ZINC AND ΑLPHA-LIPOIC ACID ALLEVIATE CYPERMETHRIN INDUCED REPRODUCTIVE TOXICITY IN MATURE MALE WISTAR RAT ABSTRACT

Transcription

1 ZINC AND ΑLPHA-LIPOIC ACID ALLEVIATE CYPERMETHRIN INDUCED REPRODUCTIVE TOXICITY IN MATURE MALE WISTAR RAT MADHUBANTI BEPARI, PRALAY MAITY, TUHINA DAS AND SUJATA MAITI CHOUDHURY* Department of Human Physiology with Community Health, Vidyasagar University, Midnapore, West Bengal, India, Pin ABSTRACT Cypermethrin (α- Cyano-3-Phenoxybenzyl (+ is, trans) 3-(2, 2-Dihlorovinyl)-2, 2-dimethyl ylopropane arboxylate) is an ative pyrethroid whih intensively ontrols a wide range of pests in agriulture and an eto-parasitiide in humans and animals. It has moderate arinogeni, mutageni, immunosuppressive and hepatotoxi effets. It auses brain and loomotor disorders, polyneuropathy. The present study was onduted to evaluate the adverse effets of ypermethrin on the reprodutive system of male Wistar rats, and also to assess the alleviating role of zin and α-lipoi aid under these toxi onditions. Male Albino Wistar rats reeived oral ypermethrin treatment at two dose levels(40 mg/kg body wt,80 mg/kg body wt) and ombined ypermethrin, zin (227 mg/l in drinking water), and α-lipoi aid (35 mg/kg body wt) treatments for onseutive 14 days. The effets of the treatments were studied on some reprodutive markers in rat testis inluding antioxidant parameters. Cypermethrin redued the testiular index, sperm motility of rats of ypermethrin treated groups. Signifiant inrease in seminal frutose onentration, testiular glutamate oxaloaetate transaminase (GOT), glutamate pyruvate transaminase (GPT), testiular malon-dialdehyde (MDA) level were seen whereas redued glutathione (GSH) level and superoxide dismutase (SOD) ativity of testis were dereased signifiantly in a dose-dependent manner ompared to the ontrol group. The present study showed that zin and α-lipoi aid supplementation restored the altered reprodutive parameters towards normal levels. The results revealed the benefiial influenes of zin and α-lipoi aid in reduing the harmful effets of ypermethrin on male reprodutive system of rat. Keywords: Cypermethrin; Zin and α-lipoi aid; Sperm motility; Seminal frutose onentration Testiular glutamate pyruvate transaminase; Antioxidant parameters. 1. INTRODUCTION Pestiides are widely used agriultural hemials whih are the largest group of poisonous substanes being disseminated throughout our environment in spite of their use in publi health to ontrol insets, weeds, animals, and vetors of disease (Eobihon DJ. 1994; Eddleston M et al. 2002). Inreasing interest has been seen among health and environmental institutions regarding the potential reprodutive effets due to exposure to these agriultural and environmental hemials (Dalsenter PR et al. 1997). Exposure to suh P - 9 environmental ontaminants auses redution of sperm motility, dereased fertilization ability, produing abnormal sperm in men and wildlife (Alm H et al. 1996). It has been reported that, pestiides with suh properties have been shown to ause overprodution of reative oxygen speies (ROS) in both extra- and intraellular spaes, produing infertility in wildlife and human (Sharpe RM and Skakkebaek NE, 1993). The antioxidant system plays an effetive role in proteting testes and thus preventing testiular dysfuntion

2 (Oshsendorf FR. 1999). Syntheti pyrethroid insetiides have been used in agriulture, animal husbandry and indoor inset management and aount for approximately one-fourth of worldwide insetiide market (Casida JE et al. 1998). Pyrethroids are used widely in an inreasing order during the last two deades with the delining use of organophosphate pestiides, whih are more autely toxi to fishes, birds and mammals than pyrethroids (Bateman DN. 2000). Cypermethrin is an ative syntheti pyrethroid insetiide is ommonly used to ontrol various pests in agriulture, publi health and in veterinary pratie against etoparasites (WHO, 1989;Bhunya SP and Pati PC, 1988). It is hemially (+/-) alphayano-(3-phenoxyphenyl) methyl (+)-is, trans-3- (2, 2-dihloroethenyl)-2, 2- dimethylylopropanearboxylate. (Bhunya SP and Pati PC, 1988). It is highly toxi to bees, fishes, and water insets but has lower toxiity to birds. It has moderate arinogeni, mutageni, immunosuppressive and hepatotoxi effets. Low levels of ypermethrin in the aquati environment produed a signifiant effet on mature male Atlanti salmon populations through disruption of reprodutive funtions ( Moore A and Waring CP, 2001). Fenvalerate and ypermethrin were reported to impair male rat reprodutive funtion, induing signifiant redutions in epididymal sperm ount (Song L et al. 2008). However, it is not lear whether fenvalerate and ypermethrin might affet spermatogenesis via interation with androgens or their reeptors. Fenvalerate and ypermethrin diretly influene mature rat sperm motility. Cypermethrin indued adverse effets on sperm head shape morphology of mouse as well as lastogeni effets on the root tip ells of Allium epa. Besides generalized toxi effets of ypermethrin, dereased number of implantation sites, number of viable fetuses and weight gain of fetuses in rabbits treated with ypermethrin have been reported (Elbetieha A et al. 2001). Semen ejaulatory volume, sperm motility perentage, mass ativity, and onentration of spermatozoa were signifiantly (P<0.01) dereased by ypermethrin in dwarf goats (Ahmed M et al. 2009). Moreover, it is observed (Manna et al. 2004) that high dose of ypermethrin aused oedema between seminiferous tubules, in addition to vauolation and hyalinization in the tubules of rat testis. It has been also reported that the proportion of dead and abnormal sperms were signifiantly inreased in mie after exposure to the ypermethrin like pyrethroid (Bhunya SP and Pati PC, 1988). More reently, muh attention has been foused on the possible role of essential trae elements in providing the neessary preventive effiay with less toxiity and side effets (Xiu YM. 1996; Kang YJ and Zhou Z 2005; Zhou Z et al. 2005). Zin is a key onstituent or ofator of over 300 mammalian proteins. It is intensively being studied for its protetive effiay in various models of animal toxiity. Zin is a benefiial agent in reduing the damage arising in inreased oxidative stress (Cagen SZ and Klaassen CD, 1979; Cabre M et al. 1999; Zhou Z et al. 2005). Alpha-lipoi aid, a biologial antioxidant, shows benefiial effets in oxidative stress beause of its synergisti ation with other antioxidants (Suzuki YJ et al. 1993). Based on the above fats, the present study was onduted to investigate the ombined effets of zin and α- lipoi aid in the amelioration of the damage inflited on the reprodutive system of the rats intoxiated with ypermethrin. 2. MATERIALS AND METHODS 2.1. Chemials and Reagents Cypermethrin 10% Emulsifiable Conentrate (EC), ommerial name (Ustad). Αlpha-lipoi aid was purhased from Sigma Aldrih. Indole, Sodium hydroxide( NaOH), Dinitrophenyl hydrazine (DNPH), Thio-barbutari aid (TBA), Trihloro aeti aid (TCA), Sodium hloride (NaCl), Hydrohlori aid (HCl), Sulfosaliyli aid(ssa), 2,4,4-dithionitrobenzoi aid(dtnb), Pyrogallol, Tris HCl, Potassium dihydrogen phosphate (KH 2 PO 4 ), Zin sulphate (ZnSO 4 ),Benzoi aid were purhased from Merk Ltd., SRL Pvt. Ltd., Mumbai, India. All other hemials used were analytial grade and obtained from Merk Ltd., SRL Pvt. Ltd., Mumbai, India Animal are and treatment The study was onduted on 36 mature Wistar male albino rats weighing gm. The animals were housed in polypropylene ages in the departmental animal house at an ambient temperature of 25 ±2 C with 12 hrs light-dark yle and were alimatized for at least 1 week before prior to different treatments. The animals P - 10

3 were maintained on the standard laboratory feed and water ad libitum, throughout the period of experimentation. Thirty six albino mature male rats were randomly divided into six groups, of six animals eah. The groups and treatments were designed as: 1. Group I: Control (5 ml /kg body wt.) 2. Group II: Zin(227 mg/l in drinking water)and α- lipoi aid( 35 mg/kg body wt.) ontrol 3. Group III: Cypermethrin-treated (Low dose, 40mg/body wt.) group 4. Group IV: Zin + α- lipoi aid+ Cypermethrin-treated (Low dose, 40mg/body wt.) group 5. Group V: Cypermethrin-treated (High dose, 80mg/body wt.) group 6. Group VI: Zin + α- lipoi aid+ Cypermethrin-treated (High dose, 80mg/body wt.) group After one hour of the treatment of Zin (227 mg/l in drinking water; Goel A et al. 2006) and α- lipoi aid (35 mg/kg body wt.; Selvakumar E et al. 2005), Cypermethrin was administered at two dose levels. All animals were observed at least one daily to notie whether there was any behavioural hange or signs of intoxiations. Mortality, morbidity, and food and water onsumption were also monitored daily. Animal s weight was taken daily and the dose was adjusted aordingly. Oral treatment at the dose level of 40mg, and 80mg of ypermethrin /kg body weight were done for onseutive 14 days Sample olletion Total body weights of rats in eah group were taken before the treatment period and before sarifie. 24 hrs after the last dose all rats were sarified by rapid deapitation. Blood was olleted using heparin as antioagulant (10 UI/ml) from the animals and serum was separated and kept at -20 C for biohemial studies. The testes and other male aessory sex organs (epedidymis and seminal vesiles) were removed immediately and adhering fats were leaned. Then testiular weights were reorded and stored properly for the biohemial estimation. Then, epididymis and seminal vesiles prepared for fertility evaluation and testiular tissues were taken for the determination of oxidative biomarkers Estimation of Testiular index Testes of sarified male albino rat (Wister) were disseted from its body and all fats were removed from the testes. Then their weights were taken. Testiular index was measured by the following formula: Testiular index = Testiular weight/body weight x Epididymal sperm motility analysis (WHO, 1999) Assessment of epididymal sperm motility was done by the method of WHO Epididymal sperm morphology analysis (Laing JA.1979) This was determined by adding two drops of warm Wells and Awa stain to the semen on a prewarmed slide. Then a uniform smear was made and airdried. The slide whih was stained, immediately examined under the mirosope using x400 magnifiation (Laing, 1979). The types and number of abnormal spermatozoa were alulated from the total number of spermatozoa in the randomly seleted five mirosopi fields. The numbers of abnormal spermatozoa were expressed as a perentage of the total number of spermatozoa 2.7. Estimation of seminal frutose onentration Frutose in seminal fluid was measured ( Karronen MJ and Malam M, 1995) by taking 0.5 ml tissue homogenate (20mg/ml, as sample), 0.5 ml frutose (0.14 mm and 0.28 mm frutose, as two standards) and 0.5 ml distilled water (as blank) with 0.5 ml indole reagent separately. They were mixed and then 5 ml onentrated HCl was added to eah test tube. All test tubes were overed with stoppered and inubated at 50 for 20 min., ooled in ie water and then in room temperature. After that the reading was taken at 470 nm Testiular glutamate oxaloaetate transaminase (GOT) and glutamate Pyruvate transaminase (GPT) (Reitman S and Frankel S, 1957) One ml buffer substrate (GOT/GPT ml) was given in test tube and waited for 5 minutes at 37 C. After that 0.2 ml tissue homogenate (20 mg/ml) was added to the test tube and inubated at 37 C for 60 minutes. DNPH (1 ml) solution was mixed to the test tube and waited for 20 minutes. In 0.2 ml working pyruvate standard solution was added to P - 11

4 0.8 ml and 1 ml buffer substrate.and in blank, 1 ml buffer substrate were taken. Distilled water (0.2 ml) and 1 ml DNPH solution were added to eah test tube and mixed well and waited for 20 minutes. Mixing 10 ml of 0.4 (N) NaOH solution to all the test tubes, readings were taken at 520 nm. after 10 minutes Estimation of oxidative stress parameters Testiular Malon dialdehyde (Ohkawa H et al. 1979) 0.5ml of testiular homogenates (50 mg /ml), 0.5 ml normal saline (0.9%), 2 ml TBA TCA mixture were mixed. It was boiled for 10 min and was ooled at room temperature and entrifuged in 4000 r.p.m for 10 min. Reading was taken at 535 nm Testiular Redued Glutathione (Grifth Mindr P. 1998) 200µl of testiular homogenates (50 mg /ml) mixed with sulfosaliyli aid and entrifuged for 10 min at 3000 rpm. The supernatant was added with DTNB and was shaken well. Reading was taken at nm Testiular Superoxide dismutase (Marklund S and Marklund G, 1974) Testiular homogenates (50 mg /ml) in ie-old 100 mm Tris-HCl buffer were entrifuged at 10,000 g for 20 min at 4 o C. The SOD ativities of the supernatants were estimated by measuring the perentage inhibition of the pyragallol autooxidation by SOD (Marklund S and Marklund G, 1974). In a spetrophotometri uvette, 2 ml of buffer, 100 µl of 2 mm pyragallol and 10 µl of supernatant were poured and the absorbane was noted in a spetrophotometer at 420 nm for 3 min Statistial analysis The results were expressed as the Mean ± Standard error of mean (SEM). Statistial analysis of the olleted data were performed by Analysis of variane (ANOVA) followed by multiple omparison two-tail t-test. The differene was onsidered signifiant when p< RESULTS Testiulo-somati index *** Figure1 shows the effet of Zin and α- lipoi aid on Testiulo-somati index in Cypermethrin indued male albino rat (N=6). Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Supersript a, Group-I versus all other groups; Supersript Group-V versus Group-VI. Asterisks represents the different level of signifiane (*** indiates p<0.001). P - 12

5 Sperm Motility b*** 50 *** (%) Figure 2 shows the effet of Zin and α- lipoi aid on Epididymal sperm morphology in Cypermethrin indued male albino rat (N=6). Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group-V versus Group-VI. Asterisks represents the different level of signifiane (*** indiates p<0.001) Table 1 shows the effet of Zin and α- lipoi aid Cypermethrin on Sperm Morphology in Cypermethrin indued male albino rat. Group Saline Control (%) Zn+ Lipoi aid ontrol (%) Cyp (Low) (%) Cyp(L)+ Zn+ Lipoi aid (%) Cyp (High) (%) Cyp(H)+Zn+ Lipoi aid (%) Tailless head 4.1± ± ± ± ± ±0.78 Headless tail 4.7± ± ± ± ± ±0.58 Bent tail 5.2± ± ± ± ± ±0.38 Curve tail 6.2± ± ± ± ± ±0.42 Bent mid piee 4.1± ± ± ± ± ±0.23 Coiled tail 0.3± ± ± ± ± ±0.02 Looped tail 0.12± ± ± ± ± ±0.03 Rudimentary tail 0.23± ± ± ± ± ±0.04 Curve mid piee 6.24± ± ± ± ± ±0.63 Total 33.9± ± ± ±1.42 b*** 61.55± ±3.48 *** Values represents Mean± SEM (N=6). Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Probability values with different supersripts (a,b,) differ from eah other signifiantly. Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group-V versus Group-VI. Asteriks represents the different level of signifiane (*** indiates p<0.001). P - 13

6 mm of frutose/lit of seminal plasma Seminal Plasma Frutose Conentration b*** *** 0 Figure 3 shows the effet of Zin and α- lipoi aid on Seminal Plasma frutose Conentration in Cypermethrin indued male albino rat (N=6). Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group-V versus Group-VI. Asterisks represents the different level of signifiane (*** indiates p<0.001). IU/Lit Testiular Glutathione pyruvate transaminase *** b*** Figure 4 shows the effet of Zin and α- lipoi aid on Testiular Glutamate Pyruvate Transaminase (GPT) in Cypermethrin indued male albino rat. Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t- tests. Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group-V versus Group-VI. Asterisks represents the different level of signifiane (*** indiates p<0.001). P - 14

7 Testiular Glutathione oxaloaetate transaminase *** a** b** a** IU/lit Figure 5 shows the effet of Zin and α- lipoi aid on Testiular Glutamate Oxaloaetate Transaminase (GOT) in Cypermethrin indued male albino rat. Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group- V versus Group-VI. Asterisks represents the different level of signifiane (** indiates p<0.01,*** indiates p<0.001) Testiular Malon-di-aldehyde a* a* nmol/mg protein Figure 6 shows the effet of Zin and α- lipoi aid on Testiular Malon-di-aldehyde in Cypermethrin indued male albino rat. Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Supersript a, Group-I versus all other groups. Asterisks represents the different level of signifiane (* indiates p<0.05, *** indiates p<0.001). P - 15

8 Testiular Redued Glutathione b*** ug/mg protein *** Figure 7 shows the effet of Zin and α- lipoi aid on Redued Glutathione in Cypermethrin indued male albino rat. Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group-V versus Group-VI. Asterisks represents the different level of signifiane (*** indiates p<0.001) Testiular Super- oxide- dismutase unit/mg protein b** a** *** C Figure 8 shows the effet of Zin and α- lipoi aid on Super-oxide dismutase in Cypermethrin indued male albino rat. Results are expressed as Mean±SEM. Analysis is done by ANOVA followed by multiple omparison two-tail t-tests. Supersript a, Group-I versus all other groups; Supersript b Group-III versus Group-IV; Supersript Group-V versus Group-VI. Asterisks represents the different level of signifiane (** indiates p<0.01,*** indiates p<0.001). Figure 1 shows the effet of zin and α-lipoi aid on testiulo-somati index in ypermethrin indued male albino rat. The Testiular index has been dereased signifiantly (p<0.001) in ypermethrin high dose treated group whih elevated by the ombined treatment of zin and α-lipoi aid. The effet of zin and α-lipoi aid on rat epididymal sperm motility in ypermethrin intoxiated male albino rat is shown in Figure 2. In ypermethrintreated group, epididymal sperm motility dereased signifiantly (p<0.001) ompared to the ontrol group. Treatment of zin and α- lipoi aid dereased the ypermethrin toxiity and restored the normal status of the testiular tissue to a great extent. Table 1 shows the effet of zin and α- lipoi aid on epididymal sperm morphology of P - 16

9 ypermethrin indued male albino rat. From this table it is observed that abnormal sperms were inreased signifiantly in ypermethrin treated groups whih were alleviated by zin and α- lipoi aid. The effet of zin and α- lipoi aid on the seminal plasma frutose onentration of ypermethrin indued male albino rat is shown in Figure 3. Seminal vesiular frutose levels were signifiantly higher in ypermethrin treated group ompared to that of ontrol rats. Zin and α- lipoi aid restored it to a good extent. Testiular glutamate pyruvate transaminase (GPT) (Figure 4) and testiular glutamate oxaloaetate transaminase (GOT) (Figure-5) levels were inreased in ypermethrin indued male albino rat in a dosedependent manner whih were altered after the treatment of zin and α- lipoi aid. In testis inreased testiular GPT and GOT levels suggest that ypermethrin auses testiular damage whih was prevented by zin and α- lipoi aid. Figure 6 shows the effet of Zin and α- lipoi aid on testiular malon-di-aldehyde (MDA) in ypermethrin indued male albino rat. The Testiular MDA level inreased signifiantly (p<0.05, p<0.001) in ypermethrin-treated groups in a dose dependent manner. Zin and α- lipoi aid treatment dereased the ypermethrin toxiity. Super-oxide dismutase (SOD), in Cypermethrin indued male albino rat dereased signifiantly (p<0.001) in ypermethrin high dose-treated group ompared to the ontrol group (Figure 7). Treatment of zin and α- lipoi aid dereased the ypermethrin toxiity and restored the oxidative status of the testiular tissue to some extent. Figure 8 shows the effet of zin and α- lipoi aid on redued glutathione in ypermethrin indued male albino rat. The Testiular GSH on. has been dereased signifiantly (p<0.001) in high dose ypermethrin-treated group. GSH onjugation is reognized as a major detoxifiation mehanism in reprodutive tissue. Administration of zin and α- lipoi aid redued ypermethrin mediated oxidative stress signifiantly. 4. DISCUSSION The present study was onduted to evaluate the adverse effets of ypermethrin on the reprodutive system of male Wistar rats, and also to assess the alleviating role of zin and α-lipoi aid under this toxi ondition. Organ weight is a basi benhmark for the toxiologial studies (Yavasoglu A et al. 2008). The testiular weight is basially dependent on the mass of the differentiated spermatogeni ells. Dereased number of germ ells, inhibition of spermatogenesis and steroidogeni enzyme ativity may be the auses of redution in testiular weight (Takahashi O and Oishi S, 2001). The testiular index dereased by ypermethrin effetively elevated by the ombined treatment of zin and α- lipoi aid. This may be due to the preventive role of zin and α-lipoi aid on testiular damage. It was reported (Song L et al. 2008) that ypermethrin derease sperm motility in vitro. In this study, redued sperm motility dereased may be due to mitohondrial enzyme ativity of the spermatozoa, altered frutose synthesis and seretion by the aessory glands. Zin and Lipoi aid ombination ameliorated the ypermethrin indued redution in sperm motility. Morphologial alteration of sperm ell aused by ypermethrin an be grouped into primary or seondary abnormalities aording to the lassifiation by Noarkes DE et al.2004.the only primary sperm abnormality was rudimentary tail abnormality. Some seondary abnormalities i.e. bent mid-piee,urved mid-piee,bent tail,urved tail,normal tail without head,normal head without a tail,looped tail and oiled tail were also found in this study.. The presene of rudimentary tail sperm abnormality was observed higher in ypermethrin treated group rats relative to the ontrol. An aberration in the proess of spermatogenesis is the main ause of primary sperm abnormality Hafez ESE. 1987). It was observed that urved mid-piee sperm ell abnormality had the highest ourrene, followed by, urve tail, bent mid piee, bent tail, head less tail, tail less head. Sperm ells whih are matured from damaged seminiferous tubules show seondary abnormalities (Thomas MJ and Thomas JA, 2001). But treatment of zin and α- lipoi aid derease sperm abnormality signifiantly (p<0.001).it may be due to the ombined ameliorative effet of zin and α- lipoi aid in the reprodutive system. Frutose prodution is a marker of seminal vesiular funtion due to its inverse relationship with spermatozoa motility. Frutose levels in seminal vesile homogenates were signifiantly higher in ypermethrin treated group ompared to ontrol rats. Zin and α- lipoi aid redued it effetively whih indiates the ability of zin and α-lipoi aid to restore the P - 17

10 normal testiular status. In testis inreased testiular GPT and GOT levels suggest that ypermethrin auses testiular damage. Inrease of transaminase ativity along with the dereased of ontent of free radial (O. 2 ) savengers are probably the onsequene of ypermethrin indued pathologial hanges in testis. Oxidative stress defines an imbalane between the formation of reative oxygen speies (ROS) and antioxidative defense mehanisms. During pyrethroid metabolism, reative oxygen speies (ROS) were generated and aused oxidative stress in intoxiated animals (Gupta A et al. 1999). In oxidative stress, lipid peroxidation is ourred due to exessive free radial prodution and is onsidered a primary mehanism of ell membrane destrution and ell damage. Malondialdehyde (MDA) is the end produt of lipid peroxidation. Cypermethrin inreased the testiular MDA; simultaneously it dereased testiular GSH ontent and SOD ativity in this study. But zin and α- lipoi aid dereased the levels of lipid peroxidation (MDA) in male Wister rats. GSH plays a major role in proteting the ell against oxidative damage by reating with ROS. In healthy human ells normally ~98% of the total GSH exists in the redued form while a muh smaller fration (~1%) exists in the oxidized forms GSSG. (Griffith OW. 1981; Dringen R and Hirrlinger J, 2003; Kennett E et al. 2005). Superoxide dismutase (SOD) is involved in the learane of superoxide. In the present investigation, dereased levels of SOD level were observed in ypermethrin indued male Wistar rats. In ypermethrin-intoxiated animals the derease in the ativity of superoxide dismutase may be due to the onsumption of this enzyme in onverting. - superoxide (O 2 ) to hydrogen peroxide (H 2 O 2 ). H 2 O 2 is then onverted to H 2 O by glutathione-stransferase (GST) and atalase (CAT) enzymes (Mates JM and Sanhez- Jimenez F, 1999). Super oxide dismutase (SOD) protets tissues from oxidative stress and damage by atalyzing the. onversion of (O - 2 ) to H 2 O. SOD ontains both opper and zin, zin is known to indue the prodution of metallothionein, whih is very rih in ysteine and is an exellent savenger of OH (Prasad AS. 1993).After the treatment of zin and α-lipoi aid, the ativities of the enzyme were signifiantly inreased. The present study demonstrated that zin and α-lipoi aid dereased the lipid peroxidation and thereby augmented the endogenous antioxidant enzyme in ypermethrin indued male rats and the observed properties may be attributed to the antioxidant properties present in zin and α-lipoi aid, whih is one of the most essential properties of any pestiide antidote. The dereased GSH ontent and SOD ativities and inreased MDA level in testis suggest that ypermethrin aused testiular damage. Zin and α- lipoi aid were used to prevent oxidative damage by interrupting the propagation of the oxidation of polyunsaturated fatty aids. Zin is not distributed uniformly in tissues; it an show a protetive effet espeially when administered prior to ypermethrin. From a reent study it has been ome to know that when lipoi aid is absent, interation of other antioxidants are not well, thereby their ability to protet ells are redued (Paker L et al. 1995). Zin and lipoi aid tried to normalize the reprodutive parameters at a good extent. These observations might also indiate that zin and lipoi aid has therapeuti effets on ypermethrin -indued male reprodutive toxiity. 5. CONCLUSION From the above disussion it an be onluded that ypermethrin-indued testiular toxiity, lipid peroxidation, oxidative stress in male rats and onjuntion supplementation and treatment with zin and α-lipoi aid has resulted in pronouned ameliorating effet espeially at the end of the experiment emphasizing its antioxidant role. CONFLICT OF INTEREST Authors delare that there are no onflits of interests. ACKNOWLEDGEMENT The authors are thankful to University Grants Commission, New Delhi for finanial assistane and Vidyasagar University, Midnapore for providing laboratory failities to exeute these studies. P - 18

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