w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Original
article
Foxo3
gene
expression
and
oxidative
status
in
beta-thalassemia
minor
subjects
Sandra
Stella
Lazarte
∗,1,
María
Eugenia
Mónaco
1,
Magdalena
María
Terán,
Ana
Cecilia
Haro,
Miryam
Emilse
Ledesma
Achem,
Blanca
Alicia
Issé
UniversidadNacionaldeTucumán,SanMigueldeTucumán,Argentina
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3October2016
Accepted5January2017
Availableonline22February2017
Keywords:
Beta-thalassemiatrait
Oxidativestress
FoxO3
Thiobarbituricacidreactivespecies
Erythrocytecatalaseactivity
a
b
s
t
r
a
c
t
Background:Oxidativestressmayaggravatesymptomsofhemolyticanemiassuchas
beta-thalassemia.FoxO3activationresultsinresistancetooxidativestressinfibroblastsand
neuronalcellcultures.
Objective:ThepurposeofthisresearchwastostudyFoxO3geneexpressionandoxidative
statusinbeta-thalassemiaminorindividuals.
Methods:Sixty-three subjects (42 apparently healthy individuals and 21 with
beta-thalassemiaminor)wereanalyzed attheUniversidadNacionaldeTucumán,Argentina,
betweenSeptember2013andJune2014.Acompletebloodcount,hemoglobin
electrophore-sis in alkaline pHand hemoglobin A2 levels were quantified.Moreover, thiobarbituric
acidreactivespecies,erythrocytecatalaseactivityandironstatuswereevaluated.
Beta-thalassemiamutationsweredeterminedbyreal-timepolymerasechainreaction.FoxO3gene
expressionwasinvestigatedbyreal-timereversetranscription-polymerasechainreaction
usingmononuclearcellsfromperipheralblood.
Results:Subjectsweregroupedaschildren(≤12years),andadultwomenandmen.The
analysisoferythrocytecatalaseactivity/hemoglobinratiorevealedasignificantdifference
(p-value<0.05)betweenhealthyandbeta-thalassemiaminoradults,butnosignificant
dif-ference wasobservedin thethiobarbituricacid reactivespecies levelsand FoxO3gene
expression(p-value>0.05).Thiobarbituricacidreactivespeciesandtheerythrocyte
cata-laseactivity/hemoglobinratiowerenot significantlydifferentoncomparingthetypeof
beta-thalassemiamutation(0or+)presentincarriers.
Conclusions: Thelackofsystemicoxidativeimbalancedemonstratedbythiobarbituricacid
reactivespeciesiscorrelatedtotheobservationofnormalFoxO3geneexpressionin
mono-nuclearcellsofperipheralblood.However,animbalancedantioxidantstatewasshownby
theerythrocytecatalaseactivity/hemoglobinratioinbeta-thalassemiaminorcarriers.It
wouldbenecessarytostudyFoxO3geneexpressioninreticulocytestoelucidatetheroleof
FoxO3inthispathology.
©2017Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published
byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:InstitutodeBioquímicaAplicada,Balcarce747,CP4000,SanMigueldeTucumán,Argentina.
E-mailaddress:slazarte@fbqf.unt.edu.ar(S.S.Lazarte).
1 Authorscontributedequallytothiswork.
http://dx.doi.org/10.1016/j.bjhh.2017.01.005
1516-8484/©2017Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Thisisan
Introduction
Theoxidative status ofcells isdetermined bythe balance
betweenpro-oxidants andantioxidants. Oxidants are
com-poundsthatcanacceptelectrons,asopposedtoreductants
thatdonateelectrons.Pro-oxidantsarereferredtoasreactive
oxygenspecies(ROS).ThetoxicityofROSdependsontheir
reactivityandlifespan.Longerlifespanpermitsthemto
dif-fuse,interactwithsensitivebiologicalsubstrates andcause
damagetovariousorganelles.Mostofthetransitionmetals,
suchasiron,canconvertrelativelystableoxidantsinto
power-fulradicals.Excessironcatalyzeshydroxylradicalgeneration
fromactivatedoxygenspeciesbytheHaber–WeissandFenton
reactions.1
Theintracellularresponsetooxidative-stressin
erythro-poiesisinvolves the transcription factor,Forkheadbox O3a
(FoxO3a), which controls pathway(s) regulating erythroid
maturation and the levels of oxidative stress in murine
erythropoiesis.2 ActivationofFoxO3ahasbeenproposed as
aprotectivemechanisminpathologicalerythropoiesis
char-acterizedbyabnormalROSlevelssuchasin-thalassemia.
ThisFoxO3aeffectismediatedbytheup-regulated
transcrip-tionofthe ROSscavengingenzymes,superoxidedismutase
2,alsoknownasmanganesesuperoxidedismutase(MnSOD),
andcatalase.3
Thalassemiais a globin gene disorder that results in a
diminishedrate of synthesisof oneor more ofthe globin
chains and, consequently, a reduced rate of synthesis of
hemoglobin(Hb)orHbsofwhichthatchainconstitutesapart.
Beta()-thalassemiaisoneofmostcommonautosomal
reces-sivedisorders worldwide,and is causedbyreduced (+)or
absent(0)synthesisoftheglobinchainsoftheHbtetramer,
whichismadeup oftwoalpha(␣)globinandtwoglobin
chains(␣22).4
Aquantitativereductioninglobin resultsinthe
accu-mulationofanexcessof␣globinchainsthatareresponsible
for the pathophysiology of the disorder. The excess of ␣
globinchainsformsunstable tetramersthatdissociate into
monomersandthen,followedbyachangeintheirtertiary
structure,areoxidized,first tomethemoglobinandthen to
hemichromes which precipitate with time. The following
stepsarereleaseofhemandfreeiron(labileironpool),and
precipitation ofthe proteinmoiety ofthe globin,including
on the plasma membrane. Cell damage occurs during the
production of red blood cell (RBC) precursors in the bone
marrow (ineffective erythropoiesis) and in mature RBC in
theperipheralblood resulting inshortenedlifespans.
Fac-torsresponsible fortheoxidative stressinthalassemia are
hemoglobininstabilityandironinexcess.5
-Thalassemiaminor(BTM)isaclinicaldefinitionapplied
tothalassemiapatientswhopresentamilderclinicalcourse
than those with -thalassemia major, with a moreor less
markedanemiathatdoesnotrequiretreatmentwithregular
bloodtransfusions.6Previousreportswerefocusedexclusively
onoxidativestress inpatients with-thalassemiamajor7,8
and -thalassemia intermedia.9,10 In severe -thalassemia,
it is difficult to evaluate oxidative stress because of the
high proportionof normalred cells dueto multiple
trans-fusions. However, oxidative stress has been found to be
increasedinboth-thalassemiasubgroups.Incontrast,
lit-tle is known about the oxidative status in BTM subjects,
althoughSeleketal.6andLabibetal.11concludedthat
oxida-tivestressisincreased.Furthermore,littleisknownaboutthe
relationofoxidativestatusinBTMsubjectswithdifferent
-thalassemiamutations.Thisevaluationisimportantdueto
thelargegenotypicandphenotypicheterogeneityofdifferent
populations.
The purposeof this work was to study the FoxO3 gene
expressionandoxidativestatusinindividualswithBTM,and
tocorrelateitwiththetypeof-thalassemiamutation(0or
+).
Methods
A descriptive cross-sectional study was carried out at the
Instituto de Bioquímica Aplicada (Universidad Nacional de
Tucumán,Argentina)fromSeptember2013toJune2014.
Subjects
This study was approved by Bioethics committee of the
Facultad de Medicina (Universidad Nacional de Tucumán),
Tucumán, Argentina. Informedconsentwas obtained from
all participants of the study. The sample consisted of 21
BTM subjects(BTM group)and 42 apparently healthy
indi-viduals(Healthygroup).Theinclusioncriteriawerepatients
who were diagnosed with BTM and healthy subjects who
wereolderthanoneyearold.Individualswhohadingested
vitamins, smokers, diabetics, and patients with coronary
heartdisease,rheumatoidarthritis,dyslipidemia,
hyperten-sion,malignancy,chronicliverdiseaseorrenaldysfunction
wereexcludedfromthestudy.Furthermorepatients
submit-tedtoirontherapywithin21dayspriortoanalysis,andthose
whohadreceivedtransfusionsforanyreasonotherthantheir
thalassemiawithinthethreemonthsleadinguptothestudy
werenotincluded.
Allproceduresfollowedwereinaccordancewiththeethical
standards ofthe committeeresponsible forhuman
exper-imentation and with the Helsinki Declaration of 1975, as
revisedin2008.
Hematologicalstudies
Blood samples of -thalassemia carriers were collected
in K2-ethylenediaminetetraacetic acid (EDTA). RBC count,
meancorpuscularhemoglobin(MCHb)andmeancorpuscular
volume(MCV)wereanalyzedwithaSysmexKX-21N
hema-tological counter (Kobe, Japan). DecreasedMCV and MCHb
allowed an individual to be considered as a potential
car-rier. Thecarrier status was confirmedby celluloseacetate
membraneelectrophoresisatalkalinepH,andtheHbA2level
bymicrocolumnchromatography(BioSystemS.A.,Barcelona,
Spain). If Hb A2 was ≥3.5%, then the subjects were
diag-nosed as-thalassemia carriers.Serumiron(Fe),totaliron
bindingcapacity(TIBC)andtransferrinsaturation(SAT)were
measured by a colorimetric method (Wiener Lab, Rosario,
Beta-thalassemiamutationsbyDNAanalysis
GenomicDNA wasisolated withthe HighPurePolymerase
ChainReaction(PCR)TemplatePreparationKit(Roche
Diag-nostics)from250LofK2-EDTAanticoagulatedwholeblood.
PCRandmutationdetectionbymeltingcurveanalysiswere
performed in a LightCycler system 2.0 (Roche
Diagnos-tics), which can simultaneously measure signals emitted
from two different fluorophores. PCR primers (forward: 5′
-gctgtcatcacttagacctca-3′; reverse: 5′-cacagtgcagctcactcag-3′)
weredesignedtoamplifya587-bpregionofthe-globingene
surroundingthemostcommon-thalassemiamutationsin
theArgentinianpopulation,asreportedpreviously.12The
clus-teringofmanymutationswithinsmall distancespermitted
theuseoftwocombinationsofprobestoanalyzefivecommon
-thalassemiamutations.
FoxO3geneexpression
The FoxO3 gene expression was analyzed using real-time
reversetranscription-PCR(RT-qPCR).Thedataobtainedfrom
the real time PCR were compared with the expression of
theglyceraldehyde3-phosphatedehydrogenase(GAPDH)geneas
anendogenouscontrol(relativequantification).RT-qPCRisa
powerfultooltoquantifygeneexpression.Thequantitative
endpointforreal-timePCRisthethresholdcycle(Ct).TheCt
isdefinedasthePCRcycleatwhichthefluorescentsignalof
thereporterdyecrossesanarbitrarilyplacedthreshold.
RNAwasextractedfrom1mLofK2-EDTAanticoagulated
bloodusingTRIzolReagentaccordingtothemanufacturer’s
protocol(Invitrogen). ThePCRdissociationcurves and
sub-sequent analyses were performed in the LightCycler 2.0
equipment (Roche Diagnostics). The amplicons were
sub-jectedtoananalysisof‘melting’temperaturetoestablishthe
specificityoftheamplifiedfragmentsandidentitythem.The
primersandPCRconditionsweretakenfromKajiharaetal.13
TheFoxO3geneexpressionwascalculatedusingthefollowing
formula:FoxO3=2−Ct,i.e.[2−(CtFoxO3−CtGAPDH)].
Oxidativestressanalysis
OxidativedamagetobiologicalmacromoleculessuchasDNA,
proteinsandlipidscanbemeasuredbyquantifyingspecific
reaction end products. During peroxidation, the peroxides
are decomposed to malondialdehyde (MDA), which can be
detectedbythiobarbituricacidinacolorimetricreactionthat
produces thiobarbituric acid reactive species (TBARS). The
TBARSlevelwasmeasuredspectrophotometricallyat532nm
underacidicconditionsinplasmaobtainedafterthe
centrifu-gationofanticoagulatedwholeblood.14TBARSisexpressedin
mol/L.
In order to defend themselves against the deleterious
effectsofROS,cellsmaintainaneffectiveantioxidantsystem
consistingofwaterorlipid-solubleantioxidantsandenzymes
thatremoveROSbymetabolicconversion.H2O2isconverted
towaterandO2bycatalase.Catalaseactivitydiffersgreatly
indiverse human tissues.The highestactivity isfound in
RBC,whichis3600-foldhigherthaninserum.Thus,the
cata-laseactivityofbloodispracticallyallattributabletoRBCs.15
Erythrocytecatalaseactivity(ECAT)wasanalyzedinK2-EDTA
anticoagulated wholeblood usingthe Góth techniquewith
minormodifications tocalculatetheresults.16 TheECATto
bloodhemoglobinratio(ECAT/Hb)wascalculated.
Statisticalanalysis
TheStatisticalPackagefortheSocialSciences(SPSS)software
version20.0wasusedthroughouttocarryoutallthestatistical
analyses.Hematologicalandoxidativestressparameterswere
comparedbytheMann–WhitneyUtestforpairwise
compar-isonsbetweengroups.Thetestswereperformedwithp-value
<0.05asthelevelofsignificance.Theinfluenceofserumiron
onTBARSvaluesandFoxO3expression,independentofthe
group(HealthyorBTM),wasevaluatedbysimpleregression
analysis.
Results
Forty-twohealthysubjectsand21BTMindividualswere
stud-ied.Table1showsthe resultsofhematologicalparameters,
TBARSandtheECAT/Hbratioinadultgroupsstratifiedby
gen-der.ThesameparametersareobservedinTable2forhealthy
and BTM children. As expected, significant differences (p
-value<0.05)inhematocrit,hemoglobin,MCVandMCHbwere
foundbetweenBTMandHealthygroups.
Lipidperoxidation,asdeterminedbytheTBARSlevel,did
not detectsignificant differences betweenthe Healthy and
BTMgroups(p-value=0.083).NormalTBARSlevelswere
deter-minedbythe5thand95thpercentiles,andarangefrom0.42
to0.88mol/Lwasobtained.OnlyoneBTMsubjectexceeded
theTBARSupperlimit.
Thecomparativeanalysisofantioxidantstatususingthe
ECAT/Hbratioshowedasignificantdifference(p-value=0.000)
betweenhealthyandBTMadults.ThenormalECAT/Hbratio
(from0.50to0.91MU/g)wasestablishedusingthe5thand95th
percentiles.
TheFoxO3geneexpressionwasmeasuredinBTMsubjects
andin21ofthe42healthysubjects;asinthecaseofTBARS
andECAT/Hb,the5thand95thpercentilesdetermineda
nor-malrangeoffrom 0.19to9.47.OnlytwowomenwithBTM
(9.5%),onewiththeIVSI-1G>Amutationandtheotherwithan
undeterminedmutation,exceededtheupperlimit(Figure1).
There was no influence of iron levels on TBARS level
(R2=0.098)orFoxO3expression(R2=0.019).TwoBTMsubjects
hadlowiron(onewomananda1-year-oldchild)butthisdid
notimpedetheobservationofanincreasedHbA2level.
The -thalassemia mutations detected in order of
fre-quencywereIVS-I-1G>A,codon39C>T, IVS-I-110G>Aand
IVS-I-6 T>C.To evaluatethe possible effectof thalassemia
mutationsonHb A2,ironstatus,TBARS,ECAT/Hb ratioand
FoxO3expression,samplesweresubdividedaccordingtothe
typeofmutation(Table3).TheIVSI-6T>C(mildphenotype)
and IVSI-110G>A(more severephenotype)mutationswere
discriminated in the + group. Differences between 0 (11
carriers) and + (7 carriers) groups were analyzed, without
anysignificantdifferencesbeingfound(HbA2:p-value=0.891;
Fe:p-value=0.556;TIBC:p-value=0.821;SAT:p-value=0.751;
TBARS: p-value=0.079;ECAT/Hb ratio:p-value=0.342;FoxO3
Table1–Hematologicalparameters,thiobarbituricacidreactivespecies(TBARS)anderythrocytecatalaseactivity
(ECAT)/hemoglobin(Hb)ratioinHealthyandbeta-thalassemiaminor(BTM)adults.
Group n Age[years] HTOa[L/L] MCVa[fL] MCHa[pg] Hba[g/dL] TBARS[mol/L] ECAT/Hba[MU/g]
Healthy
Female(range) 14 34(13–75) 0.42b(0.37–0.46) 88.1(81.8–96.9) 29.4(26.2–31.4) 11.5b(9.3–14.2) 0.61(0.29–0.90) 0.68(0.50–1.08)
Male(range) 25 37(14–64) 0.45(0.42–0.51) 86.2(81.2–93.0) 29.1(25.6–31.2) 14.8(13.0–17.6) 0.68(0.42–0.81) 0.59(0.46–0.88)
BTM
Female(range) 12 33(22–76) 0.36(0.33–0.41) 65.2(63.0–76.9) 19.3(18.3–24.1) 10.6(10.0–12.1) 0.56(0.38–0.79) 0.82(0.66–1.42)
Male(range) 3 33(16–65) 0.39(0.32–0.47) 67.6(66.9–74.3) 20.0(19.8–22.5) 11.5(9.3–14.2) 0.58(0.40–0.90) 0.86(0.76–1.18)
HTO:hematocrit;MCV:meancorpuscularvolume;MCH:meancorpuscularhemoglobin.
a p-Value<0.05betweenHealthyandBTMgroups.
b p-Value<0.05betweenHealthyfemaleandmaleindividuals.
Table2–Hematologicalparameters,thiobarbituricacidreactivespecies(TBARS)anderythrocytecatalaseactivity
(ECAT)/hemoglobin(Hb)ratioinHealthyandbeta-thalassemiaminor(BTM)children.
Group(n) n Age[years] HTO[L/L] MCV[fL] MCH[pg] Hb[g/L] TBARS[mol/L] ECAT/Hb[MU/g]
Healthy(range) 3 11(8–12) 0.39a(0.37–0.39) 81.8a(74.6–84.8) 26.3a(23.3–28.0) 122a(121–124) 0.47(0.45–0.75) 0.77(0.50–0.88)
BTM(range) 5 11(1–12) 0.35(0.32–0.38) 63.0(58.7–65.1) 19.2(17.3–19.5) 105(93–112) 0.51(0.38–0.58) 0.83(0.72–0.90)
HTO:hematocrit;MCV:meancorpuscularvolume;MCH:meancorpuscularhemoglobin.
a p-Value<0.05betweenHealthyandBTMchildren.
Discussion
The aim of this study was to investigate the relationship
betweenFoxO3geneexpressionandoxidativestatusin
indi-viduals withthe -thalassemiatrait. In severalstudies, an
increase of oxidative stress has been reported in both 
-thalassemiamajorandintermedia.Theincreaseofoxidative
damageinthalassemiahasbeenrelatedtothegenerationof
freeradicalsbyanexcessofdenatured␣-or-globinchains,
intracellularironoverloadandlowconcentrationofnormal
hemoglobin.7–11
OxidativestatushasbeeninvestigatedinBTMsubjectsby
onlyafewresearchers.6,11,17Allofthemshowedanincrease
inoxidativestressandanalterationinantioxidantcapacityin
300,000
200,000
100,000
0,000
BTM N
F
o
xO3
* 20
* 24
62
Figure1–FoxO3 geneexpressioninnormaland beta-thalassemiaminorgroups.Abbreviations:BTM: beta-thalassemiaminor(21subjects);N:Healthy(21 subjects).
-thalassemiaheterozygotes.Werecentlyreportedthat
cata-lase activity,akey enzymeofthe antioxidantsystem, was
increasedin only14% ofBTMsubjects, withno significant
differences between-thalassemiamutations.16 Inorderto
sharpentheresults,theECAT/Hbratiowascalculated,which
was significantlyhigherinBTMindividuals.IntheHealthy
group,thelevelofthisratiowassimilartothatobtainedby
VitaiandGóth.18However,Kósaetal.describedadecreased
ratiobetweencatalaseactivityinbloodandhemoglobininthe
BTMgroup.Theyproposedthatthedecreaseincatalase
activ-itymightbeduetothedamagingeffectsoffreeradicalson
thecatalaseprotein.19However,otherauthorshavereported
increasedcatalaseactivityinthalassemiaindividuals.8
Over 200different mutationshave been foundto cause
-thalassemia, with splicing mutations among the most
common. Most of these mutations activate aberrant
cryp-tic 5′-donor or 3′-acceptor splice sites without completely
abolishingnormalsplicing.Thesemutationsleadtothe
pro-duction of variable amounts of normal transcripts. Some
mutationsallowasignificant levelofnormalsplicing(such
asIVSI-6 T>C),leading tothalassemiaintermedia, whereas
othersreducenormalsplicingtolowlevels(suchasIVSI-110
G>A)orverylowlevels(suchasIVSI-5G>AandIVSII-654C>T),
causingatransfusion-dependentdiseaseinhomozygotes.20
Therefore, IVS-I-110G>A, althoughconsidered+, isavery
severegenotypewithaminimalamountofHbA(generally,
lessthan5%).Sothelackofanysignificantdifferencebetween
0and+inthepresentstudycouldberelatedtothefactthat
onlytwopatientsaregenuinely+(IVS-I-6T>C).
Inrespecttoincreasedoxidativestress,ourfindingswere
notconsistentwithpreviousstudies.Theoxidantstatusof
BTM subjects showed no significant differences with the
Healthygroup. Selek etal., bymeasuringthe totaloxidant
status(TOS),totalantioxidantcapacity(TAC),lipid
hydroper-oxide(LOOH)levelsandoxidativestressindex(OSI),detected
T able 3 – Results of beta-thalassemia minor subjects accor ding to the beta-thalassemia m uta tions. Type of m utation a n Hb [g/L] Hb A2 [%] Ir on [ g/dL] TIBC [ g/dL] SA T [%] TB ARS [ mol/L] EC A T/Hb [MU/g] FoxO3 g ene e xpr ession a 0(r ang e) 11 108 b(93–121) 4.8 (4.0–5.6) 83 (29–259) 266 (180–347) 31 (8–97) 0.60 (0.40–0.90) 0.92 b(0.66–1.42) 1.84 (0.08–37.01)  +IVSI-110 G>A (r ang e) 5 106 c(100–111) 4.7 (3.8–5.3) 80 (75–103) 297 (220–380) 32 (20–45) 0.51 (0.38–0.54) 0.87 (0.72–1.15) 2.01 (0.01–7.52)  +IVSI-6 T>C (r ang e) 2 108 (103–112) 5.2 (5.1–5.3) 64 (63–66) 224 (203–244) 29 (27–31) 0.55 (0.52–0.58) 0.82 (0.81–0.83) 1.96 (0.53–3.39) Undetermined (r ang e) 3 132 (121–142) 4.4 (4.2–5.3) 80 (58–144) 247 (247–318) 32 (23–45) 0.71 (0.48–0.79) 0.76 (0.73–0.79) 156.29 (1.75–310.83) TIBC: total ir on binding capacity; SA T : tr ansferrin satur ation; TB ARS: thiobarbituric acid reacti v e species; EC A T/Hb: er ythr oc yte catalase acti vity/hemo globin ratio . a  0gr oup wa s composed of 6 IVSI-1 G>A and 5 CD39 C>T subjects. b p -V alue <0.05 betw een  0and undetermined gr oups. c p -V alue <0.05 betw een  +IVSI-110 G>A and undetermined gr oups.
analyzingTBARSandTroloxequivalenttoantioxidant
capac-ity (TEAC), identified anincrement of oxidativestress and
antioxidantcapacityin-thalassemiaheterozygotes,mainly
incarriersofthecodon39mutation.17Labibetal.measured
TACandTBARS.TheyfoundthatTACwassignificantlylowerin
BTMsubjectsthanincontrols,whileTBARSwassignificantly
higher,withsignificantdifferencesbetweenthe0and+
tha-lassemiatraits.11Inaddition,Laietal.reportedhigherlevels
ofthepro-oxidant/antioxidant ratioin-thalassemia
inter-mediaandBTM,comparedtocontrols.21Differenceswiththe
presentstudymaybeduetomethodologicaldifferencesand
sensitivityofthemethods.
In this study,therewas no relationshipbetween serum
iron,TBARSandFoxO3expression.Inaccordance,Ondeietal.
didnotdetectaninfluenceofironlevels, SATorferritinon
TBARSandTEACvalues.17Al-Hakeimetal.reportedno
statis-ticallysignificantcorrelationbetweenironstatusparameters
and MDA or total carbonyl in patients with-thalassemia
major.22 Excessive iron concentrations can stimulate lipid
peroxidationinthalassemiamousemodels,whichresemble
thalassemiapatientsreceivingbloodtransfusions,23however
thesestudies haveindicatedno directassociationbetween
serumironparametersandMDA.22,23 Instead,thestrongest
predictor of increased MDA in patients with severe
tha-lassemiawouldbetheliverironconcentration.24
Themammalianforkheadtranscription factors ofthe O
class(FoxOs) hasfourmembers:FoxO1, FoxO3, FoxO4, and
FoxO6.FoxO1andFoxO3areexpressedinnearlyalltissues.
FoxOs transcriptionally activate or inhibit downstream
tar-getgenes,thereforeplayinganimportantroleinproliferation,
apoptosis,autophagy,metabolism,inflammation,
differentia-tion,andstressresistance.FoxO1andFoxO3playanimportant
roleinprotectionofcellsagainstoxidativestress.25Wangetal.
foundthatoxidativestressenhancestheexpressionofFoxO3a
but not FoxO1and FoxO4 inendothelialprogenitor cells.26
FoxO3increasesthelevelsofMnSODinmitochondria,which
removessuperoxideradicals.Invivoexperimentssuggestthat
FoxO3protectsagainstoxidativestressbyincreasingMnSOD
expression and production of catalase and peroxiredoxin
III.27 FoxO3 is also critical for hematopoietic self-renewal.
In vivo experiments have established that FoxO3 deletion
in hematopoietic stem cells increases ROS and impairs
theirhematopoieticcapacity.28FoxO3alsoprotects
erythro-poiesisagainstoxidativestress.2Resveratrol,anovelpotential
therapy that ameliorates oxidative stress in circulating 
-thalassemiaredcells,activatesFoxO3transcriptionalfactor.29
The use of rapamycin, a mechanistic target of rapamycin
(mTOR)inhibitor,hasbeentestedin-thalassemiamiceand
remarkablyimprovederythroidcellmaturation,-globin
pro-ductionand anemiathrough FoxO3activation.30Therefore,
up-regulationofFoxO3couldbeatargettocontroloxidative
stressin-thalassemiasubjects.31
OurdatashowthatFoxO3geneactivationwasincreasedin
onlytwoBTMindividuals.FoxO3geneexpressionwasnormal
inmostBTMsubjects,andthisobservationcouldberelated
to the lack ofoxidative imbalance shown byTBARS levels
inplasma. AlthoughFoxO3expressioninperipheral
mono-nuclearcellsisnotstrictlyrepresentativeofoxidativestressin
theerythroidlineage,theirassessmentisvalidbecauseboth
Alimitationofthisworkisthatsuperoxidedismutasewas
notmeasured;thiscouldhavecontributedtotheevaluation
oftheantioxidantstatusofthecarriers.Thelownumberof
samplesisanotherlimitationofthisstudy.
Conclusions
Oxidative stress plays an important role in the
pathophy-siology of -thalassemia, and its control could ameliorate
theclinicalsignsofthedisease.FoxOtranscriptionproteins
modulateseveralcellularfunctionsthroughregulationof
mul-tiple transcriptional targets. Therefore, regulation of FoxO
transcriptionfactors byantagonistsinsomediseasestates,
oragonistsinnormal anddisease conditions, maybe
use-fulintreatingor preventingawide varietyofdisorders.In
thepresentwork,thepresenceofsystemicoxidativestress
wasnotprovedinBTMindividuals,andinmost,theFoxO3
gene expressionwas notup-regulated. However,the study
of antioxidant activity in RBC demonstrated an increased
ECAT/Hbratio.Ananalysisofreticulocytesseparatedfromthe
bloodofBTMsubjectsmayclarifytheroleofFoxO3inthis
con-dition.Furthermore,itwouldbenecessarytoperformacohort
study of-thalassemiaheterozygotes overalong periodof
timetorecognizetheiroxidantstatusandits relationwith
thetypeofmutation.
Funding
Thisworkwasrealizedwithgrantsgrantedbythe Consejo
deInvestigacionesde laUniversidadNacionalde Tucumán
(CIUNT26/D520).
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
ThisworkcountedonthesupportoftheConsejode
Investiga-cionesdelaUniversidadNacionaldeTucumán(CIUNT26/D520),
whograntedthefundsfortheresearch.Theauthorsthank
Biochemistry Specialist Guillermo Fabián Vechetti and the
Laboratorio Tucumán for the use of its molecular biology
equipment.
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