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    PJ BS Bosxiczs so Pakistan Journal of Biological Sciences ANSIez n Network for Scientific Information 308 Lasani Towns Sargodha Road, Faisalabad - Pakistan Pakistan Joumal of Biological Seiences 10 (1): 90-95, 2007 ISSN 1028-8880 (© 2007 Asian Network For Seientifie Information An in vivo Evaluation of Induction of Abnormal Sperm Morphology by Ivermectin MSD (Mectizan") 0.4. Otubanjo, “A.A. Mosuro and 'T-F. Ladipo "Department of Zoology, University of Lagos, Lagos, Nigeria "Department of Basie Sciences, Botswana College of Agriculture, Gaborone, Botswana USA Abstract: The i vivo effets of orally administered ivermectin (Mestizan® on sperm head moepolegy of albino nice were evaluated Four different dese levels of 0.28, 0.5, 1.0and1.5x the human therapetsic dese of 150g kg body weight, were administered to the animals ‘The sperm of the mice from the eauda epididymes were ‘The animals were exposed toa single oral treatment. ‘examined 5 and 7 wecks after treatment. Ivermectin (OMectizan®) induced sperm head abnormalities; however, the induction was not significanly elevated above the negative control value, Furthermore, the induction of the sperm head abnormalities was not strictly dos dependent and there was also no correlation between dose level of administered drug and incidence of abnormal sperms, This indiostos thatthe drug might not be mutagenic, Key words: Ivermectin, mectizan, mutagenicity, sperm abnormality, onchocereiasis. in vivo INTRODUCTION Onchooerciants oF river blindness 8 a debilitating sono of public helt importance in sub-Saharan Aion, being the leading cnne of blindness, Ninety five percent ‘of cases of the cisease oocur in Africa.The main too in te glotal contol of onchocercasi is the long term mass fanvaual or bicannal adininistation of ivermectin (Mectiza®). The Aftioan Programme ox Onchocercasis ‘Control (APOC) is involved in control hreugh community drected weatment (WHO, 1995), The coverage, ‘compliance and sustainability of dig distribution and ‘administration are thus vital to accomplishing the goals of APOC. Ivermectin is distributed fice under the auspices of the giant pharmaceutical company, Merck, Sharp and Dole. It is a broad spectnan antiparasitic agent of ‘medical and veterinary importanee (Campbell ¢ a, 1983; Campbell and Ber, 1983). Trealment is usually ‘administered as a single orl dose given with water: The suggested doss interval for most patents is 12 months; at some sites, itmay be preferable to use a 6 month interval pending on sich considerations as density or prevalence of skin microfilrine. Certain studies have recommended quarterly or half yearly administration of ivemectin in the hyperendemic eemmunitis (dowu, 2004) The World Health Organization supervises the ‘mass administration of dhe drug in endemic areas. I 8 thorefore an ius of interest and conseqvenly desirable that this drug be sate 10 wie due to the widespread ‘exposin tot Recently, however, serious aide offsets and ‘complications in the wse of ivermectin have been seported in areas with endemic loaisis (Chippaux ef al, 1996: Garon af, 1997; Boussiesque etal, 1997). though adverse events {© ivermectin in some cominitics affect ivermectin compliance (Oyibo and. Fagberro- Beyiokw 2003), community awareness, participation ‘and compliance to ivermectin administration ate on the inrease. Ivermectin, a miarfilaicide is beloved to bee sats well tolerated and nor-exie, producing mild or transient side effects in the hnaman body (Dadzie et a. 1 Desole et a, 1589, Alexander eal, 1993). The efficacy and tolenmee of ivermectin in onchocerciasis is documented (Aziz etal 1992), Ivemeatin at repeated doses of 0.4 mg kg was found to have no effect om reproduction in cate, sheep, hoses, pigs dogs and as A single 0.4 mg kg™ dose of ivermectin given to bls ‘rama and ewes and (6 me. kg given to stallions and Ihoars had no ill-efféets on breeding performance or om semen quality (Campbell and Benz, 1683). Ivemectin inistred ceally at 600 yg kg (6. kg") ently ‘over 8 eatments had no adverse effects on spematogeness, fertility or reproductive performance ‘of Beagle dogs (Daurio ef a, 1987) Corresponding Author: 0. Otubano, Department of Zoolepy, Univesity of Lagos, Lagos, Nigeria 30 Pale J. Biol Sot, 10(1): 90-95, 2007 ‘The effects on embryogenesis have not been bwerved in higher animals in pre-clinia! safety assessment. studios Retrospective states in Mali of pregrant women accdertaly treated with ivermectin Proticed no malformations, utero morality and new tem morality (Duco eraf, 1990) Singer doses of 200 pe he "(02m every fw mera are three expected to be relively safe for bth men ar were However, the drug was not recommended for pregnant women, rising mothers with T wee od inf and Children under 3 yea of age bec safety hd not ten acrid Laas ta. (1985) reported the effects fivermectnen reproduction and neoates in rat Most ene ares ofthe work ncoting Nigeria and mom of sub Saberan Afi, are chiscterized by abjoa poverty and large tceming populations with ew trained medical personel. Consequently. mest medical sient cannot pobly be bought nde Sect medal Spervinion A large percentage ofthe population reso toslmediation wih campleteigeraoe of te corel prescriptions (Othanj and Mosure, 200), One cao, ompletely rule out the posit of indisesmssate tre and abe of digs an the atentant over expotne to drags. This long standing indscriminte we of ddugs B indeed a habit in mang developing outes; Taty drugs being, purchased without prescription from ebemis During the past two decades, increasing stenton ts been dicted. towards the deeminain and valuation of the mutagenic and catcinogenie poeta Sf drugs even thovgh tir ise chemotterpetic cary camol be underestimated: Ic is desirable en tht these indispensable chemotherapeuse agents ae fee frm any deleterious effects, pri when the du > distributed free and also considering its long term Proposed wage to reduce trsmision and eby ontol the dscse. The eamequcace of eng th Proposed wage of the rg in the contol of ‘ichoceriss at the multiple snl administration of tnesmectn in onchocrcisis conzl noe be evaluated for mulageniaty poeta In ths sta the spem of vermectnreated mise were analyzed to determine whether the drug is Iulagenic. An inrease inthe incidence of sbnermal Spe morphology. according to te enter of Wyrebek aad Broce (1975 and 1978) inate of mutagenicity MATERIALS AND METHODS Experimental animals: Male albino mice were obtained from the animal breeding unit of the National Institute of ‘Medical Research, Lagos (NIMR), (3H/Hel strain). Mice o (12-14 weeks old) wore aoquired and quarantined in a pathogen-free, well ventilated room in order to enable the ‘mice to acclimatize to their environment and also to avoid the transitory increases in abnormal sperm seen at the ‘onset of mouse spermatogenssis in young mioe, The ‘mice were maintained in the same room throughout the andy. Only: mice of 14 weeks and above were tested Drinking water and food (pelleted feeds) were supplied ad libitam The study was tndertaken inthe Parasitology Laoratony a the University of Lagos, Lagos Drug: Ivermectin MSD (Mectizan*) was supplied by ‘Mere: and Co. Ino. The dng was dissolved in disiled ‘water which was used as the solvent vehicle. The deug readily distolved in ditilled water. Doses used in this study were selected according tothe Human Therapeutic Dose (HTD) of 150 41 kg" body weight based on the average hnman weight of 65 kg “Assay of sperm abnormalities: Induction of sperm-head abnormalities was tested according to the oriteria of ‘wyrobek and Brice (1975 and 1978), Four different dose level treatments were considered for the drug, Four doses ‘were used to treat the mice, 1.25, 2.5, 5 and 7.5 pa, ‘corresponding to 0.25, 05, 0.1 and 1.5 the human therapeutic dose. The drug was administered as a single coral dese, A single orl dese was given, Two exposure periods ‘of S and 7 weeks from drug treatment were considered Four mice were treated for each dose level and each ‘exposure peticd, Twomice, foreach exposure period, were treated with the solvent vehicle alone as a negative ‘control. The positive control or dhe study was 100 mg of ‘methyl methane sulphonatekke per day, ip. fora period (of 5 cays Sperms were sampled and analyzed from the cada epididymes at 5 and 7 weeks following the end of ‘exposure te the 4 different dose levels of the drug, The Implication of abnormal sperm heads observed 5 and 7 weeks alter drug treatment is that the drug may have had an effect on sperm which had arisen from exposed spermatogonial cell, causing damage to the pre-meiotic stages of spermatogenesis, Thus, the abnormally shaped sperm heads observed far these exposure periods may Ihave been due to induced point mutations in the early spermatocytes and spermatogonia at the pre-meictic stages of spermatogenesis, This view is in support of those of earlier workers (Bruce etal, 1974, Wyrobek and Bruce, 1975, Soares ef al, 1979). ‘The mice were sacrificed by cervical dislocation. The ‘epididymes were excited and minced with fine seissors in plysiological saline. Smears were prepared on clean, Pak. J. Biol. Sei, 10 (1): 90-95, 2007 _grease-fe slides after staining the cells with a mixture of. normal saine and 1% eosin-Y (9:1) for 4S min. The slides were air dried and coded for subsequent examination under il. Cytological evaluation for sperm-head abnormalities was carried out using a binocular microscope at 1000*magnification. Four separate slides were prepared for each mouse, ie, wo for cach epididymes out of which two were randomly selected for scoring, The slides were read blinded to treatment, The sperms were assessed for morphological abnormalities of sperm head shape according to the criteria of Wyrobek and Bruce (1975). For each animal, 750 sperms were assessed for morphological damage. Statistical analysis: Differences between the control and. experimental groups were analyzed by means of the student's test. The test was considered postive when the frequency of abnormal sperm heads was at least double the negative control level, with p<0.05 as the criterion of significance. Furthermore, the test must have yielded statistically significant increases at a minimum of two consecutive dose levels, be reproducible in separate experiments and finally, show evidence of a dose-related rease in abnormalities. RESULTS Analyses of sperm head abnormalities were made Sand 7 weeks following the end of exposure to 4 different dose levels of ivermectin, Sperms observed at these times were presumably exposed to the drug while they were spermatocytes and spermatogonia, Figure 1 shows normal sperm and the different sperm-head abnormalities recorded from the prepared slides from the treated animals. during microscopic study. Photomicrographs of each type of sperm head shape were taken and reproduced. In the course of scoring the abnormalities, it was observed that no specific type of abnormal sperm head was predominant as they all occurred with different frequencies in both tweated and contol mice. ® |O|O/ 9/0 ® ® elo Fig 1: Observed shapes of abnormal. sperm heal. A- sperm with normal head (the morphology of a normal sperm head of a mouse consists of a definite head shape accented by a marked hook, a rectangular mid-piece attachment site and single tail; B-No hook; C-2-tail; D-knobbed hook; E+ Amorphous head; F- Bent hook; G- Hook at ‘wrong angle: H- Tall folded over heads I-Pin-heat 4J- Banana-shaped head ‘Table 1 shows the effect of different dase levels of rmectin on sperm head morphology after $ and 7 weeks exposure. The negative controls showed 2.53 and 2.30% abnormalities, respectively. The positive control gave a statistically significant elevation of abnormal sperm heads (lata not shown). Figure 2 presents the effects of different exposure periods of ivermectin on sperm head abnormality. Iwermectin did not induce statistically significant increases in sperm-head abnormality over the controls as, the ertera for positive response were not satisfied. There was an increase in the frequency of abnormal sperm heads but the increase was not significant at the p0.05 level While Fig. 2 shows increase in percentage sperm abnormality with increase drug concentration for the different exposure periods. The range in frequencies of sperm abnormality. recorded were 2.40-3.27, 2.53-3.50, 267-3.77,297-416 for 1.25, 25, 80 and 7.0 me wbwt “he | Eft of enmetn on spam mario in IMR 31) mie fer S nd 7 wees export ‘vemectn dove Seca Tweeks Inemestin dose ogo weg) att) ‘ealainomalig Toalainomaliy en as is ha aan ® 201 Tn prensa te meas ox ops of four ike Tor eh pit 92 Pale J. Biol Sot, 10(1): 90-95, 2007 12s ‘Concentra cent) Fig. 2 Relationship between percentage abmormality and different and dose levels Se ptmarmatty S$ 7 3 5 7 Tine (wed) Fig. 3: The effect of different exposure periods if ivermectia on head of mice dose levels, respectively over the 7 weeks period [Figure 3 shows dhat abnormality decreased with length of| drug exposure. DISCUSSION Onchocerciasis afflicts over 17.7 million indivicaals in sub Saliaran Attica, where the disease is endemic WHO, 1987), In the absence of a viable anti- ‘onchocereiasis vaccine, the only effective means of 93 prevention and control lies in chemotherapy. Ivermectin js a potent microfilaricide, effective in a single dose therapy and fas been widely adopted as the dug of choice to treat and control onchocerciasis, Indeed, control ‘and prevention campaigns in endemic areas ofthe warld involve the free distribution of the drug, annually or annually, to atlicted populations. Certain peroentage of sperm shows abnormal ‘morphology in mice of different strains and age. The froqueney of sperm abnoemality varies among, diferent inbred strains of mice, being constant for adult mice, with incidence ranging from 2-5Y (Soares et al, 1979). Age infhionoes the degree and frequency of abnormality. By the 13th week of age the percentage of morphologically abnormal sperm in mice would have reached the level ‘characteristic of its strain (Krzanoskwa, 1981). The control ‘mice sed in the present study showed 230-2.53% sperm abnormality Spermatogenesis in mice takes about 5 weeks to ‘complete. Its known that during spermatogenesis, DNA synthesis occurs before the pre-meiotic phase and no further synthesis occurs throughout the duration of spermatogenesis in the cell eyele (Monesi, 1962). Also sperm-head abnormality has been indicated to result from sliverse factors which include, errors in the differentiation process during spermatelecss, errors in the packaging ‘of the genetic material, induced mutations in the spermatogenic cells, the cccurence of point mutations during spermatogenesis and exposure to imadiation or ‘chemical mutagens (Bruce era, 1974; Topham, 1980a-c; Wyrobek et al, 1983) There are no available evaluation reports om the mutagenicity of ivermectin in mice. In thie sty, using the spemshead abnormality test, orally administered ivermectin induced an increase in sperm head abnormalities in albino mice over the negative controls, although the increase was not statistically significant ‘at po005, The criterion for @ positive response or ‘mutagenicity is based on evidence of statistically significant occurence of abnormal spemm at p-0.05, Furtiemore, there should be evidence of a dose-related increase in abnormalities and reproducibility in separate ‘experiments. Ivermectin, may thus not be considered to be a mutagen as the criteria for mutagenicity were not stictlysatistied, Although an increase in the frequency of abnormal sperm heads was observed in more than two consecutive dose levels except the highest dose level for both exposure periods, this was not enough to confer positive response on the drug. The drug may therefore rot be adjudged @ positive inducer of abnormal sperm ‘heads and may not be mutagenic Pale J. Biol Sot, 10(1): 90-95, 2007 ‘One plausible reason for the inability of ivermectin to Ihave induced statistically significant increases in abner sperm in mice 8 that the testes of the treated fanimals probably did not accumulate enough concentrations of the drugs, s0 as to ater the ‘hfrentiation ofthe spermatozos, Ivermectin is readily ‘metabolized and excreted from the body: the maximum uration of plasma concentration of the drug is 6-7 h (Oectizan*-Ivermectin MSD product Monograph, 1985). Furthermore, the ineidene of sperm head abnonalities tended to decrease with exposure period, as more normalities were recorded with the 5 week exposire petiod than the 7 week exposure period ‘There have been no epars of ivermectin binding DNA, which can ultimately lead to the fal

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