Fluoride Vol. 32 No. 4 215-229 1999 Research Report
215
REVERSIBLE TOXICITY OF FLUORIDE AND ALUMINIUM IN
LIVER AND GASTROCNEMIUS MUSCLE OF FEMALE MICE
NJ Chinoya and Trupti N Patel
Ahmedabad, India
SUMMARY: Effects of administration of sodium fluoride and aluminium
chloride at doses of 10 mg and 200 mg/kg body weight, respectively, were
studied for 30 days on the liver and muscle of female mice (Mus musculus).
The possible therapeutic effects of vitamin C (AA, 15 mg/animal/day), Ca (25
mg/animal/day) or vitamin E (2 mg/animal/day) administered alone or in
combination on NaF+AlCl 3-treated animals during the withdrawal period
were also investigated. A decrease in the protein levels of liver and gastrocnemius muscle by F and Al might be due to alteration in metabolism. A
significant accumulation of glycogen levels in liver and muscle could be
correlated with reduced activity of phosphorylase. Hence carbohydrate metabolism was altered in these organs. The decline in the activity of SDH in
the gastrocnemius muscle and liver suggests reduction in their oxidative
metabolism which could be related to structural alterations in the mitochondria. A decrease in activity of cholinesterase in liver and muscle may result
from toxic effects of NaF, AlCl 3, and NaF+AlCl3, which might have led to alteration in the utilization of acetylcholine thus affecting the transmission of
nerve impulses in these tissues. Elevated levels of SGPT and SGOT may
indicate hepatic damage and changes in its function by the treatments. Upon
withdrawal of combined treatment, a significant recovery occurred in most
of the parameters of liver, muscle and serum. However, none of the values
were comparable to control. On the other hand, cholinesterase activity in
liver and muscle as well as phosphorylase in liver recovered partially. The
toxic effects of NaF+AlCl3 on all the parameters were reversed significantly
by the administration of AA, Ca, or vitamin E alone, probably due to the antioxidant action of AA and vitamin E and the role of calcium in metabolism.
By the combined treatment with these antidotes, complete recovery occurred and values were comparable to control in all the parameters studied
which might be the result of their additive or synergistic effect.
Keywords: Aluminium toxicity, Ascorbic Acid (vitamin C), Calcium, Enzyme disturbances,
Female mice, Gastrocnemius muscle, Liver intoxication, Muscle effects, Protein metabolism, Toxicity reversal, Sodium fluoride toxicity, Tocopherol acetate (vitamin E).
INTRODUCTION
Aluminium and fluoride are both known to be potential environmental
hazards. They are found together in the earth’s crust in the form of cryolite
(Na3AlF6).
Studies on the combined effects of aluminium (Al) and fluoride (F) in biological systems are very controversial. Aluminium and fluoride are mutually
antagonistic in competing for absorption in the gut. Aluminium is known to
decrease the intestinal absorption of fluoride and to increase its excretion in
humans, thereby reducing its toxicity. 1 However, fluoride enhances digestive
———————————————
aFor Correspondence: Reproductive Toxicology and Endocrinology Unit, Department of
Zoology, School of Sciences, Gujarat University, Ahmedabad-380 009, India.
216
Chinoy, Patel
absorption of aluminium.2 Dai et al have shown that the combined effects of
aluminium and fluoride aggravated toxicity in the blood and femur bone of
male chicks.3 Changes in the behavioral pattern of rats following chronic aluminium fluoride (AlF3) administration are known. 4 The aluminium levels in
brain and kidney of rats were found to be high and the extent of alterations in
neuronal density as well as cerebrovasculature were greater in animals exposed to AlF3 than to NaF and greater in the NaF group than in controls. 5 F-Al
combined toxicosis is endemic in the severe fluorosis regions of the
Shuicheng area of Guizhou province in South China. 6
In a differing view, consumption of drinking water containing elevated but
non-toxic levels of fluoride has been proposed for the prevention of Alzheimer’s Disease(AD) as aluminium is one of the contributing factors in the
occurrence of this disease.7
It is now well established that ingestion of fluoride not only affects the
teeth and bones but also other organs. Structural and biochemical changes of
several soft tissues have been reported in male and female rats and mice with
different doses of fluoride. 8-14 Fluoride causes neurotoxicity in rats, and the
effects were greater in early stage of intoxication. 15,16 In a study on male and
female rabbits, the lipid metabolism in the brain was altered due to fluoride
intoxication, and the neurons of the cerebellar cortex showed degeneration. 17
The animals had retarded development, paraplegia, and quadriplegia. Fluoride
has a specific effect on soluble, basic, and total protein in rabbit brain which
may lead to degenerative changes, viz., ballooning neurons, various degrees of
loss of Nissl substance, and changes in the Purkinje cells of the cerebellar cortex. These changes in the brain may lead to paralysis of limbs in fluorosed
animals. In another study, sodium fluoride treatment (20-40 mg/kg/day for 60
days) of adult female rats suppressed spontaneous motor activity, but no
change was observed in the motor coordination of these animals.18
Aluminium is known to be a neurotoxic agent and causes neurofibrillary
degeneration and neuronal loss. Aluminium accumulation has been associated
with various human diseases such as renal dialysis, senile dementia, dialysis
osteomalacia, microcytic hypochronic anemia, gastrointestinal toxicity, and
Alzheimer’s Disease (AD). In experimental animals, aluminium toxicity is
also characterized by progressive neurological impairment of learning and
memory performance as well as altered motor function. Aluminium is thus
known to cause epilepsy, information processing defects, cognitive dysfunction, and motor neuron disease. 19
Previous reports from our laboratory have revealed that aluminium chloride treatment caused alterations in the metabolism of male reproductive organs.20,21 Histological studies by Roy et al with aluminium salts [Al 2(SO4)3
and KAl(SO4)2] at different doses revealed degenerative changes in rat tissues
like liver, brain, kidney, and bone. 22
In light of the above data it was thought worthwhile to investigate the effects of sodium fluoride and aluminium chloride alone or in combination on
Fluoride 32 (4) 1999
F and Al toxicity in mice liver and gastrocnemius muscle
217
liver and muscle of female mice and the possible reversal of the effects by
some antidotes (vitamin C, calcium, and vitamin E).
MATERIALS AND METHODS
Healthy adult female albino mice (Mus musculus) of Swiss strain, weighing between 30-35 g were obtained from the National Institute Of Occupational Health (NIOH), Ahmedabad, India. The animals were divided into nine
groups as shown in the following protocol table. They were housed in an airconditioned animal house at a temperature of 26 ± 2°C and exposed to 10 to
12 h of daylight. They were maintained on standard chow and water was given ad libitum.
EXPERIMENTAL PROTOCOL
Group Treatment and Dose
IA
IB
IC
ID
IE
II
III
IV
V
VI
VII
VIII
IX
Control, Untreated
Control + Distilled water
Vehicle Treated
Control + Olive oil (0.2 mL/animal/day)
Positive Control
Control + ascorbic acid(AA) (15 mg/animal/day)
Control + calcium (Ca2+ ) (25 mg/animal/day)
Control + Vitamin E (2 mg/animal/day)
NaF (10 mg/kg body weight/mice/day)
AlCl3 (200 mg/kg body weight/mice/day)
NaF + AlCl3 (doses as in group II and III)
NaF + AlCl3 as in group IV,
on 31 day withdrawal for 30 days
Treatment withdrawal of group IV
Treatment + AA (dose as in group IC)
Treatment withdrawal of group IV + Ca2+
(dose as in group ID)
Treatment withdrawal of group IV + vitamin E
(dose as in group IE)
Treatment withdrawal of group IV + AA + Ca2+
+ Vitamin E (doses as in group IC, ID, IE)
Duration Day of Au- No. of
(Days)
topsy
Animals
Sacrificed
with treated
20
30
31st
20
30
30
30
30
30
30
30+30
31st
31st
31st
31st
31st
31st
61st
20
20
20
20
20
20
20
30+30
61st
20
30+30
61st
20
30+30
61st
20
30+30
61st
20
All treatments were given orally with a hypodermic syringe attached to an
angular needle. Animals in Groups IA-E served as controls (untreated, vehicle
treated, and positive controls). Sodium fluoride (NaF) (Loba Chemie, Mumbai,
99% purity) was administered in water (0.2 mL) to Group II animals at a dose
of 10 mg/kg body weight for 30 days. Aluminium chloride (AlCl3) (S.D. Fine
Chem. Ltd. Boisor 401501, 99.5% purity) was administered in water to Group
III animals at a dose of 200 mg/kg body weight for 30 days. The above doses
were established on the basis of LD50 values, viz. 51.6 mg/kg body weight for
NaF and 4 g/kg body weight for AlCl3, respectively.21,23 The Group IV animals
were fed NaF and AlCl3 orally in combination (doses as above) for 30 days.
Fluoride 32 (4) 1999
218
Chinoy, Patel
To study the reversibility of induced effects, the treatment of Group IV animals was withdrawn for 30 days. These became Group V animals. They were
maintained on standard food and water ad libitum. Animals in Groups VI, VII,
VIII and IX were administered ascorbic acid (AA) (15 mg/0.2mL distilled water/animal/day) (Loba Chemie, Mumbai, 99% purity), calcium (as calcium
phosphate suspension) (25 mg Ca2+/0.2 mL distilled water/animal/day) (Glaxo,
India, 99% purity) or vitamin E (2 mg/0.2 mL olive oil/animal/day) (Tocopherol
acetate, E. Merck, India Ltd. Mumbai, 99% purity) alone and in combination
(Group IX) during the withdrawal period for 30 days. The doses of ascorbic
acid, calcium, and vitamin E were based on earlier work.12,14
At the end of each treatment, the animals were weighed on an animal
weighing balance (Ohaus, USA) and sacrificed by cervical dislocation. For
serum, the blood was collected by cardiac puncture and kept at room temperature for one hour. It was stored in the refrigerator and serum was separated after 24 hours by centrifugation. The liver and gastrocnemius muscle were dissected out carefully, blotted free of blood, and weighed on a Roller Smith Torsion Balance (USA) to the nearest milligram and utilized for the study.
BIOCHEMICAL STUDY
1 Protein levels of liver and gastrocnemius muscle of control and all treated
groups of mice were estimated by the method of Lowry et al24 and expressed as mg/100mg fresh tissue weight.
2 Succinate Dehydrogenase (SDH) (E.C. 1.3.99.1) activity in both liver and
gastrocnemius muscle of control and all the treated groups was assayed according to the method of Beatty et al.25 The activity was expressed as g
formazan formed/15 min/mg protein.
3 Glycogen concentration in liver and gastrocnemius muscle of control and
treated groups of mice was estimated by the method of Seifter et al26 and
expressed as g/100 mg fresh tissue weight.
4 Phosphorylase (E.C. 2.4.1.1) activity in liver and gastrocnemius muscle of
control and all treated groups was assayed by the method of Cori et al27
and expressed as g phosphorus released/mg protein/15 min.
5 Cholinesterase (ChE) (E.C.3.1.1.7) activity was assayed in liver and gastrocnemius muscle of control and treated animals by the method of Huerga
et al28 and expressed as activity of ChE/mg protein.
6 Serum Transaminases (E.C. 2.6.1.2) Photometric determination of serum
glutamate pyruvate transaminase (SGPT) and serum glutamate oxalate
transaminase (SGOT) was carried out by the method of Reitman and
Frankel29 and expressed as mU/mL.
For all biochemical determinations, a minimum of 8-10 replicates were
performed for each parameter. The data were statistically analysed by Student’s ‘t’ test and Analysis of Variance (ANOVA).
Fluoride 32 (4) 1999
F and Al toxicity in mice liver and gastrocnemius muscle
219
RESULTS
Protein Levels In Liver: The protein levels in liver showed a significant
(p<0.001) decline after the treatments with NaF, AlCl 3 and NaF+AlCl3
(Groups II-IV) as compared to control (Groups IA-IE). Of all three treatments,
in Groups II-IV, the combined treatment (Group IV) brought about the greatest significant decline. Withdrawal (Group V) of the combined treatment
(Group IV) resulted in a significant (p<0.001) recovery. On administration of
vitamin C, calcium or vitamin E (Groups VI-VII) as well as their combined
treatment (Groups IX), complete recovery was observed (Table 1).
Protein Levels In Muscle: A significant (p<0.001) decline in the protein levels
of the gastrocnemius muscle occurred after the treatments with NaF, AlCl 3
and NaF+AlCl3 (Group II-IV) for 30 days. A highly significant (p<0.001) decrease occurred after the combined administration of NaF+AlCl 3 (Group IV).
The withdrawal of the treatment in Group V mice resulted in recovery
(p<0.01). A complete reversal of the combined toxicity was obtained after
treatment with vitamin C, calcium or vitamin E as well as their combination
(Groups VI-IX), and the values were the same as controls (Table 1).
Table 1. Protein levels (mg/100 mg tissue weight) in liver and
Gastrocnemius muscle of control and treated mice. (Groups IA-IX).
Group Treatment
Liver
Muscle
Control + Distilled water
33.40  0.26
Control + Olive oil
33.21  0.11
Control + Ascorbic Acid
33.44  0.16
Control + Calcium
33.23  0.10
Control + Vitamin E
33.34  0.13
NaF
22.04  0.11*
AlCl3
18.52  0.09*
NaF+AlCl3
14.82  0.08*
Treatment withdrawal of Group IV
24.88  0.10*
Treatment withdrawal of group IV + Ascorbic Acid 32.02  0.20*
Treatment withdrawal of group IV + Calcium
31.39  0.08*
Treatment withdrawal of group IV + Vitamin E
30.25  0.31*
Treatment withdrawal of group IV +
32.73  0.19*
AA + Ca2+ + Vitamin E
Values are mean ± S.E. *p<0.001
Comparison between: Group I and Groups II, III, IV
Group IV and Groups V, VI, VII, VIII & IX
IA
IB
IC
ID
IE
II
III
IV
V
VI
VII
VIII
IX
18.34  0.09
18.83  0.09
18.47  0.15
18.81  0.09
18.72  0.16
16.91  0.07*
15.12  0.10*
12.84  0.16*
14.69  0.10*
18.20  0.07*
18.14  0.12*
18.33  0.17*
18.56  0.12*
Glycogen Levels In Liver: A significant (p<0.001) accumulation of glycogen
levels occurred in the livers of mice following all three treatments in Groups
II, III and IV as compared to the control Groups IA-IE. The values were very
significant in Groups III and IV. A significant recovery (p<0.001) was obtained after the withdrawal period of 30 days in the Group V mice. Admin-
Fluoride 32 (4) 1999
220
Chinoy, Patel
istration of ascorbic acid, calcium, or vitamin E alone and in combination
(Groups VI to IX), brought about a complete recovery in the glycogen levels
of liver which was more pronounced in group IX treatment
(AA+Ca2++vitamin E) (Table 2).
Table 1A.
Source of Variation
Liver protein
Between Groups
Within Groups
Muscle protein
Between Groups
Within Groups
SS
df
MS
F-cal
F-tab
4990.695
67.75786
12
117
415.8913
0.579127
718.1348
1.835815
453.4415
17.37403
12
117
37.78679
0.148496
254.4634
1.835815
SS-Sum of Squares
df-degree of freedom
MS-Mean sum of squares
F-cal=Fischers Calculated
F-Tab=Fischers Tabulated
Table 2. Glycogen concentration (µg/100 mg tissue weight) in liver
and gastrocnemius muscle of control and treated mice.
Group Treatment
Liver
Control + Distilled water
1068.82  21.36
Control + Olive oil
1057.79  33.86
Control + Ascorbic Acid
1092.81  17.91
Control + Calcium
1069.55  16.49
Control + Vitamin E
1085.18  18.22
NaF
1349.99  16.32*
AlCl3
2079.57  17.67*
NaF+AlCl3
2160.17  13.03*
Treatment withdrawal of Group IV
1495.11  10.24*
Treatment withdrawal of group IV +
1135.42  18.37*
Ascorbic Acid
VII
Treatment withdrawal of group IV + Calcium 1176.23  13.92*
VIII Treatment withdrawal of group IV + Vit. E
1157.86  23.27*
IX
Treatment withdrawal of group IV +
1078.96  18.20*
AA + Ca2+ + Vitamin E
Values are mean ± S.E. *p<0.001
Comparison between: Group I and Groups II, III, IV
Group IV and Groups V, VI, VII, VIII & IX
IA
IB
IC
ID
IE
II
III
IV
V
VI
Muscle
792.40  13.77
808.30  15.38
776.14  14.56
792.68  14.59
787.83  7.38
1018.79  14.09*
1187.95  14.56*
1306.47  11.55*
1049.33  7.65*
884.07  11.37*
881.87  10.08*
866.78  8.96*
795.51  6.99*
Glycogen Levels In Muscle: Accumulation of glycogen levels was also seen in
muscle after the administration of all three treatments (NaF, AlCl 3, and
NaF+AlCl3) alone as in Groups II, III, IV. The combination of NaF+AlCl 3
(Group IV) brought about a highly significant (p<0.001) increase in the gly-
Fluoride 32 (4) 1999
F and Al toxicity in mice liver and gastrocnemius muscle
221
cogen levels as compared to Group II and III treatments. The withdrawal of
treatment (Group V) revealed a significant (p<0.001) recovery in the muscle
glycogen levels. However, when ascorbic acid, calcium, or vitamin E were
administered alone and in combination (Groups VI-IX), nearly complete recovery occurred. Maximum recovery occurred with the combined treatment of
antidotes (Group IX) during the withdrawal period (Table 2).
Table 2A.
Source of Variation
Liver Glycogen
Between Groups
Within Groups
Muscle Glycogen
Between Groups
Within Groups
SS
df
MS
F-cal
F-tab
14026345
408621.5
12
117
1168862
3492.492
334.6785
1.835815
3518373
167899.8
12
117
293197.8
1435.041
204.3131
1.835815
Phosphorylase Activity In Liver: Liver phosphorylase activity showed a significant (p<0.001) decline after NaF, AlCl 3, or NaF+AlCl3 treatments. Combined treatment (Group IV) reduced the enzyme activity very significantly
(p<0.001). On withdrawal of treatment (Group V), a partial recovery occurred.
However, antidotes administered alone or in combination (Groups VI-IX)
brought about a significant (p<0.001) recovery. The activity regained normal
status after Group IX treatment (Table 3).
Table 3. Phosphorylase activity (µg phosphorus released/mg protein/15 min) in
liver and gastrocnemius muscle of control and treated mice.
Group Treatment
Liver
Muscle
Control + Distilled water
15.66  0.11
Control + Olive oil
15.80  0.13
Control + Ascorbic Acid
15.82  0.08
Control + Calcium
15.73  0.13
Control + Vitamin E
15.63  0.12
NaF
7.32  0.14†
AlCl3
6.61  0.14†
NaF+AlCl3
5.20  0.25†
Treatment withdrawal of Group IV
8.25  0.19*
Treatment withdrawal of group IV +
15.25  0.09†
Ascorbic Acid
VII
Treatment withdrawal of group IV + Calcium
14.71  0.13†
VIII
Treatment withdrawal of group IV + Vit. E
15.14  0.18†
IX
Treatment withdrawal of group IV + AA +
15.56  0.13†
2+
Ca + Vitamin E
Values are mean ± S.E. *p<0.01 †p<0.001
Comparison between: Group I and Groups II, III, IV
Group IV and Groups V, VI, VII, VIII & IX
IA
IB
IC
ID
IE
II
III
IV
V
VI
12.06  0.08
11.96  0.11
12.03  0.12
11.82  0.16
11.88  0.13
9.01  0.08†
6.71  0.15†
4.68  0.10†
6.94  0.10†
11.17  0.12†
10.85  0.13†
11.13  0.17†
12.06  0.07†
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Chinoy, Patel
Phosphorylase Activity In Muscle: Activity of phosphorylase declined significantly (p<0.001) with NaF, AlCl 3, and their combined treatments (Groups IIIV). NaF+AlCl3 treatment bought about a highly significant (p<0.001) decline. After withdrawing the treatment, the enzyme recovered significantly
(p<0.001) in comparison to Group IV. Administration of vitamin C, calcium,
or vitamin E alone or in combination (Groups VI-IX) brought about a significant (p<0.001) recovery. The combined treatment of antidotes (Group IX)
caused a more pronounced recovery (Table 3).
Table 3A.
Source of Variation
Liver phosphorylase
Between Groups
Within Groups
Muscle Phosphorylase
Between Groups
Within Groups
SS
df
MS
F-cal
F-tab
2124
25.4483
12
117
177
0.217507
813.7674
1.835815
757.2165
16.65053
12
117
63.10137
0.142312
443.4009
1.835815
SDH Activity In Liver: Oral administration of NaF, AlCl 3, or NaF+AlCl3
(Groups II-IV) for 30 days caused a significant (p<0.001) decline in the activity of succinate dehydrogenase in liver. In the withdrawal animals (Group V)
the recovery of enzyme activity was not very significant (p<0.01). The treatments of vitamin C, calcium, or vitamin E alone restored the enzyme activity
significantly (p<0.001). The values were comparable to controls (Groups IAIE) after the combined treatment (Group IX) with all three antidotes (Table 4).
Table 4. SDH activity (µg formazan formed/15 min/mg protein)
in liver and gastrocnemius muscle of control and treated mice.
Group Treatment
Liver
Control + Distilled water
5.46  0.15
Control + Olive oil
5.06  0.05
Control + Ascorbic Acid
4.98  0.06
Control + Calcium
5.07  0.03
Control + Vitamin E
5.05  0.05
NaF
3.53  0.05*
AlCl3
2.85  0.11*
NaF+AlCl3
2.18  0.05*
Treatment withdrawal of Group IV
3.09  0.03*
Treatment withdrawal of group IV + Ascorbic Acid
4.17  0.03*
Treatment withdrawal of group IV + Calcium
4.29  0.11*
Treatment withdrawal of group IV + Vitamin E
4.17  0.02*
Treatment withdrawal of group IV + AA + Ca2+
5.02  0.05*
+ Vitamin E
Values are mean ± S.E. *p<0.001
Comparison between: Group I and Groups II, III, IV
Group IV and Groups V, VI, VII, VIII & IX
IA
IB
IC
ID
IE
II
III
IV
V
VI
VII
VIII
IX
Fluoride 32 (4) 1999
Muscle
19.50  0.21
19.02  0.17
19.51  0.22
19.31  0.17
19.28  0.31
15.80  0.17*
12.86  0.10*
10.69  0.13*
14.96  0.28*
17.21  0.21*
17.58  0.20*
17.02  0.15*
18.88  0.10*
F and Al toxicity in mice liver and gastrocnemius muscle
223
SDH Activity In Muscle: On administration of NaF, AlCl 3 alone, or combined
(Groups II-IV), the muscle SDH activity was reduced significantly (p<0.001).
Here also, the combined treatment (Group IV) was most effective. Withdrawal
of NaF+AlCl3 treatment did not bring about complete recovery in the enzyme
activity, although it was significant (p<0.001) as compared to the treated
Group IV. AA, Ca2+, or vitamin E alone and in combination (Group VI-IX)
resulted in nearly complete recovery of the enzyme activity (Table 4).
Table 4A.
Source of Variation
Liver SDH
Between Groups
Within Groups
Muscle SDH
Between Groups
Within Groups
SS
df
MS
F-cal
F-tab
126.8855
6.07714
12
117
10.5738
0.051941
203.5718
1.835815
928.4478
44.21072
12
117
77.37065
0.377869
204.755
1.835815
Cholinesterase Activity In Liver: All three treatments of Groups II, III, and IV
brought about a significant (p<0.001) decline in the activity of cholinesterase
in liver. The combined treatment of NaF and AlCl 3 reduced the enzyme significantly (p<0.001). Withdrawing treatment caused a significant (p<0.01) recovery in the enzyme activity. Administration of ascorbic acid, calcium and vitamin E alone or in combination resulted in a significant (p<0.001) recovery.
Recovery was maximum with the combined treatment of antidotes (Table 5).
Table 5. Cholinesterase (ChE/mg protein) in liver
and muscle of control and treated mice.
Group Treatment
Liver
Control + Distilled water
4.92  0.28
Control + Olive oil
4.65  0.24
Control + Ascorbic Acid
4.28  0.16
Control + Calcium
4.68  0.13
Control + Vitamin E
4.34  0.15
NaF
3.75  0.10†
AlCl3
2.87  0.05†
NaF+AlCl3
2.31  0.09†
Treatment withdrawal of Group IV
3.06  0.06*
Treatment withdrawal of group IV + Ascorbic Acid 4.04  0.06†
Treatment withdrawal of group IV + Calcium
3.90  0.13†
Treatment withdrawal of group IV + Vitamin E
3.91  0.08†
2+
Treatment withdrawal of group IV + AA + Ca
4.29  0.08†
+ Vitamin E
Values are mean ± S.E. *p<0.01 †p<0.001
Comparison between: Group I and Groups II, III, IV
Group IV and Groups V, VI, VII, VIII & IX
IA
IB
IC
ID
IE
II
III
IV
V
VI
VII
VIII
IX
Muscle
6.31  0.23
6.28  0.21
6.25  0.16
6.33  0.18
6.06  0.20
4.20  0.16†
3.62  0.16†
2.98  0.17†
3.82  0.21*
5.66  0.28†
5.75  0.29†
5.70  0.23†
6.17  0.23†
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Chinoy, Patel
Cholinesterase Activity In Muscle: The activity of gastrocnemius muscle cholinesterase declined significantly (p<0.001) after 30 days of NaF, AlCl 3, or
NaF+AlCl3 treatments (Groups II-IV). Upon withdrawal of Group IV treatment (Group V), there was a significant (p<0.01) recovery which was greater
with the treatment of antidotes (Group VI-IX). Combined treatment of vitamin
C, calcium, and vitamin E resulted in a synergistic effect for the complete recovery of the enzyme activity (Table 5).
Table 5A.
Source of Variation
Liver Cholinesterase
Between Groups
Within Groups
Muscle Cholinesterase
Between Groups
Within Groups
SS
df
MS
F-cal
F-tab
65.71029
21.37496
12
104
5.475858
0.205528
26.64282
1.846427
173.4157
52.63648
12
117
14.45131
0.449884
32.12227
1.835815
Activities of SGPT and SGOT: The activities of SGPT and SGOT were elevated significantly (p<0.001) following NaF, AlCl 3, or NaF+AlCl3 treatments.
Combined treatment of NaF and AlCl 3 caused a maximum increase in these
enzyme activities. Withdrawal of the combined treatment and administration of
ascorbic acid, calcium, and vitamin E alone or in combination brought about a
significant (p<0.001) recovery. The combined treatment with antidotes brought
about a complete recovery of both enzymes in serum (Table 6).
Table 6. SGPT and SGOT activities (mu/ml) in serum of control and treated mice.
Group
Treatment
SGPT
Control + Distilled water
12.2  1.20
Control + Olive oil
13.6  1.80
Control + Ascorbic Acid
13.8  1.56
Control + Calcium
13.6  0.68
Control + Vitamin E
12.8  1.32
NaF
23.8  1.50*
AlCl3
34.4  1.66*
NaF+AlCl3
45.0  2.73*
Treatment withdrawal of Group IV
32.0  2.49*
Treatment withdrawal of group IV + Ascorbic Acid 15.2  1.53*
Treatment withdrawal of group IV + Calcium
16.6  1.96*
Treatment withdrawal of group IV + Vitamin E
14.4  2.46*
Treatment withdrawal of group IV + AA +
13.4  0.68*
Ca2+ + Vitamin E
Values are mean ± S.E. *p<0.001
Comparison between: Group I and Groups II, III, IV
Group IV and Groups V, VI, VII, VIII & IX
IA
IB
IC
ID
IE
II
III
IV
V
VI
VII
VIII
IX
Fluoride 32 (4) 1999
SGOT
21.4  3.15
23.2  3.00
22.2  2.26
20.6  3.67
22.0  1.87
35.4  1.40*
38.8  3.70*
41.4  2.54*
34.2  2.05*
21.2  2.39*
25.6  3.79*
22.2  2.76*
22.0  1.00*
F and Al toxicity in mice liver and gastrocnemius muscle
225
Table 6A.
Source of Variation
SGPT
Between Groups
Within Groups
SGOT
Between Groups
Within Groups
SS
df
MS
F-cal
F-tab
6608.554
793.2
12
52
550.7128
15.25385
36.10321
1.943619
3436.954
1928.8
12
52
286.4128
37.09231
7.721623
1.943619
DISCUSSION
Aluminium and fluoride are reported to cause altered protein levels in various tissues of treated animals independently or in combination 12,14,20,21,30,31
probably by forming complexes with proteins or by inhibiting its synthesis or
due to alteration in protein metabolism. 19,32
It is known that aluminium and fluoride individually alter carbohydrate
metabolism.33 Reports from our laboratory have also revealed that a significant accumulation of glycogen occurred in liver, gastrocnemius muscle, vas
deferens, and uterus concomitant with a significant decline in phosphorylase
activity in mice after treatment with sodium fluoride (NaF) or aluminium
chloride (AlCl3).11,20,31 In the present study, the glycolytic pathway in liver and
gastrocnemius muscle of NaF, AlCl 3, and NaF+AlCl3-treated mice was also
altered thus disturbing their carbohydrate metabolism.
Succinate dehydrogenase (SDH) is a mitochondrial enzyme, which catalyses reversible oxidation of succinate to fumarate. A reduction in activity of
SDH occurred in liver and gastrocnemius muscle of treated mice. The decline
was most significant with the combined treatment of NaF and AlCl 3. Similar
results have been reported in mice gastrocnemius muscle and liver from our
laboratory and the results have been correlated with alteration in mitochondrial structure.11,30 Severe structural alterations in mitochondria of guinea pig
liver as well as in fluorotic mice ovary and uterus were also reported. 34,35 In
female albino mice, Singh36 found a dose-dependent reduction in SDH activity
in liver and kidney with sodium fluoride. Ultrastructural studies are called for
in the future.
Cholinesterases are enzymes which hydrolyze esters of choline. The reduced enzyme activity in serum and liver is an index for recognizing liver
damage and neuronal activity and reflects on the metabolic rate of an animal.
In the present study, a decrease in activity of cholinesterase in gastrocnemius
muscle and liver was observed with NaF, AlCl 3, or NaF+AlCl3 treatments.
The combined complex of aluminium and fluoride (AlF 3) is also known to
inhibit the activities of key synaptosomal enzymes, viz., ATPase and acetylcholinesterase (AChE), which are involved in transmission of nerve impulses.
The toxic effect may lead to altered utilization of acetylcholine thus affecting
the transmission of nerve impulses in liver and muscle tissues. 37 In vitro stud-
Fluoride 32 (4) 1999
226
Chinoy, Patel
ies by Cimasoni38 revealed inhibition of cholinesterase with fluoride ions.
Considerable evidence indicates that aluminium alters the second messenger
system of c-AMP and G-protein. Increased c-AMP concentration in brain results in hyperphosphorylation of proteins. The abnormal change in phosphorylation may impair the axonal transport of cytoskeletal proteins. Aluminium
also causes alteration in transport systems of the blood-brain barrier increasing the permeability of a number of peptide and steroid hormones. 19 These
mechanisms need further study in liver and muscle.
SGPT and SGOT are markers of liver function. NaF, AlCl3, or NaF +
AlCl3 treatments caused an increased activity of both transaminases, suggesting hepatic damage and changes in its function. Other reports corroborate our
findings.9,11,31
In the present study, the combined treatment of NaF+AlCl 3 caused more
toxicity than the individual treatments in all the parameters studied in liver,
gastrocnemius muscle, and serum. However, muscle glycogen and activities
of SDH and cholinesterase were affected more severely than in liver, but the
liver protein levels declined comparatively more than in muscle. Thus, the
NaF+AlCl3 treatment caused severe toxicity to liver as well as muscle.
Upon withdrawal of the combined treatment, some recovery occurred in
most of the parameters of liver, muscle, and serum, but none of the values returned to those of the control. The activities of cholinesterase in liver, gastrocnemius muscle as well as liver phosphorylase also recovered partially.
Previous studies in our laboratory on male and female rodents treated with
sodium fluoride or aluminium chloride or their combination and withdrawal of
treatment thereafter showed a non-significant recovery.10,12-14,31
The induced effects of NaF+AlCl3 on all the parameters of liver, gastrocnemius muscle and serum were partially reversed by the administration of
ascorbic acid, calcium, or vitamin E alone. However, with the combined treatments, complete recovery from the induced toxicity occurred in all the parameters of liver, muscle, and serum, and the values were comparable to control.
Ascorbic acid is known to be an antioxidant and has active detoxification
properties.39 It activates adenyl cyclase but inhibits phosphodiesterase (PDE),
which will result in an increase in c-AMP levels and cause activation of several
enzymes, viz., SDH, phosphorylase, 3β-HSD, 17-βHSD, etc.40 Calcium is also
known to act in a similar manner. Earlier work from our laboratory has revealed
that supplementation with calcium brought about significant recovery in several
enzyme activities in fluoride-intoxicated mice, rabbits, and guinea pigs.8,9,41,42
The reversal of NaF-induced toxicity in soft tissues of mice with the combined
treatment of AA and Ca2+ has also been reported in other studies.11,34
Vitamin E, a potent antioxidant, exerts its protective effect primarily
through destruction of cell-damaging free radical oxygen species. Significant
recovery of the reproductive functions of fluorotic male mice treated with vitamin E was reported previously. 14 In the present study, vitamin E also helped
to reverse NaF+AlCl 3-induced toxicity in liver and gastrocnemius muscle.
Fluoride 32 (4) 1999
F and Al toxicity in mice liver and gastrocnemius muscle
227
The combined administration of antidotes is more beneficial. As reported
elsewhere, the possible explanation for this effect might be that both AA and
Ca2+ act synergistically to activate several enzymes.12,30 Together with AA and
Ca2+, vitamin E provides an additional antioxidant effect, since tocopherol interacts non-enzymatically with ascorbic acid. Furthermore, it is known that, in
microsomal membrane preparations, the radical scavenging activity of tocopherol is enhanced in the presence of ascorbic acid.43 Hence, it is clear that
ascorbic acid and vitamin E together have an additive or synergistic effect.
In summary, interaction of all these antidotes resulted in reversal of NaFand AlCl3-induced toxicity in liver and gastrocnemius muscle of female mice.
ACKNOWLEDGEMENTS
Financial support provided by Sir Dorabji Tata Trust, Mumbai, to one of
the authors (TNP) is gratefully acknowledged.
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Fluoride 32 (4) 1999