Journal of Behavioral and Brain Science, 2013, 3, 74-84
http://dx.doi.org/10.4236/jbbs.2013.31008 Published Online February 2013 (http://www.scirp.org/journal/jbbs)
Protective Effect of Curcumin on Anxiety, Learning
Behavior, Neuromuscular Activities, Brain
Neurotransmitters and Oxidative Stress Enzymes in
Cadmium Intoxicated Mice
Gasem M. Abu-Taweel1, Jamaan S. Ajarem2, Mohammad Ahmad3*
1Department of Biology, College of Education, Dammam University, Dammam, KSA
2Department of Zoology, College of Science, King Saud University, Riyadh, KSA
3Department of Medical Surgical Nursing, College of Nursing, King Saud University, Riyadh, KSA
Received November 13, 2012; revised December 15, 2012; accepted December 22, 2012
ABSTRACT
Cadmium (Cd) exposure can induce acute lethal health-related threats to humans since it has an exceptional ability to
accumulate in living organisms and cause toxicological effects. Curcumin (Cur) on the other hand has a wide variety of
biological activities and several animal studies have suggested for a potential therapeutic or preventive effects against
several ailments and infections. To study the effect of Cur on the toxicity of Cd, sixty Swiss-Webster strain male mice
were divided into 6 groups of ten each at random. Group-1 served as the naïve control and received no treatment.
Group-2, 3 and 4 were the experimental controls and were administered once a day with a single oral dose of 50% di-
methyl sulphoxide (DMSO), Cur (300 mg/kg) or Cd (100 mg/kg) respectively, for 2 weeks. Group-5 and 6 received Cur
and Cd in combination once a day orally for 2 weeks except that Cur in a dose of 150 and 300 mg/kg to group 5 and 6
respectively, was administered one hour before Cd (100 mg/kg) administration to both groups. After treatment period,
the animals were subjected to behavioral tests and thereafter, the animals were sacrificed for the estimation of neuro-
transmitters like serotonin (5-HT), dopamine (DA) and it’s metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) as
well as oxidative stress enzymes like lipid peroxides in the form of thiobarbituric acid–reactive substances (TBARS)
and total glutathione (GSH) in the forebrain tissue. Cd reduced significantly the body weight gain, the locomotor activ-
ity, anxiety behavior in the plus maze and the learning capability (cognitive effect) in the shuttle-box test. Biochemical
analysis further revealed that Cd exposure significantly altered the brain neurotransmitters and the oxidative stress en-
zymes. However, administration of Cur along with Cd had an ameliorating effect on all the behavioral and biochemical
parameters studied herein and reduced the toxicity of Cd significantly and dose-dependently. Thus, Cur may be benefi-
cial for anxiety, neuromuscular, and cognitive problems and protect from Cd intoxication.
Keywords: Curcumin; Cadmium; Male Mice; Anxiety; Cognitive Behaviors; Neurotransmitters; Oxidative Stress
1. Introduction
Cadmium (Cd) represents one of the most toxic and car-
cinogenic heavy metal [1]. It is considered as a serious
environmental and industrial pollutant and may represent
as a serious health hazard to humans and other animals
[2-4]. Some important sources of Cd exposure for hu-
mans can be emissions from industries of batteries, metal
plating, pigments, plastics, toys and alloy, cigarette smo-
king and through dietary consumption [5-7]. Exposure to
Cd may cause lesions in many organs such as the liver,
kidney and testis [8-11], leading to various possible pa-
thological conditions such as hepatic, renal and testicular
dysfunction, respiratory and nervous system disorders
[12,13]. Cd is reported to induce the generation of reac-
tive oxygen species (ROS), and this oxidative stress was
found to result in mitochondrial dysfunction and apop-
tosis, both in vivo and in vitro [14,15]. The oxidative
damage within the tissues and DNA damages is consid-
ered to be an early manifestation of Cd toxicity and car-
cinogenicity [16,17].
Curcumin (Cur) is a well known biologically active
natural phytochemical phenolic compound (diferuloylme-
thane) found as a major component in turmeric, a yellow
curry spice, extracted from the rhizome of Curcuma
longa L. (family Zingiberaceae). Cur is well absorbed in
the body system and has exceedingly low toxicity [18]. It
*Corresponding author.
C
opyright © 2013 SciRes. JBBS

G. M. ABU-TAWEEL ET AL. 75
possesses many beneficial activities in the body and is
effective in several disorders including anorexia, coryza,
cough, hepatic diseases, and sinusitis [19,20]. Recent
studies provide scientific evidence regarding the poten-
tial pharmacological, prophylactic or therapeutic use of
Cur, as anti-inflammatory, anticarcinogenic, antiviral, an-
tifungal, antiparasitic, antimutagen, antiinfectious and an-
tioxidant compound [21-26]. The multiple beneficial ef-
fects of Cur have also been elaborated in neurogenesis
process which in turn has been reported for its neuropro-
tective effects in age-related neurodegenerative diseases
[27]. Several studies have shown that Cur exhibits pro-
tective effects against oxidative damage and has antioxi-
dant property exerting powerful oxygen free radical sca-
venging effects and increased intracellular glutathione
concentration, thereby protecting lipid peroxidation [28-
32]. Commercial Cur contains 77% curcumin, 17% de-
methoxycurcumin and 3% bisdemethoxycurcumin [33]
and virtually all these three components in Cur are bio-
logically active and possess protective properties [34].
In the light of the above information it appears that
Cur may prove beneficial in several ways for Cd toxici-
ties and this aspect needs more and more research work.
Thus, the present study was undertaken to explore the ef-
fects of Cur against the Cd induced behavioral deficits
and biochemical toxicity in the brain of male mice.
2. Materials and Methods
2.1. Experimental Animals
Sixty male Swiss-Webster strain mice (8 - 10 weeks old)
were housed in opaque plastic cages under hygienic con-
ditions in the animal facility of the Zoology Department,
King Saud University, Riyadh, Saudi Arabia. All animals
were maintained under reversed lighting conditions with
white lights on from 22.00 to 10.00 hours local time. The
ambient temperature was regulated between 20˚C and
22˚C. Food (Pilsbury’s Diet) and water were available ad
libitum, unless otherwise indicated. All procedures were
carried out in accordance with the ethical guidelines for
care and use of laboratory animals, and all protocols
were approved by the local Ethics and Care of Experi-
mental Animals Committee.
All animals were divided into six different groups with
ten animals in each. Group I consisted of untreated mice
and served as naïve controls. Group II was treated with
50% DMSO (solvent of Cur). Group III was treated with
300 mg/kg Cur dissolved in 50% DMSO. Group IV was
treated with Cd (100 mg/kg). Groups V and VI consisted
of mice administered with Cur as well as Cd in combina-
tion in the doses of 150 + 100 and 300 + 100 mg/kg re-
spectively. All exposures were through oral administra-
tion, once a day, for two weeks, except that in groups V
and VI, Cur was administered one hour before Cd expo-
sure.
2.2. Cur and Cd Administration
Cur of analytical grade, Sigma Chemical Company, USA,
was dissolved in 50% DMSO to give a dose of 150 and
300 mg/kg body weight and diluted further with drinking
water in 1.0 ml volume and was administered orally once
a day. Cd was also administered orally once a day in the
form of cadmium chloride (analytical grade, Riedel de
Haen, Germany) dissolved in drinking tap water at a dose
of 100 mg/kg body weight in 1.0 ml volume. In the fifth
and sixth groups of animals where Cur and Cd were ad-
ministered together orally once a day, Cur was adminis-
tered one hour before Cd administration. The naïve con-
trol group received 1.0 ml plain tap water only. The
doses of Cur and Cd used in this study are at par with the
effective doses reported in the literature for such studies.
The factor for the possibility of presence of Cd traces in
food and tap water was not taken into account for calcu-
lating the daily Cd intake. However, this factor was mi-
nimized by giving the same source of food and tap water
to all experimental groups including the controls.
2.3. Body Weight
The body weight of all animals from each experimental
group was recorded on day 1 of the treatment and on
days 5, 10 and 15 of the total treatment period. Thereaf-
ter, the animals were subjected to behavioral tests and
subsequently were sacrificed for the isolation of fore-
brain tissue for the biochemical estimations.
2.4. Behavioral Studies
Anxiety, learning capability and locomotor behavior in
all animals were measured in the same order in plus maze,
shuttle-box and in automated activity meter respectively.
2.4.1. Anxiety Behavior in the Elevated Plus-Maze
Test
The elevated plus-maze (with 2 opened and 2 enclosed
arms) is frequently used as a measure for evaluating the
risk assessment and anxiety behavior of an ethologically
derived animal model [35]. The plus-maze was elevated
to a height of 80 cm above the floor. The mice were in-
dividually placed onto the central platform facing one of
the open arms and were observed for 5 min while freely
exploring the maze. The animal was considered to have
entered an arm when all four limbs were inside the arm.
Duration of time spent in open and enclosed arms and
number of entries in open and enclosed arms were meas-
ured during the test period. On completion of the test, the
maze was cleaned with a 10% ethanol solution to control

G. M. ABU-TAWEEL ET AL. 76
for any possible olfactory cues.
2.4.2. Learning Capability in the Shuttle-Box Test
(Active Avoidance Responses)
The active avoidance responses were measured in the
animals using an automatic reflex conditioner “shuttle
box” (Ugo Basile, Comerio-Varese, Italy). The rectangu-
lar shaped shuttle-box was divided into two chambers of
equal size by a stainless steel partition with a gate pro-
viding access to the adjacent compartment. Before start-
ing the trial sessions, each animal was allowed to adapt
and acquaint itself with the shuttle box for 2 min without
any stimulus. A light bulb (21 W) for 6 s duration and a
buzzer (670 Hz and 70 dB) was switched on consecu-
tively and used as a conditioned stimulus (CS). The CS
preceded the onset of the unconditioned stimulus (US) by
5 s. The US was an electric scrambler shock (1 mA for 4
s) applied to the metallic grid floor. If the animal avoided
the US by running into the other compartment within 5 s
after the onset of the CS, the microprocessor recorder unit
of the shuttle box recorded an avoidance response and
this was considered as conditioned avoidance response to
avoid the electric shock. Each animal was given 50 trials
with a fixed inter trial interval of 15 s. During the 50 tri-
als session of the individual animal, the total number of
avoidance was measured. The total time taken until the
animal entered the other compartment to avoid the shock
treatment (latency of avoidance response or escape la-
tency in seconds) was also measured for each animal.
The recorder unit of the automated shuttle box continu-
ously recorded these parameters during the whole expe-
rimental period (50 trials) of each animal.
2.4.3. Motor Activity Test in Automated Activity
Meter
Motor activity was measured using automated electronic
activity meter (Ugo Basile, Comerio-Varese, Italy). The
horizontal and vertical motor activities were detected by
arrays of infrared beams located above the floor of the
testing arena. Each interruption of the beams on the x or
y axis generated an electric impulse which was recorded
on a digital counter. Each animal was tested separately
and the motor activity was recorded for a period of 2 min
in the activity meter.
2.5. Biochemical Studies
Immediately after completing the behavioral tests, the
animals were sacrificed by decapitation, the brains were
dissected on ice, the fore brain areas (containing the hip-
pocampus and striatum areas) were removed and frozen
in liquid nitrogen and stored at −70˚C for determination
of monoamines, lipid peroxides (TBARS) and glutathi-
one content.
2.5.1. Determination of Monoamines
The monoamines were estimated using the modified me-
thod of Patrick et al. [36]. A 10% homogenate of fore
brain tissue was prepared by homogenizing the tissues
for 10 s in 0.1 M HClO4 containing 0.05% EDTA, cen-
trifuged at 17,000 rpm at 4˚C for 5 min. The supernatants
were filtered using 0.45 µm pore filters and analyzed by
high performance liquid chromatography (HPLC). The
mobile phase consisted of 32 mM citric acid monohy-
drate, 12.5 mM disodium hydrogen orthophosphate, 7%
methanol, 1 mM octane sulphonic acid and 0.05 mM
EDTA. The mobile phase was filtered through 0.22 µm
filter and degassed under vacuum before use. µBondpak
C18 column was used at a flow rate of 1.2 ml/min and
the injection volume of the sample was 20 µl. The levels
of dopamine (DA), DOPAC and serotonin or 5-hydroxy-
tryptamine (5-HT) were calculated using a calibration
curve and results were expressed as ng/mg tissue weight.
2.5.2. Determination of Lipid Per oxides
Lipid peroxides (LP) in fore brain tissue were determined
spectrophotometrically as thiobarbituric acid-reactive sub-
stances (TBARS) according to the method of Ohkawa et
al. [37]. The tissue samples were homogenized in 1.15%
cold KCl with an Ultraturax homogenizer. After centrifu-
gation at 3000× g for 5 min, an aliquot of supernatant
was mixed with 2 ml of reaction mixture (containing
15% trichloroacetic acid and 0.375% thiobarbituric acid
solution in 0.25 N HCl) and heated for 5 min in a boiling
water bath. The tubes were cooled at room temperature
and centrifuged at 1000× g for 10 min. The absorbance
of supernatant was read at 535 nm against a blank that
contained all reagents except homogenate. Tissue lipid
peroxide levels were quantified using extinction coeffi-
cient of 1.56 × 105 m−1·cm−1 and expressed as nanomoles
of TBARS formed per g tissue weight.
2.5.3. Determination of Glutathione
Total glutathione (GSH) level in fore brain tissue was
measured enzymatically in the brain tissues by a slightly
modified method of Mangino et al. [38]. Briefly, about
50 mg of isolated brain tissues were homogenized with 1
ml 0.1 M perchloric acid plus 0.005% EDTA. The ho-
mogenates were centrifuged at 4000 rpm for 10 min and
the supernatants were used for GSH assay. The reaction
mixture consisted of the following three freshly prepared
solutions: solution I, 0.3 mM NADPH; solution II, 6 mM
5,5’-dithio-bis(2-nitrobenzoic acid) and solution III, 50
U/ml glutathione (all chemicals from Sigma). All three
solutions were prepared with a stock buffer consisting of
125 mM NaH2PO4 and 6.3 mM EDTA at pH 7.5. At the
time of glutathione assay, 800 µl of solution I, 100 µl of
solution II, and 10 µl of solution III were mixed in a
quartz cuvette and placed in a dual beam UV-VIS spec-

G. M. ABU-TAWEEL ET AL. 77
trophotometer (Shimadzu UV160) at 30˚C. The enzyma-
tic reaction was started by the addition of 100 µl of the
supernatant and the absorbance was monitored for 3 min
at 412 nm. The slope of the change in absorbance was
used for quantitative estimation of total GSH by com-
paring the slope of the samples with a standard curve
prepared with pure glutathione (Sigma).
2.6. Statistical Analysis
The data were analyzed for variance (Bartlett’s test for
equal variance) and normality (Gaussian-shaped distribu-
tion) using the Kolmogorov-Smirnov goodness-of-fit test.
As the data passed the normality test (p > 0.10), group
means were compared with the ANOVA with post-hoc
testing using Tukey-Kramer Multiple Comparisons Test
or Student-Newman-Keuls Multiple Comparisons Tests.
All results were expressed as means ± SEM and the sig-
nificance were defined as p < 0.05 for all tests.
3. Results
3.1. Body Weight
Exposure to Cd for two weeks showed a significant (p <
0.001) depletion in the body weight gain of the treated
rats whereas Cur alone showed no significant changes in
the body weight as compared to the control animals.
However, significant and dose-dependent ameliorating
effect of Cur was found in Cd-induced alterations in the
body weight gain when the animals were treated with Cd
and Cur in combination (Figure 1).
3.2. Behavioral Studies
3.2.1. Elevated Plus-Maze Test
The time spent in open arms was significantly (p < 0.001)
lower, whereas time spent in enclosed arms was signifi-
cantly (p < 0.001) higher in animals treated with Cd as
compared to controls (Figure 2(a)). The number of en-
tries in open arms was significantly (p < 0.001) lower
and in enclosed arms was significantly (p < 0.001) higher
in Cd treated groups exhibiting more anxiety related ex-
ploratory activity as compared to controls (Figure 2(b)).
Cur alone had no effects in any of the parameters as com-
pared to the controls (Figures 2(a) and (b)). However, in
the Cur and Cd combination treated group, Cur pre-
treatment significantly (p < 0.01) and dose-dependently
attenuated the Cd induced anxiety and behavioral abnor-
malities (Figures 2(a) and (b)).
3.2.2. Shuttl e-Box Test
In the shuttle-box active avoidance test, the Cd-exposed
animals, showed a statistically significant (p < 0.001) de-
crease in the number of avoidances during the trial period
as compared to the control group (Figure 3(a)). The total
1510 15
0
10
20
30
40 Contr ol
DMSO
Cur 300 mg/kg
Cd 100 mg/kg
Cd + C ur 150 mg/kg
##
#
** ** **
Days
Body weight ( i n gm )
Cd + C ur 300 mg/kg
Figure 1. Ameliorating effect of curcumin on the declining
body weight gain in the cadmium treated mice. ***repre-
sents statistically significant (p < 0.001) from the control
group whereas # and ## represent significantly different (p <
0.05 and p < 0.01 respectively) from the cadmium treated
group by ANOVA and student’s t-test.
0
10
20 Control
#
#
**
**
Mean number of entrance ? SE M
[A]
DMSO
Cur 300 mg/ kg
Cd 100 mg/kg
Cd + Cur 150 mg/kg
Cd + C ur 30 0mg/kg
Open Arm Closed Arm
(a)
0
50
100
150
200
250
O
p
en arm Midd le s
p
ace Closed arm
Me an time spent in
seconds ?SEM
#**
#**
[B]
(b)
Figure 2. Protective effect of curcumin on the cadmium-in-
duced anxiety in the mice measured in a plus maze activity
meter by estimating the total time spent (a) and the number
of entries (b) in the open and enclosed arms of the plus
maze. ***represents statistically significant (p < 0.001) from
the control group whereas ## and ### represents significantly
different (p < 0.01 and p < 0.001 respectively) from the
cadmium treated group by ANOVA and student’s t-test.
time taken during the entire trials by the Cd treated ani-
mals to enter the other compartment to avoid the shock
treatment (latency of avoidance or escape latency re-
sponse in seconds) was significantly (p < 0.001) greater
as compared to the controls (Figure 3(b)). Animals ex-
posed to Cd were poor learners and took significant time
in avoiding the shock treatment as compared to the con-
trols (Figures 3(a) and (b)). Cur alone had no effect on
the active avoidance performances, however, Cur in com-
bination with Cd showed a significant (p < 0.05) and

G. M. ABU-TAWEEL ET AL. 78
0
50
100
150
200 Contr ol
DMSO
Cur 3 000 mg/kg
Cd 100 mg/kg
Cd + Cur 150 mg /kg
Cd + Cur 300mg/kg
[A]
#**
Latency to avoid shock
t reat ment i n avoi dance
t est ( M ean values i n
seconds ?SEM )
(a)
0
10
20
30
40
Exper imental gr oups o f male mice
#**
[B]
Number of reinforced
crossings( M ean valu es
?SEM )
(b)
Figure 3. Protective effects of curcumin on the cadmium-
induced cognitive (learning) performance in shuttle box test
for the mice to take time (latency) in avoiding the shock
treatment (a) and the number of reinforced crossing the
chamber (b) for avoiding the shock treatment during sti-
mulus of light and sound. ***represents statistically signifi-
cant (p < 0.001) from the control group whereas # and ##
represent significantly different (p < 0.05 and p < 0.01 re-
spectively) from the cadmium treated group by ANOVA
and student’s t-test.
dose-dependent attenuating effect of Cur pretreatment on
the Cd-induced poor learning capabilities (Figures 3(a)
and (b)).
3.2.3. M otor Activi t y
Treatment with Cd significantly affected the vertical as
well as the horizontal motor activity (p < 0.001) as com-
pared to the control (Figure 4). Cur alone had no signi-
ficant effect on these activities but concomitant treatment
with Cur and Cd significantly (p < 0.01) and dose-de-
pendently attenuated the Cd-induced motor impairment
(Figure 4).
3.3. Biochemical Studies
3.3.1. Levels of Monoamines in Forebrain Tissue
There was a significant (p < 0.001) depletion of DA and
DOPAC in the forebrain areas of mice treated with Cd as
compared to the control group (Figures 5(a) and (b) re-
spectively). Similarly, there was a significant (p < 0.001)
depletion of 5-HT in the forebrain tissue of the Cd treated
group as compared to the control (Figure 5(c)). Exposure
to Cur alone had no alteration in the levels of these neu-
rotransmitters as compared to the controls. However, in
the group that was administered Cur and Cd in combina-
0
100
200
300
400
500 Control
DMSO
Cur 300 mg/ kg
Cd 100 mg/kg
Cd + C ur 15 0 mg/kg
#**
#**
Ver t ical activity
Activity ?SEM
Hor iz ont al activity
Cd + C ur 30 0 mg/kg
Figure 4. Attenuating effect of curcumin on the declining
locomotor (horizontal and vertical) activity of mice treated
with cadmium measured in electronic activity meter. ***re-
presents statistically significant (p < 0.001) from the control
group whereas # and ## represent significantly different (p <
0.05 and p < 0.01 respectively) from the cadmium treated
group by ANOVA and student’s t-test.
0
1
2
3
4Control
[A]
DMSO
Cur 300 mg/kg
Cd 100 mg/kg
Cd + Cur 150 mg/kg
Cd + Cur 300 mg/kg
#
**
5-HT ( ng / mg tissue wt )
(a)
[B]
0
10
20
30
40
50
60
Dopamine (ng/mg tissue wt)
#
**
(b)
0
25
50
75
100
125
Experimental groups of male mice
#
**
DOPAC (ng/mg tissue wt)
[C]
(c)
Figure 5. Ameliorating effect of curcumin on the depleting
levels of the neurotransmitters like (a) serotonin (5-HT), (b)
dopamine (DA) and (c) the byproduct of DA (DOPAC), due
to cadmium treatment in the fore brain area of the male
mice. ***represents statistically significant (p < 0.001) from
the control group whereas # represents significantly differ-
ent (p < 0.05) from the cadmium treated group by ANOVA
and student’s t-test.

G. M. ABU-TAWEEL ET AL. 79
[A]
tion, pretreatment of animals with Cur, significantly (p <
0.001) and dose-dependently attenuated Cd-induced de-
pletion of DA and DOPAC (Figures 5(a) and (b) respec-
tively) and 5-HT (Figure 5(c)) in the forebrain tissue as
compared to the Cd treated groups.
3.3.2. Lipid Peroxidation (TBARS) Levels in the
Forebrain Tissue
The lipid peroxidation (TBARS) level in the forebrain
tissues (Figure 6(a)) were markedly (p < 0.001) increa-
sed in the Cd treated group as compared to the control.
Cur alone had no effect on the level of TBARS, however,
in the Cur and Cd combination group, Cur pre-treatment
significantly (p < 0.05) and dose-dependently attenuated
Cd-induced increase in TBARS level (Figure 6(a)) as
compared to the Cd group.
3.3.3. Glutathione (GSH) Levels in th e Fo rebrain
Tissue
A highly significant (p < 0.001) depletion in the GSH
level was observed in the forebrain tissue of Cd treated
group (Figure 6(b)). However, Cur alone had no altera-
tion on the normal level of GSH. In the combination
group (Cur + Cd), Cur pre-treatment significantly (p <
0.05) and dose-dependently attenuated the Cd-induced
depletion of GSH in the forebrain tissue (Figure 6(b)) as
compared to Cd group.
4. Discussion
The present results suggest that exposure of male mice to
Cd is toxic and influences various behavioral activities as
well as the levels of enzyme activities in the brain tissues
of the treated animals. The rodents exposed to Cd in ear-
lier studies also are reported to display lowered body
weight [39,40], impaired behavioral activity and wors-
ened conditioned reflex response [41,42], and impaired
neurobehavioral [43] and neurotoxicological [42,44] de-
velopments. It is therefore likely that the above factors
may singly or together ultimately produce behavioral
effects of Cd [45] in the present study also.
The major target organs that are reported for the acute
oral toxicity of Cd are liver [46] and central nervous tis-
sue [47]. Cd has been recognized as one of the most toxic
environmental and industrial pollutants that may induce
oxidative damage by disturbing the prooxidant-antioxi-
dant balance in the tissues. A significantly increased ac-
cumulation of Cd in liver, kidneys and other organs have
been reported with the severity of their intoxication de-
pendent on the route, dose, and duration of the exposure
to the metal [48-50]. Previous investigations show that
oral intake of Cd induces its accumulation in the red
blood cells [51], heart [52] and skeletal muscle of rats
[53], which was accompanied by considerable alterations
of enzymatic and non-enzymatic component of antioxi-
0
5
10
15
20 Control
DMSO
Cur 300 mg/kg
Cd 100 mg/kg
Cd + Cur 150 mg/kg
#
**
TBARS (nmol / g tissue wt )
Cd + Cur 300 mg/kg
(a)
[B]
#
**
5
4
3
2
1
0
Experimental groups of male mice
GSH (nmol / g t issue wt)
(b)
Figure 6. Protective effect of curcumin on the cadmium-
induced oxidative stress depicted by increased level of
TBARS (a) and decreased level of GSH (b) in the forebrain
of the mice. ***represents statistically significant (p < 0.001)
from the control group whereas # represents significantly
different (p < 0.05) from the cadmium treated group by
ANOVA and student’s t-test.
dant defense system (AOS). At cellular level also it has
been reported that, Cd mainly accumulates in the cytosol
(70%), followed by the nucleus (15%) and lowest in mi-
tochondria and the endoplasmic reticulum [54]. Such ac-
cumulation of Cd mainly in cytosol might have lead to
variations in the phosphate pool of the animals which ul-
timately lead to disturbed energy source with consequent
disturbance in their metabolism [55], and this is probably
reflected in the form of disturbed behavioral activities.
The results of the present study showed that the levels
of neurotransmitters DA, DOPAC and 5-HT were deple-
ted significantly by Cd treatment in the forebrain (cere-
bral part containing hippocampus and striatum) tissue of
the Cd-exposed mice. There is evidence of an inhibitory
role of DA mediated receptor (D2 type) in depressing the
hyperexcitability of hippocampal and striatal neurons [56,
57]. A number of 5-HT receptor subtypes have been re-
ported for having different roles in the functions of sero-
tonergic neurotransmission, including the functions con-
nected with learning and memory processes [58]. The
mice exposed to Cd performed badly in plus maze para-
meters and also resulted in decreased number of avoid-
ances (escapes) in the automatic reflex conditioner as
compared to the control animals. This suggests for a ten-
dency towards decreasing of the exploratory and memory

G. M. ABU-TAWEEL ET AL. 80
effect of Cd under conditions of reduced functional ca-
pacity of serotonergic neurotransmission as also reported
earlier for aluminum toxicity [59]. Recently, a growing
body of research has focused on the participation of se-
rotonin (5-HT) in the neurochemical mechanisms of cog-
nition and especially of learning and memory. Potential
toxic mechanisms of action for Cd may include disrup-
tion in serotonergic neurotransmission through disturbed
levels of neurotransmitters in the brain hippocampus
[60].
Other studies have also shown that Cd inhibits the ac-
tivity of majority of enzymes involved in AOS [61-63]
inducing an increased production of free radicals, lipid
peroxidation, and destruction of cell membranes [51,54,
64]. Cd is also reported to inhibit the activities of many
enzymes by binding to their sulfhydryl groups or by in-
hibiting the protein synthesis [65,66].
Cur in the present study had a significantly ameliorat-
ing effect on the Cd-induced deficits in the body weight,
anxiety behavior, learning capability (cognitive effect)
and muscular activity. Biochemical analysis in forebrain
tissue also revealed that Cur significantly attenuated Cd-
induced neurotransmitters (reportedly associated with lo-
comotor and cognitive activities) and the Cd-induced
oxidative stress related enzymes (associated with behav-
ioral and cognitive deficits). Furthermore, the ineffective-
ness of Cur alone to cause any behavioral and biochemi-
cal deficits, clearly suggest that Cur alone is non-toxic
and further supports for the ameliorating effect of Cur on
the behavioral and biochemical toxicity induced by Cd.
The biochemical damage may be due to the fact that Cd
induces an oxidative stress that results in oxidative dete-
rioration of biological macromolecules [40,67]. Cur re-
portedly has potent antioxidant activities [68,69], anti-in-
flammatory [70] and chemoprotective properties [71]. It
has been shown to have a neuroprotective effect in mod-
els of cerebral ischemia [72,73], ethanol induced brain
damage [74] and reduced amyloid pathology in trans-
genic mice of Alzheimer’s disease [75].
Lipid peroxidation (LP) is one of the main manifesta-
tions of oxidative damage, which plays an important role
in the toxicity of many xenobiotics [76,77]. Our results
confirm that intoxication with Cd causes a significant in-
crease of lipid peroxide concentration in forebrain tissue
of mice. Since it causes LP in numerous tissues both in
vivo and in vitro [6,51,62,65], it has been suggested that
Cd may induce oxidative stress by producing hydroxyl
radicals [78], superoxide anions, nitric oxide and hydro-
gen peroxide [79,80]. Moreover, it has been shown that
various antioxidants and AOS protect cells from Cd-in-
duced toxicity [81-83].
Co-treatment with Cur in the present study was effec-
tive in the prevention of oxidative damage induced by Cd,
which resulted in significantly lower lipid peroxides con-
centration in the form of TBARS in the forebrain tissue.
This can be explained by the important role of Cur in
preventing LP and in protection of integrity and func-
tioning of tissues and cells. The prevention of LP is es-
sential for all aerobic organisms and so the organism is
well equipped with antioxidants that directly or indirectly
protect cells against the adverse effects of xenobiotics,
carcinogens and toxic radicals [84]. The role of antioxi-
dants in reversing this oxidative stress has been a matter
of deep interest to basic scientists and clinicians [85].
The decreased activity of GSH due to Cd exposure in
the present study suggests for the disturbed oxidant and
antioxidant system in the brain tissue. Furthermore, pres-
ence of Cur along with Cd ameliorates significantly the
GSH level and tries to increase the level of GSH indicat-
ing for the role of Cur as an antioxidant. It has been well
established that the antioxidants such as Vit E, Vit C and
GSH protect the membrane from oxidative damage [64,
84,85]. In the present study, Cur reduced the cellular to-
xicity caused by Cd-induced ROS and protected the brain
antioxidant system. Thus, Cd accumulation in brain tis-
sue is most likely due to chronic dietary intake of Cd,
and it is associated with marked alteration of neurotrans-
mitters and enzyme GSH of AOS. These results also sug-
gest that LP is associated with Cd toxicity in brain tissue.
Our results showed that the antioxidant Cur ameliorated
oxidative stress and loss of cellular antioxidants and sug-
gested that Cur efficiently protect forebrain from Cd-in-
duced oxidative damage. However, for asserting this state-
ment, further studies are needed to measure other enzy-
matic components like superoxide dismutase (SOD), ca-
talase (CAT), glutathione peroxidase (GSH-Px) and glu-
tathione-S transferase (GST) and nonenzymatic compo-
nents such as vitamin C (Vit C) and vitamin E (Vit E) of
AOS.
5. Conclusion
It is concluded from the present study that although Cur
has many possible reported benefits, the full effects
however, are not yet fully understood and more and more
research work is needed. The results indicate that Cur
possesses several multifold beneficial effects that may in-
clude better cognitive performance, better muscular ac-
tivity and protection from externally induced oxidative
stress and neurotransmitters dysfunction in the brain.
Thus, inclusion of Cur in our normal diet and its use as a
nutritional supplement may have a tremendous potential
for health improvement and protection from Cd intoxica-
tion.
6. Acknowledgements
The authors are grateful to the The Excellence Center of
Science and Mathematics Education, King Saud Univer-

G. M. ABU-TAWEEL ET AL. 81
sity, Riyadh, for their support and encouragement.
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