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

pH 7.6). I00 pl of 200 mM NADPH, IW pI of 10
mM EDTA. 50 p1 of 20 mM glutathione oxidized
and 50 MI enzyme soum. Disappearance of
NADPH was measured immcd~ately at 340 nm
seainst blank containing all tho componcnts exccpt
thc enzymc at 10 s mtcrvals for 3 min on a
Systrotl!u S~trophotometer. The enzyme activ-
~ty was cxprcsscd In nmole of NADPH oxidized
pcr min mg-' protein.
Ci1ut;tlhionc pcroxidasc (EC 1 .I 1.1.9) was nsr;tycJ
by tine mclllod of l'ugl~;t and Valcntlac
(1967). linefly. thc assay mirturc contamed 1.59
ml of phosphatc bufTcr (100 mM. pH 7.6). 100 p1
of 10 mM EDTA. 100 g1 of sod~um azide, 50 pl of
glutathlonc rcductasc, 100 pl of glutathione reduced
100 p1 of 200 mM NADPH. 10 pl of H,O,
and 10 el cnzyme sourcc. Disappearance of
NADPH was rncasurcd immcd~atcly at 340 nm
ng.lmst blank containing all the components exccpt
thc cnzymc at 10 s intcrvdls for 3 min on a
Systron~cs Spatrophotomcter. The enzyme actlv-
~ty was cxprcsscd in nmolc of NADPH oxidized
pcr znln mp- ' prutcitl.
2.9. Hydrogen pcroxrde generalton assoy
11,0, pocr;trton was aswycd by thc mcthod or
I'lch and Kcisr~ (1981). Drlcfly, the incubatson
mtrriurc wntaincd 1.64 ml phosphate bukr (50
mM. pH 7.6). 54 4 of horseradish peroxidasc (8.5
Vlml). 30 pl of 0.28 nM phenol red. 5.5 nM of
165 pl of dextrose and 100 pl of enzyme aource
was done at 35 'C for 30 min. The reaction was
tcrmlnatcd by the addition of 60 p1 of 10 N
sodtum hydrondc. Thc absorbance was read at
610 nM against a reagent blank on a Syrtromu
Spcctrophotomctcr. The quantlty of H,O, produccd
was exprcsxd as nmolc H,O, pcnentcd pr
min per mg protcln at 35 'C. For standard curve.
known amount or H,O, and all the above
rcagcnb except cnzymc source wcrc incubated for
30 mrn at 35 'C and then added 60 14 of 10 N
saltum hydroxide and optical dsnsbty WM read at
610 nM.
2.10. Lipid pcrox~dadorion
A break-down product of lipid peroxidation
thiobarbituric actd reactive subslance (TEARS)
was measured by the method of Bucp and Aust
(1976). Ensfly. the stock solution wntained equal
volumes of trichloroaoct~c actd 15% (wlv) in 0.25
N hydrochloric acid and 2-thiobarbituric acid
0.37% (wlv) in 0.25 N hydrmhloric add. h e
volume of the test sample and two volumes of
stock reagent were mixed in a screwsopped centrifuge
tube. vortexcd and heated for IS min on a
boiling water bath. After cooling on ia the precipilalc
was rcmovcd by centrifugation at 1000 x
g for I5 man and absorbanes of the supernalant
was measured at 532 nM against blank conlaming
all the reagents except test sample. A standard
CUPS was Constructed extrapolating the amount
of commercially bought product malondialdchyde
to the rneasurcd absorbance. The value is cxpressed
In pmole of malond~aldchydc formed pr
mg protctn.
Data wcrc crprcrrcd as mean *S.D, lor four
anlmals per dose and analyrcd statistically using
one-way analysis of varlana (ANOVA) followed
by Tukey's 161. Probability valun Ins than 0.05
wcrc dcternllned to be statistically rtgn!ficant
agalnst control.
3.1. Body weaghr and organ wctghfs
Thc body weight of methoxychlor-lrcatsd rats
d~d not change r~gnificantly when wmparod wlth
the corresponding group of control animals
(Tabk I), the weights of the tutia, cpid~dymir,
=mind ves~lcr and ventral prmtate decreased
significantly in the rau treated with 100 mg
mcthoaychlor. However, the weighta remained
unchanged in the animals treated with melhory.
shlor at lower do= (Table I).