Europen Journl of Biologil Sienes 4 (3): 73-82, 2012 ISSN 2079-2085 IDOSI Pulitions, 2012 DOI: 10.5829/iosi.ejs.2012.4.3.65104 Assessment of 3, 4-Dihloroniline Toxiity s Environmentl Pollutnt in Mle Mie 1 2 3 1 F.I. Eiss, A.I. El Mkwy, M.I. Br n O.H. Elhmlwy 1 Deprtment of Environment n Bio-Agriulture, Fulty of Agriulture, Al-Azhr University, 11884, Ciro, Egypt 2 Deprtment of Cell Biology, Ntionl Reserh Center, Dokki, Giz, Egypt 3 Deprtment of Animl Proution, Fulty of Agriulture, Al-Azhr University, 11884, Ciro, Egypt Astrt: The effet of 3,4-Dihloroniline (3,4-DCA) on hromosome errtions in oth one-mrrow ells n spermtoytes, sperm qulity n histopthologil hnges in mle mie ws investigte. Eighty mle Swiss lino mie were ivie into four groups (n = 20), one ws onsiere s ontrol n the other three groups were ministere 3,4-DCA y gvge t the ose levels of 13.83, 27.67 n 55.33 mg/kg w/y for 30 onseutive ys. The oses were orrespon, respetively, to pproximtely 1/32, 1/16 n 1/8 of LD 50 vlue etermine for mle mie (442.6 mg.i./kg w). Results inite tht orl ministrtion of 3,4-DCA signifintly inrese the frequeny of struturl n numeril types of hromosoml errtions in oth one-mrrow ells n spermtoytes. Similrly, there ws signifint inrese in ount of norml spermtozo in ll 3,4-DCA trete groups of mie ompre to respetive ontrol. On the other hn, 3,4-DCA use signifint erese in the sperm ount n motility s ompre to the ontrol. Furthermore, histopthologil hnges in liver n testis were oserve. All verse effets were in ose-epenent mnner. In light of the forementione results, it n e onlue tht 3,4-DCA hs the potentil to proue genotoxi effets, reproutive toxiity n histopthologil ltertion in mle mie. Aoringly, strit limittions on the use of this ompoun must e put. Key wors: 3,4-DCA Chromosome errtions Sperm qulity Histopthology Mie INTRODUCTION onentrtion inrese ~10 times fter hlormintion within full sle rinking wter tretment plnt tht use The romti mine, 3,4-Dihloroniline (3,4-DCA) hlorinte gents s isinfetnts in Spin [5]. It hs is wiely use s hemil intermeite in the synthesis een etete s n environmentl ontminnt in surfe of severl heriies (e.g. iuron, linuron n propnil), wters n in the effluents from ye-mnufturing plnts zo yes for polyester fris, textile n phrmeutils [8]. DCA hs een etete more frequently in [1-3]. It is lso egrtion prout of environmentl smples thn its prent ompouns [9]. trihlorornilie, hemil use s tive gent in It is not reily egre y miro-orgnisms n, ue to the osmeti inustry [4]. Diuron, linuron, oxyfluorfen, its persistene in the environment, is onsiere to e 2-methyl-4-hlorophenoxyeti i, teruonzol n referene xenoioti [10]. DCA n persist in the ifenoonzol n le to the formtion of hloronilines environment s insolule resiues in soil n plnts n inluing 4-hloroniline n 3,4-ihloroniline [5]. n lso photoimerise to form rinogens [11]. DCA Thus, 3,4-DCA is moel environmentl ontminnt [6]. enngers growth, evelopment n propgtion of 3,4-DCA often gets into the environment from quti orgnisms n furthermore is potentil hrmful to griulturl tivities, either susequent to the pplition humns [12, 13]. Stuies in vitro n in vivo hve shown or to the inustril proution proesses of hemils [7]. tht DCA hs nephrotoxiity n reproutive toxiity to 3,4-DCA ws etete in rw wter t low levels ut its mmmls [14, 15]. Consequently, DCA hs een lssifie Corresponing Author: Fwzy Eiss, Deprtment of Environment n Bio-Agriulture, Fulty of Agriulture, Al-Azhr University, 11884, Nsr ity, Ciro, Egypt. Tel: +2 0100 318 328 4. 73
Europ. J. Biol. Si., 4 (3): 73-82, 2012 s ompoun of environmentl onern. Furthermore, n oserve for 14 ys. The LD 50 vlues were ue to its toxiity oth to invertertes n vertertes lulte oring to the sttistil metho of Weill n to its high proution rte, it ws inlue in the [25]. Results emonstrte tht, The ute orl LD 50 for Europen Union priority List one of hemils onsiere mle Swiss lino mie ws etermine s 442.6 mg.i./kg prtiulrly troulesome for the environment [16]. w, with 95% onfiene limits of 342.58 to 571.78 mg/kg. Chromosoml errtions my e use s n erly wrning signl for ner evelopment n it hs een Experimentl Design: Eighty mle Swiss lino mie were suggeste tht the etetion of n inrese in ivie into four groups (n = 20), oring to hromosoml errtions, relte to n exposure to pproximtely equl men oy weight. The first group genotoxi gents, my e use to estimte ner risk serve s vehile ontrol n ws ministere orlly [17]. Bone mrrow, with its rpily renewing ell y orn oil, while the other 3 groups were ministere popultions, is one of the most sensitive tissues to 3,4-DCA y gvge t the ose levels of 13.83, 27.67 n ytotoxi gents [18]. In humns, the eline n well 55.33 mg/kg w. The oses were orrespon, respetively, known regionl ifferenes in semen qulity [19] n the to pproximtely 1/32, 1/16 n 1/8 of LD 50 vlue inresing iniene of testiulr ners [20], uring the etermine for mle mie (442.6 mg.i./kg w). Dose lst five ees, hve een relte to environmentl onentrtions were juste to men oy weight ontminnts [21]. Moreover, iuron, linuron, 3,4-DCA (10 ml/kg w). All groups orlly ministere 3,4-DCA or n 3,4-ihloroetnilie (3,4-DCA) hve the pity orn oil, one y for 30 suessive ys n then in vitro to onnet to nrogen reeptors, permitting 10 mie from eh group were srifie for tion s enorine isruptors [22, 23]. On the other hn, histopthologil n hromosoml errtion nlysis of very little informtion is ville regring sperm qulity, oth somti (one mrrow) n germ (spermtoytes) ytogeneti n histopthologil hnges inue y ells. The remining nimls (10 mie) from eh group this ompoun. were left without treting for 35 ys from the lst ose Therefore, this work hs een esigne to evlute (urtion of spermtogensis), then the nimls were the potentil effet of 3,4-ihloroniline on hromosome srifie for nlysis of sperm ount, motility n errtions in oth one-mrrow ells n spermtoytes, normlities. sperm qulity n histopthologil hnges in mie. Mitoti Inex Determintion: The mitoti inex ws use MATERIALS AND METHODS to etermine the rte of ell ivision. The slies prepre for the ssessment of hromosoml errtions were lso Chemils: 3,4-Dihloroniline (3,4-DCA, 98% ) ws use for lulting the mitoti inex. Rnomly selete purhse from Tokyo Chemil inustry Co, LTD, Jpn. views on the slies were monitore to etermine the All other hemils were of nlytil gre n were numer of iviing ells (metphse stge) n the totl purhse from stnr ommeril suppliers. numer of ells. At lest 1000 ells were exmine in eh preprtion. The mitoti inex ws lulte s the rtio Animls: Mle Swiss lino mie weighing 28±5 g were of the numer of iviing ells to the totl numer of ells, purhse from the Theoor Bilhrz Reserh Institute multiplie y 100 [26]. n were house in polypropylene ges (43 30 15m, five mie per ge). Mie were mintine uner Chromosoml Aerrtion Anlysis: At the en of the ontrolle temperture (23±1 C), 50 55% reltive tretment, nimls of ll trete groups were injete humiity, photoperio of 12 h light: 12 h rk n intrperitonelly with olhiine to rrest ell ivision t permitte to freely onsume wter n foo liitum. metphse. Two hours fter injetion, nimls were The mie were limtize for 2 weeks efore osing. srifie y nek verter luxtion for preprtion of the hromosomes of one mrrow n spermtoyte ells. Assessment of Orl LD 50: An ute orl toxiity stuy Chromosomes of one-mrrow ells were prepre y ws performe in orne with OECD [24]. Twenty using the methoology of Yosi n Amno [27]. nimls were ivie into four groups, eh group Both femurs were issete out n lene of ny ontins five mie. 3,4-DCA ws issolve in orn oil n hering musle tissue. Bone-mrrow ells were ollete ministere orlly y gvge t four ifferent oses i.e., from oth the femurs y flushing in sline solution n 1 222.2, 333.3, 500 n 750 mg kg w. Following osing then inute t 37 C in hypotoni solution (KCl 0.56%) the nimls were llowe free ess to foo n wter for 35min, fixe in methnol:glil eti i (3:1). 74
Europ. J. Biol. Si., 4 (3): 73-82, 2012 The ells were resuspene in smll volume of fixtive, roppe onto hille slies, flme-rie n stine with 10% uffere Giems (ph 6.8). Spermtoytes were prepre oring to Brewen n Preston [28]. The tuni of the testis ws remove n the tuules were trnsferre to smll Petri ish ontining isotoni solution (2.2% soium itrte) n tese out with urve foreps on piee of mesh. The ell suspension ws trnsferre into onil tue n entrifuge. The superntnt ws remove n the pellet ws re-suspene in hypotoni solution (1.1% trisoium itrte) for 20 minutes n fixe in ol fixtive. The ells were resuspene in smll volume of fixtive, two or three rops of ell suspension were roppe on len slie store in ol 70% ethnol n rie on hot plte t 60 C. Slies were stine with 10% uffere Giems (ph 6.8). One hunre goo metphses for somti n germ ells of eh niml were exmine mirosopilly for soring ifferent types of errtions. Sperm Chrteristis Sperm Colletion: After 35 ys of the lst ose (urtion of spermtogenesis), the nimls were srifie y nek verter luxtion. The epiiymies from eh mouse were remove n sperm ws ollete s quikly s possile when eh mouse ws issete. The sperm ount ws ssesse from right u epiiymies while sperm motility n morphology were nlyze from the left one. Epiiymis ws exise n mine in 1 ml of phosphte uffere sline (ph 7.2) to otin sperm suspension. The suspension ws filtere through nylon mesh [29]. Sperm Count: The u epiiyml sperm ount ws performe oring to Nryn et l. [29] using Neuuer hemoytometri hmer. Lyere slies with semen were viewe y right-fiel mirosope with mgnifition of 400. The totl sperm ount in squres of 2 1 mm eh ws etermine to express the numer of sperm/epiiymis. Epiiyml sperm ounts were expresse s numer of sperms per epiiymis. To minimize the error, the ount ws repete three times on eh smple [30]. Sperm Motility: Approximtely 10 µl of sperm suspension with miropipette ws lyere onto wrme mirosope slie. Sperm motility ws ssesse y ounting ll progressive motile (effetive), the non progressive motile (non-effetive or sluggish) n the immotile (ormnt) spermtozo in the sme mirosopi fiel (400 ). In eh semen smple, 10 mirosopi fiels were exmine with t lest 100 sperm/fiel ws ounte. The numer of motile sperm ells in eh fiel ws ivie y the totl numer n the verge of the fiels ws ssye. The perentge of motile spermtozo ws etermine [31]. Sperm Anormlities: To ssess the spermtozo morphologil normlities, rop of sperm suspension ws smere on slie n ir-rie n me permnent. The smere slie ws stine with 1% eosin Y n 5% nigrosin. Morphologil sperm efets were evlute n exmine on optil mirosope using 400 mgnifition [32]. At lest 100 spermtozo from ifferent fiels in eh slie were exmine n lssifie for riteri of morphologil normlities (he, til n til-he) oring to Filler [33]. Anorml sperm ells were ounte n the perentge ws lulte. Histologil Stuy: At the en of experiment, liver n testis from eh srifie mouse were issete out; trimme of exess ft. All tissues were fixe in 10% uffere formlin n were proesse for prffin setioning y ehyrtion in ifferent onentrtions of lohol, lere with xylol n emee in prffin loks. Setions of out 5µm thikness were stine with Hrris hemtoxylin n eosin (H&E) for histologil stuy [34]. Sttistil Anlysis: Sttistil nlyses were performe with SPSS softwre. Dt were nlyze using one wy nlysis of vrine (ANOVA) followe y Dunn s post ho test for multiple omprisons etween pirs. Results were reporte s men vlues ± S.D. n ifferenes were onsiere s signifint when (P 0.05). RESULTS AND DISCUSSION There ws no mortlity, moriity or istintive linil signs oserve in ny of the experimentl niml groups uring the stuy perio. Chromosoml Aerrtions: The results of the present stuy showe tht the tretment of mle mie with 3,4-DCA inue signifintly ose epenent reution in the tivity of mitoti inex in oth one mrrow ells n spermtoytes (Figure 1). The mitoti inex ftor is use to isover the toxiity for severl physil n hemil gents whih mostly ffet the verge of ivision or levge [35]. 75
Europ. J. Biol. Si., 4 (3): 73-82, 2012 250 200 150 100 Mitoti inex in one mrrow 249.2 217.4 222.2 198.6 Mitoti inex in spermtoytes 156.6 144.8 101.4 100.8 50 0 Control 1/32 LD50 1/16 LD50 1/8 LD50 Fig. 1: Men vlues of mitoti inex in one mrrow n spermtoytes of ontrol n 3,4-DCA trete mle mie Tle 1: Men vlues of ifferent types of hromosoml errtions in one mrrow ells of ontrol n 3,4-DCA trete mle mie Numeril normlities Struturl normlities ------------------------- Totl Totl ------------------------------------------------------------------------------------------------------ Totl struturl Eno- Hypo- Poly- numeril hromosoml Tretment Gp Brek Deletion Centri fusion En to en Centro meri Frg-ment Ring form errtions mitosis ploiy ploiy errtions errtions Control 2.0 ±0.7 2.0 ±0.7 2.2 ±0.8 1.0 ±1.0 0.2 ±0.4 2.4 ±0.5 1.2 ±1.1 11.6 ±4.0 0.6 ±1.3 0.6 ±0.5 1.8 ±2.5 13.4 ±4.2 1/32 LD 4.2 ±0.4 4.2 ±0.8 3.2 ±0.4 1.2 ±0.4 1.4 ±0.5 1.0 ±1.0 4.4 ±1.1 2.8 ±0.8 22.4 ±3.3 1.0 ±2.2 1.4 ±0.5 3.0 ±1.9 25.4 ±4.6 50 1/16 LD 5.4 ±0.5 6.4 ±0.5 3.8 ±1.0 2.2 ±0.4 2.6 ±0.5 1.4 ±1.3 5.8 ±0.4 5.0 ±1.2 32.6 ±1.6 1.0 ±1.0 1.8 ±1.1 2.8 ±0.8 5.6 ±1.7 38.2 ±1.9 50 1/8 LD 6.6 ±0.5 6.8 ±1.6 6.8 ±0.4 3.6 ±0.5 5.2 ±0.8 2.4 ±0.9 7.0 ±1.5 6.4 ±1.9 44.8 ±5.6 1.4 ±1.3 3.0 ±1.7 3.6 ±1.1 8.0 ±2.0 52.8 ±5.9 50 Vlues with ifferent supersript letters within olumns represent signifint sttistil ifferenes (P < 0.05) Tle 2: Men vlues of ifferent hromosoml errtions inue in spermtoytes of ontrol n 3,4-DCA trete mle mie Struturl normlities Numeril normlities -------------------------------------------------- Totl strutrl ---------------------------------- Totl numeril Totl hromosoml Tretment X-Y univlent Autosoml Chin errtions Hypoploiy Polyploiy errtions errtions Control 1.8 ±0.4 2.4 ±0.5 1.4 ±0.5 0.4 ±0.5 1.8 ±0.8 A 4.2 ±0.4 1/32 LD50 3.2 ±0.8 2.2 ±0.4 5.4 ±0.5 2.0 ± 0.7 1.0 ±1.0 3.0 ±1.2 A 8.4 ±1.5 1/16 LD50 5.2 ±0.8 5.4 ±0.5 10.6 ±0.5 5.4 ±1.5 4.0 ±1.2 9.4 ±2.7 B 20.0 ±2.5 1/8 LD50 9.6 ±1.8 6.8 ±1.1 17.0 ±3.6 9.2 ± 1.9 6.0 ±1.2 15.2 ±3.0 C 32.2 ±6.6 Vlues with ifferent supersript letters within olumns represent signifint sttistil ifferenes (P < 0.05) Fig. 2: Different types of hromosoml errtions inue y 3,4-DCA in one mrrow ells: (A) Fig. 3: Different types of hromosoml errtions ontrol metphse (B) gp n rek (C) eletion inue y 3,4-DCA in spermtoytes of (D) entri fusion, ring n en to en ssoition mle mie: A) ontrol, B) utosoml univlent, (E) entromeri ttenution (F) frgment n C) x-y univlent, D) hypoploiy n hypoploiy (G) enomitosis (H) polyploiy E) polyploiy 76
Europ. J. Biol. Si., 4 (3): 73-82, 2012 Buhinger et l. [8] teste the lstogeniity of 3,4-DCA in humn lymphoytes in vitro. They inite tht 3,4-DCA might inue neuploiy in mmmlin ells y intertion with the mitoti pprtus. Menwhile, there re very few pulishe reports on the potentil in vivo ytogeneti effets of 3,4-DCA. The results of the present stuy illustrte tht orl ministrtion of mle mie y 3,4-DCA for 30 suessive ys inue ose epenent inreses in the frequenies of struturl n numeril n totl hromosoml errtions in oth one mrrow ells n spermtoytes (Tle 1&2). The oserve struturl hromosoml errtions in one mrrow ells were gps, reks, eletions, entri fusion, en to en ssoition, frgments, ring n entromeri ttenution (Figure 2A-E). The oserve types of numeril errtions were enomitosis, hypoploiy n polyploiy (Figure 2F-H). The most oserve types of struturl hromosoml errtions in spermtoytes were utosoml univlent n X-Y univlent, while numeril errtions were hypoploiy n polyploiy (Figure 3A-E). The inution of struturl hromosoml errtions my result from: (i) iret DNA rekge, (ii) replition on mge DNA templte, (iii) inhiition of DNA synthesis n other mehnisms suh s topoisomerse II inhiition [36]. The hromosome mlformtions re either quntittive whih inlue erese or inrese of the numer of the hromosomes, or qulittive suh s the exhnge of the hromti [37]. Our finings re in greement with the result of Feerio et l. [38] who showe tht fenuron, hlorotoluron, iuron n ifenoxuron t high onentrtions inue n inresing numer of hromosoml errtions in non-metolising Chinese hmster ovry ells. In ition, the four heriies t the lowest ose-level inue hromosoml errtions in the expose epithelil liver ell line. Also, iuron hs use geneti mge in eveloping emryos n in one mrrow ells in mie [39]. Sperm Qulity: Sperm ount n motility were signifintly reue in mie following tretment with ifferent oses of 3,4-DCA when ompre to tht of respetive ontrols. There ws lso signifint ifferene in sperm ount n motility mong ifferent 3,4-DCA tretment groups. Lowest sperm ount n motility were foun in high ose, followe y meium n low ose (Tle 3). Sperm ount is one of the most sensitive tests for ssessment of spermtogenesis n fertility [40]. A erese in sperm ount in men over the lst few ees is orrelte with stey inrese in Tle 3: Men vlues of sperm ount n motility perentge in ontrol n 3,4-DCA trete mle mie groups Tretment 6 Count 10 /ml Motility (%) Control 504.0 ±29.8 75 ±3.5 1/32 LD50 359.8 ±51.4 67 ±6.7 1/16 LD50 290.0 ±46.8 58 ±8.4 1/8 LD50 220.8 ±24.5 36 ±5.5 Vlues with ifferent supersript letters within olumns represent signifint sttistil ifferenes (P < 0.05) use of pestiies n other environmentlly tive hemils [41]. It is possile tht stey eline in the sperm ount over the yers in the future might le to oligospermi n infertility [42]. Suppression of gonotrophins might hve use erese in sperm ensity in testes [43]. Sperm motility is one of the most importnt prmeters use in the evlution of sperm qulity [44]. Sperm motility is lso n importnt funtionl mesurement to preit sperm fertilizing pity. Any negtive impt on motility woul seriously ffet fertilizing ility [45]. Mrke inhiition of sperm motility in 3,4-DCA trete groups my e ttriute to the low level of ATP ontent [46]. Sperm motility my e ffete y ltere enzymti tivities of oxitive phosphorolyti proess. Oxitive phosphorolyti proess is require for ATP proution, soure of energy for the forwr movement of spermtozo. Full ATP pool is ruil for norml spermtozo movement n slight eprivtion of ATP les to reution in motility, whih my use infertility [47]. Results showe lso tht there ws ose epenent inrese in the frequenies of sperm he n til normlities in ll 3,4-DCA trete groups of mie ompre to respetive ontrols. The frequeny of norml spermtozo lso iffere signifintly mong ifferent 3,4-DCA trete groups, the highest numer ws in high ose followe y meium n low ose. Trete mie with 3,4-DCA t high n meium oses showe signifint inrese in the frequenies of ll types of norml spermtozo, wheres the low ose trete mie showe signifint inreses in morphous he, pin he n oile til type (Tle 4 & Figure 4). These results re in greement with those of Bo et l. [48] who reporte tht 3,4-DCA ministere to mle rts showe ose epenent erese in sperm onsisteny, viility, motility n inrese the sperm errtion rte. Sperm normlity ssy, sensitive n relile enpoint is wiely use to ientify germ ell mutgen [49]. The presene of norml sperm hes suggests inution of geneti mge in the mle germ ells. 77
Europ. J. Biol. Si., 4 (3): 73-82, 2012 Tle 4: Sperm normlities in ontrol n 3,4-DCA trete mle mie groups Type of sperm normlities -------------------------------------------------------------------------------------------------------------------------------------- Tretment Hook less he Amorphous he Smll he Doule he Pin he Coile til Totl sperm normlities Control 1.0 ±0.7 4.4 ±0.5 0.4 ±0.5 1.2 ±0.8 A 7.0 ±2.2 1/32 LD50 1.4 ±0.5 8.4 ±0.5 0.6 ±0.5 3.2 ±0.4 2.0 ±0.0 B 15.6 ±1.5 1/16 LD50 4.4 ±0.5 15.6 ±3.8 2.4 ±0.5 8.2 ±0.8 4.6 ±0.5 C 35.2 ±4.8 1/8 LD50 7.8 ±0.8 23.4 ±1.1 4.2 ±0.8 10.4 ±1.7 10.4 ±1.1 D 58.8 ±2.6 Vlues with ifferent supersript letters within olumns represent signifint sttistil ifferenes (P < 0.05) Fig. 4: Grph of sperm normlities of mle mie trete with 3,4-DCA show A) norml sperm, B&C) morphous he sperm, D) hok less he sperm, E) pin he sperm, F&G) oile til sperm Fig. 5 I: Setion in the liver of ontrol mouse showing (): norml rhiteture of heptoyte riting from entrl vein (C.V) with entrl vesiulr nulei (rrows). The heptoytes re seprte y nrrow loo sinusois (irregulr rrows) n kupffer ells (he rrows). (): Portl re etween hepti loules with rnh of ile utule (B) n rnh of the portl vein (Pv). II) Setion in the liver of mouse trete with 1/8 of 3,4-DCA LD 50 vlue showing (): ggregtion of lymphoyte infiltrtion (irregulr rrow), lrge size of kupffer ells (he rrows), some inflmmtory ells (rrow) epresse in ongeste loo vessels (Bv). (): ilttion of portl vein (Pv) n prolifertion of ile utules (B). III) Setion in the liver of mouse trete with 1/16 of 3,4-DCA LD 50 vlue showing (): egenertion of heptoyte (*), kryolysis of mny nulei (rrows). (): prolifertion of ile utules (B), ilttion n ongestion of portl vein (Pv) n epression of some inflmmtory ells insie it (rrows). IV) Setion in the liver of mouse trete with 1/32 of 3,4-DCA LD 50 vlue showing (): norml pperne of heptoyte (rrows) n ongeste entrl vein (C.V). (): norml portl re; ile utule (B) n rnh of the portl vein (PV). (H&E X 400) 78
Europ. J. Biol. Si., 4 (3): 73-82, 2012 Fig. 6 A: Cross setion in testis of ontrol mouse showing norml struture of the seminiferous tuules (st) with ifferent types of spermtogeni ells (Sp) n spermtozo (S). Also, interstitil tissue with Leyig ells (L) n loo vessels (Bv) re notie. B) Cross setion in testis of mouse trete with 1/8 of 3,4-DCA LD 50 vlue showing irregulr ounries (short rrows) of seminiferous tuules (ST), egenertion of spermtogeni ells (*) ompnie with sene of spermtozo n prolifertion of Leyig ells (rrows). C) Cross setion in testis of mouse trete with 1/16 of 3,4-DCA LD 50 vlue showing isrrngement of spermtogeni ells (*) n reution in some seminiferous tuules (he rrow). Thikening wll (rrow), ilttion n ongestion of loo vessels (C) re lso notie. D) Cross setion in testis of mouse trete with 1/32 of 3,4-DCA LD 50 vlue showing norml pperne of primry spermtoytes (he rrows) n mny of spermtozo (rrows). Also, reution of some spermtogeni ells (*) is notie. (H&E, X 400) Sperm he normlities my rise ue to smll eletions This rekown is rrie out y the enoplsmi or point muttions, physiologil, ytotoxi or geneti retiulum of the heptoytes n s result the hepti mehnisms [50], or ltertion in testiulr DNA whih in ells get mge severely [52]. 3, 4-DCA ws foun to turn isrupts the proess of ifferentition of spermtozo inue free ril genertion n ntioxint epletion [51]. n use oxitive stress n lipi peroxition in liver of ruin rp [1]. Histopthologil Exmintion: The evlution of No histopthologil hnges were oserve in the histopthologil ltertions in liver n testes of trete speimens ollete from the ontrol mie s testes whih mle mie silly onfirme the geneti results s well. exhiite norml testiulr struture (Fig. 6 A). Testes of The results of the histopthologil exmintion revele the high ose tretment exhiite egenertion of tht ontrol mie show norml rhiteture of the spermtogeni ells ompnie with sene of liver (Fig. 5 I). Liver of the mie ministere with 1/8 of spermtozo n prolifertion of Leyig ells (Fig. 6 B). 3,4-DCA LD 50 vlue exhiite ggregtion of lymphoyte Similrly, testes of meium ose tretment showe infiltrtion, lrge size of kupffer ells, some inflmmtory isrrngement of spermtogeni ells n reution in ells, ilttion of portl vein n prolifertion of ile some seminiferous tuules, ilttion n ongestion of utules (Fig. 5 II). At meium ose tretment, the liver loo vessels (Fig. 6 C). In ontrst, the low ose showe egenerte heptoytes, kryolysis of mny tretment isplye norml pperne of primry nulei, prolifertion of ile utules, ilttion n spermtoytes n reution of some spermtogeni ongestion of portl vein (Fig. 5 III). At 3,4-DCA low ells (Fig. 6 D). It is well-known ft tht the ose, the liver revele pprently norml portl trt testosterone serete y the Leyig ells is essentil for n norml heptoytes (Fig. 5 IV). The liver is n the growth, ivision n ifferentition of the germinl orgn of etoxifition whih rekown toxi sustnes ells [52]. A testosterone efiit is expete to interfere n metolites of the ministere sustnes. with the ompletion of meiosis y iret tion on the 79
Europ. J. Biol. Si., 4 (3): 73-82, 2012 germ ells [53]. Thus, it seems possile tht 3,4-DCA 5. Juro-Snhez, B., E. Bllesteros n M. Gllego, might hve ffete the Leyig ells whih in turn 2012. Ourrene of romti mines n N- reue the proution of the testosterone s result the spermtogenesis gets inhiite. Also, toxints hve iret effet on sertoli ell funtion, whih ppers to e involve in the ontrol of spermition n when isture 6. nitrosmines in the ifferent steps of rinking wter tretment plnt. Wter reserh, 46: 4543-4555. Monteiro, M., C. Quintneiro, M. Pstorinho, M.L. Pereir, F. Morgo, L. Guilhermino n use epithelil isorgniztion n susequent A.M.V.M. Sores, 2006. Aute effets of 3,4- tuulr trophy [54]. 3,4-DCA use some histologil ihloroniline on iomrkers n spleen histology mges in Esturine mysi Mesopoopsis sleri, of the ommon goy Pomtoshistus mirops. espeilly on eye, gons n musulr tissue n the Chemosphere, 62: 1333-1339. presene of 3,4-DCA umultions, using rther ovl 7. Klütten, B., N. Kuntz n H. Rtte, 1996. Comine to spheril forms, whih re visile in some essentil effets of 3,4-Dihloriniline n foo onentrtion strutures. The estrution use on the struture n on lifetle t of two relte Cloerns, orgniztion of the musulr tissue n on gons ws Dphni mgn n Ceriophni qurngulr. lso onsierle [12]. Chemosphere, 32: 2015-2028. CONCLUSION 8. Buhinger, M., U. Kulk n E. Shmi, 1989. Cytogeneti effets of 3,4-ihloroniline in humn Our t inite tht 3,4-ihloroniline inues hromosome errtions in oth the one mrrow ells n spermtoytes of mie. The repression in mitoti inex inites the potentil for 3, 4-ihloroniline to inue growth rrest or to inhiit ell growth. These finings emonstrte tht 3,4-ihloroniline hs strong lstogeni/genotoxi potentil. Also, our stuy inite tht 3, 4-ihloroniline showe reproutive toxiity n histopthologil ltertion in mle mie. Aoringly, strit limittions on the use of this ompoun must e put. REFERENCES 1. Li, W., D. Yin, Y. Zhou, S. Hu n L. Wng, 2003. 3,4-Dihloroniline-inue oxitive stress in liver of ruin rp (Crssius urtus). Eotoxiology n Environmentl Sfety, 56: 251-255. 2. Kim, Y.M., K. Prk, W.C. Kim, J.H. Shin, J.E. Kim, H.D. Prk n I.K. Rhee, 2007. Cloning n hrteriztion of tehol-egring gene luster from 3,4-ihloroniline egring terium Pseuomons sp. KB35B. J. Agri. Foo Chem., 55: 4722-4727. 3. Wng, S., K. Poon n Z. Ci, 2012. Bioegrtion n removl of 3,4-ihloroniline y Chlorell pyrenoios se on liqui hromtogrphyeletrospry ioniztion-mss spetrometry. Environmentl Siene n Pollution Reserh. Jun 6, DOI: 10.1007/s11356-012-0995-9. 4. Guilhermino, L., A.M.V.M. Sores, A.P. Crvlho n M. Celeste Lopes, 1998. Aute effets of 3,4- ihloroniline to mle wistr rts. Chemosphere, 37(4): 619-632. lymphoytes n V79 Chinese hmster ells. Muttion Reserh Letters, 226(3): 197-202. 9. Giomzzi, S. n N. Cohet, 2004. Environmentl impt of iuron trnsformtion: review. Chemosphere, 56: 1021-1032. 10. Lo, S., 2003. Detoxifition of 3,4-ihloroniline in soyen y n-mlonyltion. Ph.D. thesis, University of Durhm, UK. 11. Hrvey, P.J., B.F. Cmpnell, P.M.L. Cstro, H. Hrms, E. Lihtfouse, A.R. Shffner, S. Smrek n D. Werk-Reihhrt, 2002. Phytoremeition of polyromti hyrorons, nilines n phenols. Environ. Si. Pollut. Res., 9: 29-47. 12. Sro, A.M., U.M. Azeiteiro, L. Pereir, F. Morgo n A.M.V.M. Sores, 2005. Histologil evlution of the exposure to 3,4-ihloroniline in the esturine mysi Mesopoopsis sleri, uner experimentl onitions. Fresenius Environmentl Bulletin, 14(7): 579-583. 13. Sheil, V., C. Kienle, R. Osteruer, A. Gerhrt n H.R. Köhler, 2009. Effets of 3,4-ihloroniline n izinon on ifferent iologil orgnistion levels of zerfish (Dnio rerio) emryos n lrve. Eotoxiology, 18(3): 355-363. 14. Hong, S.K., D.K. Anestis, J.G. Bll et l., 2002. In vitro nephrotoxiity inue y hloronitroenzenes in renl ortil slies from Fisher 344 rts. Toxiology letters, 129(1-2): 133-141. 15. Zhng, B. n S. Lin, 2009. Effets of 3,4- Dihloroniline on testile enzymes s iologil mrkers in rts. Biomeil n Environmentl Sienes, 22: 40-43. 80
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