Journal of Medicine and Medical Sciences Vol. 3(4) pp. 247-253, April 2012
Available online@ http://www.interesjournals.org/JMMS
Copyright © 2012 International Research Journals
Full Length Research Paper
Effects of acute oral administration of sodium fluoride
on the activities of sialidase and brain of adult mice
Wilson JI1., Nok AJ2., Hambolu JO3., Esievo KAN4
1
Department of Anatomy and Cell Biology, Delta State University, Abraka. Nigeria
2
Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria
3
Department of Vet. Anatomy, Ahmadu Bello University, Zaria, Nigeria
4
Department of Vet. Pathology, Ahmadu Bello University, Zaria, Nigeria
Abstract
The use of fluorine and its compounds is widespread and adverse effects have been documented.
Fluoride compounds which are put in water (fluoridation), toothpaste and supplement tablets cross the
blood brain barrier are said to be mutagenic and destroys brain cells. Aim: Determination of sialic acid
levels in the brain tissues, haemoglobin-free erythrocytes and serum, activities of the enzyme substrate
4-Methylumbelliferyl-Neu5Ac and the histological changes of the hippocampus of mice following acute
oral administration of Sodium Fluoride. Forty eight mice were subdivided into eight groups of six with
weights ranging between 20 and 34 grams. Daily oral dose of 0mg, 0.5mg, 1mg, 5mg, 10mg, 15mg, 20mg
and 30mg/kg BW/day of fluoride was administered to the mice in groups I, II, III, IV, V, VI, VII and VIII
respectively for fourteen days. Blood samples and brain tissues were collected for sialic acid analyses
and histological changes. The data obtained were subjected to statistical analysis and showed
statistically significant differences between the experimental groups (p < 0.05). The dose of 20mg/kg b
wt./day of fluoride showed sparse distribution of pyramidal cells in the pyramidal cell layer of
hippocampus. 20mg/ml of Sodium fluoride even in acute use may be toxic to the brain.
Keywords: Fluoride, Brain, Erythrocytes, Serum, Sialidase.
INTRODUCTION
Fluorine is one of the 92 naturally occurring elements. It
is a member of the halogen family, which includes
chlorine, bromine and iodine. . The colour arises as a
result of absorption of light on promoting an electron from
ground state to excited state. As a result it is never found
free in nature but only combined with other elements.
These compounds are called fluorides. Fluorine readily
forms compounds with all elements except helium and
neon. Fluorine has a symbol F, atomic number of 9, and
an atomic weight (mass number) of 18.984 (Lee, 1996).
Varner et al (1998) found out that even 1ppm.fluoride, the
amount purposely added to United States water supplies,
facilitated uptake of aluminium into rats’ brain causing
amyloidal deposits similar to that found in Alzheimer’s
*Corresponding Author: Wilson JI; E-mail: docwiliju@yahoo.com
patients. Fluoride has been shown to be mutagenic. It
causes chromosome damage and interferes with the
enzymes involved in DNA repair in a variety of cell and
tissue studies (Mihashi and Tsutsui, 1996). In minute
doses, it accumulates in and is damaging to the
brain/mind development of children, i.e. it produces
abnormal behaviour in animals and reduces intelligence
quotient (IQ) in humans (Mullenix, 1995; Zhao et al,
1996). Where fluoridation has been discontinued in
communities from Canada, the former East Germany,
Cuba and Finland, dental decay has not increased but
has actually decreased (Maupome, 2001; Kunzel and
Fischer, 1997; Kunzel and Fischer, 2000; Seppa, 2000).
Sialic acid is a derivative of a nine-carbon
monosaccharide and is widely distributed throughout
human tissues and found in several fluids, including
serum, cerebrospinal fluid, saliva, urine, amniotic fluid,
and mother's milk. In experimental mammals, it is found
in high levels in the brain of mice (Useh et al., 2005). This
248 J. Med. Med. Sci.
informed the decision to evaluate the sialic acid levels in
the brain, heamoglobin-free erythrocytes and serum of
mice after acute oral administration of sodium fluoride for
fourteen days.
± 0.309, 1.325 ± 0.130, 1.470 ± 0.125 and 1.543 ± 0.175
respectively.
Brain bound sialic acid
MATERIALS AND METHODS
Enzyme substrate - 4 – myethylumbelliferyl α -D-N–
acetylneuraminate i.e. 4–MU–Neu5Ac was purchased
from Sigma, U. S. A. and Sodium fluoride, a product of
Hopkins and Williams, Essex. England, were used for the
experiment. Forty eight mice were subdivided into eight
groups of six with weights ranging between 20 and 34
grams. Daily oral dose of 0mg, 0.5mg, 1mg, 5mg, 10mg,
15mg, 20mg and 30mg/kg BW/day of fluoride was
administered to the mice in groups I, II, III, IV, V, VI, VII
and VIII respectively for fourteen days.
The mice in each group were sacrificed by cervical
dislocation and brain tissues for histological investigation
were fixed in Bouin’s fluid. Other brain tissues were
carefully removed whole and 1.00g was homogenized
with 1ml of deionized for determination of both brain free
and bound sialic acid. Blood samples were collected for
Haemoglobin-free erythrocytes membranes (Ghosts)
sialic acid which represents the bound and serum free
sialic acid levels were measured using the thiobarbituric
(TBA) assay according to Aminoff (1961). The distribution
of sialic acid is [SA]B + [SA]F = [SA]T; where SA
represents sialic acid, B, F, and T stand for bound, free
and total respectively.
Preparation of Haemoglobin-Free Erythrocyte
Membranes (Ghosts) was done according to Dodge et al
(1963). Sialidase Activity Assay was carried out as
described by Kleinadam et al., (2001).
The process of preparation of brain tissue for
histological examinations was separated into a number of
stages. These stages included: Fixation, Tissue
Processing, Sectioning, Staining and Photomicrography.
The tissues were stained using routine (H & E) staining
and special (silver) staining techniques as outlined by
Gurr (1962) and Gomori (Gomori, 1937 as cited by Cook;
1974).
P ≤ 0.05 was considered to be statistically
significant.
RESULTS
The mean values of sialic acid contained in figure 2 in
groups I (control), II, III, IV, V, VI, VII, and VIII are 7.325 ±
0.773, 10.465 ± 1.371, 3.970 ± 0.849, 8.477 ± 0.544,
9.997 ± 0.691, 11.293 ± 1.236, 11.022 ± 1.253 and 8.945
± 0.917 respectively. There was a statistically significant
difference between groups VI and III, VII and III, II and III,
V and III and VIII and III.
Sialic Acid of Haemoglobin-Free
Membranes (Ghosts cells)
Erythrocyte
The mean values of sialic acid as contained in figure 3
in groups I (control), II, III , IV, V, VI, VII, and VIII are
0.687 ± 0.0497, 0.850 ± 0.107, 0.638 ± 0.140, 0.457 ±
0.0969, 0.802 ± 0.0899, 0.767 ± 0.459, 1.078 ± 0.122 and
1.127 ± 0.238 respectively. There was a statistically
significant difference between groups VIII and IV, VII and
IV.
Serum Free Sialic Acid
The mean values of serum free sialic acid contained in
figure 4 in groups I (control), II, III, IV, V, VI, VII, and VIII
are 3.477 ± 2.087, 1.127 ± 0.102, 1.503 ± 0.280, 1.340 ±
0.200, 1.56 ± 0.151, 2.090 ± 0.344, 1.697 ± 0.549 and
2.717 ± 0.0617 respectively. When comparing the control
group I with groups II-VIII, the differences in the sialic
acid levels was not statistically significant..
ENZYME SUBSTRATE ACTIVITY
The mean values of enzyme activity as presented in
figure 5 in groups I (control), II, III, IV, V, VI, VII, and VIII
are 1.285 ± 0.186, 0.820 ± 0.061, 0.947 0.0229, 1.023 ±
0.495, 0.866 ± 0.160, 0.885 ± 0.270, 1.543 ± 0.719 and
0.685 ± 0.0952 respectively. The highest point of activity
of the enzyme substrate is at group VII with a mean value
of 1.543 ± 0.719 as can be seen from figure 5.
SIALIC ACID ANALYSES
HISTOLOGICAL FINDINGS
Brain free sialic acid
The mean values of sialic acid contained in figure 1 in
groups I(control), II, III, IV, VI, VII, and VIII are 2.868 ±
0.658, 1.765 ± 0.163, 4.410 ± 1.567, 1.570 ± 0.200, 1.700
The destruction of the pyramidal cells in the pyramidal
cell layer included loss of pyramidal shape of the cells
and sparse distribution of cells Figures 6, 7 and 8). The
pyramidal cells in the pyramidal cell layer of hippocampus
Wilson et al. 249
5
Brain Free sialic acid of mice (mg/ml)
4.5
4
3.5
3
2.5
Free sialic acid of
Brain tissue of mice
2
1.5
1
0.5
(3
0m
g)
G
8
(2
0m
g)
G
7
(1
5m
g)
G
6
(1
0m
g)
G
5
G
2
G
3
G
4
(5
m
g)
(1
m
g)
(0
.5
m
g)
G
1(
C
on
tro
l)
0
Figure 1: Brain Free Sialic Acid of mice after administration of sodium fluoride for 14 days.
NB: The decreased level of brain free sialic acid from groups IV – VIII shows the high activity of the enzyme sialidase. There
are differences between the experimental groups and the control. P value = 0.031
14
Brain Bound Sialic Acid(mg/ml)
12
10
8
6
4
2
(3
0m
g)
G
8
G
7
(2
0m
g)
(1
5m
g)
G
6
(1
0m
g)
G
5
G
2
G
3
G
4
(5
m
g)
(1
m
g)
(0
.5
m
g)
G
1(
C
on
tr
ol
)
0
Figure 2. Brain Bound Sialic Acid of mice after administration of sodium fluoride for 14 days.
NB: There is increased activity of the enzyme sialidase from groups II – III and the inhibitory process of
sialidase as shown in groups IV – VII. P value < 0.001
1.6
Sialicacidof Hb. Freeerythrocyte (mg/ml)
1.4
1.2
1
Sialic acid of Hb.
Free erythrocyte
0.8
0.6
0.4
0.2
(3
0m
g)
G
8
(2
0m
g)
G
7
(1
5m
g)
G
6
(1
0m
g)
G
5
(5
m
g)
G
4
(1
m
g)
G
3
(0
.5
m
g)
G
2
G
1(
C
on
tr
o
l)
0
Figure 3. Sialic Acid of Haemoglobin--Free Erythrocyte Membranes of mice after administration of sodium fluoride for
14 days.
NB: The increased activity of the enzyme sialidase from groups III – IV and the inhibitory process of sialidase as shown in
groups V – VIII. . P = 0.010
250 J. Med. Med. Sci.
4
3.5
SerumFree Sialic acid (mg/ml)
3
2.5
2
1.5
FSSA
1
0.5
G
8
(3
0m
g)
(2
0m
g)
G
7
G
6
(1
5m
g)
(1
0m
g)
G
5
G
4
(5
m
g)
(1
m
g
)
G
3
(0
.5
m
g)
G
2
G
1(
C
on
tro
l)
0
Figure 4. Serum Free Sialic Acid of mice after administration of sodium fluoride for 14 days.
NB: The decreased level of serum free sialic acid from groups II – VII shows the high activity of the enzyme sialidase.
Inhibition sets in at group VIII. P value = 0.637.
1.8
1.6
-12
4Mu-Neu5Ac(x10 m
M/hr)
1.4
1.2
1
0.8
0.6
4Mu-Neu 5Ac
0.4
0.2
(3
0m
g)
G
8
(2
0
m
g)
G
7
(1
5
m
g)
G
6
(1
0m
g
)
G
5
G
4
(5
m
g)
(1
m
g
)
G
3
(0
.5
m
g)
G
2
G
1(
C
o
nt
ro
l)
0
Figure 5. Enzyme substrate, 4 MU-Neu5Ac activities of mice after administration of sodium fluoride for 14 days.
NB: The highest point of activity of the enzyme substrate was at group VII. Otherwise there is low activity in all the other
groups when compared with the control. P value = 0.649.
P. L.
L - ML
N
Figure 6: Coronal section of Hippocampus. GROUP I (CONTROL) H & E Stain.
NB: L - ML (Lacuna-Molecular layer), N – Nuclei of pyramidal cells, P. L. – Pyramidal layer x 100
Wilson et al. 251
P. L
N
L - ML
LPC
SPC
Figure 7: Coronal section of Hippocampus. GROUP I (CONTROL) Silver Stain
(Gomori, 1937).
NB: L - ML (Lacuna-Molecular layer), N – Nuclei of pyramidal cells, P. L. – Pyramidal
layer, SPC. – Small pyramidal cell, LPC – Large pyramidal cell. X 100
Figure 8: Coronal section of Hippocampus.GROUP VII (20mg of
Fluoride).Silver Stain (Gomori, 1937)
NB: L – ML – Lamina-Molecular layer, N – Nuclei of pyramidal cells, P. L. –
Pyramidal cell layer. x 100
have undergone chromatolysis and eventually cell death
when a dose of 20mg/kg b. wt/day was orally
administered to the mice. The sparse distribution of
pyramidal cells in the pyramidal cell layer of group VII
(20mg/kg bwt/day can be seen when compared with the
control group I (0mg/kg b.wt/day).
252 J. Med. Med. Sci.
DISCUSSION
There were significant differences in both the brain free
and brain bound sialic acid levels between the
experimental groups. As can be seen in figure 1, the
brain free sialic acid in group II was lower when
compared with the control group. Group III had a higher
level of sialic acid. Lower levels were recorded from
groups IV-VIII. Observation in figure 2, showed that the
brain bound sialic acid level had an increase in group II,
decrease in group III. Thereafter, there were steady
increases of the levels of sialic acid from groups IV-VII.
There was an activation of sialidase at lower
concentrations of fluoride. But at higher concentrations,
there was inhibition of sialidase activity. This may be the
first report to the best of our knowledge in connection
with fluoride toxicity. There may likely be an activation of
sialyltransferase at higher concentrations of fluoride as
can be seen in figure 2 of brain bound sialic acid.
In group II that had 0.5mg of fluoride, the level of sialic
acid increased, which indicates a decreased activity of
the enzyme sialidase. There were decreases in the sialic
acid levels of mice in groups III and IV that had oral
administration of fluoride of 1mg and 5mg respectively,
indicating a high activity of sialidase. There were notable
increases in the levels of sialic acid from groups V – VIII
that had oral administration of fluoride of 10mg, 15mg,
20mg and 30mg of fluoride respectively showing a
decrease in the activity of sialidase.
The serum free sialic acid levels were not statistically
affected between the experimental groups. But there
were notable differences when the groups were
compared with the control. The sialic acid levels
decreased from groups II – VII, showing a high activity of
the enzyme sialidase. In group VIII, there was a decrease
in sialidase activity, i.e. it became inhibitory.
It was expected that with the decrease in the sialic acid
levels of haemoglobin-free erythrocyte membranes, it will
lead to anaemia because the dense coat of sialic acid
molecules covering the red blood cell is being removed
by deregulated sialidase action. Once the sialic acid is
removed, the galactose residues are demasked on the
red blood cell surface after removal of the sialic acid,
giving a signal for degradation by hepatocytes, leading to
anaemia. But this did not occur as reported by Wilson et
al., (2009). There was a steady increase in the sialic acid
levels of groups V-VIII as shown in figure 3. The liver has
the ability to synthesize sialic acid from Nacetylmanosamine and pyruvate and can release sialic
acid when there is requirement. This also may have
contributed to the high levels of sialic acid at higher
concentrations of fluoride, leading to no anaemia when
acute oral sodium fluoride was administered. This means
that there was an activation of sialidase at lower
concentrations of fluoride. At higher concentrations, there
was inhibition of sialidase activity. There was likely an
activation of sialyltransferase at higher concentrations of
fluoride. The quantitative and qualitative composition of
cell surface gangliosides plays an important role in cell
behaviour and results from the activity of sialidases and
sialyltranferases, enzymes that cleave and attach,
respectively, sialic acid (Schauer, 1985).
There were decreases in enzyme activity in groups II –
VI when compared with the control group I. The highest
point of activity of the enzyme substrate was at group VII
that had 20 mg of fluoride. The effect seen in group VII of
high sialidase activity was contrary to what was seen invivo. It may be associated with the difference in the
substrate used for the assay.
The destruction of the pyramidal cells in the pyramidal
cell layer included loss of pyramidal shape of the cells,
sparse distribution of cells in the pyramidal cell layer. The
pyramidal cells in the pyramidal cell layer of hippocampus
have undergone chromatolysis and eventually cell death
when a dose of 20mg/kg b. wt/day was orally
administered to the mice. The dose of 20mg/kg b wt./day
of sodium fluoride showed
sparse distribution of
pyramidal cells in the pyramidal cell layer when
compared with the control group.
CONCLUSION
In conclusion, 20mg of fluoride may be toxic to the blood
and brain and that acute oral administration of sodium
fluoride may lead to loss of pyramidal cells. This in turn
may lead to diseased conditions such as temporal lobe
epilepsy, Alzheimer’s disease and probably may lead to
recent memory loss, although this was not investigated.
ACKNOWLEDGEMENT
We sincerely thank Dr. N. Useh and Dr. Sani Adamu of
the Department of Vet. Pathology, Faculty of Vet
Medicine who assisted with the methodology of sialic acid
research. Dr. Emmanuel Balogun of the Department of
Biochemistry and the techlonogists Mr. Yakubu and
Bashiru of the Postgraduate laboratory, Department of
Biochemistry, Dr. S. B Danborno and Dr. J. Timbuak of
Department of Human Anatomy, Ahmadu Bello
University, Zaria. Nigeria, for their contributions.
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