Mol. Cells, Vo l. 4, pp. 343-348
DNA Strand Break Repair Involvement in Hydrogen Peroxide
Induced Damage
Daryl W. Fairbairn* and Kim L. O'NeiU
Department of Microbiology, Brigham Young University, Provo, Utah 84602, USA.
(Received on July 20, 1994)
DNA repair is an essential function in maintaining proper cellular control. A loss or impairment
of repair is associated with many human diseases including malignant diseases. We present
here an evaluation of the repair of DNA damage induced by radicals generated from hydrogen
peroxide. The comet assay (single cell gel) was used to analyzed damage and its repair in
individual cells. DNA damage induced by hydrogen peroxide was heterogeneous, but still displayed dose response relationship with strand break detectability. Rejoining of strand breaks was
completed within 60 min, but the appearance of increased breaks consistent with the excision
step of excision repair was also observed. Additionally, inhibition of the enzyme poly (ADPribose) polymerase by the nicotinamide analog 3-aminobenzamide (0.1 mM and 1.0 mM) was
inhibitory of DNA strand rejoining. The study of DNA single strand breaks and repair should
provide meaninful insight into the mechanisms of normal and abnormal cellular events.
DNA damage induction and modification events
have many different consequences with regard to cellular processes. The alteration of normal cellular functions that ultimately result from these events are dependent upon the machinery within the cell to correctly
indentify and repair DNA damage. Hydrogen peroxide is a reactive oxygen species that has been shown
to be mutagenic (Imlay et al., 1988), and cause malignant transformation of cultured cells (Weitzman et aI.,
1985) in addition to other important reactions within
the context of cellular distribution. Although hydrogen
peroxide is a highly reactive molecule, it does not damage DNA directly, but resulting hydroxyl radicals
(OH ' ) from the interaction of peroxides with transition metals produce oxidative lesions (Guyton and
Kensler, 1993). A wide array of DNA damage can
be induced by the indiscriminate action of the OH·
radical, including single and double strand breaks and
oxidized bases (Satoh and Lindahl, 1994). Oxidant activated cells can also stimulate the formation of DNA
strand breaks I;>y nuclease activity (Nicotera et al.,
1990). Hydrogen peroxide is responsible for the induction of single strand breaks in the DNA of cells (Martins et al., 1991). The measurement of oxidative damage induced by hydrogen peroxide is complicated
in part by the comprehensive array of defenses cells
have developed to limit the damaging effects of free
radical formation, including enzymatic decomposition
mechanisms and sequestering proteins (Cheeseman
and Slater, 1993), and other mechanisms which alter
cellular sensitivity to hydrogen peroxide. Nonetheless,
* To whom correspondence should be addressed.
hydrogen peroxide is widely used to study the effects
of reactive oxygen species on cellular metabolism.
Defective repair of oxidative lesions produces a wide
array of biological consequences. The repair of single
strand breaks is generally rapid in human cells (repair
is virtually complete within about an hour). The study
of DNA single strand breaks and repair can provide
meaningful insight into the mechanisms of normal
cellular events such as senescence, as well as abo normal cellular events like mutagenesis and carcinogenesis (McKelvey et al., 1992; Olive et al., 1993). The abundant nuclear enzyme poly(ADP-ribose) polymerase
is extensively involved in the repair of single strand
breaks, and a mechanism for its involvement has recently been proposed (Satoh and Lindahl, 1992).
Laser scanning microscopic analysis of the single
cell gel assay (Ostling and Johanson, 1984) can provide rapid and accurate descriptions of low levels of
single strand DNA breaks and alkali-labile sites in
individual cells (O'Neill et al., 1993). Briefly, cells embedded in low melt point agarose are lysed and electrophoresed under DNA denaturing conditions. DNA
with higher levels of single strand breaks migrates farther from the bulk of the cellular DNA during alkaline electrophoresis, generating a characteristic comet
appearance. Increased DNA damage appears in this
assay syntem as increased migration of DNA comets
in the direction of electrophoresis. The potential use
for this assay is increasing in application (McKelveyMartin et aZ., 1993). We recently described in this joural some of the parameters that influence the results
of the assay (Fairbairn et al., 1993a). We presented
information about how the exposure limits and electrophoresis conditions can alter interpretation of the
© 1994 The Korean Society for Molecular Biology
344
DAN Repair
In
Hydrogen Peroxide Induced Damage
results.
In this paper, we demonstrate the applicability of
the single cell gel assay to the study of single strand
break repair as we had proposed in our previous paper. Using hydrogen peroxide as the single strand
break inducing agent (Schraufstatter et al., 1988), we
established positive and negative controls. We then
propose a simple technique to allow for the sensitive
measurement of DNA repair, and demonstrate how
DNA repair inhibitors might be used in this technique
to examine their effects in DNA repair. DNA damage
induced by hydrogen peroxide is heterogeneous because of the range of defense mechanisms, but still
demonstrated a dose response relationship with strand
break detectability. Rejoining of strand breaks were
completed within 60 min, but the appearance of increased breaks consistent with excision repair was also
observed.
Experimental Procedures
Cells and reagents
Human Raji cells, mouse myeloma FO cells, and
RG-l (an IgM monoclonal antibody producing hybrido rna cell line; Grigsby et al., 1993) cells were maintained at exponential growth in RPMI 1640 plus 10%
fetal calf serum (Hyclone Laboratories, Logan Utah).
All cells were washed with PBS before use in experiments. All chemicals and reagents were obtained from
Sigma (St. Louis, MO). Cell viability was routinely
determined by the trypan blue exclusion assay. Trypan
blue was brought -into solution in PBS, and was filtered through a 0.2 11m pore-sized fIlter before use, and
stored at ambient temperature. Cells in PBS were mixed with an equal volume of trYpan blue solution,
and the fraction not staining were calculated to represent viable cells.
Comet assay
The assay was conducted essentially as described
previously (Fairbairn et al., 1994b) with some minor
modifications. Cells were embedded in 0.75% agarose
on frosted microscope slides and treated with varying
concentrations of hydrogen peroxide as indicated. The
cells were lysed in a solution consisting of 0.03 M
NaOH, 2.5 M NaCl, O.l % Na-Iauroyl sarcosine for
60 min. The slides were equilibrated in three successive solutions of 0.03 M NaOH and 2 rnM EDTA
for 20 min each to remove salt and detergents which
are inhibitory to DNA migration, as well as to allow
complete unwinding of the DNA for accurate damage
detection. Electrophoresis was executed for 20 min at
0.7 V/crn, and the DNA was stained with ethidium
bromide for fluorescent visualization. All experiments
were repeated at least three times, and the results given are the averages of the replicate experiments. Statistical comparisons were performed against relevant
controls using the student's t-text where applicable,
and p < 0.01 was considered statistically significant.
Mol. Cells
DNA repair
Cells were allowed to repair in pre-warmed fresh
growth media at 37 °C 5% CO 2 incubator for the designated repair times immediately after hydrogen peroxide treatment. We used cold temperatures (0-4 °C)
to control the time for repair both after H 20 2 treatment and before lysis (Ward et al., 1985). Cells were
repaired in media containing the indicated concentrations of the DNA repair inhibitor 3-aminobenzamide
in some of the assays where noted.
Fluorescent image analysis
A Carl Zeiss laser scanning microscope was used
to analyze the length of the comet taus as previously
described (Fairbairn et aZ., 1993b). This technology
provides clearly defined images for measuring DNA
migration patterns. We also used a Zeiss Axioskop
to analyze percent DNA in the tail of the comet, and
comet tail moment. Comet tail moment is calculated
as the product of the tail length and the fraction of
total DNA in the tail, and is considered more informative than tail length alone. Comets were viewed
using a 32 X objective, and the images were digitized
using a CCD camera (Dage-MTI, Inc., Michigan City,
IN) attached to a computer with an ITEX imaging
board (Imaging Technology, Inc., Bedford, MA) for
analysis. Comet tail was calculated as previously described by Dr. Peggy Olive using a program written by
Dr. Ralph Durand (Olive et aZ., 1991). Between 50 and
200 comets were analyzed for each slide.
Results and Discussion
Increased levels of DNA damage are clearly correlated in this assay with increased DNA migration during electrophoresis. Measuring the resulting comets
using laser scanning microscopic analysis is an effective way of evaluating damage. The detection limits
of the assay are dependent upon the ability to correctly define the borders of the comet, which the laser
sca nning' analysis improves upon greatly. As increased
damage correlates with increased migration, repair of
induced DNA damage can be traced as a decreased
DNA migration. As repair nears completion to basal
levels, the migration patterns of the repaired 'comet'
is virtually the same as the undamaged controls.
In order to establish positive and negative controls,
we treated Raji cells with hydrogen peroxide (varying
from 10 11M to 10 mM) or PBS for 30 min on ice
to minimize DNA repair (Ward et al., 1985) (Table
Table 1.. Hydroge n peroxide increases DNA migration in
a dose dependent manner.
Concentration
o [1M
Tail length
45.7 ~m 52.7 ~m 64.9 ~m 74.8 fm1 94.2 flm 109.8 flm
3.56% 12.5% 24.6% 38.2% 58.7% 61.7%
14.60
9.34
4.34
0.49
2.50
0.42
DNA in tail
Moment
I [1M
\0 [1M 100 [1M I mM 10 mM
Vol. 4 (1994)
Daryl W. Fairbairn & Kim L. O'Neill
A.
60
~
•a
0
u
....0
.
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40
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•
20
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~
10
100
•u..
•
•a
0
20
u
10
:!a
3
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100
E.
0
200
1.0 mM
F.
40
!l
•a
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10 mM
30
0
u
20
.•
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.•
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20
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;
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20
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100 uM
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....0
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n
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JJa
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345
•.u
•
10
~
~
O+---~~-----.--~~~---.
o
100
200
10
o+-~~~~-.~--~~---,
o
100
200
Figure 1. Effect of hydrogen peroxide on the length of DNA comets.
I). Clearly, the hydrogen peroxide treated-DNA migrated further in a dose-dependent manner. The extent
of damage presented in the table can be obtained by
any experiments which provide information about the
damage in a population of cells. For example, similar
conclusions about the dose dependency of oxidative
damage, such as that induced by peroxide in this case,
were obtained using the DNA precipitation assay
(Olive et ai., 1988; data not shown).
H owever, one of the difficulties with assays that
examine whole cell populations is that subtle or statistically insignificant changes in detectable damage may
be the result from either of, or a combination of, two
possibilities. First, damage may be limited in all cells.
Alternatively, extensive damage may also be restricted
to a small number of cells, but the overall damage
may not be sufficient to allow detectability. This can
be the case for apoptotic cells. Analysis of individual
cellular damage can provide a means for distinguishing the two possibilities. Although hydrogen peroxide induces damage in a heterogeneous fashion not
attributable to cell cycle differences (Ward et al., 1993),
as expected, it does not produce extensive damage in
only a few cells (Fig. 1).
The most definitive biochemical event in most apoptotic cells is nucleosomal fragmentation which precedes membrane permeability (Arends et al., 1990;
Brown et al., 1993; Wyllie, 1992). Because of the sensitivity of the comet assay to detect DNA damage, ap
ptotic DNA fragmentation has been an obvious can -
346
DAN Repair in Hydrogen Peroxide Induced Damage
didate for evaluation using the comet assay, and it
has been applied successfully to this end (Fairbairn
et al., 1994a; Olive et aI., 1993). Mild hyperthermia is
known to induce apoptosis in a variety of cell lines
(Ghibelli et al., 1992; Papadimitriou et al., 1993; Sikora
et al., 1993; Takano et al., 1991; van Bruggen et al.,
1991). Experiments in our laboratory using both heat
shock-induced apoptosis in Raji cells, and dexamethasone-induced apoptosis in mouse thymocytes indicate
that apoptotic DNA fragmentation resulting from endogenous nuclease activation produces comets with
extensive DNA migration into the comet tail (Fairbairn and O'Neill, 1994). The tail of the comet is separated completely from the head, and most of the
DNA (up to 95%) is contained in the tail of the comet.
The comets of a population of apoptotic cells appear
as either control-like cells with little or no migration,
or as comets with separated heads and tails, with the
bulk of the DNA in the tail of the comet. These patterns are not demonstrated by peroxide-treated cells
(Fig. 1).
It is likely that radiation-induced apoptotic biochemical events and morphology are initially caused by
the DNA damaging event. A very early subcellular
requirement for apoptosis induction in this type of
a model is the formation of radicals resulting from
water radiolysis r.:w arters, 1992). Physiological cell
death which includes apoptosis (Vaux, 1993; White,
1993), is restricted to the death of individual cells, in
contrast with necrotic death which is physiologically
active on adjoining cells. The distribution of DNA
damage is not reminiscent of apoptotic cells in the
peroxide-treated Rajis. In addition, the cells remained
> 90% viable for all concentrations tested under the
exposure conditions used as determined by trypan
blue e·xclusion. The single exception to this was the
cells treated with 10 mM hydrogen peroxide, but the
viability was still calculated to be > 85%. These results
are in agreement with the results for cell killing observed in CHO and V-79 cells by Blakely and coworkers
(lliakis et aI., 1992; Ward et al., 1985) in which significant cytotoxicity is observed at concentrations far
greater than that necessary to induce single strand
breaks (> 20 mM). Observable damage, then, can not
be attributed to either apoptotic death or necrotic
death-associated arbitrary nuclease activity. Because all
treatments were performed on ice, it is unlikely that
the lesions detected by the assay were produced by
nuclease activity, but resulted from the indirect production of single · strand breaks in the backbone of the
DNA or alkali-labile oxidative lesions that were converted to strand breaks without enzymatic activity. It
should be noted, however, that necrotic death is obvious at high concentrations (> 10 mM), and apoptotic nuclear fragmentation is detectable in hydrogen
peroxide-treated cells that have been allowed to repair
after an incubation time of at least two hours (DWF,
unpublished observations).
Repair of hydrogen peroxide-induced damage was
Mol. Cells
70
,.-.,
'"C
8u
!
" " " 0.•.•
0 ...... . \.
' ~ '-
60
" .
".
..........
00
8 202 Exposed
Control
... ...
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..c
......
00
c
II)
....:l
50
......
II)
E
0
U
40
0
20
40
60
Repair Time (min)
Figure 2. Tail length decreases with rejoining of strand
breaks induced by 10 ~ hydrogen peroxide treatment.
evaluated using 10 ~ concentrations, and a 60 min
repair time. The average length of the comet tail was
observed to decrease significantly with time in the
whole media, indicating that induced single strand
breaks were being repaired (Fig. 2). The comet length
decreased from initial damage levels to that displayed
by undamaged controls exposed to PBS alone within
the 60 min time period. Similar observations have
been made in other cell lines. Mouse myeloma FO
cells have slow kinetics for rejoining hydrogen peroxide-induced strand breaks. Cells treated with 100 f1M
hydrogen peroxide demonstrate a 50% reduction in
tail length within 20 min; while an additional 35 to
40 min is required to complete the rejoining of strand
breaks in this cell line. In a hybridoma fusion line
of the FO cells with B cells producing antibodies, repair is completed much more efficiently. Damage by
exposing to 100 f1M hydrogen peroxide is reduced as
displayed by a reduction in tail length by 50% within
5 min following treatment, and is virtually complete
within an additional 10 min. These results, taken together, provide evidence that tail length is an adequate
endopoing for following the course of DNA strand
break rejoining.
However, we also observed that in some cases, the
length of the comet tail actually remained unaffected,
but the intensity of the tail decreased with repair time.
The decreased fluorescent intensity represents the repair of strand breaks as well, and it precedes tail length decreases, although the average tail length decreased with time. DNA migration patterns in the comet assay are based upon two principles, which are
related in part to the migration of DNA through agarose in a conventional sense (Norden el al., 1991). The
ability of unwound DNA to migrate depends upon
(a) the number of broken ends of DNA which can
migrate from the head of the comet, and (b) the size
of the DNA molecules. The comet tail stretches at
Daryl W. Fairbairn & Kim L. O'Neill
Vol. 4 (1994)
3
H202 Exposed
---0---
347
Table 3. Inhibition of poly(ADP-ribose) polymerase by 3amino benzamide inhibits the rejoining of strand breaks.
Moment
Tail Length
Control
Time (min)
o peroxide
10 J.1m peroxide
0.1 mM 3AB
1.0 mM 3AB
1
60
0
45.7
64.9
65.3
64.2
44.7
46.8
J.1m" 55.4
J.1m" 61.7
J.1m
J.1m"
J.1m
J.1m
J.1m"
J.1m"
0
60
0.42
2.41 "
2.35"
2.46"
0.41
0.54
0.73"
1.98"
Significantly different (p < 0.01 ) from respective control
value.
a
O+---~---.r---~---.----~--,
o
40
20
60
Repair Time (minutes)
Figure 3. Reduction of comet tail moment with rejoining
of strand breaks induced by 10 11M hydrogen peroxide treatment.
Table 2. Tail moment increases after 15 min of repair in
whole media. The increased moment is attributed to activation of the excision step in excision repair of oxidized based
damage.
o J.1m
Time (min)
DNA in tail
Moment
o
4.28%
0.39
Peroxide
15
5.1 7%
0.35
10 J.1m Peroxide
o
23.2%
2.41
15
31.9%
3.25
low damage levels, and increasing numbers of breaks
allow more pieces of DNA to migrate freely. The intensity of DNA in the tail of the comet provides additional information about damage degree which is not
accounted for by tail length alone.
We attempted to account for a full description of
DNA migration changes due to single strand break
repair by using a measurement referred to as 'tail moment'. We found that a similar decrease in tail moment was observable with incubation in repair media
following induction of damage (Fig. 3). In addition
to single strand break repair, which is expected to
follow the large number of single strand breaks produced by the OH· radical resulting from a Fentonlike reaction, we observed evidence for the activation
of excision repair activities after 15 min of repair. The
percentage of DNA in the tail of the comet, representing damaged DNA, actually increased after the first
15 min incubation in repair media (Table 2). The
same obsrvation was made for tail moment. This was
not represented by an increase in the length of the
comet, on the other hand. The increased tail intensity
is proposed to be the result of the excision step of
base excision repair, which causes a temporary increase in the degree of detectable damage (Table 2).
Agents such as UV-irradiation induce excisable
DNA damage which is not detected directly. In the
comet assay system, damaged DNA does not migrate
without incubation first in repair media. This is because strand breaks form as damaged lesions are excised and remain detectable through the polymerization
steps. It is only after the ligation step that the strand
breaks appear rejoined in this system. For example,
Xeroderma pigmentosum patient's have been demonstrated to be deficient in excision repair using the comet assay (Gedik et ai., 1992; Green et al., 1992). In
a HeLa cell exposure system, UV-induced DNA damage repair is not detectable without inclusion of
DNA synthesis inhibitors such as aphidicolin (Gedik
et ai., 1992). Moreover, the inclusion of exogenous endonucleases can increase the sensitivity of the assay
by converting damaged bases to strand breaks (Collins
et aI., 1993). The results presented here are consistent
with the observance of the excision step of excision
repair in individual cells.
In order to further examine the repair of the single
strand breaks, we incubated repairing cells in the presence of the compound 3-aminobenzamide. This compound is known to inhibit the enzyme poly(ADP-ribose) polymerase, which has been shown to be intimately involved with the repair 'of single strand DNA
breaks, such as those produced by radicals derived
from hydrogen peroxide (Ikejima et al., 1990; Satoh
and Lindahl, 1992). Poly(ADP-ribose) polymerase modulates the repair of single strand breaks, and is rapidly activated following DNA damage to recruit repair
enzymes to the damaged sites (Satoh and Lindahl,
1994). We found that the return of damaged cells to
control tail length and tail moment values was inhibited by treatment with 3-arninobenzarnide (Table 3).
These results provide an additional line of evidence
that the lesions detectable by the comet assay are either direct single strand breaks, or oxidized base damage which is converted to strand breaks in the presence of high pH, during either lysis or the pre-electrophoresis wash steps. The inclusion of 3-aminobenzamide in the repair media did not, however, appear
to change the 15 min comet moment significantly
(data not shown). This may be the result of several
possibilities. Poly(AD P-ribose) polymerase-independent repair is demonstrated by nucleotide excision repair, in contrast to base excision repair (Satoh and
Lindahl, 1994). However, base-excision repair is chara-
348
DAN Repair in Hydrogen Peroxide Induced Damage
cteristic of DNA damaged by oxygen free radicals.
It may be that the lesions excised in the initial 15
min may be reminiscent of cross-links. Similar to
HeLa cells in which high nucleotide pool levels preclude detection without ligation inhibition, the analysis
of excision repair is probably complicated by the rapid
occurrence of rejoining, disallowing accurate determination of repair. Hydrogen peroxide induced damage
displays a wide array of important DNA lesions not
necessa rily lethal to cell. The lesions examined in
these experiments do not appear to be related directly
with necrotic or apoptotic death, and are reparable
with characteristics of base excision repair, and repair
involving the activity of poly(ADP-ribose) polymerase.
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