410884

s-Gutirrez et alJournal of Histochemistry & Cytochemistry

The Author(s) 2010

JHCXXX10.1369/0022155411410884Cort

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Journal of Histochemistry & Cytochemistry 59(7) 655660

The Author(s) 2011
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DOI: 10.1369/0022155411410884
http://jhc.sagepub.com

New Application of the Comet Assay: ChromosomeComet Assay

Elva I. Corts-Gutirrez, Martha I. Dvila-Rodrguez, Jos Lus Fernndez, Carmen
Lpez-Fernndez, Altea Goslbez, and Jaime Goslvez

Divisin de Gentica, Centro de Investigacin Biomdica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, Mxico (EICG,MIDR), INIBIC
Unidad de Gentica, Complejo Hospitalario Universitario Juan Canalejo, As Xubias, La Corua, Spain (JLF), and Universidad Autnoma de Madrid,
Madrid, Spain (CLF,AG,JG)

Summary
The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA
damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze
white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including
buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can
be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome
isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant
of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole
nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA
damage produced.This protocol has great potential for the highly reproducible study of DNA damage and repair in specific
chromosomal domains. (J Histochem Cytochem 59:655660, 2011)
Keywords
comet assay, chromosome, DNA damage

The alkaline comet assay is widely used in various research

areas, including biomonitoring, routine genotoxicity assessment, and studies of DNA repair processes. Ostling and
Johanson (1984) were the first to quantify DNA damage in
cells using a microgel electrophoresis technique known as
single-cell gel electrophoresis or the comet assay.
However, the neutral conditions they used only allow the
detection of double-stranded DNA breaks. Later, the protocol was adapted by Singh et al. (1988) for use under alkaline conditions, producing a sensitive version of the assay
that could assess both double- and single-stranded DNA
breaks and detect alkali-labile sites, which are expressed as
frank strand breaks in DNA. Since its initial development,
the assay has been modified at various steps (lysis and electrophoretic conditions) to make it suitable for assessing different types of DNA damage in different cell types (Collins
2004; Speit and Hartmann 2005). The assay is now a wellestablished, simple, versatile, visual, rapid, and sensitive
tool, used extensively to assess DNA damage and DNA

repair in individual cell populations (Olive and Banath

2006). Some other lesions involved in DNA damage, such
as DNA crosslinks (e.g., thymine dimers) and oxidative
DNA damage, can also be assessed using lesion-specific
antibodies or specific DNA repair enzymes in the comet
assay. The assay has gained wide acceptance as a valuable
tool in fundamental DNA damage and repair studies (Speit
and Hartmann 2005), genotoxicity testing (Moller 2005),
and human biomonitoring (Kassie et al. 2000; Moller
2006a). White blood cells or lymphocytes are the cell types
most frequently tested with the comet assay in human biomonitoring studies (Angerer et al. 2007; Faust et al. 2004;
Received for publication December 13, 2010; accepted April 6, 2011.
Corresponding Author:
Elva I. Corts-Gutirrez, Centro de investigacin Biomdica del Noreste,
IMSS, 2 de Abril # 501. Col. Independencia, C. P. 64720 Monterrey, N. L.
Mxico
Email: elvacortes@cibinmty.net

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Corts-Gutirrez et al.

Moller 2006b). However, other cells have also been assayed,

including buccal (Szeto et al. 2005), nasal (Mussali-Galante
et al. 2005), epithelial (Emri et al. 2004; Graham-Evans
et al. 2004; Rojas et al. 2000), and placental cells (Augustowska et al. 2007) and spermatozoa (Delbes et al. 2007; Fraser 2004; Schmid et al. 2007; Singh et al. 2003), with the
simultaneous detection of single- and double-stranded breaks
(Enciso et al. 2009). In all these cases, the final level of analysis is the cell nucleus and there have been no attempts to
investigate the capacity of the comet assay to analyze DNA
damage in individualized subnuclear units, such as the chromosomes, although specific genomic regions can be probed
using a combination of the comet assay in whole nuclei and
fluorescence in situ hybridization (FISHcomet; Fernndez et
al. 2001a). This methodological variant allows DNA breakage
to be sensitively screened after its exposure to various kinds
of DNA-damaging agents and is a good tool for assessing the
distribution of region- or locus-specific DNA damage in the
whole genome (Hovhannisyan 2010; Spivak 2010).
The present study was conducted to adapt the method of
the alkaline comet assay to visualize putative DNA damage
in subnuclear structures such as metaphase chromosomes.

Materials and Methods

HeLa Cell Cultures
HeLa cells were obtained from the European Collection of
Cell Cultures (Wiltshire, UK). The cells were cultured with
standard procedures in flasks at 37C in RPMI medium
supplemented with 15% fetal calf serum, 1% glutamine,
and antibiotics (streptomycinpenicillin) in an atmosphere
supplemented with 5% CO2.

Chromosome Isolation
Chromosomes were isolated according to the method
described by Goyanes and Mndez (1982), developed to
isolate metaphase chromosomes for whole mounting for
electron microscopy. The cells were arrested at metaphase
by treatment with 0.5 g/ml colchicine for 3 hr, harvested,
and treated for 10 min with 0.075 M Kcl at 37C. After the
hypotonic treatment, the cells were centrifuged at 1200 rpm
(200 g) for 7 min and the pellet was covered in acid isolation buffer (1% citric acid, 1% Triton X-100, and 6 mM
MgCl2) for 30 min at room temperature. The cells were
gently syringed 710 times through a 22-gauge needle, and
the presence of isolated chromosomes was monitored by
phase-contrast microscopy. At this stage, the isolated chromosomes can be classified, using florescence microscopy
and image analysis, accordingly with the chromatin mass
(CM). The CM is the resulting value after the area of the
region of interest (ROI) selected after image analysis is correlated with the mean gray value obtained for the threshold

region (CM = ROI area mean gray value): for whole

cells, CM > 200E10400E10; for large chromosomes, CM >
100E10200E10; for medium chromosomes, CM > 10E10
100E10; and for small chromosomes, CM = 5E510E10.
Image processing included a fixed exposure time and current protocols for single-image background subtraction.
The image analysis and processing for quantitative microscopy were performed using the Leica Qwin software (Leica
Qwin; Leica Microsystems, Barcelona, Spain) to compare
the CM and tail lengths generated for each chromosome
and whole nucleus.

Comet Assay
The isolated chromosomes and cells were processed
at room temperature by suspending the cells in RPMI
medium to a final concentration of 5 106 cells/ml. This
chromosomenuclei suspension was then mixed with lowmelting-point agarose (0.7%) at 37C, and a 15-L aliquot
was placed onto a precoated glass slide (Halotech SL,
Madrid, Spain). The cellagarose mixture was then covered
with a cover slip (10 mm 60 mm) and allowed to solidify
at 4C for 5 min. After the gel had solidified, the cover slips
were gently removed and the slides were then ready for
further processing.
The alkaline comet assay was performed using the basic
rationale of Singh et al. (1988), with modifications. The
slides were immersed in two lysis solutions for 30 min each
at room temperature. The first solution contained 0.4 M
Tris-HCl (pH 7.5) and 1% SDS, and the second solution
contained 0.4 M Tris-HCl (pH 7.5), 1% SDS, and 0.05 M
EDTA. The slides were washed in 1 TBE buffer (89 mM
Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) for 10 min
and then treated with fresh alkaline solution (0.03 M NaOH,
1 M NaCl) for 2.5 min to cleave the alkali-labile sites. The
slides were placed horizontally in an electrophoresis tray,
which was filled with fresh alkaline electrophoresis solution (0.03 M NaOH, pH 13). Electrophoresis was performed
at 20 V for 12.5 min at room temperature. After electrophoresis, the slides were gently removed from the tray and
washed with neutralizing buffer (0.4 M Tris-HCl, pH 7.5)
for 5 min. The slides were washed in distilled H2O for 5 min
and then dehydrated in a sequential series of 70%, 90%, and
100% ethanol. Finally, the slides were stained with propidium iodide (20 g/ml) and visualized under a Leica DMLA
model motorized epifluorescence microscope controlled
with software for automatic scanning and image digitization (Leica Microsystems). A Leica EL6000 fluorescence
light source equipped with a metal halide lamp and a
PL-Fluotar 40 objective was used for routine analysis, and
a PL-Fluotar 60 objective was used to capture the images.
The images were captured with a CCD DFC-350-FX Leica
(scientific grade) for 8-bit gray-level imaging. A fluorescence filter block from Semrock (595-615 LF561-A;

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New Application of the Comet Assay: ChromosomeComet Assay

Figure 2. Chromosomes of HeLa cells after the comet assay,

showing isolated chromosomes without DNA damage (A) or
displaying different-sized tails according to the level of DNA
damage: mild (B), moderate (C), and severe (D).

Figure 1. An overview of the chromosome-comet assay. I. Nuclei

(A) and chromosomes (B, C, D) isolated from HeLa cells before
protein depletion (I) and after protein depletion and alkaline
electrophoresis (II and III). Level II includes nuclei and isolated
chromosomes without DNA damage, whereas level III includes
selected nuclei and chromosomes with clearly apparent DNA
damage.

Semrock, Inc., Rochester, NY) was used for propidium

iodide visualization.
The slides produced contained isolated chromosomes
and whole nuclei or cells at interphase that were still recognizable on the slides after the chromosome isolation. In
each experiment, 100 whole nuclei and 150 isolated chromosomes (50 large chromosomes, 50 medium chromosomes, and 50 small chromosomes) were included in the
analysis and were classified into two categories: nuclei or
chromosomes showing (a) slight chromatin displacement or
no comet (no DNA damage) or (b) a prominent comet (DNA
damage; see Figure 1).

Positive Control
Microgel slides with isolated chromosomes and whole interphase cells were treated with 20 M hydrogen peroxide for
30 min to produce massive single-stranded DNA damage.
The slides were gently washed with PBS at 37C for 30 min
and processed for the comet assay as previously described.

Statistical Procedures
Differences in CM among the chromosomes were assessed
with one-way analysis of variance. A two-sided probability
level of 0.05 was deemed significant. All analyses were
performed with SPSS for Windows 13.0 (SPSS Inc.,
Chicago, IL).

Results and Discussion

HeLa cells treated with isolation buffer, when processed for
electron microscopy and immobilized in a low-meltingpoint agarose support, produce a morphology good enough
to distinguish whole nuclei (Figure 1, IA) or discrete isolated chromosomes in which the centromeric regions can be
recognized (Figure 1, IIB, IIC, IID). This morphology varies substantially after protein depletion and electrophoresis.
For example, the centromeric region of each individual
chromosome can no longer be identified (Figure 1, IIB, IIC,
IID). After controlled protein depletion and electrophoresis,
both the whole nuclei (Figure 1, IIA) and the isolated chromosomes (Figure 1, IIIB, IIIC) displayed visible comets,
with longer tails as the DNA damage became more intense
(compare level II with level III in Figure 1). Thus, this
method allows the discrimination of chromosomes without
DNA damage from chromosomes with different levels of
DNA damage, which can then be classified as mild, moderate, or severe (Figure 2).
Under our experimental conditions, the percentage of damaged nuclei at interphase after the cells were harvested was
8%. The level of DNA damage in the isolated chromosomes
was similar: large chromosomes were affected in 12% of cases,
medium chromosomes in 9% of cases, and small chromosomes in 11% of cases. In contrast, the positive controls treated
with H2O2 showed an appreciable increase in DNA damage,
ranging from 87% of the whole nuclei to 93%, 89%, and 96%,
respectively, of the isolated chromosomes.
Image analysis was used to compare the CMs and tail
lengths distributed on each chromosome and in whole cells
displaying a comet (Figure 3). We found no correlation
between the whole DNA content and the level of DNA breaks
calculated from the size of the tail on the comet (r2 = 0.18; p
0.05). Therefore, the DNA damage produced by H2O2 treatment did not seem to be induced at random, nor was it dependent on the chromosome size, but rather occurred at specific

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Corts-Gutirrez et al.

Figure 3. Alkaline comet assay of a whole nucleus and a small chromosome from HeLa cells. The graphic on the right shows the
densitometric relationship between the chromatin mass and the comet tail length.

preferred sites or hot spots for this agent. These results are
consistent with previous observations that, for example, these
specific sites are not homogeneously disseminated within the
genome in human and mouse cells. In the human genome, the
most sensitive sites are associated with specific DNA
sequences, such as satellite DNA 1, and 5 bp classical satellite
DNA sequences on chromosome 1 (D1Z1 locus), chromosome 9 (D9Z3 locus), and the Y chromosome (DYZ1 locus)
(Fernndez et al. 2001b; Vzquez-Gundn et al. 2002). In the
mouse, the major satellite DNA families, localized in the pericentromeric regions of each chromosome, are particularly sensitive to damage (Fernndez et al. 1995).
These particular regions of the genome, which also
include telomeric-like or subtelomeric DNA regions, are
considered hot spots for the formation of symmetric
exchanges between homologous chromatids, and cryptic
aberrations in these regions are associated with human congenital abnormalities (Fernndez et al. 1995; Obe et al.
2002). An inverse relationship has been observed between
the density of active genes and the UV light sensitivity of
DNA, insofar as gene-poor chromosomes seem to be more
damaged than gene-rich ones. For example, the more highly
sensitive X chromosome in female nuclei is probably the

inactive X chromosome, which has a low density of actively

expressed genes. Conversely, in humans, chromosomes 1
and 19, which have high densities of active genes, show low
susceptibility to DNA damage. In contrast, X-ray-induced
DNA damage occurs preferentially in gene-rich regions,
indicating a different overall damaging mechanism, which
is probably related to different subchromosomal DNA
protein interactions (Tucker and Senft 1994).
In future studies, the chromosomecomet assay, using
the same rationale reported here but coupled to different
whole-chromosome-painting DNA probes or singlechromosome DNA probes, may target DNA damage induced
in specific chromosomal regions. In this way, we can gain precise information about putatively random or localized distributions of DNA damage. This information should extend our
understanding of the chromosomal architecture, subchromosomal organization, and the role of DNAprotein interactions
in chromosomal receptivity to DNA damage.

Conclusions
We have developed a new technique, based on chromosome
isolation and the comet assay, to detect DNA damage in

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New Application of the Comet Assay: ChromosomeComet Assay

human chromosomes. This protocol has great potential for
the highly reproducible investigation of DNA damage and
repair in specific chromosomes or chromosomal domains.
Once the technique has been established, we must investigate its sensitivity in other experimental contexts, for
example (a) to record the susceptibility to DNA damage of
different subchromosomal domains, using specific DNA
probes, and/or (b) to establish chromosomal doseresponse
curves for classical damaging agents, such as different
doses of ionizing radiation.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the authorship and publication of this article.

Funding
The author(s) received no financial support for the research and
authorship of this article.

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