ASTM E2186-02a(2023)
(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 2023)
Standard Guide for
Determining DNA Single-Strand Damage in Eukaryotic Cells
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. A number 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).
forming a single-cell gel electrophoresis assay (SCG) or Comet
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 DNA of individual cells. The
priate safety, health, and environmental practices and deter-
majority of studies utilizing the Comet assay have focused on
mine the applicability of regulatory limitations prior to use.
medical applications and have therefore examined DNA dam-
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
toxicity of a variety of compounds on cells in vitro and in vivo
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
this method, significantly elevated levels of DNA damage have
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 DNA damage correspond with
1.5 This international standard was developed in accor-
higher order effects such as decreased growth, survival, and
dance with internationally recognized principles on standard-
development, and correlate with significant increases in con-
ization established in the Decision on Principles for the
taminant body burdens (13, 16).
Development of International Standards, Guides and Recom-
1.2 This guide presents protocols that facilitate the expres-
mendations issued by the World Trade Organization Technical
sion of DNA alkaline labile single-strand breaks and the
Barriers to Trade (TBT) Committee.
determination of their abundance relative to control or refer-
ence cells. The guide is a general one meant to familiarize lab
2. Referenced Documents
personnel with the basic requirements and considerations
2.1 ASTM Standards:
necessary to perform the Comet assay. It does not contain
E1706 Test Method for Measuring the Toxicity of Sediment-
procedures for available variants of this assay, which allow the
Associated Contaminants with Freshwater Invertebrates
determination of non-alkaline labile single-strand breaks or
E1847 Practice for Statistical Analysis of Toxicity Tests
double-stranded DNA strand breaks (8), distinction between 4
Conducted Under ASTM Guidelines (Withdrawn 2022)
different cell types (13), identification of cells undergoing
3. Terminology
apoptosis (programmed cell death, (1, 17)), measurement of
3.1 The words “must,” “should,” “may,” “can,” and “might”
have very specific meanings in this guide. “Must” is used to
This guide is under the jurisdiction of ASTM Committee E50 on Environmental
Assessment, Risk Management and Corrective Action and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Dec. 1, 2023. Published December 2023. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2002. Last previous edition approved 2016 as E2186–02a(2016). DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E2186-02AR23. the ASTM website.
The boldface numbers in parentheses refer to the list of references at the end of The last approved version of this historical standard is referenced on
this standard. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2186 − 02a (2023)
express the strongest possible recommendation, just short of an 3.2.10 eukaryotic cell, n—cell with a membrane-bound,
absolute requirement. “Must” is only used in connection with structurally discrete nucleus and other well-developed subcel-
factors that relate directly to the acceptability of the test. lular compartments. Eukaryotes include all organisms except
“Should” is used to state that the specific condition is recom- viruses, bacteria, and cyanobacteria (blue-green algae).
mended and ought to be met if possible. Although violation of
3.2.11 ocular micrometer, n—a graduated grid placed be-
on “should” is rarely a serious matter, the violation of several
tween the viewer’s eye and an object being observed under a
will often render the results questionable. Terms such as “is
microscope, to measure the object’s size.
desirable,” “is often desirable,” and “might be desirable” are
3.2.12 single-stranded DNA, n—linear polymers of DNA
used in connection with less important factors. “May” is used
resulting from the breaking of hydrogen bonds between
to mean “is (are) allowed to,” “can” is used to mean “is (are)
complementary base pairs in double-stranded DNA.
able to,” and “might” is used to mean “could possibly.” Thus
the classic distinction between “may” and “can” is preserved
3.3 Definitions of Terms Specific to This Standard:
and “might” is never used as a synonym for either “may” or
3.3.1 comet, n—name based on the appearance of individual
“can.”
stained nuclear DNA and associated relaxed or fragmented
3.2 Definitions:
DNA migrating out from the nuclear DNA observed under the
3.2.1 CCD camera, n—charge coupled device (CCD) cam- microscope following these assay procedures.
era is a light sensitive silicon solid state device composed of
3.3.2 DNA migration distance, tail length, comet tail length,
many small pixels. The light falling on a pixel is converted into
n—distance in microns between the leading edge of electro-
a charge pulse which is then measured by the CCD electronics
phoretically migrating DNA and the closest edge of the
and represented by a number. A digital image is the collection
associated nuclear DNA (head).
of such light intensity numbers for all of the pixels from the
3.3.3 head, comet head, n—portion of a comet comprised of
CCD. A computer can reconstruct the image by varying the
the intact/immobile nuclear DNA.
light intensity for each spot on the computer monitor in the
proper order. Such digital images can be stored on disk,
3.3.4 tail, comet tail, n—portion of a comet comprised of the
transmitted over a computer network, and analyzed using
DNA migrating away from the intact/immobile nuclear DNA.
image processing techniques.
3.3.5 tail moment, n—a calculated value used to express the
3.2.2 cell lysis, n—the process of breaking open a cell by
distribution of DNA migrating from the comet head. Image
disruption of the plasma membrane.
analysis software applies an algorithm to the digitized image of
stained DNA and associated migrating DNA tail, which in
3.2.3 DNA, n—acronym for deoxyribonucleic acid, the sub-
stance that is the carrier of genetic information found in the essence defines the limits of the comet, subtracts background,
and determines the boundaries and staining intensity of the
chromosomes of the nucleus of a cell.
nucleus and comet tail. The calculated product of the percent of
3.2.4 DNA denaturation, n—refers to breaking hydrogen
DNA in the tail and the tail length is defined as the tail moment.
bonds between base pairs in double-stranded nucleic acid
molecules to produce two single-stranded polynucleotide poly-
4. Summary of Guide
mers.
4.1 Cells collected from organisms under different levels or
3.2.5 DNA lesion, n—a portion of a DNA molecule which
types of stress are dispersed and immobilized in agarose gel on
has been structurally changed.
microscope slides. The slides are placed in a solution to lyse
3.2.6 DNA supercoiling, n—the condition of DNA coiling
and disperse cell components, leaving the cellular DNA
up on itself because its helix has been bent, overwound, or
immobilized in the agarose. The DNA is denatured for a
underwound.
specified period of minutes by immersing the slides in an
alkaline solution. Strand breaks in the denatured cellular DNA
3.2.7 DNA supercoil relaxation, n—upon denaturation,
results in higher degree of supercoil relaxation: the more
DNA strand breaks allow the supercoiled DNA to unwind or
breaks, the greater the degree of relaxation. Given a sufficient
relax.
degree of relaxation, the application of an electric field across
3.2.8 double-stranded DNA, n—a structural form of DNA
the slides creates a motive force by which the charged DNA
where two polynucleotide molecular chains are wound around
may migrate through the surrounding agarose, away from the
each other, with the joining between the two strands via
immobilized main bulk of cellular DNA. Following
hydrogen bonds between complementary bases.
electrophoresis, the alkaline conditions are neutralized by
3.2.9 electrophoresis, n—a method of separating large mol- rinsing the slides in a neutral pH buffer and fixation of slide and
ecules (such as DNA fragments or proteins) from a mixture of its contents in ethanol. The DNA in the fixed slides is stained
similar molecules. An electric current is passed through a with fluorescent DNA stain and visualized using a fluorescent
medium containing the mixture, and each kind of molecule microscope. Migration distance of DNA away from the
travels through the medium at a different rate, depending on its nucleus, comet tail length, can be measured by eye using an
electrical charge and size. Separation is based on these differ- ocular micrometer. Comet tail length, percent DNA in tail, tail
ences. Agarose and acrylamide gels are the media commonly moment, and other DNA migration values can be calculated
used for electrophoresis of proteins and nucleic acids. with the use of image analysis software.
E2186 − 02a (2023)
5. Significance and Use use of T4 endonuclease V (3). Modifications of this type vastly
expand the utility of this assay and are good examples of its
5.1 A common result of cellular stress is an increase in DNA
versatility.
damage. DNA damage may be manifest in the form of base
5.2 A sufficient knowledge of the biology of cells examined
alterations, adduct formation, strand breaks, and cross linkages
using this assay should be attained to understand factors
(19). Strand breaks may be introduced in many ways, directly
affecting DNA strand breakage and the distribution of this
by genotoxic compounds, through the induction of apoptosis or
damage within sampled cell populations. This includes, but is
necrosis, secondarily through the interaction with oxygen
not limited to, influences such as cell type heterogeneity, cell
radicals or other reactive intermediates, or as a consequence of
cycle, cell turnover frequency, culture or growth conditions,
excision repair enzymes (20-22). In addition to a linkage with
and other factors that may influence levels of DNA strand
cancer, studies have demonstrated that increases in cellular
damage. Different cell types may have vastly different back-
DNA damage precede or correspond with reduced growth,
ground levels of DNA single-strand breaks due to variations in
abnormal development, and reduced survival of adults,
excision repair activity, metabolic activity, anti-oxidant
embryos, and larvae (16, 23, 24).
concentrations, or other factors. It is recommended that cells
5.1.1 The Comet assay can be easily utilized for collecting
representing those to be studied using the SCG/Comet assay be
data on DNA strand breakage (9, 25, 26). It is a simple, rapid,
examined under the light or fluorescent microscope using
and sensitive method that allows the comparison of DNA
stains capable of differentially staining different cell types.
strand damage in different cell populations. As presented in this
Morphological differences, staining characteristics, and fre-
guide, the assay facilitates the detection of DNA single strand
quencies of the different cell types should be noted and
breaks and alkaline labile sites in individual cells, and can
compared to SCG/Comet damage profiles to identify any
determine their abundance relative to control or reference cells
possible cell type specific differences. In most cases, the use of
(9, 16, 26). The assay offers a number of advantages; damage
homogenous cell populations reduces inter-cell variability of
to the DNA in individual cells is measured, only extremely
SCG/Comet values. The procedures for this assay, using cells
small numbers of cells need to be sampled to perform the assay
from many different species and cell types, have been pub-
(<10 000), the assay can be performed on practically any
lished previously (1, 2, 3, 5, 8, 10, 13, 14, 17, 18, 32-38). These
eukaryotic cell type, and it has been shown in comparative
references and others should be consulted to obtain details on
studies to be a very sensitive method for detecting DNA
the collection, handling, storage, and preparation of specific
damage (2, 27).
cell types.
5.1.2 These are general guidelines. There are numerous
5.3 The experimental design should incorporate appropriate
procedural variants of this assay. The variation used is depen-
controls, reference samples, and replicates to delineate the
dent upon the type of cells being examined, the types of DNA
influence of the major sources of experimental variability.
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 6. Equipment and Reagents
DNA can be
...
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