Romanian Biotechnological Letters
Copyright © 2015 University of Bucharest
Vol. 20, No. 6, 2015
Printed in Romania. All rights reserved
ORIGINAL PAPER
Experimental research regarding the impact
of aluminum salts on locomotor activity in mice
Received for publication, April 21, 2014
Accepted, September 20, 2015
Isabel Ghiță, Aurelian Zugravu, Ana Segarceanu,
Marinela Chirilă, Claudia Handra, Ion Fulga
University of Medicine and Pharmacy “Carol Davila”, Bucharest, Department of
Pharmacology and Pharmacotherapy, Romania
*Corresponding author: Aurelian Zugravu MD, PhD, University of Medicine and
Pharmacy “Carol Davila”, Bucharest, Department of Pharmacology and Pharmacotherapy
Phone:0723.233.243; Email: [email protected]
Abstract
Aluminum is one of the most common metals in the earth's crust with numerous uses in various
fields. The ingestion of aluminum occurs through swallowing of various drugs, water and food, the
brain being the primary target organ for its toxic action. Existing studies have linked abnormal
concentrations of aluminum present in different areas of the brain with several neurological diseases,
such as dementia, Alzheimer's disease and Parkinson's disease. This paper aims to highlight the
relationship between aluminum and locomotor activity in experimental animals that received various
aluminum compounds in single dose or chronic administration. The locomotor activity of male albino
rats was tested using an Activity Cage and the Simple Exploration Test. High doses of aluminum salts
showed a decrease in locomotor activity in the studied groups compared to the control, whilst low doses
did not produce a significant reduction in activity. Chronic exposure to aluminum salts did not
significantly influence locomotor activity. This effect set in 30 minutes after the substances were
administered and lasted for at least 2 hours. Interference with the neuronal transmission systems
(serotonergic or GABA- ergic) is presumed to be the mechanism through which the aluminum ion exerts
its effects.
Keywords: aluminum chloride, aluminum sulfate, locomotor activity
1. Introduction
Aluminum is one of the most common metals in the earth's crust with numerous uses in
various fields such as metallurgy, electrical and chemical industry. The mostly used aluminum
compounds are aluminum chloride and aluminum sulfate. Aluminum chloride (AlCl3) is a
catalyst acid used in the manufacture of rubber and lubricants and can be found in the
composition of antiperspirants. Aluminum sulfate (Al2 (SO4) 3) is used as a flocculant in
purification and wastewater treatment. It is also used in the paper industry, in the manufacture
of fire- and water-resistant clothing, in leather processing, in decanting oils and fats, as an
intermediate for the production of other compounds, as fertilizer for plants, as a component of
pesticides, soaps and in the cosmetics industry. Lately, aluminum salts have become a standard
adjuvant for vaccines. The concentration of aluminum in the soil ranges from 700 mg / kg of
soil up to 100,000 mg / kg of soil. The concentration in water is generally less than 0.01 mg / L.
In urban areas the aluminum concentration in the air is around 2,000 ng / m3, residents could
inhale up to 40 mg aluminum / day.
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Romanian Biotechnological Letters, Vol. 20, No. 6, 2015
Experimental research regarding the impact
of aluminum salts on locomotor activity in mice
The ingestion of aluminum occurs through swallowing of various drugs (antacids,
buffered analgesics), water, food and food additives. Aluminum occurs naturally in many
edible plants and is also added during food processing. Concentrations in foods and beverages
vary depending on type of preparation and processing. Most foods contain very low quantities
of aluminum, below 5 mg / kg. Generally, foods with the highest amount of aluminum are
those which contain additives. In processed foods the amount of aluminum could reach up to
2,300 mg / kg. Occupational exposure to aluminum is produced during the refining process of
metal and in various other branches of industry. Studies have established that the values to
which workers from the aluminum industry are exposed are below 1 mg / 8 hours in 10 m3 of
air inhaled / exchange. Target organs of aluminum are lungs, bones and the central nervous
system. The brain is a primary target organ for the toxic action of aluminum. Normally the
blood-brain barrier permeability is selective for certain molecules [1]. Due to the low
permeability of the blood-brain barrier, aluminum passes it through a mechanism dependent
on transferrin and with the help of lightweight molecular complexes. Aluminum affects the
transport of different drugs through the blood-brain barrier into the central nervous system,
mainly with those that dissociate as anions [2]. In patients diagnosed with dementia
researchers have been observed that the metal binds to the endothelial cells of the blood-brain
barrier and is deposited along the blood vessels. Also, aluminum can increase the passage of
other compounds through transmembrane diffusion and selectively affects the saturable
transport [3]. Cells in the central nervous system (neurons, dendrocytes, astrocytes, and
choroid plexus cells) are rich in receptors for transferrin [1]. Aluminum interferes with
cellular iron homeostasis and influences the iron-dependent processes from the brain [2].
Aluminum could influence the binding rate of ferritin - protein that ensures intracellular
storage of iron. It was observed that ferritin from the brain tissues of patients with
Alzheimer's disease contains 6 times more aluminum than in normal human brains.
Accumulation of aluminum in microglial and astroglial cells leads to the apoptosis of
astrocytes and causes neuronal loss. In addition, aluminum inhibits neurotransmitters which
play roles in nerve transmission (gamma-aminobutyric acid, glutamate, choline,
norepinephrine and serotonin). It is responsible for the degeneration of nerve fibers by
decreasing the number of microchannels in affected neurons, dendrites and cells involved in
the process of memory. Other studies have shown involvement of aluminum in the
pathogenesis of Parkinson's disease [4]. Recent studies have shown that chronic aluminum
exposure causes toxic effects on serotonin neurotransmission in the dorsal raphe nucleus and
the subcomissural organ likely through increased synthesis or decreased release. Aluminum
exposure was also shown to decrease RF glycoprotein involved in the detoxification of the
cerebrospinal fluid [5]. Effects caused by chronic exposure to aluminum affect the spatial
learning and memory abilities of both young and old rats. Aluminum intake also produces
stress-related damage to lipids, membrane associated proteins, and endogenous antioxidant
enzyme activity in the brain, with a markedly higher degree of alteration of cognitive
functions being observed in younger rats [6]. Electrophysiology studies have demonstrated
that aluminum chloride affects the activation and inactivation courses of sodium and
potassium currents in the CA1 hippocampal neurons in rats, which could explain to some
extent the damage this compound produces to the central nervous system [7]. Based on this
data gathered from the literature, this paper aims to highlight the relationship between
aluminum and locomotor activity in experimental animals that were treated with various
aluminum compounds administered in single doses or chronically for 2 weeks.
Romanian Biotechnological Letters, Vol. 20, No. 6, 2015
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ISABEL GHİȚĂ, AURELİAN ZUGRAVU, ANA SEGARCEANU,
MARİNELA CHİRİLĂ, CLAUDİA HANDRA, ION FULGA
2. Materials and Methods
For the study of locomotor activity three experiments were conducted. The first two
experiments aimed to evaluate locomotor activity at 30 and 120 minutes after administration
of a single dose of AlCl3 and Al2(SO4)3 to the test animals. Testing was performed using a
cage of locomotor activity - Activity Cage. The third experiment aimed to assess locomotor
activity after administering AlCl3 and Al2(SO4)3 chronically for 2 weeks. Testing was
performed using the simple exploration cage. For the first two experiments, batches of 15
albino male mice were used, with a weight between 25-35 grams. For the final experiment,
groups consisting of more than 25 male albino mice were used, due to the increased mortality
caused by the stress of daily administering of substances. All the animals were provided by
the "Carol Davila" University of Medicine and Pharmacy Bucharest biobase. The mice were
brought to the laboratory within 24 hours before the start of the tests being maintained in
standard environmental conditions and having access ad libitum to food and water. The
animals were housed 12 per each cage in Plexiglas cages (bed of wood chips). Ambiental
temperature was between 21 and 24 ° C and the relative humidity was maintained between
45-60%. All experiments were performed in accordance with European Directive 86/609 /
EEC / 11.24.1986 and GD 37 / 30.01.2002 regarding the protection of animals used for
experimental or other scientific purposes. Tests were conducted with the approval of Ethics
Committee UMF „Carol Davila” Bucharest. During the first two experiments the substances
administered to the mice were: aluminum chloride (AlCl3) in a dose of 9 mg×kg-1 and 18
mg×kg-1, aluminum sulfate (Al2(SO4)3) 3.6 mg×kg-1 and 7.2 mg×kg-1 respectively and saline
solution 0.1 ml / 10 g mouse. All substances were injected intraperitoneally and the tests were
conducted after 30 and 120 minutes after administering the substances. During the final
experiment, the substances used were: AlCl3 in a dose of 0.2 mg×kg-1 and 1 mg×kg-1.,
(Al2(SO4)3) 0.1 mg×kg-1,and 0.5 mg×kg-1 and saline solution 0.1 ml / 10 g mouse. These
substances were administered by gavage, two doses at first, in the morning and in the evening
during the first week, and then single doses at the same time each day for the following week.
Testing was carried out 2 hours after the final dose of the substance was administered. Doses
were chosen to be 1/5 and 1/10 of LD50.
Assessment of locomotor activity was done in case of the first two experiments by
measuring spontaneous movements. A Ugo Basile 374331 Locomotor Activity Cage with a
size of 41/41/33 cm was used. Emitters of infrared radiation and sensors that detect horizontal
and vertical movements made by each animal were placed on the sides of the cage which
allow us to measure movement. Discontinuation of the radiation beam was interpreted as a
movement of the animal in the action area of that beam. The sensors were connected to a unit
allowing automatic counting of motion and printing the final test result. The duration of each
test was 5 minutes and between testing the box was cleaned with a 10% alcohol solution.
During each test background noise was minimized so as to eliminate possible error factors
(noise can generate the occurrence of an inhibition behavior, „freeze" or „extreme agitation").
Each mouse was placed in the same corner of the cage and that was called the „start corner”.
A reduction in the number of spontaneous horizontal or vertical movements made by each
mouse was interpreted as an indicator for decreased locomotor activity.
To assess locomotor activity in the third experiment the simple exploration test was used.
For this test we used a plexiglass box with a size of 40 x 30 cm, whose floor was marked with
20 squares with a size of 8 x 7.5 cm. Each mouse was placed in a corner of the box (same
corner for all mice) and the squares through which the mouse passed with all 4 paws were
counted for 5 minutes. Between two tests the box was cleaned with a 10% alcohol solution.
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Experimental research regarding the impact
of aluminum salts on locomotor activity in mice
Results were analyzed using Microsoft Office Excel. Means and standard deviations for each
batch were calculated and then the Student t-test was applied. Results were considered
statistically significant if p <0.05.
3. Results and discussion
Experiment 1: Influence on locomotor activity at 30 minutes after administration of
single doses of AlCl3 and (Al2(SO4)3)
The group that received a dose of 9 mg×kg-1 of AlCl3 (Fig. 1) showed a decrease in the
number of movements made on the horizontal axis, the average number of movements being
461.57 compared with the control group who presented an average of 528.57. Also the dose
of 18 mg×kg-1 of AlCl3 led to a reduction in the number of horizontal movements compared
with the control group, with an average of 330.07.
At 30 minutes the average number of horizontal movements recorded by the group that
received 3.6 mg×kg-1 of Al2(SO4)3 was 390.64, much lower than the average achieved by the
control group. The group that received 7.2 mg×kg-1 of Al2(SO4)3 obtained an average of
418.43 horizontal movements, also markedly reduced compared to the control group.
According to the results, it can be concluded that the two aluminum salts administrated in
two doses decreased the locomotor activity at 30 minutes and the effect was statistically
significant compared to the control group (p <0.05).
In the case of vertical movements (Fig. 2) at 30 minutes the group that received the low
dose of AlCl3 had a mean number of vertical movements of 45.21, significantly lower than
the control that had average of 56.21. The group injected with high dose AlCl3 showed an
average of 23.21 vertical movements.
Both groups that received Al2(SO4)3 showed a significant decrease in the number of
vertical movements with an average of 11 and 36.71 respectively, significantly lower
compared with the values obtained by the control group.
In conclusion it was observed that the two substances based on aluminum decreased
significantly the number of vertical movements of the treated animals compared with controls
30 minutes after administering the substance (p <0.05).
Figure 1. Activity Cage Test - 30 minutes
- horizontal movement
Figure 2. Activity Cage Test - 30 minutes
- vertical movement
Romanian Biotechnological Letters, Vol. 20, No. 6, 2015
10979
ISABEL GHİȚĂ, AURELİAN ZUGRAVU, ANA SEGARCEANU,
MARİNELA CHİRİLĂ, CLAUDİA HANDRA, ION FULGA
Experiment 2 - Influence on locomotor activity at 120 minutes after administration
of single doses of AlCl3 and Al2(SO4)3 (Fig. 3 and 4)
At 120 minutes all groups showed a significant decrease in the mean number of horizontal
movements compared with the control who presented a mean value of 480.67 (p <0.05).
The group that received a low-dose of AlCl3 recorded an average number of horizontal
movements of 388.25, a low value compared to 432.92 for the group who received high-dose
AlCl3.
The group that received the small dose of Al2(SO4)3 had an average of 412.33 horizontal
movements and the group that received Al2(SO4)3 in high dose had an average of 399.83 (Fig. 3).
Vertical movements (Fig. 4) recorded at 120 minutes after administering the substances
also registered a noticeable reduction compared to the control. The group that received the
low-dose of AlCl3 achieved a value of 20.25, those who received the high-dose of AlCl3 had a
value of 8.92, the group with the small dose of Al2(SO4)3 recorded a value of 36.92, and those
who received the high dose of Al2(SO4)3 had an average of 40.83. Only results recorded in
those groups who received AlCl3 were significantly lower compared to control group. In the
case of the groups that received Al2(SO4)3 the results had borderline significance.
Figure 3. Activity Cage Test - 120 minutes
- horizontal movement
Figure 4. Activity Cage Test - 120 minutes
- vertical movements
Conclusions
The two aluminum salts, administered in high doses decreased the number of movements
both horizontally and vertically compared to control group. Influence on locomotor activity
debuted in 30 minutes and lasted 2 hours. The decrease in locomotor activity with high doses
of studied compounds can be attributed to the aluminum ion.
This effect obtained by administration of high doses of AlCl3 and Al2(SO4)3 may be due
to the interference with the neuronal transmission system (serotonergic or GABAergic).
Low doses of the studied aluminum compounds did not alter locomotor activity, the
cause probably being that extremely high doses of aluminum ions which cross the blood-brain
barrier are needed so that the aluminum ion manages to interfere with the transmission path of
various neurotransmitters from the brain.
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of aluminum salts on locomotor activity in mice
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