1、Designation: E 2186 02aStandard Guide forDetermining DNA Single-Strand Damage in Eukaryotic CellsUsing the Comet Assay1This standard is issued under the fixed designation E 2186; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the
2、year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers the recommended criteria for per-forming a single-cell gel electrophoresis assay (SCG) or C
3、ometassay for the measurement of DNA single-strand breaks ineukaryotic cells. The Comet assay is a very sensitive methodfor detecting strand breaks in the DNA of individual cells. Themajority of studies utilizing the Comet assay have focused onmedical applications and have therefore examined DNA dam
4、-age in mammalian cells in vitro and in vivo (1-4).2There isincreasing interest in applying this assay to DNA damage infreshwater and marine organisms to explore the environmentalimplications of DNA damage.1.1.1 The Comet assay has been used to screen the geno-toxicity of a variety of compounds on c
5、ells in vitro and in vivo(5-7), as well as to evaluate the dose-dependent anti-oxidant(protective) properties of various compounds (3, 8-11). Usingthis method, significantly elevated levels of DNAdamage havebeen reported in cells collected from organisms at pollutedsites compared to reference sites
6、(12-15). Studies have alsofound that increases in cellular DNA damage correspond withhigher order effects such as decreased growth, survival, anddevelopment, and correlate with significant increases in con-taminant body burdens (13, 16).1.2 This guide presents protocols that facilitate the expres-si
7、on of DNA alkaline labile single-strand breaks and thedetermination of their abundance relative to control or refer-ence cells. The guide is a general one meant to familiarize labpersonnel with the basic requirements and considerationsnecessary to perform the Comet assay. It does not containprocedur
8、es for available variants of this assay, which allow thedetermination of non-alkaline labile single-strand breaks ordouble-stranded DNA strand breaks (8), distinction betweendifferent cell types (13), identification of cells undergoingapoptosis (programmed cell death, (1, 17), measurement ofcellular
9、 DNA repair rates (10), detection of the presence ofphotoactive DNA damaging compounds (14), or detection ofspecific DNA lesions (3, 18).1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to e
10、stablish appro-priate safety and health practices and determine the applica-bility of regulatory requirements prior to use.1.4 This guide is arranged as follows:SectionScope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Equipment and Reagents 6Assay Procedures 7Treatmen
11、t of Data 8Reporting Data 9Keywords 10Annex A1References2. Referenced Documents2.1 ASTM Standards:3E 1706 Test Methods for Measuring the Toxicity ofSediment-Associated Contaminants with Fresh Water In-vertebratesE 1847 Practice for Statistical Analysis of Toxicity TestsConducted Under ASTM Guideline
12、s3. Terminology3.1 The words “must,” “should,” “may,” “can,” and “might”have very specific meanings in this guide. “Must” is used toexpress the strongest possible recommendation, just short of anabsolute requirement. “Must” is only used in connection withfactors that relate directly to the acceptabi
13、lity of the test.“Should” is used to state that the specific condition is recom-mended and ought to be met if possible. Although violation ofon “should” is rarely a serious matter, the violation of severalwill often render the results questionable. Terms such as “isdesirable,” “is often desirable,”
14、and “might be desirable” are1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.02 on Terrestrial Assessment and Toxicology.Current edition approved April 10, 2003. Published June 2003. Originally
15、approved in 2002. Last previous edition approved 2002 as E218602.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book o
16、f ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.used in connection with less important factors. “May” is usedto mean “is (are) allowe
17、d to,” “can” is used to mean “is (are)able to,” and “might” is used to mean “could possibly.” Thusthe classic distinction between “may” and “can” is preservedand “might” is never used as a synonym for either “may” or“can.”3.2 Definitions:3.2.1 CCD camera, ncharge coupled device (CCD) cam-era is a li
18、ght sensitive silicon solid state device composed ofmany small pixels. The light falling on a pixel is converted intoa charge pulse which is then measured by the CCD electronicsand represented by a number. A digital image is the collectionof such light intensity numbers for all of the pixels from th
19、eCCD. A computer can reconstruct the image by varying thelight intensity for each spot on the computer monitor in theproper order. Such digital images can be stored on disk,transmitted over a computer network, and analyzed usingimage processing techniques.3.2.2 cell lysis, nthe process of breaking o
20、pen a cell bydisruption of the plasma membrane.3.2.3 DNA, nacronym for deoxyribonucleic acid, thesubstance that is the carrier of genetic information found in thechromosomes of the nucleus of a cell.3.2.4 DNA denaturation, nrefers to breaking hydrogenbonds between base pairs in double-stranded nucle
21、ic acidmolecules to produce two single-stranded polynucleotide poly-mers.3.2.5 DNA lesion, na portion of a DNA molecule whichhas been structurally changed.3.2.6 DNA supercoiling, nthe condition of DNA coilingup on itself because its helix has been bent, overwound, orunderwound.3.2.7 DNA supercoil re
22、laxation, nupon denaturation,DNA strand breaks allow the supercoiled DNA to unwind orrelax.3.2.8 double-stranded DNA, na structural form of DNAwhere two polynucleotide molecular chains are wound aroundeach other, with the joining between the two strands viahydrogen bonds between complementary bases.
23、3.2.9 electrophoresis, na method of separating large mol-ecules (such as DNA fragments or proteins) from a mixture ofsimilar molecules. An electric current is passed through amedium containing the mixture, and each kind of moleculetravels through the medium at a different rate, depending on itselect
24、rical charge and size. Separation is based on these differ-ences. Agarose and acrylamide gels are the media commonlyused for electrophoresis of proteins and nucleic acids.3.2.10 eukaryotic cell, ncell with a membrane-bound,structurally discrete nucleus and other well-developed subcel-lular compartme
25、nts. Eukaryotes include all organisms exceptviruses, bacteria, and cyanobacteria (blue-green algae).3.2.11 ocular micrometer, na graduated grid placed be-tween the viewers eye and an object being observed under amicroscope, to measure the objects size.3.2.12 single-stranded DNA, nlinear polymers of
26、DNAresulting from the breaking of hydrogen bonds betweencomplementary base pairs in double-stranded DNA.3.3 Definitions of Terms Specific to This Standard:3.3.1 comet, nname based on the appearance of individualstained nuclear DNA and associated relaxed or fragmentedDNA migrating out from the nuclea
27、r DNA observed under themicroscope following these assay procedures.3.3.2 DNA migration distance, tail length, comet tail length,ndistance in microns between the leading edge of electro-phoretically migrating DNA and the closest edge of theassociated nuclear DNA (head).3.3.3 head, comet head, nporti
28、on of a comet comprised ofthe intact/immobile nuclear DNA.3.3.4 tail, comet tail, nportion of a comet comprised ofthe DNA migrating away from the intact/immobile nuclearDNA.3.3.5 tail moment, na calculated value used to express thedistribution of DNA migrating from the comet head. Imageanalysis soft
29、ware applies an algorithm to the digitized image ofstained DNA and associated migrating DNA tail, which inessence defines the limits of the comet, subtracts background,and determines the boundaries and staining intensity of thenucleus and comet tail. The calculated product of the percent ofDNAin the
30、 tail and the tail length is defined as the tail moment.4. Summary of Guide4.1 Cells collected from organisms under different levels ortypes of stress are dispersed and immobilized in agarose gel onmicroscope slides. The slides are placed in a solution to lyseand disperse cell components, leaving th
31、e cellular DNAimmobilized in the agarose. The DNA is denatured for aspecified period of minutes by immersing the slides in analkaline solution. Strand breaks in the denatured cellular DNAresults in higher degree of supercoil relaxation: the morebreaks, the greater the degree of relaxation. Given a s
32、ufficientdegree of relaxation, the application of an electric field acrossthe slides creates a motive force by which the charged DNAmay migrate through the surrounding agarose, away from theimmobilized main bulk of cellular DNA. Following electro-phoresis, the alkaline conditions are neutralized by
33、rinsing theslides in a neutral pH buffer and fixation of slide and itscontents in ethanol. The DNAin the fixed slides is stained withfluorescent DNA stain and visualized using a fluorescentmicroscope. Migration distance of DNA away from thenucleus, comet tail length, can be measured by eye using ano
34、cular micrometer. Comet tail length, percent DNA in tail, tailmoment, and other DNA migration values can be calculatedwith the use of image analysis software.5. Significance and Use5.1 Acommon result of cellular stress is an increase in DNAdamage. DNA damage may be manifest in the form of basealtera
35、tions, adduct formation, strand breaks, and cross linkages(19). Strand breaks may be introduced in many ways, directlyby genotoxic compounds, through the induction of apoptosis ornecrosis, secondarily through the interaction with oxygenradicals or other reactive intermediates, or as a consequence of
36、excision repair enzymes (20-22). In addition to a linkage withcancer, studies have demonstrated that increases in cellularDNA damage precede or correspond with reduced growth,abnormal development, and reduced survival of adults, em-bryos, and larvae (16, 23, 24).E 2186 02a25.1.1 The Comet assay can
37、be easily utilized for collectingdata on DNA strand breakage (9, 25, 26). It is a simple, rapid,and sensitive method that allows the comparison of DNAstrand damage in different cell populations.As presented in thisguide, the assay facilitates the detection of DNA single strandbreaks and alkaline lab
38、ile sites in individual cells, and candetermine their abundance relative to control or reference cells(9, 16, 26). The assay offers a number of advantages; damageto the DNA in individual cells is measured, only extremelysmall numbers of cells need to be sampled to perform the assay(10 000), the assa
39、y can be performed on practically anyeukaryotic cell type, and it has been shown in comparativestudies to be a very sensitive method for detecting DNAdamage (2, 27).5.1.2 These are general guidelines. There are numerousprocedural variants of this assay. The variation used is depen-dent upon the type
40、 of cells being examined, the types of DNAdamage of interest, and the imaging and analysis capabilities ofthe lab conducting the assay. To visualize the DNA, it is stainedwith a fluorescent dye, or for light microscope analysis theDNA can be silver stained (28). Only fluorescent stainingmethods will
41、 be described in this guide. The microscopicdetermination of DNA migration can be made either by eyeusing an ocular micrometer or with the use of image analysissoftware. Scoring by eye can be performed using a calibratedocular micrometer or by categorizing cells into four to fiveclasses based on the
42、 extent of migration (29, 30). Imageanalysis systems are comprised of a CCD camera attached to afluorescent microscope and software and hardware designedspecifically to capture and analyze images of fluorescentlystained nuclei. Using such a system, it is possible to measurethe fluorescence intensity
43、 and distribution of DNAin and awayfrom the nucleus (8). Using different procedural variants, theassay can be utilized to measure specific types of DNAalterations and DNArepair activity (1, 3, 8, 10, 13, 14, 17, 18).Alkaline lysis and electrophoresis conditions are used for thedetection of single-st
44、randed DNA damage, whereas neutral pHconditions facilitate the detection of double-strand breaks (31).Various sample treatments can be used to express specific typesof DNA damage, or as in one method, to preserve stranddamage at sites of DNA repair (10). Nuclease digestion stepscan be used to introd
45、uce strand breaks at specific lesion sites.Using this approach, oxidative base damage can be detected bythe use of endonuclease III (18), as well as DNA modificationsresulting from exposure to ultraviolet light (UV) through theuse of T4 endonuclease V (3). Modifications of this type vastlyexpand the
46、 utility of this assay and are good examples of itsversatility.5.2 A sufficient knowledge of the biology of cells examinedusing this assay should be attained to understand factorsaffecting DNA strand breakage and the distribution of thisdamage within sampled cell populations. This includes, but isno
47、t limited to, influences such as cell type heterogeneity, cellcycle, cell turnover frequency, culture or growth conditions,and other factors that may influence levels of DNA stranddamage. Different cell types may have vastly different back-ground levels of DNA single-strand breaks due to variations
48、inexcision repair activity, metabolic activity, anti-oxidant con-centrations, or other factors. It is recommended that cellsrepresenting those to be studied using the SCG/Comet assay beexamined under the light or fluorescent microscope usingstains capable of differentially staining different cell ty
49、pes.Morphological differences, staining characteristics, and fre-quencies of the different cell types should be noted andcompared to SCG/Comet damage profiles to identify anypossible cell type specific differences. In most cases, the use ofhomogenous cell populations reduces inter-cell variability ofSCG/Comet values. The procedures for this assay, using cellsfrom many different species and cell types, have been pub-lished previously (1, 2, 3, 5, 8, 10, 13, 14, 17, 18, 32-38). Thesereferences and others should be consulted to obtain details onthe collection, handlin
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