ASTM E2186-02a(2016)
(Guide)Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
SIGNIFICANCE AND USE
5.1 A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand breaks may be introduced in many ways, directly by genotoxic compounds, through the induction of apoptosis or necrosis, secondarily through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes (20-22). In addition to a linkage with cancer, studies have demonstrated that increases in cellular DNA damage precede or correspond with reduced growth, abnormal development, and reduced survival of adults, embryos, and larvae (16, 23, 24).
5.1.1 The Comet assay can be easily utilized for collecting data on DNA strand breakage (9, 25, 26). It is a simple, rapid, and sensitive method that allows the comparison of DNA strand damage in different cell populations. As presented in this guide, the assay facilitates the detection of DNA single strand breaks and alkaline labile sites in individual cells, and can determine their abundance relative to control or reference cells (9, 16, 26). The assay offers a number of advantages; damage to the DNA in individual cells is measured, only extremely small numbers of cells need to be sampled to perform the assay ((2, 27) .
5.1.2 These are general guidelines. There are numerous procedural variants of this assay. The variation used is dependent upon the type of cells being examined, the types of DNA damage of interest, and the imaging and analysis capabilities of the lab conducting the assay. To visualize the DNA, it is stained with a fluorescent dye, or for light microscope analysis the DNA can be silver stained (28). Only fluorescent staining methods will be described in this guide. The microscopic determination of DNA migration can be made either by eye using an ocular micrometer or with the use of image analysis software. Scoring by eye can be performed using a calibrated ocular...
SCOPE
1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4).2 There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.
1.1.1 The Comet assay has been used to screen the genotoxicity of a variety of 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). Using this method, significantly elevated levels of DNA damage have been reported in cells collected from organisms at polluted sites compared to reference sites (12-15). Studies have also found that increases in cellular DNA damage correspond with higher order effects such as decreased growth, survival, and development, and correlate with significant increases in contaminant body burdens (13, 16).
1.2 This guide presents protocols that facilitate the expression of DNA alkaline labile single-strand breaks and the determination of their abundance relative to control or reference cells. The guide is a general one meant to familiarize lab personnel with the basic requirements and considerations necessary to perform the Comet assay. It does not contain procedures for available variants of this assay, which allow the determination of non-alkaline labile single-strand breaks or double-stranded DNA strand breaks (8), distinction between different cell types (13), identification of cells undergoing apoptosis (programmed cell death, (1, 17)), measurement of cellular DNA repair...
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Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E2186 − 02a (Reapproved 2016)
Standard Guide for
Determining DNA Single-Strand Damage in Eukaryotic Cells
1
Using the Comet Assay
This standard is issued under the fixed designation E2186; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope cellular DNA repair rates (10), detection of the presence of
photoactive DNA damaging compounds (14), or detection of
1.1 This guide covers the recommended criteria for per-
specific DNA lesions (3, 18).
formingasingle-cellgelelectrophoresisassay(SCG)orComet
1.3 This standard does not purport to address all of the
assay for the measurement of DNA single-strand breaks in
safety concerns, if any, associated with its use. It is the
eukaryotic cells. The Comet assay is a very sensitive method
responsibility of the user of this standard to establish appro-
for detecting strand breaks in the DNAof individual cells.The
priate safety and health practices and determine the applica-
majority of studies utilizing the Comet assay have focused on
bility of regulatory requirements prior to use.
medical applications and have therefore examined DNAdam-
2
1.4 This guide is arranged as follows:
age in mammalian cells in vitro and in vivo (1-4). There is
increasing interest in applying this assay to DNA damage in
Section
freshwater and marine organisms to explore the environmental
Scope 1
implications of DNA damage.
Referenced Documents 2
1.1.1 The Comet assay has been used to screen the geno-
Terminology 3
Summary of Guide 4
toxicityofavarietyofcompoundsoncellsinvitroandinvivo
Significance and Use 5
(5-7), as well as to evaluate the dose-dependent anti-oxidant
Equipment and Reagents 6
(protective) properties of various compounds (3, 8-11). Using
Assay Procedures 7
Treatment of Data 8
thismethod,significantlyelevatedlevelsofDNAdamagehave
Reporting Data 9
been reported in cells collected from organisms at polluted
Keywords 10
sites compared to reference sites (12-15). Studies have also Annex Annex A1
References
found that increases in cellular DNAdamage correspond with
higher order effects such as decreased growth, survival, and
2. Referenced Documents
development, and correlate with significant increases in con-
3
2.1 ASTM Standards:
taminant body burdens (13, 16).
E1706TestMethodforMeasuringtheToxicityofSediment-
1.2 This guide presents protocols that facilitate the expres-
Associated Contaminants with Freshwater Invertebrates
sion of DNA alkaline labile single-strand breaks and the
E1847Practice for Statistical Analysis of Toxicity Tests
determination of their abundance relative to control or refer-
Conducted Under ASTM Guidelines
ence cells. The guide is a general one meant to familiarize lab
personnel with the basic requirements and considerations
3. Terminology
necessary to perform the Comet assay. It does not contain
3.1 Thewords“must,”“should,”“may,”“can,”and“might”
proceduresforavailablevariantsofthisassay,whichallowthe
have very specific meanings in this guide. “Must” is used to
determination of non-alkaline labile single-strand breaks or
expressthestrongestpossiblerecommendation,justshortofan
double-stranded DNA strand breaks (8), distinction between
absolute requirement. “Must” is only used in connection with
different cell types (13), identification of cells undergoing
factors that relate directly to the acceptability of the test.
apoptosis (programmed cell death, (1, 17)), measurement of
“Should” is used to state that the specific condition is recom-
mended and ought to be met if possible.Although violation of
on “should” is rarely a serious matter, the violation of several
1
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
will often render the results questionable. Terms such as “is
Assessment, Risk Management and CorrectiveAction 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. Originally
3
approved in 2002. Last previous edition approved 2010 as E2186–02a(2010). DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E2186-02AR16. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
2
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
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