…As can be seen in Table 1, the fluoride exposure brought
about a significant decrease in the testicular zinc
concentration and an increase in lipid…
Zinc Protection From Fluoride-Induced
Testicular Injury in the bank vole
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Article in Toxicology Letters · April 2004
Impact Factor: 3.26 · DOI: 10.1016/j.toxlet.2003.11.012 · Source: PubMed 3 authors, including: Tadeusz Włostowski University of Bialystok
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Toxicology Letters 147 (2004) 229–235
Zinc protection from fluoride-induced testicular injury in
The Bank Vole (Clethrionomys glareolus)
Alicja Krasowska∗, Tadeusz Włostowski, El˙zbieta Bonda
Institute of Biology, University of Białystok, ´ Swierkowa 20B, 15-950 Białystok, Poland
Received 6 October 2003; received in revised form 30 October 2003; accepted 6 November 2003
Previous work has shown that a high fluoride intake in rodents leads to histopathological changes in the germinal epithelium
of testes that is associated with zinc deficiency. The purpose of this study was to determine whether supplemental dietary Zn
would protect against testicular toxicity induced by fluoride in a small rodent, the bank vole. The 4-month exposure period to
fluoride (200_g/ml of drinking water) induced histopathological changes (hemorrhage in interstitium, necrosis and apoptosis
in seminiferous tubule epithelium) which were accompanied by decreased testicular zinc concentration and increased lipid
peroxidation. Supplemental dietary zinc (110–120_g/g) together with fluoride treatment resulted in complete reversal of the
fluoride-mediated effects. However, supplemented dietary Zn did not affect the accumulation of fluoride in the testes and
bone. These data suggest that a zinc-enriched diet protects seminiferous tubules against fluoride toxicity by preventing the
fluoride-induced testicular zinc deprivation.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fluoride; Zinc; Testes; Histopathology; Apoptosis
Fluoride (F) is an essential trace element which has
a very high affinity for bone mineral where it is incorporated
into the apatite crystal structure by substitution
for hydroxyl ion (Zipkin, 1970). This substitution
confers protective effects against mineral dissolution,
with important implications for animals and human
demineralizing diseases (Guo et al., 1988; Machoy,
1991). However, at higher doses fluoride becomes
toxic and adversely affects a number of physiological
∗ Corresponding author. Fax: +48-857457302.
E-mail address: firstname.lastname@example.org (A. Krasowska).
processes including reproduction (Weber and Reid,
1969; Messer et al., 1973; Zeiger et al., 1993; Freni,
1994). It has been suggested that impaired reproduction
may be directly related to an injury of the germinal
epithelium of testicular tubules induced by fluoride
exposure (Kour and Singh, 1980a; Krasowska, 1989).
Previous studies from our laboratory revealed that
testicular necrosis caused by fluoride is accompanied
primarily by a reduction of zinc concentration
in the testes of rats and bank voles (Krasowska and
Włostowski, 1992, 1996). Because a zinc-deficient
diet produces similar histopathological changes in the
germinal epithelium as those brought about by fluoride
exposure (Millar et al., 1958; Mason et al., 1982;
0378-4274/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
230 A. Krasowska et al. / Toxicology Letters 147 (2004) 229–235
Merker and Günther, 1997), it is conceivable that fluoride
affects spermatogenesis through changes in zinc
metabolism. If this is the case, then protection against
fluoride-induced testicular Zn deprivation should prevent
injury to the organ. Therefore, this study was
designed to determine whether supplemental dietary
Zn would protect against histopathological changes
in the testes of bank voles exposed chronically to fluoride.
Since Zn deficiency has been shown to induce
apoptosis and lipid peroxidation in the testes (Oteiza
et al., 1995; Nodera et al., 2001), in the present study
the two processes were also examined.
Materials and methods
2.1. Animals and experimental design
Forty male bank voles (1 month old, weighing
11–13 g) from our own laboratory stock were used
throughout the study. The animals were housed in
stainless steel cages (two per cage) under controlled
environmental conditions (18–20 ◦C temperature,
50%–70% relative humidity, 12-h light/dark cycle).
The bank voles were divided into four groups
according to drinking water and dietary zinc: (1)
control—receiving distilled water and food containing
20–25_g Zn/g; (2) fluoride—receiving distilled
water containing 200 _g F/ml as NaF (Krasowska
and Włostowski, 1996) and food containing 20–25 _g
Zn/g; (3) fluoride + zinc—receiving distilled water
containing 200 _g F/ml as NaF and food containing
110–120_g Zn/g, and (4) zinc—receiving distilled
water and food containing 110−120_g Zn/g. For 4
months, bank voles received ad libitum fluoride in
their drinking water and control or zinc-supplemented
wheat grains which are considered to be an adequate
quality food for the bank vole (Włostowski et al.,
1996). The grains supplemented with zinc (soaked in
ZnSO4 solution) and containing 15–20 _g F/g were
prepared prior to the experiment. Atomic absorption
spectrophotometry (AAS) analysis of the grain
revealed that actual levels of Zn were between 20
and 25 _g Zn/g dry weight (control) and 110–120 _g
Zn/g dry weight (Zn-enriched diet). In addition, an
identical amount of apple was offered to all animals
(3 g per vole per week) which ate it completely. The
water intake was measured weekly. The experimental
protocols were approved by the local ethical committee
for performing and experimental study on
laboratory animals (Medical Academy in Białystok).
At the end of the 4-month exposure period, the bank
voles were weighed, euthanized by cervical dislocation
and both testes and femur were removed. The
left testis was transferred to 2.0 ml chilled 0.25M sucrose
and homogenized with a Teflon pestle in a glass
homogenizer. Aliquots (0.2 and 1.0 ml) of the homogenate
were taken for determination of lipid peroxidation
and zinc concentration, respectively. The
right testis was fixed in Bouin’s fluid. One half of the
testis was processed for histological examination and
immunohistochemistry. The other half was dried and
used for fluoride measurement.
Lipid peroxidation was assessed by measuring
malondialdehyde (MDA) formation, using the thiobarbituric
acid (TBA) assay (Ohkawa et al., 1979).
To 0.2 ml of the tissue homogenate, 0.2 ml of 8.1%
sodium dodecyl sulfate, 1.5 ml of 20% acetic acid,
1.5 ml of 0.8% thiobarbituric acid, and 0.6 ml of distilled
water were added and vortexed. The reaction
mixture was placed in a water bath at 95 ◦C for 1 h.
After cooling, 1.0 ml of distilled water and 5.0 ml
of butanol/pyridine mixture (15:1, v/v) were added
and vortexed. After centrifugation, absorbance
of the organic phase was determined at 532 nm.
Tetraethoxypropane was used to prepare a calibration
curve. The results were expressed as TBARS (nmol/g
Zinc determination was performed as described previously
(Włostowski et al., 1996). Briefly, the homogenate
(1.0 ml) was placed in a glass tube with
2.0 ml of concentrated nitric acid. After 20 h of sample
digestion at room temperature, 72% perchloric
acid (0.5 ml) was added and the mixture was heated
at 100 ◦C for 3 h. Finally, the temperature was raised
to 150 ◦C and digestion continued for another 4 h.
Deionized water was added to the residue after digestion
to a volume of 3.0 ml. Zinc in these solutions
was measured on a flame absorption spectrophotometer
(AAS 3, Zeiss Jena). Samples of bovine liver 1577b
(NIST) were also analyzed in an identical manner to
check accuracy of the method. The recovery of Zn was
Krasowska et al. / Toxicology Letters 147 (2004) 229–235 231
Fluoride determination was performed spectrophotometrically
at 622 nm by using modified lanthanum/
alizarin complexone reagent (Culik, 1986).
The separation of fluoride from dry portion of the
testis (30–35 mg) and bone (6–7 mg) was achieved by
microdiffusion from perchloric acid and absorption
by sodium hydroxide on filter paper in disposable
One half of each fixed testis was processed by
standard histological methods, stained with hematoxylin
and eosin, and analyzed by light microscopy
for histopathological changes.
Apoptosis in the testes was demonstrated in situ by
the TUNEL (TdT-mediated dUTP-fluorescein Nick
End Labeling) assay, using a kit from Roche Diagnostics
(Mannheim, Germany) according to their
instructions. Briefly, sections were dewaxed in xylene,
hydrated in graded alcohol series and permeabilized
in 0.1% Triton X-100/0.1% sodium citrate for
8 min. Terminal deoxynucleotidyl transferase (TdT)
enzyme and fluorescein-labeled nucleotides were applied
to the sections for 60 min at 37 ◦C. Sections
were washed with PBS and treated with alkaline
phosphatase-conjugated anti-fluorescein antiboby for
30 min at 37 ◦C. They were next treated with substrate
solution (NBT/BCIP) for 10 min in dark. Apoptotic
activity was quantified by counting the number of cells
The effect of fluoride exposure on body and testis weights, testicular zinc and lipid peroxidation (TBARS), testicular and bone fluoride
and testicular injury in the bank vole fed diets supplemented and not supplemented with zinc
Control Fluoride Fluoride + Zinc Zinc
Body weight (g) 21.5 °æ 2.8a 22.1 °æ 2.5a 21.7 °æ 2.4a 23.2 °æ 3.0a
Left testis wet weight (mg) 330 °æ 79a 300 °æ 60a 291 °æ 80a 348 °æ 60a
Testicular zinc (_g/g wet weight) 32.5 °æ 3.8a 18.0 °æ 2.0b 31.5 °æ 2.5a 33.0 °æ 2.0a
TBARS (nmol/g wet weight) 52.6 °æ 9.1a 75.0 °æ 11.1b 56.5 °æ 10.6a 51.0 °æ 9.7a
Testicular fluoride (_g/g dry weight) 4.00 °æ 1.50a 11.90 °æ 2.96b 9.00 °æ 1.54b 3.90 °æ 1.35a
Bone fluoride (_g/g dry weight) 247 °æ 38a 4872 °æ 687b 4790 °æ 488b 210 °æ 75a
Apoptosis 0.23 °æ 0.12a 1.16 °æ 0.49b 0.23 °æ 0.09a 0.15 °æ 0.03a
Histopathology − + − −
Values represent the mean °æ S.D. for n = 10. Apoptosis is expressed as TUNEL-positive cells per seminiferous tubule (see Fig. 2).
Histopathology: normal morphology (−), histopathological changes (+) (see Fig. 1). Means in the same row marked with different
superscript letters (a and b) are significantly different (P < 0.05).
positive for TUNEL staining within entire testis cross
section, as proposed by Young et al. (1999). Apoptosis
was expressed as number of TUNEL-positive
cells per total number of seminiferous tubules within
each testis cross.
2.5. Statistical analysis
Data were expressed as means °æ S.D. The values
were analyzed by two-way analysis of variance
(ANOVA) followed by the Duncan’s multiple-range
test (MS Statistica). Differences at P < 0.05 were
considered statistically significant.
During the 4-month period of observation, fluoride
loading (200_g F/ml) did not affect the consumption
of water, which amounted to 3–4 ml per animal per
day. There were also no significant differences in the
final body and testes weights among the four groups
of bank voles (Table 1).
Histopathological changes (hemorrhage in interstitium,
vacuolization and necrosis of seminiferous epithelium)
(Fig. 1) and increased incidence of apoptosis
(Fig. 2) occurred in the testes of all bank voles exposed
to fluoride alone (Table 1); notably, no lesions
were produced by fluoride in the presence of the extra
As can be seen in Table 1, the fluoride exposure
brought about a significant decrease in the
testicular zinc concentration and an increase in lipid
232 A. Krasowska et al. / Toxicology Letters 147 (2004) 229–235
Fig. 1. Representative photomicrographs of testis section from (A) control bank voles and (B, C) bank voles that received water containing 200 _g F/ml (fluoride group)
for 4 months. Hemorrhage in interstitium (B) (arrows), and vacuolization and necrosis of the seminiferous epithelium (C) in the fluoride-treated voles were observed. No
histopathological changes were seen in bank voles from the control, fluoride + zinc and zinc groups (not shown). H & E staining, 200°ø.
Krasowska et al. / Toxicology Letters 147 (2004) 229–235 233
Fig. 2. Immunohistochemical demonstration of apoptotic cells in testes by the TUNEL technique. (A) Control bank voles showing the
normal level of apoptosis. (B) Bank voles received drinking water containing 200 _g F/ml, for 4 months showing the increased number
of apoptotic cells (arrows); 200°ø.
peroxidation; supplemental dietary Zn resulted in
complete reversal of the fluoride mediated effects.
The two-way analysis of variance revealed that the
accumulation of fluoride in the testes and bone of bank
voles was significantly affected by the fluoride exposure
(P < 0.0001), but supplemental dietary Zn had
no influence on the fluoride concentrations (Table 1).
Zinc has been shown so far to protect against toxicity
of various chemicals (Cagen and Klaassen, 1979;
Miranda et al., 1982; Szyma´nska et al., 1991; Kaji
et al., 1993; Zhou et al., 2002). The mechanism of
zinc protection has been attributed to the alteration
234 A. Krasowska et al. / Toxicology Letters 147 (2004) 229–235
in pharmacokinetics of xenobiotics, stabilization of
membranes, inhibition of P-450-dependent monooxygenase
activity, stabilization of cellular thiols or the
activation of gluthatione-associated enzymes, inhibition
of lipid peroxidation and sequestration of reactive
moieties, free radicals and metal ions, by the
Zn-induced metallothionein (Chvapil et al., 1972;
Cagen and Klaassen, 1979; Goering and Klaassen,
1984; McMillan and Schnell, 1984; Cho and Fong,
The results of the present study indicate that concurrent
administration of elevated but physiologic level of
dietary Zn can protect seminiferous tubules from the
toxic effects of fluoride. This protection was probably
not due to an alteration in pharmacokinetics of fluoride,
as supplemental dietary Zn did not change the
tissue distribution of this element (Table 1). It is thus
unlikely that spermatogenesis in the bank vole can be
directly affected by fluoride. The assumption in confirmed
by results of Sprando et al. (1996) who have
demonstrated that spermatogenesis in the adult rat is
not adversely affected by direct exposure to fluoride
even at levels 200 times greater than those under normal
The present work showed, however, that supplemental
dietary Zn prevents a reduction in the testicular
zinc concentration induced by fluoride exposure
(Table 1). The exact mechanism by which fluoride decreases
the testicular zinc is unknown at present. It
cannot be ruled out that the decrease may have been
due to an inhibition of Zn absorption and/or an increase
of its excretion in the urine under the influence
of fluoride. The latter possibility may be involved because
kidneys are also adversely affected by prolonged
use of NaF (Kour and Singh, 1980b). Moreover, the sequestration
of zinc from the plasma by fluorotic bone
may also account, at least to some degree, for the deprivation
of Zn in the testes as well as in the liver and
kidneys (Krasowska and Włostowski, 1992).
Several lines of evidence indicate that reactive oxygen
species (ROS) are involved in fluoride-induced
impairment of soft-tissue function (Zhi-Zhong et al.,
1989; Rzeuski et al., 1998; Shivarajashankara et al.,
2001). Lipid peroxidation is considered as an indirect
measure of ROS generation (Suzuki et al., 2000).
In the present study, lipid peroxidation increased
in the testes of bank voles exhibiting at the same
time the tissue injury and apoptosis (Table 1). Thus,
it is possible that fluoride induced oxidative stress
could be responsible for these processes. Notably,
the fluoride-induced testicular lipid peroxidation,
histopathological changes and increased incidence
of apoptosis were associated with testicular Zn depletion,
and supplemental dietary Zn resulted in
complete reversal of the fluoride-mediated effects
(Table 1). These data suggest that the Zn depletion in
testes caused by fluoride exposure may be a causal
factor in inducing testicular injury in the bank vole.
The assumption is supported by the fact that both
lipid peroxidation and apoptosis in testes have been
demonstrated to increase in animals fed Zn-deficient
diets (Oteiza et al., 1995; Nodera et al., 2001) and
dietary Zn deficiency produces similar histopathological
changes in testes as those observed in this
study (Millar et al., 1958; Mason et al., 1982; Merker
and Günther, 1997). It is also worth noting that both
a Zn-deficient diet (Gilabert et al., 1996) and fluoride
exposure (Tokar and Savchenko, 1977) induce
a reduction in serum testosterone, which in turn can
lead to an inhibition of spermatogenesis, further supporting
the notion that zinc plays an important role
in the pathogenesis of fluoride-induced testicular
In conclusion, the results from the present study
demonstrate that a zinc-enriched diet protects seminiferous
tubules against fluoride toxicity. This protection
appears to be due to the prevention of fluoride-induced
testicular zinc deprivation.
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_ _ _ _ _
♦ Note: Since then a few species, including humans, have acquired
a spanner somewhere in the genetic works that prevents them
synthesising vitamin C. Another oft-quoted exception is the
guinea pig, but similar defects are found in bats, fish,
birds and many of our closest primate cousins.
Most animals make ascorbic acid to order.