 2.22±0.10 3.25±0.27 Deferasirox 24 10 6.45±0.34a1 5.00±0.70b2 0.050±0.001b2 2.26±0.04 2.84±0.16a1 24 20 5.62±0.46 16.25±1.18a2b1 0.172±0.004a2b1 2.05±0.03a2b1 2.78±0.19a1 24 40 6.43±0.46 18.75±0.85a3b2 0.190±0.004a2b2 1.90±0.09a1b1 1.83±0.17a2b2 DMSO 48 3.7␮L/mL 4.99±0.23 7.25±0.75 0.070±0.003 2.51±0.08 3.84±0.38 Deferasirox 48 10 6.33±0.21a2b1 10.50±1.32a1 0.105±0.002a1 2.31±0.03b1 2.83±0.22a1b1 48 20 6.56±0.45a1 13.25±1.31a2b1 0.132±0.002a2b1 1.63±0.06a2b2 1.51±0.17a3b3 48 40 8.18±0.66a1 22.00±2.86a2b1 0.270±0.008a2b1 1.37±0.11a2b2 0.99±0.10a3b3

a:signiﬁcantfromcontrol;b:signiﬁcantfromsolventcontrol(DMSO).a1b1:P≤0.05;a2b2:P≤0.01;a3b3:P≤0.001.

Table2–Theinvitrosisterchromatidexchange,abnormalcellpercentage,CA/cellratio,proliferationindex(PI),andmitoticindex(MI)valuescausedbythedeferasirox inthehumanperipherallymphocytesinthepresenceofmetabolicactivator(S9mix).

Testsubstance Treatment SCE/cell±SE Abnormalcell±SE% CA/cell±SE Cytotoxicity

Period(h) Conc.(␮g/mL) PI±SE MI±SE Control – – 5.53±0.23 5.50±0.28 0.055±0.003 2.09±0.09 2.91±0.35 DMSO 3 3.7␮L/mL 5.04±0.47 6.25±0.94 0.068±0.008 2.25±0.08 3.82±0.66 Deferasirox+S9mix 3 10 6.04±0.59 10.00±0.70a2b1 0.100±0.004a2b1 2.20±0.06 3.64±0.21a1 3 20 6.81±0.77 12.00±0.40a3b3 0.120±0.002a3b3 2.20±0.04 3.70±0.23a1 3 40 7.50±0.53a1b1 11.75±0.47a3b3 0.138±0.009a3b3 2.19±0.05 3.09±0.20b1

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791

Table3–Theinvivoabnormalcellpercentage,CA/cellratio,andmitoticindex(MI)valuescausedbythedeferasiroxin thebonemarrowcellsofrats.

Testsubstance Treatment Abnormalcell±SE% CA/cell±SE MI±SE

Period(h) Conc.(mg/kg) Control – – 2.75±0.25 0.0275±0.001 1.43±0.10 DMSO 12 16mL/kg 1.75±0.25 0.0175±0.001 1.93±0.19 Deferasirox 12 250 2.75±0.47 0.0275±0.001 2.53±0.27a1 12 500 4.75±0.25a2b3 0.0475±0.001a2b2 1.66±0.06a1b1 12 1000 4.75±0.47a1b2 0.0475±0.001a1b2 1.41±0.10b1 DMSO 24 16mL/kg 3.25±0.25 0.0375±0.002 1.48±0.11 Deferasirox 24 250 2.00±0.40 0.0200±0.001 1.93±0.36 24 500 2.75±0.25 0.0275±0.001 1.22±0.06a1b1 24 1000 3.50±0.28 0.0354±0.001 1.67±0.13

a:signiﬁcantfromcontrol;b:signiﬁcantfromsolventcontrol(DMSO).a1b1:P≤0.05;a2b2:P≤0.01;a3b3:P≤0.001.

inﬂuenceoftheexogenousmetabolicactivator(P<0.05). How-ever,theMI valuedetermined inthe highestconcentration herewasstatisticallylowerincomparisontothesolvent con-trol(P<0.05)(Table2).

In the in vivo tests, the deferasirox had heterogeneous

effectsonmitoticdivisionofbonemarrowcells.Inthe12h

application,thedeferasiroxledtosigniﬁcantMIdecreasesin

highdosesincomparisontothesolventcontrol.Inthe24h

application,the testsubstancedecreasedthemitoticindex

signiﬁcantly incomparison to the control and the solvent

controlonlyinoneconcentration (500mg/kgb.w.)(P<0.05)

(Table3).

Inaddition,itwasdeterminedthatthedifferences

occur-ringincytotoxicityvaluesasaresultofincreaseindosewere

notstatisticallysigniﬁcant(P>0.05).

Tosumupthepropertiesofthedeferasiroxregarding

cyto-toxicity,itsloweddownreplicationanddivisionratesinthe

in vitro tests when no metabolic activator was used, but

increasedthem,thoughslightly, incomparisontothe

con-trolintheteststhatcontainedmetabolicactivator.Although

ﬂuctuationswereobservedinMIvaluesintheinvivotests,

thevalueswereparallelwiththeresultsoftheinvitrotests

includingS9mix.

4. Discussion

4.1. Clastogeniceffectofdeferasirox

There are limited studies about genotoxic effects of iron

chelators. Manystudies based ondifferent testingsystems

reportedthatthedeferoxamin(DFO),amemberofiron

chela-tors including deferasirox, removed the metal in the cell,

and thus had an antigenotoxic effect on the DNA

frag-mentsderiving from hydroxylradical occurringasa result

ofFenton/Haber–Weissreaction(Cooganetal.,1986;Stinson

etal.,1992;Bealletal.,1996;Zhangetal.,1996;Andersonetal., 2000;Witteetal.,2000;Chakrabartietal.,2001).Tothe

con-traryoftheseﬁndings,astudyindicatedthatDFOwerehighly

toxicandmutagenicirrespectiveofthepresenceorabsence

ofS9inL5178Yratlymphomacells (Whittakeretal.,2001).

Itwasfoundthatdeferipronedidnothaveasmuch

clasto-geniceffectasDFOhad,andledtochromosomefractionless

frequentlythandeferoxamine(Marshalletal.,2003).In

addi-tion,itwasdeterminedthatDFOwasnotclastogenicbyitself,

butincreasedacentricfragmentandringchromosome

forma-tionfrequencyalongwithgammarays(Juckettetal.,1998).

In general, iron chelators remove the excessive iron in

the cell, and thus minimize iron-mediated hydroxyl

radi-cal formation reactions, which reduce the DNA fragments

and proliferative effects caused by the said radical.

How-ever, infew studiesit wasreported thatthe ironchelators

were genotoxic (Juckett et al., 1998;Whittaker et al., 2001;

Marshalletal.,2003).Accordingtotheresultsofthecurrent

study,thedeferasiroxgenerallyincreasedtheSCEfrequency.

Theseincreasesoccurredinthe48htreatmentperiodwithout

metabolicactivatorandathighconcentrationswithmetabolic

activator.Theseresultssuggestthatthetestsubstanceleads

toDNA fragments.ThehighCAfrequenciesemerging

irre-spectiveofthepresenceofmetabolicactivatorsupportthis

theory,too.However,CAvaluesbecameevidentinratbone

marrowcellsinacellcycle(12h),butinthe24hprocess,the

CAsdisappeared.Itcanbeexplainedbyeitherthatthetest

substanceeliminatedfromthebodythroughmetabolization

(thehalf-lifeofthedrugis8–16hfollowingtheoral

applica-tion)orthattheabnormalcellsunderwentselection.

4.2. Cytotoxiceffectofdeferasirox

Accordingtotheproliferationindex(PI)data,thedeferasirox

decreased the replication rate in the absence ofmetabolic

activation most probably as a result of genotoxicity, but

the decrease in the PI value disappeared in the presence

ofthe metabolic activation.Metabolic activation decreased

theantiproliferativeeffectofdeferasirox.Wethinkthatthe

decrease in the replication rate was a slowing down

aris-ing from DNA damage. In connection with this situation,

mitotic index wasdecreased. A similar situation was seen

in thefollowing study (Sedigh-Ardekani et al., 2013). In all

concentrations and application periods of deferasirox, the

mitoticindexesdecreasedconsiderably.Suchslowingdownis

stronglyconsistentwiththeCAresults.However,these

ﬁnd-ingschangedsomewhatinthetestcarriedoutwithS9mix.In

invivotest,MIvaluesexhibitinverseproportiontotheCA

for-mations.Theseresultsareparallelwiththeresultsofthe

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of chelators. For example, deferiprone functioned as an

antioxidantinastudyonhumanumbilicalveinendothelium

cellsunderinvitroconditions,andthusitwassuggestedthat

itmightreduceatherogenesis(Matthewsetal.,1997).

ItwasfoundthattheapplicationofironsalttotheKaposi

sarcomacell culturesstrongly stimulated the development

inthesaidcells.Thestimulated growthwasinhibitedwith

deferiprone and DFO inthe same culture(Simonart et al.,

1998).

Itwas foundthatsuchchelatorsas1,10-phenanthroline

(OP),desferrioxaminemesylate,and deferipronehada

pro-tectiveeffectagainstthetoxicitycausedbythecompoundof

9,10-phenanthraquinone(PQ)(maincomponentinthediesel

exhaustparticles)inhumanlungepithelialcells.Here,PQled

toiron-mediated oxidative damage(Sugimoto et al., 2005).

Whenthe antiproliferativeeffectsofsuchironchelatorsas

DFXandO-trensoxonhumanhepatocarcinomacelllineand

humanhepatocyteculture were compared, the deferasirox

wasfoundtoplayamoreactiverolethanO-trensoxinthat

itinducedtheDNAfragmentation,inhibitedDNAreplication,

and reduced cell vitality. Itwas reported that these

chela-torsblockaded thecell cycleatthe phasesofG0–G1andS

respectively. Based on theseresults, it was suggestedthat

thedeferasiroxmighthaveaverystrongantitumoraleffect

incancertreatment(Chantrell-Groussardetal.,2006).

Simi-larly,inanotherstudy,itwasstatedthattheprogressofthe

cellcycledependedonintracellularironlevel,andthe

chela-torsreducedcellproliferationthroughremovingiron.Itwas

mentionedthatsuchantiproliferativeeffectcouldbeinhibited

inthe presence ofexogenous iron(Pires et al., 2006). DFO

activatedp53-mediated check points inthe cultured blood

lymphocytes, and stimulated apoptosis in human

periph-eralbloodlymphocytesviamitochondriadamage(Kimetal.,

2007).

Apartfromthat,theHIV-1replicationinducedbyexcessive

ironisinhibitedbyDFObecausethechelatorpreventsthe

pro-liferationofthevirusthroughremovingtheironintheinfected

cells.Itisstronglyrecommendedtofocusonthedevelopment

ofironchelatorsforanti-retroviraldrugsinthefuture(Debebe

etal.,2007).Anotherstudyreportedthatsuchironchelatorsas

DFO,deferasirox,andciclopiroxolamineblockedWntsignal

andcelldevelopmentinthecolorectalcancercellline(Song

etal.,2011).

Accordingtoliterature,DFXismostprominentiron

chela-torascomparedwithotherones.However,thereisnotenough

informationonthegenotoxicproﬁleofthedeferasirox.Toﬁll

thisgap,thesetestswereperformedandimportantﬁndings

wereobtained.Amongsuchﬁndings,themostimportantone

isthatthedeferasiroxhasaclastogeniccharacter.Apartfrom

that,theagentledtoevidentreductionsinPIandMIvaluesin

parallelwiththegenotoxicityvalues.Basedontheseresults,it

canbesaidthatthetestsubstanceexhibitedan

antiprolifera-tivecharacterassociatedwithgenotoxicity.Thischaracterwas

similarwithsomechemicalagents(mitomycinC,bleomycin,

etc.)usedinthecancertreatment.Itmustberememberedthat

adrugmaytreatthetargetdiseaseononehand,butstimulate

genotoxiceffectsontheotherhand.Althoughsomeimportant

ﬁndingswereobtainedinthepresentstudy,thedeferasirox

shouldalsobetestedthroughothertestingsystemsandalong

withtumorcellsinparticular.

Whiledeferasiroxisusedasachelator,owingto

antipro-liferativeeffect, it canbeagoodcandidatetotreatmentof

tumors.

Conﬂict

of

interest

Theauthorsdeclarethattherearenoconﬂictsofinterest.

Transparency

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Acknowledgements

WewishtothanktheCukurovaUniversityScientiﬁcResearch

Commissionforsupportingourstudythroughprojectgrants

no.FEF2010D11.WewouldliketothankEbrahimValipourfor

helpingtoeditourmanuscriptlanguagecarefully.

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