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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

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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).
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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.

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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).


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.


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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