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DNA damage induced by hydrogen peroxide in cultured tobacco cells is

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Article  in  Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis · March 2002

DOI: 10.1016/S1383-5718(01)00330-8 · Source: PubMed

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 Mutation Research 514 (2002) 147–152

DNA damage induced by hydrogen peroxide in cultured

tobacco cells is dependent on the cell growth stage
Diana A. Stavreva, Tomáš Gichner ∗
Institute of Experimental Botany, Academy of Sciences of the Czech Republic,
Na Karlovce 1a, 160 00 Prague 6, Czech Republic
Received 23 July 2001; received in revised form 23 October 2001; accepted 6 November 2001

Abstract
The level of hydrogen peroxide (H2 O2 )-induced genomic DNA damage measured by the Comet assay in tobacco suspension
cells (TX1) increased as a function of the age of the culture. After treatment of TX1 cells with 15 mM H2 O2 , the average (±S.E.)
median tail moment value was only 4.85 ± 1.00 ␮m in nuclei isolated from 2-day-old cells compared to 72.33 ± 1.40 ␮m in
nuclei isolated from 12-day-old cells. By contrast, nuclei first isolated from 2 and 12-day-old cells and then treated with H2 O2 ,
expressed the same level of DNA damage. The activity of catalases was markedly higher in 2-day-old TX1 cells compared to
12-day-old cells. The results indicate that the reaction of the H2 O2 with nuclear DNA is modified by the presence of the plant
cell wall, and enzymes and macromolecules present in the cytosol, and is not connected with changes in the nuclear DNA
sensitivity during cell suspension growth. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Comet assay; Ethyl methanesulphonate; Nicotiana tabacum; Single cell gel electrophoresis

1. Introduction affected in plants by enzymes such as the peroxidases

Previously, we reported a method using cultured
tobacco cells for the Comet (single cell gel electro-
phoresis) assay. We demonstrated that the genomic
DNA damage induced by the direct acting muta-
gen ethyl methanesulphonate (EMS) was uniform
throughout the cell growth curve of the suspension [1].
Hydrogen peroxide (H2 O2 ) can induce DNA damage
as measured by the Comet assay [2]. Its activity is
and catalases in the cytosol, and by the cell wall [3,4].
The purpose of this study was to determine if
H2 O2 -treated tobacco TX1 cells express uniform
genomic DNA damage throughout the growth curve
as after EMS treatment, and to determine if a differ-
ence in the pattern of DNA damage is expressed when
intact cells or isolated nuclei are treated with H2 O2 .
2. Materials and methods

Abbreviations: H2 O2 , hydrogen peroxide; EMS, ethyl methane- 2.1. Chemicals

sulphonate; SCGE, single cell gel electrophoresis; TX1, Nico-
tiana tabacum plant cell suspension culture; ROS, reactive oxygen
species
∗ Corresponding author. Tel.: +42-2-243-10109;
fax: +42-2-243-10113.
E-mail address: gichner@ueb.cas.cz (T. Gichner).
Hydrogen peroxide (H2 O2 , CAS no. 7722-84) was
purchased from Lachema, Neratovice (Czech Repub-
lic), reagents for electrophoresis and catalase assay
were purchased from Sigma (St. Louis, MO, USA).

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 3 3 0 - 8
148 D.A. Stavreva, T. Gichner / Mutation Research 514 (2002) 147–152
2.2. Tobacco cell suspension cultures
Long-term tobacco (Nicotiana tabacum) plant cell
suspension cultures (line TX1) were maintained in
MX medium, a modified liquid medium developed
by Murashige and Skoog [5]. All cultures were
grown at 26 ◦ C, under dark conditions with shaking
at 150 rpm. The fresh weights (mg ml−1 ) of the TX1
cells reported in the experiments were determined
from three repeated measurements. The viability of
the TX1 control and treated cells was determined
using the phenosafranine dye exclusion method
[6,7].
2.3. TX1 cell treatment
TX1 cells in mid-log-phase (6–8-day-old) were har-
vested by sieving, washed with MX medium, and
suspended in MX medium at 400 mg ml−1 . For the
growth curve experiments, lag- and stationary-phase
TX1 cells were also employed. Each reaction tube con-
tained a 2-ml volume of TX1 cell suspension adjusted
to 400 mg ml−1 , a known concentration of H2 O2 and
MX medium added to a final volume of 2.5 ml. The
reaction tubes were incubated for 2 h at 26 ◦ C.
2.4. Isolation of TX1 nuclei
After the treatment period, the cell suspension
was poured onto a pad of 10 layers of cheese-cloth
which was placed upon an absorbent paper. The cells
were rinsed with 5 ml of cold, modified Sörensen
extraction buffer (50 mM sodium phosphate, pH 6.8,
0.1 mM ethylene-diaminetetraacetic acid (EDTA),
0.5% dimethylsulfoxide (DMSO)). Using a spatula
approximately 400 mg of TX1 cells were scraped from
the cheese-cloth and placed into a cold microfuge tube
to which 500 ␮l of cold modified Sörensen extrac-
tion buffer and approximately 100 mg of washed sea
sand were added. The cells were gently agitated three
times with a metal spatula. After the sand settled to
the bottom of the tube, the mixture was poured into a
cold microfuge tube that was previously modified by
slicing the bottom off with a razor and fusing it with
a 53 ␮m mesh nylon filter (Spectra/Mesh, Spectrum,
Houston, TX, USA). The cell/nuclei suspension
was agitated 20–30 s with the end of a flat-blade
metal spatula; the nuclei passed through the filter
and were collected in an attached microfuge tube
on ice.
2.5. Comet assay
SCGE slides were made by dipping regular micro-
scope slides into a solution of 1% normal melting
point agarose prepared with distilled water at 50 ◦ C.
Immediately after isolation, 45 ␮l of the nuclei suspen-
sion were mixed with 45 ␮l of 1% low melting point
agarose (LMA prepared in PBS) upon a SCGE micro-
scope slide and covered with a coverslip. The slides
were placed on an iced surface for 5 min after which
the coverslip was removed and a final layer of 90 ␮l
0.5% LMA was placed upon the slide. A coverslip was
placed upon the LMA and the slide was kept on an
iced surface for 5 min. The coverslips were removed
and the slides were immersed in freshly prepared
cold lysing solution (2.5 M NaCl, 100 mM Na2 EDTA,
10 mM Tris, 1% sodium sarcosinate, 1% Triton X-100
and 10% DMSO). The slides were kept overnight at
4 ◦ C in the lysing solution, then placed in a horizontal
gel electrophoresis tank (Horizon 20 25 GIBCO-BRL)
with a freshly prepared cold electrophoresis solution
(1 mM Na2 EDTA and 300 mM NaOH, pH >13.5),
incubated for 20 min to allow the DNA to unwind and
to resolve alkali-labile sites, and electrophoresed at
0.75 V cm−1 (26 V, 300 mA) (Bio-Rad Power Pac 300)
at 4 ◦ C for 30 min. After electrophoresis, the slides
were rinsed three times in 700 mM Tris buffer, pH 7.5,
stained with 80 ␮l of 20 ␮g ml−1 ethidium bromide so-
lution and covered with a coverslip, and analyzed with
a fluorescence microscope that contained an excitation
filter of BP 546/10 nm and a barrier filter of 590 nm.
For each slide, 25 random nuclei were scored. Within
each experiment, three slides were analyzed for each
treatment and control group. The experiments were
repeated two to four times. We used a computerized
CCD camera digital image analysis system (Komet
version 3.1, Kinetic Imaging Ltd., Liverpool, UK) to
measure the tail moment values (integrated value of
tail density multiplied by the migration distance).
2.6. Treatment of nuclei
SCGE slides with nuclei from untreated TX1 cells
were prepared as outlined above and the slides were
immersed in solutions of 400 mM Tris–HCl buffer,
 D.A. Stavreva, T. Gichner / Mutation Research 514 (2002) 147–152 149

pH 7.5 containing different concentrations of H2 O2 3. Results

(0–1 mM) for 2 h at 26 ◦ C. After the treatment period,
the slides were rinsed three times for 5 min by im-
mersion in cold distilled water, kept in lysing solution
overnight at 4 ◦ C and electrophoresed and analyzed as
described above.
2.7. Catalase assay
Catalase activity was determined by measuring the
initial decomposition of H2 O2 at 240 nm (ε: 0.036
mM−1 cm−1 ) [8]. Spectrophotometric analyses were
conducted on a Hitachi U-3300 spectrophotometer.
TX1 cells were harvested by sieving and resuspended
in cold extraction buffer (polyvinylpyrrolidone 0.25%,
0.1 M Tris–HCl, 10−3 M dl-dithiothreitol, 10−3 M
EDTA, pH 7.8) at concentration of 1 g cells (fresh
weight) to 5 ml buffer. The cells were disrupted by
sonication (Vibra CellTM sonificator) 2× for 30 s,
and centrifuged at 4000 × g at 0 ◦ C for 10 min. The
supernatant fluid was recovered and kept on ice. The
reaction mixture consisted of 1 ml of the supernatant,
1.5 ml phosphate buffer (50 mM, pH 7.0) and 0.5 ml
of 0.1 M H2 O2 . For each sample, 1 ml of the super-
natant and 2 ml of the phosphate buffer were used as
spectrophotometric blank.
Catalase activity was expressed in units (U) mg−1
protein (1 U = ␮mol min−1 substrate degradation).
The protein content of each supernatant was deter-
mined using the Bio-Rad protein assay according to
manufacturer’s instructions (Bio-Rad Laboratories,
Richmond, CA, USA).
3.1. H2 O2 concentration response
of TX1 cells
Mid-log-phase TX1 cells (6–8-day-old) were trea-
ted for 2 h with H2 O2 that ranged in a concentra-
tion range from 2 to 70 mM. Comet analysis resulted
in a significant concentration response above 5 mM
(F8,27 = 33.735; P < 0.001) (Fig. 1). The control
average median (±S.E.) tail moment was 2.10 ±
0.37 ␮m and increased to 82.04 ± 4.01 ␮m for cells
treated with 70 mM H2 O2 . No decrease in the per-
cent of viable TX1 cells was observed throughout the
H2 O2 concentration range.
3.2. H2 O2 treatment of TX1 cells at different
stages within their growth curve
A Comet analysis was conducted of control TX1
cells from lag-phase (day 2) to stationary-phase (day
12) cultures, treated with 15 mM H2 O2 for 2 h (Fig. 2).
Throughout the growth curve, control or H2 O2 -treated
cells did not express acute cytotoxicity (data not
shown). From day 2 to 12, the average median tail

2.8. Statistical analysis

Data were analyzed using the statistical and

graphical functions of SigmaPlot 4.01 and SigmaStat
2.03 (SPSS Inc., Chicago, IL). From the repeated
experiments, the average median tail moment value
was calculated for each treatment group from the
median tail moment value from each SCGE slide
[9]. The median tail moment values were used in
a one-way analysis of variance test. If a significant
F-value of P ≤ 0.05 was obtained, a Dunnett’s mul-
Fig. 1. The concentration response curve of the average median
tiple comparison versus the control group analysis tail moment values for nuclei isolated from TX1 cells treated with
was conducted. Differences between two groups were different concentrations of H2 O2 for 2 h at 26 ◦ C. The error bars
statistically evaluated by the Paired t-test. represent the S.E. of the mean.
150 D.A. Stavreva, T. Gichner / Mutation Research 514 (2002) 147–152
Fig. 2. Growth curve of TX1 cells over a 12-day period at 26 ◦ C
with the corresponding average median tail moment values of the
nuclei isolated from untreated control cells and cells treated with
15 mM H2 O2 or 20 mM EMS for 2 h at 26 ◦ C. EMS data were
published previously [1]. The error bars represent the S.E. of
the mean.
moment values for the negative control cells ranged
from 2.89 to 3.56 ␮m and there was no significant
difference among the groups (F5,63 = 0.667; P =
0.650). The level of the H2 O2 -induced DNA damage
was dependent on the growth stage of the TX1 suspen-
sion. From 48 to 288 h, the average (±S.E.) median
tail moment significantly increased from 4.85 ± 1.00
to 72.33 ± 1.40 ␮m (F5,35 = 315.484; P < 0.001).
The EMS data were published previously [1].
3.3. H2 O2 treatment of nuclei isolated
from TX1 cells
TX1 cell nuclei were isolated from 5-day-old
cells, imbedded in 0.5% LMA on SCGE slides and
treated for 2 h with H2 O2 that ranged from 0.1 to
1 mM (Fig. 3). The control average median (±S.E.)
tail moment was 6.84 ± 0.68 ␮m and increased
to 50.79 ± 1.54 ␮m for the 1 mM H2 O2 treatment
group. There was a significant increase in the tail mo-
ment values as compared to the negative control with
all H2 O2 concentrations analyzed (F4,25 = 192.463;
P < 0.001).
Fig. 3. The concentration response curve of the average median
tail moment values for H2 O2 -treated isolated TX1 nuclei for 2 h
at 26 ◦ C. The error bars represent the S.E. of the mean.
3.4. H2 O2 treatment of TX1 cells or nuclei
at day 2 or 12 of the growth cycle
Experiments were conducted with TX1 cells
that were harvested after 2 or 12 days of growth.
TX1 cells were treated with 5 or 15 mM of H2 O2
(Fig. 4A) or isolated nuclei were treated with 0.1 or
0.25 mM (Fig. 4C). Hydrogen peroxide treatment of
the 12-day-old stationary-phase TX1 cells induced
higher average median tail moment values as com-
pared with 2-day-old lag-phase cells. With 15 mM
H2 O2 , the lag-phase TX1 cells expressed an aver-
age median (±S.E.) tail moment of 5.15 ± 0.89 ␮m
while the tail moment values of the stationary-phase
cells were 14 times higher (72.36 ± 1.39 ␮m). With
the H2 O2 -treated nuclei, isolated from lag- and
stationary-phase TX1 cells, there were no significant
differences in the tail moment values (the t-values
ranged from P = 0.199 to P = 0.721).
3.5. Catalase activity of 2- and 12-day-old
TX1 cells
The catalase activity of lag-phase TX1 cells
was of 48.95 ± 5.33 U mg−1 protein, and for the
stationary-phase cells the catalase activity was reduced
to 8.9 ± 0.25 U mg−1 protein (Fig. 4B). The catalase
 D.A. Stavreva, T. Gichner / Mutation Research 514 (2002) 147–152
Fig. 4. (A) A comparison between the sensitivity of 2- and
12-day-old TX1 cells to the DNA damaging effect of 5 and 15 mM
H2 O2 applied for 2 h at 26 ◦ C. (B) Catalase activity (units mg−1
protein) determined in the extracts prepared from 2- and 12-day-old
TX1 cells. (C) A comparison between the sensitivity of nuclei
isolated from 2- and 12-day-old TX1 cells to the DNA damaging
effect of 0.1 and 0.25 mM H2 O2 applied for 2 h at 26 ◦ C. The
error bars represent the S.E. of the mean.
151
activities are mean values from three experiments
(±S.E.).
4. Discussion
Organisms produce a range of reactive oxygen
species (ROS), including superoxide (O2 • ), the hy-
droxyl radical (OH• ), and hydrogen peroxide (H2 O2 ),
during the course of aerobic metabolic processes. If
not effectively and rapidly removed from the cells,
ROS can damage a wide range of macromolecules,
including DNA. Both enzymatic and non-enzymatic
mechanisms have evolved to protect cells from oxy-
gen toxicity, these include ROS-scavenging enzymes
such as superoxide dismutase, catalase, and peroxi-
dase [10].
The occurrence of endogenous DNA oxidation, via
ROS released during normal metabolism, has been
suggested as a possible cause of aging, cancer and
other diseases [11]. In the presence of transition met-
als, H2 O2 may give rise to highly reactive hydroxyl
radicals leading to DNA damage and mutations
[12].
In a previous paper [1], we demonstrated that
tobacco TX1 cells were uniformly sensitive to the
DNA damaging effects of EMS throughout the growth
curve of the cell suspension. In contrast, the DNA
damaging effect of H2 O2 in tobacco cells is depen-
dent on their stage in the growth curve. Lag-phase
DNA damage is significantly lower than in the log or
stationary phase (Fig. 2).
In mammalian cells H2 O2 induces base oxidation
and SSBs [13]. Hydrogen peroxide is a potent inducer
of DNA migration in the SCGE (Comet) assay [2,14].
In murine fibroblast cells, the DNA damaging effect
of H2 O2 was higher in stationary than in exponentially
growing cells [15].
Catalase functions as a cellular sink for H2 O2 .
Excised leaves from catalase-deficient tobacco
seedlings removed external H2 O2 to a much lower
extent than excised leaves from wild type tobacco
plants [4]. The catalase activity of lag-phase TX1
cells was significantly higher than of stationary-phase
cells. This time-dependent decrease in catalase acti-
vity explains the increased sensitivity of log- and
stationary-phase cells to external H2 O2 (Fig. 4B).
By contrast, the activity of peroxidases, secreted into
 152 D.A. Stavreva, T. Gichner / Mutation Research 514 (2002) 147–152
the cultivation medium by TX1 cells increased as a
function of the age of the culture [16].
Isolated TX1 cell nuclei (Fig. 3) were more sensi-
tive to the DNA damaging effect of H2 O2 than intact
TX1 cells (Fig. 1). After a treatment of 0.1 mM H2 O2 ,
the isolated nuclei expressed a tail moment (±S.E.)
value of 25.6 ± 1.33 ␮m, while at a 20 time higher
concentration of H2 O2 in the TX1 cells, the tail mo-
ment value was 6.30 ± 1.30 ␮m. Fig. 4A presents
data on the DNA damaging effect of H2 O2 on intact
TX1 cells, which have catalase in the cytosol. An
inverse correlation of catalase levels with DNA dam-
age was demonstrated. Fig. 4C presents data of the
effect of H2 O2 on isolated nuclei not having cytosol,
thus, without catalases. As a result, no significant
differences in DNA damage was observed in nuclei
isolated from 2- and 12-day-old TX1 cells.
In addition, the differential sensitivities of the
intact cells and isolated nuclei may be the result of the
cellular defense to protect genomic DNA. Structural
defense such as the plant cell wall, and most cellular
macromolecules (lipids, proteins, carbohydrates) may
pose barriers to H2 O2 . During preparation of SGE
slides oxidative damage may occur in isolated nuclei,
but it will be rather small, as the DNA damage in the
controls, expressed by the tail moment value is less
than 5 ␮m. However, ROS are generated by aerobic
metabolism and must be neutralized by intracellular
mechanisms, such as by ROS-scavenging enzymes
like catalases and peroxidases.
In summary, the level of H2 O2 -induced genomic
DNA damage measured by the Comet assay in tobacco
suspension cells (TX1) increased with the increased
age of the cell culture. This increase is explained by
the higher activity of catalases in lag-phase compared
to log- and stationary-phase cells.
Acknowledgements
This research was funded by Grant Agency of the
Czech Republic Grant no. 521/99/0532. We thank
Drs. A. Collins (Aberdeen, UK) and M.J. Plewa
(Urbana, USA) for discussions and assistance with
this manuscript.
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