Food and Chemical Toxicology 50 (2012) 315319

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

The effects of chronic aluminum exposure on learning and memory of rats

by observing the changes of Ras/Raf/ERK signal transduction pathway
Xin Cui, Biao Wang, Zhihong Zong, Suyuan Liu , Wei Xing
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences of China Medical University, No. 92, Beier Road, Heping District, Shenyang 110001, China

a r t i c l e

i n f o

Article history:
Received 16 September 2011
Accepted 27 October 2011
Available online 6 November 2011
Keywords:
Aluminum
Ras/ERK signal pathway
LTP
Learning
Memory

a b s t r a c t
Objective: To investigate the effects of chronic aluminum (Al) exposure on learning and memory function
of rats by observing the changes of Ras/Raf/ERK (Ras/ERK) signaling pathway.
Methods: Eighty weaned Wistar rats were divided into four groups ad libitum, 20 rats in each group. The
four groups were fed with drinking water containing 0% (control), 0.2%, 0.4% and 0.6% (Al exposure) AlCl3
for 3 months individually to set up aluminum exposure models. The laboratory was maintained at
1823 C and 4555% relative humidity. Graphite furnace atomic absorption spectrometry was used to
detect the content of Al in brain and blood. Western blot and real-time PCR (RT-PCR) were used to determine the protein and mRNA expression levels for Ras, Raf1, ERK2 and CREB.
Results: Chronic Al exposure increased the content of Al in rats blood and brain. It increased expression
of Ras in the hippocampi compared with the control but the expression decreased along the Al exposure
groups (p < 0.05). Similarly, Raf1, ERK2 and CREB expressions decreased compared to the control in a
dose-dependent manner (p < 0.05).
Conclusion: Chronic Al exposure may affect learning and memory through impact on Ras/ERK signal
pathway.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Al is the most abundant metal in the earth crust which exists
widely and is pathogenic to humans (Peto, 2010). With the development of Al industry, the chance of Al exposure to people increase
gradually. Its trivalent ionic form is extremely toxic to all organisms. The normal reference range of Al concentration in human
blood is 05.4 lg/L and in urine is 530 lg/L (Dhatrak and Nandi,
2009). Research has shown that Al can damage the cognitive function when its concentration reaches 135 lg/L in urine (MeyerBaron et al., 2007). Al enters the brain tissues via Ca2+ channels
and voltage-gated channels of the cells (Stevanovic et al., 2009).
Al has been reported to be involved in various neuronal diseases
including Alzheimers disease, dialysis encephalopathy, the disease
of Guam, Parkinsons disease, epilepsy, learning and memory dysfunction, the function of exercising common sense and language
dysfunction, myoclonus, mental disorders, etc. (Bolognin et al.,
2011; Mudge et al., 2011; Domingo et al., 2011; Pohl et al., 2011;
Abdel-Aal et al., 2011; Komatsu et al., 2011; Shi-Lei et al., 2005).
Chen et al. (2002) found that compared with the control rats, Alexposed rats showed a decrease in LTP amplitude of 58% and 20%

Corresponding authors. Tel.: +86 24 23256666x5477.

E-mail addresses: cuixin163@126.com (X. Cui), liusuyuan0305@126.com (S. Liu),
xingwei0305@163.com (W. Xing).
0278-6915/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2011.10.072

for PS and EPSP, respectively, which implies that the Al-induced

impairment of LTP of PS was more serious than that of EPSP. Animal experiments show that calcium channel is a target of Al.
Sub-chronic exposure of Al could impair the ability of learning
and memory in rats during development, inhibit the expression
of NMDARa and reduce Ca2+ concentration, suggesting that the
disorder of Ca2+ signaling system might be one of the mechanisms
of Al damaging the ability of learning and memory (Jin et al., 2010;
Li et al., 2006; Sarin et al., 1997). Xing et al. (2007) showed that Al
goes into the cell via calcium channels and leads to (Ca2+)i decrease, which affect the development and maintenance of LTP.
In recent years, many scholars showed that Ras/ERK signaling
pathway plays an important role in the consolidation process of
cognition which affects the brain information storage and memory,
related to the formation of LTP (Liu et al., 2011; Jin and Feig, 2010).
Research by Hyman et al. (2005) suggests that the defects of Ras/
ERK signaling pathway can reduce learning ability in hippocampus.
Sato et al. (2006) and Arguin et al. (2010) pointed out that, the
cAMP-responsive element-binding protein (CREB) is related to
the inux of Ca2+. When the L-type voltage-dependent Ca2+ channels open, a huge amount of extracellular Ca2+ enters into the cell.
And then intracellular Ca2+ increase which can activate the CaMK
or ERK dependent pathways (Ras/ERK). The effects of Al on the
Ras/ERK pathway have not been reported. Our previous study,
Wang et al. (2010) studied effect of Al exposure to learning and
memory and detected changes in ERK1/2 in the Al exposed rats.

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2. Materials and methods

2.1. Animal model
Wistar rats were obtained from the Department of Laboratory Animals of China
Medical University (CMU) and treated according to the guidelines approved by the
CMU Animal Care and Use Committee. The Wistar rats after weaning (about 30 g),
were randomly divided into four groups, each group containing 20 animals. AlCl3
was dissolved in distilled water to the nal concentrations of 0.2%, 0.4% and 0.6%
Al. The average daily AlCl3 solution intakes of each Al-exposed group were 25 2,
20 2 and 15 2 mL per rat per day, respectively. Control rats consumed
50 2 mL of distilled water per day. Rats maintained on the Al diet remained
healthy and their average body weight was 90 6% of control rats. The laboratory
room temperature was maintained at 1823 C and relative humidity at 4555%.
Three months after Al administration, rats were subjected for analyses.
2.2. The content of Al in rats brain and blood
Immediately after the electrophysiological experiments, the blood of rats was
extracted from the abdominal aorta and then sacriced. A portion of brain tissues
were weighed and put into polytetrauoroethene (PTFE), then added 0.5 mL nitric
acid and 2 mL H2O2 per 500 g tissue, and incubated at 120 C for 2 h. The concentration of Al was measured by graphite furnace atomic absorption spectrophotometer.

Table 1
Effects of chronic exposure to Al on total body weight and brain weights (x SE, n = 10).
The values are signicantly different from controls (p < 0.01) are indicated by.
Group (%)

Total body weight

(g)

Brain weight
(g)

Brain of total body

weight (%)

Control
0.2% AlCl3
0.4% AlCl3
0.6% AlCl3

263.39 33.15
252.72 26.66
250.83 25.09
247.94 25.82

1.31 0.12
1.18 0.12
1.11 0.11
1.06 0.09

0.62 0.10
0.47 0.05
0.42 0.08
0.43 0.07

Table 2
Accumulation of Al in selected tissues of rats upon chronic exposure to Al (x SE,
n = 10). The values are signicantly different from controls (p < 0.01) are indicated by.
Group (lg g1)

Al accumulation
in blood (lg L1)

Al accumulation
in brain

Control
0.2% AlCl3
0.4% AlCl3
0.6% AlCl3

16.73 1.49
67.71 9.94
68.37 12.10
85.11 14.14

5.79 2.21
9.62 1.99
10.00 1.86
10.96 1.66

2.3. Western blot

Equivalent amounts of Ras, Raf1, ERK2 and CREB were resolved in 10% sodium
dodecylsulfatepolyacrylamide gel electrophoresis. After electrophoresis, proteins
were transferred to PVDF membranes. Membranes were incubated in Tris-buffered
saline with 0.5% Tween 20 (TTBS) containing 5% non-fat milk overnight at 4 C to
block non-specic binding. The blots were reacted with primary antibodies diluted
in 5% BSA for 4 h at room temperature (Ras 1:800, Raf1 1:200, ERK2 1:500, CREB
1:200). After washing in PBS 3  10 min the blots were incubated with the secondary antibody (1:2000, 3% BSA diluted) solution for 2 h. Membranes were then
washed three times with PBS. The bands were visualized by enhanced chemiluminescence system (ECL, Amersham Biosciences, Little Chalfont, UK) according to the
manufacturers instructions.
2.4. RT-PCR method for detection of CREB and real time-PCR method for detection of
Ras, Raf1, ERK2 in hippocampus
Total RNA was extracted from hippocampus using Trizol (Invitrogen). PCR primers for Ras, Raf1, ERK2, CREB and b-actin were as follows: Ras, forward: 50 TTTGCCATCAACAACACC-30 and reverse: 50 -TATGCTGCCGAATCTCAC-30 ; Raf1, forward: 50 -CAA TGGTTTCGGACTCAA-30 and reverse: 50 -GCGTCGGTGTTCCAATCT-30 ;
ERK2, forward: 50 -CACCCAGGCCTTCTCAATAG-30 and reverse: 50 -GCTGGCTAACTGAGGGTTCA-30 ; and CREB, forward: 50 -CACCCAGGCCTTCTCAATAG-30 and reverse:
50 -GCTGGCTAACTGAGGGTTCA-30 ; b-actin, forward: 50 -GCCAACCGTGAAAAGATG-30
and reverse: 50 -CCAGGATAGAGCCACCAAT-30 . The RNA was reverse-transcribed
with PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Gel image analysis system (Smartview 200l, Japan) was used to analyze each gray value for CREB
and b-actin. SYBR Green II-based detection was carried out for Ras, Raf1, ERK2 and
b-actin in ABI 7500 real-time PCR machine with thermal cycler conditions of: 95 C
for 30 s, followed by 40 cycles (95 C for 5 s and 60 C for 34 s).

group and the differences between the groups were statistically

signicant (p < 0.05).
3.2. The expression of Ras, Raf1, ERK2 and CREB at protein level
Western blot (Fig. 1A and B) results detected the expression of
hippocampal Ras. The expression of Ras was high in 0.2%, 0.4% and
0.6% Al exposure groups compared to the control group (p < 0.05).
The expression of Ras increased compared to control group, but
showed a decreasing trend along the exposure groups and the
differences between the groups were statistically signicant
(p < 0.05). The expression of Raf1 (Fig. 2A and B) showed a decreasing trend in the hippocampi of Al exposure groups compared with
control group and the differences between the groups were statistically signicant (p < 0.05). The expression of ERK2 (Fig. 3A and B)
showed a decreasing trend in the hippocampi of Al exposure
groups compared with control group and the differences between
the groups were statistically signicant (p < 0.05). The expression
of CREB (Fig. 4A and B) showed a decreasing trend in the hippocampi of Al exposure groups compared with control group and
the differences between the groups were statistically signicant
(p < 0.05).
3.3. The expression of Ras, Raf1, ERK2 and CREB at mRNA level

2.5. Statistical analyses

Data were compared between each Al exposed group and the control. SPSS statistical analysis software was used for data analysis. A p-value of less than 0.05 was
considered signicant. The statistical analysis was performed with one-way analysis of one way ANOVA followed by post hoc Dunnetts test.

3. Results
3.1. The results of effects of chronic exposure to Al on total body weight
and brain weights
As shown in Table 1, the total body weights for Al exposure
groups were not different compared to the control group
(p > 0.05). However, accumulation of Al signicantly reduced the
brain weights (816%). In addition, the ratios of brain to total body
weight were also signicantly reduced in groups of rats fed with
higher concentration of Al (0.4% or 0.6%). As shown in Table 2,
the accumulation of Al in selected tissues of rats upon chronic
exposure to Al showed an increasing trend compared with control

Real-time PCR was performed for Ras, Raf1 and ERK2, and general RT-PCR was used to detect the expression of CREB mRNA. To
measure mRNA expression levels of Ras, Raf1, ERK2 and CREB,
we used b-actin as internal reference. The mRNA expression levels
of Ras mRNA (Fig. 5A) in Al exposure groups signicantly increased
compared with the control group and showed a decreasing trend
between the exposure groups. The mRNA expression levels of
Raf1 mRNA (Fig. 5B) in Al exposure groups signicantly decreased
compared with the control group. The mRNA expression levels of
ERK2 were signicantly reduced in Al exposure groups when compared with the control group (Fig. 5C). The mRNA expression levels
of CREB signicantly reduced in Al exposure groups when compared with the control group (Fig. 5D).
4. Discussion
In this study, we added different concentrations of AlCl3 (0.2%,
0.4%, and 0.6%) to the drinking water for weaned rats (this

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317

Fig. 1. Expression of Ras protein in hippocampi of Al exposed rats. (A) Western blot results. (B) Graphical representation.

Fig. 2. Expression of Raf1 protein in hippocampi of Al exposed rats. (A) Western blot results. (B) Graphical representation.

Fig. 3. Expression of ERK2 protein in hippocampi of Al exposed rats. (A) Western blot results. (B) Graphical representation.

Fig. 4. Expression of CREB protein in hippocampi of Al exposed rats. (A) Western blot results. (B) Graphical representation.

situation is more likely to the actual human situation of intaking Al

through water or food). We found that compared to the control
group, the brain weight of Al-exposure rats were signicantly
changed. As the Al increased, the weights of rat brains decreased
and the differences were statistically signicant (p < 0.05). The

brain factors of 0.4% and 0.6% groups (brain weight/body weight 

100%) also decreased compared with the control which means that
Al exposure can affect brain development of rats. The concentration of Al in blood and brain increased with the increase of Al
concentration, indicating that Al can deposit in the brain through

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X. Cui et al. / Food and Chemical Toxicology 50 (2012) 315319

Fig. 5. Graphical representations for expressions of Ras, Raf1, ERK2 and CREB mRNA in hippocampi of Al exposed rats. (A) Ras, (B) Raf1, (C) ERK2 and (D) CREB.

the bloodbrain barrier. Al can disturb the balance of brain ion

metabolism which has close relationship with the development
of the central nervous system.
Learning and memory is a complex process. The process of human cognition involves synaptic plasticity changes which affect
information storage and memory consolidation process of brain
(Zhang and Luo, 2011; Dhatrak and Nandi, 2009). It includes the
synaptic transmission, the genes involved in synaptic plasticity,
and post-transcriptional synthesis of new proteins. The typical
mode of synaptic plasticity and the formation mechanisms of
learning and memory (Castillo et al., 2011) may be the LTP. The formation of LTP is induced by high frequency stimulation, which promotes the release of pre-synaptic glutamate neurotransmitter
which depolarizes the postsynaptic nerve cell and gradually relieves the Mg2+ blockade of NMDA receptors causing Ca2+ inux
through the NMDA receptor channels. The increased Ca2+ concentration can trigger a series of chemical reactions in cells.
We have proved that (Wang et al., 2010) upon chronic administration of Al for three months, the population spike (PS) amplitudes
after high-frequency stimulation(HFS), which is used to determine
the changes of LTP in the CA1 area of Al-treated rats, were dramatically decreased. The amplitude of PS in these rats did not increase
even 45 min after HFS. It is suggested that Al exposure induces
changes of LTP, with the increase of Al concentration, LTP decreased and lead to decline in the function of learning and memory
(Fig. 6). Al exposure causes the brain metabolic disorder and leads
to changes in expression or activation of a number of intracellular
signal transduction molecules related to the LTP, including protein
kinase A, protein kinase C, Ca-calmodulin dependent protein kinase II (CaMKII), extra-cellular signal regulated kinase (ERK), etc.
Many scholars have conrmed Ras/ERK signaling pathway
being related to the formation of LTP. Ca2+ inux into cells through
Ca2+ channels converts the non-activated Ras-GDP form into the
activated Ras-GTP form. Ras-GTP, then through Raf1/MEK, activates ERK2. After being activated, ERK2 transfers into the nucleus
where it phosphorylates some transcription factors such as CREB.
In this study, we used Western blot and real-time PCR method to

detect Ras. The results showed that compared to the control group,
the protein and mRNA levels of Ras in Al exposure groups increased. But, among the Al groups, the expression of Ras decreased
as the concentration of Al increased (p < 0.05), similar to the results
of Denayer et al. (2008). According to the original case, Ras activation leading to c-amino butyric acid (GABA)-mediated signal
enhancement resulted in learning dysfunction. LTP inhibition in
Al exposure groups seen in our results could also be due to increased expression of Ras protein and mRNA causing GABA-mediated signal enhancement, which nally induces. However, gradual
increase in Al exposure caused decrease in protein and mRNA levels of Ras.
The protein and mRNA expression levels of Raf1 (highly expressed in brain tissue) among the Al exposure groups decreased
with the increase in concentration of Al compared to the control
group (p < 0.05). Based on the experimental results, we can infer
that expression of Raf1, which is the downstream molecules of
Ras, is not affected by Ras in Al exposure groups, but is affected
by the concentration of Al as unlike Ras, the protein and mRNA
expression levels of Raf1 in Al exposure groups decreased.
The protein and mRNA expression levels of ERK2 (highly expressed in brain tissue) in Al exposure group decreased with the
increase in concentration of Al compared to the control group
(p < 0.05). ERK2 which plays an important role in cell activation,
migration, neuronal apoptosis, synaptic plasticity and memory is
a downstream molecule of Raf1 and is regulated by it (Schaeffer
and Weber, 1999). We can infer that expression levels of ERK2 in
Al exposure groups decreased as Raf1 decreased along the Al exposure groups. CREB is the downstream molecules of ERK2 and regulated by it (Davis et al., 2000). Affected by ERK2, the protein and
mRNA expression levels of CREB in Al exposure groups decreased.
CREB is a critical component of the neuroprotective transcriptional
network, and CREB dysregulation contributes to an array of neuropathological conditions (Sakamoto et al., 2011).
In summary, according to the chronic Al exposure in rats, as the
Al increase, the brain weight of rats decreased, LTP in the CA1 area
of hippocampus dropped, the protein and mRNA expression of Ras

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319

Fig. 6. (A) Percent changes of PS amplitudes. Baselines prior to stimulation are reported as 100%. The values are signicantly different from controls (p < 0.05) are indicated
by. (B) Comparison of PS amplitude 5 min prior and 30 min after HFS. A representative single curve for each group was shown. (a) Comparison in the control group. (b)
Comparison in the 0.2% AlCl3 treated group. (c) Comparison in the 0.4% AlCl3 treated group. (d) Comparison in the 0.6%AlCl3 treated group.

were increased (compared with the control group but decreased

between the Al exposure groups), and the protein and mRNA
expression of Raf1, ERK2 and CREB decreased (compared with
the control group and along the Al exposure groups). Therefore,
Al exposure can affect the normal function of Ras/Raf/ERK2/CREB
pathway and may lead to learning and memory impairment.
Conicts of Interest
The authors declare that there are no conicts of interest.
Acknowledgement
This work was supported by the Higher Research Project of
Liaoning province of China: No.2008848, No. 2008737 and No.
2009A761.
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