Impact of Aluminum Sub-Chronic Toxicity on Body Weight and Recognition Memory of Wistar Rat
The aims of this study was to investigate the impact
of aluminum nitrate administered in drinking water during 90 days (sub-chronic
toxicity), on body weight gain, motor activity, brain aluminum accumulation
and especially in recognition memory of wistar rats. Two groups of young
female wistar rats were used. Treated rats received (80 mg L-1)
of aluminum nitrate diluted in drinking water, while control rats received
a drinking water only, for 3 months. An evolution of body weight, a motor
activity, object recognition memory (NOR) and brain aluminum concentration
has been evaluated. The body weight was taken weekly, whereas the memory
abilities and the motor activity are measured once every fortnight alternatively,
by submitting rats to the open field test and to the novel object recognizing
memory test. The results have showed a significant decrease in rats` body
weight (p<0.05). Though, no significance was registered for motor activity.
Nevertheless, a high significance is showed for recognition memory compared
to control rats (p<0.01), especially at the end of testing period,
even the difference between control and aluminium treated rats in brain
aluminum levels was not significant.
Aluminum (Al) is the 3rd abundant metallic element in the nature after
oxygen and silicon; it constitutes about 8% of the Earth`s crust. It is
present in numerous sources, including air, food, drugs, cosmetics, vaccines,
household materials and water. In Europe, 5% of total aluminum quantity
ingested by human comes from drinking water and 95% comes from aliments.
However, the dissolved aluminum in the water is under a particularly bioavailable
shape, very easily absorbed by the digestive mucous membranes. Human exposure
to aluminum is thus inevitable but neither cases of Al deficiency nor
any physiological function for Al have been described as yet. For many
years, Al was thought to be innocuous and largely unabsorbed from the
gastrointestinal tract. Its toxicity was first recognized in 1972 and
its association with a neurological syndrome in patients on prolonged
hemodialysis reported. This syndrome included progressive dementia, speech
difficulties, facial grimacing and motor abnormalities. Since then, further
neurological syndromes have been attributed to aluminum (Arnaud and Favier,
1991; Struys-Ponsar et al., 1997; Terken et al., 2003).
In animals and in the absence of overt encephalopathy or neurohistopathology,
animals exposed to soluble aluminum salts in the diet or drinking-water
presented a behavioural impairment. Both rats (Commissaris et al.,
1982; Connor et al., 1988) and mice (Yen-Koo, 1992) have demonstrated
such impairments at doses exceeding 200 mg of aluminum per kg of body
weight per day. Although, significant alterations in acquisition and retention
of learned behaviour were documented (Bilkei-Gorzo, 1993; Lal et al.,
In addition, the motor activity and body weight were also altered by
aluminum intake. However, the findings in these aspects are still divergent
and unclear. Colomina et al. (2005) and Roig et al. (2006)
found that exposing rats to aluminum drinking water did not alter significantly
the motor activity in the open field test. Nevertheless, other researcher
showed that oral studies with aluminum in rats, during short-term and
sub-chronic exposure, produced accumulated aluminum levels in the brain
as well as altered general motor activity and impaired motor coordination
(Golub et al., 1989; Sahin et al., 1995).
Concerning the body weight, Golub and Germann (2001) showed significant
decreases in mice pup body weight after aluminum exposure during mother`s
gestation/lactation and offspring exposure. Nevertheless, Colomina et
al. (2005) found that no significant alterations in body weight, food
consumption, or water consumption were observed during gestation in the
dams exposed to aluminum.
The aims of this study is to contribute on elucidation of the impact
of aluminum nitrate administered in drinking water during 90 days (sub-chronic
toxicity), on body weight gain, motor activity, brain aluminum accumulation
and especially in recognition memory of wistar rats, because in our knowledge,
all the studies realized in memory and investigate the spatial memory
MATERIALS AND METHODS
Animals and treatment: Female wistar rats, 3 months of age and
179.6±4.28 g in weight (Means±SEM, n = 14) at the beginning
of the treatment, were used in this study. They were reproduced in colony
room of Biology Department, Faculty of Sciences, Kenitra Morocco. The
rats were housed in propylene cages under standards conditions (20°C,
50-70% humidity and 12L: 12D cycle). They were given free access to food
(SNV, Temara, Morocco) and tap water. The control rats (n = 7) were given
tap water and the aluminum intoxicated rats (n = 7) received 80 mg L-1
of aluminum nitrate (Merck). Aluminum nitrate was diluted in tap water
and given to animals during 90 days.
Open field behavior: An open field test was conducted between
09:00 and 11:00 am to examine the possible effect of sub chronic aluminum
intoxication on behavior in a novel environment every fortnight from the
beginning to the end of intoxication period.
Apparatus consisted of an open top wooden box (100x100x40 cm) covered
by white consistent plastic. Floor area was marked into 25 squares and
illuminated in the center by a 60 W halogen bulb suspended 100 cm above.
Animals were placed in the center of the open field and behavior was
videotaped for 7 min for each rate. Open field behaviors were scored by
a trained observer who was blind to the treatment conditions. The measures
scored consisted on horizontal activity (number of squares crossed).
Novel Object Recognition (NOR) memory task: The apparatus and
procedures for NOR training have been described elsewhere (Ennaceur et
al., 2004; De Lima et al., 2005). The task took place in a
40x50 cm2 open field surrounded by 50 cm high walls, made of
plywood covered by black fine plastic layer. All animals were given a
habituation session where they were left to freely exploring the open
field for 5 min. No objects were placed in the box during the habituation
trial. Twenty-four hours after habituation, NOR training was conducted
by placing individual rats for 5 min into the field, in which two identical
objects (objects A1 and A2) were positioned in two adjacent corners, 10
cm from the walls. In a long-term retention test given 24 h after training,
the same rats explored the field for 5 min in the presence of familiar
object (A) and a novel object (B).
A single set of three objects was used for all animals. All objects presented
similar textures, colours and sizes, but distinctive shapes. The index
of recognition memory was defined as ratio of exploration object B number
and the sum of exploration object A and B number. Between trials the objects
were washed with 10% ethanol solution.
Exploration of an object was defined as directing the nose to the object
at a distance≤1 cm and/or touching it with the nose; conversely, turning
around or sitting on the object was not considered as exploratory behaviour.
NOR procedures were conducted in a presence of luminescent source (60
w) from 1 m in the top of the apparatus.
The test took place every fortnight alternatively with the open field
Brain aluminum evaluation: The day after the last test, aluminum
concentration was estimated in control and treated rat`s brain by graphite
furnace atomic absorption spectrometry (Perkin Elmer 1100) with deuterium
back ground correction. Rats were anesthetized by the chloral 7% and killed
by decapitation. The whole brain was extracted from the skull. Tissues
samples (0.1-0.3 g) were dried, milled and digested by HNO3
acid (4 mL) 65% (Merck). All the analyses were performed in triplicate
and the results were expressed in μg g-1 tissue wet weight.
All the vessels and their caps used were previously washed in hydrochloric
acid and then in 1% nitric acid (Merck) for a week and rinsed in ultrapure
water, to prevent any contamination (Pinta, 1980; Struys-Ponsar et
Statistical analyses: Data obtained was expressed as Mean±SEM.
To evaluate the differences between control and treated groups, the non
parametric Mann-Whitney U-test was used. A p-value smaller than 0.05 was
considered to reflect a statistically significant difference.
The body weight gain: There was no significant difference in body
weight between control (C) and aluminium treated rats (A) in the first
eleven weeks. However, the significance was showed at the end of testing
period (W12 and W13, p<0.05) (Fig. 1).
||The record of weight gain shown for the 13 weeks of
aluminum exposure. *Statistically difference was showed in the end
of test (W12 and W13, p<0.05)
||Mean of recognition memory index (ratio of object B
exploration number and the sum of object A and B exploration number)
of every week respecting a fortnight period between recognition memory
and open field test. *, **A higher difference between control (C)
and treated groups (A) was registered at week 8 (p<0.05) and week
10 (p<0.01) of recognition memory test
Motor activity in open field test (OF): The mean crossed squares
exploring by both control (C) and treated rat by aluminum (A) in (OF)
test was not significantly different (p>0.05) in each fortnight testing
and during the Whole period (90 days) (Table 1).
Index of recognition memory: The effect of aluminium on rat recognition
memory, during the whole period, was significant (p<0.05). The mean
of the index of recognition memory was 0.63±0.04 in control (C),
against 0.45±0.06 in the aluminium treated rats (A) (Table
|Mean of squares crossed number (SC number), of recognition
memory index and of aluminum brain levels, during 90 days in control
rats (C) and in aluminum treated rats
|Values with different superscript
letter(s), for each parameter, are significantly different (p<0.05)
The study of this effect in each fortnight showed that the significant
effect appears at week 8 (p<0.05) and week 10 (p<0.01) (Fig.
Brain aluminum evaluation: The aluminium concentration didn`t
show any signification between both groups p>0.05 (Table
The current findings showed that aluminum affects significantly the rats`
body weight at the end of the experiment (after 90 days exposure). This
result is consistent with others researches which found that exposure
of different aluminum salts decreased rats` and hamsters` body weight
(Drew et al., 1974; Stone et al., 1979). In addition, Golub
and Germann (2001) showed significant decreases in mice pup body weight
after aluminum exposure during mother`s gestation/lactation and offspring
exposure. This decrease found accompanied by reduction in water consumption
of rats (Hicks et al., 1987).
In this study, the body weight` decrease may be explained by a possible
aluminum effect on brain and kidneys which control drinking behaviour.
Actually, many studies have shown that aluminum is found to accumulate
in these organs (Alfrey et al., 1980; Greger et al., 1986;
Domingo, 1987). Otherwise, the decrease of serum triglycerides and mitochondrial
energy metabolism after different rats aluminum salts exposure, can be
another hypothesis (Panda et al., 2008; Sugawara et al.,
In this study, the aluminum exposure didn`t show any significant effect
on motor activity in open field test either in short-term and sub-chronic
aluminum exposure. These results are in accordance with other studies
that found no alterations in open field behaviour (Connor et al.,
1988; Jope and Johnson, 1992; Domingo et al., 1996; Colomina et
With respect to the effect on memory, this study demonstrates that recognition
memory was affected by this intoxication. This impact was clearly proved
in the end of the testing period, suggesting that the administered dose
is weak to induce a quick effect. This suggestion is supported by the
brain aluminum content which showed no significant difference between
control and intoxicated rats.
In the other researches, it is reported that aluminum decreased the rats`
maze-learning ability after 90 days of aluminum salts treatment (Bilkei-Gorzo,
1993). In addition, Lipman et al. (1988) showed that memory impairment,
particularly for short term memory, is characteristic of the aluminum-associated
encephalopathies including Alzheimer`s disease, suggesting a deficit in
memory acquisition (learning) and consolidation.
The findings results concerning the impairment in recognition memory
may be explained by the aluminum effect on hippocampus functioning and
on long-term potentialisation impairments (Platt et al., 1995;
Gilbert and Shafer, 1996).
Furthermore, a long retention interval studies have shown that hippocampus
is implicated in object recognition memory. In that report, it was observed
a delay-dependant hippocampal involvement (Vnek and Rothblat, 1996; Clark
et al., 2000; Hammond et al., 2004).
In conclusion, this study demonstrates that the aluminum sub-chronic
toxicity, at the used concentration, didn`t affect the motor activity
and brain aluminum amounts, but it decreases body weight and recognition
memory faculties. Though, the process is still ambiguous and requires
more detailed studies.
All the authors of this study dedicate it to the memory of Professor
Elhanbali M. whom contributes a lot to this work. This research was supported
by Program of support for the scientific research (PROTARS III: D63/01),
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