www.jped.com.br
ORIGINAL
ARTICLE
Nuclear
abnormalities
in
cells
from
nasal
epithelium:
a
promising
assay
to
evaluate
DNA
damage
related
to
air
pollution
in
infants
夽
,
夽夽
Michelle
Mergener
a,∗,
Cláudia
R.
Rhoden
b,
Sérgio
L.
Amantéa
baUniversidadeFederaldeCiênciasdaSaúdedePortoAlegre(UFCSPA),PortoAlegre,RS,Brazil bUniversidadeFederaldoRioGrandedoSul(UFRGS),PortoAlegre,RS,Brazil
Received13January2014;accepted3April2014 Availableonline18July2014
KEYWORDS
Micronucleiassay; Nasalcells; Environmental health; Infants; Toddlers
Abstract
Objectives: Thisstudyintendstoprovideaquick,easy,andinexpensivewaytoassessnuclear
abnormalitiessuchasmicronucleiandbudfrequencies;binucleated,karyorrhectic,karyolytic,
pycnotic,andcondensedchromatincellsinnasalscrapingsofinfants,whichareparticularly
important for conducting genotoxic studies related to the inhaledatmosphere in pediatric
populations.
Methods: Nasalswabsampleswerecollectedfrom40infantsunder12monthsofageusinga
smallcytobrush.2,000cellsfromeachinfantsamplewereanalyzedandclassifiedaccordingto
thefrequencyofnuclearabnormalities.
Results: Ratesofnuclearabnormalitiesfoundagreewithvaluesreportedinotherstudiesof
neonatesandchildren.Thisstudyfound0.13%ofcellswithmicronuclei;1.20%karyorrhexis;
0.03% pyknosis; 10.85%karyolysis; 1.11% condensed chromatin; 0.54binucleated cells;and
0.02%nuclearbud.Differenceswerenotobservedbetweengendersorenvironmentalpassive
smoking,norwasanyagecorrelationfound.
Conclusion: Theassayproposedhereissuitableforassessingthefrequencyofnuclear
abnor-malitiesfromnasalcellsininfants.
©2014SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.Allrightsreserved.
夽 Pleasecitethisarticleas:MergenerM,RhodenCR,AmantéaSL.Nuclearabnormalitiesincellsfromnasalepithelium:apromisingassay
toevaluateDNAdamagerelatedtoairpollutionininfants.JPediatr(RioJ).2014;90:632---6. 夽夽
StudyconductedatUniversidadeFederaldeCiênciasdaSaúdedePortoAlegre(UFCSPA),PortoAlegre,RS,Brasil. ∗Correspondingauthor.
E-mail:michimerg@yahoo.com(M.Mergener).
http://dx.doi.org/10.1016/j.jped.2014.04.009
PALAVRAS-CHAVE
Ensaiode micronúcleos; Célulasnasais; Saúdeambiental; Neonatos;
Crianc¸asdeumatrês anos
Anormalidadesnuclearesemcélulasdoepitélionasal:umatécnicapromissorapara avaliardanonoDNArelacionadoàpoluicãoatmosféricaemneonatos
Resumo
Objetivos: Este estudo pretendeu fornecer uma forma rápida, fácil e barata de avaliar
alterac¸õesnucleares,comofrequênciasdemicronúcleosebrotosnucleares,células
binucle-adas,núcleoscariorréticos,cariolíticos,picnóticosecomcromatinacondensada,emesfregac¸os
nasaisdeneonatos,particularmenteimportanteparaarealizac¸ãodeestudosgenotóxicos
rela-cionadoscomapoluic¸ãoambientalempopulac¸õespediátricas.
Métodos: Foramcoletadasamostrasdeesfregac¸onasalde40neonatoscommenosde12meses
deidade,utilizandoumapequenaescovacitológica.Foramanalisadas2.000célulasdaamostra
decadaneonatoeclassificadasdeacordocomafrequênciadeanormalidadesnucleares.
Resultados: Astaxas deanormalidadesnuclearesencontradasnesteestudo saocompatíveis
comos valores relatadosem outrosestudosde neonatose crianc¸as.Encontramos 0,13%de
células commicronúcleos,1,20%comcariorrexe, 0,03%compicnose, 10,85%comcariolise,
1,11%comcromatinacondensada,0,54comcélulasbinucleadase0,02%decélulascombrotos
nucleares. Nãoobservámos diferenc¸as entreos gênerose otabagismopassivo, assim como
nenhumacorrelac¸ãoentreidades.
Conclusão: Oensaiopropostonesteestudoéadequadoparaavaliarafrequênciade
anormali-dadesnuclearesdecélulasnasaisemneonatos.
©2014SociedadeBrasileiradePediatria.PublicadoporElsevierEditoraLtda.Todososdireitos
reservados.
Introduction
Studyingtheeffectsofenvironmentalimpactonexposures intendstoquantifytheagentspresentintheenvironment, toinvestigatetheireffectonchronicdiseaseoccurrencein exposedindividuals,andtoclarifythemanymechanismsby whichtheseprocessesoccur.Inthisregard,therehasbeen anincreasingeffortworldwidetodeterminetheimpactof environmental, genetic,and life-stylefactors ongenomic stability.
Both in adults and children, evaluation indexes that reflect DNAdamage, suchasthe presence of micronuclei (MN), have shown to be a useful tool that can monitor changesoverincreasingaccidentalexposures,bothrelated toenvironmentalandlifestylefactors.Specialfocuson chil-drenorinfants isimportant,sincetheymayhaveahigher sensitivity togenotoxic agents when compared toadults. Furthermore,earlyagegeneticdamagemayaffectthe life-timeriskofadversehealthoutcomes.Becauseofthat,the micronucleusassaymethodhasbeen widelyusedtostudy genome damage in children afterin utero andpost-natal exposuresinavarietyofruralandurbanenvironmental sett-ings,resultingfrommaternalsmokingaswellasaccidental industrialortechnologicaloverexposures.1---3
Through a survey of the current literature, it was observedthatthefrequencyofnuclearabnormalitiesisvery lowatbirth.However,incasesofexposure,theincreaseis muchmorepronouncedwhencomparedtoadults.This cor-roboratestheideathatthereisagreatersensitivityamong exposedchildren.Thestudiesshowedsignificantincreases inthefrequencyofMNandothernuclearabnormalitiesin casesofchildrenexposedtotobaccosmokeindoors,living nearchemicaldeposits,orexposedtoarsenic-contaminated waterandheavymetals,inadditiontoindustrialpollutants.
Despitethis,thelong-termactionofcertainchronicdiseases andstrongtreatmentslikechemotherapyhasnotyetbeen conceptualized.
TheuseofMNasameasureofchromosomedamagewas firstproposedbyCountrymanandHeddle(1976)4in
periph-eralbloodlymphocytes,anditisthemethodofchoicefor evaluationofgenotoxicityinhumanpopulations.5However,
mostotherinvestigatorswereinterestedinanalysesof exfo-liatedcellsfrombuccalornasalmucosaeinordertogather dataforsurveysofoccupationalexposure.6Theauthors
sug-gestedthesuitabilityofthesecollectionsitesbecausethey areinclosecontactwithharmfulenvironmentalagentsand canbecollectednon-invasively,whichisparticularly impor-tantwhenconductingpediatricstudies.Inexfoliatedcells, thepresence of MN indicates extra-nuclearcytoplasmatic bodiesassociatedwithchromosomalaberrations.Theseare inducedbyavarietyof substances,includingcarcinogenic compoundspresentintobaccosmoke.Theinductionbycells withMNofcarcinogensandmutagensisasignofthe geno-toxiceffectofsuchsubstances.7
Throughthemicronucleusassayinexfoliatedcells,itis alsopossibletoevaluatesomenuclearabnormalitiessuchas breakageorlossofgeneticmaterialbymeasuringthe pres-enceofMNornuclearbuds(alsoknownas‘‘brokeneggs’’), cytokinesisdefectsrepresentedbybinucleatecells,andcell deathwhenpresentingverysmallnuclei(pyknosis)or con-densedchromatin,orevencompletelylosingtheirnuclear materials(knownaskaryorrhecticandkaryolyticcells).8
buccalcellsand/orperipheralbloodlymphocytesinspecific situationsofexposure,suchasionizingradiation,9biomass
burning,10 or in cases of Down syndrome.11 Others used
bloodcollectioninordertoassessDNAdamageinhealthy children.12
Arecentstudyfoundnoincreaseofanomaliesthatreflect chromosomaldamage(MN,binucleates,nuclearbuds),but significantly higher rates of nuclear aberrations that are indicative for cytotoxicity (karyolysis, karyorrhexis, con-densed chromatin) were observed in the workers. These effectsweremorepronouncedinnasalcellsthaninbuccal cells.13
Presently, the few existing studies have shown incon-clusive results in cytogenetic toxicity in humans due to different exposure levels, misleading factors, and proto-coldifferences amonglaboratories. Thus, considering the importanceofevaluatingtheeffectsoftheenvironmenton thehealth, focusingmainlyonair pollutionin veryyoung children,andalsotheneedtostandardizeafeasiblemethod for researching the presence of nuclear abnormalities in exfoliatedcells,thisstudyaimedtopresentaquick,easy, andinexpensivewaytoassessnuclearabnormalitiesinnasal scrapingsofinfants.
Methods
AfterapprovalbytheEthicsCommittee(CEP/UFCSPA),nasal swab samples were collected from 43 infants under 12 monthsofage whowerehospitalizedfor other conditions not affecting the airways. Children with underlying asso-ciated diseases were excluded, such as congenital heart disease, immunodeficiency, chronic lung disease, neuro-muscular diseases, cytogenetic syndromes, or those who requiredmechanicalventilation.
Aquestionnairewasappliedtotheparentswho accom-panied the infant at the time of sample collection. The questions were developed based on the ‘‘Global Adult Tobacco Survey (GATS): Core Questionnairewith Optional Questions’’,14 created by the World Health Organization
(WHO),validatedin2010,andwithchangesrelatedto preg-nancyandinfantexposuretotobaccosmoke.
The toolchosenfor collectingthesamples wasa small cytobrush,commerciallymanufacturedfordentalpractices. Itwasusedspecificallybecauseofitsnylonbristles (impor-tantforbreakingoffcellseasily)andsuitablesizeforbeing insertedinthenostrilsofsuchsmallchildren,andalsotofit entirelyinamicrotube.Thecellcollectionwasperformed bypediatricresidents,underpreceptordoctorsupervision. The brusheswereinserted in thenostrils ofinfants up to themiddleturbinate, sothatthebristlesfacingthenasal septumwerebentandthebristlesontheothersidecame incontactwiththewingofthenose.Inthisposition,they wererotatedinthenasalcavityinordertoobtainthelargest possibleamountofmaterialcellsfromtheepithelium.Later, thecytobrusheswerestoredinmicrotubescontaining1.0mL ofsalinesolution(0.08NaCl)andidentifiedaccordingly.The materials werekept refrigerated(4◦C)for a maximum of 12hours.
In thelaboratory, themicrotubes were centrifugedfor 10minutesat1,500rotationsperminute(RPM),followedby threewashesofthepelletswith1.5mLofCarnoy’sfixative
solution(methanol3:1aceticacid),alwayssuspendingthe material.Inthelastwashing,thesuspensionofthepellet wasperformed with only1.0mLof Carnoy’s fixative solu-tioninordertofurtherconcentratethecells.Attheendof theprocess,threeslidesweremadeforeachpatient,each withapproximately0.3mLofthecellsuspensionmaterial. The slideswere pre-washedand stored in 70% ethanol in ordertoobtainbetteradhesion.Wheninuse,theslideswere washedagainwithtapwaterandthenwithdistilledwater. Whiletheywerestillwetwithdistilledwater,0.3mLofthe solutioncontainingcellsinsuspensionwasdrippedontothe slides.Then,theywereallowedtodryatroomtemperature and,later,theywerestainedwitha10%Giemsasolutionin phosphatebuffered saline(PBS)pH 7.0.Finally,theslides wererinsedintapwaterandair-dried.
Theslideanalyseswereperformedwithanoptical micro-scopeat400Xand1,000Xmagnificationwiththeadditionof mineraloil.Onlybasalanddifferentiatedcellswere consid-eredintheanalysis.Twothousandbasalanddifferentiated cellsfromeachinfantsamplewereanalyzedandclassified according to the frequency of micronuclei,nuclear buds, binucleated, pycnotic, karyorrhectic and karyolytic cells, andcondensedchromatin,followingtheevaluationcriteria suggestedbyKnasmuelleretal.8andThomasetal.15
Thestudiedvariabledepartedsignificantlyfrom normal-ityandthereforethenon-parametricMann---WhitneyU-test wasappliedtodata.The associations betweentwo varia-bles wereanalyzed bySpearman correlation.The level of significancewasconsidered asp ≤0.05.Allanalyses were conductedusingtheSPSSforWindows(IBMCorp,Armonk, USA),version17.0.
Results
The age ofthe infants ranged from14daysto12 months (mean4.28±3.14months).28(70.0%)weremales.No reac-tionsofinfantstotheprocedureswereobserved.Nocases ofirritationwereobserved.Discomfortatthetimeof collec-tionoccurredduetotheinfant’sfrightand,inmostcases, crying.
The mean frequency of nuclear abnormalities is pre-sented in Table 1. Only three slides were ruled out for theirlowcellularity(<2,000;thus,theanalysisconsidered 40 infants. There was no significant correlation between nuclearabnormalitiesandage(inmonths)(Spearman’s cor-relation),nordifferencesbetweengenders(Mann-Whitney U-test).
Regardingexposuretopassivesmoking,accordingtothe questionnaire data, only 11 mothers indicated cigarette smoking before and after pregnancy, including nine who admitted smoking during pregnancy. In the family room, 20mothersadmittedthattherewereotherhousehold resi-dentswhosmoke.Statisticalanalysis(Mann-WhitneyU-test) alsoshowednodifferencesonnuclearabnormalitiesamong infantswhowerepassivelyexposedtocigarettesmokeand thosewhowerenot.
Discussion
Table1 Frequencyofnuclearabnormalitiesfrominfantnasalcells.
Nuclearabnormalities Frequency/2,000cells Standarddeviation Percentage
Micronuclei 2.70 1.53 0.13%
Karyorrhexis 24.02 10.36 1.20%
Pycnotic 0.65 0.83 0.03%
Karyolytic 217.10 64.41 10.85%
Condensedchromatin 22.27 11.28 1.11%
Binucleated 10.95 3.63 0.54%
Bud 0.40 0.74 0.02%
andthebuccalepithelium.Reticulocytesornasalcellsare rarelyused.1Instead,thisstudyassessedexfoliativecells,
sincetheyhavebeenmoreutilizedduetothenon-invasive collectionmethods and tothe fact that thefrequency of nuclei abnormalities directly reflects the damage rate in thetargettissues.15,16 Thenasalcellsanalysisthroughthe
micronucleusassaypresented itself asadequate oncethe visualizationofcompletecellsbecamepossible,capableof reflectingnuclearalterations.
In the present study, nuclear abnormalities were eas-ily observed in cells from smears stained with the 10% GiemsasolutioninPBSpH7.0.However,bacteriaandcell debris are misleading factors that may mask the effect of the micronucleior buds ascomparedtoother staining testslikePapanicolau’s, Feulgen’s,or Wright’sstain.6,17---19
Nevertheless, this difficulty can be resolved using micro-scope resources. The bacteria stained with Giemsa and observedthroughthemicroscopelightdifferfrom micronu-clei or buds becausethey look brighter,are smaller, and aregroupedtogetherwitha strongercolor. Likewise, cell debris can bedifferentiated from micronucleior budsby addingPBS,which providesaneutralpHandalsobecause thedebrisreactsdifferentlywhenstainedcomparedtothe mainnucleus.
Nuclear abnormality frequency was analyzed in 2,000 cells similar to other studies, which have analyzed 2,000 cells ormore.19 Only threeslideswereruled outfor their
lowcellularity(<2,000).Despitethelowernumberofcells, inthepresentstudyitwaspossibletoassessthepresence ofnuclearabnormalitiesincellsfromthenasalcavitiesof infants,which aresmaller thanthoseofadultindividuals, andalsoaremoredifficult tocollectsincethe brushmay beuncomfortableforthechildrenandtheymayhavetobe controlledbytheirparents.
Many other studies employed micronucleus assay in child populations using invasive methods such as blood samples,1,20,21orinnewbornsusingumbilicalcordblood.22
Despite using Giemsa as stain, the findings presented here correspondwithothersinthe literature,considering comparisonswitholderchildrenwhoalreadyweremore sig-nificantlyexposedtoenvironmentalfactors,becausenone of thecurrent studieslinkedthe presenceof MNor other nuclearabnormalitiesinthefirstyearoflifewith environ-mentalfactors.
At the present time, it is difficult to define the real biological significance of nuclei abnormality frequency in infants.Comparisonsregardinggenderonlyemphasize the lackofdifferencesbetweenmalesandfemales,especially amongchildren soyoung andnot yet suffering theaction
of hormonal factors. However, their predictive value for diseaserequiresstudiescombiningdatafromdifferent expo-sure,such astheenvironmental factorsof the infantand themother’slifestyle.1Therefore,thefrequencyofnuclei
abnormalitiesinnasalcellsfrominfantswasnotassociated withsmokinghabitsofsame-residenceinhabitants.
Therearenostudiesofnasalcellsfrominfants,although ratesofnuclearabnormalitiesfoundhereagreewithvalues reportedinotherstudies19 ofneonatesandchildren(0.13%
micronuclei; 1.20% karyorrhexis; 0.03% pyknosis; 10.85% karyolysis; 1.11% condensed chromatin; 0.54 binucleated cells;0.02%nuclearbud).Evaluatinglymphocytesand buc-calcells,otherauthorssuggestthatmicronucleifrequency waslowatbirthandincreasedinchildren1-4yearsofage.23
Accordingtoliteraturedata,thereisstillcontroversyover the baseline frequency of micronuclei and other nuclear abnormalities,anditislessclearwhenevaluatedin exfoli-atedepithelialcells,mainlyinregardtoageandgender.24
The meanmicronucleilevelsin exfoliativebuccal cells reportedbytwootherstudies25,26in0-to6-year-oldchildren
differedbymorethan0.2-fold,suggestingamajorimpact ofenvironmental factorsandtechnicalvariability on buc-calcellmicronucleifrequencyinhumanstudies.Inanother studythatinvolvedlymphocytesandbuccalcellsofchildren andtheirmothers,nostatisticallysignificantdifferenceby agewasobserved,duetoabroadrangeofinter-individual variability.15
Atmosphericairpollutioneffectsappeartobeespecially significantinchildren,whoaremoresensitivethan adults because their bodies are still in a development stage.27
Interestingly,inastudy withchildrenofvariousages,the strongest effectofair pollution onmicronucleifrequency wasobserved in the youngest individuals.28 This
observa-tionisconsistentwiththepotentiallygreatersensitivityof childrentoenvironmentalexposures.29
Thisisthefirstinfantbiomonitoringstudytoidentifyand quantifynucleiabnormalitiesinnasalcellsfrominfants.The resultspresentedheremaybeprecursorstonew perspec-tivesin understanding the environmental effects in nasal cells,andeffectsrelatedtorespiratorydiseasesinthe pedi-atricpopulation.Althoughthisstudydidnotfinddifferences inthefrequencyofnuclearabnormalitiesamonginfantswho livedwithexposure tocigarette smoke,the micronucleus assayofnasal cells wasalsoabletodetect other nuclear abnormalities.
changesintheelucidationofdiseaseprocessesand popula-tionmonitoring,providingbetterhealthconditionsforboth infantsandtheelderly.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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