growth restriction the of erythrocytes pre- and full-term in neonates withintrauterine Major the differences levels of redox in status and antioxidant defencemarkers Reproductive Toxicology

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Reproductive Toxicology

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / r e p r o t o x

Major differences in the levels of redox status and antioxidant defence markers in the erythrocytes of pre- and full-term neonates with

intrauterine growth restriction

Ágnes Ferencz

^a

, Hajnalka Orvos

^b

, Edit Hermesz

^a,∗

aDepartmentofBiochemistryandMolecularBiology,FacultyofScienceandInformatics,P.O.Box533,H-6701Szeged,Hungary

bDepartmentofObstetricsandGynaecology,UniversityofSzeged,P.O.Box533,H-6701Szeged,Hungary

a r t i c l e i n f o

Articlehistory:

Received30September2014

Receivedinrevisedform16February2015 Accepted17February2015

Availableonline24February2015

Keywords:

Antioxidantdefence IUGR

Oxidativestress Peroxynitrite Umbilicalcordblood

a b s t r a c t

Intrauterinegrowthrestriction(IUGR)isapleiotropiccomplicationofpregnancy.Prematurityandgrowth abnormalitiesarecommonriskfactorsforperinatalmorbidityandmortality.Freeradicaldamagehas beenrecognizedasacommonpathogenicmechanismofmanyneonataldiseases.Theaimofthepresent studywastocharacterizethepossiblelinksbetweenthelevelofmaturity,thebirthweightandthe antioxidantstatusofneonatesbornwithIUGR.Ourdatasuggestthatthestressmarkersmeasuredon thecordbloodofneonateswithIUGRandmature,healthyneonatesdonotnecessarilyreﬂecttheextent ofoxidativestress.However,signiﬁcantcorrelationswerefoundbetweenthematurityoftheneonates withIUGRandtheoxidativedamage.ThematureIUGRsexhibitedONOO⁻accumulationandincreased lipidperoxidationmorefrequentlyascomparedwiththepre-termgroup.Theresultssuggestthatthe oxidativeinjuryinIUGRmaydependonthelevelofmaturityandthebirthweight.

1. Introduction

Pregnancyisaphysiologicalstateassociatedwithanenhanced metabolism and an increased demand for oxygen. Premature infants are at particular risk from oxidative stress, as neither theendogenous northe passively acquiredexogenous antioxi- dantdefencesystemaccelerates inmaturationuntil late inthe third trimester [1,2]. Intrauterine growth restriction (IUGR) is one of themajor complications of pregnancy and accounts for signiﬁcant neonatal mortality and morbidity [3]. The develop- mentofIUGRmaybeaconsequenceofvariousfactors,including an abnormal fetomaternal blood circulation, genetic disorders, pregnancy-inducedhypertension,pregestationaldiabetes,alean umbilicalcord,previousintrauterineinfections,apoornutritional stateofthemother,frequentcigarettesmoking[4]andtoxinor drugexposure[5].However,inthevastmajorityofthecasesthe causeremainsidiopathic.

IUGRis often complicated by intrauterinehypoxia and may inducethegenerationofreactiveoxygenspecies(ROS)andfoetal oxidativestress,resultinginseriousconsequencesforthefoetus, suchaslowbirthweightandprematurity[6].NeonateswithIUGR

∗Correspondingauthor.Tel.:+3662544887;fax:+3662544887.

E-mailaddress:hermesz@bio.u-szeged.hu(E.Hermesz).

aremoresusceptibletoROS-inducedoxidativedamagebecause theirenzymaticandnon-enzymaticantioxidantdefencesystems andtheabilitytoundergoinductionduringahyperoxicchallenge areimpaired[7].

To eliminate the harmful effects of ROS, cells are equipped withanefﬁcientantioxidantdefencesystem,includingenzymes suchas superoxide dismutase (SOD), catalase (CAT),hemeoxy- genases (HOs),and low-molecular weightantioxidants suchas glutathione (GSH)and metallothioneins (MTs) [8,9]. SOD catal- yses thereduction of thesuperoxide anion (O₂˙−)tohydrogen peroxide(H2O2).Inasubsequentstep,CATstimulatesthedegra- dationofH2O2tomolecularoxygenandwater[10].TheMTsare smallproteinswithunusuallyhighcysteinecontent.Thisconfers theirhighmetal-bindingand ROS-reducingproperties[11].The HOsplayrolesinhemedegradation,yieldingequimolarquantities ofbiliverdin,carbonmonoxide(CO)withimportantfreeradical- scavenging properties and free Fe ions. In mature neonates, a considerableproportionoftheplasmatotalantioxidantcapacity originatesfrombilirubin[12].HO-2isaconstitutive,andHO-1an inducibleisoformofHO,anantioxidative,anti-inﬂammatoryand cytoprotectiveenzymethatisinducedinresponsetocellularstress, includingoxidativestress[13].

Nitricoxide(NO)maybeanimportantfactorfortheregulation ofbloodpressureandoxygendeliverytothefoetus[14].Umbili- calcordbloodvesselslackinnervations,andendothelialcellsmust http://dx.doi.org/10.1016/j.reprotox.2015.02.008

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Table1

Clinicalparametersofthestudygroupsandthematernalage.Dataareexpressedasmeans±SD.Theminimumandthemaximumvaluesoftheparametersaregivenin parentheses.

Full-termneonateswithnormalweight Full-termneonateswithIUGR Pre-matureneonateswithIUGR Gestationalageatdelivery(weeks) 39.2±0.77(38–40+2) 38.741.31(37–40+4) 34.01±1.62(30–36)

Birthweight(g) 3409±455(3190–4340) 2354238.8(2090–2490) 1518±460.5(980–2120)

ThepHofbloodsamples 7.25±0.11(7.04–7.42) 7.240.088(7.1–7.36) 7.20±0.142(7.05–7.3)

1minAPGAR 8.83±1.37(6–10) 8.361.65(6–10) 7±1.63(4–9)

Maternalage(years) 29.9±5.74(22–42) 28.96.55(20–41) 29.3±7.22(21–42)

thereforeplayamajorroleinthelocalcontrolofbloodﬂow[15].

NOderivedfromendothelialnitricoxidesynthase(eNOS)isconsid- eredthemainvasodilatoragentinfetoplacentalvessels[16].The simultaneousgenerationofNOandO₂˙−insufﬁcientlyhighcon- centrationsinthesamecompartmentfavourstheproductionofa toxicreactionproduct,peroxynitriteanion(ONOO⁻).ONOO⁻and otherreactivenitrogenspeciescanaffectthecellfunctionsthrough theoxidationornitrationofvariouscellulartargets[17].

Understressconditions,genescodingformoleculesinvolved inbiologicaldefenceandcellularrepairaremarkedlyupregulated, andthechangesingeneexpressioncanbecharacteristic,sensi- tiveandmeasurableendpoints[18].Membersofthisantioxidant defencesystemareusefulbiomarkersoftheoxidant–antioxidant statusofneonateswithIUGR.Theaimofthepresentstudywas tocharacterizethepossiblelinksbetweenthelevelofmaturity, thebirthweightandtheantioxidantstatusofneonatesbornwith IUGR.Wereportdataonmacromoleculardamage,theaccumula- tionofpowerfuloxidantssuchONOO⁻,H2O2,theactivitiesofthe antioxidantenzymesSODandCAT,andtheexpressionsofaset ofgenescodingformembersofantioxidantdefencesystem(sod1, sod2,cat,mt-1,mt-2,ho-1,ho-2andenos)fromtheaspectsofthe levelofmaturityandthebirthweightofneonateswithIUGR.

2. Materialsandmethods 2.1. Humansubjects

The blood samples were obtained from the Department of ObstetricsandGynaecologyattheUniversityofSzeged,Hungary.

The Ethics Committee of the Department of Obstetrics and Gynaecologyapprovedthestudyprotocol(149/2012).24mature neonateswithnormalweightand28matureand28premature neonateswithIUGRofeithersexwereexamined.Theneonates wereconsideredprematureiftheywerebornbeforethegesta- tionalageof37weeksandfull-termifthedeliveryoccurredafter37 weeks.Newbornsthathadahistoryofdifﬁcultdeliveryandfoetal distress,orshowedmalformationsorevidenceofgeneticdisorders wereexcluded.Thenutritionalstatusofthemothersduringpreg- nancywassatisfactory;nocaseofmalnutritionoccurred.Smoking mothersandtheirneonateswerealsoexcludedfromthisstudy.

Bloodwastakenfromtheumbilicalartery,beforethebirthof theplacenta.BloodcoagulationwasinhibitedbyEDTA.Theblood sampleswerecentrifugedat3000rpmfor20minat4^◦C,andthe plasmaandthebuffycoatwereremoved.Theredbloodcell(RBC) phasewaswashedtwicewith2volumesofisotonicsalinesolu- tionatpH7.0.Thesampleswerestoredat−80^◦Cuntilprocessing (Table1).

2.2. RNAextraction,reversetranscriptionandPCRampliﬁcation

Approximately,100mgoffrozenRBCwerehomogenizedinRNA Beereagent(Tel-Test,Inc.)andtotalRNAswerepreparedaccord- ingtotheproceduresuggestedbythemanufacturer.TotalRNAwas routinelytreatedwith100URNAse-freeDNAseI(ThermoScien- tiﬁc)toavoidanyDNAcontamination.

For the quantification of mt-1and mt-2,sod1 and sod2, cat, enos,ho-1and ho-2mRNAs,reverse transcription followedPCR amplifications(RT-PCRs)wereperformed.First-strandcDNAswere synthesizedbyusing5␮gtotalRNAsastemplates,200pmolof each dNTP(ThermoScientific),200UMaximaH MinusReverse Transcriptase(ThermoScientific)and500pmolrandomhexamer primers(Sigma)inafinalvolumeof20␮L,andincubatedfor10min at37^◦C,followedby1hat52^◦C.Onemicroliterreversetranscrip- tionproductwasaddedto25␮LDreamTaqGreenPCRMasterMix 2x(ThermoScientific).AmplificationwasperformedinaPTC200 PeltierThermalCycler(MJResearch)using15cyclesof95^◦Cfor 30s,55^◦Cfor30s,and72^◦Cfor30sforthe18SrRNA,usedasinter- nalreferenceand30cyclesformt-1andmt-2,sod1andsod2,cat, enos,ho-1andho-2mRNAs,respectively.Theamplifiedproducts weredetectedona2%agarosegel.TherelativelevelsofmRNAsare expressedasratios(mRNA/18SrRNA).

2.3. Primers

The following primer sets were selected: sod1: F: aagatg- gtgtggccgatgtg and R: ctacagctagcaggataacag; sod2: F: caag- gctcaggttggggttgandR:gctgggatcattagggtagtatg;cat:F:cacagaa- gatggtaactgggandR:ggcgatgtccatctggaatc;enos:F:cactgagcccgtg- gcagtagandR:ggcaggcagcgccaccgacg;mt-1:F:atggaccccaactgctcctg and R: gttcccacatcaggcacagc; mt-2: F: atggaccccaactgctcctg and R:cggtcacggtcagggttgtac;ho-1: F:gctgctggtggcccacgcttand R: ctctggtccttggtgtcatgg; ho-2: F: tggcccacgcatacacccgc and R:

ggtctctctggccagtgtgga.Forthenormalizationofsods,cat,enos,mts andhosmRNAs,thelevelofcarp18SrRNAwasusedasinternal standard, detectedwithprimerpairs F:gaaacggctaccacatccaagg, andR:ccgctcccaagatccaactacg.

2.4. Densitometry

Imagesoftheethidiumbromide-stainedagarosegelsweredig- itizedwithaGDS7500GelDocumentationSystemandanalyzed withtheGelBase/GelBlot^TMProGelAnalysisSoftware(UVP).

2.5. Enzymeactivitymeasurement

TheRBCswerehemolysedbytheadditionofdistilledwaterata ratioof1:9.ExceptforSODactivitydeterminations,thealiquotsof thehemolysateswereuseddirectly.Thequantityofproteinwas determinedwithFolin reagent, using bovineserumalbuminas standard[19].Biomate5Double-BeamUV–visphotometerrecor- ding (ThermoSpectronic)wasusedforSOD measurementsand GENESYS10SUV–visspectrophotometer(ThermoScientiﬁc)was usedforalltheotherparameters.

SOD(EC1.15.1.1)activitywasdeterminedonthebasisofthe inhibition of the epinephrine–adrenochrome autoxidation [20].

Spectrophotometricmeasurementwascarriedoutat480nm.The resultswereexpressedinU/mgprotein.

CAT(EC1.11.1.6)activitywasdeterminedspectrophotometri- callyat240nmbythemethodofBeersandSizer[21]andspeciﬁc

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Fig.1.ThelevelsofONOO⁻,H2O2,lipidperoxidation(TBARS),andtheCATactivity inthecordredbloodcellsofmature,healthyneonates(C)andneonateswithIUGR.

Datawereexpressedasmeans±SEMfrommeasurementson24/56samples.

CATactivitywasexpressedinBergmeyerunits(BU)/mgprotein(1 BU=decompositionof1gH2O2/minat25^◦C).

2.6. Lipidperoxidationestimationassay

Thelevelofthiobarbituricacid-reactivesubstances(TBARS)is regardedasanappropriateindicatoroftheextentoflipidperoxi- dation(LPO)[22].LPOwasestimatedbyaTBARSassayat532nm againstablankthatcontainedthethiobarbituricacid(TBA)reagent (0.15g/mLTCA,3.75×10⁻³g/mLTBAand0.25MHCl)asdescribed bySerbinovaetal.[23].

2.7. DeterminationofH₂O₂production

FortheassayofH₂O₂,0.05mg/mLhorseradishperoxidaseand 0.1mg/mLo-dianisidineinsodiumphosphatebuffer(100mM,pH 6.5) was used. The H2O2 concentration was determined spec- trophotometrically at 400nm and was calculated as nmol/mg protein[24].

2.8. DeterminationofONOO⁻production

ONOO⁻wasassayedbydilutingsamplesinto1.0MNaOH(60:1) andmeasuringtheincrease inabsorbanceat302nm.Asacon- trol,sampleswereaddedto100mMpotassiumphosphate(pH7.4) (60:1).ThedecreaseinabsorbancewasmeasuredatneutralpHas ONOO⁻decomposes[25]

2.9. Statisticalanalysis

Statisticaldifferenceswerecalculatedwithone-wayanalysis ofvariance(ANOVA)(MedCalcStatisticalSoftwareversion9.4.2.0,

Fig.3.FoldofchangeinthelevelsofONOO^-,H2O2,lipidperoxidation(TBARS),and theCATactivityinthecordredbloodcellsofmatureandprematureneonateswith IUGR.Fornormalizationthelevelsofmature,healthyneonateswereusedasrefer- ence.Significantdifferencewasacceptedatâ/bp≤0.05.(a)Asignificantdifference betweenthemature,healthyneonatesandneonateswithIUGR.(b)Asignificant differencebetweenthematureandprematureneonateswithIUGR.

Broekstraat,Belgium)withaStudent–Newman–Keuls follow-up test.Significantdifferencewasacceptedatâ,bp≤0.05,âap≤0.01 andâaap≤0.001.

3. Results

3.1. Comparativestudiesofmature,healthyneonatesand neonateswithIUGR

Thelevelsofoxidantmolecules(H2O2andONOO⁻)andthio- barbituricacid-reactivesubstances(TBARS)andtheactivityofthe H₂O₂-degradingCATdidnotdiffersigniﬁcantlybetweentheIUGR andcontrolgroups(Fig.1).

AsconcernsthemRNAlevelsofgenescodingforantioxidant molecules, significantly lower amounts were measured for cat (∼20%),ho-1(∼40%)andho-2(∼50%)intheIUGRgroupthaninthe controls.Themt-2mRNAlevelwasapproximately1.5-foldhigher intheneonateswithIUGRthaninthecontrolgroup(Fig.2).As aconsequenceoftherelativelyhighindividualfluctuations,the averagesoftheamountsofmt-1,sodsandenosmRNAdidnotdiffer significantly(Fig.2).

3.2. Comparativestudiesofmatureandprematureneonateswith IUGR

TherewasnosigniﬁcantdifferenceinthelevelofH₂O₂orthe activityofCATbetweenthematureandpre-termneonateswith IUGR.However,thematuregroupexhibiteda1.5-foldhigheraccu- mulationofONOO⁻,andalsoahigherTBARSlevel(Fig.3).Thelevels ofONOO⁻ andTBARSforthepre-termIUGRneonateswerewas moresimilartothoseforthenormalcontrols.

2 2.5

Relative level of mRNA

C IUGR

mt-1 mt-2 ho-1 ho-2 sod-2 sod-1 cat enos a

a

aa

aa C

IUGR C IUGR

0 0.5 1 1.5

C IUGR

Fig.2. mRNAexpressionlevelofantioxidantgenesinthecordredbloodcellsofmature,healthyneonates(C)andneonateswithIUGR.Fornormalizationthelevelof18S rRNAwasusedasaninternalstandard.Datawereexpressedasmeans±SEMfrommeasurementson24/28samples.^ap<0.05,^aap<0.01.1.

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mt-1 mt-2 ho-1 ho-2 sod-2 sod-1 cat enos 0

0.5 1 1.5 2 2.5 3 3.5 4.5

Fold of induction

mature IUGR

premature IUGR ^ab

aa aa

b aaa

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mature IUGR

premature IUGR ab

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Fig.4. FoldofchangesinthemRNAlevelsofantioxidantgenesinthecordredbloodcellsofmatureandprematureneonateswithIUGR.Levelsofmature,healthyneonates wereusedasreferences.Significantdifferencewasacceptedatâ,bp≤0.05,âap≤0.01,âaap≤0.001.(a)Asignificantdifferencebetweenthemature,healthyneonatesand neonateswithIUGR.(b)AsignificantdifferencebetweenthematureandprematureneonateswithIUGR.

MarkeddifferenceswerealsoobservedbetweenthemRNAlev- elsofselectedparametersinthegroupsofmatureandpremature IUGRneonates.Therewasa∼60%differenceinthelevelofthemt-1 andmt-2mRNAs.ThemRNAlevelsofbothgeneswereapproxi- matelydoubledinthematureIUGRneonatesascompared with thepre-termIUGRsandalsothenormalcontrols(Fig.4A).Thetran- scriptionproductsofthehogeneswereconsiderablylowerinboth IUGRgroups;inthematureIUGRs,thelevelsofho-1andho-2mRNA were∼50%and40%ofthoseforthecontrol.IntheprematureIUGR group,bothmRNAlevelswerelowerby25%,thoughthesechanges werenotsigniﬁcant.ThelevelofcatmRNAwaslikewiselowerin bothIUGRgroups:asigniﬁcant∼25%decreasesweredetected.The transcriptsofthesodgenesinthematureIUGRgroupwere∼1.5- foldthosemeasuredinthecontrolandprematureIUGRgroups.In theprematureIUGRgroup,theenosmRNAwas∼3-foldthatinthe matureIUGRgroup(Fig.4B).Inthiscase,thematureneonateswith orwithoutIUGRexhibitedsimilarmRNAlevels.

4. Discussion

Since IUGRis postulatedto bea pleiotropic complicationof pregnancy,theidentificationofunitmarkermoleculesorreaction pathwaysconnectedwiththisdisorderposesanumberofdifficul- ties.VariousstudieshavefocusedontheantioxidantstatusofIUGR orprematureneonates,butonlybriefaccountshavebeengivenas concernsthecomparisonofprematureandfull-termneonateswith IUGRfromanyaspect.Themajorfindingofthepresentstudyisthe importanceoftheage-basedgroupingofneonateswithIUGR.Sig- nificantdifferencesinthelevelsofspecificstressmarkersrelative tothecontrolgroupwereobservedonlywhenthefull-termand pre-termIUGRneonatesweregroupedseparately.

Ahigherdegreeofoxidativestresswasdetectedinthecord bloodofmatureIUGRneonatesascomparedwiththecontroland pre-termIUGRgroups;thelevelofONOO⁻wasalmosttwiceas high,indicatingincreasedO2˙⁻andNOproduction.Thesimulta- neousgenerationofNOandO₂˙⁻inrelativelyhighconcentrations inthesamecompartmentfavoursproductionofthetoxicreaction productONOO⁻[17].AlthoughONOO⁻itselfisnotafreeradical,it isapowerfuloxidant,whosetoxicityismanifestedamongothers inLPO[17]andwhichplaysaroleinthepathophysiologyofIUGR [26].

TheelevatedONOO⁻andO₂˙⁻levelsinthecordbloodofmature neonateswithIUGRwereclearlyreﬂectedbyanincreasedlevelof TBARS.NO,however,isaJanus-facedmolecule.Sincetheumbili- calcordbloodvesselslackinnervations,theNOproductioninthe endothelialcellsplaysamajorroleinthelocalcontrolofbloodﬂow andinoxygendeliverytothefoetus[15].Inthisstudy,wehave

demonstratedasignificantlyhigherlevelofthemRNAofeNOSin thecordbloodsamplesofunderdevelopedneonateswithIUGR.In thesecases,theincreaseineNOSexpressionwasnotparalleledby ONOO⁻accumulation,suggestinganunalteredlevelofO2˙⁻.Thisis inaccordwiththefactthattherewerenosignificantdifferencesin theexpressionsofthesodgenes,andtheactivityofSODwasalso unaltered(datanotshown).Additionally,nosignificantchanges wereobservedintheTBARSlevelinthecordbloodofthepremature neonateswithIUGR.Aninsufficientlevelofvasodilatationofthe umbilicalvesselsandreducedeNOSactivitywereearlierreported tobeassociatedwithneonateswhoweresmallforgestationalage [16].

EvidencehasrecentlybeenaccumulatingthatCO,bestknown foritstoxicity,canfunctioninasimilarmannertoNO[27]ormay eveninteractwiththeNO-producingpathway[28].COisformed primarilyasaproductofhemedegradation,whichiscatalysedby theHOs.Theobservationof substantialHOactivityin thecord tissuessupportsthepossibilityoftheaccumulationofCOinsuf- ficientlyhighamountstoplayaroleinfetoplacentalbloodflow regulation[29].Ourstudypresentsevidencethattheexpressions ofthehogenesaremarkedlylowinIUGRneonates(regardlessof thelevelofmaturity)versuscontrols.Thoughthelowexpressions ofbothhogeneswerecharacteristicforbothIUGRgroups,asig- nificantdifferencewasobservedonlyforthefull-termneonates withIUGR.Deficienciesin HO-1 havepreviously beenfoundto beassociatedwithpregnancydisorders,suchasrecurrentmiscar- riages,IUGRandpre-eclampsia[30].Ourresultssupportthis:the lowerexpressionofhoinIUGRneonatesmightresultinadecreased COlevelandunsatisfactoryfetoplacentalbloodflow.Moreoverour datarelatingtotheselectedantioxidantmoleculessuggestthatho couldserveasanindicativemarkerprovidingapossiblelinktothe IUGRphenotype.

ThepresenceofIUGRinfull-termneonateswasaccompaniedby significantlyhigherlevelsofexpressionofbothmtgenes.MT,an importantproteinwhichbindsbivalentmetals,playsasignificant roleinnumerous cellularmetabolicprocesses,suchasinmain- tainingZnandCuhomeostasisandin CdandHgdetoxification.

Additionally,anincreasedMTlevelwasdemonstratedbyZapata etal.in theerythrocytesofpregnantwomen[31].ElevatedMT levelshavebeenattributedtotheproliferationanddifferentiation ofbloodcellsintheprocessoferythropoiesis,theprotectiverole oferythrocytesagainsttheactionoffreeradicals,andincreased levelsofestrogenandprogesterone,hormoneswhichinducethe synthesisofMT[32].Astatisticallysigniﬁcantincreaseinthelevel ofMTproteinhasadditionallybeenrevealedin theplasmaand RBCs of pregnantwomen and of theirneonateswith IUGR[4].

ThepossiblephysiologicalfunctionsofMTintheplacentainclude

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temporaryZnstorageandregulationoftheZnﬂowtothefoetus, whilerestrictingtoxicmetaltransfer[33]andprotectionagainst theembryotoxic andteratogeniceffects ofa Zndeﬁciency [34], whichmaybecausedbycigarettesmoke,alcohol,gestationalinfec- tionandexposuretoenvironmentalcontaminants,includingheavy metalsandendocrinedisrupters[35].

Froman analysisof theparameters relating tothedifferent IUGRgroups,wecanconcludethatfull-termneonateswithIUGR areatespeciallyhighrisk.Pre-termneonates,atgestationalages of33–36weeksdemonstratelessdamageintheintegrityofthe lipidmolecules,asigniﬁcantaccumulationoftheharmfuloxidant ONOO⁻cannotbedetected,andtheexpressionsofgenescodingfor antioxidantmarkersarelessaffected.ThesigniﬁcantlylowerLPOin theprematureneonatesmaybecorrelatedwiththedecreasedsen- sitivityoftheRBCstooxygenradicals,orwiththefactthattheRBCs ofprematurenewbornscontainasmallerquantityofunsaturated fattyacids[36].Thisphenotypeisnotwithoutprecedent.TheGSH andNADPHlevelsprovedtobelowerinthosewithgestationalages oflessthan33weeksthaninpre-terminfantswithgestationalages of33–36weeks,andthelevelsofthesemarkerswereaslowinfull- term,small-for-ageinfantsasinpre-terminfantswithgestational agesoflessthan33weeks[7].

5. Conclusions

Ourdatafurnishevidencethatthereisextremeheterogeneityin ROSproductionandintheactivationoftheantioxidantdefencesys- temwithinneonateswithIUGR,whichisbluntedbytheuseofmean measurements.Intergroupingbasedongestationalageandbirth- weight,yieldedevidencethatthemolecularresultsinthepre-term IUGRgroupwereoftensimilartothoseforthemature,appropriate- for-ageneonates.However,thebackgroundoftheIUGRphenotype ispleiotropic,andmeasurementsofvariousantioxidantsprovide onlyapartiallyadequatepictureoftheoverallconditionofthe neonates.Data emergingfromdifferentapproaches tothetopic present evidenceonly of theinvolvement of additionalmarker moleculesinthedevelopmentofIUGRdisorders.Ourstudyindi- catesthatthelevelsofexpressionofthemtandhogenesaregood candidatesthroughwhichtocharacterizetheIUGRphenotype.

Conﬂictofintereststatement

Theauthorsdeclarethattherearenoconﬂictsofinterest.

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