Mutagenesis, 2015, 30, 21–28
doi:10.1093/mutage/geu047
Original Article
Original Manuscript
Does the duration of lysis affect the sensitivity
of the in vitro alkaline comet assay?
José Manuel Enciso, Oscar Sánchez, Adela López de Cerain and
Amaya Azqueta*
Department of Pharmacology and Toxicology, University of Navarra, Irunlarrea 1, 31009 Pamplona, Spain
*To whom correspondence should be addressed. Tel: +34 948425600 ext. 806343; Fax: +34 948425652; Email: [email protected]
Received May 14 2014; Revised August14 2014; Accpeted August 14 2014.
Abstract
The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in
cells. Several attempts have been made to identify and evaluate the critical points affecting the comet
assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able
to compare the results obtained in different laboratories. However, reports on the effect of modifying
the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control
HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or
H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine
DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA
damage levels were detected in the absence of lysis or after 1 h of lysis when the standard comet
assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer
lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was
necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after
5 min or 1 h of lysis and the response increased up to 24 h. In conclusion, the time of lysis can be varied
depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should
be used if results from different experiments or laboratories are to be compared.
Introduction
The alkaline comet assay is now the method of choice for measuring
different kinds of DNA damage in cells (1). This assay is sensitive,
detecting DNA break frequencies down to a few hundred per cell,
and it can be applied to almost all cell types, as long as a singlecell or nuclear suspension can be obtained (i.e. including animal and
human tissues and plants). Briefly, cells are immobilised in agarose on
a support (a glass microscope slide or a plastic film), lysed to obtain
nucleoids and then DNA is unwound, electrophoresed, stained and
visualised under a fluorescence microscope. If strand breaks (SBs) or
alkali-labile sites (ALS) are present, they relax the supercoiled DNA,
allowing it to migrate towards the anode. The amount of DNA that
is able to migrate is quantified and it reflects the frequency of the
aforementioned lesions. Other lesions can be detected with the use
of specific endonucleases (e.g. oxidised and alkylated bases, misincorporated uracil, pyrimidine dimers) (2) or with some modification
of the protocol (e.g. DNA–DNA and DNA–protein cross-links) (3).
Moreover, it can also be used to measure DNA repair (4). Finally, the
comet assay is easily learned, quick and economical both in equipment and in materials.
The comet assay is used in genotoxicity testing where sensitive,
specific and reliable methods are required. Its sensitivity in detecting
low levels of DNA damage and its specificity have been widely demonstrated, but its reliability is still under debate; a high inter-laboratory
variation is often reported and has been specifically investigated in the
European Comet Assay Validation Group project (5). The variation is
most likely due to the many different protocols that are in use and the
lack of understanding of the critical steps. Attempts have been made to
control for the variation by including reference standard cells (treated
with an appropriate agent) in each experiment alongside experimental
samples, or even within the same gel as the sample, in which case a
way of distinguishing sample and standard cells is needed (6).
The standard alkaline version of this technique was first described
by Singh et al. (7). They embedded untreated, X-ray- or H2O2treated human lymphocytes in agarose on a glass microscope slide
(untreated, X-ray- or H2O2-treated lymphocytes) and lysed them for
© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.
For permissions, please e-mail: [email protected].
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1 h in a buffer at pH 10 containing a high concentration of salts
and detergents (2.5 M NaCl, 100 mM Na2-EDTA, 10 mM Tris, 1%
sodium sarcosinate and 1% Triton X-100), unwound the DNA for
20 min in an alkaline solution (1 mM Na2-EDTA and 0.3 M NaOH,
pH > 13) and then electrophoresed it in the same buffer at 25 V (they
did not specify the voltage gradient, V/cm) for 20 min. This protocol is essentially the most widely used nowadays for performing the
alkaline comet assay, with unwinding and electrophoresis carried
out at pH > 13 (so the alkali-labile damage is converted into breaks
and detected), but several small details vary between protocols (and
laboratories).
The high inter-experimental variation with this assay may be
ascribed to these small variations in protocols: different concentrations of agarose for embedding the cells (even the use or not of an
extra layer of agarose on top of the layer containing the cells), different compositions of the lysis solution (e.g. with or without sodium
sarcosinate, with or without dimethylsulphoxide, DMSO), different
duration of lysis, different duration and temperature of the unwinding period and electrophoresis, different voltages in the electrophoresis, different ways of staining comets, different methods of scoring
(e.g. different image analysis software) and even different ways of
expressing the results (e.g. using the % tail DNA, tail moment, tail
length or proportion of damaged cells). When enzymes are used to
detect different lesions, we can add at least two more parameters to
this list: concentration of the enzyme and time of incubation.
Vijayalaxmi et al. (8) studied the effect of different duration of
the alkaline unwinding treatment (20, 40 or 60 min) and of electrophoresis (20 or 40 min at 25 V—they did not specify the V/
cm—and 300 mA) and they observed that increasing the duration
of the alkaline treatment and electrophoresis enhanced the length
of DNA migration in both control and γ-irradiated human lymphocytes. Some years later, Yendle et al. (9) checked the effect of the
duration of alkaline incubation using longer times (0.3, 1, 2, 4, 8 or
18 h) in mouse keratinocytes exposed in vitro to N-methyl-N-nitroN-nitrosoguanidine or to the solvent DMSO. The extent of the DNA
damage measured using different parameters increased as the alkaline incubation time increased: control cells substantially increased
their DNA damage after 4 h of alkaline incubation (until then a very
slight increase was observed) while treated cells increased their DNA
damage from 0.3 to 1 h. After 18 h of alkaline incubation, treated
cells presented all the DNA in the tail. Similar experiments were performed by Speit et al. (10). They tested the duration of the alkaline
incubation (20 or 40 min) and of electrophoresis (20, 30 or 40 min at
0.68 V/cm) with whole blood and V-79 cells (Chinese hamster lung
fibroblasts) showing an increase in DNA damage when longer alkaline incubation or electrophoresis was carried out. The duration of
the alkaline incubation was also studied in γ-irradiated blood cells
showing an increase of DNA damage with time. Interestingly, they
performed the two steps, alkaline incubation and electrophoresis, at
4 and 20°C and they observed an increase in the DNA damage when
the steps were carried out at 20°C. Quite recently, Azqueta et al. (11)
studied the effect of the final agarose concentration in gels (0.4, 0.6,
0.8 or 0.95%), the duration of the alkaline incubation (10, 20, 40
or 60 min) and different voltages (0.16, 0.49, 0.83, 1.15 or 1.48 V/
cm), duration (5, 10, 20, 30 and 40 min) and current (210, 270, 325
and 400 mA) during the electrophoresis of control and H2O2-treated
TK-6 cells (human lymphoblast cell line) and human lymphocytes.
The different concentrations of agarose did not have an effect in
control cells, but there was a decrease in the level of DNA damage
detected in the treated cells as the agarose concentration increased.
The apparent DNA damage increased with longer alkaline incubation, higher voltages and longer electrophoresis. While slightly
J. Ge et al., 2015, Vol. 30, No. 1
affected by changes in conditions, control cells presented relatively
low values of DNA damage in almost all tested conditions. Ersson
and Möller (12) published in the same year very similar results in
γ-irradiated THP 1 cells (human acute monocytic leukaemia cell
line), H2O2-treated A549 cells (human type II alveolar epithelial
cell line) and untreated cells. They also studied the duration of the
enzyme and alkaline incubations in A549 cells treated with the photosensitiser Ro 19-8022 (Ro) plus light (to induce 8-oxoGua) using
the alkaline comet assay in combination with formamidopyrimidine
DNA glycosylase (FPG). The detection of net FPG-sensitive sites was
affected by both the duration of the alkaline and the enzyme incubations (saturation was seen after 30 min with FPG). There have also
been some studies of comet staining; Olive et al. (13) observed that
different concentrations of propidium iodide influenced the sensitivity of the comet assay in X-irradiated V-79 cells and controls. This
group also concluded that the same sensitivity to detect DNA damage was observed when comets from the same cell type, X-irradiated
V-79 cells and controls, were stained with propidium iodide (an
intercalating DNA-binding dye), Hoechst 33342 and 4,6-diamidino2-phenylindole (DAPI; both non-intercalating DNA-binding dyes)
(14). They also tested the option of incorporating bromodeoxyuridine (BrdUrd) into replicating DNA and using fluorescein isothiocyanate-conjugated anti-BrdUrd antibodies to visualise the comets;
this was as sensitive as the use of conventional dyes. This group
uses only an alkaline lysis solution (pH > 13, see below), but results
regarding different staining methods also apply when the standard
alkaline comet assay protocol is used.
Complete standardisation of the alkaline comet assay has not
been achieved and although some important factors that influence the results have been identified, there is still some work to
do. Among other parameters, the duration of the lysis step has
not been thoroughly studied though several times of lysis are normally used. Very often, comet assay protocols specify that cells
should be lysed for at least 1 h, this time being the most widely
used (15,16). Nevertheless, overnight or 24-h lysis periods are
also very common and practical; lysis has been considered as
the step where the operator can safely and conveniently pause
the comet assay. Longer lysis periods are occasionally employed.
Although cell lysis is normally performed in a buffer containing
a high concentration of salts and detergents at pH 10 (to destabilise the cellular and nuclear membranes and deproteinise the
cell including the removal of histones), a few researchers perform
the standard alkaline comet assay by lysing cells in an alkaline
solution (pH > 13), so lysis of the cells and unwinding of DNA
are performed at the same time (13,14,17,18). (This last option
is not appropriate when the comet assay is used in combination
with enzymes to detect specific DNA lesions.) Besides, some cell
types such as human or animal keratinocytes (19) and human
buccal cells (20), used in biomonitoring studies, need extensive
lysis including digestion with proteinase K to remove residual
proteins. Moreover, in the case of sperm, which present a tight
packaging of DNA, not only proteinase K but also dithiothreitol
is used (21).
The aim of this work was to study the effect of changing the
duration of lysis of cells with a buffer containing a high concentration of salts and detergents at pH 10, followed by alkaline unwinding and electrophoresis (pH > 13), i.e. the most common protocol.
We have performed the experiments using the standard alkaline
comet assay in untreated and methyl methanesulfonate (MMS)- or
H2O2-treated HeLa cells and the alkaline comet assay combined
with FPG in the same cell line, untreated or treated with Ro plus
visible light.
Duration of lysis in the in vitro alkaline comet assay, 2015, Vol. 30, No. 1
Materials and methods
Cells
HeLa cells, derived from human cervical cancer and originally from
the American Type Culture Collection, were thawed using standard
procedures and grown in Minimum Essential Medium with l-glutamine supplemented with 10% foetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin in a humidified atmosphere with
5% CO2 at 37°C. The cells were used while in exponential growth
phase.
Treatment of cells, preparation of the slides
and lysis
MMS treatment and different times of lysis
Taking into account the results obtained in a previous dose–response
experiment, 90 and 180 µM of MMS, a known monofunctional
alkylating agent, were chosen to perform this experiment. DMSO
was used as the solvent and so provided the negative control (the
final concentration in the cell culture was 1% DMSO in all cases).
Cells were incubated with different concentrations of either
MMS or the solvent for 3 h at 37°C. After that, cells were washed
twice with Dulbecco’s phosphate buffered saline (PBS), trypsinised,
counted and resuspended in PBS to obtain a cellular suspension of
~1 × 106 cells/ml. Thirty microlitres of the cellular suspension were
mixed with 140 μl of 1% low melting point agarose in PBS at 37°C.
Immediately, two drops of 70 μl were placed on a glass microscope
slide (pre-coated with 1% normal melting point agarose in water
and dried) and covered with a 20 × 20 mm coverslips. Gels were set
on a metal plate on ice for 3 min and the coverslips were removed.
Ten different times of lysis were tested for each of the three concentrations (0, 90 and 180 µM MMS): 0 min (no lysis), 5, 10, 20 and
40 min, 1, 24, 48 and 72 h, and 1 week. Consequently, 10 identical slides were prepared from each cell suspension. To make this
experiment possible, just after preparing the slides and removing the
coverslips, the 1, 24, 48 and 72 h and 1 week slides were placed in a
Coplin jar containing lysis solution (2.5 M NaCl, 0.1 M Na2-EDTA,
10 mM Tris, pH 10.0, 1% Triton X-100). The other slides (from 0 to
40 min) remained on the metal plate on ice and were placed in lysis
at appropriate times so that all five slides, together with the 1 h of
lysis slide, finished the lysis step at the same time and were consequently analyzed in the same run.
H2O2 treatment and different times of lysis
Different concentrations of H2O2 were tested using PBS as the solvent
and negative control. From a dose–response curve previously made,
the concentrations of 10 and 40 μM were chosen; H2O2 working
solutions were prepared just before performing the assay. Cells were
washed twice with PBS, trypsinised, counted and resuspended in PBS
to obtain a cellular suspension of ~1 × 106 cells/ml. After preparing
the slides as previously described, the coverslips were removed and
the slides were dipped into their corresponding H2O2 solution or PBS
for 5 min at 4°C. Afterwards, slides were thoroughly washed with
PBS at 4°C. Again, 10 different times of lysis were tested for each
one of the three concentrations (0, 10 and 40 μM H2O2) from 0 min
(no lysis) to 1week. The same lysis strategy explained in the previous
section was followed.
Ro 19-8022 treatment and different times of lysis
Ro 19-8022 (Ro), which specifically produces oxidised purines
(mainly 8-oxoguanine) in the presence of visible light, was kindly
23
given by Hoffmann-La Roche. According to a previous dose–
response curve, 1 µM of Ro was chosen. Since this compound is
photosensitive, Ro solutions were prepared in darkness just before
treating the cells. PBS was used as the solvent and so acted as the
negative control.
HeLa cells were incubated with 0 and 1 µM of Ro 30 cm below
a 500 W source of light for 5 min on ice. After washing the cells with
PBS, they were trypsinised, counted, centrifuged and resuspended in
PBS to obtain a cellular suspension of ~1 × 106 cells/ml. Besides, for
each concentration, seven different times of lysis were tested: 0 min
(no lysis), 5, 15 and 30 min, 1 and 24 h and 1 week. Slides were prepared exactly as explained above, but two identical slides were prepared for each condition (Ro concentration and lysis time): ‘Buffer
F’ slide, where gels would be incubated with FPG reaction buffer
(Buffer F), and ‘FPG’ slide, where gels would be incubated with FPG.
The same strategy explained for the other experiments was followed
in this one so that the 1 h, 24 h and 1 week slides were placed in one
Coplin jar with lysis solution, while the other slides were transferred
from the cold metal plate at staggered intervals.
Effect of holding cells on ice
Experiments were carried out to see how the time the slides remained
on ice affected the results. Cells were treated with 0, 90 or 180 µM
MMS or 0, 10 and 40 µM H2O2 or 0 and 1 µM Ro plus light, as
explained in the previous sections. In the case of MMS- and H2O2treated cells, two electrophoresis runs were carried out: one with
the 0 and 5 min lysis samples and the other with the 1 h lysis samples; thus, it was possible to place slides in lysis immediately after
treatment, avoiding any holding on ice. In the case of Ro plus lighttreated cells, only 5 min and 1 h of lysis were tested in two independent electrophoresis runs, since in the previous experiment, no
damage had been detected in the absence of lysis (with FPG).
Rest of the comet assay: from lysis to scoring
After the appropriate times of lysis, slides of cells treated with either
MMS or H2O2 were immersed in an alkaline buffer (0.3 M NaOH,
1 mM EDTA, pH > 13) at 4°C for 40 min. After that, electrophoresis
was performed at 1.2 V/cm in the same buffer at 4°C and ~300 mA
for 20 min. Following electrophoresis, slides were neutralised with
PBS for 10 min at 4°C, washed in distilled water for another 10 min
at 4°C and air-dried at room temperature.
In the case of Ro plus light-treated cells, before the alkaline treatment was performed, the slides were washed three times (5 min each)
with the FPG reaction buffer (40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml
bovine serum albumin, pH 8.0—Buffer F) at 4°C and then incubated
with either Buffer F or FPG in a humidified atmosphere at 37°C for
30 min. FPG was a crude protein extract prepared from overproducing bacteria and used at a dilution determined in a titration experiment to give maximum detection of 8-oxoguanine (induced with Ro
plus light) with minimal non-specific strand breakage.
Afterwards, DNA in each gel was stained with 1 μg/ml DAPI, and
comets were visualised under a fluorescence microscope (NIKON
Eclipse 50 i). DNA damage was quantified in 100 randomly selected
comets per slide (50 comets in each gel) by measuring the % tail
DNA using the image analysis software Comet Assay IV (Perceptive
Instruments Ltd). For each slide, the median value of the % tail DNA
was calculated. In the case of Ro-treated cells, the net oxidative damage induced was calculated by subtracting the median value of the
‘Buffer F’ slide from the one obtained in the ‘FPG’ slide for each
condition.
J. Ge et al., 2015, Vol. 30, No. 1
24
Statistics
Three independent experiments were carried out in each case and
the means of the median values of % tail DNA and the standard
deviation were calculated. Further descriptive statistical analysis was
not performed since the results obtained (i.e. differences and effects)
are clear enough and our conclusions would not be affected by the
statistical analysis.
Results
Experiments with MMS
The results obtained performing the alkaline comet assay with
10 different times of lysis in HeLa cells treated with 0, 90 and
180 µM MMS for 3 h are shown in Figure 1. Similar, low values
of % tail DNA were obtained for cells treated with DMSO, the
negative control, regardless of the time of lysis employed. A dose
response was observed for each time of lysis tested. In the case
of MMS-treated cells, with from 0 to 40 min of lysis, the % tail
DNA for each dose was similar, and the % tail DNA for cells
treated with 180 µM MMS was higher than the % tail DNA
after 1 h of lysis (these last samples did not remain on ice after
being prepared). Besides, it can be clearly seen that the % tail
DNA for 90 and 180 µM MMS increases gradually from 1 h to
1 week of lysis.
The experiment with HeLa cells treated with MMS in the
absence of lysis, and with 5 min and 1 h of lysis, was repeated, but in
this case, all samples were processed immediately after preparation
of the slides, so that they did not spend any time on ice (Figure 2).
A dose response was also observed for each time of lysis and even in
its absence. For the three tested concentrations (0, 90 and 180 µM
MMS), similar results were observed in the absence of lysis and after
5 min and 1 h of lysis. A very slight increase in the % tail DNA was
observed when increasing the time of lysis. On the other hand, the
results obtained in the absence of lysis or 5 min of lysis are similar
but lower than seen in the previous experiment for the same lysis
condition, suggesting that the time the slides of the first experiment
remained on ice (Figure 1) was a factor affecting the yield of DNA
damage.
Experiments with H2O2
The results obtained performing the alkaline comet assay with 10
different times of lysis in HeLa cells treated with 0, 10 and 40 µM
H2O2 for 5 min are shown in Figure 3. Results are similar to the ones
obtained with MMS in that a dose response was observed for each
time of lysis tested, and there were no relevant differences in the
DNA damage observed in HeLa cells treated with PBS (the negative
control) for the different times of lysis. For cells treated with each
concentration of H2O2, similar levels of damage are observed from 0
to 40 min of lysis; they do not differ from the damage levels seen after
1 h of lysis (with no time on ice)—in contrast with the results with
MMS. However, we observed that as lysis was performed for more
than 1 h, the DNA damage detected increased for the treated cells.
The results obtained performing the alkaline comet assay with
HeLa cells treated with H2O2 after no lysis, 5 min and 1 h of lysis,
with no time spent on ice before lysis, are shown in Figure 4. A dose
response was also observed here in all the cases including the absence
of lysis. Similar results were obtained in the absence of lysis and after
5 min and 1 h of lysis.
Experiments with Ro 19-8022
The results obtained performing the comet assay in combination
with FPG with different times of lysis in HeLa cells treated with 0
and 1 µM Ro (plus light) are shown in Figure 5.
First of all, no DNA damage was detected in the absence of
lysis. Secondly, there were essentially no differences between the
DNA damage levels observed in HeLa cells between 5 min and 1 h
of lysis. Finally, if lysis was performed for either 24 h or 1 week,
the detected DNA damage increased for both the negative control
and the Ro-treated cells, but it did not increase from 24 h to 1 week
of lysis.
The results obtained executing the comet assay with HeLa cells
treated with Ro after 5 min and 1 h of lysis with no time spent on ice
Figure 1. Effect of different times of lysis on the DNA damage detected by the comet assay in HeLa cells treated with different concentrations of MMS. The
slides from 0 min (no lysis) to 40 min of lysis remained on ice for different times. Mean and standard deviation obtained in three independent experiments are
represented.
Duration of lysis in the in vitro alkaline comet assay, 2015, Vol. 30, No. 1
25
Figure 2. Effect of no lysis, 5 min and 1 h of lysis on the DNA damage detected by the comet assay in HeLa cells treated with different concentrations of MMS.
Samples were processed immediately after preparation of the slides. Mean and standard deviation obtained in three independent experiments are represented.
Figure 3. Effect of different times of lysis on the DNA damage detected by the comet assay in HeLa cells treated with different concentrations of H2O2. The
slides from 0 min (no lysis) to 40 min of lysis remained on ice for different times. Mean and standard deviation obtained in three independent experiments are
represented.
are shown in Figure 6. Values of % tail DNA did not differ between
5 min and 1 h of lysis for both treated and untreated cells. There were
no difference between the slides that remained on ice (Figure 5) and
the slides that did not (Figure 6).
Discussion
Standardisation of the in vitro comet assay would be a very important step to overcome the inter-laboratory variability that this assay
presents. In particular, it would be very helpful in order to achieve
acceptance by regulatory bodies and—ultimately—an Organisation
for Economic Co-operation and Development (OECD) guideline for
the testing of chemicals. At present only the in vivo comet assay has a
draft OECD guideline (22) and is recommended in the strategy to test
the genotoxicity of pharmaceuticals published by the International
Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (23). This in vivo
version of the comet assay has also been proposed for performing
genotoxicity studies for the risk assessment of substances in food
and animal feedstuffs by the European Food Safety Authority (24).
However, the in vitro comet assay is widely used for screening novel
cosmetics (where only in vitro testing is allowed) and pharmaceuticals, and it is recommended as an appropriate test for use under the
Registration, Evaluation, Authorisation and Restriction of Chemical
Substances programme of the European Commission. The Japanese
Center for the Validation of Alternative Methods in collaboration
with the European Centre for the Validation of Alternative Methods
and the Interagency Coordinating Committee on the Validation of
Alternative Methods are now focused in the in vitro comet assay to
achieve an OECD guideline in the future.
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J. Ge et al., 2015, Vol. 30, No. 1
Figure 4. Effect of no lysis, 5 min and 1 h of lysis on the DNA damage detected by the comet assay in HeLa cells treated with different concentrations of H2O2.
Samples were processed immediately after preparation of the slides. Mean and standard deviation obtained in three independent experiments are represented.
Figure 5. Effect of different times of lysis on the net FPG-sensitive sites detected by the comet assay in HeLa cells treated with Ro 19-8022 (plus light). The
slides from 0 min (no lysis) to 30 min of lysis remained on ice for different times. Mean and standard deviation obtained in three independent experiments are
represented.
Taking into account this scene, it is important to detect the critical points of the comet assay that influence its results and sensitivity. It is known that several variables have marked effects on comet
assay outcome, but the duration of lysis, which is normally at least
1 h but can be as long as weeks, has not been thoroughly studied.
In this article, we have studied the effect of different times of lysis
in untreated and MMS- or H2O2-treated HeLa cells in the standard
alkaline comet assay to detect SBs and ALS as well as in untreated
and Ro-treated HeLa cells in the alkaline comet assay in combination with FPG to detect oxidised bases. Lysis at pH 10 leaves the
DNA undenatured and the ALS are not converted into breaks.
Our results showed that the duration of lysis has a clear effect on
the results obtained with the standard alkaline comet assay, where
SBs and ALS are detected. The duration of lysis (from none to 1
week) does not have any effect in untreated cells (Figures 1–4), but it
has a marked effect in H2O2- and MMS-treated ones. Yendle et al. (9)
also affirm that leaving untreated cells (rat hepatocytes or nasal cells,
L5178Y cells—a mouse lymphoma cell line—and keratinocytes) for
18 h in lysis failed to produce comet tails. However, the detection of
SBs and ALS increased with increasing time of lysis from 1 h to at
least 1 week (Figures 1 and 3). Performing the alkaline comet assay
without the lysis step also gave reliable results; similar results were
Duration of lysis in the in vitro alkaline comet assay, 2015, Vol. 30, No. 1
27
Figure 6. Effect of 5 min and 1 h of lysis on the net FPG-sensitive sites detected by the comet assay in HeLa cells treated with Ro 19-8022 (plus light). Samples
were processed immediately after preparation of the slides. Mean values and standard deviation, based on three independent experiments, are shown.
obtained in both H2O2- and MMS-treated cells in the absence of lysis
or after 1 h of lysis (the most widely used), probably due to the lysing
effect of the alkaline treatment (Figures 2 and 4). Nevertheless, lower
sensitivity is observed compared with the use of longer times of lysis
(i.e. 24, 48, 72 and 1 week of lysis) (Figures 1–4). Vivek Kumar et al.
(25) evaluated whether the alkaline treatment of cells could replace
lysis. They performed the standard alkaline comet assay and the
modified version (removing the lysis step) in lymphocytes irradiated
with different doses of γ-rays and controls. Adding 0.02 M Trizma
to the alkaline solution was necessary to avoid an increase in the
background level of DNA damage in control cells when the modified version was applied. We did not observe such an effect when the
lysis step was removed from the protocol (Figures 2 and 4). On the
other hand, they found good correlation and satisfactory sensitivity
of the DNA damage detected with both protocols; nevertheless, the
values obtained with the modified version, without the lysis of the
cells, were slightly lower. In our case, the sensitivity of the alkaline
comet assay when we removed the lysis step was the same as performing the lysis of the cells for 1 h, but we incubated the cells in the
alkaline buffer for 40 min while Vivek Kumar et al. did it for only
20 min (20).
Looking at the experiment where the slides remained on ice during different times to be able to test different times of lysis (from
0 to 40 min) in the same electrophoresis run (Figures 1 and 3), it
is clear that holding on ice has an effect in MMS-treated cells, but
this is not so clear for the H2O2-treated cells. Cells that were not
lysed at all but that remained on ice during 1 h before the alkaline
treatment showed a clear dose response in both H2O2- and MMStreated cells (Figures 1 and 2: 0 min of lysis), but, in the latter case,
the sensitivity was higher than if the cells were in the lysis solution
for 1 h (Figure 1: 0 min and 1 h of lysis). In this case, the alkaline
treatment is also helping in the lysis of the cells, but a clear difference
is seen compared with cells that have not been on ice. More experiments are needed to be able to explain this interesting phenomenon
and to check if shorter times on ice have the same effect. A possible
hypothesis is that the MMS inside the cells could keep on damaging
DNA while the cells remained on ice. The H2O2 is a very unstable
compound that exerts its effect in a very short time, so there is probably no H2O2 inside the cells to induce more damage while the cells
remain on ice.
When carrying out the alkaline comet assay in combination
with FPG to detect the Ro-induced oxidised bases, it is clear that
the enzyme is not capable of getting into the cells to digest FPGsensitive sites without prior lysis (Figure 5). Nevertheless, 5 min of
lysis was enough for the FPG to enter into the cells and to detect
the Ro plus light-induced oxidised bases; there were no differences
between 5 min and 1 h of lysis (Figure 6). After 24 h of lysis, a higher
level of lesions was detected (Figure 5); however, the frequency of
FPG-sensitive sites increased not only in Ro-treated cells but also in
the negative control. Similar levels of damage were seen after 1 week
and after 24 h of lysis (Figure 5). Lacoste et al. (26) studied the influence of the duration of the lysis on the FPG-sensitive sites induced by
an alkylating agent. The FPG-sensitive sites detected with the comet
assay after alkylation treatment correspond to N7-methylguanine
(N7-mG) (27,28). In fact, FPG detects formamidopyrimidines (fapy)
that are produced after a strong alkali treatment of N7-mG. Lacoste
et al. (26) wanted to check if lysis at pH 10.0 could make this transformation efficiently. To do so, they performed the alkaline comet
assay in combination with FPG and using different times of lysis
(0.5–24 h) in U937 cells (human histiocytic lymphoma) treated with
dimethyl sulphate, a source of N7-mG. They observed that the number of FPG-sensitive sites detected increased gradually with the time
of lysis up to 12 h. Holding the slides on ice for different times did
not have any effect on the yield of FPG-sensitive sites—in contrast
with the effect seen here with MMS-treated cells and consistent
with an explanation in terms of damage from residual MMS within
the cells; Ro, even if still present, would not induce damage in the
absence of strong light.
Our results demonstrate that using different times of lysis affects
the results of both the standard alkaline comet assay and the alkaline
comet assay in combination with FPG. This condition could be used
to increase or decrease the sensitivity of the assay for detection of
28
certain lesions, but a constant time of lysis should be used if results
from different experiments or laboratories are to be compared. On
the other hand, different cells and different types of lesions may
require different conditions in order to achieve the desired sensitivity.
Acknowledgements
We thank Andrew R. Collins for the gift of the enzyme FPG and F. Hoffmann-La
Roche for the gift of Ro 19-8022.
Conflict of interest statement: None declared.
References
1. Azqueta, A. and Collins, A. R. (2013) The essential comet assay: a comprehensive guide to measuring DNA damage and repair. Arch. Toxicol., 87,
949–968.
2. Collins, A. R. (2011) The use of bacterial repair endonucleases in the
comet assay. In Gautier, J.-C. (ed.), Methods in Molecular Biology 691,
Drug Safety Evaluation. Humana Press, New York, USA. pp. 137–147.
3. Pfuhler, S. and Wolf, H. U. (1996) Detection of DNA-crosslinking agents
with the alkaline comet assay. Environ. Mol. Mutagen., 27, 196–201.
4. Azqueta, A., Langie, S. A., Slyskova, J. and Collins, A. R. (2013) Measurement of DNA base and nucleotide excision repair activities in mammalian
cells and tissues using the comet assay—a methodological overview. DNA
Repair, 12, 1007–1010.
5. Møller, P., Möller, L., Godschalk, R. W. and Jones, G. D. (2010) Assessment and
reduction of comet assay variation in relation to DNA damage: studies from
the European Comet Assay Validation Group. Mutagenesis, 25, 109–111.
6. Zainol, M., Stoute, J., Almeida, G. M., Rapp, A., Bowman, K. J. and Jones,
G. D.; ECVAG (2009) Introducing a true internal standard for the Comet
assay to minimize intra- and inter-experiment variability in measures of
DNA damage and repair. Nucleic Acids Res., 37, e150.
7. Singh, N. P., McCoy, M. T., Tice, R. R. and Schneider, E. L. (1988) A simple
technique for quantitation of low levels of DNA damage in individual
cells. Exp. Cell Res., 175, 184–191.
8. Vijayalaxmi, Tice, R. R. and Strauss, G. H. S. (1992) Assessment of radiation-induced DNA damage in human blood lymphocytes using the singlecell gel electrophoresis technique. Mutat. Res., 271, 243–252.
9. Yendle, J. E., Tinwell, H., Elliott, B. M. and Ashby, J. (1997) The genetic
toxicity of time: importance of DNA-unwinding time to the outcome of
single-cell gel electrophoresis assays. Mutat. Res., 375, 125–136.
10.Speit, G., Trenz, K., Schütz, P., Rothfuss, A. and Merk, O. (1999) The
influence of temperature during alkaline treatment and electrophoresis on
results obtained with the comet assay. Toxicol. Lett., 110, 73–78.
11.Azqueta, A., Gutzkow, K. B., Brunborg, G. and Collins, A. R. (2011)
Towards a more reliable comet assay: optimising agarose concentration,
unwinding time and electrophoresis conditions. Mutat. Res., 724, 41–45.
12.Ersson, C. and Möller, L. (2011) The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments. Mutagenesis, 26, 689–695.
J. Ge et al., 2015, Vol. 30, No. 1
13.Olive, P. L., Banáth, J. P. and Durand, R. E. (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured
using the “comet” assay. Radiat. Res., 122, 86–94.
14.Olive, P. L., Wlodek, D., Durand, R. E. and Banáth, J. P. (1992) Factors
influencing DNA migration from individual cells subjected to gel electrophoresis. Exp. Cell Res., 198, 259–267.
15.Tice, R. R., Agurell, E., Anderson, D. et al. (2000) Single cell gel/comet
assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen., 35, 206–221.
16.Collins, A. R., Oscoz, A. A., Brunborg, G., Gaivão, I., Giovannelli, L.,
Kruszewski, M., Smith, C. C. and Stetina, R. (2008) The comet assay: topical issues. Mutagenesis, 23, 143–151.
17.Banáth, J. P., Kim, A. and Olive, P. L. (2001) Overnight lysis improves the
efficiency of detection of DNA damage in the alkaline comet assay. Radiat.
Res., 155, 564–571.
18. Olive, P. L. and Banáth, J. P. (2006) The comet assay: a method to measure
DNA damage in individual cells. Nat. Protoc., 1, 23–29.
19.Decome, L., De Méo, M., Geffard, A., Doucet, O., Duménil, G. and Botta,
A. (2005) Evaluation of photolyase (Photosome) repair activity in human
keratinocytes after a single dose of ultraviolet B irradiation using the
comet assay. J. Photochem. Photobiol. B, 79, 101–108.
20. Rojas, E., Valverde, M., Sordo, M. and Ostrosky-Wegman, P. (1996) DNA
damage in exfoliated buccal cells of smokers assessed by the single cell gel
electrophoresis assay. Mutat. Res., 370, 115–120.
21.Baumgartner, A., Cemeli, E. and Anderson, D. (2009) The comet assay in
male reproductive toxicology. Cell Biol. Toxicol., 25, 81–98.
22. OECD (2013) Draft: In vivo Mammalian Alkaline Comet Assay. OECD
Guidelines for the Testing of Chemicals, Section 4. OECD Publishing,
Paris, France.
23.ICH Harmonised Tripartite Guideline S2 (R1) (2011) Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for
Human Use. http://www.ich.org/products/guidelines/safety/article/safetyguidelines.html. International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use. ICH
Publishing, Geneva, Switzerland.
24.EFSA Scientific Committee (2011) Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment. EFSA J., 9,
2379 [69 pp.].
25.Vivek Kumar, P. R., Cheriyan, V. D. and Seshadri, M. (2009) Could a
strong alkali deproteinization replace the standard lysis step in alkaline
single cell gel electrophoresis (comet) assay (pH>13)? Mutat. Res., 678,
65–70.
26.Lacoste, S., Castonguay, A. and Drouin, R. (2006) Formamidopyrimidine
adducts are detected using the comet assay in human cells treated with
reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK). Mutat. Res., 600, 138–149.
27.Angelis, K. J., Dusinská, M. and Collins, A. R. (1999) Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity.
Electrophoresis, 20, 2133–2138.
28. Speit, G., Schütz, P., Bonzheim, I., Trenz, K. and Hoffmann, H. (2004) Sensitivity of the FPG protein towards alkylation damage in the comet assay.
Toxicol. Lett., 146, 151–158.