Pharmacology & Pharmacy, 2011, 2, 127-135
doi:10.4236/pp.2011.23017 Published Online July 2011 (
Copyright © 2011 SciRes. PP
Protective Effect of Resveratrol against Oxidation
Stress Induced by 2-Nitropropane in Rat Liver
Maura Lodovici, Elisabetta Bigagli, Cristina Luceri, Elena M. Manni, Mohamed Zaid
Department of Pharmacology, University of Florence, Florence, Italy.
Received March 29th, 2011; revised April 25th, 2011; accepted May 17th, 2011.
We investigated the effect of resveratrol on oxidation damage and variation of antioxidant defences induced by
2-nitropropane (2-NP) in rat liver. One group of five rats was given resveratrol (50 mg/kg/d body weight) in the diet
until the end of the experiment. After 14 days, 2-NP (100 mg/kg) was injected i.p. into two groups of animals (2-NP +
Res and 2-NP groups) while control animals were treated with vehicle alone. Animals were killed by decapitation 15 h
after 2-NP injection. The levels of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) were significantly increased by
2-NP injection, but resveratrol restored 8-oxodGuo to levels similar to those measured in controls. Superoxide dismu-
tase (SOD) and xanthine oxidase (XO) activities in the liver were significantly increased by 2-NP, but were similar to
those found in the group treated with resveratrol and 2-NP (2-NP + Res). We also observed that 2-NP injection sig-
nificantly reduced GSH/GSSG ratio in the liver and this change was partially reversed by resveratrol treatment. More-
over, an increased (p = 0.06) expression of the oxoguanine glycosylase (OGG1) gene was found in 2-NP rats, whereas
pre-treatment with resveratrol restored OGG1 expression to control levels. An up-regulation of caspase-3 was also
observed in 2-NP group, but resveratrol significantly reduced the activation of caspase-3. An inverse correlation was
found between GSH/GSSG and 8-oxodGuo in the 2-NP group. On the contrary, 8-oxodGuo levels, GSH/GSSG ratio,
XO and SOD activities in the colon mucosa of 2-NP rats were similar to those of controls confirming that the colon is
not a target of oxidation damage 2-NP induced. In conclusion, our results indicate that oxidative DNA damage and
apoptosis are the main mechanisms of cell death in a model of chemically induced severe acute hepatic injury and in
this early stage of damage pharmacological doses of resveratrol can ameliorate hepatic oxidation damage by its anti-
oxidant and scavenging properties through a reduction of XO activity, a partial restoration of GSH/GSSG ratio in addi-
tion to its capacity to inhibit apoptosis.
Keywords: 2-Nitropropane, 8-Oxodguo, lipoperoxidation, MDA, XO, SOD, Resveratrol, Apoptosis
1. Introduction
It is well documented that DNA damage induced by re-
active oxygen species (ROS) plays an important role in
aging and in a number of human pathological processes,
such as chronic inflammation, atherosclerosis, diabetes,
ischemia-reperfusion injury and cancer [1-5]. Resveratrol
is a natural phenolic compound with free radical scav-
enging and antioxidant properties [6-9]. Many studies
have demonstrated the anti-inflammatory and anticancer
effects of resveratrol in various organs [10-12]. In addi-
tion, Harikumar and Aggarwal [13] reported that res-
veratrol is capable of binding to numerous macromole-
cules involved in cell function, such as multidrug resis-
tance proteins, topoisomerase II, aromatase, DNA poly-
merase, estrogen receptors and tubulin. Resveratrol can
also activate various transcription factors (NF-kB, STAT3,
β-catenin and PPAR-γ), inhibit many protein kinases,
induce antioxidant enzymes [13] and reduce alterations
in the protein expression of mitochondria-mediated
apoptosis markers [14]. Recently, Tunali-Akbay et al.
[15] found that resveratrol protects against methotrexate-
induced hepatic injury in rats by reversing the oxidative
toxic damage. In addition, has been reported that res-
veratrol is able to suppress oxidative stress and inflam-
matory response in rat hepatocarcinogenesis induced by
diethylnitrosamine [16].
A specific nitroalkane, 2-nitropropane (2-NP), used as
a constituent of paints and inks and present in tobacco
smoke, can cause hepatic damage in humans and animals
[17,18]. The mechanism by which 2-NP induces hepa-
toxicity is not clearly defined, but since many studies
128 Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver
have demonstrated its effect as a potent inducer of oxi-
dative DNA damage in liver tissue, its toxicity is inter-
preted as a consequence of ROS generation [18,19].
Thus, 2-NP has been widely used as a model compound
for studies of oxidation damage in the liver of rodents
On the basis of these considerations, the present stud-
ies were carried out to determine the effects of resvera-
trol on 2-NP-induced oxidative stress and apoptotic
changes in the rat liver.
2. Materials and Methods
2.1. Animals and Treatments
All experiments were carried out in accordance with the
European Communities Council Directive of 24 No-
vember 1986 (86/609/EEC) for experimental animal
Male Fisher 344 rats (180 - 200 g) were purchased
from Nossan (Milan, Italy). After their arrival from the
supplier, 15 animals were quarantined for 1 week and fed
standard lab chow and water, ad libitum. The rats were
then randomly divided into three groups of 5 rats each.
One group (2-NP + Res) was given resveratrol (50
mg/kg/d body weight) in the diet until the end of the ex-
periment. The dose of resveratrol was selected by taking
into account the safety of this compound in animal stud-
ies [23] and based on previously reported chemopreven-
tive doses of resveratrol in rodents [24]. A second group
was administered only 2-NP and the third group served
as control (C). Taking into account that approximately 15
g of diet were consumed by each rat every day, we added
resveratrol in the diet (600 ppm) in order to reach a dose
of 50 mg/kg/day. After 14 days, 100 mg/kg of 2-NP in
saline supplemented with 0.1 % Tween80 was injected
i.p. into 2 groups of animals while the control group (C)
was treated with the vehicle alone. Animals of all 3
groups were killed by decapitation 15 h after 2-NP injec-
tion. The liver and colon were excised and frozen at
–80˚C. Liver and colon tissues were homogenized in 50
mM phosphate buffered solution (PBS) containing 0.1M
dithiothreitol and then centrifuged at 4˚C for 20 min at
2000 × g. Pellets were used for 8-oxo-7,8-dihydro-2’-
deoxyguanosine (8-oxodGuo) determination, while mal-
onyldialdehyde (MDA), protein carbonyl residues, su-
peroxide dismutase (SOD), xanthine oxidase (XO) were
measured in the liver and colon mucosa supernatants. In
addition, in the liver supernatant reduced (GSH) and
oxidized glutathione (GSSG), were also measured.
2.2. Materials
2-NP, resveratrol and all chemicals were purchased from
Sigma, Milan, Italy. HPLC Shimadzu (10 AD). Cou-
lochem II electrochemical detector (ESA). UV (Perkin
Elmer) detector.
2.3. 8-Oxodguo Assay
Liver pellets were re-suspended and DNA was isolated
with the method recommended by ESCODD group [25].
Purified DNA was hydrolysed with P1 nuclease (14 IU)
and alkaline phosphatase (10 IU). The hydrolysed mix-
ture was filtered using Micropure-EZ enzyme remover
(Amicon, MA, USA) and 50 µl were injected into an
HPLC apparatus. The nucleosides were separated using a
C18 reverse-phase column (Supelco, 5 µm, I.D. 0.46 ×
25 cm). The 8-oxodGuo and 2dG levels in DNA were
measured using an ESA Coulochem II electrochemical
detector in line with a UV detector as previously de-
scribed [26].
2.4. Carbonyl Residues Assessment
Carbonyl residues were determined by the method of
Correa-Salde and Albesa [27]. Liver supernatant (0.35
ml) was treated for 1 h with 1 ml of 0.1% dinitrophenyl-
hydrazine in 2 M HCl and precipitated with 10% tri-
chloroacetic acid before being centrifuged for 20 min at
10,000 x g. The pellets were extracted with 1 ml of an
ethanol:ethyl acetate mixture (1:1) three times and then
dissolved in 2 ml of 6 M guanidine HCl in 20 mM potas-
sium phosphate buffer (PBS) pH 7.5. The solutions were
incubated at 37˚C for 30 min and insoluble debris was
removed by centrifugation. The absorbance was meas-
ured at 364 nm.
2.5. MDA Determination
MDA was determined after derivatization with 2,4-dini-
trophenylhydrazine (DNPH) as described by Mateos et al.
[28]. Briefly, 50 µl of 6M NaOH were added to an ali-
quot of 500 µl of liver supernatant. Alkaline hydrolysis
of protein bound MDA was achieved by incubating this
mixture in a 60˚C water bath for 30 min and protein pre-
cipitated with 250 µl of 30% (v/v) trichloroacetic acid. A
250 µl volume of supernatant was mixed with 25 µl of 5
mM DNPH in 2M hydrochloric acid. Finally, this reac-
tion mixture was incubated for 30 min at room tempera-
ture protected from light. An aliquot of 100 µl was in-
jected into a Shimadzu LC-10AD HPLC system with a
Waters Spherisorb RP-18 column (4.6 mm × 150 mm,
ODS 25 um, Supelco).
Samples were eluted with a mixture of 0.2% (v/v) ace-
tic acid and acetonitrile (62:38, v/v) at a flow rate of 0.8
ml/min . Chromatograms were acquired at 310 nm. The
calibration curve, prepared by diluting a standard solu-
tion of tetraethoxypropane (TEP), was reported in Fig-
ure 1.
opyright © 2011 SciRes. PP
Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver 129
Figure 1. Calibration curve of MDA.
2.6. SOD Activity
SOD was determined in the liver supernatant using the
nitroblue tetrazolium (NBT) reaction [29]. The reaction
mixture, containing 0.2 ml of liver supernatant, 2.4 ml of
50 mM Na2CO3, 0.1 ml of 3 mM EDTA, 0.1 ml of 3 mM
xanthine, 0.1 ml of 0.8 mM NBT and 0.1 ml of XO (140
mU/ml initial concentration), was incubated for 30 min
at 24˚C. The inhibition of NBT reduction in each sample
was determined spectrophotometrically at 470 nm. SOD
content, expressed as U/mg protein, was evaluated by
relating to inhibition by the SOD standard measured at
the same time.
2.7. XO Activity
XO was determined in the liver supernatant according to
Corte and Stirpe [30]. XO activity, expressed as U/mg
protein, was assayed by measuring uric acid production
at 280 nm.
2.8. Determination of GSH and GSSG
The procedure was performed following the method by
Cereser et al. [31] with few modifications. Briefly, 100
µl of liver supernatant were diluted by addition of 500
mM sodium phosphate pH 7; 100 µl of this solution was
mixed with 100 µl ortho-pthalaldehyde (OPA) (5 mg/ml)
and 100 µl was injected into an HPLC system to deter-
mine reduced GSH; total glutathione (GSHt) was evalu-
ated by performing a reduction step of GSSG with di-
thiotreitol. The GSSG concentration was obtained by
subtraction of the GSH from the GSHt. Chromatography
of GSH and GSHt after their derivatization with OPA
was accomplished using isocratic elution in a Discovery
C18 (150mm × 4mm i.d.) 5µm analytical column (Su-
pelco, USA) at 37˚C. The mobile phase consisted of 8%
acetonitrile in 50 mM sodium acetate pH 6.2. The flow
rate was set at 1 ml/min. An optimum response of the
fluorescent derivate was observed when the excitation
and emission wavelengths in the detector were set at 350
and 420 nm, respectively.
2.9. Oxoguanine Glycosylase (OGG 1) mRNA
Levels in the Liver
Total liver RNA was extracted using the RNeasy Midi
kit with DNase step (Qiagen, Milan, Italy), according to
the manufacturer’s instructions. For first-strand cDNA
synthesis, 1 mg of total RNA from each sample was re-
verse-transcribed using 100 units of RT Super-Script II
(Life Technologies, San Giuliano, Milan, Italy) and 1X
random hexamers (Roche Diagnostics, Monza, Italy).
The primers used were: OGG1 (NM_030870) F 5’-CAC
CCA CTC GAA GC-3’ (346 bp); β-actin F 5’-ACC
CTT TAC GGA TGT CAA CG-3’ (281 bp).
The PCR reactions were carried out in a 25 µL volume
containing 1X PCR buffer, 1.2 mmol/L MgCl2, 0.5
mmol/L dNTPs, 1.6 µmol/L of OGG1 primers, 0.04
µmol/L of the b-actin primers and 1.25 U of Taq poly-
merase (Sigma-Aldrich, Italy). The PCR conditions were:
95˚C for 7 min and then 35 cycles at 95˚C for 30 sec,
60˚C for 30 sec and 72˚C for 55 sec. PCRs were per-
formed in a Perkin Elmer 9700 thermal cycler (Perkin
Elmer, Foster City, CA, USA). The PCR products were
separated on 1.6% agarose gel and visualized by ethi-
dium bromide staining. Gel images were captured by a
digital photocamera (UviDoc) and the intensity of the
bands was analysed with Quantity-One software (Bio-
Rad, Segrate, Milan, Italy). The relative amount of mRNA
in the samples was normalised using b-actin co-amplified
as internal standard [32].
2.10. Western-Blot
Liver samples were homogenized in lysis buffer of the
following composition (in mM): 50 Tris·HCl pH 7.5, 1
EDTA, 150 NaCl, 1 Na3VO4, 10 NaF and complete
protease inhibitor cocktail tablet (Sigma-Aldrich, St.
Louis, MO, USA). The homogenate was centrifuged
(1000 × g) for 10 min at 4˚C to remove cell debris.
Proteins were then separated on a 12% (w/vol) SDS-
PAGE, transferred to PVDF membranes, blocked with
Blocker Non-Fat Dry Milk (Bio-Rad, Richmond, CA)
and probed with mouse anti-rabbit caspase-3 polyclonal
antibodies (1:1000 dilution; St. Cruz Biotechnology Inc.,
CA, USA) or with polyclonal anti-rabbit glyceraldehyde-
3-phosphate dehydrogenase (GAPDH; 1:1000 dilution;
Sigma-Aldrich, St. Louis, MO, USA) overnight at 4˚C.
After extensive washings, a monoclonal goat anti-rabbit
peroxidase conjugated antibody was added (1:10000 di-
lution; Sigma-Aldrich, St. Louis, MO, USA) and immu-
Copyright © 2011 SciRes. PP
130 Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver
Figure 2. Levels of 8-oxodGuo in the liver DNA of control
rats (C), rats treated with 2-NP and those administered 2-
NP and resveratrol (2-NP + Res). Data are expressed as
means ± S.E.M; n = 5 in each group. *p < 0.05 vs. C.
nodetected bands were visualized by ECL. Densitometric
analysis of autoradiographic bands referred to GAPDH
expression, taking into account the size and area of the
band (Scion software Image Corp).
2.11. Statistical Analysis
Parametric variables were compared using the t-test.
Correlations were performed using linear regression
analysis, and the significance level was considered as p <
0.05. The statistical analysis was carried out using the
Stata statistical package (Stata, Collage Station, TX).
3. Results
To evaluate the effect of resveratrol on oxidation damage
in the rat liver, we used 2-NP to induce oxidative DNA
Administration by i.p. of 100 mg/kg 2-NP generated
ROS and considerably increased 8-oxodGuo in the rat
liver DNA, 15 h after injection (Figure 2). Pre-treatment
of rats with resveratrol (50 mg/kg/d) for 14 days before
2-NP administration restored 8-oxodGuo in DNA to lev-
els similar to those of controls (Figure 2). Although the
average protein oxidation measured as carbonyl residues
was not significantly modified by 2-NP injection (Table
1), a correlation between carbonyl residues and 8-oxod-
Guo levels in the liver of rats treated with 2-NP was
found (Figure 3).
2-NP induced an increase (about 2.5-fold) in SOD ac-
tivity and a slight, but significant increase in XO acti-
vity, a pro-oxidant enzyme; however, in the group treated
with resveratrol, SOD and XO levels were similar to
controls (Table 1). On the contrary, lipoperoxidation,
measured as MDA levels, was similar in the three groups
(Table 1). An inverse correlation existed between MDA
and SOD activity in rats from 2-NP group (Figure 4).
We examined whether 2-NP injection affected the ex-
Table 1. SOD and XO activities, carbonyl residues in pro-
teins, MDA and OGG1 expression levels in the liver of
control rats (C), rats treated with 2-nitropropane (2-NP)
and in those administered 2-nitropropane and resveratrol
(2-NP + Res).
C 2-NP 2-NP + Res
SOD (U/mg) 4.2 ± 1.3 10.3 ± 1.9* 5.3 ± 1.5
XO (U/mg) 2.3 ± 0.38 3.6 ± 0.41* 2.8 ± 0.75
Carbonyl residues
(nmol/mg) 0.34 ± 0.03 0.41 ± 0.12 0.35 ± 0.02
MDA (µM) 0.14 ± 0.01 0.10 ± 0.01 0.13 ± 0.01
OGG1/b-actin expres-
sion (Arbitrary Unit)0.72 ± 0.16 1.18 ± 0.07$ 0.99 ± 0.35
Data are ex pressed as means ± S.E.M. n = 5 in each group ; *p < 0.05 vs.
C; $p = 0.060 vs. C.
Figure 3. Correlation between carbonyl residues and 8-
oxodGuo levels in liver of 2-NP rat group.
Figure 4. Correlation between MDA levels and SOD activ-
ity in liver of rats treated with 2-NP.
pression of the 8-oxoguanine-DNA glycosylase (OGG-1)
which catalyses the removal of mutagenic 8-oxo-7,8-di-
hydroguanine from DNA. We observed a borderline sig-
nificant increased expression of the OGG1 gene in the rat
opyright © 2011 SciRes. PP
Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver 131
Figure 5. Example of the analysis of OGG1 gene expression
by semi-quantitative RT-PCR, using total RNA extracted
from livers of controls (C, n = 2), treated with 2-NP (n = 2)
and those administered 2-NP and resveratrol (2-NP + Res,
n = 2) and b-actin as an internal standard.
Figure 6. GSH/GSSG ratio in the liver DNA of control rats
(C), rats treated with 2-NP and those administered 2-NP
and resveratrol (2-NP + Res).
liver of 2-NP group (p = 0.06) whereas, rats pre-treated
with resveratrol showed a hepatic OGG1 expression si-
milar to that observed in controls (Table 1 and Figure
Treatment with 2-NP also significantly reduced GSH/
GSSG ratio (75%) in comparison to controls and this
change was partially reversed by resveratrol (Figure 6).
An inverse correlation existed between the GSH/GSSG
ratio and 8-oxodGuo in 2-NP group (Figure 7). Figure 8
shows Western blot analysis for the 2-NP induced altera-
tions and preventive response of resveratrol on the ex-
pression of specific marker protein (activated caspase-3)
of apoptosis in the rat liver. Animals responded to 2-NP
(100 mg/kg) injection by up-regulating the activation of
caspase-3 (about 40-fold) in comparison to controls.
Figure 7. Correlation between GSH/GSSG ratio and 8-
oxodGuo levels in the liver of rats treated with 2-NP.
Figure 8. Levels of caspase-3 in the liver of control rats (C),
rats treated with 2-NP and those administered 2-NP and
resveratrol (2-NP + Res).
Pre-treatment with resveratrol (50 mg/kg/d) for 14 days
before the 2-NP injection significantly reduced the acti-
vation of caspase-3 (Figure 8).
We saw no oxidation damage on the colon mucosa af-
ter 2-NP, in fact, all measured markers of oxidative stress
and antioxidant response were similar to those of con-
trols (Table 2).
4. Discussions
Resveratrol is a phenolic compound with free-radical
scavenging and antioxidant properties [8,9]. Our data
show that treatment with resveratrol before 2-NP injec-
tion significantly reduces oxidative stress and apoptosis
in the liver of rats.
These results are consistent with previous studies
demonstrating resveratrol does prevent the increased of
Copyright © 2011 SciRes. PP
132 Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver
Table 2. Levels of 8-oxodGuo, SOD and XO activities, car-
bonyl residues in proteins and MDA in the colon mucosa of
control rats (C), rats treated with 2-nitropropane (2-NP)
and in those administered 2-nitropropane and resveratrol
(2-NP + Res).
C 2-NP 2-NP + Res
8-oxodGuo/106 dG
SOD (U/mg)
9.2 ± 1.4
1.3 ± 0.3
10.8 ± 1.5
1.2 ± 0.2
11.93 ± 1.7
1.5 ± 0.1
XO (U/mg) 0.6 ± 0.09 0.4 ± 0.04 0.5 ± 0.04
Carbonyl residues
(nmol/mg) 0.15 ± 0.030.17 ± 0.02 0.15 ± 0.02
MDA (µM) 0.10 ± 0.010.09 ± 0.01 0.11 ± 0.02
Data are expressed as means ± S.E.M. n = 5 in each group.
8-oxodGuo in kidney DNA induced by toxic compounds
[33] and the increased excretion of 8-oxodGuo in urine
of genetically hypertensive rats [34]. Recently, resvera-
trol has also been shown to ameliorate hepatic injury in
rats with severe acute pancreatitis, suppress oxidative
stress and inflammatory response in diethylnitrosamine-
initiated rat hepatocarcinogenesis [9,16] and protect
PC12 cells against 4-hydroxynonenal induced oxidative
stress-mediated apoptotic neurodegeneration [17].
It has been suggested that 2-NP metabolism in the
liver does produce a variety of metabolites, including the
anionic tautomer propane 2-nitrate, nitric oxide, lipid
hydroperoxide radicals and nitrogen dioxide radicals,
capable of causing cellular damage [33-35]. In particu-
larly, as we and others reported, 2-NP at dose of 100
mg/kg is able to induce a powerful oxidative DNA dam-
age in the rat liver tissue [18,20,21,36]. Resveratrol does
seem to be able to protect cells against oxidative injury
through different mechanisms [37,38]. Leonard et al.,
[39] have showed that resveratrol did scavenge the O2
radical produced by cells after exposure to Cr(VI) and
observed an inhibition of DNA damage due to OH· radi-
cals produced by the Fenton reaction [39]. The protective
effects of resveratrol on oxidative DNA damage in vivo-
induced found in our experiments confirm the radical
scavenger ability of resveratrol earlier found in vitro and
in ex vivo assays [40,41]. In addition, the normalization
of XO activity induced by resveratrol in our experiments
does suggest that a reduced superoxide generation
through XO may explain another mechanism by which
resveratrol exercts a protective effect, in line with results
previously presented by Ates et al. [42] in traumatic
brain injury. We also found that resveratrol tended to
normalize the GSH/GSSG ratio which was inversely
correlated with the variations of 8-oxodGuo in DNA.
This last observation does suggest that the reduction of
oxidative DNA damage operated by resveratrol might be
possibly due, amongst other causes, to an attenuation of
2-NP-mediated GSH depletion, as reported by Kode et
al., [43], who demonstrated that resveratrol does protect
against cigarette smoke-mediated oxidative stress in hu-
man lung epithelial cells inducing GSH synthesis.
Lipoperoxidation measured as MDA levels, in the
liver of rats killed 15 h after 100 mg/kg 2-NP, did not
undergo any significant change in our experiments. On
the contrary, an increase of MDA was reported by
Wilhelm in rats who had been administered with 120
mg/kg 2-NP. Lipoperoxidation products (MDA +
4-hydroxyalkenals) was found by others in rat liver
treated with 2-NP at 165 mg/kg [44] and at our dose of
100 mg/kg but measured 48 h after 2-NP administration
[21]. Therefore, the lack of lipoperoxidation in our ex-
periments may have been cause by variations in dose and
sampling times. However, in our experiments 2-NP did
enhance SOD activity, probably as an antioxidant re-
sponse. Consequently, lipoperoxidation did not occur, as
suggested by the inverse correlation found between
MDA and SOD in 2-NP rat group. We suppose that
pre-treatment with resveratrol protecting liver by injury it
is capable of blocking anti-oxidant defensive response,
consequently, SOD activity in 2-NP+Res group is similar
to that of controls. Borges et al. [21] did not observe a
change in SOD activity in the liver of rats sacrificed 48h
after 2-NP injection. Once more the difference in lag
time might explain such discrepancy. Like change in
SOD activity, a tendency (p = 0.06) of increase in the
expression of the OGG1 gene, absent in the 2-NP rat
group treated with resveratrol, was detected in the liver
of 2-NP rats, indicating that 2-NP injection may induce a
protective response also through an increasing of DNA
repair pathway. Although higher OGG1 gene expression
and SOD activity were found in the liver of 2-NP rats
than controls, 8-oxodGuo levels were enhanced in their
liver DNA. On the contrary, 2-NP+Res rats had
8-oxodGuo levels similar to those found in controls sug-
gesting that resveratrol is capable to reduce oxidation
damage 2-NP-induced in the liver DNA. The induction
of oxidative DNA damage 15 h after 100 mg/kg 2-NP
injection was accompanied with a relevant activation of
caspase-3, so informing of an induced apoptosis. Res-
veratrol at the dose of 50 mg/kg/day for 14 days before
2-NP injection was able to almost prevent completely
(98%) the induction of apoptosis and to reduce oxidative
DNA damage at levels similar to those detected in the
liver of control rats. This observation was consistent with
the results obtained by Sha et al., [9] in rats with acute
hepatic injury who found that resveratrol (10 mg/kg),
injected through the Vena Dorsalis of the penis amelio-
rated hepatic injury, via the mitochondrial pathway, in
rats with chemically induced acute pancreatitis [9]. Re-
cently, similar observations were reported by Siddiqui et
al., [16] in PC12 cells exposed for 2 h to 4-hydroxynone-
opyright © 2011 SciRes. PP
Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver 133
nal using resveratrol at 25 µM concentration.
Coming to a conclusion, our results prove oxidative
DNA damage and apoptosis to be the main mechanisms
of death cell in a model of chemically induced acute he-
patic injury. At this early stage of damaging processes,
resveratrol at dose of 50 mg/kg/day administered 14 days
long can ameliorate hepatic injury by its antioxidant and
scavenging properties, through a reduction of XO activ-
ity, a partial restoration of GSH/GSSG ratio in addition
to its capacity to inhibit apoptosis.
5. Acknowledgements
This work has been receiving financial support from the
University of Florence. The authors express their grati-
tude to Mary Forrest for her linguistic revision.
6. References
[1] H. Wei, “Activation of Oncogenes and/or Inactivation of
Anti-Oncogenes by Reactive Oxygen Species,” Medical
Hypotheses, Vol. 39, No. 3, 1992, pp. 267-267.
[2] T. Takeuchi and K. Morimoto, “Increased Formation of
8-Hydroxydeoxyguanosine, an Oxidative DNA Damage,
in Lymphoblasts from Fanconi’s Anemia Patients Due to
Possible Catalase Deficiency,” Carcinogenesis, Vol. 14,
No. 6, 1993, pp. 1115-1120.
[3] R. Shimoda, M. Nagashima, M. Sakamoto, N. Yamagu-
chi, S. Hirohashi, J. Yokota and H. Kasai, “Increased
Formation of Oxidative DNA Damage, 8-Hydroxydeoxy-
guanosine, in Human Livers with Chronic Hepatitis,”
Cancer Research, Vol. 54, No. 12, 1994, pp. 3171-3172.
[4] M. B. Reddy and L. Clark, “Iron, Oxidative Stress, and
Disease Risk,” Nutrition Reviews, Vol. 62, No. 3, 2004,
pp. 120-124. doi:10.1301/nr.2004.mar.120-124
[5] A. M. Shah and K. M. Channon, “Free Radicals and Re-
dox Signalling in Cardiovascular Disease,” Heart , Vol.
90, No. 5, 2004, pp. 486-487.
[6] Y. J. Cai, J. G. Fang, L. P. Ma, L. Yang and Z. L. Liu,
“Inhibition of Free Radical-Induced Peroxidation of Rat
Liver Microsomes by Resveratrol and Its Analogues,”
Biochimica et Biophisica Acta, Vol. 1637, No. 1, 2003,
pp. 31-38.
[7] A. Cavallaro, T. Ainis, C. Bottari and V. Fimiani, “Effect
of Resveratrol on Some Activities of Isolated and in
Whole Blood Human Neutrophils,” Physiological Re-
search, Vol. 52, No. 5, 2003, pp. 555-562.
[8] G. C. Yen, P. D. Duh and C. W. Lin, “Effects of Res-
veratrol and 4-Hexylresorcinol on Hydrogen Peroxide-
Induced Oxidative DNA Damage in Human Lympho-
cytes,” Free Radical Research, Vol. 37, No. 5, 2003, pp.
509-514. doi:10.1080/1071576031000083099
[9] H. Sha, Q. Ma, R. K. Jha, F. Xu, L. Wang, Z. Wang, Y.
Zhao and F. Fan, “Resveratrol Ameliorates Hepatic In-
jury via the Mitochondrial Pathway in Rats with Severe
Acute Pancreatitis,” European Journal of Pharmacology,
Vol. 601, No. 1-3, 2008, pp. 136-142.
[10] D. Delmas, A. Lançon, D. Colin, B. Jannin and N. La-
truffe, “Resveratrol as a Chemopreventive Agent: A
Promising Molecule for Fighting Cancer,” Current Drug
Targets, Vol. 7, No. 4, 2006, pp. 423-442.
[11] E. Tili, J. J. Michaille, H. Alder, S. Volinia, D. Delmas, N.
Latruffe and C. M. Croce, “Resveratrol Modulates the
Levels of microRNAs Targeting Genes Encoding Tumor-
Suppressors and Effectors of TGFβ Signaling Pathway in
SW480 Cells,” Biochemical Pharmacology, Vol. 80, No.
12, 2010, pp. 2057-2065.
[12] S. Pervaiz, “Chemotherapeutic Potential of the Chemo-
preventive Phytoalexin Resveratrol,” Drug Resistance
Updates, Vol. 7, No. 6, 2004, pp. 333-344.
[13] K. B. Harikumar and B. B. Aggarwal, “Resveratrol: A
Multi Targeted Agent or Age-Associated Chronic Di-
seases,” Cell Cycle, Vol. 7, No. 8, 2008, pp. 1020-1035.
[14] M. A. Siddiqui, M. P. Kashyap, V. Kumar, A. A.
Al-Khedhairy, J. Musarrat and A. B. Pant, “Protective
Potential of Trans-Resveratrol against 4-Hydroxynonenal
Induced Damage in PC12 Cells,” Toxicology in Vitro,
Vol. 24, N. 6, 2010, pp. 1592-1598.
[15] T. Tunali-Akbay, O. Sehirli, F. Ercan and G. Sener,
“Resveratrol Protects against Methotrexate-Induced He-
patic Injury in Rats,” Journal of Pharmacology and
Pharmacy Science, Vol. 13, No. 2, 2010, pp. 303-310.
[16] A. Bishayee, K. F. Barnes, D. Bhatia, A. S. Darvesh and
R. T. Carroll, “Resveratrol Suppresses Oxidative Stress
and Inflammatory Response in Diethylnitrosamine-Initi-
ated Rat Hepatocarcinogenesis,” Cancer Prevention and
Research, Vol. 3, No. 6, 2010, pp. 753-763.
[17] R. Harrison, G. Letz, G. Pasternak and P. Blanc, “Fulmi-
nant Hepatic Failure after Occupational Exposure to
2-Nitropropane,” Annals of Internal Medicine, Vol. 107,
No. 4, 1987, pp. 466-468.
[18] E. S. Fiala, C. C. Conaway and G. E. Mathis, “Oxidative
DNA and RNA Damage in the Livers of Sprague-Daw-
ley Rats Treated with the Hepatocarcinogen 2-Nitropro-
pane,” Cancer Research, Vol. 49, No. 20, 1989, pp.
[19] R. S. Sodum, G. Nie and E. S. Fiala, “8-Aminoguanine:
A Base Modification Produced in Rat Liver Nucleic Ac-
ids by the Hepatocarcinogen 2-Nitropropane,” Chemical
Research of Toxicology, Vol. 6, No. 3, 1993, pp. 269-276.
[20] C. Casalini, M. Lodovici, C. Briani, G. Paganelli, S.
Remy, V. Cheynier and P. Dolara, “Effect of COMPLEX
polyphenols and Tannins from Red Wine (WCPT) on
Copyright © 2011 SciRes. PP
134 Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver
Chemically Induced Oxidative DNA Damage in the Rat,”
European Journal of Nutrition, Vol. 38, No. 4, 1999, pp.
190-195. doi:10.1007/s003940050061
[21] L. P. Borges, C. W. Nogueira, R. P. Panatieri, J. B. T.
Rocha and G. Zeni, “Acute Liver Damage Induced by
2-Nitropropane in Rats: Effect of Diphenyl Diselenide on
Antioxidant Defences,” Chemico-Biological Interactions,
Vol. 160, No. 2, 2006, pp. 99-107.
[22] A. Unnikrishnan, J. J. Raffoul, H. V. Pate, T. M.
Prychitko, N. Anyangwe, L. B. Meira, E. C. Friedberg, D.
C. Cabelof and A. R. Heydari, “Oxidative Stress Alters
Base Excision Repair Pathway and Increases Apoptotic
Response in Apurinic/Apyrimidinic Endonuclease 1/Re-
dox Factor-1 Haploinsufficient Mice,” Free Radical Bi-
ology and Medicine, Vol. 46, No. 11, 2009, pp. 1488-
1499. doi:10.1016/j.freeradbiomed.2009.02.021
[23] L. D. Williams, G. A. Burdock, J. A. Edwards, M. Beck,
J. Bausch, L. D. Williams, G. A. Burdock, J. A. Edwards,
M. Beck and J. Bausch, “Safety Studies Conducted on
High-Purity Trans-Resveratrol in Experimental Animals,”
Food and Chemical Toxicology, Vol. 47, No. 9, 2009, pp.
2170-2182. doi:10.1016/j.fct.2009.06.002
[24] A. Bishayee, “Cancer Prevention and Treatment with
Resveratrol: From Rodent Studies to Clinical Trials,”
Cancer Prevention Research, Vol. 2, No. 5, 2009, pp.
409-418. doi:10.1158/1940-6207.CAPR-08-0160
[25] C. M. Gedik, A. Collins, ESCODD (European Standards
Committee on Oxidative DNA Damage), “Establishing
the Background Level of Base Oxidation in Human Lym-
phocyte DNA: Results of an Interlaboratory Validation
Study,” FASEB Journal, Vol. 19, No. 1, 2005, pp. 82-84.
[26] M. Lodovici, C. Casalini, R. Cariaggi, L. Michelucci and
P. Dolara, “Levels of 8-Hydroxydeoxyguanosine as a
Marker of DNA Damage in Human Leukocytes,” Free
Radical Biology and Medicine, Vol. 28, No. 1, 2000, pp.
13-17. doi:10.1016/S0891-5849(99)00194-X
[27] V. Correa-Salde and I. Albesa, “Reactive Oxidant Spe-
cies and Oxidation of Protein and Haemoglobin as Bio-
markers of Susceptibility to Stress Caused by Chloram-
phenicol,” Biomedical Pharmacotherapy, Vol. 63, No. 2,
2009, pp. 100-104. doi:10.1016/j.biopha.2008.05.001
[28] R. Mateos, E. Lecumberri, S. Ramos, L. Goya and L.
Bravo, “Determination of Malondialdehyde (MDA) by
High-Performance Liquid Chromatography in Serum and
Liver as a Biomarker for Oxidative Stress. Application to
a Rat Model for Hypercholesterolemia and Evaluation of
the Effect of Diets Rich in Phenolic Antioxidants from
Fruits,” Journal of Chromatography B-Analytical Tech-
nologies in the Biomedical and Life Science, Vol. 827,
No. 1, 2005, pp. 76-82.
[29] C. Beauchamp and I. Fridovich, “Superoxide Dismutase:
Improved Assays and an Assay Applicable to Acrylamide
Gels,” Analytical Biochemistry, Vol. 44, No. 1, 1971, pp.
276-287. doi:10.1016/0003-2697(71)90370-8
[30] E. D. Corte and F. Stirpe, “Regulation of Xanthine Oxi-
dase in Rat Liver: Modifications of the Enzyme Activity
of Rat Liver Supernatant on Storage at 20 Degrees,” Bio-
chemical Journal, Vol. 108, No. 2, 1968, pp. 349-351.
[31] C. Cereser, J. Guichard, J. Drai, E. Bannier, I. Garcia, S.
Boget, P. Parvaz and A. Revol, “Quantitation of Reduced
and Total Glutathione at the Femtomole Level by
High-Performance Liquid Chromatography with Fluo-
rescence Detection: Application to Red Blood Cells and
Cultured Fibroblasts,” Journal of Chromatography B
Biomedical Sciences and Applications, Vol. 752, No. 1,
2001, pp. 123-132.
[32] C. Luceri, G. Caderni, A. Sanna and P. Dolara, “Red
Wine and Black Tea Polyphenols Modulate the Expres-
sion of Cycloxygenase-2, Inducible Nitric Oxide Syn-
thase and Glutathione-Related Enzymes in Azoxyme-
thane-Induced f344 Rat Colon Tumors,” Journal of Nu-
trition, Vol. 132, No. 6, 2002, pp. 1376-1379.
[33] X. S. Deng, J. Tuo, H. E. Poulsen and S. Loft, “Preven-
tion of Oxidative DNA Damage in Rats by Brussels
Sprouts,” Free Radical Research, Vol. 28, No. 3, 1998,
pp. 323- 333. doi:10.3109/10715769809069284
[34] G. Barja, “Resveratrol, Melatonin, Vitamin E, and PBN
Protect against Renal Oxidative DNA Damage Induced
by the Kidney Carcinogen KBrO3,” Free Radical Biology
and Medicine, Vol. 26, No. 11-12, 1999, pp. 1531-1534.
[35] K. Mizutani, K. Ikeda, Y. Kawai and Y. Yamori,” Protec-
tive Effect of Resveratrol on Oxidative Damage in Male
and Female Stroke-Prone Spontaneously Hypertensive
Rats,” Clinical and Experimental Pharmacology and
Physiology, Vol. 28, No. 1-2, 2001, pp. 55-59.
[36] B. Halliwell and J. M. Gutteridge, “Role of Free Radicals
and Catalytic Metal Ions in Human Disease: An Over-
view,” Methods in Enzymology, Vol. 186, 1990, pp. 1-85.
[37] W. Bors, C. Michel, C. Dalke, K. Stettmaier, M. Saran
and U. Andrae, “Radical Intermediates during the Oxida-
tion of Nitropropanes. The Formation of NO2 from
2-Nitropropane, Its Reactivity with Nucleosides, and Im-
plications for the Genotoxicity of 2-Nitropropane,”
Chemical Research in Toxicology, Vol. 6, No. 3, 1993, pp.
302-309. doi:10.1021/tx00033a008
[38] C. Kohl, P. Morgan and A. Gescher, “Metabolism of the
Genotoxicant 2-Nitropropane to a Nitric Oxide Species,”
Chemico-Biological Interactions, Vol. 97, No. 2, 1995,
pp. 185-194.
[39] S. S. Leonard, C. Xia, B. H. Jiang, B. Stinefelt, H. Klan-
dorf, G. K. Harris and X. Shi, “Resveratrol Scavenges
Reactive Oxygen Species and Effects Radical-Induced
Cellular Responses,” Biochemical and Biophysical Re-
search Communications, Vol. 309, No. 4, 2003, pp. 1017-
1026. doi:10.1016/j.bbrc.2003.08.105
[40] S. Bradamante, L. Barenghi and A. Villa, “Cardiovascu-
lar Protective Effects of Resveratrol,” Cardiovascular
and Drug Reviews, Vol. 22, No. 3, 2004, pp. 169-188.
[41] P. S. Ray, G. Maulik, G. A. Cordis, A. A. Bertelli, A.
Bertelli and D. K. Das, “The Red Wine Antioxidant Res-
opyright © 2011 SciRes. PP
Protective Effect of Resveratrol against Oxidation Stress Induced by 2-Nitropropane in Rat Liver
Copyright © 2011 SciRes. PP
veratrol Protects Isolated Rat Hearts from Ischemia
Reperfusion Injury,” Free Radical Biology and Medi-
cine, Vol. 27, No. 1-2, 1999, pp. 160-169.
[42] O. Ates, S. Cayli, E. Altinoz, I. Gurses, N. Yucel, M.
Sener, A. Kocak and S. Yologlu, “Neuroprotection by
Resveratrol against Traumatic Brain Injury in Rats,” Mo-
lecular and Cellular Biochemistry, Vol. 294, No. 1-2,
2007, pp. 137-144. doi:10.1007/s11010-006-9253-0
[43] A. Kode, S. Rajendrasozhan, S. Caito, S. R. Yang, I. L.
Megson and I. Rahman, “Resveratrol Induces Glutathione
Synthesis by Activation of Nrf2 and Protects against
Cigarette Smoke-Mediated Oxidative Stress in Human
Lung Epithelial Cells,” American Journal of Physiologi-
cal Lung Cellular and Molecular Physiology, Vol. 294,
No. 3, 2008, pp. L478-L488.
[44] S. J. Kim, R. J. Reiter, M. V. Rouvier Garay, W. Qi, G. H.
El-Sokkary and D. X. Tan, “2-Nitropropane-Induced
Lipid Peroxidation: Antitoxic Effects of Melatonin,”
Toxicology, Vol. 130, No. 2-3, 1998, pp. 183-190.