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Abstract

6thInternationalConferenceonEnvironmentalMutagensinHumanPopulations,Doha,Qatar,

March26-29,2012

Automatedimageanalysisofthemicronucleuscytomeassayandcometassayforhighthroughputhumanbiomonitoringandinvitro/invivogenotoxicitytestinginindustrystudies:IMSTARsystem

F.Soussaline(1),A.Papine(1),E.Tatarinova(1),I.Decordier(2),J.Dumont(3),A.Azqueta-Oscoz(4),A.K.Sharma(5),M-L.Binderup(5),A.Collins(4),E.Lorge(3),M.Kirsch-Volders(2),
(1)IMSTARS.A.60rueNotre-Dame-des-Champs,75006Paris,France;(2)LaboratoriumvoorCellulaireGenetica,VrijeUniversiteitBrussels,Belgium;(3)BiologieServierGroup,routedeServan45520Gidy,France;(4)DepartmentofNutrition,UniversityofOslo,Blindern,0316Oslo,Norway;(5)TechnicalUniversityofDenmark,NationalFoodInstitute,MørkhøjBygade2860Søborg,Denmark

GenetictoxicologytestssuchasmicronucleiandcometassaysareprovidingcrucialinformationaboutDNAdamagesinenvironmentalriskassessment,biomonitoringandcompoundtesting.Theyrequirebothsubstantialtimeandexpertisewhenperformedbyconventionalscreening.

Analysisofmicronuclei(MN)isthemostwidelyusedforbiomonitoringorinvitro/invivogenotoxicitytestingtoassessgeneticdamage[1].IthasbeenshownthatscoringMNfrequenciesinbinucleatedandmononucleatedcellsincytokinesis-blockedhumanlymphocytesisapredictivebioMarkerforcancerriskandareliable,sensitivemethodforbiomonitoringofearlygeneticeffects[2].However,automationofMNanalysisisneededformorereliable,fasterdetection,andimprovedscoringaccuracy.Italsoallowslargescaleanalysisformulti-centrescohortstudies.WithintheframeworkoftheEUprojectNewGeneris[3|,anautomatedimageanalysissystemforscoringMNfrequenciesinhumanlymphocyteswasdevelopedincollaborationbetweenVUBCellulaireGeneticaLabandIMSTAR[4,5].TheIMSTARPathfinderTMautomatedimagingsystemisbasedonsolidandspecificalgorithmsforimageanalysis,startingfromthedetectionofeachindividualcellinthewholesample;then,withineverycell,nucleiandmicronucleiareaccuratelydetected,andthescoringinmono,biandpolynucleatedcellsisproposedandvalidatedbyanexperttechnician.

Inparallel,inordertoincreasethescoringthroughputandrobustness,andtodecreaseoperator-dependencyofMNscoringforindustrialcompoundscreeningofseveralcelllines,e.g.L5178Y,V79,CHO,TK6,andHepG2,weimplementedanautomatedimagingmoduletobeusedinfurtherregulatoryinvitromicronucleusstudiescompliantwithGoodLaboratoryPractice(OECDPrinciplesofGPL[C(97)186/Final]).MouselymphomaL5178Ycellswereexposedtoknowngenotoxiccompoundsatvariousconcentrationsaccordingtodifferenttreatmentconditions.Thescoresofmicronucleatedcellswerecomparedbetweenvisualandautomatedscoringintermsoftime-saving,repeatability,reproducibilityandaccuracy.TheresultsshowedthatthePathfinderTMimagingsystemwasabletodetectmicronucleatedcellsasaccuratelyasvisualscoring,whilesavingscoringtimeby10to15fold.Moreover,inthisstudy,abetterhomogeneitywasfoundbetweentwoculturesofasametreatedgroupwiththeautomatedmethod.

Thecometassayorsingle-cellgelelectrophoresisisawell-establishedgenotoxicitytestandasimpletousemethodtodetectabroadspectrumofDNAdamagewithhighsensitivity.Itiswidelyusedinregulatory,mechanisticandbiomonitoringstudies[6].Moreover,ithasbeenrecentlyrecommendedthattheinvivocometassaycouldbepreferredtotheOECDguidelineratliverUDSassayforpharmaceuticalsintendedforhumanusetesting,asasecondinvivoassaywhenpositiveresultsareobtainedinvitrobuttheinvivomicronucleustestisnegative[9].

WithintheCOMICSEuropeanproject[7],wevalidatedthePathfinderCOMETsystem[8]anddemonstratedtheadded-valueofhigh-contentscreeningmethodsusingtheIMSTARautomatedscoringsystemintermsoftimesavingand,moreimportant,providingnewinformationforempoweredsensitivityandversatilityofthetest,asagenuinealternativetoanimaltesting.

Morespecifically,automatedimageanalysisandscoringalgorithmsweredevelopedbyIMSTARincollaborationwiththeNationalFoodInstituteofDenmark,tofitawiderangeofcelllinesforinvitrocometassay:HEK,V79,keratinocytes,fibroblasts,HelaandCaco2.Asforinvivocometassay,dedicatedmodulesforpreparationsfromrat’sbonemarrow,liver,lungandstomachcellsarenowavailable.Instudieswherefullyautomatedwascomparedtoconventionalprocedure,thescoringwasfaster,sensitivityhigher,variabilitylower,whiletheseveralthousandsofcometsscoredpersampleenablestoworkwithparameterdistributions.

References
[1]Kirsch-Voldersetal.,ArchToxicol.2011,85:873-899:[2]Kirsch-VoldersandFenech,Mutagenesis2001,16:51-58;[3]EUIntegratedProject“NewGeneris(Contractn°FOOD-CT-2005-016320);[4]Decordieretal.,Mutagenesis,2009,24:85-93;[5]Decordieretal.,Mutagenesis,2011,26:163-168;[6]WiklundSJ,etal.Mutagenesis2003;18:167-175;[7]EUSTREPCOMICSproject,(contractn°LSHB-CT-2006-037575);[8]AzquetaA.etal.,Mutagenesis2011;26:393-399;[9]OCDE486Guideline.

全自动微核扫描分析系统在遗传毒理学大会的研究论文/高通量人类生物监测的研究论文：该论文在2012年卡塔尔多哈第六届国际会议上发表，由IMSTAR提供。

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