Original Paper

Vitamin C prevents DNA damage

induced by renovascular
hypertension in multiple organs
of Wistar rats

Human and Experimental Toxicology

29(7) 593599
The Author(s) 2010
Reprints and permission:
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DOI: 10.1177/0960327109358267
het.sagepub.com

Erika Emy Nishi1, Ruy Ribeiro Campos1, Cassia Toledo Bergamaschi1,2,

Vitor Rossi de Almeida2 and Daniel Araki Ribeiro2

Abstract
The aim of this study was to investigate, through the single-cell gel (comet) assay, whether vitamin C is able to
protect against renovascular hypertension-induced genotoxicity in multiple organs. A total of 32 male Wistar
rats were divided into four groups: negative control (n 6); animals treated with vitamin C (n 6); hypertensive rats (n 10) and hypertensive rats and treated with vitamin C (n 10). Hypertension was induced as a
result of partial obstruction of the left renal artery by means of a silver clip during 6 weeks. Vitamin C was
administered at 150 mg/kg during 7 consecutive days before the end of the experimental period. The results
showed that vitamin C was able to protect blood cells against hypertension-induced genotoxicity. Brain, liver
and heart cells were also protected by vitamin C following hypertension-induced genotoxic damage. Regarding
blood pressure, vitamin C reduced the hypertensive state. In conclusion, our results suggest that vitamin C can
prevent hypertension-induced DNA damage in blood, liver, brain and heart cells as well as to normalize the
blood pressure of rats.
Keywords
hypertension, vitamin C, rats, DNA damage, single cell gel (comet) assay

Introduction
The last 100 years have been characterized by
dramatic changes in the epidemiological scenario,
especially regarding the main causes of death among
the population.1 The prolongation of life expectancy
and the decline in mortality from infectious diseases
led to the replacement of the latter with chronic
degenerative diseases as prevailing causes of death
among the population.2 The incidence of such kind
of diseases grows exponentially with age. To date,
hypertension is a major public health problem in the
world and one of the leading risk factors contributing
to cardiovascular diseases, which remain the foremost
cause of death in developing and developed countries.
This serves to characterize a group of patients who
bear the risk of acquiring cardiovascular diseases high
enough to merit medical concern.3 From among the
putative causes available in the literature, stress,
obesity, alcohol consumption, cigarette smoking,
pregnancy, hypercholesterolemia are some of the risk

factors of hypertension due to their ability to induce

oxidative stress.4
Oxidative stress is a state in which excess reactive
oxygen species (ROS) overwhelm endogenous
antioxidant systems.5 Particularly, previous studies
conducted by our group have demonstrated that an
increase in oxidative stress plays a significant role
in maintaining high arterial blood pressure and sympathetic drive during experimental hypertension.6
Oxidative stress is an important cause of DNA
1

Department of Physiology, Cardiovascular Division, Paulista

Medical School, Federal University of Sao Paulo, UNIFESP, SP,
Brazil
2
Department of Biosciences, Federal University of Sao Paulo,
UNIFESP, SP, Brazil
Corresponding author:
Daniel Araki Ribeiro, Departamento de Biociencias, Av. Ana
Costa, 95 Vila Mathias, Santos SP, Brazil.
Email: daribeiro@unifesp.br

594

damage, because oxidative DNA damage has been

recognized as a major cause of cell death and mutations in all aerobic organisms.7 Some studies have
provided evidence that peripheral lymphocyte DNA
damage is caused by multiple factors, including,
mainly, oxidative stress as well as other exogenous
and/or endogenous sources.8,9 Moreover, some
studies have shown that subjects suffering from
hypertension have decreased antioxidant capacity and
increased levels of ROS.10 ROS-induced DNA
damage occurs more in hypertensive patients than in
normal ones.11
Biological compounds with antioxidant properties
contribute to the protection of cells and tissues against
deleterious effects of ROS and other free radicals.12
Ascorbic acid (vitamin C) is known to act as an efficient antioxidant, either directly by reacting/through
reaction with aqueous peroxyl radicals or indirectly
by restoring the antioxidant properties of vitamin
E.13 In light of such considerations, it would be useful
to find out whether, and to what extent, vitamin C can
modulate hypertension-induced genotoxic stress, particularly due to lack of previous reports.
Over the past decade, the single-cell gel (comet)
assay in its alkaline version has been a rapid, simple,
and reliable biochemical technique for evaluating
DNA damage in mammalian cells.14 The basic
principle of the single-cell gel (comet) assay is the
migration of DNA in an agarose matrix under
electrophoretic conditions. When viewed under a
microscope, the damaged cell has the appearance of
a comet, with head (nuclear region) and tail containing DNA fragments or strands migrating towards the
anode.15 Previous studies conducted by our group
have proved that the single-cell gel (comet) assay is
a suitable tool to investigate in vivo genotoxicity.16-19
As a result of limited evidence, the aim of this
study was to investigate whether vitamin C, a
well-established antioxidant, might exert some antigenotoxicity during renovascular hypertension by the
single-cell gel (comet) assay. Certainly, such data will
contribute to our better understanding of hypertension
and vitamin C upon the cellular system.

Material and methods

Animals and experimental design
All experimental procedures were conducted according to the National Health Institute guidelines on the
use and care of animals, and the study protocol was

Human and Experimental Toxicology 29(7)

approved by the Research Ethics Committee of the

Medicine School of Sao Paulo Federal University.
Male Wistar rats weighing approximately 250 g were
obtained from Sao Paulo Federal University (UNIFESP), Brazil, and kept under controlled conditions
of temperature (24 + 2 C), 12-hour light/dark periods, with free access to commercial diet (Purina1)
and water. In order to bring the animals to develop
a hypertensive state, the left renal artery was partially
obstructed by means of a 0.2-mm-wide silver clip during 6 weeks.20 Surgical procedure was performed in
20 male rats (150180 g). Vitamin C (150 mg/kg/day;
Bayer S.A., Rio de Janeiro, Brazil) or vehicle (NaCl
0.9%) was orally administered by gavage for 7 consecutive days, between 12:00 and 14:00 hours, by the
sixth week after clipping. Vitamin C dose was chosen
based on previous experiments from our group.21 A
total of six animals served as negative control (nontreated group). In addition, a total of six animals were
treated with vitamin C following the same procedures
described above.

Surgical instrumentation for the acquisition of the

blood pressure
Six weeks after renal surgery, the rats were anesthetized with inhalatory halotane (Tanohalo(R), Cristalia,
SP, Brazil, thymol 0.01% final concentration) and
instrumented with femoral venous and arterial catheters for drug injection and arterial pressure recording,
respectively. Arterial blood pressure was recorded in
conscious rats. Adequate depth of anesthesia was
monitored by blood pressure supervision, and, if
required, additional anesthetic was administered
(5% of initial dose).

Sample collection
After completing the experimental design and subsequently performing the blood pressure measurement
to confirm the hypertensive state in the experimental
and control groups, blood sample was collected from
the heart into a lightly heparinized syringe. In addition,
central fragments from the heart, liver and brain were
collected and minced in 0.9% NaCl. The supernatant
was removed and the cellular suspensions (*10 mL)
were used for the single-cell gel (comet) assay.

Single-cell gel (comet) assay

The alkaline version of the single-cell gel (comet)
assay used in this study is sensitive for a wide variety

Nishi EE et al.

of DNA lesions, among which we can point out

single- and double-strand breaks, oxidative DNA base
damage, alkali-labile sites including abasic and
incomplete repair sites and DNA-DNA/DNA-protein/DNA-drug cross-linking in any eukaryotic cell.14
The protocol used for peripheral blood, liver, heart
and brain cells followed the guidelines proposed by
Sasaki et al.,22 with some modifications. Namely, a
volume of 5 mL of peripheral blood was added to
120 mL 0.5% low-melting-point agarose at 37 C,
layered onto a slide pre-coated with 1.5% regular
agarose and covered with a coverslip. The supernatant
(cellular suspension; 10 mL) of the liver, heart and
brain was added to 120 mL 0.5% low-melting-point
agarose at 37 C, layered onto a slide pre-coated with
1.5% regular agarose and covered with a coverslip.
After brief agarose solidification in a refrigerator, the
coverslip was removed and slides immersed in lysis
solution (2.5 M NaCl, 100 mM EDTA, 10 mM
TrisHCl buffer, pH 10, 1% sodium sarcosinate with
1% Triton X-100 and 10% DMSO) for about 1 hour.
Prior to electrophoresis, the slides were left in alkaline
buffer (pH > 13) for 20 min and electrophoresed for
another 20 min, at 0.7 V/cm, 300 mA. After electrophoresis, the slides were neutralized on 0.4 M Tris
HCl (pH 7.5), fixed in absolute ethanol and stored
until analysis in a fluorescent microscope at 400
magnification.
Independent positive controls, using cells from peripheral blood, liver, brain and heart, were treated in
vitro with 10 mg/mL MMS (methylmethasulfonate)
for 10 min at 37 C, in order to ensure assay reproducibility and sensitivity as established in previous studies conducted by our group investigating noxious
activities of chemicals or biological agents in multiple
organs of rats.23,24

Genotoxicity data
A total of 50 randomly captured comets per animal
(25 cells from each slide)25 were blindly examined
by one expertise observer at 400 magnification,
making use of a fluorescence microscope (Olympus1)
connected through a black and white camera to an
image-analysis system (Comet Assay II1 Perceptive
Instruments, Sufolk, Haverhill, UK) previously calibrated according to the manufacturers instructions.
The computerized image-analysis system acquires
images, computes the integrated intensity profiles for
each cell, estimates the comet cell components and
then evaluates the range of derived parameters.

595

Undamaged cells have an intact nucleus without a

tail and damaged cells have the appearance of a
comet. To measure DNA damage, two imageanalysis system parameters were considered: tail
intensity (% migrated DNA) and tail moment (the
product of the tail length and the fraction of DNA
in the comet tail).23 Since none of the groups showed
significant difference between such parameters, we
chose tail moment for the presentation of the results.
Tail moment is a virtual measure calculated by the
computerized image-analysis system considering
both the length of DNA migration in the comet tail
and the tail intensity. Such a parameter is one of the
best indices of induced DNA damage among the various parameters calculated by this method.

Statistical methods
Statistical analysis for blood pressure was carried out
by one-way analysis of variance (ANOVA) followed
by Tukeys test. Tail moment data were assessed by
Kruskall-Wallis non-parametric test followed by
Dunns test using Sigma Stat/SigmaStat1 for Windows
(Jadel Scientific, USA). p < .05 were considered for
statistical significance.

Results
There were statistically significant differences
(p < .01) in blood pressure between control and
hypertensive rats. Animals treated with vitamin C
did not increase blood pressure when compared to
negative control. However, subchronic exposure
of hypertensive rats to vitamin C was able to
diminish blood pressure. The aforementioned data
are summarized in Figure 1. No animals died
during the experiment.
With regard to genotoxic parameters, statistically
significant differences (p < .05) were found in the
blood cells of hypertensive rats when compared to
negative control (Figure 2). The same occurred to
other assessed organs, such as the liver, which displayed an increased DNA migration in hypertensive
rats (Figure 3). Also, the heart and brain showed
strong genotoxicity in hypertensive rats, with a more
pronounced effect when compared to blood or even
liver cells. Such results are shown in Figures 4 and
5, respectively.
Exposure to vitamin C did not induce genotoxic
damage in all assessed organs in this study, that is,
blood, brain, liver or heart cells. However, such antioxidant compound was able to negatively modulate

596

Human and Experimental Toxicology 29(7)

Tail moment (arbitrary units)

6
*

5
4

3
2
1
0

Negative
control

Vit C

HPT

HPT + Vit C

Positive
control

Groups

Figure 1. Arterial blood pressure from hypertensive rats.

Values are expressed as mean + SD. Vit C, vitamin C;
2K-1C, hypertension; Positive control: 10 mg/mL MMS
(methylmethasulfonate).

Figure 3. DNA damage expressed as the mean tail

moment in rat liver cells submitted to hypertension and
treated with vitamin C. Values are expressed as mean +
SD. Vit C: vitamin C; HPT: hypertension; Positive control:
10 mg/mL MMS (methylmethasulfonate). *p < .05 when
compared to negative control.

4
3
2
1

8
7

Negative
control

Vit C

HPT

HPT + Vit C Positive

control

6
5
4
3
2
1
0

9
Tail moment (arbitrary units)

Tail moment ( arbitrary units)

Negative
control

Vit C

HPT

HTTP + Vit C Positive

control

Groups

Groups

Figure 2. DNA damage expressed as the mean tail

moment in rat blood cells submitted to hypertension and
treated with vitamin C. Values are expressed as mean +
SD. Vit C: vitamin C; HPT: hypertension; Positive control:
10 mg/mL MMS (methylmethasulfonate). *p < .05 when
compared to negative control.

Figure 4. DNA damage expressed as the mean tail

moment in rat heart cells submitted to hypertension and
treated with vitamin C. Values are expressed as mean +
SD. Vit C: vitamin C; HPT: hypertension; Positive control:
10 mg/mL MMS (methylmethasulfonate). *p < .05 when
compared to negative control.

Discussion
renovascular hypertension-induced DNA damage in
blood cells. Brain, liver and heart cells were also protected by vitamin C following hypertension-induced
genotoxic damage.
Rat blood, liver, brain and heart cells were further
assayed with MMS to ensure assay sensitivity.
Distinct sensitivity was observed (p < .05) when compared to the negative control group.

The aim of this study was to evaluate putative antigenotoxicity exerted by vitamin C during renovascular hypertension. The investigation was conducted
through the single-cell gel (comet) assay. To the best
of our knowledge, such approach has not been demonstrated so far.
On the basis of tail moment data, the results of this
study displayed that the alkaline single-cell gel

Nishi EE et al.

597

Tail moment (arbitrary units)

8
7

6
5
4
3
2
1
0

Negative
control

Vit C

HPT

HPT + Vit C

Positive
control

Groups

Figure 5. DNA damage expressed as the mean tail

moment in rat brain cells submitted to hypertension and
treated with vitamin C. Values are expressed as mean +
SD. Vit C: vitamin C; HPT: hypertension; Positive control:
10 mg/mL MMS (methylmethasulfonate). *p < .05 when
compared to negative control.

(comet) assay, in the experimental conditions thereof,

was able to detect the presence of DNA damage on
blood and liver cells in hypertensive rats. Furthermore, our results showed that vitamin C was able to
reduce the hypertensive state in rats as a result of
decreasing blood pressure. Some authors have argued
that vitamin C decreased the blood pressure in hypertensive rats even under very high blood-pressure condition, as high as 250 mm Hg.26 Interestingly, vitamin
C was able to prevent genotoxic damage on these
organs as well. Such findings, fully in line with others,
suggest that liver may be protected by vitamin C as it
improves total antioxidant status, and thus might prevent high blood pressure and its complications to
experimental hypertension.27 It seems that ascorbic
acid may also work effectively to protect liver functions. Taken together, there seems to be evidence that
hypertension can induce genetic damage on blood and
liver cells, vitamin C being a potent anti-genotoxic
compound.
Taking into consideration that hypertension is considered as a state of oxidative stress contributing to
the development of atherosclerosis28 and other
hypertension-induced damage to organs,29 the heart,
an important target for hypertension, was assessed
in such a setting. Our results showed that genetic
damage was induced by hypertension in that organ.
Some authors have argued that hypertension significantly increased 8-oxo-dG accumulation in heart
cells. It has been established that accumulation of

8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dG) in
DNA is associated with genotoxicity and mutagenesis.30 In addition, vitamin C was able to prevent
hypertension-induced genotoxic damage as well. By
comparison, recent studies conducted by our group
have revealed that ascorbic acid can prevent myocardial infarction in rats.21,31
To further elucidate the possible outcomes of
hypertension in the central nervous system, we were
able to assess genotoxicity on the brain. Our results
revealed that hypertension was able to exert strong
genetic damage on brain cells. An early study conducted by our group has pointed out high oxidative
stress within the brains of rats suffering from hypertension.5 It plays a major role in maintaining high
arterial blood pressure and sympathetic drive in
hypertension, and consequently inducing genetic
damage to brain cells. Taken as a whole, such findings
strongly suggest that oxidative DNA damage may be
involved in the neuronal damage in the brains of rats
submitted to hypertension.4 Vitamin C exerted potent
anti-genotoxicity on that organ. Newaz et al.27 have
assumed that the brain maybe protected by vitamin
C, which improves total antioxidant status, thus being
able to prevent high blood pressure and its complications in hypertensive rats. Our findings are in agreement with such results.
In summary, our results suggest that hypertension
may contribute to DNA damage in the blood, liver,
brain and heart, bearing a strong effect in the last ones.
Vitamin C can prevent renovascular hypertensioninduced genotoxic damage for all assessed organs.
Certainly, such findings offer new insights into the
mechanisms underlying the relation between oxidative
stress, antioxidant status and hypertension.
Acknowledgements
This work was supported by FAPESP (Fundacao de
Amparo a` Pesquisa do Estado de Sao Paulo (Grant number:
07/01228-4). RRC, CTB and DAR are recipients of the
CNPq fellowships.

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