ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

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t 02 ELSEVIER Toricology OM) (2W1) DM-wo wwv.el~vicr.com/lo~~le~loii~)l Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis C. Latchoumycandane, P.P. Mathur I* S'hwl v/ LII Scicnrrr. Pvldtchrrry Un~urriry. Pondrdcrry 605 014, Indra Rccctvcd 21 N~vcmbcr ZmI, rccetvcd m rcvn~d iorm 19 h m h r 2001. a~cc~~cd 28 March 2W1 Abstract Mcthoaychior. an cnvironmenlal contdmlnanl, which is widely used as a pesticide in many countries and has k n shown lo ~nducc rcproductivc abnormalities ~n mule rats The precise nature and mechanism ot action of methoxy cllior on lllc lniblc rcproducllve ryslcm is no1 clwr In tho prcxnt study, we have sought to invesligille the induction of o*ldat~vc stress In thc tesl~l of rat after expoure to methorychlor Melhoiychlor (I, 10, and IW mg kg-' body wcnghl wr Jsy) was adrninisttrsd ormlly to the rat for 45days Aitcr 24 h of the last treatment the anlmals were killed wing :~ne\.thct~c clhcr. The body welght of thc anlmalr adm~n~slcrcd with methorychlor d~d not show any s~gn~flcam chungc Thc wcaghts of the testis, cpadtdymis, seminal vn!cles and ventral prostate dscreawd significantly in 100 mg dox but rcma~ncd unchsngtd In I and 10 ma do~s Miloehondrial and microsome-rich fract~ons of the test~s were oblatned by the method or d~llcrcntlal centrifugation. The activitles of anttoridant enzymes such as superoxide dlsmutasc. cslnlarc, plutath~one rcducmre and glutnthlone prox*dasc decreued s~gnificantly ,n the animals treated well8 tnc~horyci~lor In a dose-dependent manner In thc m~lochondilal and micromme-rich fraetloer of rat testis The lcvclr of hydrogcn peroiidc pncmllon (H,OI) and lip~d peroxtdatlon increased in m~~ochondriul and m~crorome-rich lracl~ons of the lcstts of the rats tmtd with melhoxychlor The rcsulu suppaled that the low to medlum dosn ol rncthuxyci,lor clcn deplet8on of antloxtdanl enzymes and concom~lant increase In the levels or HIO* and lipid prortdiltian dtllcrcnl\ally in mitochondrial and micro~omc-nch fractions of rat testis In conclusion, the adverse cflccl 01 mcthoxychlor on malc rsproducuon could be duc to lhc lnduclion of oxidal~ve stress in lestis. Q 2002 Publl,hd by Elrvicr Sclsncc Ireland Ltd lcywvrdr Mc~horychior, Tuur, Anboridanl cnqmn. L~ptd pcrondalaon. Mllochondnsl fnclionr, Mncroromcrich Irlclionl: Rat The exposure of humans to cnvironmcntal contaminants that negatlvely affccl the male reproductive svstcm is increasing -(Sham, . 1993: Chia, 'Cormpondlny author Tel. +91413615.211, 81. +91. 2000,, 11;~ important to notc that many cnviron. 413.bJJ.lll K.n~~a1~~d~,rcu pp~~~;~~I~ut~~!l~olm~~I,com (P.P Muthur) mcnta' like organochlor'ne ' B.mwl vpnnlhurgpl pon.n~.tn, cider, accumulate in fatty tissues. Therefore,
continuous exposure of humans to increasing numbcn and amounts of thns cnvtronmcntal contaminants niay have cumulative efTects that lcad to reproductive disorders (Lafuentc et al., 2000). Mctlioiychlor is onc of the cnv~ronmentnl contamlnonts wli~clt as w~dcly urd as a pesticide in many countries that was developed to replau dichloro-diphenyl-tnchloroethane (DDT), and its chemical name is 1.l.l-trichloro-2.2-his@- mcthoxy phenyl) cthanc. Mcthoxychlor displays both ntrogcnic and antrandrogcnic activities in vivo and In vitro (Maness el nl.. 1998). Methoxychlor 1s wnstdcred to be a proatrogcn that 1s mctabolired into mono- and bs-hydroxy melabohtcs, wh~ch have been shown to have more estrogenic properties than the parental compounds (Bulger el al., 1978). Due to the estrogcnictty of mcthoxychlor metabolites. exposure of either neonatal or adult organlsm to high doses of mclhoxychlor may place the male reproductive system at risk. It has been reported that methoxychlor can affect malc rcprodvction of rats by Jccrcnsina spc-rm counts (Chnpin ct al.. 1997). Mctlioxychlor has also been shown to inducc ox~dat~ve strm In the ep~didymal sperm of goat (Gnligudhar~a ct al.. 2001). It has bcen rcportcd th;it mcthoxyclilor undcrgo hcpatic m~crosomal nmnooxygcnax mcdiatcd actwallon and the resultant rcactlve metabol~tes possibly free radicals bind wvalcnlly to microsomal components (Bulger et al.. 1983). Ant$ox~dants/froe radical scavengers, and sulfhydryl containing compounds inhibat covalent bindlng of methoxychlor in human liver microsomes, suggesting that the rcastive intermediate is a frec radlcal (Bulgcr and Kupfur, 1989). It has also been reported that human cy. tmhrome P-450 enzymes responsible for wnversion of methoxychlor into its major metaboliter, the mono-o-dcmethylatcd dcrivatlvn and CYPIA2, havc bcen shown lo play predomrnant role tn thas rcaction (Strcrxr and Kupfcr. 1998). Thc abklity of cylmhrorne P-450 system to mduu rcacuvc oxygcn spx~es (ROS) has ban reported (Bondy and Naderi. 1994). ROS are formed in both phys~ological and patholog~cal wnditions in mammalran ttssun, duc to lhcir high reactivity thcy s,;ty tmcrilct with hiomol~zuln inducing oxidat~ve stress (Ochxndocrf, 1999). Mitochondr~al respiration is the main bioloaical source of suoeror~dc anion radicals und~r-~h~siolo~iul conditions. F m radiFalsiROS generated in tissues mnd subcellular wmpMmonts are cfficicntly rcavcngcd by the antioxidant defence system, which co~ts(itutcs antioxidant enzymes such as supcror. ide dismutaac, &lase. xlutathione rcduclnse and glutathlonc peroxidase. Under normal physiological conditions ires radiulaIROS are generated in subcellular compartments of testis which are subsequently mvcngd by the antioxidant defence system of the corresponding cellular compartments. The suixzllular membranes an more sus- ceptible lo l~p~d pcroxidat~ons, which are rich in unslaturated fatty acids and has bcen shown to contain low amount of antiox~dants. The subcellular membranes have been reported to undergo pcrmeab~lity changes following enhanced lip~d pcroxidation and glutathione depletion (Chance ct al.. 1979). The testicular production or free radicals and function of the antioxidant dcfcna systcm have ken reported upon exposure to toxlc chcm~calr (Peltola ct sl.. 1994; Sujotha ct al.. 2WI. Latchoumvcandanc el al.. 2W2a) Prevaous stud~es from ou; laboratory havc shown that the cnvironmcntal wntamrnants lake Ilndanc. mcthoxychlor and 2.3.7.8-tetrachlorodibenzo.pdioxin indua oxidauvc suns in the epid~dymrs and eptdidymal sperm of rats (Chttra el al.. 2WI; Latchournycdndane el al., 2002b,c). In order to explain the mechanism of action of methoxychlor at suixzlluhr levels In testis, the present studies wen undertaken to evaluate the eNm of methoxychlor an the antioxidant systcm in mitochondrial and microsome-rich frlclions of rat testlS. 2. Material and arthods Methoxychlor (I.l.I-lrichloro-2.2.b~~[~ mcthoxyphenyl] cthanc), Appror. 95% purc, Sigma Chemical Company, was obta~ncd as gift from Dr Ute Ticmann. Rescarch institute for the Biology of Farm An~mals. Dummcntorf, Germany. Th~obarbitunc acid and malondialdchyde
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FUNCTIONAL STUDIES ON THE EFFECTS O
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ACKNOWLEDGEMENTS I wish to express
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TABLE OF CONTENTS Page No ACKNOWLED
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TABLE OF CONTENTS (Continued) Deter
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TABLE OF CONTENTS (Continued) Effec
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TAULIC OF CON'I'ISN'I'S (C'untinued
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xi LIST OF TABLES (Continued) I'agc
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Table 24a Table 24b Table 24c Table
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Fig. 1 Fig. 2 Effect of TCDD on the
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Fig. 12c Fig. 12d Fig. 13 Fig. 14a
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LIST OF FIGURES (Continued) Fig. 23
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LH M PL mg mRNA N AD NADH NADP NADP
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layers, an outer layer of visceral
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tubular epithelium and continually
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lmn~unization of monkeys against FS
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and this reaction is catalyzed by t
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extratubular environment or synthes
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the most active in protein secretio
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1958). A rapid fall in serum testos
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have also been reported in the rats
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Effect of TCDD on heart muscle has
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1.4 Effect of oxidative stress on m
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2 SCOPE OF THE PRESENT STUDY There
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3 MATERIALS AND METHODS 3.1 Animals
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Subgroup III: Administration of TCD
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choppcd with the help of sharp razo
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anti-FSH antibody. After incubation
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lust sped (500 to 700 rpm) on an or
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3.13.4 Quantitative determinntiun u
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The plates were dried by inverting
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centrifuged at 3,500 g for I5 min o
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3.17 Preparation of tiasue humogena
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supcrnutant was taken. Hlunk was pr
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3.21.4 Assay of glutathione peroxid
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4 RESULTS 4.1 Administration of TCD
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seminiferous tubules was found to b
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fractions of rat testis as compared
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4.1.11.1 Effect of TCDD on the prod
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4.2 Co-administration of TCDD and v
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4.2.8 Effect of co-administration o
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4.2.11.5 Effect of co-administratio
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Table I b. Effect of TCDD on the we
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Table 4. Effect of TCDD on the seru
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Table 7b. Effect of TCDD on the pro
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Table 8c. Effect of TCDD on the lev
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Table 9d. Effect of TCDD on the act
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Table 13. Effect of TCDD on the ant
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Table 15. Effect of TCDD on the lev
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Table 16c. Effcct of TCDD on the an
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Table 18a. Effect of co-administrat
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Table 21. Effect of co-administrati
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Table 24b. Effect of co-administrat
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Table 25c. Effect of co-administrat
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Table 26d. Effect of co-administrat
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Table 30. Effect of co-administrati
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Table 32. Effect of caadrninistrati
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Table 33c. Effcct of co-administrut
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5 DISCUSSION 5.1 Significance of th
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Measurement of weights of the acces
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compartment of the cells (Chainy el
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the reduction in daily sperm produc
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eported in the TCDD-treated rats (M
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lilnction is to ililcrccpt lipid pc
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in the animals administered with TC
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protective effects on serum testost
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suygcstccl that vivamin 1: conld im
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Fig.4~. ENect of TCDD or TCDD and v
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tE W d In w it I I (,OCX) 3s PUB (,
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Fig. 7c. Effect of TCDD or TCDD and
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u 3 f ~ = m w * N O .- ~Slew aew en
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Fig.lOb. ENmt of TCDD or TCDD and v
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Fig. 1Ir Effect of TCDD or TCDD md
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Fig. 14c. Effect of TCDD or TCDD an
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Fig. 16b. Effect of TCDD or TCDD an
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Fig.18~. EKect of TCDD or TCDD and
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1 1.2 l4 Fig. 19b. ENect of TCDD or
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Fig. ZOb. Elkt of TCDD or TCDD and
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Fig. 21. Effect olTCDD or TCDD and
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Fig. 22c. Effect of TCDD or TCDD an
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Fig. 24b. Eflect ofTCDD or TCDD and
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7 REFERENCES Aitken RJ, Irvine DS,
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Chitm KC, Latchoumycandanc,C, Mathu
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Flsher JS, Turner KJ, Brown D, Shar
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Hornsby PJ. Regulation of cytochrom
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Latcboumycandane C, Chitra KC, Math
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Moore RW, Panona JA, Bookstaff RC,
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Rommerts FFG, Cooke BA, Van Der Kem
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Slaunwhite WR, Burgett MJ. h virro
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1.1 Preparation of dosing solution
Page 195 and 196: 1.4.6 Wash concentrate One bottle,
Page 197 and 198: 1.7.3 Estradiol controls Two vials.
Page 199 and 200: 1.1 1.2 Orcinol(6%) 6 g of orcinol
Page 201 and 202: 1.16.2 Horseradish peroxidase (8 Un
Page 203 and 204: 1.20 Superoxide dismutase 1.20.1 Tr
Page 205 and 206: 1.24.2 Thiobarbituric acid (TBA; 0.
Page 207 and 208: Chronic effect: of enclosulfan on t
Page 209: enbw1h1.W mu IUY Wcab unpiliml~t at
Page 212 and 213: cbniuis 11~11 b"nci:~lc ~wlive oxyg
Page 214 and 215: DNA, RNA ;uxl Wcin wcrr abarval, wh
Page 216 and 217: Mt~~m(10-lZsu&)vacob- Wfmn Ihehml ~
Page 218 and 219: New Delhi, llrliil Ib SCI~KK Rcm& M
Page 220 and 221: tiii~lc rcprmluction or rut (Cllitr
Page 222 and 223: wciglit (b.wt.) per day for 45 days
Page 224 and 225: 0.1 00 01 " 0.8 I . .lo Dl* rcnn IW
Page 226 and 227: Chilr:~. K.C.. L!olnn!myci!~dum. C.
Page 228 and 229: C. hteboumyeradlos . KC. CMtn . P.P
Page 230 and 231: I. 1 Ilal 01 2 1.7 # I c I ~ ~ L I
Page 232 and 233: Fin. L Corrclatwn bclwfcn "perm cou
Page 235: Iionshii nepmdvctivc mxicity ad upt
Page 240 and 241: o. Glutathione reductage mg pmWn mg
Page 242 and 243: IOWAME -. Tor. #558517 PME: 9 SESS:
Page 244 and 245: JOBNAME: Rqm. Tm. M5W7 PAOB: 11 SBS
Page 248 and 249: ---- - wcre obtu~ncd from E-Monk. G
Page 250: 3.2. A,III~AILNI syrlo~t of ,nIroch
Page 253 and 254: Uc,#!dy. SC. N;tdcru. S , 1% Conlrb
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