See also  HERE

...As can be seen in Table 1, the fluoride exposure
 about a significant decrease in the
testicular zinc
 concentration and
an increase in lipid…


 Some 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 [some] bats, fish, some birds and
many of our closest primate cousins.
Most animals make ascorbic acid to order.

Zinc Protection From Fluoride-Induced
Testicular Injury In The Bank Vole
(Clethrionomys glareolus)


Myodes glareolus

Original ⇒ HERE

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



Elżbieta Bonda-Ostaszewska
University of Bialystok



All in-text references underlined in blue are linked to publications on Research Gate, letting you access and read them immediately.

Available from: Elżbieta Bonda-Ostaszewska

Retrieved on: 24 June 2016

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: (A. Krasowska).

processes including reproduction (Weber and Reid,

1969; Messer et al., 1973; Zeiger et al., 1993; Freni,


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.

  1. .
  2. 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).


2.2. Assays

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

wet weight).

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


  1. 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

polypropylene vials.

2.3. Histopathology

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.

2.4. Immunohistochemistry

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


Table 1

The effect of fluoride exposure on body and testis weights, testicular zinc and lipid peroxidation (TBARS), testicular and bone fluorideVand 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.

  1. .
  2. Results

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

dietary Zn.

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.

  1. 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).

  1. .
  2. Discussion

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,1990).

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 conditions.

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 injury.


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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[? * ] F. is a serious environmental pollutant – we need to reduce our intake – and stop contaminating our water supply with it.
This research is therefore significant.

We thank you for this paper.

Since then a few species,
including humans, have acquired
 a spanner somewhere in the genetic
works that prevents them
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.

Birds are known to synthesize vitamin C
in sufficient amounts, many feel it
is not necessary in their diets.
We have noticed that in times of stress,
and that includes at breeding times,
our birds consume larger
 amounts of
foods containing this vitamin;
thus, we feel it to be especially
useful at these times.