Biol Trace Elem Res (2007) 118:260–268 DOI 10.1007/s12011-007-0036-6
Effect of Long-term Fluoride Exposure on Lipid Peroxidation and Histology of Testes in First- and Second-generation Rats Meral Öncü & Ahmet Kocak & Erdal Karaoz & Hakan Darici & Emin Savik & Fatih Gultekin
Received: 20 June 2006 / Revised: 21 February 2007 / Accepted: 2 March 2007 / Published online: 9 May 2007 # Humana Press Inc. 2007
Abstract This experiment was designed to investigate the histological and lipid peroxidation effects of chronic fluorosis on testes tissues of first- and secondgeneration rats. Sixteen virgin female Wistar rats were mated with eight males (2:1) for approximately 12 h to obtain first-generation rats. Pregnant rats were divided into two groups: controls and fluoride-given group, each of which containing five rats. Pregnant rats in the fluoride-given group were exposed to a total dose of 30 mg/l sodium fluoride (NaF) in commercial drinking water containing 0.07 mg/l of NaF throughout the gestation and lactation periods. After the lactation period, the young animals (first generation, F1) were exposed to the same dose of NaF in drinking water for 4 months. At the end of the 4 months of experimental period, nine randomly chosen male rats (F1) were killed and testes tissues were taken for histopathological and biochemical analysis. The remaining eight female rats were mated with four males (2:1) for approximately 12 h to obtain second-generation rats. Six female were identified as pregnant and treated with similarly throughout the gestation and the lactation periods. After the lactation period, the young male animals (second generation, F2) were also treated in the same way for 4 months. At the end of the 4 months of experimental period, nine randomly chosen
The study was carried out in Suleyman Demirel University.
M. Öncü (*) : A. Kocak : H. Darici Department of Histology and Embryology, Faculty of Medicine, Suleyman Demirel University, Morfoloji Binasi, Isparta 32040, Turkey e-mail:
[email protected] E. Karaoz Health High School, Kocaeli University, Kocaeli, Turkey E. Savik : F. Gultekin Faculty of Medicine, Department of Biochemistry, Suleyman Demirel University, Isparta, Turkey
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male rats (F2) were killed and testes tissues were collected for histopathological and biochemical analysis. The rats in the control group were applied the same procedure without NaF administration. In biochemical analysis of the fluoride given F1 and F2 rats, it has been found that plasma fluoride levels and testes thiobarbituric acid reactive substance levels were significantly increased when compared with the control group. In F1 and F2 rats, similar histopathological changes were observed. In both groups, spermatogenesis was severely reduced. Spermatogonia and primary spermatocytes were normal, however, there was a widespread degeneration in other spermatogenic cell lines of the seminiferous epithelium. The histological structures of the Sertoli and interstitial Leydig cells were normally observed. It is concluded that chronic fluorosis exposure leads to a remarkable destruction in testes tissues of F1 and F2 rats via lipid peroxidation.
Keywords Fluoride . Testes . Histology . Lipid peroxidation . Rat
Introduction Fluoride is a robust hard anion and a cumulative toxic agent [1]. Fluoride intake can be either by ingestion or inhalation. Being highly soluble in water, environmental fluoride is absorbed easily from the stomach and gut. Although high amount of fluoride in plasma is in bound form, a small fraction is in ionic form. Fluoride passes easily through the cell membranes in its ionic form. Therefore, primary involvement of bone and teeth in chronic fluorosis is attributed to the ability of these tissues to accept fluoride ion in exchange for other anions. Fluoride is mainly excreted from the kidneys. In a healthy individual, urinary excretion of fluoride is approximately 0.1 to 0.5 mg/day. Many researchers have reported a positive correlation between the intake and the urinary loss of fluoride. In endemic fluorosis, urinary fluoride excretion has been found to be 1.2 to 10.0 mg/day [2]. Chronic fluorosis is a slow and progressive process, which can lead to some symptoms regarding several systems, especially musculoskeletal and dental tissues [2]. Metabolic, functional, and structural damages caused by chronic fluorosis have been reported in many tissues including kidneys [3], liver [4], thyroid [5], parathyroid [6], pituitary gland [7], testis [8], muscle [2], and brain [9]. Excessive fluoride consumption can produce severe damage in many systems, especially in dental and skeletal systems [2]. High doses of oral fluoride intake produces the signs of acute fluorosis in many tissues including stomach [10], lung [11, 12], heart [13], and kidney [14]. Fluoride exposure also causes some biochemical changes including increase in blood potassium level [15], decrease in blood calcium level [16, 17], and deterioration in mitochondrial enzyme activity [18]. Treatment of rats with sodium fluoride (NaF) at the dose of 20 mg kg−1 day−1 for 28 days resulted in significant diminution of testicular Delta [5], 3 beta-hydroxysteroid dehydrogenase, and 17 beta-hydroxysteroid dehydrogenase activities and low plasma levels of testosterone, follicular-stimulating hormone, and luteinizing hormone [19]. Shashi and Kaur [20] stated that the testicular structural, nuclear, and total proteins, and testicular DNA were significantly depleted in rats exposed to experimental fluoride. The city of Isparta is located in the southwest Turkey and it is an endemic area for fluorosis. The residents of Isparta are chronically exposed to fluoride by drinking water containing 3.5– 4.9 ppm fluoride. In this study, we aimed to investigate the biochemical and histopathological effects of chronic fluorosis on first- and second-generation rat testis tissues.
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Methods Animals Sixteen female and eight male Wistar albino rats, aging 5 months and weighing 120–140 g, were obtained from Suleyman Demirel University, Laboratory of Animal Science (Isparta, Turkey). The rats were housed in macrolon cages under standard laboratory conditions (light period 7.00 A.M. to 7.00 P.M., 21±1°C) and received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health.
Treatment Sixteen female Wistar albino rats were mated with eight males (2:1) for approximately 12 h to obtain first-generation rats. Mating was confirmed by the presence of sperm in vaginal smears. Sperm in vaginal smears was observed in 10 of 16 rats (day 0). These rats were identified as pregnant and were included in this experiment. Pregnant rats were divided into control and fluoride-given groups, each of which containing five rats. In some previous studies, it has been reported that membrane lipid composition was changed in rats exposed to NaF chronically at a dose of 30 mg kg−1 day−1 [4, 9]. Also, in our study, we aimed to investigate the lipid peroxidation effect of NaF, which was chronically administered to Wistar albino rats at a dose of 30 mg kg−1 day−1. The pregnant rats in fluoride given group were exposed to a total dose of 30 mg/l NaF (Sigma, St. Lois, MO, USA) in commercial drinking water containing 0.07 mg/l NaF throughout gestation and lactation periods. After the lactation period, young animals (first generation, F1) were exposed to the same amount of NaF in drinking water for 4 months. At the end of the 4 months of experimental period, nine randomly chosen male rats (F1) were killed, and testis tissues were taken for histopathological and biochemical analysis. The remaining eight female rats were mated with four males (2:1) for approximately 12 h to obtain second generation rats. Six female rats were identified as pregnant and treated similarly throughout gestation and lactation periods. After the lactation period, the young male rats (second generation, F2) were also treated similarly for 4 months. At the end of the 4 months of experimental period, nine randomly chosen male rats (F2) were killed and testis tissues were taken for histopathological and biochemical analysis. The rats in the control groups underwent the same procedure without NaF treatment. Histopathological Analysis The testes were removed from each rat and divided into the lobes after cleaning. The lobes were fixed in 10% neutral buffered formalin. The routine light microscopic techniques were used on the tissue samples. The paraffin-embedded blocks were cut to 4–5 μm and stained with hematoxylin–eosin (H–E). The slides were then examined using a light microscope (Nikon Optiphot II, Japan) and photographed. Plasma Fluoride Levels Before rats were killed, 6 ml of blood was taken by cardiac puncture under ether anesthesia for the fluoride measurement. The plasma fluoride level was determined by an ion
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electrometer containing fluoride selective electrode (Orion Research, Inc., 500 Cummings Center, Beverly, MA 0 1915-6199, USA). Biochemical Analysis After the sacrification, the left testes were removed, and washed with physiological saline solution. Then, they were homogenized in cold phosphate-buffered solution for 3 min (Ultra-Turrax T25, Staufen, Germany) to provide a 10% homogenate. The homogenates were then centrifuged at 6,000×g for 10 min to obtain supernatants. Finally, the levels of thiobarbituric acid reactive substance (TBARS) were determined in the supernatants. The protein content of homogenates was determined by the method of Lowry et al. [21]. TBARS was estimated by the double-heating method of Draper and Hadley [22]. The principle of the method was the spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with malondialdehyde. For this purpose, 2.5 ml of 100 g/l trichloroacetic acid solution was added to 0.5 ml homogenate in a centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1000×g for 10 min, and 2 ml of the supernatant was added to 1 ml of 6.7 g/l TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured at 532 nm. The concentration of TBARS was calculated by the absorbance coefficient of malondialdehyde– TBA complex of 1.56×105 cm−1 M−1 and expressed in nanomoles per gram protein. Statistical Analysis The data were expressed as mean ± SD. The significance between the means was determined by Student’s t test. P values smaller than 0.05 were considered as meaningful.
Results Fluoride Levels The plasma fluoride levels were increased significantly (p
