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Journal of
Trace Elementsrace Elementsin Medicine and Biology
Journal of Trace Elements in Medicine and Biology 19 (2005) 203208
TOXICOLOGY
Oxidative damage following chronic aluminium exposure in adultand pup rat brains
Bimla Nehru, Priya Anand
Department of Biophysics, Panjab University, Chandigarh 160014, India
Received 2 February 2005; accepted 26 July 2005
Abstract
Aluminium is known to cause neurotoxic effects. In the past few years there has been an upsurge of interest in
aluminium exposure through diet and environment, which might impair the development of mammals. The present in
vivo study was designed to investigate the potential of aluminium to participate in either antioxidant or pro-oxidant
processes in both developed and developing rat brain. Markers of oxidative stress were determined in rat brains
exposed to AlCl3 (100 mg/kg body weight) for 8 weeks. The aluminium dose was given to adult rats for 8 weeks and in
another group, exposure of aluminium for 60 days was done postnatally, 21 days to the feeding mother (lactation
period) and 39 days to the rat pups. The results showed a statistically significant (pp0:01) increase in lipid
peroxidation (LPx) as measured by production of malondialdehyde in both cerebrum and cerebellum of pup brains.
A significant increase (pp0:001) in LPx was also observed in the adult group. Furthermore, aluminium exposure
resulted in a significant decrease in superoxide dismutase and catalase activity in both regions of the brain of
developing and developed rat brain.Thus the results of the present study suggest that in rats, aluminium (100 mg/kg body weight) has a pro-oxidant
effect and thus acts as a neurotoxin.
r 2005 Published by Elsevier GmbH.
Keywords: Aluminium; Neurotoxicity; Lipid peroxidation; Catalase; Superoxide dismutase
Introduction
Aluminium (Al) is the third most abundant element in
the earth crust and is ubiquitous in nature and in the
domestic environment [1,2]. There are many studieswhich have linked aluminium to Alzheimers disease [3].
Aluminium-induced neurotoxicity was related to ele-
vated brain aluminium levels and neurofibrillary tangles
(NFT) which are also characteristics of Alzheimers
disease. Aluminium exposure inhibits the prenatal and
postnatal development of the brain in humans and
experimental animals, resulting in symptoms including
growth retardation, delayed ossification, and malforma-
tion at high doses, leading also to reduced maternal
weight gain [4]. Investigations on neurotoxic effects of
aluminium on developing brains are still inconclusive,since the incorporation of aluminium into fetuses and
suckling animals during gestation and lactation has not
been clarified due to the non-availability of suitable
tracer isotopes of aluminium (Tables 13).
The brain is a target of aluminium toxicity. Al-
transferrin and Al-citrate complexes or disruption of the
blood brain barrier (BBB) mediate the aluminium
transport to the brain. Another possibility is that
physiological ligands present at these barriers get altered
ARTICLE IN PRESS
www.elsevier.de/jtemb
0946-672X/$ - see front matterr 2005 Published by Elsevier GmbH.
doi:10.1016/j.jtemb.2005.09.004
Corresponding author. Tel.: +91 172 534119.
E-mail address: [email protected] (B. Nehru).
http://www.elsevier.de/jtembhttp://www.elsevier.de/jtemb -
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in several disease states, thus leading to aluminium
exposure [5]. Aluminium exposure also results in the
production of free radicals [6], being responsible for
neurotoxicity. The pro-oxidant effects of aluminium
damage the neuronal membrane by altering the physical
properties of membrane, interfering with the functioning
of voltage-activated ionic channels or altering the
secretion of transmitters [7].
Several factors seem to influence the neurotoxic effects
of aluminium. These include the integrity of the BBB, the
clearance from the brain and the developmental stage of
the brain. Studies have indicated that the lactation
period is most susceptible to aluminium, since this is theperiod of maximum synaptic plasticity. It is known that
in humans, the maximum development of brain occurs in
the prenatal period. On the contrary, in rats it occurs
mostly in the postnatal period [8]. Accordingly, the
aluminium exposure in the developing stage has been
carried out postnatally in the present study in rats.
The clearance of aluminium from the brain is likely
to depend upon several factors such as metal binding
proteins, scavenging molecules like glutathione, the
status of the redox enzyme system superoxide
dismutase (SOD), catalase, glutathione peroxidase as
well as its interactions with other micronutrients.
In the present study, the toxic manifestations of
aluminium in terms of status of lipid peroxidation (LPx)
and redox enzymes have been evaluated in both
cerebrum and cerebellum of developing and developed
rat brain following AlCl3 exposure (100 mg/kg body
weight (b.w.)) for 60 days.
ARTICLE IN PRESS
Table 1. Effect of aluminium on body weight and brain
weight
Body weight (g) Brain weight (g)
Group A: Adults
Control 225725 1.67670.07
Aluminium
treated
224720 1.67970.21
Group B: Pups
Control 12570 1.74270.007
Aluminium
treated
12073 (1.3%) 1.43970.08 (17.4%)
Values are mean7SD of 67 determinations.No significant value was obtained as there was no change in body
weight.
Table 2. Alterations in lipid peroxidation (LPx), superoxide dismutase and catalase activity in the brain of adult rats following
aluminium exposure
Control Aluminium treated
Cerebrum Cerebellum Cerebrum Cerebellum
LPx (nmol MDA/mg protein/3 min) 0.19270.01 0.20970.06 0.26470.02*** (+37.8%)* 0.36870.03** (+76.1%)*
Superoxide dismutase (U/mg protein) 0.81570.004 0.81270.006 0.58770.005*** 0.51770.03***
Catalase (U/mg protein) 0.25570.01 0.18770.03 0.18770.13* (26.6%)* 0.13570.04* (34.5%)*
Values are mean7SD of 56 determinations.
Values in brackets are % increase (+) or % decrease ().
*po0:05, **po0:01, ***po0:001: treatment vs. control group.
Table 3. Alterations in lipid peroxidation (LPx), superoxide dismutase and catalase activity in the brain of rat pups following
aluminium exposure
Control Aluminium treated
Cerebrum Cerebellum Cerebrum Cerebellum
LPx (nmol MDA/mg
protein/3 min)
0.14970.001 0.09370.01 0.23870.06 ** (+59.4%)* 0.25970.09** (+178.4%)*
Superoxide dismutase
activity (U/mg protein)
0.72870.006 0.77270.008 0.51170.05*** 0.50670.05***
Catalase (U/mg protein) 0.27370.02 0.19470.08 0.14370.01*** (47.6%)* 0.12770.01* (34.5%)*
Values are mean7SD of 56 determinations.
Values in brackets are % increase (+) or % decrease ().
*po0:05, **po0:01, ***po0:001: treatment vs. control group.
B. Nehru, P. Anand / Journal of Trace Elements in Medicine and Biology 19 (2005) 203208204
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Material and methods
Experimental design
Healthy Sprague Dawley (SD) rats were procured
from the central animal house of Panjab University,Chandigarh, India. The animals were 6 months old and
their body weight was in the range of 150200 g. They
were acclimatized under hygienic conditions and were
fed on standard pelleted rat feed (Hindustan Lever Ltd.,
Mumbai, India) and water ad libitum. The diet had
adequate quantities of micronutrients as well as macro-
nutrients. The animals were kept and cared for at all
stages in compliance with the applicable guidelines and
regulations of the institute.
The breeding of animals was done in the animal house
of Panjab university by keeping the ratio of males to
females 3:2 in the plastic cages. The females were
checked daily for vaginal plaque and the day on which
the vaginal plaque was seen was marked as day one. The
female rats were separated, divided into two groups, and
placed in separate cages for the gestation period. The rat
litter obtained from the parents was allowed to survive
on their mothers milk.
One group of mothers served as control and the
other group was treated with AlCl3. The litter size
of the control subgroup was 810 rats which was
decreased to 56 rats after aluminium treatment. Weekly
weight changes were recorded, and the dose was
adjusted accordingly. The rats were monitored for
their health, general behaviour, and daily food in-take. AlCl3 and other reagents were purchased from
Merck.
The animals were assigned to two groups of 12
animals each: group A (the adult rats) served as
developed brain model and group B (the rat pups) as
developing brain model. Group A was further divided
into two subgroups: subgroup I served as control and
was given free access to water and diet. Subgroup II
received aluminium chloride (in water) by oral gavage
daily with a dose of 100 mg elemental Al/kg b.w. over a
period of 8 weeks. Similarly in group B, the subgroup III
served as control and the subgroup IV received
aluminium (100 mg/kg b.w.) as aluminium chloride (21
days to feeding mothers and 39 days to pups) orally for
8 weeks.
For the different biochemical measurements, the
animals of the different groups were sacrificed by
decapitation. Their brain was removed immediately
and two brain regions, i.e. cerebral cortex and cerebel-
lum, were dissected out, rinsed in ice cold water normal
saline and blotted dry. All processes were carried out
under cold conditions. The two brain regions were
homogenized in 10 mmol/L phosphate buffer saline
(10% w/v) of pH 7.4.
Body weight
A careful record of body weight changes of both
group A and group B was kept throughout the study.
The animals were weighed at the beginning of the
experiment, then twice a week, and finally before
sacrificing them.
Biochemical measurements in the brains
In the cerebrum and cerebellum samples, the follow-
ing parameters were investigated: LPx, SOD and
catalase.
Lipid peroxide assay
Lipid peroxide formation was assayed by the method
of Wills [9]. The results were expressed as nmol ofmalondialdehyde (MDA)/mg of protein. MDA is a
degradation product of peroxidized lipids. The pink
colour of the TBA-MDA chromophore has been taken
as an index of LPx (absorption maximum at 532 nm).
Superoxide dismutase
The activity of SOD was determined by the method of
Kono [10]. The reaction is designed to observe the
inhibition of the oxidation rate of nitroblue tetrazolium
(NBT) using hydroxylamine hydrochloride as electron
donor. The result is expressed in units: one unit ofenzyme is defined as the inverse amount of enzyme
required for 50% inhibition.
Catalase
Catalase was determined by means of the UV
spectrophotometric method described by Luck [11].
H2O2 was used as substrate. The UV absorption of the
H2O2 solution was recorded at 240 nm after reaction of
H2O2 with catalase. From the decrease in optical
density, the enzyme activity was calculated. The amount
of H2O2 decomposed was calculated on the basis of the
molar extinction coefficient of H2O2 (0.71 L/mol/cm)and the results were expressed as mmol of H2O2decomposed/min/mg protein.
The protein content in the different samples was
estimated by the method of Lowry et al. [12].
Statistical analysis
The data were expressed as mean7SD and differences
between the controls and aluminium treated animals
were determined by students t-test.
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Results
Body and brain weight
In aluminium treated adult animals, no significant
decrease in the body weight was observed and also no
decrease was observed in the brain weight as comparedto the control animals. On the other hand, a significant
decrease in body weight as well as brain weight was
observed in the aluminium treated pup group as
compared to their control group.
Lipid peroxidation
Following aluminium treatment for 8 weeks, the
MDA levels were significantly elevated (pp0:001) in the
adult group as compared to controls in both cerebrum
and cerebellum regions of the brain. In cerebellum, the
increase extended to 76.5%, whereas that in cerebrumwas 37.8%. Similarly, in pups, there was a significant
increase (pp0:01) in LPx in both brain regions,
cerebellum showing an increase of 178% as compared
to 59.4% in cerebrum.
Superoxide dismutase
The enzyme activity in both cerebrum and cerebellum
was significantly decreased (pp0:001) in the adult group
after aluminium exposure for 8 weeks as compared to
the animals of the control group. Similarly, in rat pups,
there was a significant decrease (po0:001) in SOD
activity in both brain regions.
Catalase
The catalase activity was significantly decreased
(pp0:05) in the adult group after aluminium exposure
for 8 weeks in both cerebrum and cerebellum as
compared to the animals of the control group. The
decrease was similar in both brain regions. Similarly, in
rat pups, there was a significant decrease (po0:001) in
both brain regions.
Discussion
The physiological effects of aluminium have been
extensively investigated, but the mechanism by which
aluminium causes neuronal damage in brain is still
unknown. One possible mechanism involves oxidative
injury, which has been suggested to contribute to some
neurodegenerative disorders. Although aluminium is
not a transition metal and therefore cannot initiate
peroxidation, many investigations have searched for a
correlation between aluminium accumulation and oxi-
dative damage in the brain. In vitro studies have
indicated that aluminium greatly accelerates iron-
mediated LPx under acidic and neutral conditions
[1315]. LPx rates were elevated in autopsy samples
from the cortex of Alzheimers disease patients [16], and
aluminium has been reported to accumulate in the brain
in Alzheimers disease [17,18].Ginkel et al. reported a significant increase in brain
aluminium levels in animals having received four
injections of AlCl3 (5mg/kg), either alone or with
equimolar citric acid or malton over a 7-day period
[19]. In the present experiment, aluminium in the form
of AlCl3 (100 mg Al/kg b.w.) was administered orally
over a period of 8 weeks, which was a higher dose than
that reported to cause aluminium accumulation in the
brain of experimental animals.
The primary effects of aluminium on the cerebral
functions are thought to be mediated via damage to cell
membranes. LPx of biological membranes results in the
loss of membrane fluidity, changes in membrane
potential, an increase in membrane permeability and
alterations in receptor functions. In the present experi-
ment, there was a significant increase (pp0:001) in LPx
after aluminium exposure in the adult group, measured
in terms of MDA levels in both cerebrum and
cerebellum. Julka and Gill [20] also reported a
significant increase in whole brain thiobarbituric acid
reactive substances after stimulation by aluminium salts.
The ionic radii of Al3+ most closely resemble those of
Fe3+ [21]; therefore, the appearance of Al3+ in Fe3+
sites is probable. Aluminium is known to be bound by
the Fe3+
carrying protein transferrin thus reducing thebinding of Fe2+. The increase in free intracellular Fe2+
causes the peroxidation of membrane lipids and thus
causes membrane damage.
Moreover, Flemming and Joshi [22] have reported
that the amount of aluminium found in ferritin
extracted from Alzheimers disease affected brains
was 5.6 times higher than in ferritin from matched
control samples. The increase may have been due
to a general increase in the availability of aluminium
to the brain of patients with Alzheimers disease, and
raised the possibility that aluminium releases iron
as Fe3+.
A similar result was found in the rat pups group:
aluminium exposure (21 days to the feeding mothers and
39 days to the pups) significantly increased (pp0:001)
the LPx measured in terms of MDA levels in both
cerebrum and cerebellum, with the higher increase in the
cerebellum. This could be due to the fact that cerebellar
functions depend upon the inputs from several regions
of the brain, provided by a large number of tracts, that
are quickly communicating to several regions of brain
stem nuclei after integration. These tracts are rich in
lipids and thus are susceptible to peroxide damage
following aluminium exposure.
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The more pronounced effects of aluminium exposure
on rat pups as compared to adult rats might be related
to the cell biology of the BBB. The BBB is formed by
brain capillary endothelial cells. In the late embryonic
and early postnatal period, these cells respond to
inducing factors found in the brain environment by
adopting a set of defined characteristics, including high-electrical-resistance tight junctions [23]. The presence of
aluminium at the postnatal stage might affect the
formation of BBB in pups, resulting in a higher exposure
to the neurotoxic metal compared to the same dose
given to the adult group, and resulting in increased LPx.
Reports related to the effect of aluminium on the
different biochemical parameters in pups are very
limited till date.
The increased LPx is, at least in part, due to an
inhibition of SOD in the brain. The result is a
substantial increase in the rate of phospholipid perox-
idation in brain cells, leading to membrane damage and
neuron death. SOD presents the first line of defence
against superoxide, as it dismutases the superoxide
anion to H2O2 and O2 [24]. Because the SOD enzyme
generates H2O2, it works in collaboration with H2O2removing enzymes. Catalase converts H2O2 to water and
oxygen. Catalase is present in the peroxisomes of
mammalian cells, and probably serves to destroy H2O2generated by oxidase enzymes located within these
subcellular organelles.
However, the brain is an organ that is especially
susceptible to peroxide damage because of several
factors, such as high lipid content, high oxygen turn-
over, low mitotic rate as well as low antioxidantconcentration. These factors may explain why alumi-
nium exposure affects the brain more than any other
organ. This is the reason why the antioxidants (Cu/Zn)-
SOD and catalase have been studied after 8 weeks
aluminium exposure.
In the present experiment, aluminium altered the
cellular redox state by inhibiting the enzymes involved in
antioxidant defense, that is SOD and catalase, which
function as blockers of free radical processes. We
observed a significant decrease (pp0:05) in the enzymes
in both cerebrum and cerebellum of the adult group.
The results are in accordance with Dua and Gill [25]
who observed a significant decrease in the activities of
SOD and catalase in cerebrum, cerebellar and brain
stem after aluminium treatment.
Similarly, the decrease was significant (pp0:001) in
the rat pup group. The decrease in both could be either
the result of a decrease in the substrate levels of H2O2 or
of a reduced synthesis of the enzyme itself as a result of
higher intracellular concentrations of aluminium. The
decrease was more significant in the pup group, which
may correspond to the fact that aluminium (at a dose of
100 mg/kg) was highly neurotoxic and inhibited the
development of brain.
Conclusion
In conclusion, the present study indicates that oral
administration of aluminium (100 mg/kg b.w.) is neuro-
toxic to both developing and developed rats. The
aluminium-induced damage in rat brain in our study
seemed to be due to the decreased activity of SOD andcatalase through a yet unconfirmed mechanism, result-
ing in oxidative damage. Lipid peroxidation may be
another biochemical key process responsible for neuro-
nal dysfunction and neuronal death related to metal
toxicity.
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