2009 The Authors Doi: 10.1111/j.1742-7843.2009.00404.

x
Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104

Effect of N-Acetyl Cysteine against Aluminium-induced Cognitive

Dysfunction and Oxidative Damage in Rats
Atish Prakash and Anil Kumar
Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
(Received 13 October 2008; Accepted 4 January 2009)

Abstract: Aluminium is a potent neurotoxin involved in the initiation and progression of various cognitive disorders like
Alzheimer's disease. Chronic aluminium exposure induces oxidative stress and increases amyloid beta levels in vivo. The
role of oxidative stress has been well-suggested in these cognitive problems. Therefore, the present study was designed to
explore the possible role of N-acetyl cysteine against aluminium mediating cognitive dysfunction and oxidative stress in
rats. Aluminium chloride (100 mg/kg, p.o.) was given to rats daily for 6 weeks. N-acetyl cysteine (per se; 50 and 100 mg/kg,
i.p.) pre-treatment was given 30 min. before aluminium daily for 6 weeks. On the third (21st day) and sixth week (42nd day)
of the study, various behavioural tests (Morris water maze and elevated plus maze task paradigms) and locomotion
(photoactometer) were done to evaluate cognitive tasks. The rats were killed on the 43rd day following the last behavioural
test, and various biochemical tests were performed to assess the extent of oxidative damage. Chronic aluminium chloride
administration resulted in poor retention of memory in Morris water maze, elevated plus maze task paradigms and caused
marked oxidative damage. It also caused a significant increase in the acetylcholinesterase activity. Chronic administration
of N-acetyl cysteine significantly improved memory retention in tasks, attenuated oxidative damage and acetylcholinesterase
activity in aluminium-treated rats. The study suggests a neuroprotective effect of N-acetyl cysteine against aluminium-
induced cognitive dysfunction and oxidative damage.

Increasing evidence has indicated that excessive aluminium
in selective regions of the brain generates cytotoxic free
radicals formation and thereby implicates in the aetiology
of several neurodegenerative disorders [1]. Experimentally,
it has been demonstrated that chronic aluminium exposure
causes neurological signs and results in intraneuronal neuro-
filamentous aggregation of proteins akin to neurofibrillary
tangles (in the hippocampus, cerebral cortex, brain stem,
spinal cord) and biochemical changes as seen in patients with
cognitive disorders [1]. Aluminium has easy access to the
brain via the specific high affinity receptors for transferrin
expressed in the blood–brain barrier. Upon entering the brain
through the blood–brain barrier, aluminium can interact with
various enzyme pathways of brain. Aluminium is a non-redox
active metal which is capable of increasing the cellular
oxidative milieu by potentiating pro-oxidant properties of
transition metals such as iron and copper [2]. Aluminium
causes progressive deterioration of mitochondrial functions
due to excessive free radical generation. This further damages
other cellular molecules including deoxy ribonucleic acid
damage, nitration of protein residues and lipid peroxidation
[3]. A report has demonstrated that aluminium supplemen-
tation causes an increase in acetylcholinestrase level in the
brain [4]. Aluminium is a potent cholinotoxin [5] and causes
apoptotic neuronal loss which is a characteristic symptom of
Author for correspondence: Anil Kumar, Pharmacology division,
University Institute of Pharmaceutical Sciences, Panjab University,
Chandigarh 160014, India (fax + 91 172 2534114, e-mail kumaruips@
yahoo.com).
Alzheimer's disease [6]. At the molecular level, it influences
DNA topology, gene transcription [7] and cellular energy
metabolism. It induces misfolding and self-aggregation of
highly phosphorylated cytoskeleton proteins such as neuro-
filaments or microtubule-associated proteins and Ab which
are implicated in Alzheimer's disease [8,9].
Several compounds with antioxidant properties are used for
therapy of neurodegenerative disorders including cognitive
disorder. N-acetyl cysteine is precursor of glutathione that
plays an essential role in oxidative damage. Its antioxidant
properties have recently been reviewed [10]. In particular,
N-acetyl cysteine is known to increase the intracellular
stores of glutathione thereby enhancing the endogenous
antioxidant level. It is well-reported that the thiol group in
N-acetyl cysteine interacts directly with reactive oxygen
species leading to cellular protection against oxidative
damage in vivo and in vitro. [11]. N-acetyl cysteine is freely
filterable with a ready access to the blood–brain barrier and
intracellular compartment [12]. N-acetyl cysteine has been
shown to attenuate neuroinflammation in various disease
models such as ischaemia-reperfusion injury [13,14], lethal
endotoxemia [15], multiple sclerosis [16,17] and hypoxic
ischaemic brain injury in neonatal brains [18,19]. In addition,
N-acetyl cysteine could prevent apoptotic cell death of cultured
neuronal cells [20,21] and promote survival of PC12 cells as
a neuroprotective, due to its ability to scavenge free radicals
[22]. Based on this, the present study was designed to investi-
gate the neuroprotective effect of N-acetyl cysteine against
aluminium-induced cognitive impairment and associated
oxidative damage in rats.
 N-ACETYL CYSTEINE IN NEUROBEHAVIOURAL ALTERATION
Materials and Methods
Animals. Young male Wistar rats (Central Animal House, Panjab
University, Chandigarh) weighing 180–200 g at the start of the study
were used. Animals were acclimatized to the laboratory conditions
at room temperature before the experiments. Animals were kept
under standard conditions of a 12-hr light/dark cycle with food and
water ad libitum in plastic cages with soft bedding. All manipu-
lations were carried out in the light phase between 09.00 a.m. and
17.00 p.m. The protocol was approved by the Institutional Animal
Ethics Committee and was carried out in accordance with the
Indian National Science Academy Guidelines for the use and care
of animals.
Drugs and treatment schedule. Aluminium chloride (CDH, India)
and N-acetyl cysteine (Sigma Chemicals Co., St Louis, MO, USA)
solutions were made freshly at the beginning of each experiment.
For oral administration, aluminium chloride was dissolved in drinking
water and N-acetyl cysteine was dissolved in 0.5% carboxymethyl
cellulose and administered in a dose of 0.5 ml/100 g body weight.
Animals were randomized into six groups based on their body
weight:
Group I (control group): Rats were administered distilled water
for 6 weeks.
Group II (aluminium-treated group): Rats were administered
aluminium chloride (100 mg/kg) for 6 weeks through oral gavages.
Group III (low-dose N-acetyl cysteine group): Animals were
administered intraperitoneally with 50 mg/kg daily for 6 weeks.
Group IV N-acetyl cysteine (high-dose N-acetyl cysteine group):
Rats were administered intraperitoneally with 100 mg/kg daily for
6 weeks.
Group V (aluminium + low dose N-acetyl cysteine group): N-acetyl
cysteine was administered with 50 mg/kg intraperitoneally 1 hr before
aluminium chloride administration for 6 weeks.
Group VI (aluminium + high dose of N-acetyl cysteine group):
N-acetyl cysteine was administered with 100 mg/kg intraperito-
neally 1 hr before aluminium chloride administration for 6 weeks.
The doses of N-acetyl cysteine and aluminium chloride were
selected on the basis of those reported in the literature [23,24]. The
study lasted 42 days (6 weeks).
Behavioural assessments.
Assessment of cognitive performance.
Spatial navigation task. The acquisition and retention of a spatial
navigation task was evaluated by Morris water maze [25]. Animals
were trained to swim to a visible platform in a circular pool (180 cm
in diameter and 60 cm in height) located in a test room. In principle,
rats can escape from swimming by climbing onto the platform and
over time the rats apparently learn the spatial location of the platform
from any starting position at the circumference of the pool. The
pool was filled with water (28 € 2) to a height of 40 cm and movable
circular platform (9 cm diameter), mounted on a column was placed
in a pool 2 cm above the water level during the acquisition phase. A
similar platform was placed in the pool 2 cm below the water level
for the maze retention phase. During both phases, the platform was
placed in the centre of one of the quadrants. The water was made
opaque by adding a non-toxic dye. Four equally spaced locations
around the edge of the pool (N, S, E and W) were used as starting
points and this divided the pool into four equal quadrants.
1. Maze acquisition phase (training). Animals received a training
session consisting of four trials on day 20. In all four trials, the
starting position was different. A trial began by releasing the
animal into the maze facing the wall of the pool. The latency to
find the escape platform was recorded to a maximum of 90 sec. If
the rat did not escape onto the platform within this time, it was
guided to the platform and was allowed to remain there for 20 sec.
The time taken by the rat to reach the platform was considered the
initial acquisition latency. At the end of the trial, the rats were
 2009 The Authors
Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104
99
returned to their home cages and a 5-min. gap was given between
the subsequent trials.
2. Maze retention phase (testing for retention of the learned
task). Following 24 hr, retention was assessed on 21st and 42nd
day. The rat was released randomly at one of the edges facing the
wall of the pool and tested for retention of response. The time
taken to find the hidden platform on days 21 and 42 following start
of aluminium chloride administration was recorded and termed as
first retention latency (1st RL) and second retention latency (2nd
RL), respectively.
Elevated plus maze paradigm. The elevated plus maze consisted of
two opposite black open arms (50 · 10 cm), crossed with two closed
walls of the same dimensions with 40 cm high walls. The arms were
connected with a central square of dimensions 10 · 10 cm and the
entire maze was placed 50 cm above the ground. Acquisition of
memory was tested on day 20 from the start of aluminium chloride
administration. Rats were placed individually at one end of the
open arm facing away from the central square. The time taken by
the animal to move from the open arm to the closed arm was
recorded as the initial transfer latency. Animals were allowed to
explore the maze for 20 sec. after recording the initial transfer
latency and were then returned to the home cages. If the animal did
not enter the enclosed arm within 90 sec., it was pushed on the
back into one of the enclosed arms and the initial transfer latency
was recorded as 90 sec. Retention of memory was assessed by
placing the rat in an open arm and the retention latency was noted
on days 21 and 42 of the initial transfer latency and was termed as
the first retention transfer latency (1st RTL) and second retention
transfer latency (2nd RTL), respectively [25].
Assessment of gross behavioural activity. Gross behavioural activity
was observed at the end of each week for a total of 6 weeks since
the initiation of aluminium chloride treatment. Each animal was
placed in a square (30 cm) closed arena equipped with infra-red
light-sensitive photocells using digital photoactometer. The animals
were observed for 5 min. and the values were expressed as counts/
5 min. The apparatus was placed in a darkened, light and sound-
attenuated and ventilated test room [26].
Biochemical assessment. Biochemical tests were conducted 24 hr after
the last behavioural test. The animals were killed by decapitation.
Brains were removed and rinsed with ice-cold isotonic saline. Brains
were then homogenised with ice-cold 0.1 mmol/l phosphate buffer
(pH 7.4). The homogenate (10% w/v) was then centrifuged at
10,000 ·g for 15 min. and the supernatant so formed was used for
the biochemical estimations.
Measurement of lipid peroxidation. The extent of lipid peroxidation
in the brain was determined quantitatively by performing the method
by Wills [27]. The amount of thio barbituric acid reactive substances
(TBARS) was measured by reaction with thiobarbituric acid at
532 nm using Perkin Elmer Lambda 20 spectrophotometer. The
values were calculated using the molar extinction co-efficient of
chromophore (1.56 · 105 (mol/l))1 cm)1).
Estimation of nitrite. The accumulation of nitrite in the supernatant,
an indicator of the production of nitric oxide, was determined by a
colorimetric assay with Greiss reagent (0.1% N-(1-napththyl) ethylene
diamine dihydrochloride, 1% sulphanilamide and 5% phosphoric
acid.) [28]. Equal volumes of the supernatant and the Greiss reagent
were mixed and the mixture was incubated for 10 min. at room
temperature in the dark. The absorbance was measured at 540 nm
using Perkin Elmer Lambda 20 spectrophotometer. The concen-
tration of nitrite in the supernatant was determined from sodium
nitrite standard curve.
Estimation of reduced glutathione. Reduced glutathione was estimated
according to the method described by Ellman [29]. One millilitre of
100 ATISH PRAKASH AND ANIL KUMAR
Table 1.
Effect of N-acetyl cysteine on spatial navigation task in aluminium chloride-treated rats.
Treatment (mg/kg) IAL
Control 45.2 € 1.42
AlCl3 (100) 110.0 € 2.5*
NAC (50) 61.33 € 1.51
NAC (100) 58.5 € 1.077
NAC (50) + AlCl3 69.6 € 2.470†
NAC (100) + AlCl3 63.4 € 1.438†‡
NAC ¼ N-acetyl cysteine, AlCl3 ¼ aluminium chloride
The initial acquisition latencies (IAL) on day 20 and retention latencies on days 21 (1st RL) and 42 (2nd RL) following aluminium chloride
treatment were observed in Morris water maze. Values are mean € S.E.M. *P < 0.05 as compared to control group; †P < 0.05 as compared to
AlCl3-treated group; ‡P < 0.05 as compared to N-acetyl cysteine (50) + AlCl3 group; (Repeated measures two-way anova followed by Tukey's
test for multiple comparisons).
supernatant was precipitated with 1 ml of 4% sulphosalicylic acid
and cold-digested for 1 hr at 4. The samples were then centrifuged at
1200 ·g for 15 min. at 4. To 1 ml of the supernatant obtained,
2.7 ml of phosphate buffer (0.1 mmol/l, pH 8) and 0.2 ml of 5, 5’
dithio-bis (2-nitrobenzoic acid) was added. The yellow colour the
developed was measured at 412 nm using Perkin Elmer Lambda 20
spectrophotometer. Results were calculated using the molar extinction
coefficient of the chromophore (1.36 · 104 (mol/l))1 cm)1).
Estimation of antioxidant enzyme activities.
Superoxide dismutase activity. Superoxide dismutase activity was
assayed by the method of Kono [30]. The assay system consisted of
EDTA 0.1 mM, sodium carbonate 50 and 96 mM of nitro blue
tetrazolium. In the cuvette, 2 ml of the above mixture, 0.05 ml of
hydroxylamine and 0.05 ml of the supernatant was added and the
auto-oxidation of hydroxylamine was measured for 2 min. at 30-sec.
intervals by measuring the absorbance at 560 nm using Perkin
Elmer Lambda 20 spectrophotometer.
Catalase activity. Catalase activity was assessed by the method of
Luck [31], wherein the breakdown of hydrogen peroxide is measured.
Briefly, the assay mixture consisted of 3 ml H2O2 phosphate buffer
and 0.05 ml supernatant of the tissue homogenate. The change in
absorbance was recorded for 2 min. at 30-sec. intervals at 240 nm
using Perkin Elmer Lambda 20 spectrophotometer. The results
were expressed as micromoles of H2O2 decomposed per min. per mg
protein.
Estimation of acetylcholinesterase activity. Acetylcholinesterase is a
marker of extensive loss of cholinergic neurons in the forebrain. The
acetylcholinesterase activity was assessed by the Ellman method
[32]. The assay mixture contained 0.05 ml supernatant, 3 ml sodium
phosphate buffer (pH 8), 0.1 ml acetylthiocholine iodide and 0.1 ml
5, 5’ dithio-bis (2-nitrobenzoic acid) (Ellman reagent). The change in
absorbance was measured for 2 min. at 30 sec. intervals at 412 nm
using Perkin Elmer Lambda 20 spectrophotometer. Results were
expressed as micromoles of acetylthiocholine iodide hydrolyzed per
min. per mg of protein.
Protein estimation. The protein content was estimated by the Biuret
method [33] using bovine serum albumin as standard.
Statistical analysis. Values are expressed as mean € S.E.M. The
behavioural assessment data were analysed by a repeated measures
two-way ANOVA with drug-treated groups as between and sessions as
the within-subjects factors. The biochemical estimations were separately
 2009 The Authors
Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104
Mean latency (in sec.)
1st RL 2nd RL
18.5 € 1.2 12.2 € 2.16
84.0 € 2.2* 72.5 € 2.5*
13.83 € 1.945 11.16 € 2.16
12 € 1.09 9.66 € 1.421
39.65 € 1.577† 32.15 € 0.475†
24.0 € 0.569†‡ 20.5 € 1.576†‡
analysed by two-way ANOVA. Post hoc comparisons between groups
were made using Tukey's test. P < 0.05 was considered significant.
Results
Effect of N-acetyl cysteine on memory performance in spatial
navigation task paradigm in aluminium chloride-treated rats.
In the spatial navigation task, the control and N-acetyl cysteine
(50 and 100 mg/kg, per se) group of animals quickly learned
to swim directly to the platform in the Morris water maze
on day 21. Aluminium chloride-treated rats showed an initial
increase in escape latency, which declined with continued
training during the acquisition of a spatial navigation task
on day 20. There was a significant difference in the mean
initial acquisition latency of the aluminium chloride-treated
group when compared to the control group on day 20
indicating that chronic administration of aluminium chloride
impaired acquisition of spatial navigation task (P < 0.05). In
contrast, concomitant administration of N-acetyl cysteine
(50 and 100 mg/kg, i.p.) with aluminium chloride significantly
decreased the initial acquisition latency to reach the platform
in the pre-trained rats as compared to aluminium chloride-
treated rats on day 20 (table 1). Following training, the mean
retention latencies (1st and 2nd RL) to escape onto the
hidden platform were significantly decreased in the control
group on days 21 and 42, respectively, as compared to initial
acquisition latency on day 20 since the initiation of aluminium
chloride treatment. On the contrary, the performance in the
aluminium chloride-treated rats had changed after initial
training in the water maze on days 21 and 42, with significant
increase in mean retention latencies compared to retention
latencies on days 21 and 42 of the control group. The results
suggest that aluminium chloride caused significant cognitive
impairment. However, chronic N-acetyl cysteine treatment
(50 and 100 mg/kg, i.p.) in aluminium chloride-treated rats
showed a significant decline in the 1st and 2nd retention
latencies as compared to aluminium chloride-treated rats on
days 21 and 42, respectively (table 1), and improved the
retention performance of the spatial navigation task.
 N-ACETYL CYSTEINE IN NEUROBEHAVIOURAL ALTERATION
Table 2.
Effect of N-acetyl cysteine on memory performance in elevated plus
maze paradigm in aluminium chloride-treated rats.
Mean Transfer Latency (in sec.)
Treatment (mg/kg) ITL 1st RTL 2nd RTL
Control 56.5 € 1.5 22.5 € 2.2 15 € 2.5
AlCl3 (100) 68.25 € 1.5* 79.5 € 1.33* 75.25 € 1.20*
NAC (50) 62.5 € 1.5 20.5 € 0.95 16.25 € 1.5
NAC (100) 58 € 1.5 17.5 € 1.7 10.5 € 0.84
NAC (50) + AlCl3 64.5 € 2.25 45.5 € 1.83† 38.25 € 0.5†
NAC (100) + AlCl3 63 € 1.23 30.5 € 1.85†‡ 24.25 € 1.3†‡
NAC ¼ N-acetyl cysteine, AlCl3 ¼ aluminium chloride
The initial transfer latencies (ITL) on day 20 and retention transfer
latencies on days 21 (1st RTL) and 42 (2nd RTL) following aluminium
chloride treatment were observed. Values are mean € S.E.M. *P < 0.05
as compared to control group; †P < 0.05 as compared to aluminium
chloride-treated group; ‡P < 0.05 as compared to N-acetyl cysteine
(50) + AlCl3 group; (Repeated measures two-way anova followed
by Tukey's test for multiple comparisons).
Effect of N-acetyl cysteine on memory performance in
elevated plus maze task paradigm in aluminium
chloride-treated rats.
In the elevated plus maze task, mean initial transfer latency on
day 20 for each rat was relatively stable and showed no
significant variation. All the rats entered the closed arm within
90 sec. Following training, control and N-acetyl cysteine-
treated (50 and 100 mg/kg, i.p.) rats entered the closed arm
quickly and mean retention transfer latencies (1st RTL and
2nd RTL) to enter the closed arm on days 21 and 42 were
shorter as compared to initial transfer latency on day 20
of each group, respectively. In contrast, aluminium chloride-
treated rats performed poorly throughout the experiment
and did not show any change in the mean retention transfer
latencies on day 21 s and 42 as compared to pre-training
latency on day 20, demonstrating that chronic aluminium
chloride administration induced marked memory impair-
ment. Chronic administration of N-acetyl cysteine (50 and
100 mg/kg, i.p.) following aluminium chloride adminis-
tration significantly decreased the mean retention latencies
on days 21 and 42 (P < 0.05 versus the aluminium chloride-
treated group) (table 2). The mean transfer latencies of
N-acetyl cysteine (50 and 100 mg/kg, i.p.) + aluminium
chloride-treated groups were significantly different from
that of N-acetyl cysteine per se groups on days 21 and 42
(P < 0.05) (table 2).
Effect of N-acetyl cysteine on locomotor activity in
aluminium chloride-treated rats.
In the present series of experiments, the mean scores of locomotor
activity for each rat were relatively stable and showed no sig-
nificant variation. The mean scores in control and aluminium
chloride-treated rats remained unchanged. Chronic adminis-
tration of N-acetyl cysteine (50 and 100 mg/kg, per se, i.p.)
had no effect on locomotor activity as compared to control
rats throughout the study period (fig. 1). Furthermore, N-acetyl
 2009 The Authors
Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104
101
Fig. 1. Effect of N-acetyl cysteine on locomotor activity in aluminium
chloride-treated rats. Values are mean € S.E.M. Data were analysed
by two-way anova. NAC ¼ N-acetyl cysteine, AlCl3 ¼ aluminium
chloride.
cysteine (50 and 100 mg/kg, i.p.) pre-treatment in aluminium
chloride-treated rats did not cause any alterations in the
locomotor activity as compared to aluminium chloride-
treated rats (fig. 1).
Effect of N-acetyl cysteine on brain lipid peroxidation, nitrite
and reduced glutathione levels in aluminium chloride-treated
rats.
Chronic administration of aluminium chloride caused a
marked increase in TBARS levels, nitrite concentration
and depletion of reduced glutathione levels as compared to
control rats (P < 0.05). Furthermore, there were no alteration
in TBARS levels, nitrite concentration and reduced glutathione
levels in N-acetyl cysteine (50 and 100 mg/kg, i.p.) per se
treatment as compared to control rats. However, chronic
N-acetyl cysteine (50 and 100 mg/kg, i.p.) administration to
aluminium chloride-treated rats significantly prevented the
increase in TBARS, nitrite concentration and depletion of
reduced glutathione (table 3).
Effect of N-acetyl cysteine on brain antioxidant enzyme
activities in aluminium chloride-treated rats.
Chronic administration of aluminium chloride caused sig-
nificant decrease in the antioxidant enzyme activities namely
superoxide dismutase and catalase as compared to control rats
(P < 0.05). Furthermore, N-acetyl cysteine (50 and 100 mg/kg,
per se, i.p.) treatment did not cause any significant alteration
in the superoxide dismutase and catalase activities when
compared to control rats. However, concomitant chronic
N-acetyl cysteine (50 and 100 mg/kg, i.p.) administration
to aluminium chloride-treated rats caused a significant
increase in the levels of superoxide dismutase and catalase
activities (table 3).
Effect of N-acetyl cysteine on brain acetylcholinesterase
activity in aluminium chloride-treated rats.
Chronic aluminium chloride administration in rats showed sig-
nificant increase in the brain regarding acetylcholinesterase
activity as compared to control rats. However, chronic N-
acetyl cysteine (50 and 100 mg/kg, i.p.) treatment significantly
attenuated acetylcholinesterase activity as compared to the
aluminium chloride-treated group (P < 0.05) (fig. 2).
102 ATISH PRAKASH AND ANIL KUMAR

Table 3.
Effect of N-acetyl cysteine on aluminium chloride-induced oxidative stress parameters in rat brain.

TBARS levels Nitrite levels Reduced-glutathion Catalase (lmol of H2O2 SOD

(nmol MDA/mg protein) (lmol/mg protein) (nmol/mg protein) decomposed/min/mg (Units/mg protein)
Treatment (% control) (% control) (% control) protein) (% control) (% control)
Control 0.174 € 0.010 264 € 12 0.65 € 0.01 6.75 € 0.012 0.72 € 0.14
(100) (100) (100) (100) (100)
AlCl3 (100) 0.589 € 0.021* 606.32 € 12* 0.15 € 0.06* 1.1 € 0.12* 0.096 € 0.05*
(339) (229.67) (23.37) (16.43) (13.45)
NAC (50) 0.2 € 0.016 288.42 € 18 0.61 € 0.01 5.83 € 0.13 0.6 € 0.15
(115.2) (109.25) (95.35) (86.5) (84.25)
NAC (100) 0.187 € 0.012 262.68 € 8 0.7 € 0.015 6.1 € 0.14 0.66 € 0.16
(107.5) (99.5) (108.25) (90.45) (92.5)
NAC (50) + AlCl3 0.391 € 0.027† 475.86 € 16† 0.44 € 0.05† 3.72 € 0.08† 0.27 € 0.12†
(224.81) (180.25) (68) (55.25) (38.65)
NAC (100) + AlCl3 0.299 € 0.016†‡ 391.9 € 12†‡ 0.53 € 0.09†‡ 4.89 € 0.12†‡ 0.49 € 0.14†‡
(172.26) (148.45) (82.45) (72.5) (68.5)
NAC ¼ N-acetyl cysteine, AlCl3 ¼ aluminium chloride.
Values are mean € S.E.M. *P < 0.05 as compared to control group; †P < 0.05 as compared to AlCl3-treated group; ‡P < 0.05 as compared to
N-acetyl cysteine (50) + AlCl3 group; (Repeated measures two-way anova followed by Tukey's test for multiple comparisons).

Discussion The results of our study indicate that chronic administration

Aluminium is a ubiquitous metal that has been implicated
in the aetiology of neurodegenerative disorders where it
exacerbates brain oxidative damage [23], neuroinflammation
and Ab deposition. Alzheimer's disease is characterized
by impairment in working memory [34], visuoperception,
attention and semantic memory. A decrease in escape latency
time during ongoing acquisition trial denotes normal acqui-
sition of memory, and marked decrease in retention latency
indicates retrieval of memory in control rats in both test
paradigms (Morris water maze and elevated plus maze task).
Fig. 2. Effect of N-acetyl cysteine on acetyl cholinesterase activity in
aluminium chloride-treated rats. Values are mean € S.E.M. aP < 0.05
as compared to control group; bP < 0.05 as compared to the aluminium
chloride-treated group (Repeated measures two-way anova followed by
Turkey's test for multiple comparisons). NAC ¼ N-acetyl cysteine,
AlCl3 ¼ aluminium chloride.
of aluminium chloride results in progressive deterioration of
spatial memory in both Morris water maze and elevated
plus maze task paradigms as demonstrated by the increase
in retention latency as compared to the control group. Exper-
imentally, it has been shown that intracerebral adminis-
tration of aluminium chloride causes learning deficits in
Morris water maze task in rabbits [35] which is in concordance
with our findings. Chronic aluminium treatment leads to
impairment of glutamate–NO–cGMP pathway in the cere-
bellum of rats [36], which explains the memory impairment
and neurobehavioural deficits. Chronic administration of
N-acetyl cysteine was able to reverse the cognitive deficit,
suggesting its potential role as a neuroprotectant against
aluminium-induced neurotoxicity.
Aluminium causes marked oxidative damage by increasing
the redox active iron concentration in the brain mainly via
the Fenton reaction [37]. In our study, chronic administration
of aluminium chloride resulted in marked oxidative stress as
indicated by an increase in lipid peroxidation, nitrite levels,
and decrease in reduced glutathione levels, catalase and
superoxide dismutase level. This could be due to the reduced
axonal mitochondria turnover, disruption of Golgi and reduction
of synaptic vesicles induced by aluminium treatment which
results in release of oxidative products like malondialdehyde,
carbonyls, peroxynitrites and enzymes like superoxide dis-
mutase within the neurons [1].
In the present study, N-acetyl cysteine alone has no sig-
nificant effect on the markers of oxidative stress in the brain of
normal animals; however, it significantly attenuated the
aluminium chloride-induced oxidative damage. The beneficial
effect of N-acetyl cysteine administration on oxidative damage
is related to its activity as a direct and potent, free radical
scavenger. First, N-acetyl cysteine enhances the levels of
endogenous glutathione by increasing intracellular cysteine
and subsequently potentiates the natural anti-oxidative

 2009 The Authors

Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104
 N-ACETYL CYSTEINE IN NEUROBEHAVIOURAL ALTERATION
cellular defence mechanism [38]. There is another culture
study that supports that inhibition of cysteine uptake causing a
decrease of cellular glutathione and accumulation of cellular
oxidant leads to cell death [39]. More recent data by Aguir
et al. have addressed that N-acetyl cysteine treatment after
exercise-induced oxidative damage was effective in lipid
peroxidation, carbonyl compound and antioxidant enzyme
activity. In this study, we found that N-acetyl cysteine adminis-
tration following aluminium chloride toxicity could reduce
oxidative damage by increasing the reduced glutathione level,
catalase and superoxide dismutase level also, by reducing
lipid peroxidation and nitrate concentration. In our study,
N-acetyl cysteine attenuated behavioural alterations in
aluminium chloride-treated rats by decreasing the retention
latency to escape to the platform in Morris water maze. The
observed protective effect of N-acetyl cysteine in prevention
treatment might be associated with the total glutathione
that could block the cytotoxicity activity by reactive oxygen
species [40]. N-acetyl cysteine can act by direct reaction
between its reducing thiol group and reactive oxygen species.
However, in our study, we found that N-acetyl cysteine
alone exerts a mild effect in oxidative stress. The oxidative
metabolism of N-acetyl cysteine can generate thiol-free
radicals that have been increasingly considered as intermediates
in the process that may be involved in the development of
biological damage resulting from oxidative stress. N-acetyl
cysteine increases hydroxyl radical generation in a system
with Fe (III)-citrate and H2O2 in vitro [41].
AD affects mainly the cholinergic system resulting in increased
activity of acetylcholinesterase [42] and choline acetyl trans-
ferase [43]. Experimentally, aluminium has been shown to
increase acetylcholinesterase in mouse brain [44]. In fact, it
causes a biphasic effect on the acetylcholinesterase activity,
with an initial increase in the activity of this enzyme during
4–14 days of exposure followed by a marked decrease. This
has been attributed to the slow accumulation of aluminium
in the brain [45]. This also explains the fact that aluminium
chloride treatment caused a significant increase in the
acetylcholinesterase activity which was attenuated by chronic
N-acetyl cysteine treatment. Sun et al. 2006 showed that
N-acetyl cysteine attenuated the increase in acetylcholine
esterase and decrease in choline acetyltransferase activity,
acetyl choline level in b-amyloid toxicity rats. This lends
support to our findings that N-acetyl cysteine caused a
decrease in the increase in acetylcholine esterase levels in
the aluminium toxicity rats. In conclusion, the present
study suggests the therapeutic potential of N-acetyl cysteine
against aluminium-induced behavioural alterations and
oxidative damage. It provides a hope that N-acetyl cysteine
could be used as an effective agent in the management of
cognitive and related disorders such as Alzheimer's disease.
Acknowledgement
The authors are thankful for the financial support from
the Indian Council of Medical Research (ICMR), New Delhi
and University Institute of Pharmaceutical Sciences for pro-
viding infrastructure facilities to carry out this study.
 2009 The Authors
Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104
103
References
1 Bharathi P, Shamasundar NM, Sathyanarayana Rao TS,
Naidu MD, Ravid R, Rao KSJ. A new insight on Al-maltolate-
treated aged rabbits as Alzheimer's animal model. Brain Res
Rev 2006;52:275–92.
2 Bjertness E, Candy JM, Torvik A, Ince P, McArthur F, Taylor GA
et al. Content of brain aluminium is not elevated in Alzheimer's
disease. Alzheimer Dis Assoc Disord 1996;10:171–4.
3 Becaria A, Bondy SC, Campbell A. Aluminium and copper
interact in the promotion of oxidative but not inflammatory events:
implications for Alzheimer's disease. J Alz Dis 2003;5:31–8.
4 Ravi SM, Prabhu BM, Raju TR, Bindu PN. Long term effects of
post natal aluminium exposure on acetylcholinesterase activity
and biogenic amine neurotransmitters in rat brain. Indian J
Physiol Pharmacol 2000;44:473–8.
5 Gulya K, Rakonczay Z, Kasa P. Cholinotoxic effects of alumin-
ium in rat brain. J Neurochem 1990;54:1020–6.
6 Ghribi O, Dewitt DA, Forbes MS, Herman MM, Savory J.
Co-involvement of mitochondria and endoplasmic reticulum in
regulation of apoptosis: changes in cytochrome-c, Bcl-2 and Bax
in the hippocampus of aluminium treated rabbits. Brain
Res 2001;8:66–73.
7 Lukiw WJ, LeBlanc HJ, Carver LA, McLachlan DR, Bazan
NG. Run on gene transcription in human neocortical nuclei.
Inhibition by nanomolar aluminium and implications for
neurodegenerative diseases. J Mol Neurosci 1998;11:67–78.
8 Kawahara M, Muramoto K, Kobayashi K, Kuroda Y. Functional
and morphological changes in cultured neurons of rat cerebral
cortex induced by long-term application of aluminium. Biochem
Biophys Res Commun 1992;189:1317–22.
9 Kawahara M, Muramoto K, Kobayashi K, Mori H, Kuroda Y.
Aluminium promotes the aggregation of alzheimer's b-
amyloid protein in vitro. Biochem Biophys Res Commun
1994;198:531–5.
10 Harvey BH, Joubert C, Preez JL, Berk M. Effect of chronic N-
Acetyl cysteine administration on oxidative status in the presence
and absence of induced oxidative stress in rat striatum. Neuro-
chem Res 2008;33:508–17.
11 Aruoma OI, Halliewell B, Hoey BM, Butler J. The antioxidant
action of N-acetyl cysteine: its reaction with hydrogen peroxide,
hydroxyl radical, superoxide, and hypochlorous acid. Free Radic
Biol Med 1989;6:593–7.
12 Farr SA, Poon HF, Dogrukol-Ak D, Drake J, Banks WA,
Eyerman E et al. The antioxidants alpha-lipoic acid and N-
acetylcysteine reverse memory impairment and brain oxidative
stress in aged SAMP8 mice. J Neurochem 2003;84:1173–83.
13 Sekhon B, Sekhon C, Khan M, Patel SJ, Singh I, Singh AK.
N-acetyl cysteine protects against injury in a rat model of focal
cerebral ischemia. Brain Res 2003;971:1–8.
14 Khan M, Sekhon B, Jatana M, Giri S, Gilg AG, Sekhon C
et al. Administration of N-acetylcysteine after focal cerebral
ischemia protects brain and reduces inflammation in a rat
model of experimental stroke. J Neurosci Res 2004;76:519–27.
15 Victor VM, Rocha M, De la Fuente M. N-acetylcysteine
protects mice from lethal endotoxemia by regulating the redox
state of immune cells. Free Radic Res 2003;37:919–29.
16 Lehmann D, Karussis D, Misrachi-Koll R, Shezen E, Ovadia H,
Abramsky O. Oral administration of the oxidant-scavenger
N-acetyl-L-cysteine inhibits acute experimental autoimmune
encephalomyelitis. J Neuroimmunol 1994;50:35–42.
17 Stanislaus R, Gilg AG, Singh AK, Singh I. N-acetyl-L-cysteine
ameliorates the inflammatory disease process in experimental
autoimmune encephalomyelitis in Lewis rats. J Autoimmune
Dis 2005;2:4–12.
18 Jatana M, Singh I, Singh AK, Jenkins D. Combination of
systemic hypothermia and N-acetylcysteine attenuates
104 ATISH PRAKASH AND ANIL KUMAR
hypoxic-ischemic brain injury in neonatal rats. Pediatr Res
2006;59:684–9.
19 Wang X, Svedin P, Nie C, Lapatto R, Zhu C, Gustavsson M
et al. N-acetylcysteine reduces lipopolysaccharide-sensitized
hypoxic-ischemic brain injury. Ann Neurol 2007;61:263–71.
20 Ferriari G, Ran CI, Greene LA. Acetylcysteine prevent apoptotic
death of neuronal cells. J Neurosci 1995;15:2857–66.
21 Olivieri G, Baysang G, Meier F, Muller-Spahn F, Staneli HB,
Brockhaus MN. N-acetyl-L-cysteine protects SHSY5Y neuroblas-
toma cells from oxidative stress and cell cytotoxicity: effects on
beta-amyloid secretion and tau phosphorylation. J Neurochem
2001;76:224–33.
22 Yan CI, Ferrari G, Greene LA. N-Acetylcysteine promoted
survival of PC12 cells is glutathione independent but transcription
dependent. J Biol Chem 1995;270:26827–32.
23 Nehru B, Bhalla P, Garg A. Further evidence of centrophenoxine
mediated protection in aluminium exposed rats by biochemical
and light microscopy analysis. Food Chem Toxicol 2007;45:2499–
505
24 Fu AL, Dong JH, Sun MJ. Protective effect of N-acetyl-L-cysteine
on amyloid b-peptide-induced learning and memory deficits in
mice. Brain Res 2006;109:201–6.
25 Kumar A, Seghal N, Padi SV, Naidu PS. Differential effects of
cyclooxygenase inhibitors on intracerebroventricular colchicine-
induced dysfunction and oxidative stress in rats. Eur J Pharmacol
2006;551:58–66.
26 Reddy DS, Kulkarni SK. Possible role of nitric oxide in the
nootropic and antiamnesic effects of neurosteroids an aging
and dizocilpine – induced learning impairment. Brain Res
1998;799:215–29.
27 Wills E D. Mechanism of lipid peroxide formation in animal
tissues. Biochem J 1966;99:667–76.
28 Green LC, Wagner DA, Glagowski J. Analysis of nitrate, nitrite
and (15 N) nitrate in biological fluids. J Biol Chem 1982;193:265–75.
29 Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys
1959;82:48670–7.
30 Kono Y. Generation of Superoxide radical during auto-oxidation
of hydroxylamine and an assay for superoxide dismutase. Arch
Biochem Biophys 1978;186:189–95.
31 Luck H. Catalase. In: Bergmeyer HU (ed.). Methods of Enzymatic
analysis. Academic Press, New York. 1978;885–93.
32 Ellman GL, Courtney KD, Andres V, Featherstone RM. A new
 2009 The Authors
Journal compilation  2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 105, 98–104
and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol 1961;7:88–95.
33 Gornall AG, Bardawill CT, David MM. Determination of
serum proteins by means of Biuret reaction. J Biol Chem
1949;177:751–66.
34 Germano C, Kinsella GJ. Working memory and learning in early
Alzheimer's disease. Neuropsychol Rev 2005;15:1–10.
35 Rabe A, Moon HL, Shek J, Wisniewski HM. Learning deficit in
immature rabbits with aluminium induced neurofibrillary changes.
Exp Neurol 1982;76:441–6.
36 Canales JJ, Corbaln R, Montoliu C, Llansola M, Monfort P,
Erceg S et al. Aluminium impairs the glutamate-nitric oxide-
cGMP pathway in cultured neurons and in rat brain in vivo:
molecular mechanisms and implications for neuropathology. J
Inorgan Biochem 2001;87:63–9.
37 Pratico D, Uryu K, Sung S, Tang S, Trojanowski JQ, Lee VMY.
Aluminium modulates brain amyloidosis through oxidative stress
in APP transgenic mice. FASEB J 2002;16:1138–40.
38 Pocernich CB, La Fontaine M. In-vivo glutathione elevation
protects against hydroxyl free radical-induced protein oxidation in
at brain. Neurochem Int 2000;36:185–91.
39 Murphy TH, Schnaar RL, Coyle JT, Immature cortical
neurons are uniquely sensitive to glutamate toxicity by inhibition
of cysteine uptake, FASEBJ 1990;4:1624–38.
40 Froissard P, Monrocq H, Duval D. Role of glutathione
metabolism in the glutamateinduced programmed cell death of
neuronal-like PC12 cells. Eur J Pharmacol 1997;326:93–9.
41 Sagrista ML, Garcia AE, Africa De Madariaga M, Mora M.
Antioxidant and pro-oxidant effect of the thiolic compounds
N-acetyl-L-cysteine and glutathione against free radical-induced
lipid peroxidation. Free Radic Res 2002;36:329–40.
42 Dai J, Buijs RM, Kamphorst W, Swaab DF. Impaired axonal
transport of cortical neurons in Alzheimer's disease is associated
with neuropathological changes. Brain Res 2002;948:138–44
43 Gibson GE, Peterson C. Aging decreases oxidative metabolism
and the release and synthesis of acetylcholine. J Neurochem
1981;37:978–84
44 Zatta P, Ibn-Lkhayat M, Zambenedetti P, Kilyen M, Kiss T.
In vivo and in vitro effects of aluminium on the activity of mouse
brain acetylcholinesterase. Brain Res Bull 2002;59:41–5.
45 Kumar S. Biphasic effect of aluminium on cholinergic enzyme of
rat brain. Neurosci Lett 1998;248:121–3.