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Use of the Comet Assay in Environmental Toxicology

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11

Use of the Comet Assay in Environmental Toxicology

Loren D. Knopper and James P. McNamee

Summary
The comet assay, also known as the single-cell gel electrophoresis assay, is a method
of detecting DNA damage in virtually any nucleated cell. The comet assay has significant
advantages over other genotoxicity tests, but it is very sensitive to subtle changes that
can yield appreciable variability in results. The purpose of our chapter is to present
background information and detailed standard operating procedures for the use of the
alkaline comet assay in environmental genotoxicity assessment. We address pitfalls and
concerns associated with conducting the comet assay, and briefly discuss modifications
of the general alkaline procedure that can be used to address different issues relevant to
environmental toxicology.

Key Words: Alkaline comet assay; DNA damage; environmental toxicology;

genotoxicity.

1. Introduction
The comet assay, also known as the single-cell gel electrophoresis assay, is
a method of detecting DNA damage (including DNA single- and double-strand
breaks, DNA–DNA crosslinks, DNA–protein crosslinks, and alkali-labile DNA
damage) in virtually any nucleated cell. Significant advantages of the comet
assay over other genotoxicity tests include its fairly simple methodology,
sensitivity, requirement for small numbers of cells, and rapid production of
data (1). However, researchers need to be aware that the technique is very
sensitive to subtle changes (e.g., temperature, buffer volume, and pH) that can
yield appreciable variability in results.

From: Methods in Molecular Biology, vol. 410: Environmental Genomics

Edited by: C. Cristofre Martin © Humana Press, Totowa, NJ

171
172 Knopper and McNamee

The general technique begins by obtaining a single-cell suspension of

nucleated cells of interest. The cells should be assessed for viability, diluted
to the appropriate cell concentration, and then mixed with molten agarose
and “cast” onto frosted glass slides (1,2) or into individual plastic chambers
affixed to a piece of Gelbond acetate film (Fig. 1) (3). We suggest the use
of the Gelbond technique because it eliminates the main technical problems
associated with using glass slides (e.g., lack of agarose adhesion to slides,
shrinking of gels during storage) and results in increased productivity and
efficiency without decreasing assay reliability. After the solidification of the

Fig. 1. Comet assay setup using the GelBond technique. (a) Lab Tek II chambers
are affixed to Gelbond; (b) casting agarose-cell suspension mixture into chambers;
(c) removing chambers from Gelbond; (d) placing GelBond-agarose complex into lysis
buffer.
Comet Assay in Environmental Toxicology 173

cell-agarose suspension, the entire Gelbond–gel or glass slide–gel complex

is placed in a lysis buffer consisting of salts and detergents to degrade the
cellular membrane and expose the nucleus. This is followed by submersion of
the complex in an alkaline or neutral buffer (see later) that allows for DNA
unwinding before electrophoresis, which is conducted for a given time based
on cell type and/or desired sensitivity. After electrophoresis, the complex is
placed in a neutralizing buffer to stabilize the gel, and then dehydrated in
ethanol. This technique is common to most researchers but cell dilutions, buffer
pH, and electrophoresis conditions (voltage and time) are specific to cell type,
desired sensitivity, and equipment being used. (See Notes 1–3.)
The National Institute of Health maintains a Listserve for the Comet Assay
Interest Group (www.cometassay.com), where ongoing discussion and debate
takes place regarding all aspects of the comet assay. The Listserve is regularly
updated and all correspondence is archived so past postings can be viewed.

2. Materials
2.1. Solutions
1. 10× Ca2+ - and Mg2+ -free phosphate-buffered saline (PBS): 1.31 M NaCl, 50
mMNa2 HPO4 , 16 mMKH2 PO4 . Store in the dark at room temperature. Dilute to
1× before use and adjust to pH 7.4.
2. 0.75% Agarose: 750 mg of low melting point (LMP) agarose, 1× Ca2+ -and Mg2+ -
free PBS. Stir together on stirring plate with heat on medium/high and then dispense
1.0-mL aliquots into 1.5-mL Eppendorf tubes. Store at 4°C until needed. On the
day of experiment, melt in microwave for 10×20 s on high and place in a heater
block or water bath at 42°C.
3. 1.0% Agarose: 1.0 g of agarose, 100 mL of Ca2+ - and Mg2+ -free PBS. Stir together
on stirring plate with heat on medium/high and then dispense 4-mL aliquots into
Falcon tubes and store at 4°C until use. On the day of the experiment, melt in a
microwave for 10–20 s on high, then maintain in a heater block or water bath at
42°C.
4. Lysis buffer (See Note 4): 2.5 M NaCl, 100 mM tetrasodium EDTA, 10 mM Tris
base, 1% n-lauryl sarcosine. Cover and shake to remove clumps. Slowly add 1 L
of purified H2 0 and stir on a stir plate. Adjust the pH to 10.0 with concentrated
NaOH or HCl. Store in the dark at room temperature. Add 1% Triton X-100 to
the required volume on the day of the experiment, 30 min before use. Store at 4°C
(see later).
5. Electrophoresis buffer A (See Note 5): 0.3 M NaOH, 10 mM tetrasodium EDTA,
0.1% 8-hydroxyquinoline, 2% dimethyl sulfoxide (DMSO). Slowly add purified
H2 0 and stir on a stir plate. Add DMSO while mixing. Adjust to pH 13.1 ± 0.1 on
a calibrated pH meter with concentrated NaOH or HCl. Prepare fresh on the day
of the experiment.
174 Knopper and McNamee

6. Neutralization buffer: 1 M ammonium acetate. Adjust the pH to 7.0 with concen-

trated NaOH or HCl
7. Dual stain for cell viability: stock fluorescein diacetate (5.0 mg/mL acetone), stock
ethidium bromide (200 μg/mL Ca2+ - and Mg2+ -free PBS), adjust to pH 7.4. Store
stock fluorescein diacetate at –20°C and stock ethidium bromide, wrapped with
aluminum foil, at 4°C. For a working stain, mix in an Eppendorf: 1.2 mL of Ca2+ -
and Mg2+ -free PBS, 7.5 μL of stock fluorescein diacetate, and 50 μL of stock
ethidium bromide. Make fresh on the day of use.
8. DNA stain: Dispense 5.0 μL of SYBR Gold stain (Molecular Probes cat. no.
S-11494) into 50 mL of purified H2 O. Wrap in aluminum foil. Store in the dark
at 4°C. (See Note 6.)

2.2. Glassware and Labware

1. Pipets.
2. Pipet tips (10, 20, 100, 200, 1000μL).
3. Volumetric flasks (100 mL, 500 mL, and 1 L).
4. Graduated cylinders (100 mL, 500 mL, 1 L).
5. Beakers (250 mL, 500 mL, 1 L).
6. Petri dishes (120 mm diameter).
7. Dispensing bottle (for purified water).
8. Ice bucket.
9. Cryovials (1.0 mL).
10. Eppendorf tubes (0.5 mL, 1.5 mL).
11. Centrifuge tubes with screw caps (50 mL).
12. Plastic/glass trays (e.g., lids and bottoms of pipet tip containers).
13. Microscope slides.
14. Microscope cover slips (22 × 50 mm).

2.3. Instrumentation
1. pH meter.
2. Analytical balance.
3. Magnetic stirrer and magnetic bars.
4. Power supply capable of low voltage and high milli-amperage (e.g., 300 V, 2000 mA).
5. Horizontal gel electrophoresis units.
6. Tweezers.
7. Scissors.
8. Gelbond Film (agarose gel support medium) (Mandel cat. no. 53740 GB 1638).
9. Lab Tek II chambers (Nalge Nunc cat. no. 154461-B).
10. Heater block or water bath.
11. Computer with Comet assay imaging system (Kinetic Komet 5.5 or other).
12. Fluorescence microscope with ×40 oil immersion objective (Zeiss AxioPlan II or
other) and proper excitation/emission filters for dye (e.g., SYBR Gold: ex 300, em
495/537 nm, bound to nucleic acid).
Comet Assay in Environmental Toxicology 175

3. Methods
3.1. Calibration
Calibration of the comet assay technique can be achieved by assessing DNA
damage in cells that have been exposed to either ionizing radiation (X-ray or
-ray) or chemical exposure (hydrogen peroxide, cyclophosphamide, or methyl
methanesulfonate) (See Note 7) (1,4,5). Generating a dose–response curve can
also be used to assess the sensitivity of the technique in a given laboratory
(Fig. 2). Calibration should be conducted with each new species and cell type
being assessed. Some laboratories incorporate a concurrent positive control
(e.g., radiation or chemical exposure) for each comet assay experiment to
ensure assay conditions are consistent across experiments.

3.2. Cell Viability

Cells that are apoptotic or necrotic do not display the typical comet
appearance. Rather, they exhibit very small or nonexistent heads and very
large diffuse tails (Fig. 3 ). These cells are commonly referred to as tear-drops,
ghost cells, or hedgehog cells (1,4). Such cells can be produced after exposure
to cytotoxic agents and/or nongenotoxic agents and should be excluded from
analysis. Cells that have been exposed to genotoxins can also show this type
of appearance and should be used in data analysis. Thus, it is important to
conduct a concomitant assessment of cytotoxicity in the cell suspension in

Fig. 2. An example of a dose–response curve generated by exposing whole blood

from meadow voles (Microtus pennsylvanicus) to gradations of 137 Cs radiation. N = 4
at each dose. Notice the plateauing trend in tail length and the exponential trend in
moment with dose. (Modified from Knopper et al., 2005.) (22)
176 Knopper and McNamee

Fig. 3. Control (a), damaged (b), moderately damaged (c), and apoptotic (i.e.,
hedgehog, ghost-cell) or highly damaged nucleoids (d) as observed after 20 min of
electrophoresis at 1.5 V/cm. Cells stained with SYBR Gold DNA dye.

order to determine the cause of highly damaged cells and determine whether
they should be included in the data analysis.
Cell viability is predominantly assessed using one of two methods. The
first is with the dual stain viability assay (6). In this technique, equal volumes
of the cell solution and ethidium bromide/fluorescein diacetate working stain
(See Subheading 2.1., item 7) are mixed together, then loaded into both sides of
a hemacytometer (10 μL per side). Viable and nonviable cells are then counted
manually via fluorescence microscopy. Metabolically competent (viable) cells
will fluoresce green as a result of the conversion of fluorescein diacetate to
its fluorescent metabolite fluorescein by cellular esterases, while metabolically
incompetent (nonviable) cells fluoresce red because of membrane leakage and
staining of DNA by ethidium bromide. Another way to assess viability is by the
trypan blue exclusion assay. In this assay, equal volumes of the cell solution
and trypan blue are mixed together, loaded into both sides of a hemacytometer
(10 μL per side), then viable and nonviable cells counted manually under light
Comet Assay in Environmental Toxicology 177

microscopy after a settling period (usually between 2 and 5 min). Cells that
take up the dye are nonviable, whereas those that exclude the dye are viable.
In general, samples with viability below 70–75% of that in the control samples
should be discarded from further analysis (1).

3.3. Species Concerns

The comet assay can be conducted using virtually any nucleated cell, with
the caveat that those cells are viable. It has been found that in some species
(e.g., avian), whole blood is not appropriate for use with the comet assay
because more than 80% of the cells exhibit the “ghost cell” or “hedgehog”
appearance, presumably owing to degraded and functionally inert DNA/RNA
within nucleated, mature erythrocytes. In this situation, leukocytes need to
be separated from the nucleated erythrocytes to be used for the comet assay.
Whole blood from amphibians does not appear to display this phenomenon.

3.4. Metrics for Assessing Damage

To quantify DNA damage, gels are stained with a DNA stain (e.g., ethidium
bromide, SYBR Gold, SYBR Green) and the comets scored for DNA migration
under a fluorescence microscope using an appropriate software package such as
Komet 5.5 (Kinetic Imaging, Nottingham, UK), Comet Assay IV (Perceptive
Instruments, Suffolk, UK), or others. Damaged cells have an appearance similar
to astronomical comets, with long tails of DNA migrating from the center of the
exposed nucleoid. Damage is generally quantified using three main values: comet
tail length; tail moment (i.e., tail length multiplied by the % DNA in the tail) or
Olive tail moment (i.e., distance between the center of gravity of the tail and the
center of gravity of the head, multiplied by % DNA in the tail); and % DNA in the
tail (7,8). The most informative metric is currently under debate, mainly because
computerized imaging programs tend to compute these metrics differently.
Tail length is expected to increase quickly with low levels of exposure to
a genotoxin, but this metric will plateau at higher exposures (9). However,
the amount of DNA in the tail region can continue to increase as the dose
increases, theoretically from 0 to 100% (7). Thus, with increasing dose, it is
tail intensity that continues to increase with increasing doses, not tail length
(Fig. 2). Because tail moment is calculated based on length measurements, it
has been argued that tail intensity, or % DNA in the tail, should be the best
metric of genotoxicity (10).

3.5. Freezing Samples

When the comet assay cannot be conducted quickly after sample collection,
samples can be frozen in liquid nitrogen and stored until the appropriate time,
178 Knopper and McNamee

as long as they have been placed in a proper cryopreservant (11–13). We have

found when using blood samples, Ca2+ - and Mg2+ -free PBS plus 10% DMSO
or Ca2+ - and Mg2+ -free PBS plus 10% DMSO and 20 mM EDTA (12) are
acceptable cryopreservatives. Samples should be thawed in a room temperature
water bath and processed immediately thereafter. However, comet assay data
from frozen samples cannot be directly compared to data for unfrozen samples,
as the freezing process elevates the background level of DNA migration.
As such, all experimental controls should be handled (frozen) in a similar
fashion.

3.6. Alkaline Comet Assay on Gelbond Film

1. Remove 1.5-mL Eppendorf tubes of 0.75% low melting point agarose (LMP) and
melt in microwave for approx 10–20 s on high.
2. Once melted, place tubes in a prewarmed heating block or water-bath, set at 42°C.
3. Affix Lab-Tek II chambers to Gelbond by applying constant pressure for approx
30 s. (See Note 8.)
4. Remaining steps should be conducted under subdued lighting.
5. Remove a 30-μL aliquot from the diluted cell suspension and add to 270 μL of
liquified 0.75% LMP agarose. Mix gently by pipetting. (See Note 9.)
6. Cast a 120-μL aliquot of the cell–agarose mixture into an individual well of the
two-well chamber.
7. Cast another 120-μL aliquot into a different chamber affixed to a different Gelbond
film. Repeat steps 5–6 with each sample.
8. An internal control should be run simultaneously. (See Note 10.)
9. Once the agarose has solidified, carefully remove the Lab-Tek II chambers and
place each Gelbond film in a small plastic box filled with 75 mL of lysis buffer.
10. Place dishes at 4°C overnight.
11. The following day, calibrate the pH meter.
12. Make fresh electrophoresis buffer.
13. Remove gels from the fridge/incubator/water bath.
14. Place a Petri dish in the sink and fill with fresh water. Allow water to gently run
into dish.
15. Using tweezers, remove Gelbond film from lysis buffer and repeatedly dip into
the Petri dish for about 30 s, or until the foam from the lysis buffer has drained.
(See Note 11 and 12.)
16. Place Gelbond film into the gel electrophoresis units, filled to a volume of
electrophoresis buffer required to achieve a level approx. 1 cm above the gel (See
Note 13). Allow the gels to stand for 30 min.
17. After 30 min, electrophorese gels using constant voltage (time and voltage will
depend on sample type; See Note 3).
18. Place a Petri dish in the sink and fill with fresh water. Allow water to gently run
into dish.
Comet Assay in Environmental Toxicology 179

19. Using tweezers, gently remove the Gelbond film from the electrophoresis unit and
repeatedly dip into the Petri dish for about 30 s, or until the electrophoresis buffer
has drained.
20. Transfer Gelbond film to another tray containing 75 mL of neutralization buffer
and leave for 30 min.
21. After 30 min, rinse as described in the preceding text, and place Gelbond film into
approx. 75 mL of 85% ethanol for a minimum of 2 h.
22. Remove film and then air-dry overnight.
23. Store dried gels in labeled manila envelopes.
24. To stain gels, make SYBR Gold solution in a 50-mL tube (See Subheading 2.2.,
item 10) and fill another 50-mL tube with water.
25. Cut one of the duplicate Gelbond films into strips, so each strip contains two
samples.
26. Label and place in stain. (See Note 14.)
27. After the staining period, remove the Gelbond strip with tweezers, and dip in water
two or three times.
28. Place Gelbond, gel side up, on microscope slide and cover with a 22 × 50 mm
glass cover slip.
29. Gently press with a paper towel to remove excess water and to form a seal.
30. Add a drop of immersion oil to the cover slip and then view on the microscope.
A minimum of 50 cells, on each of two duplicate slides, should be scored using
appropriate software.

3.7. Other Comet Assay Techniques for Environmental Toxicology

Three modifications to the general alkaline version on the comet assay can
also be useful tools for studies in environmental toxicology and genomics.
First, the comet assay can be combined with fluorescence in situ hybridization
(FISH) to determine in which gene regions DNA strand breaks are occurring
(14,15). Second, the comet-DNA diffusion assay, which involves precipitating
DNA with ethanol during the comet assay without the use of electrophoresis,
can be used to discriminate between the mechanisms of cellular death (i.e.,
apoptosis (programmed cell death) and necrosis (death of cells through injury
or disease) (16). Finally, the comet assay can also be conducted using a neutral
buffer rather than alkaline buffers in order to assess only double-strand DNA
breaks (5,17). This technique is also useful for assessing DNA damage in germ
cells, which possess naturally high levels of alkali-labile sites (5,18,19). (See
Note 6.)

4. Notes
1. Because the basis of the comet assay is to measure DNA damage in individual cells
(i.e., generally no fewer than 50 per sample), it is important that cell density in
180 Knopper and McNamee

the gels is not too high; otherwise the comets may overlap, making measurements
impractical or impossible. Generally, a dilution of a sample resulting in 2–4 ×
105 cells/mL is sufficient. For example, mammalian whole blood requires a 1:10
dilution with phosphate Ca2+ - and Mg2+ -free PBS whereas amphibian whole blood
requires a 1:100 dilution.
2. Under neutral pH conditions, only double-stranded DNA breaks can be revealed
using the comet assay (17). Under alkaline pH conditions (pH > 13.1), double-and
single-strand breaks as well as alkali-labile sites (expressed as single-strand breaks
under alkaline conditions) can be detected (1,4). Compared to radiation exposure,
chemical genotoxins cause orders of magnitude fewer double-strand breaks than
single-strand breaks (1). Thus, the alkaline comet assay is a more useful tool
for biomonitoring studies than the neutral version. However, in some cases cells
(e.g., sperm) may contain inherently high levels of ambient alkali-labile sites (18).
Alkaline buffers will not be useful when using these cell types because these sites
will be expressed as DNA single-strand breaks under alkaline conditions. Thus,
control cells will exhibit substantial DNA damage. In these circumstances, the use
of neutral buffers is suggested (e.g., Tris–borate EDTA or Tris–acetate EDTA; pH
7–8). Some researchers have used electrophoresis buffers of pH 9.0 (20) and 12.5
(21) with no apparent increases in ambient DNA damage.
3. If the voltage or running time used during electrophoresis is too low, DNA will
not migrate from the comet head. Conversely, if the voltage or running time is
too high, DNA from undamaged cells will migrate extensively. Thus, conditions
need to be optimized so only true damage is detected. Generally, control cells
should exhibit no more than 5–10% DNA in the tail and have tail lengths of no
more than 15–20 μm (10). The appropriate voltage and time of electrophoresis
will vary depending on the species and cell type being assessed, the equipment
being used, and on the volume of buffer required to cover the gels. Electrophoresis
voltage (constant voltage) should be expressed as V/cm, as determined by the
running voltage divided by the distance between the anode and cathode in the
electrophoresis units. A good starting point for blood samples is 1.5 V/cm for
between 16 and 20 min of electrophoresis. It is imperative that these conditions be
optimized before an experiment is conducted and then strictly adhered to within
and across experiments.
4. This recipe for lysis buffer is the standard for many cell types, but when using
germ cells, a different lysis buffer is required to expose the nucleus. For these cells,
prepare lysis buffer containing 2.5 M NaCl (146.1 g/L), 100 mM tetra-sodium
EDTA (41.6 g/L), 10.0 mM Tris-HCl (0.61 g/L). Cover and shake to remove
clumps. Slowly add 1 L of purified H2 0 and stir on a stir plate. Adjust the pH to
10.0 with concentrated NaOH or HCl. Store in the dark at roughly 21°C. Add 1%
Triton X-100 and 4 mM DTT to required volume on the day of experiment and
stir. DO NOT refrigerate. After 1 h, decant buffer and add fresh buffer containing
0.1 mg/mL of proteinase K, and incubate at 37°C overnight. The addition of DTT
and proteinase K to this buffer is required to decondense sperm chromatin.
Comet Assay in Environmental Toxicology 181

5. This recipe for electrophoresis buffer is the standard for the alkaline version of the
comet assay. Germ cells have naturally abundant alkali-labile sites (18) and require
the use of a neutral buffer. Neutral buffers are required when only double-stranded
DNA damage is being quantified. Tris–acetate EDTA or Tris–borate EDTA (pH
7–8) buffer is appropriate for use in these circumstances.
6. When stain intensity has diminished, another 5 μL of SYBR Gold can be added
to replenish the solution. This should be replenished only one time, after which
a new stain solution should be made. Dispose of the solution by pouring through
activated charcoal.
7. When using germ cells, where the chromatin is very tightly packed, cells must
undergo lysis before chemical exposure (e.g., H2 O2 ). If in vitro damage is induced
by radiation exposure, however, lysis is not required before irradiation (5).
8. New chambers have an adhesive backing, but this adhesive backing will degrade
with use. It is possible to reuse these old chambers by dipping the bottoms of
the chambers in a 1% agarose solution. Gently dip a gloved finger in melted 1%
agarose and run along the bottom of a Lab-Tek II chamber until the bottom is
coated, then affix to the Gelbond film and allow it to solidify. The agarose on the
bottom of the chamber will form a seal, and samples can then be cast as previously
described.
9. If using frozen samples: remove samples from the –80°C freezer and place in a
room temperature water bath. Once the samples have thawed (∼1–3 min), gently
mix, and place on ice.
10. If available, use a control sample. If no control is available, human blood (dilution:
20 μL of human blood mixed with 180 μL of Ca2+ - and Mg2+ -free PBS), obtained
from finger prick, is a suitable substitute. Control samples can be frozen ahead
of time and thawed when needed, but the viability and DNA damage in these
samples should be checked regularly for consistency as time spent frozen is related
to increased DNA damage.
11. Salt from lysis buffer that remains in the gel can cause misshapen comets during
electrophoresis, so proper rinsing should be ensured.
12. Some researchers who are interested in assessing for the presence of specific types
of DNA damage (e.g., DNA–protein crosslinks and several forms of oxidative base
damage) employ a second lysis step in which DNA specific endonucleases (such
as endonuclease III) or proteases are added. These enzymes recognize and attempt
to repair certain types of DNA modifications. However, these repair complexes are
unstable, and when the gels are exposed to alkaline conditions, these repair sites
degrade to yield single-strand DNA breaks. The increased level of DNA damage
in the presence of such enzymes, relative to controls, indicates the presence of
DNA lesions specific to that recognized by the enzyme added (10).
13. Because many different models of horizontal electrophoresis chambers can be
used, each with its own relative dimensions, the optimal electrophoresis buffer
volume needs to be determined. As a general practice, gels should be covered with
approx 1 cm of buffer. It is very important to be consistent and accurate with the
volume of buffer being used in the electrophoresis units, because differing buffer
182 Knopper and McNamee

volumes will result in differing electric fields strengths for DNA migration and will
yield inconsistency in results. This is particularly important for matched samples
within an experiment, if more than one electrophoresis chamber is required.
14. When using leukocytes from mammals and birds, and mammalian sperm, leave
gels in stain for 10–15 min. Whole blood from amphibians requires approx
30 min.

References
1. Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H.,
Miyamae, Y., Rojas, E., Ryu, J.-C., and Sasaki, Y.F. (2000) Single cell gel/comet
assay: guidelines for the in vitro and in vivo genetic toxicity testing. Environ. Mol.
Mutagen. 35, 206–221.
2. Krause, G., Wolz, L., and Scherer, G. (2001) Performing the “comet assay” for
genetic toxicology applications. Life Sci. News. 7:1–3.
3. McNamee, J. P., McLean, J. R. N., Ferrarotto, C., and Bellier, P. V. (2000) Comet
assay: rapid processing of multiple samples. Mutat. Res. 466, 63–69.
4. Hartmann, A., Agurell, E., Beevers, C., Brendler-Schwaab, S., Burlinson, B., Clay,
P., Collins, A. R., Smith, A., Speit, G., Thybaud, V., and Tice, R. R. (2003)
Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis
118, 45–51.
5. Singh, N. P., Muller, C. H., and Berger, R. E. (2003) Effects of age on DNA
double-strand breaks and apoptosis in human sperm. Fertil. Steril. 80, 1420–1430.
6. Strauss, G. H. (1991) Non-random cell killing in cryopreservation: implications
for performance of the battery of leukocyte tests (BLT), I. Toxic and immunotoxic
effects. Mutat. Res. 252, 1–15.
7. Collins, A. R., Dobson, V. L., Dusinská, M., Kennedy, G., and Stetina, R. (1997)
The comet assay: what can it really tell us? Mutat. Res. 375, 183–193.
8. Schnurstein, A., and Braunbeck, T. (2001) Tail moment versus tail length-
application of an in vitro version of the comet assay in biomonitoring for genotox-
icity in native surface waters using primary hepatocytes and gill cells from
zebrafish (Danio rerio). Ecotoxicol. Environ. Safe. 49, 187–196.
9. Fairbairn, D. W., Olive, P. L., and O’Neil, K. L. (1995) The comet assay: a
comprehensive review. Mutat. Res. 339, 37–59.
10. Collins, A. R. (2004) The comet assay for DNA damage and repair: principles,
applications, and limitations. Mol. Biotechnol. 26, 249–261.
11. Anderson, D., Yu, T.-W., Dobrzynska, M. M., Ribas, G., and Marcos, R. (1997)
Effects in the comet assay of storage conditions on human blood. Teratogen.,
Carcinogen. Mutat. 17, 115–125.
12. Tice, R. R., and Vasquez, M. (1999) Protocol for the application of the pH>13
alkaline single cell gel (SCG) assay to the detection of DNA damage in
mammalian cells, in Integrated Laboratory Systems. Research Triangle Park, NC.
http://www.cometassay.com/Files/raytice.doc
Comet Assay in Environmental Toxicology 183

13. Duty, S. M., Singh, N. P., Ryan, L., Chen, Z., Lewis, C., Huang, T., and Hauser,
R. (2002) Reliability of the comet assay in crypopreserved human sperm. Hum.
Reprod. 17, 1274–1280.
14. McKelvey-Martin, V. J., Ho, E. T., McKeown, S. R., Johnston, S. R., McCarthy,
P. J., Rajab, N. F., and Downes, C. S. (1998) Emerging applications of the single
cell gel electrophoresis (Comet) assay. I. Management of invasive transitional
cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for
the identification of damaged and repaired DNA sequences in individual cells.
Mutagenesis 13, 1–8.
15. Horváthová, E., Dušinská, M., Shaposhnikov, S., and Collins, A. R. (2004) DNA
damage and repair measured in different genomic regions using the comet assay
with fluorescent in situ hybridization. Mutagenesis 19, 269–276.
16. Singh, N. P. (2000) A simple method for accurate estimation of apoptotic Cells.
Exp. Cell Res. 256, 328–337.
17. Olive, P. L., Wlodek, D., and Banáth, J. P. (1991) DNA double-strand breaks
measured in individual cells subjected to gel electrophoresis. Cancer Res. 51,
4671–4676.
18. Singh, N. P., Danner, D. B., Tice, R. R., McCoy, M. T., Collins, G. D., and
Schneider, E. L. (1989) Abundant alkali-sensitive sites in DNA of human and
mouse sperm. Exp. Cell Res. 184, 461–470.
19. Haines, G. A., Hendry, J. H., Daniel, C. P., and Morris, I. D. (2001) Increased
levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or
X-irradiation of spermatogonia. Mutat. Res. 495, 21–32.
20. Duty, S. M., Singh, N. P., Silva, M., Barr, D. B., Brock, J. W., Ryan, L., Herrick, R.
F., Christiani, D. C., and Hauser, R. (2003) The relationship between environmental
exposures to phthalates and DNA damage in human sperm using the neutral comet
assay. Environ. Health Persp. 111, 1164–1169.
21. Migliore, L., Naccarati, A., Zanello, A., Scarpato, R., Bramanti, L., and Mariani,
M. (2002) Assessment of sperm DNA integrity in workers exposed to styrene.
Hum. Reprod. 17, 2912–2918.
22. Knopper, L. D., Mineau, P., McNamee, J. P., and Lean, D. R. S. (2005) Use
of comet and micronucleus assays to measure genotoxicity in meadow voles
(Microtus pennsylvanicus) living in golf course ecosystems exposed to pesticides.
Ecotoxicology 14, 323–335.

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