Rev. Int. Contam. Ambient.

14 (12) 111114, 1998

SEGUNDOTALLERSOBRESMART:UNMTODOPARADETECTARLASACTIVIDADESMUTAGNICAY
RECOMBINOGNICAENCLULASSOMTICASDE DrosophilaENLAUNIVERSIDADFEDERALDE
UBERLANDIA(MG),BRASIL

SECONDWORKSHOPATTHEFEDERALUNIVERSITYOFUBERLANDIAUBERLANDIA(MG)(Brazil):
SMART:AMETHODFORTHEDETECTIONOFMUTAGENICANDRECOMBINAGENICACTIVITYIN
SOMATICCELLSOF Drosophila

MarioA.Span1 yUlrichGraf2

1
DepartmentodeGenticayBioqumica,UniversidadFederaldeUberlndia,CampusUmuarama,38400902Uberlndia,MG,Brasil
2
InstitutodeToxicologa,SwissFederalInstituteofTechnology(ETH)Zurich,CH8603Schwerzenbach,Switzerland

Lamoscadelafruta, Drososphilamelanogaster ,ofreceuna The fruit flyDrosophila melanogaster offers a series of

serie de ventajas como sistema de prueba eucaritico de
genotoxicidad invivo.Esfcildecultivar,suciclodegeneracin
es corto y los ensayos somticos son baratos porque slo se
requieredeequipodelaboratoriobsicoydebajocosto.Ade
ms de los sistemas de prueba bien establecidos y validados
que utilizan clulas germinales, especialmente el de letales
recesivosligadosalsexo,sehandesarrolladootroscomoelde
lasclulasdelojoydelaladelosdiscosimagales.Estaspruebas
demutacinyrecombinacinsomticas(SMART)sonensayos
deunasolageneracinyutilizanmarcadoresrecesivosquese
expresanenlasuperficiedelosojosodelasalasdelasmoscas
adultas.Lainduccindelaprdidadeheterocigosisdemarca
doresenlasclulasdelosdiscosimagalesdelaslarvasmedian
tetratamientoconcompuestosgenotxicos,conducealaforma
cindeunclondeclulasmutantesquedespusdelametamor
fosisseexpresancomomanchasenlosojosoenlasalas.La
principalventajadeestossistemasdepruebasomticosreside
enelhechodequenoslodetectanvariostiposdeeventosde
mutacin, sino que bsicamente inducen la recombinacin
mittica.Lapruebadelamanchadelalaofrecelaventajadeque
en cada experimeto es posible cuantificar la actividad
recombinognicacontralamutagnica(Frei etal.1992).Ade
msdelacruzaestndarenlapruebaSMARTdelala,esposible
realizarunacruzadebioactivacinelevada(BE).Laslarvasde
staltimasecaracterizanporunelevadonivelconstitutivode
citocromosP450,loquefacilitaladeteccindepromutgenosy
procancergenos(GrafyVanSchaik1992).GuzmnRincny
Graf(1995)yGraf etal.(1996)proporcionanmsdetallesacerca
del uso de la prueba del ala. Los ensayos somticos de
genotoxicidadseencuentranvalidadosconungrannmerode
agentesqumicosymezclascomplejas(msde400)(Graf etal.
1998a,Vogeletal.1998)yademsestnbienadaptadospara
estudiosde antigenotoxicidad(Grafetal. 1998a).La prueba
SMARTdelalade Drosophila seutilizaenunnmerocadavez
mayordelaboratorios,especialmenteenAmricaLatina.
ConelfindeintroduciraBrasillosensayossomticosde
Drosophila y para ensear las tcnicas bsicas, en 1991 se
realizuncursodeentrenamientoenlaFacultaddeFilosofa,
CienciasyLetrasdelaUniversidaddeSaoPaulo,enRibeiro
advantages for its use as an eukaryotic in vivo genotoxicity
assaysystem.Itiseasytoculture,hasashortgenerationtime,
andthesomaticassaysareinexpensivebecausetheyonlyneed
basiclaboratoryequipmentandlowcostmaterial.Inadditionto
thewellestablishedandvalidatedtestsystemsthatusegerm
cells,primarilythetestforsexlinkedrecessivelethals,newassays
weredevelopedthatusesomatictissues,i.e.cellsoftheeyeor
wing imaginal disks. These Somatic Mutation And
RecombinationTests(SMART)areonegenerationassayswhich
makeuseofappropriaterecessivemarkersthatareexpressedon
thesurfaceoftheeyesorthewingsoftheadultfly.Inductionof
lossofheterozygosityofthesemarkersinimaginaldiskcellsof
thelarvabytreatmentwithagenotoxiccompoundleadstothe
formationofacloneofmutantcellswhichwillthenexpressafter
metamorphosisasamutantspotontheeyeorthewing.The
mainadvantageofthesesomatictestsystemsliesinthefactthat
they detect not only various types of mutational events but
primarilyinducedmitoticrecombination.Thewingspottestoffers
theadvantagethatthequantificationofrecombinagenicactivity
versusmutagenic activityispossiblewithin eachexperiment
(Freietal.1992). Furthermore,in thewing SMARTa High
Bioactivation(HB)crossisavailableinadditiontothestandard
cross.ThelarvaeofthisHBcrossarecharacterisedbyahigh
constitutive level of cytochromes P450 which facilitates the
detection of promutagens and procarcinogens (Graf and van
Schaik1992).Moredetailsontheuseofthewingspottestare
giveninGuzmnRincnandGraf(1995)andGraf etal. (1996).
The somatic genotoxicity assays in Drosophila are now well
validatedwithalargenumberofchemicalcompoundsorcomplex
mixturestested(morethan400)(Graf etal. 1998aVogel etal.
1998).Furthermore,theassaysare wellsuitedforstudieson
antigenotoxicity (Graf et al. 1998a). The wing SMART of
Drosophilaisbeingusedinanincreasingnumberoflaboratories,
especiallyinLatinAmerica.
InordertointroducethesomaticassaysofDrosophilaand
toteachitsbasictechniquesinBrazil,afirsttrainingcoursewas
heldin1991attheFacultyofPhilosophy,SciencesandLetters,
University of So Paulo, in Ribeiro Preto (SP), which was
organisedbyC.S.TakahashiandpresentedbyU.Graf.Similar
112
Preto(SP).DichocursofueorganizadoporC.S.Takahashie
impartidoporU.Graf.EnMxicotambinserealizaroneven
tossimilares(Graf etal.1994).En1994,M.A.Spanpreparun
tallerdetresdasenelDepartamentodeBiocienciasdelaUni
versidadFederaldeUberlndia,enUberlndia(MG)queigual
mentefuedadoporU.Graf.DuranteelQuintoCongresoLatino
americanodeMutagnesis,CarcinognesisyTeratognesisAm
bientalesyelCursoAlexanderHollaender,realizadosenCuritiba
(PR),Brasil,del15al20denoviembrede1998,sellevacaboun
simposiotitulado Drosophila comoorganismocentinelapara
mutagnesisconlaparticipacindeH.H.deAndrade(Porto
Alegre,RS,Brasil),M.A.Span(UberlndiaM.G.,Brasil),J.
GuzmnRincn(Mxico,D.F.,Mxico)(GuzmnRincn etal.
1998)yU.Graf(Zurich,Suiza)(Graf etal.1998b).Altrminodel
Congresoserealiz,del25al26denoviembrede1998enel
DepartamentodeGenticayBioqumicadelaUniversidadFede
raldeUberlndia,elsegundotallersobre SMART:unmtodo
paraladeteccindeactividadesmutagnicayrecombinognica
enclulassomticasde Drosophila .Estareuninfueorganiza
daporM.A.SpanconelapoyodelaCoordinacindelCurso
deGenticayBioqumicaparaPosgraduadosdelCentrodeCien
ciasBiomdicasdelaUniversidadFederaldeUberlndia,as
comodelaSociedadBrasileadeMutagnesis,Carcinognesis
yTeratognesisAmbientales.Secontconlapresenciade30
estudiantes,graduadosyposgraduadosycientficosdelasuni
versidadesdeUberlndia(MG)yGoinia(GO).
LamaanadelprimerdaelTallerseinaugurconunaalegre
ceremoniaconacompaamientomusicalalaqueasistieronlos
profesoresW.E.KerryM.A.BrandenburgodelDepartamento
deGenticayBioqumica.Despusdelaceremonia,U.Grafpre
sentlaconferenciadeaperturasobreElusodeDrosophila
paraladeteccindeactividadesgenotxicas.Lasegundacon
ferenciafue deM.A. SpansobreLaprueba demutacin y
recombinacinsomticas(SMART):unanuevametodologapara
ladeteccindelasactividadesmutagnicayrecombinognica
enDrosophilamelanogaster .
Enlatarde,enelLaboratoriodeCitogenticayMutagnesis
delaUniversidadFederaldeUberlndia,tuvolugarunasesin
deentrenamientoprcticopreparadaporM.A.Spanysusco
laboradoresydirigidaporM.A.SpanyU.Graf.Duranteesta
sesinlosparticipantessefamiliarizaroncon:(1)elmanejoyel
cultivodelascepasutilizadasenlapruebadelamanchadelala,
(2)losfenotiposdelascepasconlosmarcadoresmwh(pelos
mltiplesdelala )y flr3 (flama ),(3)lacosechadelaslarvasdel
medioespecialdelevadura,(4)lasmedidasdeprecaucinparael
manejodesustanciasmutagnicasycarcinognicas,(5)laapli
cacindeloscompuestosaprobarpormediodelaalimentacin
delaslarvasconmedioinstntaneopara Drosophila oconpur
depapas,(6)lacolectadelasmoscasadultasdelosvialesde
tratamientoparaalmacenarlasenetanolal70%,(7)elmontajede
lasalasenlosportaobjetos,(8)elregistrodelaaparicinde
manchassencillas(delosfenotiposmwhflr )ydemanchas
dobles(clon mwh adyacentealclon flr )enlasalasy(9)elproce
samientodedatosysuevaluacin.Estasesinintensadeentre
namientoprcticojuntoconfructferosperiodosdediscusin
M.A. Span y U. Graf
trainingcourseswerealsoheldinMxico(Graf etal. 1994).In
theyear 1994,a threedayworkshop wasorganised byM.A.
SpanattheDepartmentofBiosciences,FederalUniversityof
Uberlndia,Uberlndia(MG),whichwasalsogivenbyU.Graf.
The Fifth LatinAmerican Congress of Environmental
Mutagenicity,CarcinogenicityandTeratogenicityandAlexander
HollaenderCoursetookplaceinCuritiba(PR),Brazil,from15to
20November1998.Duringthisevent,asymposiumwasheldon
Drosophilaasasentinelorganisminmutagenesiswiththe
participationofH.H.deAndrade(PortoAlegre,RS,Brazil),M.A.
Span(Uberlndia,MG,Brazil),J.GuzmnRincn(Mxico,D.F.,
Mxico)(GuzmnRincn etal.1998),andU.Graf(Zurich,
Switzerland)(Graf etal. 1998b).AfterthisCongress,aSecond
WorkshoponSMART:Amethodforthedetectionofmutagenic
andrecombinagenicactivityinsomaticcellsofDrosophilatook
placeon25and26November1998attheDepartmentofGenetics
and Biochemistry of the Federal University of Uberlndia,
Uberlndia(MG).ThisWorkshopwasorganisedbyM.A.Span
andreceivedsupportbytheCoordinationofthePostGraduate
CourseinGeneticsandBiochemistry,BiomedicalSciencesCen
tre, Federal University of Uberlndia. Further support was
providedbytheBrazilianSocietyforEnvironmentalMutagenesis,
CarcinogenesisandTeratogenesis.ThisWorkshopwasattended
by30graduateandpostgraduatestudentsandscientistsfrom
theUniversitiesofUberlndia(MG)andGoinia(GO).
InthemorningofthefirstdayoftheWorkshop,afestive
openingceremonywithmusicalaccompanimenttookplacein
thepresenceof Profs.W.E.Kerrand M.A.M.Brandeburgo,
DepartmentofGeneticsandBiochemistry.Thiswasfollowedby
anopeninglecturegivenbyU.GrafonTheuseofDrosophila
forthedetectionofgenotoxicactivity.Thesecondlecturewas
presented by M.A. Span on Somatic Mutation And
RecombinationTest (SMART):A novel methodology for the
detection of mutagenic and recombinagenic activity in
Drosophilamelanogaster .
Intheafternoon,apracticaltrainingsessiontookplaceinthe
LaboratoryofCytogeneticsandMutagenesis,FederalUniversity
of Uberlndia, which was organised by M.A. Span and his
collaboratorsandpresentedbyM.A.SpanandU.Graf.During
thissession,alltheparticipantsweremadefamiliarwith(1)han
dlingandculturingofthestrainsusedinthewingspottest,(2)
phenotypesofthemarkerstrains mwh (multiplewinghairs)and
flr3(flare),(3)collectionoflarvaeonspecialyeastmedium,(4)
safety precautions for the handling of mutagenic and
carcinogenicsubstances,(5)applicationoftestcompoundsby
feedingoflarvaewithDrosophilaInstantMediumorwithmashed
potatoes,(6)collectionofadultfliesfromtreatmentvialsfor
storingin70%ethanol,(7)mountingofwingsonmicroscopic
slides,(8)scoringofwingsfortheoccurrenceofsinglespots
(either mwh or flr phenotype)andtwinspots(mwh cloneadjacent
to flr clone),(9)dataprocessingandevaluation.Thisintensive
practical training session together with fruitful discussion
periodsprovedtobeverystimulatingandsuccessfulforallthe
persons involved.
InthemorningoftheseconddayoftheWorkshop,seven
 SEGUNDO TALLER SOBRE SMART
fueronmuyestimulantesyprovechososparatodoslospartici
pantes.
EnlamaanadelsegundodadelTallerserealizaronsiete
presentaciones orales sobre los avances de proyectos de las
universidadesdeUberlndia(UFU)yGoinia(UFG).N.Coelho
de Sousa present el trabajo Evaluacin de la actividad
mutagncia y recombinognica del barbatimo (Stryphno
dendronadstringensMart)(SousayCarvalho1997).Lase
gundapresentacin,acargodeE.AraujoRocha(UFG),fueEva
luacin del potencial genotxico de extractos de Hyptis
suaveolens y la tercera, de S. de Carvalho (UFG), Efectos
genotxicos del tordon. Despus del receso para el caf, B.
LassmarBuenoValadares(UFU)presentelexcelentetrabajo
Posiblesefectosantigenotxicosdelpropleoextradodepa
nalesde Apismellifera (ValadaresySpan1998).Laspresenta
ciones subsecuentes fueron sobre la actividad reguladora de
dos compuestos qumicos. E.J. Fragiorge habl de Efectos
moduladoresdelavitaminaCencombinacinconlaadriamicina
(Fragiorge etal.1998)yE.PeresRodrigues(UFU)deEfectos
moduladoresdelamelatoninasobrelaactividadgenotxicade
losrayosgamma(Rodrigues etal.1998).Laltimapresenta
cindeestasesinestuvoacargodeJ.C.Nepomucenoyfue
sobrelosavancesdelproyectoEfectosprotectoresde Moringa
oleifera sobrelaactividadgenotxicadelamitomicinaCydel
uretano(Nepomuceno etal.1998).
Enlasesinvespertinasehicierondospresentacionesfina
les:M.A.SpantratsobreLacuantificacindelosefectos
recombinognicosdelosagentesqumicosyU.Grafacercade
La prueba de la antigenotoxicidad en clulas somticas de
Drosophila (Graf etal.1998a).
ElTallersecaracterizporunaatmsferamuyestimulantey
agradable,tantoenlassesionescientficascomoduranteeltra
bajoprcticoenellaboratorio.Nohaydudadequeestetercer
eventodeentrenamientoenBrasilfueunaexperienciaenrique
cedoratantoparalosparticipantescomoparalaUniversidad.Se
agradeceampliamenteelexcelentetrabajodeorganizacinde
R.M.GonalvesSilva,L.PereiraSilvaydetodoslosotrosmiem
brosdelLaboratoriodeCitogenticayMutagnesis(UFU).Tam
binseleestmuyreconocidoaN.M.Castro(UFU)porelenor
meapoyoprcticoenesteevento.
REFERENCIAS/REFERENCES
FragiorgeE.J.,SpanM.A.andGrafU.(1998).Efeitosgenotxi
cos daadriblastina em clulas somticas deDrosophila
melanogaster.GeneticsandMolecularBiology 21 September,
Suppl.,136.
FreiH.,ClementsJ.,HoweD.andWrglerF.E.(1992).The
genotoxicityoftheanticancerdrugmitoxantroneinsomatic
andgermcellsof Drosophilamelanogaster .Mutat.Res. 279,
2133.
GrafU.andvanSchaikN.(1992).Improvedhighbioactivation
crossforthewingsomaticmutationandrecombinationtest
in Drosophilamelanogaster .Mutat.Res. 271,5967.
113
oral presentations were given on projects in progress at the
UniversitiesofUberlndia(UFU)andGoinia(UFG).N.Coelho
deSousa(UFG)gaveapaperonEvaluationofthemutagenic
andrecombinagenicactivityofBarbatimo(Stryphnodendron
adstringens Mart) (Sousa and Carvalho 1997). The second
presentationEvaluationofthegenotoxicpotentialofextracts
of HyptissuaveolenswasgivenbyE.ArajoRocha(UFG).The
thirdpaperwasbyS.deCarvalho(UFG)onGenotoxiceffects
ofTordon.Afterthecoffeebreak,B.LassmarBuenoValadares
(UFU)presentedanexcellentpaperonPossibleantigenotoxic
effectsofpropolisextractedfromhoneycombsof Apismellifera
(ValadaresandSpan1998).Thesubsequentpaperspresented
very interesting studies on the modulating activity of two
chemicalcompounds.E.J.Fragiorge(UFU)talkedonModulating
effectsofvitaminCincombinationwithadriamycin(Fragiorge
etal. 1998)andE.PeresRodrigues(UFU)onModulatingeffects
ofmelatoninonthegenotoxicactivityofgammarays(Rodrigues
etal.1998).Thelastpresentationinthissessionwasgivenby
J.C.NepomucenoontheadvancedprojectProtectiveeffectsof
Moringaoleifera againstthegenotoxicactivityofmitomycinC
andurethane(Nepomuceno etal. 1998).
Intheafternoonsession,twofinallecturesweregiven:M.A.
SpantalkedonQuantificationoftherecombinageniceffects
of chemical agents, and U. Graf gave a paper entitled
AntigenotoxicitytestinginsomaticcellsofDrosophila(Graf
etal. 1998a).
ThewholeWorkshopprofitedfromaverystimulatingand
pleasantatmospherebothduringthescientificsessionsaswell
asduringthepracticalworkinthelaboratory.Thereisnodoubt
thatthisthirdtrainingeventinBrazilwasindeedaprofitable
experiencebothforalltheparticipantsandtheUniversity.The
excellentorganisationalworkprovidedbyR.M.GonalvesSilva
andL.PereiraSilvaaswellasbyalltheothermembersofthe
LaboratoryofCytogeneticsandMutagenesis(UFU)isgratefully
acknowledged.SpecialthanksarealsoduetoN.M.Castro(UFU)
forallherpracticalsupportinconnectionwiththisevent.
GrafU.,DelgadoRodrguezA.,VillalobosPietriniR.andGmez
Arroyo S. (1994).LatinAmericanWorkshop on Genetic
Toxicology.I.Drosophilamelanogaster (meetingreport).
Rev.Int.Contam.Ambient. 10,Suppl.1,14.
GrafU.,SpanM.A.,GuzmnRincnJ.,AbrahamS.K.andde
Andrade H.H. (1996). The wing somatic mutation and
recombinationtest(SMART)in Drosophilamelanogaster .
Afr.Newslett.onOccup.HealthandSafety 6,Suppl.1,913.
GrafU.,AbrahamS.K.,GuzmnRincnJ.andWrglerF.E.
(1998a).Antigenotoxicitystudies in Drosophila melano
gaster .Mutat.Res. 402,203209.
GrafU.,AbrahamS.K.,GuzmnRincnJ.andWrglerF.E.
(1998b).TheuseofDrosophilaassomaticassayinantigeno
114
toxicitystudies(abstract).GeneticsandMolecularBiology
21 November,Suppl.,2627.
GuzmnRincnJ.andGrafU.(1995). Drosophilamelanogaster
somaticmutationandrecombinationtestasabiomonitor.
In : Biomonitor s a nd bioma r ker s as indicator s of
environmentalchange (F.M.Butterworth,C.D.Corkumand
J.GuzmnRincn,Eds.),Plenum,NewYork,pp.169181.
GuzmnRincnJ. (1998). Drosophilaassentinelorganism
in mutagenesis (abstract). Genetics and Molecular
Biology 21November,Suppl.,50.
NepomucenoJ.C.,SpanM.A.,WrglerF.E.andGrafU.
(1998).Efeitoprotetorda Moringaoleifera contraaao
genotxicadamitomicinaCem D.melanogaster (abstract).
GeneticsandMolecularBiology21 November,Suppl.,
127.
Rodrigues E. P., Span M.A. and Graf U. (1998). Efeitos
moduladoresdamelatoninaemclulassomticasde Drosophila
M.A. Span y U. Graf
melanogaster tratadascomraiosg(abstract).Geneticsand
MolecularBiology 21 November,Suppl.,128.
SousaN.C.andCarvalhoS.(1996).Avaliaodospossveis
efeitos mutagnicos e recombinognicos do barbatimo
(StryphnodendronadstringensMart)emclulassomticas
de D.melanogaster.II.Resultadosparciais(abstract).Brazilian
JournalofGenetics 19 September,Suppl.,179.
ValadaresB.L.B.andSpanM.A.(1998).Avaliaodaprpolis
pelotestededetecodemutaeserecombinaessomticas
(SMART) em clulas de asa de Drosophila melanogaster
(abstract). Geneticsand MolecularBiology21 November,
Suppl.,129.
VogelE.W.,GrafU.,FreiH.andNivardM.M.J.(1998).Theresults
ofDrosophilaassaysasindicatorsofcarcinogenexposure.
In: Shortandmediumtermcarcinogenicitytests,mutation
analysis,andmultistagemodelsinriskidentification.IARC,
Lyon,France.Inpress.