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Albino rat, Fluoride, Antifertility effects, Reproductive organs

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Albino rat, Fluoride, Antifertility effects, Reproductive organs Powered By Docstoc
					                                     Smita Tiwari et al., IJSID 2011, 1 (2), 294-302


                                                                                              ISSN:2249-5347
                                                                                                        IJSID
                          International Journal of Science Innovations and Discoveries             An International peer
                                                                                              Review Journal for Science


Research Article                                                    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
                                                                    ABSTRACT
Published: 29.12.2011
                                                The effect of chronic exposure of Sodium Fluoride
Keywords:
Albino rat, Fluoride,                    (NaF), (5, 20, 50 mg/kg b.w.) for 90days, on reproductive
Antifertility effects,
Reproductive organs                      tissue damage in young male albino rat was evaluated
*Corresponding Author                    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:                                    Epididymis , vasdeference seminal vesicle , and prostate
Dr. Smitha Tiwari
Place:                                   gland . Even the epermatogenesis seemed to be arrested
 Dehradun, Uttarakhand                              INTRODUCTION
E-mail:                                  and clumping of spermatozoa was also obsorbed. The
ctiwari_1983@rediffmail.com
                                         said effect was not observed in the control group.


                                                    INTRODUCTION




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                                                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 Paget’s 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 Kreb’s 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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         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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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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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. (Plate-
5c) 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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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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 Table-1: Effect of Sodium Fluoride on the body weight and reproductive tissue wet weight in male rats.

  Treatment     Dose     Duration   Initialb.w.   Finalb.w.   Testis   Epididydmis      Vas      Seminal   Prostate
               (mg/kg)    (Days)        (g)          (g)                             Deference   Vesicle
    Control                 90         170.1       182.29     945.61     361.32       210.59     389.10    325.10
                                       +1.20       + 1.51     + 3.42     + 6.16       + 2.49      + 6.12   + 8.13
    Sodium        5                   160.10       171.61       933      310.41       220.15     375.12    463.10
    fluoride                          + 1.51       + 2.49     + 3.40     + 1.35*      + 1.35      + 3.44   + 1.45
     Doses       20                   180.45       184.32     915.10     300.31       201.20     320.71    401.10
                                      + 1.33       + 1.63        +       + 1.41       + 1.55*    + 1.21*   + 1.71*
                                                               1.46*
                 50                  171.41        168.25     876.20     300.41       172.72     286.49    200.41
                                     + 4.21        + 6.10        +       + 3.55*      + 1.35*    + 4.18*   + 3.18*
                                                               7.40*
** 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.
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