…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
(Clethrionomys glareolus)

bank-vole-fOriginal → 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

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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

Abstract

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

  1. Introduction

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: alak@uwb.edu.pl (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.

doi:10.1016/j.toxlet.2003.11.012

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. 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

90%–95%.

  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 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.

  1. 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. 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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_  _  _  _  _

♦ 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.

bank-vole-f