1、Designation: E2186 02a (Reapproved 2016)Standard Guide forDetermining DNA Single-Strand Damage in Eukaryotic CellsUsing the Comet Assay1This standard is issued under the fixed designation E2186; the number immediately following the designation indicates the year oforiginal adoption or, in the case o
2、f revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () 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 a
3、ssay (SCG) or Cometassay 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 e
4、xamined DNA dam-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
5、 compounds on cells 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 r
6、eference sites (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 facilitat
7、e the expres-sion 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
8、containprocedures 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), measure
9、ment ofcellular 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 thi
10、s standard to establish 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 Proce
11、dures 7Treatment of Data 8Reporting Data 9Keywords 10Annex Annex A1References2. Referenced Documents2.1 ASTM Standards:3E1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater InvertebratesE1847 Practice for Statistical Analysis of Toxicity TestsConducted Und
12、er ASTM Guidelines3. 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
13、 to the acceptability 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 “is1This guide is un
14、der the jurisdiction ofASTM Committee E50 on EnvironmentalAssessment, Risk Management and Corrective Action and is the direct responsibil-ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.Current edition approved Feb. 1, 2016. Published May 2016. Originallyapproved in 2002. Las
15、t previous edition approved 2010 as E218602a(2010). DOI:10.1520/E2186-02AR16.2The 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 A
16、nnual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1desirable,” “is often desirable,” and “might be desirable” areused in conn
17、ection with less important factors. “May” is usedto mean “is (are) allowed 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 D
18、efinitions:3.2.1 CCD camera, ncharge coupled device (CCD) cam-era is a light 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 th
19、e collectionof such light intensity numbers for all of the pixels from theCCD. 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 usin
20、gimage processing techniques.3.2.2 cell lysis, nthe process of breaking open a cell bydisruption of the plasma membrane.3.2.3 DNA, nacronym for deoxyribonucleic acid, the sub-stance that is the carrier of genetic information found in thechromosomes of the nucleus of a cell.3.2.4 DNA denaturation, nr
21、efers to breaking hydrogenbonds between base pairs in double-stranded nucleic 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 beca
22、use its helix has been bent, overwound, orunderwound.3.2.7 DNA supercoil relaxation, 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 joi
23、ning between the two strands viahydrogen bonds between complementary bases.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 mo
24、leculetravels through the medium at a different rate, depending on itselectrical 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,st
25、ructurally discrete nucleus and other well-developed subcel-lular compartments. 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, t
26、o measure the objects size.3.2.12 single-stranded DNA, nlinear polymers of 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 nuclea
27、r DNA and associated relaxed or fragmentedDNA migrating out from the nuclear 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 clos
28、est edge of theassociated nuclear DNA (head).3.3.3 head, comet head, nportion of a comet comprised ofthe intact/immobile nuclear DNA.3.3.4 tail, comet tail, nportion of a comet comprised of theDNA migrating away from the intact/immobile nuclear DNA.3.3.5 tail moment, na calculated value used to expr
29、ess thedistribution of DNA migrating from the comet head. Imageanalysis software 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
30、thenucleus and comet tail.The calculated product of the percent ofDNAin the 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 slid
31、es are placed in a solution to lyseand disperse cell components, leaving the 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
32、relaxation: the morebreaks, the greater the degree of relaxation. Given a sufficientdegree 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 D
33、NA. Followingelectrophoresis, the alkaline conditions are neutralized byrinsing the slides in a neutral pH buffer and fixation of slide andits contents in ethanol. The DNA in the fixed slides is stainedwith fluorescent DNA stain and visualized using a fluorescentmicroscope. Migration distance of DNA
34、 away from thenucleus, comet tail length, can be measured by eye using anocular 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 i
35、ncrease in DNAdamage. DNA damage may be manifest in the form of basealterations, 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 w
36、ith oxygenradicals or other reactive intermediates, or as a consequence ofE2186 02a (2016)2excision 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
37、survival of adults,embryos, and larvae (16, 23, 24).5.1.1 The Comet assay can 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, t
38、he assay facilitates the detection of DNA single strandbreaks and alkaline labile 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 extremelysm
39、all numbers of cells need to be sampled to perform the assay(10 000), the assay 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 numerouspro
40、cedural variants of this assay. The variation used is depen-dent upon the type 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 anal
41、ysis theDNA can be silver stained (28). Only fluorescent stainingmethods will 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 calibratedocu
42、lar micrometer or by categorizing cells into four to fiveclasses based on the 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 nuc
43、lei. Using such a system, it is possible to measurethe fluorescence intensity 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).Alkalin
44、e lysis and electrophoresis conditions are used for thedetection of single-stranded DNAdamage, 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 stranddamag
45、e at sites of DNA repair (10). Nuclease digestion stepscan be used to introduce strand breaks at specific lesion sites.Using this approach, oxidative base damage can be detected bythe use of endonuclease III (18), as well as DNAmodificationsresulting from exposure to ultraviolet light (UV) through t
46、heuse of T4 endonuclease V (3). Modifications of this type vastlyexpand the 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 distribu
47、tion of thisdamage within sampled cell populations. This includes, but isnot 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
48、different back-ground levels of DNAsingle-strand breaks due to variations inexcision repair activity, metabolic activity, anti-oxidantconcentrations, or other factors. It is recommended that cellsrepresenting those to be studied using the SCG/Comet assay beexamined under the light or fluorescent mic
49、roscope usingstains capable of differentially staining different cell types.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 an
