Smita Tiwari et al.

, IJSID 2011, 1 (2), 294-302

ISSN:2249-5347

IJSID
International Journal of Science Innovations and Discoveries
Research Article
An International peer Review Journal for Science

Available online through www.ijsidonline.info

HISTOPATHOLOGICAL STUDIES ON THE EFFECT OF SODIUM FLUORIDE ON THE REPRODUCTIVE ORGANS AND BODY WEIGHT OF MALE ALBINO RAT
Smita Tiwari 1*and R.K.Pande2
1Prof.of

zoology & principal, S.K. (P.G.) College, Basti-272002 (U.P), 2P.G.Department of Zoology And Environmental Biology, D.B.S.(P.G.) College, Dehradun-248001(U.K.), India

Received: 09.09.2011 Modified: 16.10.2011 Published: 29.12.2011 Keywords: Albino rat, Fluoride, Antifertility effects, Reproductive organs
*Corresponding Author

ABSTRACT The effect of chronic exposure of Sodium Fluoride (NaF), (5, 20, 50 mg/kg b.w.) for 90days, on reproductive tissue damage in young male albino rat was evaluated histopathologicaly alongwith certain genital tissue wet weight . Damaging effect on testicular histoarchitecture alongwith disfigured tubular structure was recorded alongwith histological change in other organs viz.-

Name: Dr. Smitha Tiwari Place: Dehradun, Uttarakhand E-mail: ctiwari_1983@rediffmail.com

Epididymis , vasdeference seminal vesicle , and prostate gland . Even the epermatogenesis seemed to be arrested and clumping of spermatozoa was also obsorbed. The said effect was not observed in the control group.
INTRODUCTION

INTRODUCTION

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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302 INTRODUCTION Fluoride is a trace element and has clinical importance as it prevents dental caries (Robert Jr.1980) and is used for the treatment of Pagets disease (Robert Jr.1980). Besides its clinical importance , at present fluoride is considered as an important water pollutant. Fluoride contamination of ground water was noted above the tolerable limit in Sri Lanka, China , West Indies, Spain, Holland, Italy, South and North America and India (Suma Latha et al.1999). In India, fluoride contamination of the top aquifer system was also noted in Andhra Pradesh , Tamil Nadu, Karnataka, Gujrat, Rajasthan, Punjab, Haryana, Bihar, Kerala, and West Bengal (Handa 1975; Suma Latha et al.1999). The tolerable limit of fluoride in drinking water is 1ppm (Erickson 1978; Robert Jr.1980; Agrawal et al. 1997), but at various zones in India, the drinking water contains 10 to25 ppm of fluoride result in inhibition in the Krebs cycle (Bogin et al. 1976) as well as induction of muscle atrophy (Kaul and Susheela 1974; Susheela and Kharp 1990), liver toxicity (Saralakumari et al, 1988; Carlson and Suttle 1996), and kidney toxicity (Suketa and Terui 1980). There is paucity of information of fluoride on the reproductive system. Few reports available in this line stated that fluoride toxicity results in impairment of fertility (Messer et al. 1973), low birth rate (Messer et al. 1973; Freni 1994), and deflagellation of sperm both in human and experimental

animals(Chinoy and Sequeria 1989), Recently , we reported that fluoride intoxication is associated with inhibition in testicular androgenesis and gametogenesis (Ghosh et al. 2002). Fluoride at theis dose also affects the reproductive system in humans and in experimental animals (Chinoy and Narayan 1994), Therefore, from that angle, the results of this experiment have implications in the community. MATERIALS AND METHODS The study was conducted on young male albino rats (Rattus rattus ), of average age 12-14 weeks and weighing 125-200grams. They were acclimatized to laboratory conditions for 15 days prior to the commencement of the treatment. The rats were kept in open air cages (60x45x45 cm) at room temperature (Edmond,1950). The rats were fed standard rodent pellet (Hindustan Lever Ltd.) and water was allowed ad libitum. After the treatment animals were sacrificed by cervical dislocation. Test Chemicals- Sodium fluoride (ExcelaR) obtained from Qualigens Fine Chemicals, Mumbai was used. (a) Histopathological study Experimental Design: The rats were divided into the following four groups: Group 1- Control, Group 2-4-Experimental: 5,20,50mg/kg b.w. of sodium fluoride dissolved in 1ml.of double distilled water/kg.b.w./ day were given orally for the 30 days. 5 rats in each group of 5mg , 20mg and 50mg doses. Thereafter, they were sacrificed by cervical dislocation and reproductive organs were taken out, for histological studies.
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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302 The reproductive organs were removed and fixed in alcoholic Bouins Fluid. The tissues (Testes, Epididymis , Vas deferens, Seminal vesicle and Prostate) were washed in 70% alcohol and then dehydrated in alcholic series and embedded in paraffin wax, several sections were cut of 6 micron and stained with iron Hemotoxylin and Eosin for Histopathological examination. (McManus and Lowery, 1965) After conducting the above experiment, histopathological studies were conducted by preparing the stained microtome slides. Histopathological studies were observed and compared with the control, under the NaF stress. (b) Body Weight and Organ Weights Final body weight of each animal was recorded on the day of sacrifice . Testis, Epididymis , vas deferens, Seminal vesicle and prostate were dissected out , and wet weights of these organs were measured by single pan electrical balance . RESULTS AND DISCUSSION Testes Control rats consisted of highly expanded semniferous tubules with germinal epithelium consisting of germ cells, such as primary spermatocyte, secondary spermatocyte, spermatids and spermatozoa. The interstitial space was filled with loose connective tissue, made up of cell of Leydigs and blood vessels.(Plate -1) The germinal epithelial cells were found touching closely the epithelial cells of other seminiferous tubules. In the central region, spermatozoa seemed to be lesser but no significant change in the shape of seminiferous tubules were observed. (Plate- 1a) Their was significant intermingling of the seminiferous tubules. Even the spermatogonia and spermatocytes also displayed damage and disruption. (Plate- 1b) The shape of the seminiferous tubules seemed to be quite

disintegrated and the spermatogonia and spermatocytes were damaged to a very high degree. In the central region of the tubule and in between the tubules the damaged cell debris has dissolved showing vacuoles. (Plate-1c) The early stages of spermatogenesis regarding its reinitiation in hypophysectomized rats under gonadotropic hormonal stimulation are reported by Lostroh et.al, (1963); Chowdhury and Steinberger, (1975); Chemes et al. (1976, 1979).A dose dependent reduction in body weight of NaF exposed rats is indicative of impending toxicity (Chinoy and Sequeria, 1989 b). Epididymis The transverse section of Caput epididymis presented a normal histological picture. The epithelial cells of the caput were tall, columnar with nucleus arranged in a row, near the thin basement membrane. The segments of stereocilia were more profuse in the caput region than in the cauda. (Plate- 2) The epithelium of the cauda consisted of low cuboidal cells. The lumen of the ductules was larger in the cauda
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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302 and smaller in the caput. Intertubular connective tissue and vascularity were observed to be normal in cauda and caput epididymis. Both the portions of the epididymis were full of spermatozoa. (Plate- 3) The columnar cells are getting shortened and the basement membrane around the basal cells is getting shrinken. The stereocillia are getting bunched, the basal cells too are not distinguished, the muscle fibers were getting thickned and touching each other, due to which the spermatozoal movement becomes difficult. (Plate- 2a) The lumen of the ductules are showing reduction as compared to control due to shrinking of ductules, vacuolar regions became visible. Stereocilia were getting clustered. (Plate- 3a)The histological picture is further deteriorated and the celluar demarcation is lost, the muscle fibres surrounding the ductules got further thickned. Stereocilia are getting gradually lost. (Plate- 2b) Lumen of the ductules are further reduced, columnar and basal cells are not distinguished and are loosing the contact with muscle fibers on which they are attached. Due to shrinkage, large vacuoles are visible. (Plate- 3b) The muscle fibres surrounding the ductules are disintegrated. Columnar cells and basal cells lost their complete shape and due to degeneration of cells large vacuolar regions are visible. (Plate- 2c) Several disintegration in the histoarchitecture are observed. Stereocilia seems to be lost due to breakage of ductules wall, there are large spaces formed between the ductules. (Plate- 3c) The clear cells in the rat Epididymis are subdivided into two types and are concerned with the secreations of either glycoprotiens or glycolipoprotiens (Rajalakshmi, 1985). The blood epididymal barrier, constituted by zona occludens of the junctional complexes at the apical ends of the principal cells (Hooker and Gilmore, 1972) also appears to play an important role in maintaining a physiological milieu in the epididymal canal suitable for sperm maturation. Epididymal and ejaculated spermatozoa of a number of species also possessed enzymes capable of metabolizing steroids (Sheela Rani et al., 1978) Vas Deferens The transverse sections of the vas deferens of control rats showed normal histological picture with three distinct muscular patterns, i.e. the outer longitudinal, middle circular and inner longitudinal layers. The lamina propria was present between the inner longitudinal muscle layer and the pseudostratified epithelium, which contained stereocilia. The epithelial cell layer was folded so as to form a stellate lumen in which the sperm bundles were present. (Plate- 4) In 5mg/kg b.w. treated group the wall of the duct i.e. cremaster muscle are effected. The mucosal lining is getting stretched and has started loosing the shape of columnar and round cells. Muscularis appears to be thinning. The fibrosa is also reducing. (Plate- 4a) In 20mg/kg b.w. treated group the mucosal lining is loosing Stereocilia, lumen of
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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302 the ductules is reduced further, the inner lumen has started loosing the shape due to disintegration of columnar and basal cells of the mucosal epithelium. (Plate- 4b) In 50mg/kg b.w. treated group lumen shape is almost lost and due to the disintegration of mucosal epithelium, the lumen is loosing its shape and large vacuolar gaps are visible reflecting the severe damage in the hsitoarchitecture the mucosal crypts which are totally damaged. (Plate- 4c) The histoarchitecture of proximal and distal vas deferens reveals that it is not a simple connection for sperm transport, but it is a vital organ for the maintenance of sperm structure, survival and viability (Chinoy, 1985; Rajalakshmi, 1985; Chinoy et al, 1977). The vas deferens could be differentiated from the other parts on the basis of the characteristic epithelium, lamina propria, shape of lumen and the presence and absence of folds in the epithelium (Chinoy, 1985; Talwar et al., 1995;Terner and Maclaughin, 1973). Seminil vesicle The transverse sections (T.S.) of seminal vesicle of control rats showed muscosal folds extending into the lumen. The lumen was filled with secretions produced by glandular epithelium . The epithelial lining of mucosa consisted of a single layer of tall columnar cells with basal oval nuclei . The lamina propria of the mucosa comprised of cellular connective tissues containing smooth muscle cells. (Plate- 5) In 5mg/kg b.w. treated group the mucosa of the vesicle showing numerous tall narrow and complicated folds forming irregular chambers are loosing their shape. The mucosal epithelium which is pseudostratified made up of cuboidal cells are loosing their shape. The lamina propria is rich in elastic fibres and smooth muscle are getting deshaped. (Plate- 5a) In 20mg/kg b.w. treated group due to the disintegration of the mucosal lining, damage in the columnar cells, the lumen of the vesicle is getting enlarged. The circular and longitudinal mucles are also getting effected and loosing the original histoarchitecture. (Plate- 5b) In 50mg/kg b.w. treated group the mucosa of the vesicle is severely damaged and the crypts are seen damaged with large spaces in the histoarchitecture, due to the heavily damaged crypts. The mucosal epithelium is totally disintegrated. The lamina propria lost its elastic fibers and the smooth muscle support is totally damaged, loosing the basic function of seminal vesicle. (Plate5c) The fructose is an essential substrate in the anaerobic glycolysis by which the spermatozoa survive the low oxygen tensions present in the seminal fluid (Hamilton and Mossman, 1972; Roy et al.,1976). Prostate Gland The histological structure of the prostate gland in T.S. of control animals showed a number of alveoli lined by the low columnar epithelium with basal nuclei, the follicular lumen was full of secretions . There was an intervening fibromuscular stroma. The epithelium had proliferated into the crypts, having
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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302 invaded lumen. Folding of the mucosal lining was observed in smaller tubules but distended tubules had no mucosal folds. (Plate- 6) The glandular epitheliuim showed variation in shape, the alveoli have started declining in the pseudostratified epithelium and the columnar cells as well as the flat basal cells around the lumen have started disintegrating. The lumen of the alveoli and the corporo amylacea has started disintegrating. (Plate- 6a) The glandular epithelium, the alveoli of the pseudostratified epithelium have reduced. The lumen of large number of alveoli are widening and were still declining of in the histoarchitecture, as compared to 5mg stage impact.(Plate- 6b) In this treatment, the histoarchitecture is further declined and the lumen of the alveoli have lost their normal shape to a measure extent. (Plate- 6c) Mechanism of action of fluoride on exposure to laboratory animals and humans. Tissue- or organ-specific effects linked with repeated exposure to fluorides may involve metabolic pathways associated with lipid, carbohydrate, bone and energy metabolism, as well as signal transduction pathways (Kaminsky et al., 1990). Fluoride influences a number of enzymatic activities. The results of studies carried out in vitro have indicated that fluoride inhibits the synthesis of DNA and protein, inhibits cell proliferation and is cytotoxic in sufficiently high doses (Gilman, 1987; Elsair and Khelfat, 1988; Godfrey and Watson, 1988; Kaminsky et al., 1990). Effects produced by the exposure of laboratory animals and humans to fluoride may be attributable to one or more of these metabolic and biochemical effects. The stimulatory effect of fluoride on osteoblast proliferation may proceed, at least in part, through the inhibition of phosphotyrosyl protein phosphatases (Thomas et al., 1996; Lau and Baylink, 1998). The results of the present study of NaF impact on the reproductive organs suggest the possible effect of antiandrogenic agent, which may have altered the physiology and metabolism of reproductive organs. It is concluded that the fluoride exposure to rats could have induced alterations in histoarchitecture of the reproductive organs and also caused inducing hematological changes ultimately exerting its impact on fertility. (b) Effect of Sodium Fluoride (NaF) on the body weight and on reproductive organ weight. The results of the experiment are presented in table 1 Sodium Fluoride (NaF) Doses When 5mg/kg b.w. was administered for 90days mild change in the body weight was recorded. Treatment of rats with 20mg /kg of NaF for 90days no change in body weight was observed. Treatment of rats with 50mg/kg of NaF for 90days resulted in increase in body weight as compared to control group.
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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302 Table-1: Effect of Sodium Fluoride on the body weight and reproductive tissue wet weight in male rats.
Treatment Control Sodium fluoride Doses 5 20 Dose (mg/kg) Duration (Days) 90 Initialb.w. (g) 170.1 +1.20 160.10 + 1.51 180.45 + 1.33 171.41 + 4.21 Finalb.w. (g) 182.29 + 1.51 171.61 + 2.49 184.32 + 1.63 168.25 + 6.10 Testis 945.61 + 3.42 933 + 3.40 915.10 + 1.46* 876.20 + 7.40* Epididydmis 361.32 + 6.16 310.41 + 1.35* 300.31 + 1.41 300.41 + 3.55* Vas Deference 210.59 + 2.49 220.15 + 1.35 201.20 + 1.55* 172.72 + 1.35* Seminal Vesicle 389.10 + 6.12 375.12 + 3.44 320.71 + 1.21* 286.49 + 4.18* Prostate 325.10 + 8.13 463.10 + 1.45 401.10 + 1.71* 200.41 + 3.18*

50

** 90 days of treatment values are mean + SE (n= 5) * denotes significance at 5% level In each category 5 Rats/ Tissues were used. AKNOWLEDGEMENTS We wish to express our thanks to Uttarakhand Council of Science & Technology (UCOST), Dehradun, for their valuable support and to Dr. Rajendra Dobhal, Director, UCOST for inspiring the authors. REFERENCES
1. Agrawal , V., Vaish, A.K., and Vaish P, (1997)Ground water quality : Focus on fluoride and fluorosis in Rajasthan, Curr, Sci, (73), 743-746 2. Bogin, E., Abrams, M., Avida, Y., and Israeli, B., (1976).Effect of fluoride on enzymes from serum, liver, kidney, skeletal and heart muscles of mice. Fluoride (9), 42-46 3. Carlson, I. R., and Suttle, J.W., (1996).Pentose phosphate pathway enzymes and glucose oxidation in fluoride fed rats. Am. J. Physio(l),210,79-83 4. Chinoy, N.J., and Sequeriaa, E., (1989) Effects of fluoride on the histoarchitecture of reproductive organs of the male mouse. Reprod, Toxicol,(3),261-268. 5. Chemes, H.E., et al., (1976).Action of testosterone, dihydotestosterone and 5-alpha androstane-3-alpha, 17-beta diol on spermatogenesis of immature rats. Biol. Reprod., (14):332-338 6. Chemes, H.E., Dym, M. and Raj, H.G.M., (1979).The role of gonadotropins and testosterone on initiation of spermatogenesis in the immature rat. Biol. Repord., (21):241-249 7. Chinoy, N.J. (1985).Structure and physiology of mammalian vas deferens in relation to fertility regulation. J.Biosci., (7):215-221 8. Chinoy, N.J. and Sequeira, E. (1989). Effect of Fluoride on histoarchitecture of reproductive organs of male mouse. Reprod. Toxicol. (3): 261-7

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Smita Tiwari et al., IJSID 2011, 1 (2), 294-302

9. Chinoy, N.J. and Sequeira, E.: (1989).Fluoride induced biochemical changes in reproductive organs of male mice. Fluoride, (22):78-85 10. Chowdhury, A.K. and Steinberger, E., (1975).Effect of 5-alfa induced androgens on sex accessory organs, initiation and maintainence of spermatogenesis in the rat. Biol. Reprod., (12): 609-617 11. Edmond, J.F., 1950The care and Breedings of laboratory animals. John Wiley and Sons Inc., New York. Compain and Hall Ltd. London : 12. Elsair, J. and Khelfat, K., (1988).Subcellular effects of fluoride. Fluoride, (21): 9399 13. Erickson, J.D., (1978).Mortility in selected cities with fluoridated and non-fluoridated water supplies, N.Egnl.J.Med.(298),1112-1116 14. Freni, S.C., (1994).Exposure to high fluoride concentration in drinking water is associated with decreased birth rates.J.Toxicol, Environ. Health,(42),109-112 15. Ghosh, D., Das(Sarkar), S., Maiti, R., Jana,D., and Das, U.B., (2002).Testicular toxicity in sodium fluoride treated rats, Association with oxidative stress. Reprod, Toxicol.16,385-390 16. Gilman, A. G. proteins: (1987).transducers of receptor-generated signals. Annu. Rev. Biochem., (56): 615 649 17. Godfrey, P. and Watson, S., (1988). Fluoride inhibits agonist-induced formation of inositol phosphates in rat cortex. Biochem. Biophys. Res. Commun., (155): 664669 18. Hooker, R.H. and Gilmore, D.P., (1972).Obstruction of vas deferentia of male rat and rabbit with silastic medical adhesive. Int.J.Fertil., (17):14-6 19. Handa, B.K., (1975).Geochemistry and genesis of fluoride containing in ground water in India . Ground water., (13),275-281 20. Kaul, R.D and Susheela, A.K., (1974).Evidence for muscle fiber degeneration in rabbits treated with sodium fluoride. Fluoride,(7),177-181 21. Kaminsky, L., Mahoney, M., Leach, J., Melius, J. and Miller, M., (1990).Fluoride: Benefits and risks of exposure. Crit Rev. Oral Biol. Med., (1): 261281 22. Lau, K.H. and Baylink, D.J., (1998).Molecular mechanism of action of fluoride on bone cells. J. Bone Miner. Res., (13): 16601667 23. Lostroh, A.L et al., (1963).Effect of ovaine gonadotropins and antiserum to interstitial cell- simulating hormone on the testis of the hypophysectomized. Acta Endocrinol., (44):536-544 24. Mc Manus, J.F.A. and Lowry, (1965) R.W.Staining methode histological and histochemical. Herpo and Row, New York Evaniston, London 25. Messer , H.H., Armstrong, W.D., and Singer, L., (1973).Influence of fluoride intake on reproductive in mice.J.Nutr.,(103),1319-1326
International Journal of Science Innovations and Discoveries, Volume 1, Issue 2, November-December 2011

301

Smita Tiwari et al., IJSID 2011, 1 (2), 294-302

26. Rajalkshmi, M., (1985).Physiology of epididymis and spermatozoa. J.Biosci., 7(2):191-195 27. Roy, A.K., Boyal, J.Q., Biswas, N(1976)..M. and Chaudhury, A.K., Protection of spermatogenesis by a2-a globulin in rats treated with cyproterone acetate. American J. Anat., (146):369-362 28. Robert, Jr.C.H., ,(1980)Agent affecting calcification , Calcium , parathyroid hormone, calcitonin, vitamin D and other compound . In Goodman Gilmans the pharmacological basis of therapeutics, 7th ed., pp14961522. New York: Macmillan Publishing Co. 29. Saralakumari, D., Varadacharyula, N,C., and Rao, R.P., (1988).Effect of fluoride toxicity on growth and lipid in liver kidney , and serum in rat. Arogya, J. Healh. ,(15),24-29 30. Sheela Rani, C.S., Murty, G.S.R.C. and Moudgal, N.R., (1978).Effect of chronic neutralization of endogenous FSH on testicular function in the adult male bonnet monkey-assessment using biochemical parameters. Int.J.Androl.,(1):489-500 31. Suketa, Y., and Terui, Y., (1980).Adrenal function and changes of sodium and potassium in serum and urine in fluoride intoxicated rat. Fluoride ,(13),4-9 32. Suma Latha, S., Ambika, S.R., and Prasad, S.J., (1999).Fluoride contamination status of ground water in Karnataka Curr. Sci, (6),730-734 33. Susheela, A.K., and Kharjp, P., (1990).Aortic calcification in chronic fluoride poisoning, Biochemical electron microscopic evidence. Exp. Mol. Puthol,(53),72-80 34. Talwar, G.P et al., (1995).Preneem polyherbal cream and pessaries with dual properties of contraception and alleviation of genital infections. Current Science., (68): 437-440 35. Teotia, S.P.S., Teotia, M.,Singh ,D.P.,Rathore, R.S.Singh , C.V.,Tomor, N.P.S., et al., (1984).Endemic fluorosis , Change to deeper bore wells as a practical community acceptable approach to its eradication. Fluoride, (17),48-52 36. Thomas, A.B., Hashomoto, H., Baylink, D. and Lau, K-H., (1996).Fluoride at mitogenic concentrations increases the steady state phosphotyrosyl phosphorylation level of cellular proteins in human bone cells. J. Clin. Endocrinol. Metab., (81): 25702578 37. Turner, CH., Hasegawa, K., Zhang, W., Wilson, M., Li, Y. and Dunipace, A.J., (1995) Fluoride reduces bone strength in older rats. J. Dent. Res., (74): 14751481

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