【英文标准名称】:StandardGuideforDeterminingDNASingle-StrandDamageinEukaryoticCellsUsingtheCometAssay
【原文标准名称】:用彗星分析法测定真核细胞中DNA单线损伤的标准指南
【标准号】:ASTME2186-2002a
【标准状态】:现行
【国别】:
【发布日期】:2002
【实施或试行日期】:
【发布单位】:美国材料与试验协会(US-ASTM)
【起草单位】:E47.02
【标准类型】:(Guide)
【标准水平】:()
【中文主题词】:损伤;电池;核酸;遗传学
【英文主题词】:biomarker;cellular;Cometassay;DNAdamage;DNAstrandbreaks;stresseffects;toxic
【摘要】:1.1Thisguidecoverstherecommendedcriteriaforperformingasingle-cellgelelectrophoresisassay(SCG)orCometassayforthemeasurementofDNAsingle-strandbreaksineukaryoticcells.TheCometassayisaverysensitivemethodfordetectingstrandbreaksintheDNAofindividualcells.ThemajorityofstudiesutilizingtheCometassayhavefocusedonmedicalapplicationsandhavethereforeexaminedDNAdamageinmammaliancellsinvitroandinvivo(1-4).ThereisincreasinginterestinapplyingthisassaytoDNAdamageinfreshwaterandmarineorganismstoexploretheenvironmentalimplicationsofDNAdamage.1.1.1TheCometassayhasbeenusedtoscreenthegenotoxicityofavarietyofcompoundsoncellsinvitroandinvivo(5-7),aswellastoevaluatethedose-dependentanti-oxidant(protective)propertiesofvariouscompounds(3,8-11).Usingthismethod,significantlyelevatedlevelsofDNAdamagehavebeenreportedincellscollectedfromorganismsatpollutedsitescomparedtoreferencesites(12-15).StudieshavealsofoundthatincreasesincellularDNAdamagecorrespondwithhigherordereffectssuchasdecreasedgrowth,survival,anddevelopment,andcorrelatewithsignificantincreasesincontaminantbodyburdens(13,16).1.2ThisguidepresentsprotocolsthatfacilitatetheexpressionofDNAalkalinelabilesingle-strandbreaksandthedeterminationoftheirabundancerelativetocontrolorreferencecells.TheguideisageneralonemeanttofamiliarizelabpersonnelwiththebasicrequirementsandconsiderationsnecessarytoperformtheCometassay.Itdoesnotcontainproceduresforavailablevariantsofthisassay,whichallowthedeterminationofnon-alkalinelabilesingle-strandbreaksordouble-strandedDNAstrandbreaks(8),distinctionbetweendifferentcelltypes(13),identificationofcellsundergoingapoptosis(programmedcelldeath,(1,17)),measurementofcellularDNArepairrates(10),detectionofthepresenceofphotoactiveDNAdamagingcompounds(14),ordetectionofspecificDNAlesions(3,18).1.3Thisstandarddoesnotpurporttoaddressallofthesafetyconcerns,ifany,associatedwithitsuse.Itistheresponsibilityoftheuserofthisstandardtoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatoryrequirementspriortouse.1.3Thisguideisarrangedasfollows:SectionScope1ReferencedDocuments2Terminology3SummaryofGuide4SignificanceandUse5EquipmentandReagents6AssayProcedures7TreatmentofData8ReportingData9Keywords10AnnexA1References