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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
MODIFIED PROCEDURE TO ASSESS DNA BREAKAGE IN SPERMATOZOA BY MEANS OF
THE COMET ASSAY
Griset MARTÍNEZ-LUNA
1
, Julieta CASTILLO-CADENA
1
and Jorge Humberto SERMENT-GUERRERO
2
*
1
Facultad de Química, Universidad Autónoma del Estado de México, Paseo Colón esq. Paseo Tollocan s/n,
CP 50100, Toluca, Estado de México
2
Departamento de Biología, Instituto Nacional de Investigaciones Nucleares, Carretera México-Toluca s/n,
km 36.5, La Marquesa, Municipio de Ocoyoacac, CP 52750, Estado de México
* Corresponding author: jorge.serment@inin.gob.mx
(Received May 2014; accepted January 2015)
Key words: genotoxicity, DNA breakage, sperm, microelectrophoresis
ABSTRACT
The comet assay is a relatively inexpensive, fast, sensitive and reliable method to
detect DNA breakage upon individual cells. The most widely used cell type in this
technique is lymphocyte, because they are easy to obtain and handle, and because they
are continually exposed to xenobiotics that enter into the body. However, is important
to consider the possibility to use other cell types for very speciFc purposes. Sperm cells
are of special interest because they could be used as a biomonitor to risk assessment
in populations occupationally exposed to xenobiotics. Besides, the fact that there are
not functional DNA repair mechanisms in these cells could increase the sensitivity of
the system. We present here several modiFcations to the comet assay methodology to
evaluate DNA breakage in sperm cells with reliable results.
Palabras clave: genotoxicidad, rupturas de ADN, esperma, microelectroforesis
RESUMEN
El ensayo cometa es un método relativamente simple, rápido, sensible y conFable para
detectar rompimientos en el ADN en células individuales. Las células más comúnmente
utilizadas en esta técnica son los linfocitos, ya que son fáciles de obtener y manejar y
además porque se encuentran expuestas continuamente a los xenobióticos que llegan a
entrar al cuerpo. Sin embargo, es importante el considerar la posibilidad de utilizar otros
tipos celulares para propósitos especíFcos. Las células espermáticas son de especial
interés ya que pueden servir como biomonitores de riesgo en poblaciones ocupacional-
mente expuestas. Además, el hecho de que este tipo de células no tienen mecanismos
funcionales de reparación de lesiones en el material genético podría incrementar la
sensibilidad del ensayo. En el presente trabajo se muestran algunas modiFcaciones a
la técnica del cometa para evaluar rupturas en el ADN de espermatozoides con resul-
tados conFables.
Rev. Int. Contam. Ambie. 31 (1) 39-45, 2015
G. Martínez-Luna
et al.
40
INTRODUCTION
Genotoxicity is defned as any change in basic
DNA chemical structure as a result of the interac-
tion of harmful chemical or physical agents with
genetic material. These changes will fnally lead to
three possible outcomes: cell death, total repair or
imperfect repair that may result in the appearance of
mutations. Those genetic alterations would be trans-
mitted to subsequent generations through germ cells
such as sperm (Clayson and Grant 1992). Thus, it is
imperative to assess and identify xenobiotic agents
capable of producing such DNA damage. Genotoxi-
city bioassays are usually employed to evaluate the
risk to human health in order to establish prevention
and control regulations (Paules
et al.
2011).
One of the most popular genotoxicity tests is
the single cell gel microelectrophoresis, commonly
called comet assay, a sensitive, fast and inexpensive
method to examine DNA breakage in individual
cells (Berwick and Vineis 2000, Collins 2004).This
assay has positioned itself in a privileged position
into the battery of biomonitoring studies used to eva-
luate hazardous agents in environmental toxicology
(Hartman
et al.
2003), in occupational exposure to
xenobiotics and as a biomarker capable to demons-
trate dose-effect relationships (Jakubowski and
Trzcinka-Ochocka 2005).
The comet assay allows the detection of single or
double strand DNA breakage or alkaline-labile sites
in individual cells, with relatively high sensitivity.
BrieFy, cells are mixed with low melting point agarose
(LMP) to form a thin gel on top of a microscope slide
and then lysed
in situ
to remove all cellular membranes
and proteins. Then, slides are put into an electropho-
resis cell and a voltage is applied. Fragmented DNA
will migrate out of the lysed cell towards the anode
(Nadin
et al
. 2001), forming a structure similar to a
comet (Hughes
et al
. 1996, Collins 2004).
Although a wide range of cell types can be used,
lymphocytes has been traditionally used in this
technique, because of the ease to obtain and also
because these cells are circulating in the peripheral
blood as part of the immune response; therefore they
are continually exposed to xenobiotics that enter
into the body either through the skin, oral, digestive
or respiratory mucosae, so a high degree of DNA
damage could be assessed by these cells (Moller
et
al.
1989, Orson
et al.
1989, McCarthy
et al.
1990,
Rödl
et al.
1990). Indeed, the damage to individual
somatic cells such as lymphocytes, as measured by
the comet assay, detects changes in DNA structure
that can lead to genomic instability.
In germ cells, such as spermatozoa, which carry
genetic information passed from one generation to
the next, is important to evaluate individuals with
occupational exposure to detect possible DNA
damage that could lead to malformations in future
generations. Another possible advantage in using
this kind of cells is that they can be considered as
packages of DNA, so for
in vitro
test repair events
could be avoided, increasing the sensitivity of the
system. However, even though several methodolo-
gies have been reported, we couldn’t reach consis-
tent results with any of them, therefore we tried to
improve the protocol proposed by Hauser and co-
workers (Hauser
et al
. 2007) for comet assay with
human spermatozoa, making changes that permit
obtaining reliable results.
MATERIAL AND METHODS
The procedure for evaluation of DNA damage
in human sperm with the general modifcations is
shown below.
Chemicals and solutions
Glucose, potassium chloride and sodium hy-
droxide were purchased from J.T. Baker; sodium
chloride was from BDH; Dithiothreitol (DTT) was
from Gibco BRL; proteinase K was from Thermo
Lab, and the rest of chemicals were purchased from
Sigma-Aldrich. Casiopeina III-Ea was synthesized,
purifed and provided by Dr. Lena Ruiz-Azuara, ±rom
the school of Chemistry, UNAM (Ruíz-Ramirez
et
al.
1991, Ruiz-Azuara 1993) and used as a positive
control (Serment-Guerrero
et al.
2011).
Semen sample collection
Samples were obtained from three different
healthy donors who were properly informed about
the purposes of the investigation and voluntarily
consented to participate. Semen was obtained by
masturbation after a recommended abstinence period
of 48 hours, and collected in a sterile plastic cup.
Liquefaction was performed at 37 ºC for 60 minutes,
and semen was diluted 1:20 in Beltsville Thawing
Solution (BTS) (glucose 0.2M; Na
2
EDTA 3 mM;
NaHCO
3
0.015 M; citrate Na
3
.2H
2
O 0.02 M and KCl
0.01 M, pH 7.2) (Erikkson and Martínez-Rodríguez
2000, Mapeka
et al.
2012), and frozen at –20 ºC
(Pursel and Johnson 1975) for later analysis. When
needed, samples were thawed by gently shaking in
a water bath at 37 ºC for 10 minutes and semen was
immediately processed for comet assay.
DNA BREAKAGE IN SPERMATOZOA BY THE COMET ASSAY
41
In vitro
exposure and treatments
A 100 mM stock solution of the casiopeina was
prepared in 1% dimethyl sufoxide (DMSO), and sub-
sequent dilutions were made in sterile milliQ water.
To induce DNA damage in spermatozoa, 180 μL
of 1:20 sperm-BTS dilution (10 µL of semen with
190 µL of BTS) were placed in a microtube with 10 μL
of the proper dilution of Casiopeina III-Ea (each
sample was made in duplicate), the mixture was
then incubated in a 37 ºC water bath for 30 minutes
and centrifuged for 10 minutes at 2500 rpm. The
supernatant was discarded and the pellet suspended
in 180 μL of BTS.
Comet assay
Clear slides were previously prepared with 90 µL
of Type I: Low EEO agarose at 0.6 % and let to dry
at 37 ºC, then a second layer was applied and the
drying step was repeated.
Cell suspension was mixed with an equal volume
of 1% low melting point agarose to reach a Fnal con
-
centration of 0.5 %, then 90µL of this mixture were
applied on previously prepared slides and topped
with a cover slip, placed on a metal tray and put in
the refrigerator for 5 minutes to solidify. Afterwards,
cover slip was removed and slides were immersed
in cold lysis solution (2.5 M NaCl, 10 mM Tris,
100 mM Na
2
.EDTA, 10 % DMSO, 1 % N-lauroyl
sarcosine, 1% Triton X-100, pH 10.0) for one hour.
Afterwards, slides were withdraw from the lysis
solution and 60 μL of a solution of proteinase K
(1 mg/mL) and DTT (5 mM) in the same lysis solution
reported above were applied and incubated overnight
(18-20 hours) at 37 ºC in a humidity chamber. Slides
were placed on a horizontal electrophoresis cell,
covered with electrophoresis solution (0.3 M NaOH,
1 mM Na
2
.EDTA) for 20 minutes to allow DNA unwin-
ding and then a current was applied (20 V, 300 mAmp,
15 minutes at 4 ºC).
Later, slides were removed from the electrophore-
sis chamber, gently rinsed three times with neutrali-
zation buffer (0.4 M Tris-HCl; pH 7.5), staining with
60 μL of ethidium bromide (20 µg/ml) and Fnally
observed under a Hund Wetzlar epi±uorescence mi
-
croscope with a 40x objective using a 515-560 nm
(green light) exciting Flter. Two slides were prepared
for each treatment. One hundred randomly selected
cells were scored by means of the Comet Assay
IV Analyzer (Perceptive Instruments Inc.). DNA
breakage was evaluated as percentage of cells with
fragmented DNA and/or tail moment, which is the
relation between comet tail length and DNA ±uores
-
cence intensity (Collins 2004).
RESULTS AND DISCUSSION
Currently, this laboratory is interested in mo-
nitoring the effect of xenobiotics in spermatozoa
from occupationally exposed populations, so it was
essential to have a reliable comet assay procedure to
assess DNA breakage. However, when the standard
methodology report by Tice and co-workers (2000)
was used, no good results were obtained. Alter-
natively, the one reported by Hauser
et al.
(2007),
speciFcally designed for spermatic cells was tested,
unsuccessfully. Therefore, several modifications
were made to try to improve the procedure to make
it functional in these facilities.
In the Frst place, the use of clear slides instead of
frosted slides was implemented, in order to reduce
the background ±uorescence. The standard methodo
-
logy recommended the use of a Frst layer of normal
agarose, a second layer of LMP mixed with cells
and a Fnal third layer of LMP (Tice
et al
. 2000). In
this work, we used two layers of Type I: Low EEO;
once dried, the agarose crystals serve as a frosting
that allow the next layer of LMP agarose to become
Frmly attached to the slide. Another advantage of
using this technique is that the preparation of slides
is faster, so treated cells can go into the lysis solution
in less time.
The lysis solution reported by Tice and co-
workers has proved to be quite functional for
lymphocytes and some other cell types. However,
it does not work upon spermatozoa, since this
kind of cells has a plasmatic membrane with a
unique lipid and protein composition, that allows
for very little or no substance exchange, therefore
it is necessary to use a mucolytic agent capable of
lysing these cells to liberate the genetic material
(Davies-Morel 1999). Previous reports proposed
a lysis procedure in which the use of proteinase
K (Hughes
et al.
1996) and RNase (Hauser
et al.
2007) were included. At Frst, the technique was
implemented using only proteinase K, but lysis did
not occur, therefore DTT was added as a mucolytic
agent, which is able to disrupt the protein disulFde
bonds (-SS-) that are present in sperm, allowing
fully deployment and separation of protein subu-
nits of a multimeric protein. DTT is routinely used
in DNA extraction for forensic purposes to make
accessible the chromatin of the sperm head (Bart-
mazt
et al
. 1994, González-Estrella
et al
. 1994) and
was used in the comet assay before (Donnelly
et
al.
1999, Enciso
et al.
2009). First, DTT (40 mM,
20 mM and 10 mM) and proteinase K (1 mg/mL)
were added straight into the lysis solution and
G. Martínez-Luna
et al.
42
incubated at 4
ºC for 60, 90, 120 and 180 minutes
(Kaymak
et al
. 2012). However, lysis was not good.
Besides, the addition of DTT in the lysis solution in-
creased dramatically the cost of the assay, so 60 mL
of a DTT/proteinase K solution were applied di-
rectly on the slides after 1 hour in standard lysis
solution, and further incubated for 18-20 hours in a
humidity chamber at 37 ºC. Under microscope, ima-
ges resembling “smashed” cells instead of comets
were found (
Fig. 1
), indicating that lysis actually
occurred but DNA migration did not, most probably
because the electric charge of the DTT interfered
with the negative charge of DNA.
The amount of DTT showed to be very important.
In total, 11 different concentrations of DTT were
tested, but when solutions of 1 mM or lower were
used, lysis did not occurred, whereas when solutions
of 100 mM or higher were employed, DNA migration
was no good. At 5 mM of DTT lysis was excellent
and so was migration (
Fig. 2
).
Another parameter that changed in this work was
the diluent used for semen. When the classical PBS
solution was used (Hauser
et al
. 2007) controls were
not good, with more than ten percent of comets in
untreated cells, so results could not be trustworthy.
When the BTS solution reported by Pursel and
Johnson (1976) results were superb. Moreover, BTS
has a dual advantage, since it acts as a handling solu-
tion and is excellent for cryopreservation of semen.
Indeed, frozen cells with more than three weeks at
–70 ºC, produced results in the comet assay that were
highly correlated with results from freshly obtained
samples.
Cell concentration is an important issue in this
technique, since too many cells will result in comet
overlapping, whereas too few will take too long
for scoring. The cell concentration average of the
samples used in this work has 7×10
7
cells/mL, so
routinely a 1:20 dilution of the ejaculated in BTS
was prepared and from there 100 µL aliquots were
used per treatment, reaching a fnal concentration oF
1×10
5
cells/mL approximately.
The electrophoresis time was also adjusted. The
original procedure indicated 20 minutes at 20 V and
300 mA, however, spermatozoa are haploid cells
(n) with half of the genetic material contained in
any other cell type. When an electrophoresis time of
20 minutes was used the comets showed long tails,
yet unusually low tail moment values were obtained
and dose response correlation was not good. Hughes
and co-workers (1996) reported an electrophoresis
time of 10 minutes, but comets were rather small and
hence diminished the sensitivity of the assay. Finally,
the electrophoresis time was reduced to 15 minutes
with good results (
Fig. 3
).
To verify the optimal performance of the impro-
ved methodology described above experiments were
carried out using a molecule recently reported as an
actual genotoxic (Serment-Guerrero
et al
. 2011), casio-
peina III-Ea ([Cu(4,7-dimethyl-1,10-phenanthroline)
acac]NO
3
),
fgure 4a
). Casiopeina is the generic name
Fig. 1.
Images resembling smashed cells when a relatively high
concentration of DTT is used during cell lysis
Fig. 2.
Comets obtained when slides were treated with 60mL
of a DTT (5 mM)/proteinase K (1 mg/mL) solution and
incubated overnight
DNA BREAKAGE IN SPERMATOZOA BY THE COMET ASSAY
43
of a group of compounds, with a central cooper atom
bound to organic ligands designed to be used as an-
tineoplastics. Different concentrations of CasIII-Ea
were apply to a suspension of spermatozoa diluted in
BTS for 30 minutes at 37 ºC, then mixed with LMP
agarose and lysed a described above. Experiments
were scored by means of the Comet Assay IV (Per-
ceptive Instruments, UK) and tail moment was taken
into account. Under microscope, comets in which tail
increased along with the concentration were found
(
Fig. 4b
). The results confrmed the genotoxic activity
of CasIII-Ea and, moreover, showed that this improved
methodology is more sensitive, regarding the results
obtained when lymphocytes were used, most probably
due to the lack of DNA repair mechanisms in spermatic
cells (Serment-Guerrero
et al
. 2011).
Fig. 4.
a) Chemical structure of casiopeina CasIII-Ea [Cu(4,7-dimethyl-1,10-phenanthroline)acac]NO
3
(Bravo-Gómez
et al.
2009)
;
b)
DNA breakage produced by CasIII-Ea in spermatic cells or lym-
phocytes (expressed as tail moment)
14
b)
12
10
8
6
4
2
0
01
02
0
Concentration (μM)
Spermatozoa
Lymphocytes
Tail moment
30
40
50
H
3
C
a)
NO
3
NN
Cu(II)
OO
CH
3
+
Fig. 3.
Comparison between comets obtained with different times of electrophoresis. a) 20 minutes; b) 15 minutes
a)
b)
G. Martínez-Luna
et al.
44
CONCLUSIONS
Overall, the sum of the small modiFcations introdu
-
ced to the comet assay technique resulted in a reliable
methodology for the use of sperm cells. The use of the
BTS solution for both cryopreservation and dilutions
minimized the breakage upon untreated cells. To apply
a solution of DTT/proteinase K directly on the slides
permitted an optimal disruption of spermatic cells
membrane without increasing signiFcantly the cost
of the assay. Due to relatively low amount of DNA
in spermatic cells the tail moment was not a repre-
sentative parameter for genotoxicity since, as stated
above, genetic material in the tail was too dispersed.
By reducing the electrophoresis time to 15 minutes,
a good correlation between tail moment and the dose
applied were found. This was demonstrated by the use
of CasIII-Ea; results showed an increase in tail moment
along with the concentration of this compound. Under
the conditions described here, the use of spermatic
cells seems to increase the sensitivity of the comet
assay, compared with others types of cells.
ACKNOWLEDGMENTS
This project was partially funded by the UAEMex,
Project Agreement No. 3452/2013CHT and by the Ins-
tituto Nacional de Investigaciones Nucleares. Studies
were carried out according to the regulations of the
Reglamento de la Ley General de Salud en Materia de
Investigación para la Salud (OfFcial Mexican Regula
-
tions in the Feld of health research) and approved by
the corresponding institutional technical committee.
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