APPENDIX 1

PREPARATION OF REAGENTS
1.1 Preparation of Stock Solutions

1.1.1 Cis-diamminedichloroplatinum (cisplatin)

50 mg of cisplatin was dissolved in 100 ml of normal saline (0.9% sodium
chloride) to give a concentration of 0.5 mglml and stored at 4C.

1.1.2 Testosterone propionate (TP)

1 g of TP was dissolved in 20 ml of sterile olive oil to give a concentration of

50 mg/ml.

1.2 Estimation of DNA

1.2.1 Diphenylamine reagent

1.5 g of diphenylamine reagent was dissolved in 100 ml of glacial acetic acid.

To this 1.5 ml of conc sulphuric acid was added and stored at 4C in a amber coloured
bottle.
To every 20 ml of this reagent, 0.1 ml of 1.6% aqueous acetaldehyde was
added to potentiate the colour development.

1.2.2 Perchloric acid

16.66 ml of perchloric acid (60%)was made up to 100 ml with distilled water.

1.2.3 DNA (standard)

2 mg of calf thymus DNA was dissolved in 10 ml of hot 5 % trichloroacetic acid

to give a concentration of 200 pglml and stored at 4C.

1.3 Estimation of RNA

1.3.1 Orcinol reagent

1 g of ferric chloride was dissolved in 1 litre of conc hydrochloric acid and 35

ml of 6% orcinol was added.

1.3.2 Orcinol, 6%

6 g of orcinoi was dissolved in 100 ml of ethyl alcohol.

1.3.3 RNA (standard)

2 mg of RNA was dissolved in 10 ml of distilled water to give a concentration

of 200 pglml and stored at 4OC.

1.4 Estimation of Protein

1.4.1 Bradford reagent

100 mg of coomassie brilliant blue G-250 was dissolved in 25 ml of 95% ethyl

alcohol and 50 ml of 85% ortho phosphoric acid was added. This was made up to 500
mi with distilled water, filtered through a Whatman (No. 1) paper and stored in amber
coloured bottle.

1.4.2 BSA (standard)

10 mg of BSA was dissolved in 10 ml distilled water to give a concentration of
1 rnglml and stored at 4C.

1.5 RIA of Serum Testosterone

1.5.1 Gelatin-phosphate buffer saline-GPBS (pH 7.4)

1.95 g of sodium dihydrogen phosphate (2.5mM), 6.64 mg of disodium

hydrogen phosphate (7.5rnM) and 45 g of sodium chloride (0.9%) was dissolved and
made up to 5 litres with distilled water. The pH was adjusted to 7.4 with 1N sodium
hydroxide. 5 g of gelatin (0.1%) was added and stored at 4C.

1.5.2 Dextran coated charcoal

1 g of activated charcoal and 100 mg of dextran T-70 was dissolved and made
up to 100 ml with GPBS. It was stored at 4C and vortexed every time before use.

1.5.3 Scintillation fluid

10 g of PPO. 800 mg of POPOP and 42 ml of methanol was dissolved and

made up to 2 litres with toluene. It was stored at room temperature.

1.6 Radio lodination of Hormones

1.6.1 Phosphate buffer, 0.2M (pH 7.2)
Solution A
7.12 g of disodiutn hydrogen pl~osphatewas dissolved in 200 ml or distilled
water.

Solution B

3.12 g of sodium dihydrogen phosphate was dissolved in 100 ml of distilled

water.
144 rnl of solution A and 56 rnl of solution B were mixed and pH was adjusted

to 7.2 with 2N sodium hydroxide.

1.6.2 Phosphate buffer saline (PBS), 0.05M (pH 7.2)

Solution A

7.12 g of disodium hydrogen phosphate was dissolved in 200 ml of distilled

water.

Solution B

3.12 g of sodium dihydrogen phosphate was dissolved in 100 rnl of distilled

water.

Solution C
4.908 g of sodium chloride was dissolved in 600 ml of distilled water.

144 ml of solution A and 56 ml of solution B were mixed and added to 600 ml

of solution C. The pH was adjusted to 7.2 with 2N sodium hydroxide and 800 mg of
sodium azide was added as preservative.

1.6.3 Ethylenediaminetetrwtic acid-normal rabbit serum (EDTA-NRS) buffer

in 0.05M PBS, 0.05M (pH 7.2)
9.305 g of EDTA was dissolved in 500 ml of O.05M PBS and pH was adjusted
to 7.2 with 5N sodium hydroxide. To this, 500 mg (0.1%) of sodium azide was added
as preservative.
1:25 ml of NRS (1 : 400 dilution) was added to this buffer.

1.6.4 Hormones for iodination

100 ug of highly purified iodination grade rFSH and rLH were diluted with 1
ml of 0.2M phosphate buffered saline (PBS), pH 7.2 to give a concentration of 2.5 ug
of hormone/25

pi.

1 mg of chloramine T was dissolved in 1 ml of 0.05M PBS, pH 7.2. It was

prepared at the time of use.

1.6.6 Sodium metabisulphite

2 mg of sodium metabisulphite was dissolved in 1 ml of 0.05M PBS, pHt7.2. It

was prepared at the time of use.

1.6.7 Preparation of sephadex G-25 column

The prepared sephadex G-25 (PD-10 column) (Gel filtration theory and
Practice; Pharmacia, technical note, page. 9) was equilibrated with 25 ml of 0.05M

PBS (pH 7.2). Then the reaction mixture was transferred carefully on to the top of the
column. When the top layer of the solution was about to disappear into the gel bed, 3.5
ml of 0.05M PBS (pH 7.2) was added. Then 5 drop of aliquots of the eluted solution
were collected into polypropylene tubes containing 500 p1 of 1 % BSA in 0.05M PBS
(pH 7.2). The column was first eluted with 3.5 ml of 0.05M PBS (pH 7.2). As this
volume of solution disappeared, more volumes of 0.05M PBS (pH 7.2) were added on
to the column and the effluent was collected.

1.7 RIA of Serum FSH

1.7.1 FSH tracer

The iodinated [1125] FSH tracer was diluted with 1% BSA in 0.05M PBS (pH
7.2) to get approximately 20, 000 cpm per 100 pof tracer.

1.7.2 Rat FSH antiserum (rabbit)

1 ml of 1 : 12.5 diluted rat FSH antiserum (rabbit) in 2% normal rabbit serum

(NRS) in PBS containing 0.01 % merthiolate was provided. 0.1 ml of this antiserum
was suitably diluted with 0.05M EDTA-NRS buffer in 0.05M PBS (pH 7.2) to get a
final dilution of 1 : 1, 25, 000 in the assay.

1.7.3 Rat FSH reference preparation

1 vial containing 10 ug of lyophilized hormone in 1 ml of 1% BSA in PBS was

provided. This lyophilized powder was reconstituted with 1 ml of distilled water to get

a concentration of 10 ug FSHIml and serial dilutions were made from this solution to
get the concentrations of 0.25, 1, 4, 16, 32 and 64 ng of FSHIml.

1.7.4 11 Ab- goat-antirabbit gamma globulin (ARGG)

100 pl of goat-antirabbit gamma globulin (ARGG) was diluted with I ml (1 : 10

dilution) of O.05M EDTA-NRS buffer in 0.05M PBS (pH 7.2) and used for the
precipitation of immunocomplex.

1.8 R I A of Serum LH
1.8.1 LH tracer

The iodinated

1 1 ~ LH
~ ~tracer
1 was diluted with

1 % BSA in 0.05M PBS (pH

7.2) to get approximately 20, 000 cpm per 100 poi tracer.

1.8.2 Rat LH antiserum (rabbit)

1 ml of 1 : 18.75 diluted rat LH antiserum (rabbit) in 2% normal rabbit serum

(NRS) in PBS containing 0.01% merthiolate was provided. 0.1 ml of this antiserum
was suitably diluted with 0.05M EDTA-NRS buffer in 0.05M PBS (pH 7.2) to get a
final dilution of 1 : 1, 80. 000 in the assay.

1.8.3 Rat LH reference preparation

1 vial containing 5 ug of lyophilized hormone in 1 ml of 1% BSA in PBS was

provided. This lyophilized powder was reconstituted with 1 ml of distilled water to get

a concentration of 5 ug LHlml and serial dilutions were made from this solution to get
the concentrations of 0.25, 1, 4, 16, 32 and 64 ng of LHIml.

1.8.4 11 Ab-goat-antirabbit gamma globulin (ARGG)

100 pof goat-antirabbit gamma globulin (ARGG) was diluted with 1 ml (1 : 10

dilution) of 0.05M EDTA-NRS buffer in 0.05M PBS (pH 7.2) and used for the
precipitation of immunocomplex.

1.9 Alkaline Phosphatase Assay

1.9.1 Sodium carbonate-bicarbonate buffer, 0. l M (pll 10.0)

6.36 g of anhydrous sodium carbonate and 3.36 g of sodium bicarbonate was

dissolved and made up to 1 litre with distilled water. The pH was adjusted to 10.0.

1.9.2 p-nitrophenyl phosphate

1 g of p-nitrophenyl phosphate was dissolved and diluted to 250 ml with

distilled water to give a concentration of 4 mgiml and stored at 4C. It was prepared at
the time of use.

1.9.3 p-nitrophenol (standard)

The stock solution was prepared by dissolving 5.8 mg of p-nitrophenol in 10

ml distilled water to give a concentration of mmole/ml. This stock was diluted 10 times
to prepare working standard solution of concentration 10 pmlml and stored at 4C. It
was prepared at the time of use.

1.9.4 Sodium hydroxide, O.lN

0.4 g of sodium hydroxide was dissolved in 100 ml of distilled water.

1.10 fi-glucuronidase Assay

1.10.1 Acetate buffer, 0.1M (pH 4.5)
Solution A

1.16ml of glacial acetic acid was made up to 200 ml with distilled water.

Solution B

0.82g of sodium acetate was dissolved in 100 ml of distilled water.

120 ml of solution A and 80 ml of solution B were mixed and pH was adjusted
to 4.5 with IN sodium hydroxide.

1.10.2 Glycine buffer (pH 11.2)

4.08 g of glycine, 3.16 g of sodium chloride and 1.36 g of sodium hydroxide
(for 2.73 ml of 50% sodium hydroxide) was dissolved and made up to 250 ml with
distilled water. The pH was adjusted to 11.2.

1.10.3 Phenolphthalein 8-glucuronic acid, 0.005M

24.72 mg of phenolphthalein 8-glucuronic acid was dissolved in 10 rnl of
distilled water and stored at 4C.

1.10.4 Phenolphthalein (standard)

1 mg of phenolphthalein was dissolved in 5 ml of 95% ethanol and made up to

10 ml with distilled water to give a concentration of 100 pglml and stored at 4C.

1.1 1 Leydig Cells Separating Medium

MEM

- 1.02 g

Collagenase (Type 1)

HEPES
BSA

0.05 g
0.10~
0.10 g

All the above were dissolved in distilled water, pH was adjusted to 7.2 and
made up to 100 ml. It was filtered in millipore membrane filter (0.22pm) and stored at
4C.

1.12 Sertoli Cells Separating Medium

MEM

- 0.51 g

Collagenase (Type 1)

- 0.013 g

Trypsin

- 0.025 g

BSA

DNase l

- 0.05 g

0.05 g

All the above were dissolved in distilled water, pH was adjusted to 7.2 and
made up to 50 ml. It was filtered in millipore membrane filter (0.22pm) and stored at
4".

1.13 Medium 199

0.99 g of Medium 199 and 0.22 g of sodium bicarbonate were dissolved in

distilled water and pH was adjusted to 7.2. 1 ml of penicillin-streptomycin solution
was added and made up to 100 ml with distilled water.lt was filtered and stored at 4C.

1.14 Minimum nsscnlial Mcdium (Eagle Modified)

10.16 g of MEM and 2.2 g of sodium bicarbonate were dissolved in distilled

water and pH was adjusted to 7.2. 10 ml of penicillin-streptomycin solution was added
and made up to 1 litre with distilled water. It was filtered and stored at 4C.

1.15 Minimum Essential Medium (Eagle Modified)

+ Fetal Bovine Serum

10.16 g of MEM and 2.2 g of sod~umbicarbonate were dissolved in distilled

water and pH was adjusted to 7.2. 10 ml of penicillin-streptomycin solution and 70 ml
of fetal bovine serum were added and made up to 1 litre with distilled water. It was
filtered and stored at 4C.

1.16 Percoll Solution (stock)

Percoll

- 22.5 ml

BSA

MEM
Sodium pyruvate
Sodium lactate

0.075 g
0.026 g
0.0075 g
10 fi1

All the above were mixed, pH was adjusted to 7.2 and made up to 25 ml with
distilled water. It was filtered and stored at 4C.

1.17 Preparation of Stock Solutions (in vilro Study)

1.17.1 Cis-diamminedichloroplatinum (cisplatin)

5 mg of cisplatin was dissolved in 10 ml of normal saline (0.9% NaCI) to give a

concentration of 0.5 mglml. The stock solution was serially diluted with normal saline
to get the concentrations of 10 ng, 100 ng, 1 pg, 5 pg and 10 pg.

1.17.2 Testosterone
300 pg of testosterone was dissolved i n 15 pl of 95% ethanol and 5 ml of

normal saline was added to give a concentration of 60 pglml.

1.18 3%-hydroxysteroidDehydrogenase Assay

1.18.1 Tris-HCI, 0.1M (pH 8.2)
Solution A
1.21 g of Tris was dissolved and made up to 100 ml with distilled water
Solution B
0.9 ml of conc hydrochlor~cacla was maae up to 100 ml with distilled water.

62.5 ml of solution A and 50 ml of solution B were mixed and made up to 250

ml with distilled water. The pH was adjusted to 8.2 with IN sodium hydroxide.

1.18.2 Pyrophosphate buffer, lOOpM (pH 9.0)

13.30 mg of tetrasodiumpyrophosphate was dissolved and made up to 500 ml
with distilled water. The pH was adjusted to 9.0.
1.18.3 NAD
34.27 pg of NAD was dissolved in 100 ml of distilled water. It was prepared at
the time of use.

2.88 pg of dehydroepiandrosterone was dissolved in 100 ml of distilled water.

1.19 176-hydroxysteroid Dehydrogenase Assay

1.19.1 Tris-HCI, 0.1M (pH 7.2)
Solution A
1.36 g of Tris was dissolved in 100 ml of distilled water.
Solution B
0.9 ml of conc hydrochloric acid was made up to 100 ml with distilled water.
62.5 ml of solution A and 55.25 rnl or solution B were mixed and made up to
250 ml with distilled water. The pH was adjusted to 7.2 with 1N sodium hydroxide.

1.19.2 Pyrophosphate buffer. lOOpM (pH 9.0)

13.30 mg of tetrasodiumpyrophosphate was dissolved and made up to 500 ml
with distilled water and pH was adjusted to 9.0.

1.19.3 NADPH
41.67 pg of NADPH was dissolved in 100 ml distilled water. It was prepared at
the dme of use.

22.91 pg of 4-androstene 3.17-dione was dissolved in 100 ml of distilled water.

1.20 Estimation of Lactale

1.20.1 Hydrazine(0.4M)-Glycine(O.5M)buffer (pH 9.0)
19 g of glycine and 41.65 ml of hydrazine hydrate was dissolved and made up
to 500 ml with distilled water after adjusting the pH to 9.0.

1.20.2 Perchloric acid, IN

83.3 ml of perchloric acid was made up to 500 ml with distilled water.

1.20.3 Potassium carbonate, 5 M

34.56 g of potassium carbonate was dissolved and made up to 50 ml with
distilled water.

1.20.4 Methyl orange indicator

50 mg of methyl orange was dissolved in 100 ml of distilled water.

1.20.5 NAD. 40mM

1.33 g of NAD was dissolved in 100 ml of distilled water. It was prepared at
the time of use.

1.21 Estimation of Pyruvate

1.21.1 Triethanolamine buffer (pH 7.6)
Solution A

15.2 g of triethanolamine was dissolved in 200 ml of distilled water.

Solution B
4 g of sodium hydroxide was dissolved in 100 ml of distilled water.

120 ml of solution A and 66 ml of solution B were mixed and made up to 400

mi with distilled water. The pH was adjusted to 7.6 with 2 N hydrochloric acid.

1.21.2 Perchloric acid, 0.6N

5 0 ml of perchloric acid was made up to 500 rnl with distilled water.

1.21.3 Potassium hydroxide, 2 N

11.2 g of potassium hydroxide was dissolved,in 100 ml of distilled water.

1.21.4 Ethylenediaminetetraacetic acid (EDTA), 5mM

186.1 mg of EDTA was dissolved in 100 ml of distilled water.

1.21.5 NADH. 6mM

7.09 mg of NADH was dissolved in 100 ml of distilled water. It was prepared

at the time of use.

APPENDIX 2
LIST OF PUBLICATIONS RASED ON THE PRESENT WORK

1.

Malarvizhi, D. and Mathur, P.P. (1995) Effects of cisplatin on

physiological

status of normal rat testis. Ind. J. Exp. Biol. 33: 281-283.

2.

Malarvizhi, D. and Mathur, P.P. (1996) Testosterone propionate

prevents

cisplatinum induced inhibition of renal 8-glucuronidase activity in rats.

Ind. J. Exp. Biol. (in press).

3.

Malawizhi, D. and Mathur, P.P. (1996) Effects of cisplatin on testicular

functions in rats. Ind. J. Exp. Biol. (in press).

4.

Malarvizhi, D. and Mathur, P.P. (1996) Renal 8-glucuronidase activity is a

bioassay of serum testosterone levels in rats. Ind. J. Exp. Biol. (in press).