الفهرس 
Introduction
MancozebOnly 14 pages are availabe for public view
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Abstract
Environmental contaminants have adverse effects on the reproductive system of humans and animals. These toxicants cause endocrine distruption and alterations in testicular function as well as male fertility.Dithiocarbamates (DTCs) are a class of thiocarbamate derivatives which have been used in agriculture as pesticides,insecticides, and fungicides for more than 50 years.Ethylenebis-dithio-carbamates (EBDCs) are a subclass of DTCs, usedprimarily as broad spectrum organometallic fungicides. Amongthese is mancozebwhich is apolymeric complexes of EBDC.The potential adverse reproductive and developmental effects of mancozeb, especially in rabbits, have not been fully reviewed for this widely used fungicide.
This study was carried out to investigate the possible ameliorative effect of non-enzymatic antioxidant glutathioneagainst reproductive toxicity of mancozeb fungicide in male rabbits (Oryctolaguscuniculus). A total of 36 adult male White New Zeland rabbit weighting 1,9-2,6 kgof 4-5 month oldwere randomly allocated into four groups of 9 animals in each.Exposure to mancozeb and GSHwas applied orally by stomach tube for 12 weeks.
GroupI: nine rabbits were kept as control and administered corn oil only for 12 weeks.
group II:nine rabbits were exposed to 100 mg/kg b.w1\50 of LD50 that equal 5000mg\kg of mancozeb,dissolved in corn oil, given twice weekly, orally by stomach tube for 12 weeks.
Groups III:nine rabbits received both mancozeb(100mg/kgb.w) and glutathione (100mg/kgb.w),dissolved in distilled water.twice weekly, orally by stomach tube for 12 weeks
group IV:nine rabbits receivedGSH(100mg/kgb.w), dissolved in distilled water, twice weekly, orally by stomach tube for 12 weeks.
The weight of rabbits was recorded weekly.
Three rabbits from each group were killed by cervical dislocation after 24 hours of last exposure to mancozeb and glutathione.Blood, testes, epididymis and accessory sex organs (the vasa deferentia, seminal vesicles, prostate glands, Cowper’s glands, coagulatorygland) samples were collected at 4, 8 and 12 weeks post-exposure.Serum samples were obtained from clotted blood collected without anticoagulant after centrifugation. The serum samples were used for determination of testosterone, FSH and LH hormones levels. Testis, epididymis and accessory genital glands were excised, weightedrecorded to thenearest milligram.
Testiswere dissected out, trimmedoffrom adherent fatsandprepared to be homogenized for the estimation of activities of lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and 3β-hydroxysteroid dehydrogenase (3βHSD) enzymes and also for the estimation of protein, lipid and cholesterol.Caudaepididymal ducts prepared to sperm studies to evaluate the sperms vitality and morphology.Sections from the testis and accessory genital glands immediately after sacrifice were used for histopathologicalexamination.
Body weight, sex organ absolute and relative weight, biochemical parameters as well as histopathological changes were the criteria used to evaluate the reproductive efficacy of the treated male rabbits.
Gravimetric analysis of body and reproductive organ revealed that the value of final body weight ofmancozeb treated rabbits showed a significant decrease equal 13% during the whole period of experiment.While the effect on reproductive organs weight including testes and accessory genital glands (the vasa deferentia, seminal vesicles, prostate glands, Cowper’s glands andcoagulatory gland)revealed thatthe value of right and left testis absolute weight showed 18% decrease in weight while relative weight showed 7% decrease during the whole period of experiment in comparison with control.The value of accessory genital glands absolute weight showed 20% decrease while its relative weight showed 5% decrease during the whole period of the experiment in comparison with control.
The reduction in body weight may be due to high rate of protein breakdown, which might be needed to fulfill energy requirements during detoxification. Similarly, reduction in male reproductive organ weight is a sensitive endpoint in reproductive toxicity assessments including alterations in testis structure. This reduction of testis weight in the present study could be attributed to a loss of spermatogenetic elements.
Hormonal assay revealed a significant reduction in serum Luteinizing hormone (LH) 33% and follicle-stimulatinghormone (FSH)levels.17%The value of serum testosterone levels showed a highly significant decrease equal 71% during the whole period of experiment.The low-serum level of gonadotrophins may be due impairment in reproductive function by direct insult to the cell populations within the gonads resulting in impairment to feedback mechanisms to the hypothalamus and pituitary. The reduction in serum testosterone demonstrated the inhibitory effects of mancozeb on the secretion of pituitary gonadotrophins (FSH and LH).
Biochemical investigation of testiculartissue homogenate revealed that upper regulation in steroidogenic(3β-HSD) enzyme activity andshowed a significant increasein its expression inmancozeb treated rabbitsequal 33%during the whole period of experiment in comparison with control.Theupper regulation of 3β-HSD activity is one of the possible mechanism through which xenoestrogens disturb spermatogenesis and confirm mancozeb exhibited significant anti-androgenic activities.
Testicular androgen-dependent enzymes suchas LDH, ACP, and ALP are consideredfunctional indicators of spermatogenesis, showed a significantdecrease than control group.LDH showed 32 % decrease, ACP showed 36 % decrease, and ALPshowed 61 % decrease. Since the activity of these enzymes is closely associated with spermatogenesis and male testiculardevelopment, the decreased activity of these enzymes inmancozeb-administered animals represents a defect inspermatogenesis and testicular maturation.
The macromolecular contents of testis,cholesterol and triglycerides showed a significantdecreasein mancozeb-administered animals. While no significant change in the content oftesticularprotein.Decreasesin the levels of cholesterol 61%, and lipids 78% with mancozebtreatment suggest either an increased catabolism of thebiomolecules to meet the enhanced energy demand of animals under stress or reduced synthesis due to impairedtissue function.
Evaluation of sperm parametersinthis study revealeda significant decrease in sperm viability in cauda epididymisandasignificant increase in number of abnormal sperms in cauda epididymis was observed after mancozebtreatment.Sperm morphology and viability exhibited alterations inphysiological function and histo -architeture of the epididymis also showed regressive changes. Effects of mancozebin the tested dose on spermviabilitycan be summarized inthe percent of dead epididymalsperm was 32 %in comparison of 15% in control rabbits.Effects of mancozeb in the tested dose on sperm morphology can be summarized inthe percent of abnormal epididymalsperm was 32%in comparison of 10% in control rabbits. Abnormal sperms morphology as sperms with coiled tail , double tail, double heads with no tail, double head with a single tail, small tail and sperms with no tail.
Histopathologicalexamination revealedthat testis of the rabbits treated with mancozeb (100mg/kg/ twice/ week) showing vacuoles formed by exfoliation germ cells and disorganization of interstitial tissue and decreased in the number sperms in the lumen. Sertoli cells have vacuoles,reduced number of spermatogenic cells, lumen loss of sperms and disorganization of spermatogonia and spermatocytes.Interstitial cells were also affected,edema of interstitial tissue and Leydig cells degeneration. Spermatocyte death with secondary depletion of postspermatocyte germ cells, progressive apoptosis and depletion of the elongating steps of spermatid development.Accessory glands like prostate showed adenoid and stromal hyperplasia and congestion of the interstitial tissue.
On the other hand, the most prominent result in this study suggested that co-administration ofGSHin adoseof (100mg/kg/ twice / week) with mancozebin adoseof (100mg/kg/ twice aweek) alleviate reproductive toxicity caused bymancozeb exposure in male rabbits.Co-administration of GSHshowed a highly significant increase in body weight8% and reproductive organs weight including testes absolute weight 7.35 %, relative weight0.7%while accessory genital glands absolute weight 52%, relative weight 40.35% in comparison with group ofmancozeb treated rabbits. GSH co-administration enhanced testicular androgenesis, and showed a highly significant increase in concentrationsof serum testosterone88% and gonadotrophinshormonelevelsFSHincreasesd50% while LH increased 4%, in comparison of mancozebtreated group. And also, a significant increase inconcentrations of testicular androgen-dependent enzymes LDH94%, ACP 17%, ALP147% was obtained.Alteration in the levels of testicular biomolecules as protein and triglycerides was alsoenhanced, as triglycerides showed 7% increase but testicular cholesterol showed 17% decrease in comparison of mancozebtreated group.Steroidogenic (3β-HSD) enzyme activity showed a significant decrease in its expression equal 38% in comparison with mancozeb treated groupImprovedspermatogenesisreflected by significant increase in sperm viability the percent of dead epididymalsperm was 11.33 % as compared with mancozeb treated rabbits, the percent was 32 %.A significant decrease number of abnormal sperms in cauda epididymis was also observed in GSH, the percent of abnormal sperm was 12.66 % as compared with mancozeb treated rabbits the percent was 32 %).
Finally, it could be concluded that mancozeb exposure resulted in an inhibition in testicular androgenesis, and decreasing concentrations of serum testosterone and gonadotrophins, decreasing concentrations of testicular androgen-dependent enzymes.It induced alteration in the levels of testicular biomolecules. Impairment of spermatogenies is reflected by significant change in sperm viability and morphology. Histopathoogical alterations of testis showeddisruption of germinalepithelium with vacuolization of leydig cellsand decreasedspermatogenic cells, which resulted in dysfunction in the steroidogenesis process.Therefore, this study suggested thatmancozeb exposure induced a physiological impairment and disturbed the testicular function.Co-administration of GSH which is called the mother of all antioxidants can restore the reproductive toxicity resulted in mancozeb exposure on the level of previous parameters. This study proved that concurrent administration of glutathione mitigated reproductive toxic effects caused by mancozeb in male rabbits.