FERTILITY EFFECTS OF SODIUM FLUORIDE IN MALE MICE

Transcription

1 128 Fluoride Vol. 33 No Reserch Report FERTILITY EFFECTS OF SODIUM FLUORIDE IN MALE MICE Ahmed Elbetieh, Hom Drmni, Ahmd S Al-Hiyst b Irbid, Jordn SUMMARY: Sexully mture mle Swiss mice were exposed t 60 dys of ge to 100, 200 nd 300 ppm sodium fluoride (NF) in their drinking wter for 4 weeks or 10 weeks. The effect of NF exposure on fertility ws ssessed by breeding these mles with untreted femle mice fter the exposure periods. Fertility ws significntly reduced t ll three concentrtions by exposure for 10 weeks but not for 4 weeks. The number of implnttion sites nd vible fetuses ws significntly reduced in femles mted with mles tht hd ingested NF t concentrtion of 200 ppm for 10 weeks. Reltive weights of seminl vesicles nd preputil glnds were significntly incresed in mice exposed to 200 nd 300 ppm NF for 4 weeks but not in mice exposed for 10 weeks. These results indicte tht long-term ingestion of NF dversely ffects fertility in mle mice. Keywords: Fluoride nd fertility, Fertility reduction, Mle mice, Sodium fluoride. INTRODUCTION The effect of fluoride on mle nd femle fertility hs become n re of growing concern. Vrious studies show tht fluoride cuses dverse effects on both mle nd femle fertility. 1-3 Fluoride intke hs been linked to lowered humn birth rtes 1 nd decresed testosterone concentrtions. 3 Infertility ws found in n re of Indi where fluorosis is highly endemic. 2,4 Furthermore, in vitro exposure of humn sperm to fluoride produced significnt decline in sperm motility. 2 Sodium fluoride hs been tested in fertility studies in severl species of lbortory nimls. Some studies indicte no effects on reproductive function nd development 5-9 wheres others revel tht exposure to sodium fluoride cuses reproductive toxic effects The reported reproductive toxic effects include increses in numbers of bnorml spermtozo, 11 loss of spermtogenesis, 12 nd interference with steroidogenesis. 13 In order to gin further insight into the effect of sodium fluoride on mle fertility, the present study ws undertken to investigte the effects of sodium fluoride on fertility in mle mice. MATERIALS AND METHODS Animls: Eighty 60-dy-old dult mle Swiss mice were used in the experiments. They were rised in the niml house unit in the Fculty of Medicine t Jordn University of Science nd Technology t controlled temperture of 21 ± 1 C on 12-h light/drk cycle. Food (mnufctured by the Fculty of Veterinry Medicine t Jordn University of Science nd Technology, Irbid, Jordn, ccording to stndrd recipes) nd wter were supplied d libitum. The For Correspondence: Dr Ahmed Elbetieh, Deprtment of Applied Biologicl Sciences, Fculty of Science, Jordn University of Science nd Technology, P.O. Box 3030, Irbid 22110, Jordn. Phone: ,. Ext , Fx: E-mil: betieh@just.edu.jo; b Deptrtment of Restortive Dentistry, Fculty of Dentistry, Jordn University of Science nd Technology.

2 Fertility effects of sodium fluoride in mle mice 129 diet hd very low levels of fluoride (F), nd the tp wter contined 0.3 ppm F. Therefore, our clcultions of F intke re bsed solely on the sodium fluoride (NF) dded to the drinking wter. Administrtion of sodium fluoride: Sodium fluoride (Sigm Chemicl Compny, St Louis, MO, USA) ws dissolved in tp wter (0.3 ppm), t concentrtion of 100, 200 nd 300 ppm NF. The exposure of mice ws conducted in two experiments: in one experiment the mice were given NF in their drinking wter for period of 4 weeks. For this experiment, 40 of the mice were rndomly divided into four groups of 10. The first group served s the control nd ws llowed d libitum ccess to tp wter without ny dded NF for 4 weeks. The other three groups were llowed d libitum ccess to tp wter contining either 100, 200 or 300 ppm NF for the sme period. In the second experiment the other 40 mice were rndomly divided into four groups of 10 nd treted the sme s in the first experiment except tht the exposure period ws 10 weeks. Fertility test nd exmintion of fetuses: Animls were observed dily for clinicl signs of toxicity from the first dy of exposure to NF. Their wter consumption nd body weight were mesured every week. After the exposure period ech mle ws plced in n individul cge with two virgin untreted femles of the sme strin nd d libitum ccess to food nd untreted tp wter. They were left together for ten dys during which two estrus cycles should hve elpsed. 14 The mles were then removed, nd 10 dys lter the femles were killed by cervicl disloction under light ether nesthesi, nd the following mesurements were recorded: number of pregnnt femles, number of vible fetuses, nd number of embryo resorptions. Body nd orgn weights: The exposed mle mice nd their control counterprts were scrificed t the end of the exposure period (4 or 10 weeks) plus the 10- dy mting period. Body weight nd weights of pired testes, seminl vesicles (stripped of fluid), nd preputil glnds were recorded. Sttisticl nlysis: Dt re expressed s men ± SD. Differences between control nd NF-exposed groups were nlyzed using either the Fisher exct test or Student s t test. A p vlue less thn 0.05 ws considered significnt. RESULTS Effect of NF on wter consumption: Wter consumption of the exposed nd the control groups ws mesured every week, nd the verge dily wter consumption for ech niml ws clculted. The dt depicted in Tbles 1 nd 2 demonstrte tht ingestion of NF (100, 200, or 300 ppm concentrtion) did not significntly ffect the verge dily wter consumption per niml. The verge doses of NF tht the 4- nd 10-week exposed nimls received bsed on wter consumption per kg body weight per dy re shown in Tbles 1 nd 2. None of the control nor ny of the NF groups showed ny clinicl signs of toxicity when the exposure period ws 4 weeks. In the 10-week exposed groups, however, 2 nimls out of 10 nd 3 out of 10 died during the lst week of the exposure to 100 nd 300 ppm NF, respectively.

3 130 Elbetieh, Drmni, Al-Hiyst Tble 1. Effect of 4 weeks ingestion of sodium fluoride on verge dily wter consumption in mle mice nimls Wter consumption (ml/dy) NF from drinking wter (mg/kg/dy) Control ± NF (100 ppm) ± ± 3.16 NF (200 ppm) ± ± 6.45 NF (300 ppm) ± ± 5.19 Dt re expressed s mens ± SD. The clcultions of F intke re bsed solely on the NF dded to the drinking wter. Tble 2. Effect of 10 weeks ingestion of sodium fluoride on verge dily wter consumption in mle mice nimls Wter consumption (ml/dy) NF from drinking wter (mg/kg/dy) Control ± NF (100 ppm) ± ± 3.63 NF (200 ppm) ± ± 7.01 NF (300 ppm) ± ± Dt re expressed s mens ± SD. The clcultions of F intke re bsed solely on the NF dded to the drinking wter. Effect of NF on orgn weights: Mice exposed to 200 nd 300 ppm NF for 4 weeks showed sttisticlly significnt increse in the reltive weights of preputil glnds (p<0.01 nd p<0.001, respectively) (Tble 3). The reltive testes weights were lso significntly incresed in the group exposed to 200 ppm NF for 4 weeks. On the other hnd, nimls exposed to NF for 10 weeks showed no significnt effects on the reltive weights of ny reproductive orgn investigted in this study (Tble 4). Effects of NF on fertility of mle mice: Tble 5 demonstrtes tht the exposure of dult mle mice to NF for period of 4 weeks hd no effect on mle fertility (the number of pregnnt femles impregnted by exposed mles ws very close to the number impregnted by the unexposed control mles). The number of resorptions, however, ws significntly incresed in femles impregnted by mles tht hd been exposed to NF t 100 ppm (p<0.005), 200 ppm (p<0.05) nd 300 ppm (p<0.005). Tble 6 demonstrtes the effects of 10 weeks ingestion of NF on mle fertility. The number of femles tht becme pregnnt ws significntly reduced when they were mted with mles exposed to 100 ppm (p<0.0005), 200 ppm (p<0.0001) nd 300 ppm (p< ) NF. The number of implnttions ws significntly decresed in femles impregnted by mles which hd ingested

4 Fertility effects of sodium fluoride in mle mice ppm NF (p<0.01). Furthermore, the number of vible fetuses ws lso reduced in pregnnt femles impregnted by mles exposed to 200 ppm NF. Tble 3. Effect of 4 weeks ingestion of sodium fluoride on orgn weights Weight on dy of scrifice (g) Testes (mg/10 gm B.wt.) Seminl vesicles (mg/10 gm B.wt.) Preputil glnds (mg/10 gm B.wt.) Control ± ± ± ± 4.78 NF (100 ppm) ± ± ± ± 1.80 NF (200 ppm) ± ± ± 9.55* ± 4.36* NF (300 ppm) ± ± ± ± 6.39 Reltive weights. Dt re expressed s mens ± SD. *p<0.01, p<0.001 significntly different s compred to control vlues (Student's t test). Tble 4. Effect of 10 weeks ingestion of sodium fluoride on orgn weights of mle mice Weight on dy of nimls scrifice (g) Testes (mg/10 gm B. wt.) Seminl vesicles (mg/10 gm Bwt) Preputil glnds (mg/10 gm Bwt) Control ± ± ± ± 5.72 NF (100 ppm) ± ± ± ± 8.09 NF (200 ppm) ± ± ± ± 1.72 NF (300 ppm) ± ± ± ± 1.49 Reltive weights. Dt re expressed s mens ± SD. Tble 5. Effect of 4 weeks ingestion of sodium fluoride on fertility of mle mice mles femles Pregnnt femles implnttions vible fetuses Resorptions/ implnttions Control /20 (100%) 8.75 ± ± /175 NF (100 ppm) /20 (90 %) 8.67 ± ± /156 NF (200 ppm) /20 (95%) 8.11 ± ± /155 * NF (300 ppm) /20 (85%) 8.41 ± ± /143 Results re expressed s mens ± SD. * p< 0.05, p < significntly different s compred to control vlues (Fisher's exct test). Tble 6. Effect of 10 weeks ingestion of sodium fluoride on fertility of mle mice Ded Mted pregnnt vible Resorptions/ mles mles femles femles implnttions fetuses implnttions Control /20 (100%) 7.80 ± ± /156 NF (100 ppm) /16 (50%) * 8.22 ± ± /74 NF (200 ppm) /20 (45%) 5.78 ± ± /52 NF (300 ppm) /14 (35.7%) 7.20 ± ± /36 Dt re expressed s mens ± SD. *p< , p<0.0001, p< significntly different s compred to control vlues (Fisher's exct test). p<0.01, p<0.005 significntly different s compred to control vlues (Student's t test).

5 132 Elbetieh, Drmni, Al-Hiyst DISCUSSION The im of the present study ws to ssess the dverse effects on fertility nd reproduction of mle mice fter ingestion of NF in their drinking wter. The model employed in this work hs been used previously by severl investigtors to ssess the toxicity of different compounds on fertility nd reproduction in lbortory nimls. 5,15 The concentrtions of NF used in this study were chosen ccording to previous studies. 5 The drinking wter route of exposure ws chosen to mimic humn exposure nd to reflect the impct on fertility of the sustined blood levels of fluoride tht would occur from wter consumption throughout the dy. The results presented here demonstrte tht exposure of dult mle mice to NF t concentrtions of 100, 200 nd 300 ppm in drinking wter for period of 4 weeks hd no significnt effect on their fertility. There ws, however, sttisticlly significnt increse in the number of embryo resorptions in femles impregnted by these exposed mles. The increse in the number of resorbed embryos my be ttributed to n increse in peri-implnttion mortlity of fertilized ov. Furthermore, there ws sttisticlly significnt increse in reltive weights of seminl vesicles for nimls exposed to 200 ppm NF but not to the other lower or higher concentrtions. This chnge in the ccessory glnd weight might suggest n ltertion in the pttern of testosterone secretion. A reduction in serum testosterone concentrtion hs been reported in ptients with fluorosis. 3 A significnt increse in the reltive weights of preputil glnds ws lso observed in niml groups dministered NF t 200 nd 300 ppm for 4 weeks. This glnd produces behvior-modifying pheromones which lter fighting nd other behvior. 16 In contrst to these results, exposure of mle mice to NF for 10 weeks hd no significnt effects on weights of ccessory sex glnds. These results suggest tht the increse in reltive weights of these glnds observed in the 4-week exposed groups ws trnsient nd is reversed with longer exposure to NF. The min finding of the present study ws the significnt reduction in fertility of mle mice exposed to NF for 10 weeks t the three concentrtions used. This impirment in mle fertility ws dose dependent nd incresed s the concentrtion of NF incresed. Severl other niml studies of the effects of sodium fluoride on reproduction nd development hve shown decresed fertility in most niml species studied. Abnorml spermtozo were produced by mle Swiss mice dministered 8 mg/kg of fluoride by intrperitonel injection for five consecutive dys. 11 Another study conducted on mice reported tht exposure to 500 or 1000 ppm NF for 3 months resulted in cler dmge to spermtogenesis. 17 Additionlly, mice exposed to fluoride concentrtions of 100 nd 500 ppm in drinking wter showed lck of mturtion nd differentition of spermtocytes nd loss of spermtogenesis. 12 Impired fertility hs lso been reported in mice dministered NF t 125, 250, nd 500 ppm in their feed. 18 Furthermore, it hs been reported tht in vitro exposure of humn sperm to

6 Fertility effects of sodium fluoride in mle mice 133 fluoride (250 mm) results in ltered lysosoml ctivity, ltered glutthione levels, nd morphologicl nomlies, producing decline in sperm motility. 2 In rbbits, disruption nd degenertion of seminiferous tubules were observed when NF ws orlly dministered t 10 mg/kg for 29 months. 19 In contrst to the bove findings, other work hs reported tht fluoride does not ffect spermtogenesis or sperm morphology. 20 In study using B6C3F 1 mice mintined on 75 ppm NF in drinking wter for 21 weeks, no effect on spermtogenesis ws observed. 21 In summry, our work hs shown tht sodium fluoride dministered in drinking wter t verge doses of up to mg/kg/dy for 4 weeks in the mice hd no significnt effects on fertility of mle mice. However, ingestion of sodium fluoride t doses of up to mg/kg/dy for 10 weeks did dversely ffect their fertility. REFERENCES 1 Freni SC. Exposure to high fluoride concentrtions in drinking wter is ssocited with decresed birth rtes. J Toxicol Environ Helth 1994;42: Chinoy NJ, Nryn MV. In vitro toxicity in humn spermtozo. Reprod Toxicol 1994;8: Susheel AK, Jethnndni P. Circulting testosterone levels in skeletl fluorosis ptients. J Toxicol Clin Toxicol 1996;34: Neelm K, Suhsini RV, Sudhkr RY. Incidence of prevlence of infertility mong mrried mle members of endemic fluorosis district of Andhr Prdesh (Abstrct). Proceedings of Conference of the Interntionl Society of Fluoride Reserch Nyon, Switzerlnd Messer HH, Armstrong WD, Singer L. Influence of fluoride uptke on reproduction in mice. J Nutr 1973;103: To S, Suttie JW. Evidence for lck of effect of dietry fluoride level on reproduction in mice. J Nutr 1976;106: Aurlerich RJ, Npoltino AC, Bursin SJ, Olshon BA, Hochstein JR. Chronic toxicity of dietry fluorine to mink. J Anim Sci 1987;65: Heindel JJ, Btes HK, Price CJ, Mrr MC, Myers CB, Schwetz BA. Developmentl toxicity evlution of sodium fluoride dministered to rts nd rbbits in drinking wter. Fundm Appl Toxicol 1996;30: Sprndo RL, Collins TFX, Blck TN, Rorie J, Ames MJ, nd O Donnell, M. Testing the potentil of sodium fluoride to ffect spermtogenesis in the rt. Food Chem Toxicol 1997;35: Hoffmn DJ, Pttee OH, Wiemeyer SN. Effects of fluoride on screech owl reproduction: tertologicl evlution, growth, nd blood chemistry in htchlings. Toxicol Lett 1985;26(1): Pti PC, Bhuny SP. Genotoxic effect of n environmentl pollutnt, sodium fluoride, in mmmlin in vivo test system. Cryologi 1987;40: Kour K, Singh J. Histologicl finding of testes following fluoride ingestion. Fluoride 1980;13: Nryn MV, Chinoy NJ. Effect of fluoride on rt testiculr steroidogenesis. Fluoride 1994;27:7-12.

7 134 Elbetieh, Drmni, Al-Hiyst 14 Rugh R. The mouse, its reproduction nd development. Burgess, Minnepolis, Elbetieh AM, Al-Hmood MH. Long-term exposure of mle nd femle mice to trivlent nd hexvlent chromium compounds: effect on fertility. Toxicology 1997;116: Brin PF, Homdy MH, Minrdi M. Preputil glnds, dominnce nd ggressiveness in mice. Boll Zool 1983;50: DHHS. Review of fluoride: benefits nd risks. United Sttes Deprtment of Helth nd Humn Services, Public Helth Service Ridh M, Al-Jiboori N, Mehdi AW. Effect of high fluoride intke on reproductive system of the mle mice. Irqi J of Vet Med 1978;2: Susheel AK, Kumr A. A study of the effect of high concentrtions of fluoride on the reproductive orgns of mle rbbits using light nd scnning electron microscopy. J Reprod Fertil 1991;92: Li Y, Dunipce AJ, Stookey GK. Effects of fluoride on the mouse sperm morphology test. J Dent Res 1987;66: Dunipce AJ, Zhng W, Noblitt TW, Li Y, Stookey GK. Genotoxic evlution of chronic fluoride exposure: Micronucleus nd sperm morphology studies. J Dent Res 1989;68: Published by the Interntionl Society for Fluoride Reserch Editoril Office: 727 Brighton Rod, Ocen View, Dunedin 9051, New Zelnd