162
Fluoride Vol. 32 No. 3 162-170 1999 Research Report
BENEFICIAL EFFECTS OF THE AMINO ACIDS
GLYCINE AND GLUTAMINE ON TESTIS OF
MICE TREATED WITH SODIUM FLUORIDE
NJ Chinoya and Dipti Mehta
Ahmedabad, India
SUMMARY: Biochemical effects of feeding sodium fluoride (NaF, 5 mg/kg
body weight) for 30 and 45 days on testis of male mice (Mus musculus) were
investigated. The reversibility of fluoride-induced effects on testicular protein levels and the activities of 3β- and 17β-hydroxysteroid dehydrogenases
(HSD) and succinate dehydrogenase (SDH) by withdrawal of NaF treatment
were also investigated, as well as the effects of administration of glycine
and glutamine alone and in combination. Withdrawal of NaF for 30 days resulted in partial recovery of these parameters, with greater recovery after 45
days. Administration of glycine and glutamine individually during the withdrawal periods significantly enhanced recovery. In combination, these two
amino acids were even more effective and restored all parameters almost to
control levels or, in the case of protein, even higher. Testicular cholesterol
levels were not significantly affected throughout any of the treatments.
These results show that NaF affects testicular steroidogenesis, protein
levels, and HSD and SDH activities in mice. The effects, however, are transient and reversible, with the amino acids glycine and glutamine producing
marked beneficial effects. A protein-supplemented diet might therefore ameliorate the toxic effects of fluoride in endemic areas.
Keywords: Fluoride-treated mice, Glutamine, Glycine, Hydroxysteroid dehydrogenase,
Male mice, Mouse testis, Protein levels, Sodium fluoride, Succinate dehydrogenase,
Testicular steroidogenesis, Testosterone, Toxicity reversal.
INTRODUCTION
Human populations are exposed to fluoride from soil, water and air. Excess
fluoride in drinking water leads to fluorosis, a disease with a variety of symptoms which can be crippling. It is estimated that nearly 25 million people are
afflicted with fluorosis in 15 states of India.
Extensive research has been carried out during the past several decades on
skeletal and dental fluorosis.1,2 However, the effects of fluoride on the reproductive organs as well as fertility impairment are not fully understood, and the
data are conflicting. Messer et al3,4 reported that a low fluoride intake by female
mice impaired their reproductive capacity and fertility, although growth rate and
litter size were not affected. On the other hand, Tao and Suttie5 observed that,
with adequate iron intake, fluoride had no real effect on reproduction in female
mice. Earlier reports from our laboratory revealed that fluoride ingestion altered the structure and functions of reproductive organs of male rodents.6,7
The present study was undertaken to investigate the effects of fluoride ingestion for 30 and 45 days on the testes of mice to determine the degree of
recovery after withdrawal of treatment, as well as the effect of supplementa———————————————
aFor Correspondence: Reproductive Physiology and Endocrinology Unit, Department of
Zoology, School of Sciences, Gujarat University, Ahmedabad 380 009, India.
Amino acid effects on testis of NaF-treated mice
163
tion with the amino acids glycine and glutamine administered alone and in
combination during the withdrawal period.
MATERIALS AND METHODS
Adult male mice (Mus musculus) of Swiss strain weighing between 20-30
g were used. The animals were maintained on standard laboratory food and
water was given ad libitum. The animals were divided into eight groups, and
daily treatments (administered orally in water by a feeding tube attached to a
hypodermic syringe) were given as shown in Table 1.
Table 1. Experimental Protocol
Group Treatment
I
Control (untreated)
II
Control + Glycine
(1 mg/animal/day)
Control + Glutamine
(1 mg/animal/day)
NaF (5 mg/kg body wt/mouse/day)
Same as group IVa,b then withdrawal for further 30, 45 days
Same as group Va + glycine
(1 mg/animal/day) for 30 days
Same as group Va + glutamine
(1 mg/animal/day) for 30 days
Same as group Va + glycine +
glutamine for 30 days
III
IVa,b
Va,b
VI
VII
VIII
Treatment
length (days)
-
Autopsy day
No. of
animals
10
30
Sacrificed with
treated groups
31st
30
31st
10
30 + 45
30 + 30
45 + 45
30 + 30
31st, 46th
61st
91st
61st
20
10
10
10
30 + 30
61st
10
30 + 30
61st
10
10
After the respective treatments, the animals were sacrificed by cervical dislocation. The testes were excised, blotted free of blood, weighed on a micro
balance, and utilized for determining the first four of the biochemical parameters listed below. Blood collected by cardiac puncture was allowed to clot and
the serum separated by centrifugation for testosterone assay. The biochemical
parameters studied were:
1. Protein: Protein levels in the testis were determined by the method of Lowry et al8 and expressed as mg/100 mg fresh tissue weight.
2. Succinate dehydrogenase (SDH) (E.C1.3.99.1): SDH activity in the testis
was determined by the tetrazolium reduction method of Beatty et al9 and expressed as µg formazan formed/mg protein.
3. Cholesterol: Cholesterol concentrations in the testis were estimated by the
procedure of Zlatkis et al10 and expressed as mg/100 mg fresh tissue weight.
4. 3β- and 17β-Hydroxysteroid dehydrogenases (HSD) (E.C.1.1.1.51): The
activities of these enzymes were assayed in testis by the method of Talalay 11
and expressed as nmol of androstanedione formed/mg protein/minute.
Fluoride 32 (3) 1999
164
Chinoy, Mehta
5. Testosterone: Serum testosterone levels of control and treated mice were
assayed by the double antibody technique of Peterson and Swerdloff. 12 Specificity of the antibody to testosterone was 100% with 1.7% cross reactivity to
dihydrotestosterone (DHT) and 5.2 x 10 -5% and 5.9 x 10-2% cross reactivity to
progesterone and estradiol, respectively. Sensitivity of the assay was found to
be less than or equal to 15 ng/dL with 4.3% inter-assay variation. Intra-assay
variation was found to be 6.3%. The concentration of testosterone is given as
ng/mL.
Statistics: For each biochemical parameter a minimum of 5-6 replicates were
assayed, and the data were statistically analyzed by Student’s ‘t’ test and
ANOVA.
RESULTS
1. Protein: The protein content of testis showed a significant durationdependent decrease (p<0.001) after fluoride treatment (Groups IVa,b) as compared to controls (Groups I, II, and III) (Table 2).
The withdrawal of treatment for 30 days (Group Va) did not result in recovery as compared to Group IVa, whereas, withdrawal of treatment for 45
days (Group Vb) restored the protein levels significantly (p<0.001), almost to
those of the control (Group I) (Table 2).
The individual treatments with either glycine or glutamine in the withdrawal period (Groups VI and VII) resulted in significant recovery (p<0.001)
as compared to treated animals in Group IVa, and the levels were similar to
those of the respective controls (Groups II and III).
The treatment with glycine and glutamine together in the withdrawal period (Group VIII) resulted in a significant increase (p<0.001) in the protein levels compared to those in the control group (Table 2).
2. Succinate dehydrogenase (SDH): The activity of SDH in testis was decreased significantly (p<0.001) depending on the duration of NaF treatment as
compared to the control (Table 2).
The withdrawal of NaF after 45 days alone resulted in recovery of SDH activity significantly (p<0.001) as compared to treated group IVb.
In Groups VI and VII, where individual glycine and glutamine treatments
were given during the withdrawal period, the recovery in SDH activity was
better than in Group Va. However, by combined treatment with both amino
acids (Group VIII), the enzyme activity was restored almost to the control
state (Table 2).
3. Cholesterol: The levels of cholesterol in the testis were not significantly
affected throughout the treatments as compared to the controls (Table 3).
4. Testosterone: The serum testosterone levels were decreased (p<0.05) in
animals treated with NaF for 45 days (Group IVb) as compared to the control.
The withdrawal of NaF treatment did not bring about a recovery in testosterone levels. However, by giving glycine or glutamine individually (Groups
VI and VII), the recovery was significant (Group VI: p<0.05; Group VII:
p<0.01) as compared to Group IVb. In Group VIII (glycine + glutamine
Fluoride 32 (3) 1999
Amino acid effects on testis of NaF-treated mice
165
treatment given in the withdrawal period), the testosterone levels were the
same as in the control groups (Table 3).
5. 3β-Hydroxysteroid Dehydrogenase (3β-HSD): The activity of 3β-HSD was
significantly reduced (p<0.001) after 45 days of NaF treatment (Group IVb).
After withdrawal for 45 days, the activity recovered (p<0.02). A significant
restoration of 3β-HSD activity occurred by treatment with glycine alone
(Group VI) (p<0.001). In Groups VII and VIII, the activity was restored significantly (p<0.001 almost to the control state (Table 4).
6. 17β-HSD: The activity of 17β-HSD was decreased (p<0.02) by 30 and 45
days NaF treatment as compared to the control group. The recovery was insignificant after 30 days of withdrawal but significant after 45 days (p<0.01).
In Groups VI, VII and VIII, the enzyme activity was significantly (p<0.01)
restored to almost the control level (Table 4).
Table 2. Effects of NaF and amino acids on protein levels
and succinate dehydrogenase (SDH) activity in testis of mice
Group
Treatment
I
II
Control (untreated)
Control + glycine
(1 mg/animal/day)
Control + glutamine
(1 mg/animal/day)
NaF for 30 days
NaF for 45 days
(5 mg/kg body wt/mouse/day)
NaF as in group IVa
then withdrawal for 30 days
NaF as in group IVb
then withdrawal for 45 days
NaF withdrawal + glycine
(1 mg/animal/day) for 30 days
NaF withdrawal + glutamine
(1 mg/animal/day) for 30 days
NaF withdrawal + glycine +
glutamine for 30 days
III
IVa
IVb
Va
Vb
VI
VII
VIII
Protein (mg/100
mg tissue wt)
13.76 ± 0.4
13.47 ± 0.32
SDH (µg formazan
formed/mg protein)
10.83 ± 1.3
10.81 ± 0.15
13.59 ± 0.14
10.80 ± 0.13
11.04 ± 0.42*
8.69 ± 1.35*
6.79 ± 1.15*
4.34 ± 0.83*
11.29 ± 0.5†
8.99 ± 0.51†
12.42 ± 1.70*
9.44 ± 0.43*
13.35 ± 0.19*
10.44 ± 0.35*
13.48 ± 0.48*
10.06 ± 0.22*
15.34 ± 0.5*
10.74 ± 0.37*
Values are mean ± S.E. *p<0.001 †not significant
For p values, comparisons were done between:
Gr. I, II, III and IVa,b
Gr IVa and Va
Gr IVb and Vb
Gr IVa and VI, VII, VIII
Table 2A. Testis Protein ANOVA
Source of
Variation
Groups
Residual
SS
df
MSS
f(cal)
F(tab)
178.47
47.812
9
45
19.8303
1.0624
18.66405
2.096
SS Sum of Squares df degrees of freedom MSS Mean sum of squares
Fluoride 32 (3) 1999
166
Chinoy, Mehta
Table 2B. Testis SDH ANOVA
Source of
Variation
Groups
Residual
SS
df
197.21
23.053
9
36
MSS
21.9121
0.64035
f(cal)
F(tab)
34.2185
2.153
SS Sum of Squares df degrees of freedom MSS Mean sum of squares
Table 3. Testicular cholesterol and serum testosterone levels
Cholesterol
(mg/100 mg tissue wt)
0.402 ± 0.09
0.412 ± 0.15
Group Treatment
I
II
Testosterone
(ng/mL)
2.55 ± 0.024
2.54 ± 0.021
Control (untreated)
Control + glycine
(1 mg/animal/day)
III
Control + glutamine
0.418 ± 0.01
2.545 ± 0.016
(1 mg/animal/day)
IVa
NaF for 30 days
0.406 ± 0.01‡
2.304 ± 0.018‡
IVb
NaF for 45 days
0.564 ± 0.03‡
2.083 ± 0.027*
(5 mg/kg body wt/day)
Va
NaF as in group IVa
0.410 ± 0.008‡
2.335 ± 0.012‡
then withdrawal for 30 days
Vb
NaF as in group IVb
0.409 ± 0.012‡
2.39 ± 0.018‡
then withdrawal for 45 days
VI
NaF withdrawal + glycine
0.419 ± 0.01‡
2.502 ± 0.008*
(1 mg/animal/day) for 30 days
VII
NaF withdrawal + glutamine
0.417 ± 0.01‡
2.516 ± 0.012†
(1 mg/animal/day) for 30 days
VIII
NaF withdrawal + glycine
0.414 ± 0.012‡
2.545 ± 0.014†
+ glutamine for 30 days
Values are mean ± S.E. *p<0.05 †p<0.01 ‡not significant
For p values, comparisons were done between:
Gr I, II, III and IVa,b
Gr IVa and Va
Gr IVb and Vb
Gr IVa and VI, VII, VIII
Table 3A. Testicular cholesterol ANOVA.
Source of
Variation
Groups
Residual
SS
df
MSS
f(cal)
F(tab)
0.0109
0.0359
9
27
0.001211
0.001331
0.909896
2.250
SS Sum of Squares df degree of freedoms
MSS Mean sum of squares
Table 3B. Serum testosterone ANOVA
Source of
Variation
Groups
Residual
SS
df
MSS
f(cal)
F(tab)
1.9069
0.2701
8
80
0.238
0.0037
64.34
2.05
SS Sum of Squares df degrees of freedom
Fluoride 32 (3) 1999
MSS Mean sum of squares
Amino acid effects on testis of NaF-treated mice
167
Table 4. Actives of testicular 3β- and
17β-Hydroxysteroid dehydrogenases (HSD)
3 β-HSDa
17β-HSDb
Control (untreated)
Control + glycine
(1 mg/animal/day)
Control + glutamine
(1 mg/animal/day)
NaF for 30 days
NaF for 45 days
(5 mg/kg body wt/mouse/day)
NaF treatment
then withdrawal for 30 days
0.184 ± 0.015
0.183 ± 0.004
0.055 ± 0.005
0.053 ± 0.0012
0.185 ± 0.003
0.054 ± 0.0017
0.138 ± 0.03§
0.083 ± 0.01‡
0.026 ± 0.003*
0.021 ± 0.004*
0.133 ± 0.009
0.035 ± 0.007§
Vb
NaF treatment
then withdrawal for 45 days
0.142 ± 0.02*
0.046 ± 0.002
VI
NaF withdrawal + glycine
(1 mg/animal/day) for 30 days
0.164 ± 0.015‡
0.052 ± 0.003†
VII
NaF withdrawal + glutamine
(1 mg/animal/day) for 30 days
0.182 ± 0.026‡
0.050 ± 0.003†
VIII
NaF withdrawal + glycine +
glutamine for 30 days
0.183 ± 0.021
0.053 ± 0.002
Group
Treatment
I
II
III
IVa
IVb
Va
a3
β-HSD (nmols of androstanedione formed/mg protein/minute)
(nmols of androstanedione formed/mg protein/minute)
b17β-HSD
Values are mean ± S.E. *p<0.02 †p<0.01 ‡p<0.001 §not significant
For p values, comparison were done between:
Gr I, II, III and IVa,b
Gr IVa and Va
Gr IVb and Vb
Gr IVa and VI, VII, VIII
Table 4A. Testis 3β-HSD ANOVA
Source of
Variation
Groups
Residual
SS
df
MSS
f(cal)
F(tab)
0.04179
0.04099
9
27
0.00464
0.00152
3.05844
2.25
SS Sum of Squares df degrees of freedom
MSS Mean sum of squares
Table 4B. Testicular 17β-HSD ANOVA
Source of
Variation
Groups
Residual
SS
df
MSS
f(cal)
F(tab)
0.0058
0.0008
9
27
0.000643
0.0000292
21.9953
2.250
SS Sum of Squares df degrees of freedom
MSS Mean sum of squares
Fluoride 32 (3) 1999
168
Chinoy, Mehta
DISCUSSION
Earlier researchers have reported that increased fluoride intake results in a
decline in food consumption which is inversely correlated with the increased
plasma fluoride levels in rat.13 Schwarz and Milne14 reported that 1-2 µg F/g
of diet stimulated the growth of rats fed a highly purified amino acid diet and
maintained in a trace element controlled isolator. However, other workers 15,16
were not able to confirm these findings.
It is known that fluoride inhibits biosynthesis of protein in vitro and in vivo, which is probably due to interference of fluoride with the binding of the
amino acyl-t-RNA adducts to the ribosomal RNA template. 17,18 In the present
study, NaF treatment caused a significant decrease in levels of total protein in
the testis. Since testicular proteins, including inhibin and androgen-binding
proteins, are important for several functions in testis, 19 their metabolism could
be altered under fluoride treatment. The decrease in protein content could also
be related to necrotic changes in the testis. 6 A decrease in protein from NaF
treatment was also reported in the spermatozoa and reproductive organs of rat,
mice, and rabbit,7,20,21,22,23 and in the serum of a fluorotic human population of
North Gujarat.24
The significant decline in SDH activity in the testis of NaF-treated mice
found in the present study is in agreement with earlier data. 20 This fact suggests that the treatment altered the metabolism of the testis. Since SDH is
primarily a mitochondrial oxidative enzyme, it is possible that alteration in
mitochondrial structure and metabolism might also have occurred in the testes
after treatment, as observed in the ovary of NaF-treated mice (authors’ observations). Hence, histological and ultrastructural studies in this direction are
called for in order to investigate this aspect.
A hypercholesterolemic effect in the serum has been observed in NaFexposed animals,25 which may constitute a predisposition to atherosclerosis. In
the present study, however, testicular cholesterol levels were not significantly
altered, while the activities of testicular 3β- and 17β-hydroxysteroid dehydrogenases were decreased after 45 days of NaF administration. These results
suggest that fluoride interferes to some extent with testicular steroidogenesis in
mice. Similar results were reported by Narayana and Chinoy in fluorotic rats.26
It is known that testosterone concentrations decrease in males suffering
from skeletal fluorosis,27 while in another study on a human population in endemic fluorosis areas of the Mehsana district of North Gujarat, India, the circulating testosterone levels were only slightly affected. 28 This discrepancy
could be related to the degree of fluorosis in these populations. In the present
study, the serum testosterone levels were decreased after 45 days in NaFtreated mice.
The results of the present study reveal that fluoride has a definite effect on
male reproduction. The withdrawal of NaF for 30 days was not effective in
restoring the parameters to control levels. Withdrawal for 45 days, however,
led to the recovery of testicular protein levels and 17β-HSD activity. The addition of the amino acids glycine and/or glutamine was beneficial in promot-
Fluoride 32 (3) 1999
Amino acid effects on testis of NaF-treated mice
169
ing the recovery from fluoride-induced toxicity. The ameliorative effect of the
amino acids was probably due to their roles in various physiological functions,
including as biologically active antioxidants. 29
It is evident from the results that the effects of NaF may be transient and
reversible if cessation of the fluoride intake is accompanied by amino acid
ingestion. Hence, a protein-rich or supplemented diet might mitigate to a considerable extent some of the fluoride health hazards in endemic areas.
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Published by the International Society for Fluoride Research
Editorial Office: 17 Pioneer Crescent, Dunedin 9001, New Zealand
Fluoride 32 (3) 1999