Neurotoxicology and Teratology 24 (2002) 751 – 757
www.elsevier.com/locate/neutera
Chronic fluoride toxicity decreases the number of nicotinic
acetylcholine receptors in rat brain
Yi-Guo Longa, Ya-Nan Wangb, Jia Chena, Su-Fen Jianga,
Agneta Nordbergc, Zhi-Zhong Guana,c,*
a
Department of Pathology, Guiyang Medical College, Guiyang 550004, Guizhou, PR China
Department of Scientific Research Administration, Guiyang Medical College, Guiyang 550004, Guizhou, PR China
c
Department of NEUROTEC, Division of Molecular Neurophamarcology, Karolinska Institutet, S-14186 Stockholm, Sweden
b
.
Received 25 January 2002; received in revised form 23 April 2002; accepted 7 June 2002
Abstract
In order to investigate the molecular mechanism(s) underlying brain dysfunction caused by chronic fluorosis, neuronal nicotinic
acetylcholine receptors (nAChRs) in the brain of rats receiving either 30 or 100 ppm fluoride in their drinking water for 7 months were
analyzed in the present study employing ligand binding and Western blotting. There was a significant reduction in the number of
[3H]epibatidine binding sites in the brain of rats exposed 100 ppm of fluoride, but no alteration after exposed to 30 ppm. On the other hand,
the number of [125I]a-BTX binding sites was significantly decreased in the brains of rats exposed to both levels of fluoride. Western blotting
revealed that the level of the nAChR a4 subunit protein in the brains of rats was significantly lowered by exposure to 100 ppm, but not 30
ppm fluoride; whereas the expression of the a7 subunit protein was significantly decreased by both levels of exposure. In contrast, there was
no significant change in the level of the b2 subunit protein in the brains of rats administered fluoride. Since nAChRs play major roles in
cognitive processes such as learning and memory, the decrease in the number of nAChRs caused by fluoride toxicity may be an important
factor in the mechanism of brain dysfunction in the disorder.
D 2002 Elsevier Science Inc. All rights reserved.
Keywords: Brain; Fluoride; Nicotinic acetylcholine receptors; Toxicity
1. Introduction
Chronic fluoride toxicity represents a severe hazard to
human health in several developing countries [22]. Furthermore, adverse health effects in response to fluoridation of
drinking water and exceptionally high exposure to fluoride
have also been observed in certain developed countries [29].
Excessive accumulation of fluoride in the body can exert
toxic effects on many tissues and organs, giving rise to a
vast array of symptoms and pathological changes in addition
to the well-known effects on skeleton and teeth [6,39,40].
Interestingly, a link between excessive exposure to fluoride and dysfunction of the central nervous system has
been established. When exposure to high doses of fluoride
* Corresponding author. Department of NEUROTEC, Division of
Molecular Neurophamarcology, Huddinge Hospital, B84, S-14186 Stockholm, Sweden. Tel.: +46-8-5858-3898; fax: +46-8-5858-3860.
E-mail address: [email protected] (Z.-Z. Guan).
occurs for a prolonged period of time, the blood –brain
barrier no longer can prevent this ion from entering the
central nervous tissue and, consequently, fluoride accumulates in the brain [11,15]. Individuals affected in this manner
exhibit a variety of neurological symptoms, including partial
paralysis or spasticity of the arms and legs, severe headache,
visual disturbances and mental retardation [38]. In the brains
of experimental animals, a number of histopathological
changes, e.g., demyelinization, a decrease in the number
of Purkinje cells, thickening and disappearance of dendrites,
swelling of Nissl substance and pyknosis of individual
neurons have been observed following administration of
large doses of fluoride [6]. Furthermore, the severity of the
adverse effects of fluoride on the behavior of rats is directly
correlated with the concentration of this ion in the plasma
and in specific regions of the brain [28]. In previous
investigations, we detected disturbances in brain development in the offspring of rats with chronic fluorosis [15].
In addition, the latency of the pain reaction and conditioned
0892-0362/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 8 9 2 - 0 3 6 2 ( 0 2 ) 0 0 2 7 3 - 8
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Y.-G. Long et al. / Neurotoxicology and Teratology 24 (2002) 751–757
reflex are longer in rats receiving high concentrations of
fluoride in their diets than in control animals [26]. Furthermore, alterations in the density of neurons and in the
number of undifferentiated neurons were observed in the
brains of fetuses therapeutically aborted in an area characterized by endemic fluorosis, suggesting that fluorosis exerts
harmful effects on the developing human brain as well [5].
Meanwhile, the mental work capacity and the Intelligence
Quotient of children born and raised in the area with
endemic fluorosis were found to be reduced [23,24].
Recently, we have detected the alterations of cellular membrane lipids in rat brain affected by chronic fluorosis [13].
The lipid composition determines the fluidity, stability and
permeability of membranes, and thereby influences function
of enzymes, ion channels and protein receptors.
The nicotinic acetylcholine receptors (nAChRs) that
locate in the cellular membrane of neurons are transmittergated ion channels composed of a and ß subunits [12]. To
date, nine different a (a2 –a10) and three b (b2 –b4) subunits have been cloned. Different a and ß subunits or
different a subunits alone are combined in various ways
to form receptor subtypes demonstrating physiological,
pharmacological and anatomical distributions [32]. Importantly, nAChRs play major roles in cognitive processes such
as learning and memory [21,31]. Specially, based on our
recent results that the changed lipid compositions in cellular
membrane in animal brains were resulted from fluorosis, it
is very interesting to further investigate if nAChRs are
affected by the presence of excessive amounts of fluoride.
In the study, we exposed rats to two different levels of
fluoride for a prolonged period and thereafter quantitated the
numbers of these receptors employing ligand binding and
Western blotting. Fluoride toxicity was found to decrease
the number of nAChRs in the rat brain, which may be an
important factor in connection with the mechanism of brain
dysfunction due to chronic fluorosis.
2. Methods
2.1. Chemicals
Goat polyclonal anti-a4, -a7 and -b2 antibodies, and
anti-goat IgG conjugated with horseradish peroxidase were
purchased from Santa Cruz Biotechnology (USA). [3H]Epibatidine, [125I]a-bungarotoxin (a-BTX), hyper performance
chemiluminescence film and protein-site markers (Rainbow)
were purchased from Amersham (Representative Office in
China). DC protein assay kit was obtained from Bio-Rad
(USA). The other chemicals employed were generally
purchased from Sigma (Representative Office in China).
2.2. Experimental animals
Forty-eight Wistar rats, weighing 100– 120 g upon initiation of this study, were divided randomly into three groups
each containing eight male and eight female animals. The diet
of all of the animals contained low-fluoride (4 ppm fluoride)
in order to avoid possible interference from the level of
fluoride in the drinking water. Throughout the study, the
animals were housed in stainless-steel cages suspended in
stainless-steel racks, the humidity ranged from 30% to 55%
and the temperature remained between 22 and 25 C. The
control rats were given access to drinking water containing
0.6 ppm fluoride. The other two groups of rats received the
same water to which was added 30 or 100 ppm fluoride
(NaF). This diet and water were administered to ad libitum for
seven months, after which the animals were sacrificed and
their brains dissected out and stored at 80 C until further
analysis. However, food consumption was not measured due
to a difficulty to determine exactly eating-amount of the
animals during the long-term exposure of fluoride.
2.3. General examinations
Successful achievement of chronic fluorosis was monitored as described previously [13]. Briefly, the fluoride
concentrations in the urine and brain tissue were determined
[20] using a CSB-F-I fluoride ion electrode (Changsa
Analysis Instrumentation, China) and the animals’ teeth
were examined for dental fluorosis. For light microscopy,
slices of brain tissue were stained with hematoxylin-eosin
(H.E.). For examination with an electron microscope (100
CX-2, Hitachi, Japan), sections of brain tissues (mainly the
frontal cortex and temporal cortex) were stained with uranyl
acetate and lead citrate.
2.4. Assays for [3H]epibatidine and [125I]a-bungarotoxin
bindings
The [3H]epibatidine binding assay was performed as
described previously [18]. Briefly, whole brain tissues
(except cerebellum and brainstem) were homogenized in
50 mM Tris – HCl buffer (pH, 7.4) and subsequently centrifuged at 10,000 rpm for 15 min at 4 C. The resulting pellets
were washed twice and resuspended in the binding buffer.
Protein contents in the samples were measured by DC
protein assay kit. Aliquots of this cellular membrane fraction (200 mg protein) were incubated with 2.5 nM [3H]epibatidine (specific radioactivity 54.6 Ci/mmol) in the binding
buffer at 25 C for 3 h. Thereafter, the samples were passed
through Whatman GF/C glass filters (presoaked with a 0.3%
solution of polyethyleneimine for 3 –4 h) and washed three
times with assay buffer. The radioactivity trapped on the
filters was counted in a scintillation counter (LSC-3500,
Aloka, Japan). Nonspecific binding was determined in the
same manner, but in the presence of 0.1 mM ( )-nicotine.
[125I]a-Bungarotoxin (a-BTX) was employed to assay
binding to a7 receptor [17]. Whole cerebral tissues were
homogenized in ice-cold 10 mM phosphate buffer (pH 7.4)
containing 50 mM NaCl and subsequently centrifuged at
60,000 g for 20 min at 4 C. The resulting pellets were
Y.-G. Long et al. / Neurotoxicology and Teratology 24 (2002) 751–757
753
Table 1
Body weight and fluoride levels in urine and brain from rats treated with fluoride and controls
Body weight (g)
Control
30 ppm F
100 ppm F
Urine fluoride (ppm)
Brain fluoride (ppm)
Male
Female
Male
Female
Male
Female
380 ± 29 (n = 8)
330 ± 21 * (n = 8)
286 ± 22 * * (n = 8)
277 ± 21 (n = 8)
233 ± 18 * (n = 8)
195 ± 17 * * (n = 8)
2.36 ± 0.16 (n = 8)
6.57 ± 0.47 * * (n = 8)
9.72 ± 0.58 * * (n = 8)
2.24 ± 0.18 (n = 8)
5.95 ± 0.34 * (n = 8)
9.19 ± 0.66 * * (n = 8)
0.35 ± 0.04 (n = 4)
1.31 ± 0.07 * (n = 4)
1.69 ± 0.08 * * (n = 4)
0.41 ± 0.05 (n = 4)
1.28 ± 0.06 * (n = 4)
1.57 ± 0.09 * * (n = 4)
The values are the means ± S.D.
* Significant difference, P < .05 as compared with controls, employing the analysis of variance (ANOVA), followed by the Student – Newman – Keuls test.
** Significant difference, P < .01 as compared with controls, employing the analysis of variance (ANOVA), followed by the Student – Newman – Keuls
test.
The levels of the a4, a7 and b2 nAChR subunit proteins
were quantitated by Western blotting as described previously [16,19]. Briefly, whole cerebral tissues were homogenized in ice-cold 50 mM sodium phosphate buffer (pH
7.4) at 4 C containing a protease inhibitor cocktail (Com-
plete, Roche Diagnostics) and subsequently centrifuged
at 60,000 g for 60 min, after which the pellets were
washed twice by recentrifugation in the same manner.
The resulting pellets were solubilized by resuspension in
the ice-cold buffer containing 2% Triton X-100. After
centrifugation at 100,000 g for 60 min at 4 C, protein
contents in the samples were measured by DC protein assay
kit and the solubilized membrane fraction in the supernatant
separated by electropheresis on 10% SDS-PAGE gels and
the bands thus obtained then transferred onto polyvinylidene difluoride (PVDF) membranes employing a transfer
unit (Bio-Rad).
For quantitation of the a4, a7 and b2 subunits, these
PDVF membranes were incubated with goat polyclonal
anti-a4 antibody (SC 1772, 0.25 mg/ml), anti-a7 antibody
(SC 1447, 0.25 mg/ml) or anti-b2 antibody (SC 1449, 0.25
mg/ml), respectively, for 120 min. After washing, the membranes containing bound anti-a4, -a7 or -b2 antibodies were
incubated with HRP-conjugated anti-goat IgG (0.04 mg/ml)
for 60 min. Finally, these membranes were placed in ECL
Plus reagent for 5 – 15 min and the resulting signals visualized by exposure to hyper performance chemiluminescence
film. Rainbow markers were employed as molecular weight
Fig. 1. [3H]Epibatidine binding in the brains of rats subjected to chronic
fluorosis as compared to control animals. Total membrane samples
(containing 200 mg protein) from brain were incubated with 2.5 nM
[3H]epibatidine. Each determination was performed in triplicate and the
results are expressed as means ± S.D. of the values for eight rats (four males
and four females). * P < .05 compared to the corresponding control value,
as determined employing the analysis of variance (ANOVA), followed by
the Student – Newman – Keuls test.
Fig. 2. [125I]a-Bungarotoxin binding in the brains of rats subjected to
chronic fluorosis as compared to control animals. Total membrane
preparations (containing 100 mg protein) from brain were incubated with
2 nM [125I]a-bungarotoxin. Each determination was performed in triplicate
and the results are expressed as means ± S.D. of the values for eight rats
(four males and four females). * P < .05, * * P < .01 compared to the
corresponding control value, as determined employing the analysis of
variance (ANOVA), followed by the Student – Newman – Keuls test.
washed twice and resuspended in the binding buffer containing 0.1% BSA. Protein contents in the samples were
measured by DC protein assay kit. Samples of this membrane preparations (100 mg protein) were then incubated
with 2 nM [125I]a-BTX (specific radioactivity 260 Ci/
mmol) for 30 min at 25 C. Binding was terminated by
the addition of 1 ml cold binding buffer and thereafter
centrifuging the samples at 60,000 g for 5 min at 4 C and
washing the pellets twice by centrifugation. The bottom of
the microtube containing each pellet was cut off and
counted in a g-counter (Wall ACK-1261, Shengzhen,
China). Nonspecific binding of [125I]a-BTX was determined in the same manner except that the samples were
preincubated with 1 mM unlabelled a-BTX for 30 min at 37
C prior to addition of [125I]a-BTX.
2.5. Analysis of nAChR subunit proteins by Western blotting
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Y.-G. Long et al. / Neurotoxicology and Teratology 24 (2002) 751–757
Table 2
[3H]Epibatidine and [125I]a-BTX binding sites in brains from male and
female rats with fluorosis and controls
Control
30 ppm F
100 ppm F
[3H]Epibatidine
(fmol/mg protein)
[125I]a-BTX
(fmol/mg protein)
Male
Female
Male
Female
60.2 ± 5.5
54.8 ± 3.4
46.5 ± 7.6
59.2 ± 4.0
54.0 ± 2.4
45.0 ± 7.0
29.3 ± 2.1
20.4 ± 2.3
12.5 ± 5.2
29.6 ± 0.4
18.7 ± 8.8
16.3 ± 8.7
statistical significance employing the analysis of variance
(ANOVA), followed by the Student – Newman– Keuls test.
3. Results
3.1. General observations
The results are expressed as means ± S.D. of the values
for the different groups. These data were examined for
Chronic toxicity in the rats exposed to high doses of
fluoride in their drinking water for 7 months was manifested
in the same manner as observed previously [13,14]. Dosedependent increases in the concentrations of fluoride in the
urine and brain tissues and decreases in body weight were
observed in animals exposed to chronic fluorosis (Table 1).
Dental fluorosis was observed primarily in the group receiving 100 ppm fluoride, but also in some of the rats treated
with 30 ppm fluoride. With light microscopy, no abnormal
histomorphological changes were seen in any regions of the
brains of rats with chronic fluorosis. However, electron
microscopic examination revealed swelling of mitochondria
and dilation of the endoplasmic reticulum in neurons in the
frontal and temporal cortices of rats, especially those in the
group receiving 100 ppm fluoride.
Fig. 3. Level of the a4 subunit protein in the brains of rats subjected to
chronic fluorosis as compared to control animals. Solubilized membrane
fractions from brain were subjected to SDS-PAGE (30 mg protein per lane),
blotted onto PVDF and subsequently incubated with polyclonal anti-a4
antibody. (A) The size of the immunoblotted a4 band as determined by
comparison to Rainbow molecular weight markers. (B) Quantitation of the
level of the a4 subunit protein. The results (means ± S.D.) are expressed as
a percentage of the mean control value (100%) from 10 rats (5 males and 5
females). * * P < .01 compared to control value, as determined employing
the analysis of variance (ANOVA), followed by the Student – Newman –
Keuls test.
Fig. 4. Level of the a7 subunit protein in the brains of rats subjected to
chronic fluorosis as compared to control animals. Solubilized membrane
fractions from brain were subjected to SDS-PAGE (30 mg protein per lane),
blotted onto PVDF and subsequently incubated with polyclonal anti-a7
antibody. (A) The size of the immunoblotted a7 band as determined by
comparison to Rainbow molecular weight markers. (B) Quantitation of the
level of the a7 subunit protein. The results (means ± S.D.) are expressed as
a percentage of the mean control value (100%) from 10 rats (5 males and 5
females). * P < .05, * * P < .01 compared to control value, as determined
employing the analysis of variance (ANOVA), followed by the Student –
Newman – Keuls test.
Each determination was performed in triplicate and the results are expressed
as means ± S.D. of the values for four rats. No significant differences of
receptor binding sites in brains between male and female animals as
determined employing Student t test.
standards for the proteins. The signal intensity of each band
on the film was quantitated employing a computer-assisted
image system based on the public domain NIH Image
Program [33] and the values thus obtained were expressed
as percentages of the average corresponding control values.
2.6. Statistical analysis
Y.-G. Long et al. / Neurotoxicology and Teratology 24 (2002) 751–757
3.2. Binding sites for [3H]epibatidine and [125I]a-BTX in
the brains of rats subjected to fluorosis as compared to
control animals
A significant 21% reduction in the number of binding
sites for [3H]epibatidine was observed in the brains of rats
exposed to 100 ppm fluoride, but there were no changes
with 30 ppm fluoride (Fig. 1). The numbers of [125I]a-BTX
binding sites in the brains of rats exposed to 30 or 100 ppm
fluoride were decreased significantly by 34% and 48%,
respectively, compared to control animals (Fig. 2). There
were no significant differences of these ligand-binding sites
between male and female animals (Table 2).
3.3. Levels of nAChR subunit proteins in the brains of rats
subjected to chronic fluorosis as compared to control
animals
In the brains of rats, protein bands exhibiting molecular
weights of 57, 55 and 58 kDa were detected using anti-a4,
-a7 and -b2 antibodies, respectively (Figs. 3A, 4A and 5A).
The level of the a4 subunit proteins was significantly
lowered by 24% in the brains of rats exposed to 100 ppm
fluoride (Fig. 3B), but no changes was observed in the case
of 30 ppm fluoride. With respect to a7, significant decreases
Fig. 5. Level of the b2 subunit protein in the brains of rats subjected to
chronic fluorosis as compared to control animals. Solubilized membrane
fractions from brain were subjected to SDS-PAGE (30 mg protein per lane),
blotted onto PVDF and subsequently incubated with polyclonal anti-b2
antibody. (A) The size of the immunoblotted b2 band as determined by
comparison to Rainbow molecular weight markers. (B) Quantitation of the
level of the b2 subunit protein. The results (means ± S.D.) are expressed as a
percentage of the mean control value (100%) from 10 rats (5 males and
5 females). No statistically significant differences were observed.
755
were observed in the brains of rats exposed to either 30 ppm
(decreased by 21%) or 100 ppm (decreased by 36%) (Fig.
4B). In contrast, chronic fluorosis resulted in no significant
change in the level of the b2 subunit protein (Fig. 5B).
There were no significant differences of these nAChR
subunit proteins in brains between male and female animals
(data not shown).
4. Discussion
Chronic fluorosis results from excessive exposure to
fluoride for a prolonged period of time. In the case of
animals administered high doses of fluoride in their food or
drinking water, increased levels of fluoride are detected not
only in the urine and plasma, but even in soft organs such as
the brain, liver and kidney [28,35]. These findings indicate
that fluoride ions can cross the intestinal barrier and be
distributed throughout the body via the blood and deposited
in various tissues. In the present study, increased fluoride
contents in the urine and brain, decreased body weight,
specific dental lesions and abnormal brain histology were
observed in rats receiving 30 or 100 ppm fluoride in their
drinking water for 7 months. These observations are in
agreement with our previous findings [13,14], confirming
that chronic fluoride toxicity was achieved here [14,36].
nAChRs are expressed by several populations of cells
present in the cortical, hippocampal and cerebellar regions
of brain. It has been demonstrated that multiple subtypes of
functional neuronal nAChRs can be formed from various
combinations of subunits, including the a4b2, a3b2, a4b4
and a7 subtypes. However, the majority of the high-affinity
nAChRs in the brain appear to be of the heteromeric a4b2
and homomeric a7 subtypes [12]. Nicotinic receptors apparently play pivotal roles in a number of functional processes
in the central nervous system, including learning and
memory, and are involved in several diseases of the brain,
as well as mediating the addiction to nicotine observed in
chronic tobacco users [32].
In the present study, we examined the number of
nAChRs, both with respect to ligand binding sites and
subunit protein levels, in the brains of rats exposed chronically to high doses of fluoride. A decline in [3H]epibatidine
binding was observed upon exposure to 100 ppm fluoride.
Since epibatidine is the most potent agonist for a4- and a3containing subtypes of nAChR, exhibiting extremely high
affinity for such receptors [12], this decreased [3H]epibatidine binding may reflect a loss of these types of nAChRs.
Nicotinic receptors of the a7 subtype were assayed here by
[125I]a-BTX binding. Significant decreases in this binding
were observed in the brains of rats treated with 30 ppm
( P < .05) or 100 ppm fluoride ( P < .01), as compared to
control animals. Recently, the a7 subunit has received
considerable attention, due to the high density of receptors
containing a7 in the hippocampus and neocortex; the ability
of this subunit to form homooligomeric receptors; and the
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Y.-G. Long et al. / Neurotoxicology and Teratology 24 (2002) 751–757
apparent involvement of a7-containing nAChRs in several
types of learning and memory-related behavior [4,27,34].
Thus, the clear decrease in [125I]a-BTX binding observed
here might be important in connection with deficits of brain
function resulting from fluorosis. However, since the singlepoint ligand binding used here does not distinguish the
changes between receptor affinity and maximal binding
capacity (Bmax), a saturation binding experiment for [3H]epibatidine or [125I]a-BTX will be carried out in future to
determine the differences. In addition to monitoring ligand
binding, we examined the expression of nAChR subunits at
the protein level by Western blotting method. A significantly
lower level of the a4 subunit was observed in the brains of
rats exposed to 100 ppm fluoride than in control animals. In
addition, significant decreases in the level of the a7 subunit
protein were found in the brains of rats receiving both 30 or
100 ppm fluoride, i.e., more severe changes than those
detected in the case of the a4 subunit. However, there were
no any significant changes in the level of the b2 subunit
protein in association with chronic fluorosis, indicating the
expression of this subunit is more stable under these conditions [19]. These changes of nAChRs in the brains of rats
subjected to chronic fluorosis may reflect alterations in the
synthesis of these receptors at different levels, e.g., transcription, translation and posttranslational modifications, as
well as alterations in receptor insertion into the membrane
and subsequent turnover. Neuronal membranes exhibit a
highly balanced composition of specialized lipids involved
in numerous cellular functions, including receptor binding
and neurotransmission, ion transport, signal transduction and
enzyme activities [8 – 10]. In a previous investigation, we
characterized changes in the membrane lipids of the brains of
rats exposed to 30 or 100 ppm fluoride for a prolonged
period of time, which showed the decreased level of phospholipids and the modification of ubiquinone [13]. Since the
abnormal cellular membrane structure can influence function
of protein receptors, the changed lipid composition might be
associated with the mechanism of nAChR deficit in brain
affected by fluorosis. Interestingly, it has been reported that
excessive fluoride can induce lipid peroxidation and consequently decrease the proportion of polyunsaturated fatty
acids resulted in the attack by free radicals [13]. In recent
investigations by employing cultural cell lines treated with
free radical inducers, we found that lipid peroxidation
directly resulted in lower expression of nAChRs [17,18].
Therefore, it is plausible that oxidative stress induced by
excessive fluoride may play an important role in the mechanism of down-regulation of nAChRs.
Sex- and dose-specific behavioral deficits have been
observed in the rats with fluorosis [28]. Males were most
sensitive to prenatal days 17 – 19 exposure of fluoride,
whereas females were more sensitive to weanling and adult
exposures [28]. However, there were no differences of
nAChR binding sites and protein levels in brains between
male and female animals exposed to high dose of fluoride in
the study we presented.
Recently, it has indicated a connection of nAChRs with
certain brain disorders that are associated with dementia,
such as Parkinson disease, Alzheimer’s disease [25,30],
schizophrenia [1,16] and Down syndrome [7]. There always
is a large loss in the number of nAChRs in the brains
affected by these diseases. More interestingly, Down syndrome, as a phenotypic result of trisomy 21, is a complex
condition with multiple and variable neurobiologic and
neuropsychologic manifestations [3]. Early studies showed
a positive link between Down syndrome and natural fluoridation, in which the age of women bearing the children
with mongolism was decreased, relating directly to the
exposure to increasing fluoride [2]. Recently, it has been
further confirmed that fluoride from daily food may contribute to the births of Down syndrome, suggesting one of
the major causes of the disorder other than aging of mothers
[37]. Furthermore, the alterations of nAChRs observed in
the brain from Down syndrome might provide a cue for a
connection of these receptors with fluorosis.
Since nAChRs play major roles in cognitive processes
such as learning and memory, the decrease in the number of
nAChRs caused by fluoride toxicity may be an important
factor in the mechanism of brain dysfunction in the disorder.
Acknowledgements
This work was financed by grants from the National
Natural Science Foundation of China (30060026) and the
Government Foundation of Guizhou Province of China.
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