testicular damage after chronic exposure to carbendazim in male goats
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Testicular damage after chronicexposure to carbendazim in male goatsWaghe Prashantkumar a , R.S. Sethi b , Devendra Pathak c , S.Rampal a & S.P.S. Saini aa Department of Veterinary Pharmacology and Toxicology , Collegeof Veterinary Sciences , GADVASU, Ludhiana 141004 , Punjabb School of Animal Biotechnology Guru Angad Dev Veterinary andAnimal Sciences University , Ludhiana 141004 , Punjabc Department of Veterinary Anatomy College of VeterinarySciences , GADVASU, Ludhiana 141004 , PunjabAccepted author version posted online: 21 May 2012.Publishedonline: 06 Jun 2012.
To cite this article: Waghe Prashantkumar , R.S. Sethi , Devendra Pathak , S. Rampal & S.P.S. Saini(2012) Testicular damage after chronic exposure to carbendazim in male goats, Toxicological &Environmental Chemistry, 94:7, 1433-1442, DOI: 10.1080/02772248.2012.693493
To link to this article: http://dx.doi.org/10.1080/02772248.2012.693493
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Toxicological & Environmental ChemistryVol. 94, No. 7, August 2012, 1433–1442
Testicular damage after chronic exposure to carbendazim in male goats
Waghe Prashantkumara*, R.S. Sethib, Devendra Pathakc, S. Rampala and S.P.S. Sainia
aDepartment of Veterinary Pharmacology and Toxicology, College of Veterinary Sciences,
GADVASU, Ludhiana 141004, Punjab; bSchool of Animal Biotechnology Guru Angad DevVeterinary and Animal Sciences University, Ludhiana 141004, Punjab; cDepartment of VeterinaryAnatomy College of Veterinary Sciences, GADVASU, Ludhiana 141004, Punjab
(Received 30 November 2011; final version received 9 May 2012)
The present investigation was aimed to determine the effect of chronic exposureof carbendazim on cellular changes in testes of male goats. The goats wererandomly divided into two groups, control and treatment (N¼ 7 each). Thetreatment group was exposed to carbendazim at the dose rate of 50mgkg�1 bodyweight per day orally, once daily for 180 consecutive days. Testes were removedfrom control and experimental animals surgically on the 90th, 120th, and 180thday. On the 180th day of the treatment, a maximum number of seminiferoustubules became atrophic, and vacuolization of germinal epithelium, andsloughing of the germinal epithelium and sertoli cells was more marked ascompared to days 90 and 120. Some of the seminiferous tubules were devoid ofgerminal cells. These pathological findings were supported by ultrathin andelectron microscopic examination. On the basis of the present investigation itcould be concluded that chronic exposure of carbendazim in male goats did notallow proper maturation of testis.
Keywords: carbendazim; male goats; seminiferous tubules
Introduction
Goat is an important source of food for humans, its meat being relished by people fromalmost all cultural backgrounds. The damage to the reproductive organs of the goats candecrease the reproduction and breeding potential and can lead to financial losses in goatindustry. In the present world scenario, pesticides have become an indispensable tool forsustaining mankind through crop protection and control of various pests. Carbendazim(methyl-2-benzimidazole carbamate) is one of the broad spectrum fungicides currentlywidely used, as well as its major metabolite benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate] is (Vettorazzi 1976). It is used as preservative in paint, textile,papermaking, and leather industry as well as a preservative of fruits (Selmanoglu et al.2001). The fungicidal property of carbendazim is targeted toward tubulin, causingdisruption of microtubule formation and mitotic cell division (Burland and Gull 1984;Foster, Burland, and Gull 1987). Can and Albertini (1997) have shown that carbendazimdisrupts cell cycle progression in the mouse oocytes by altering meiotic spindlesmicrotubules stability and spindle pole integrity.
*Corresponding author. Email: [email protected]
ISSN 0277–2248 print/ISSN 1029–0486 online
� 2012 Taylor & Francis
http://dx.doi.org/10.1080/02772248.2012.693493
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In rats, carbendazim and benomyl have been known to induce sloughing of immaturespermatids (Hess et al. 1991) and the inhibition of microtubule assembly (Lim and Miller1997b). Several other studies conducted in rats (Rehnberg et al. 1989; Mahgoub andEl-Madany 2000; Barlas et al. 2002), mice (Correa et al. 2002), hamsters (Gray et al. 1990),and Japanese quails (Aire 2005) have elaborated the seriousness of carbendazim andbenomyl toxicity toward reproductive system. However, there is no data on the effects ofcarbendazim on the reproductive efficacy of goats. So, the present study was aimed todetermine the chronic oral exposure of carbendazim on testes of male goats.
Materials and methods
The experiments were approved and conducted under the institutional guidelines of theGuru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.Fourteen Amritsari male goats (8 months age, 12–15 kg body weight) were used in thepresent study. The animals were acclimatized in the animal shed of the department underuniform conditions for 3 weeks and were subjected to regular clinical examination andtreated with anthelmintics (Albendazole at the dose rate of 10mg per kg body weight for 4days) for deworming, 20 days before the commencement of the experiment. The animalswere maintained on green fodder and wheat straw and water was provided ad libitum.Carbendazim fungicide (Bavistin 50% WP) formulation was purchased from BASF IndiaLtd. (Mumbai, India). Goats were randomly divided in to two groups, control andtreatment (N¼ 7, each). To the animals of the treatment group, carbendazim dissolved inwater and administered by gavage, at daily dose of 50mg per kg body weight, for 180consecutive days. The dose was selected on the basis of a pilot study (data not shown).Goats treated with carbendazim were closely observed for appearance of symptoms oftoxicity. The nature, degree, and time of occurrence of various symptoms were recordedduring the experimental period. Testes were removed surgically from control andexperimental animals on 90th, 120th, and 180th day (two animals from each group) forhistopathological examination.
Histopathological studies
The tissue samples from testes removed surgically from control and experimental animalson 90th, 120th, and 180th day were fixed in 10% neutral-buffered formalin. The tissues’samples were processed to get 5 mm thick paraffin sections which were stained withhaematoxylin and eosin, and used for histological studies of testis.
Electron microscopic studies
Testes removed from control and experimental animals on 180th day for electronmicroscopic examination were cut to the size of 2–3mm and collected in 2.5%glutaraldehyde. The fixed tissue samples were processed for transmission electronmicroscopy as per standard protocol (Electron Microscope Facility, All India Instituteof Medical Sciences, New Delhi). The 60–90 nm ultrathin sections were cut frompolymerized gelatin-embedded tissues using an ultramicrotome. The ultrathin sectionswere lifted on copper grids and stained with using uranyl acetate and lead citrate (Zamboni1976).
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Results
The histoarchitecture of the testes from control goats appeared to be normal and was
formed of seminiferous tubules surrounded by tunica albuginea (Figures 1 and 2). Each
tubule was surrounded by outer flattened advential cells and an inner basement
membrane. In between the tubules, the interstitial connective tissue showed fibroblasts,
blood vessels, and interstitial cells of Leydig. The seminiferous tubules appeared uniform
in size and shape. They were lined by regularly arranged rows of spermatogenic cells in
different stages of maturation.Administration of carbendazim at the dose rate of 50mgkg�1 for 90 days showed
effects on testicular histology (Figures 3 and 4).There was disruption of the seminiferous
tubules and vacuolisation in the germinal epithelium. Few multinucleated giant cells and
proliferation of spermatogonia up to certain level were observed but differentiation of the
sparmatid and spermatogonia was lacking. Few Leydig cells also showed pyknotic
appearance.The histological details of the testes after 120 days exposure are shown in Figures 5
and 6. Vacuolisation in the germinal epithelium was more marked as compared to 90 days
of exposure. The different phases of maturation were not observed in the germinal cells,
Figure 2. Normal histology of testis. HE 40�.
Figure 1. Normal histology of testis. HE 20�.
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Figure 5. 120th day treatment group histology of testis. HE 20�.
Figure 3. 90th day treatment group histology of testis. HE 20�.
Figure 4. 90th day treatment group histology of testis. HE 40�.
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and these cells had pkynotic nuclei and interstitial thickening appeared due to increasednumber of fibers.
The maximum histopathological damage was observed in testes of goats exposed for180 days on same dose rate (Figures 7 and 8). In this group, a maximum number ofseminiferous tubules were atrophic, and vacuolisation of the germinal epithelium wasmore marked than at 90 and 120 days exposure. Sloughing of germinal epithelium andsertoli cells was observed and some of the seminiferous tubules were devoid of germinalcells.
Electron microscopic examination of ultrathin section of testes, showed normal nucleiand cytoplasm in control animals (Figures 9 and 10). Animals exposed to carbendazimexhibit fibrosis in interstitial cells of the testis, dilated endoplasmic reticulum anddegenerated swollen mitochondria (Figures 11–13). In severely affected cells the nucleiwere pyknotic and shrunken.
Discussion
Sloughing of immature germ cells and the inhibition of cell division are the first testicularalterations observed after exposure to benzimidazoles in rats (Hess et al. 1991).
Figure 6. 120th day treatment group histology of testis. HE 40�.
Figure 7. 180th day treatment group histology of testis. HE 20�.
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Atrophied seminiferous tubules, vacuolisation of the germinal epithelium, sloughing ofgerminal epithelium and sertoli cells, fibrosis in interstitial cells, dilated endoplasmicreticulum and swollen mitochondria in histological and electron microscopic findings ofthe present study are consistent with the findings of Mahgoub and El-Madany (2000),
Figure 10. Normal electron microscopy of testis. 1600m� 2m.
Figure 9. Normal ultra thin microscopy of testis. HE 40�.
Figure 8. 180th day treatment group histology of testis. HE 40�.
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Rajeswary, Mathew, et al. (2007), Barlas et al. (2002), and Nakai and Hess (1994). Theseverity of damage increased with the concentration of carbendazim at the target site, thetestis, and also with time after administration (Lim and Miller 1997a, b). The fungicidalproperty of carbendazim is targeted toward tubulin, causing disruption of microtubuleformation and mitotic cell division (Foster et al. 1987). Our histopathological observationare also supported by the observations of Hess and Nakai (2000) who found that theadministration of carbendazim at moderate to low dosage induced sloughing of germ cellsand abnormal development of the head of elongating spermatids. In the present study, thepathological changes observed in the Leydig cells might be due to increased oxidativestress in testicular Leydig and seminiferous tubules (Rajeswary, Kumaran, et al. 2007).
At the molecular level, benzimidazoles bind to the �-tubulin subunit of the � and �tubulin heterodimer, inhibiting microtubule polymerization (Quinlan, Pogson, and Gull1980). Studies of the effects of carbendazim on microtubule assembly demonstrate acolchicine-like decrease in the rate of assembly and decreased microtubule stability(Winder, Strandgaard, and Miller 2001). Age-dependent differences susceptibility tobenomyl induced testicular damage have been reported in following subchronic exposureof benomyl by oral route in rats (Carter et al. 1984). The dilatation of the endoplasmicreticulum in sertoli, spermatogenic, and Leydig cells in our study is also in agreement with
Figure 11. 180th day treatment group ultra thin microscopy of testis. HE 40�.
Figure 12. 180th day treatment group electron microscopy of testis. 1600m� 2m.
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Hess and Nakai (2000) and Mahgoub and El-Madany (2000) who assigned these changes
to the intracellular redistribution of water and ions.Carter, Hess, and Laskey (1987) reported that carbendazim caused an early, relatively
short-term infertility in 16.8% male rats and an irreversible infertility in 50% rats. They
found severe seminiferous tubular atrophy (485% of tubules were atrophic) in thosecarbendazim-treated male rats that failed to recover fertility after 245 days of exposure.
However, some atrophic tubules (13–85%) were also observed in those male rats thatrecovered fertility.
Conclusion
On the basis of the present investigation, it could be concluded that chronic exposure ofcarbendazim in male goats does not allow proper maturation of testis.
Acknowledgments
The authors are grateful to the Vice Chancellor, Guru Angad Dev Veterinary and Animal SciencesUniversity (GADVASU) Ludhiana 141004, Punjab, India, for providing the necessary facilities.
Figure 13. 180th day treatment group electron microscopy of testis. 1600m� 2m.
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