INTRODUCTION

Chemoprevention (or chemoprotection) refers to the

use of chemicals including phytochemicals, pharmaceuti-
cals and minerals to retard, reduce or reverse the onset of
cancer caused by exposure to xenobiotics such as drugs,
heavy metals and electrophiles (Wattenberg, 1985).
Among the phytochemicals examined so far, organosulfur
compounds (OSCs) found in garlic have been extensively
studied and are found to possess antioxidant (Imai et al.,
1994), anticarcinogenic (Hatono et al., 1996), antiathero-
genic (Steiner and Lin, 1998; Efendy et al., 1997), immu-
nostimulatory (Abdullah et al., 1989; Lau et al., 1991)
and antiaging properties (Moriguchi et al., 1996).
Garlic (Allium sativum), a member of the lily family,
is a perennial plant that is cultivated worldwide. The gar-
lic bulb is composed of individual cloves enclosed in a
white skin. It is the bulb, either fresh or dehydrated that
is used as a spice of medicinal herb (Block, 1985). Gar-
lic has been used throughout history for the treatment of
a wide variety of conditions and these have been exten-
sively investigated (Umar et al., 1996). Its usage predates
Antioxidant effects of heated garlic juice on
cadmium-induced liver damage in rats as compared to
ascorbic acid
Akeem Olalekan Lawal
1
, Adetola Folusho Lawal
2
, Augustine Ologundudu
1
,
Olawale Yakubu Adeniran
1
, Akhere Omonkhua
1
and Federick Obi
3
1
Department of Biochemistry, Adekunle Ajasin University, P.M.B 001, Akungba-Akoko, Ondo State, Nigeria
2
Department of Chemistry and Industrial Chemistry, Adekunle Ajasin University, P.M.B 001, Akungba-Akoko,
Ondo State, Nigeria
3
Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Edo State, Nigeria

(Received May 15, 2011; Accepted July 18, 2011)

ABSTRACT The antioxidant efcacy of heated garlic juice (HGJ) in liver was evaluated with that
of ascorbic acid (AA) in rats exposed to acute dose of cadmium (4 mg kg
-1
bd. wt). The rats were either
given HGJ (100 mg kg
-1
bd. wt) orally, daily for 4 weeks or AA (100 mg kg
-1
bd. wt) orally, daily for
4 weeks or both or cadmium (4 mg kg
-1
bd. wt) intraperitoneally for 3 days. Another group of rats
was given cadmium (4 mg kg
-1
bd. wt) intraperitoneally for 3 days after pretreatment with either HGJ
(100 mg kg
-1
bd. wt) or ascorbic acid (100 mg kg
-1
bd. wt) for 4 weeks and the liver excised. The results
obtained show that AA and HGJ signicantly reduced the level of liver malondialdehyde (MDA) induced
by cadmium compared to control (p < 0.05) but AA tends to be more potent when compared with HGJ.
The presence of either HGJ or AA also signicantly reduced the levels of ROS in the presence of cadmium
(Cd). The presence of either AA or HGJ pre-treatment produced signicant increase in liver SOD and Cat-
alase activities when compared with rats treated with Cd alone. There was no signicant reduction in the
activities of these enzymes in the presence of cadmium compared to control. Western blot shows that the
expressions of Nrf2 and NQO1 in the liver were signicantly increase by 3 and 1.7-fold respectively in the
AA pretreated mice when compared with Cd. However no signicant changes were seen in HGJ pretreated
rats. The expression of HO-1 was not signicantly increase in the AA pretreated rats. The results show that
though both ascorbic acid and HGJ are efcient in preventing Cd-induced damage in the rat liver, ascorbic
acid appeared to be a more powerful antioxidant than heated garlic juice in preventing cadmium-induced
oxidative damage in liver and its action may be mediated in parts via Nrf2-keap1 pathway.
Key words: Antioxidant, Malondialdehyde, Superoxide dismutase, Catalase, Garlic, Ascorbic acid,
Cadmium

Correspondence: Akeem Olalekan Lawal (E-mail: tolekan@hotmail.com)
Original Article
The Journal of Toxicological Sciences (J. Toxicol. Sci.)
Vol.36, No.5, 549-557, 2011
Vol. 36 No. 5
549
written history. Its use as a remedy for heart disease,
tumors and headaches are documented in the Egyp-
tian codex Ebers, dating from 1550 BC. Epidemiologi-
cal studies have suggested that garlic plays a signicant
role in the reduction of deaths caused by malignant dis-
eases (Agarwal, 1996). This has led many investigators
to examine garlic and garlic constituents for their anti-
tumor and cytotoxic actions both in vitro (Prasad et al.,
1996; Rabinkov et al., 1998)) and in Laboratory animals
(Augusti and Sheela, 1996; Iqbal and Athar, 1998). Data
suggested that garlic contains several potentially impor-
tant agents that possess antitumor and anticarcinogenic
properties (Agarwal, 1996). It contains both water soluble
compounds such as L- seleocystein (SC), S- allycysteine
(SAC) as well as lipid soluble compounds such as dial-
lytrisulde (DATS), diallydisulde (DADS) (Nisha and
Tood, 2001). Garlic also contains high level of tellurium
and selenium compounds (Ip et al., 2000; Mesheeshy et
al., 1998).
Ascorbic acid (vitamin C) is essential micronutrients in
primates. It is a water-soluble chain breaking antioxidant
that exhibits its antioxidant property by scavenging free
radicals. Ascorbic acid also recycles plasma membrane
-tocopherol via the reduction of the -tocopheroxyl
radical. Ascorbic acid has also been reported to exhib-
its prooxidant activities (Chen et al., 2005, 2007) . It was
earlier reported by Halliwell (1996) that ascorbate pro-
motes fenton reaction in vitro and may induce non enzy-
matic lipid peroxidation in various tissues. Also, Chen
et al. (2008) reported that ascorbate at pharmacological
doses produced sustained ascorbate radical and hydrogen
peroxide in mice. These and other reports have implicated
ascobate in enhancing reactive oxygen production which
may trigger-off Nrf2 protective response and this could be
an important mechanism in the protective effect of ascor-
bic acid via the Nrf2-mediated response in exposed ani-
mals.
Cadmium is widely used in industrial chemicals
(Waisberg et al., 2003). The toxicities associated with this
metal ion are well known (ATSDR, 1999). It has been
reported that cadmium depletes glutathione and protein
bound sulfhydryl groups (Waisberg et al., 2003), resulting
in enhanced production of reactive oxygen species such
as superoxide ion, hydroxyl radicals and hydrogen perox-
ide (Galan et al., 2001; Stohs et al., 1995). These reactive
oxygen species play an important role related to degen-
erative or pathological processes such as aging (Burns
et al., 2001), cancer, coronary heart disease, Alzheimer
disease (Diaz et al., 1997), neurodegenerative disorders,
atherosclerosis, cataracts and inflammation (Aruoma,
1998). Studies have also shown that cadmium elevates
the levels of lipid peroxidation in the liver (El-Maraghy
et al., 2001) and liver of mitochondrial of exposed rats
(Casalino et al., 1997).
Therefore, the present study was carried out to evalu-
ate the antioxidant effects of heated garlic extract, a nat-
ural source of antioxidant, when compared with ascorbic
acid, a convectional antioxidant, in cadmium-induced rat
liver damage and to dene a role for the involvement of
Nrf2-dependent antioxidants in this protection.
MATERIALS AND METHODS
Plant materials
Garlic (Allium sativum) bulbs were purchased from a
local market in Akure, Ondo State, Nigeria. The garlic juice
was prepared by the method of Kasuga et al. (2001).
Animals
Adult male albino rats of wister strain, weighing 160-
220 g were purchased from Department of Animal Science,
University of Ilorin, Nigeria. They were kept under stand-
ard laboratory conditions with standard pelleted diet and
water ad libitum. The animals were allowed to acclima-
tize for four weeks before the experiment.
Reagents
All chemicals used were of analytical grade and are
products of Sigma Chemical Co. (St. Louis, MO, USA).
SOD assay kit (Product code 19160) was obtained from
Fluka (Gillingham, UK). Catalase activity assay kit
(Product number CAT 100) was obtained from Sigma,
Poole, Dorset, UK. Thiobarbituric acid was obtained
from Sigma (UK). Antibodies were purchased from Santa
Cruz Biotech. Inc. (Santa Cruz, CA, USA). Goat anti-
rabbit antibody (horseradish peroxidase conjugated)
was obtained from Bio-Rad (Herts, UK). Nitrocellulose
membranes were obtained from Amersham Biosciences.
Dichlorouorescein diacetate (DCF-DA) was purchased
from Sigma-Aldrich (UK).
Dosage and treatment
Heated Garlic Juice (HGJ) and ascorbic acid were giv-
en as oral dose of 100 mg kg
-1
day body weight each for
4 weeks. Acute intraperitoneal dose of 4 mg kg
-1
of cad-
mium was given for 3 days at the last week of treatment.
The rats were sacriced 24 hr after the last dose of cadmi-
um had been administered.
Experimental procedures
The rats were divided into six groups of 5 animals
each. Treatments were done as below:
Vol. 36 No. 5
550
A.O. Lawal et al.
Group 1: Normal control
Group 2: Animals received heated garlic juice 100 mg
kg
-1
body wt orally, daily for 4 weeks.
Group 3: Animals received ascorbic acid 100 mg kg
-1

body wt orally, daily for 4 weeks.
Group 4: Animals received acute dose of cadmium 4 mg
kg
-1
body weight intraperitoneally.
Group 5: Animals received cadmium 4 mg kg
-1
body
weight intraperitoneally for 3 days + pretreat-
ment with heated garlic juice 100 mg kg
-1
body
wt orally, daily for 4 weeks.
Group 6: Animals received cadmium 4 mg kg
-1
body
weight intraperitoneally for 3 days + pretreat-
ment with ascorbic acid, 100 mg kg
-1
body
weight orally, daily, for 4 weeks.
Preparation of samples
For biochemical estimation, liver tissue was taken after
sacricing the animals by cervical dislocation. The liver
was then washed, weighed and homogenized in ice cold
0.9% normal saline solution. The homogenate was centri-
fuges at 4,000 x g for 15 min at 25C and the supernatant
was used for the studies.
Lipid peroxidation assay
The level of malondialdehyde (MDA) in the homoge-
nate was assayed by the thiobarbituric acid reactive spe-
cies (TBARS) method (Varshney and Kale, 1990). The
assay involve the monitoring of the pink coloured com-
plex formed in a reaction between MDA and thiobarbi-
turic acid (TBA). The reaction mixture consists of 500 l
homogenate, 1,000 l TBA reagent (0.375% TBA, 0.3
M Sodium acetate and 0.15% SDS, pH 3.8), 50 l 2 mM
butylated hydroxytoluene (BHT). After incubating for
60 min at 90C, the mixture was cooled and centrifuged
at 5,000 x g for 10 min. The absorbance of 1 ml of the
supernatant was read at 532 nm and the amount of MDA
formed was extrapolated from the standard curve and
expressed as mol MDA/mg protein. Protein was deter-
mined by the Biuret method of Gornall et al. (1949) using
bovine serum albumin (BSA) as standard.
ROS measurement
The level of ROS production was assessed using
dichlorofluorescein diacetate (DCF-DA) according to
the method described by Hempel et al. (1999). The
500 l of homogenates were incubated with 500 l of
2 mg/ml DCF-DA for 30 min at 37C. 100 l of 1 M
NaOH was added and the fluorescence intensity of 1
ml of the mixture was read with a fluorescence read-
er (FL6000) at excitation of 488 nm and emission of 512
nm. The results were expressed as relative uorescence
unit (RFU)/mg protein.
Enzyme assays
Superoxide dismutase (SOD) activity
Superoxide dismutase (SOD) activity was determined
by the method of Misra and Fridovich (1972) using the
SOD assay kit (Sigma). The reaction consists of 200 l
of homogenate, 400 l of water soluble tetrazolium salt
(WST) and 200 l of enzyme working solution (con-
taining superoxide anion). The mixture was incubated at
37C for 20 min and absorbance read in Labsystem iEMS
spectrophotometer. SOD activity was expressed as units/
mg protein.
Catalase activity
Catalase activity was measured by following decom-
position of H
2
O
2
according to the method of Sinha (1972)
using the catalase assay kit (Sigma). The reaction mixture
consists of 50 l of homogenate, 450 l of assay buff-
er (50 mM potassium phosphate buffer, pH 7.0) and 500
l of 20 mM H
2
O
2
. The decrease in absorbance was mon-
itored at 30 sec interval at 25C at wavelength of 240 nm.
Results were expressed as units/mg protein.
Gel preparation and Western blot analysis
Western blot analysis was carried out on the liver
homogenate. The samples were prepared in 2x lysis buff-
er [Tris-Cl (pH 6.8), sodium dodecyl sulfate (SDS) (2 g),
glycerol (9 ml), mercaptoethanol (5 ml), bromophenol
blue (0.1%) and distilled water] and heated at 100C for 5
min. The samples were centrifuged at 1,000 x g for 1 min
and equal amount of proteins were loaded on 10% poly-
acrylamide gel electrophoresis (SDS-PAGE). The pro-
teins were then transferred to nitrocellulose membrane
(Hybond ECL) at 200 mA for 2 hr and incubated in spe-
cic antibodies for 1 hr. The membranes were washed 4
times in 1X TBSTween [10X TBS (100 ml), Tween20
(2 ml) and distilled water] and then incubated with sec-
ondary antibody [Goat anti-Rabbit IG-horseradish per-
oxidase conjugate antibody (Bio-Rad)] for 1 hr at room
temperature. GAPDH was used for the normalization of
protein loading. The membranes were washed 3 times
with 1X TBSTween and once with 10X TBS and the blot
were developed using ECL luminol chemiluminescence
solutions. The proteins were quantified with an image
reader LAS 3000 and the band intensities were deter-
mined by image J software.
Statistical Analysis
Data were analyzed using one way analysis of variance
Vol. 36 No. 5
551
Hepatotoxicity of Cadmium and protective effect of heated garlic juice
(ANOVA) and unpaired students t-test by prism soft-
ware. Comparison between groups was done using Dun-
netts post test. Signicant difference were set at p* < 0.5;
p** < 0.01; p*** < 0.001 compared to control.
RESULTS
Effects of Heated garlic juice and ascorbic acid
on membrane lipid peroxidation after cadmium
exposure
In order to assess the protective effects of heated garlic
juice (HGJ) and ascorbic acid (AA) on Cadmium-induced
liver damage in exposed rats, rats were pre-treated oral-
ly with 100 mg/kg body weight of either HGJ or AA
for 4 weeks before 3 days intraperitoneal treatment with
4 mg/kg body weight of CdCl
2
. Levels of MDA were then
determined by the thiobarbituric acid reactive substance
(TBARS) as an indices of membrane lipid peroxidation.
The results show signicant 3 and 1.54-fold decrease in
MDA level in the presence of HGJ and AA respective-
ly when compared with control (Fig.1). The presence
of Cd caused a 1.42-fold increase in MDA levels when
compared with control (Fig. 1). The results also show
that HGJ significantly (1.44-fold) decrease MDA level
in the presence of Cd when compared to rats exposed to
Cd alone (Fig. 1). Also MDA level was signicant (1.66-
fold) decrease in the liver of AA pre-treated rats when
compared to rats exposed to Cd alone (Fig. 1). These set
of data seem to suggest that AA seem more potent in pre-
venting lipid peroxidation than HGJ.
Effects of Heated garlic juice and ascorbic acid
on ROS production after cadmium exposure
In order to assess the modulating effect of HGJ and AA
on the oxidative stress induced by Cadmium in the liv-
er of the exposed rats, the level of ROS was determined
in AA and HGJ pre-treated rats before cadmium expo-
sure. The results show that there was signicant decrease
in ROS production in the presence of either HGJ or AA
when compared with control (Fig. 2). The presence of
Cd however induced a signicant increase in ROS level
when compared with control (Fig. 2). There was a signif-
icant decrease in ROS level in the liver of rats pre-treated
with either HGJ (1.8-fold) or AA (2-fold) when compared
with Cd. This indicates that HGJ and AA have the poten-
tial of attenuating ROS production by Cd.
Effects of Heated garlic juice and ascorbic acid
on Superoxide dismutase activities after
cadmium exposure
In order to evaluates the effects of HGJ and AA on
antioxidant enzymes activities after Cd exposure, rats
were pre-treated with oral dose of 100 mg/kg body weight
of either HGJ or AA for 4 weeks before 3 days exposure
to 4 mg/kg body weight of CdCl
2
given intraperitoneally.
Superoxide dismutase (SOD) activities were then deter-
mined in the liver of the treated rats after Cd exposure.
The results show signicant increase of 2.07 and 3.5-fold
Fig. 1. Levels of MDA in rats liver pre-treated with either
HGJ or AA before Cd exposure. Rats were pre-treat-
ed with 100 mg/kg body weight of either HGJ or AA
for 4 weeks before 3 days intraperitoneal exposure to
4 mg/kg body weight CdCl
2
. Lipid peroxidation was
determined by TBARS method. Data represents the
mean activity (n = 5) relative to total cell protein
S.D. ***p < 0.001, **p < 0.01, *p < 0.05 (one way
ANOVA, Dunnetts post test). # represents signicant
different between pre-treated and unpre-treated rats
(unpaired student t-test).
Fig. 2. Reactive oxygen species production in the liver of rats
pre-treated with either HGJ or AA before Cd exposure.
Rats were pre-treated with 100 mg/kg body weight of
either HGJ or AA for 4 weeks before 3 days intraperi-
toneal exposure to 4 mg/kg body weight CdCl
2
. ROS
level was measured by DCF-DA. Data represents the
mean activity (n = 5) relative to total cell protein
S.D. ***p < 0.005, **p < 0.01, *p < 0.05 (one way
ANOVA, Dunnetts post test). # represents signicant
different between pre-treated and unpre-treated rats
(unpaired student t-test).
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552
A.O. Lawal et al.
in the liver of HGJ and AA treated rats respectively when
compared with control (Fig. 3). However, the presence
of Cd causes a significant decrease (1.77-fold) in SOD
activities when compared with control (Fig. 3). Also the
results show a 2.3-fold increase in SOD activity in HGJ
pre-treated rats before Cd exposure when compared with
rats exposed to Cd alone (Fig. 3). Similarly, there was
4.23-fold increase in SOD activity in AA pre-treated rats
when compared with rats exposed to Cd alone (Fig. 3).
These set of data seems to suggest that AA is more effec-
tive in enhancing SOD activities than HGJ in the pres-
ence of Cd.
Effects of Heated garlic juice and ascorbic acid
on Catalase activities after cadmium exposure
SOD is an incomplete antioxidant enzyme that
requires the presence of catalase for the complete remov-
al of superoxide anions. Therefore, in order to evaluate
whether HGJ and AA have any effects on the activities
of catalase after Cd exposure, rats were pre-treated orally
with either 100 mg/kg body weight of either HGJ or AA
for 4 weeks before 3 days treatment with 4 mg/kg body
weight of CdCl
2
. The results show signicant increase of
1.82 and 1.68-fold in catalase activities in the liver of rats
treated with HJG and AA respectively when compared
with control (Fig. 4). The results also show a 1.37-fold
decrease in catalase activity in the presence of Cd (Fig.
4). The results also show that HGJ pre-treatment caused
3.6-fold significant increase in catalase activities when
compared with rats exposed to Cd alone (Fig. 4). Similar-
ly, a 5.2-fold signicant increase in catalase activity was
observed in the liver of AA pre-treated rats when com-
pared with rats exposed to Cd alone (Fig. 4). These set of
data suggest that both HGJ and AA protects liver against
Cd toxicity by enhancing catalase activities.
HGJ and AA alter the expression of antioxidant
enzymes in the presence or absence of Cd
Nrf2 is a transcription factor that regulates the expres-
sion of antioxidant enzymes thereby attenuating ROS
level in oxidative stress. In this study, the level of Nrf2
and Nrf2-regulated genes; NQO1 and HO-1 , were deter-
mined by western blots in the liver of rats after pre-treat-
ment with either HGJ (100 mg/kg) or AA (100 mg/kg)
before Cd (4 mg/kg) exposure. The results show that AA
pre-treatment significantly enhanced the induction of
Nrf2 and NQO1 proteins when compared with Cd (Figs.
5A and B). No signicant changes were seen in HGJ pre-
treated rats when compared with control (Figs. 5A and
B). On the other hand, the pre-treatment with HGJ caused
a signicant increase in HO-1 expression when compared
with Cd (Figs. 5A and B). The presence of either HGJ,
AA and Cd alone however caused a signicant increase
in the antioxidant enzymes when compared with control
Fig. 3. Levels of superoxide dismutase activities in rats liver
pre-treated with either HGJ or AA before Cd exposure.
Rats were pre-treated with 100 mg/kg body weight of
either HGJ or AA for 4 weeks before 3 days intraperi-
toneal exposure to 4 mg/kg body weight CdCl
2
. SOD
activities were measured. Data represents the mean ac-
tivity (n = 5) relative to total cell protein S.D. ***p
< 0.005, **p < 0.01, *p < 0.05 (one way ANOVA,
Dunnetts post test). # represents signicant different
between pre-treated and unpre-treated rats (unpaired
student t-test).
Fig. 4. Levels of catalase activities in rats liver pre-treated
with either HGJ or AA before Cd exposure. Rats were
pre-treated with 100 mg/kg body weight of either HGJ
or AA for 4 weeks before 3 days intraperitoneal expo-
sure to 4mg/kg body weight CdCl
2
. catalase activities
were measured. Data represents the mean activity (n
= 5) relative to total cell protein S.D. ***p < 0.005,
**p < 0.01, *p < 0.05 (one way ANOVA, Dunnetts
post test). # represents significant different between
pre-treated and unpre-treated rats (unpaired student t-
test).
Vol. 36 No. 5
553
Hepatotoxicity of Cadmium and protective effect of heated garlic juice
(Fig. 5). These data suggest an involvement of Nrf2 and
Nrf2-regulated enzymes in mediating HGJ and AA pro-
tection against Cd toxicity in the exposed rat liver.
DISCUSSION
In this study we examined the protective effects of
heated garlic juice (HGJ) against cadmium-induced tox-
icity in liver of exposed rats and these effects were com-
pared with that of a known antioxidant, ascorbic acid
(AA). Examination of Cd toxicity, using MDA levels as
an index of oxidative stress; show that both HGJ and AA
prevent Cd-induced membrane lipid damage. Many stud-
ies have reported increase lipid peroxidation in liver of
rats exposed to Cd (El-Maraghy et al., 2001; Casalino et
al., 1997; Mller, 1986). The present study was in agree-
ment with these earlier studies as increase MDA was
observed in the liver of rats after 3 days exposure to 4 mg/
kg body weight Cd. The increase MDA levels observed in
the presence of Cd may be as a result of enhanced oxida-
Fig. 5. Western blots analysis of the expression levels of antioxidant enzymes in liver of rats homogenates pre-treated with either HGJ
or AA before Cd exposure. Rats were pre-treated with 100 mg/kg body weight of either HGJ or AA for 4 weeks before 3 days
intraperitoneal exposure to 4 mg/kg body weight CdCl
2
. Protein expression levels of (A) Nrf2, NQO1 and HO-1 were deter-
mined by western blots analysis. (B) Histogram for the expressions of these proteins. Data represents the mean band intensities
of three different experiments (n = 3) relative to -actin S.D.***p < 0.005, **p < 0.01, *p < 0.05 (one way ANOVA, Dun-
netts post test). # represents signicant different between pre-treated and unpre-treated rats (unpaired student t-test).
A
B
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A.O. Lawal et al.
tive stress due to increase production of reactive oxygen
species (Stohs et al., 1995). Lipid peroxidation is known
to induce cellular damage and it is responsible for reac-
tive oxygen species induced organ damage (Halliwell
and Gutteridge, 1989). The present study shows that the
increase MDA caused by Cd may be due to increase ROS
production. The increase in lipid peroxidation in the liver
caused by cadmium was signicantly prevented by pre-
treatment with ascorbic acid and HDJ. This may be due
to the decrease in ROS observed in the presence of HGJ
and AA. However, the results show that AA seems more
effective than HGJ in preventing Cd-induced lipid perox-
idation.
It has earlier been reported that heated garlic juice is
less effective than aged garlic extract in preventing lip-
id peroxidation (Borek, 2001). HGJ contains mainly alli-
in due to the inactivation of enzyme allinase as a result of
the heating process and this allinase was reported to be
responsible for converting alliin to allicin, the precursor of
most bioactive compounds in age garlic extract (Banarjee
and Maulik, 2002). Therefore, the presence of alliin in
HGJ may account for the less effectiveness of HGJ in
preventing lipid peroxidation caused by Cd when com-
pared with AA.
The most abundant reactive oxygen species (ROS)
generated in living cells are superoxide anion and its
derivatives, particularly highly reactive and damaging
hydroxyl radicals, which induce lipid peroxidation of cell
membrane lipids (Hemnani and Parihar, 1998). Increase
in superoxide dismutase (SOD) activity of organs appears
to be benecial in the event of increase free radical gen-
eration. However, it has been reported that a rise in super-
oxide dismutase activity without a concomitant rise in
the activity of catalase might be detrimental (Herman,
1991). Thus a simultaneous increase in catalase activity
is essential for an overall benecial effect of increase in
SOD activity. Therefore, the increase in SOD and cata-
lase activities in liver following pretreatment with heat-
ed garlic juice and ascorbic acid has a special signicance
in relation to cellular defense against oxidative stress. Cd
has been reported in many studies to inhibit the activi-
ties and expression of most antioxidant enzymes such as
SOD and catalase (Casalino et al., 1997; Hussain et al.,
1987; Liu et al., 2002; Shukla et al., 1989). The reduction
in SOD and catalase activities observed in the presence of
Cd may be responsible for the increase ROS and MDA.
This means that the enhancement of SOD and catalase
activities by HGJ and AA pre-treatment may account for
the reduction in MDA level due to the enhanced remov-
al of the superoxide anion and hydrogen peroxide. It
has earlier been reported that heated garlic juice reduces
tumor growth in mice (Kasuga et al., 2001) and this may
be due to the enhanced expressions and activities of SOD
and catalase antioxidant enzymes observed in this study.
Nrf2 is a transcription factor that regulates the expres-
sion of antioxidant enymes in response to stress (Nguyen
et al., 2009). Nrf2 is sequential in the cytosol by keap1
repressor, but in the presence of oxidative stress, the
Nrf2 dissociate from its keap1 repressor and migrate to
the nucleus where it bind to the antioxidant response ele-
ment (ARE) to activate transcription of protective enymes
(Motohashi and Yamamoto, 2004). Since Cd generates
ROS (Liu et al., 2009), it is possible that the increase
ROS induced the release of Nrf2 from keap1 repres-
sor resulting in enhanced production of Nrf2-dependent
enzymes such as NQO1 and HO-1. It has earlier be shown
that Cd-induced the migration of Nrf2 from cytosol into
the nucleus in 1321N1 cells via PKC leading to increase
expression of HO-1 and NQO1 (Lawal and Ellis, 2011).
Therefore the increase in Nrf2, NQO1 and HO-1 protein
expressions seen in this study may be due to the increase
ROS production by Cd. The increase protein levels of
Nrf2 and NQO1 in AA and HO-1 in HGJ pre-treated rats
may be responsible for the decrease MDA and ROS lev-
els observed in these rats after Cd exposure.
The result of this study suggests that both ascorbic acid
and heated garlic juice are potent in preventing cadmium-
induced liver damage in rats and ascorbic acid seems to
be more effective.
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