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APPENDIX 1

Preparation of reagents

1.1. Preparation of dosing solution


Nonylphenol
15 mg of Nonylphenol was dissolved in olive oil (10 ml) and used as stock
solution. The stock solution was serially diluted and used for treatments.

1.2. Heparin
2 mg heparin was dissolved in 0.9 % NaCl (10 ml). 200 l of heparin was
added to each 1 ml of blood to prevent coagulation.

1.3. Determination of protein

1.3.1. Alkaline copper reagent


Reagent A: 2 g of sodium carbonate was dissolved and made up to 100 ml
with 0.1 N sodium hydroxide solution.
Reagent B: 5 mg of copper sulphate was dissolved in 1.0 ml of 4 % sodium
potassium tartrate.
Reagent A of 50 ml and reagent B of 1.0 ml were mixed fresh at the time of
use.

1.3.2. Sodium hydroxide (0.1 N)


0.4 g of sodium hydroxide was dissolved and made up to 100 ml with distilled
water.

1.3.3. Sodium potassium tartrate (4%)


4 g of sodium potassium tartrate was dissolved and made up to 100 ml with
distilled water.

1.3.4. Protein standard solution


100 mg bovine serum albumin (BSA) was dissolved and made up to 100 ml
with distilled water. This solution was diluted 10 times to obtain concentration
of 0.1 mg/ ml.

1.3.5. Folin-Ciocalteau reagent (1 N)


Commercial Folin-Ciocalteau reagent was diluted with two volume of distilled
water.

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Preparation of reagents

1.4. Superoxide dismutase

1.4.1. Tris HCl buffer (50 mM) containing EDTA (1 mM; pH 8.2)
605 mg of Tris HCl was dissolved in 100 ml of distilled water. To this, 0.0372
g of EDTA was added and pH was adjusted to 8.2.

1.4.2. Pyrogallol (0.2 mM) in 50 ml of HCl (10 mM)


1.26 mg of pyrogallol was dissolved in 50 ml of 10 mM HCl.

1.4.3. HCl (10 mM)


41.6 l of concentrated HCl was made up to 50 ml with distilled water

1.5. Catalase

1.5.1. Phosphate buffer (0.05 M, pH 7.0)


Solution A: 0.89 g of disodium hydrogen phosphate was dissolved in 100 ml
of distilled water.
Solution B: 0.69 g of sodium dihydrogen phosphate was dissolved in 100 ml of
distilled water.
39 ml of Solution A and 61 ml of Solution B was mixed and made up to 200
ml with distilled water. The pH was adjusted to 7.0.

1.5.2. H2O2 (0.019 M)


58 l of hydrogen peroxide (from 30 %) was made up to 100 ml with distilled
water. Stored in cool and dark place.

1.6. Glutathione peroxidase

1.6.1. Phosphate buffer (100 mM, pH 7.6)


Solution A: 1.78 g of disodium hydrogen phosphate was dissolved in 100 ml
of distilled water.
Solution B: 1.38 g of sodium dihydrogen phosphate was dissolved in 100 ml of
distilled water.
13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml
with distilled water. The pH was adjusted to 7.6.

1.6.2. EDTA (0.01M)


37.224 mg of EDTA was dissolved in 10 ml of distilled water

1.6.3. Sodium azide

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Preparation of reagents

6.5 mg of sodium azide was dissolved in 10 ml of distilled water.

1.6.4. Glutathione reductase


31.25 mg of glutathione reductase was dissolved in 5 ml of distilled water.

1.6.5. Glutathione reduced


30.733 mg of glutathione (reduced) was dissolved in 10 ml of distilled water.

1.6.6. NADPH (0.2 M)


16.667 mg of NADPH was dissolved in 10 ml of distilled water

1.7. Glutathione reductase

1.7.1. Phosphate buffer (0.1 M, pH 7.6)


Solution A: 1.78 g of disodium hydrogen phosphate was dissolved in 100 ml
of distilled water.
Solution B: 1.38 g of sodium dihydrogen phosphate was dissolved in 100 ml of
distilled water.
13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml
with distilled water. The pH was adjusted to 7.6.

1.7.2. NADPH (0.2 M)


16.667 mg of NADPH was dissolved in 10 ml of distilled water.

1.7.3. Glutathione oxidized (2 mM)


12.252 mg of glutathione oxidized was dissolved in 1 ml of distilled water.

1.7.4. EDTA (0.01 M)


37.224 mg of EDTA was dissolved in 10 ml of distilled water.

1.8. Hydrogen peroxide generation

1.8.1. Phosphate buffer (0.05 M, pH 7.6)


Solution A: 0.89 g of disodium hydrogen phosphate was dissolved in 100 ml
of distilled water.
Solution B: 0.69 g of sodium dihydrogen phosphate was dissolved in 100 ml of
distilled water.
13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml
with distilled water. The pH was adjusted to 7.6.

155
Preparation of reagents

1.8.2. Horseradish peroxidase (8 units/ mg)


1 mg of horseradish peroxidase was dissolved in 10 ml of distilled water to
make 8 units / ml.

1.8.3. Dextrose (100 nM)


180.11mg/ ml was prepared as a stock solution (1 M) and from this 100 nM
was prepared by serial dilution with the distilled water

1.8.4. Sodium hydroxide (10 N)


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

1.8.5. Phenol red (28 nM)


354.4 mg/ ml was prepared as a stock solution (1 M) and from this 28 nM was
prepared by serial dilution with the distilled water.

1.9. Lipid peroxidation

1.9.1. Trichloroacetic acid (TCA; 15%)


15 ml of TCA from 100% was made up to 100 ml with distilled water.

1.9.2. Thiobarbituric acid (TBA; 0.37%)


0.375 mg of TBA was dissolved in 100 ml of distilled water.

1.9.3. HCl (0.25 N)


1.08 ml of HCl was made up to 50 ml with distilled water.

1.9.4. TCA: TBA: HCl (1:1:1)


Equal volumes of the above three solutions were mixed to make 1: 1: 1 ratio of
TCA: TBA: HCl.

1.10. Liver Transaminase assay

1.10.1. Phosphate buffer (0.1 M, pH 7.4


Solution A: 1.78 g of disodium hydrogen phosphate was dissolved in 100 ml
of distilled water.
Solution B: 1.38 g of sodium dihydrogen phosphate was dissolved in 100 ml of
distilled water.
13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml
with distilled water. The pH was adjusted to 7.4

156
Preparation of reagents

1.10.2. Sodium pyruvate standard


Sodium pyruvate (22 mg) was dissolved in 100 ml of phosphate buffer

1.10.3. Sodium hydroxide


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

1.10.4. AST substrate


Dissolved 1.33 g of DL-aspartate and 15 mg of alpha-ketoglutatrate in
dissolved. It was made upto 50 ml with 1 N NaOH. Adjusted pH to 7.4

1.10.5. ALT substrate


Dissolved 29.2 mg of alpha-ketoglutarate (200mM/L) and 1.78 g of DL-
alanine 2 mM/ L. it was made upto 50 ml with 1 N NaOH. Adjusted pH to 7.4

1.10.6. 2,4-Dinitrophenyl hydrazine


Dissolved 19.8 mg of 2, 4-Dinitrophenyl hydrazine in 100 ml of 1 N HCl

1.11. Hexokinase

1.11.1. Triethanolamine buffer (0.05 M; pH 7.6)


450 mg of triethanolamine was dissolved in 50 ml of distilled water and the pH
was adjusted to 7.6.

1.11.2. D-glucose (0.555 M)


2.016 g of D-glucose was dissolved in 20 ml of distilled water.

1.11.3. MgCl2 (0.1 M)


200 mg of MgCl2 was dissolved in 10 ml of distilled water.

1.11.4. NADP (0.014 M)


117 mg of NADP was dissolved in 10 ml of distilled water.

1.11.5. ATP (0.019 M)


104 mg of ATP was dissolved in 10 ml with distilled water.

1.12. Phosphofructokinase

1.12.1. Tris HCl buffer (50 mM; pH 8)

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Preparation of reagents

605 mg of Tris HCl was dissolved in 100 ml of distilled water and the pH was
adjusted to 8.

1.12.2. Fructose-6-phosphate (1 mM)


3.4 mg of fructose-6-phospahte was dissolved in 10 ml of distilled water.

1.12.3. ATP (1 mM)


5.51 mg of ATP was dissolved in 10 ml of distilled water.

1.12.4. MgCl2 (2 mM)


4.06 mg of MgCl2 was dissolved in 10 ml of distilled water.

1.12.5. NADH (0.16 mM)


1.13 mg of NADH was dissolved in 10 ml with distilled water.

1.12.6. Dithiothreitol (2.6 mM)


3.85 mg of dithiothreitol was dissolved in 10 ml of distilled water.

1.12.7. EDTA (1 mM)


3.72 mg of EDTA was dissolved in 10 ml of distilled water.

1.12.8. Ammonium sulphate (5 mM)


6.67 mg of ammonium sulphate was dissolved in 10 ml of distilled water.

1.13. Glycogen phosphorylase

1.13.1. Citrate buffer (0.1 M, pH 6.0)


Solution A: 2.94 g of sodium citrate was dissolved in 100 ml of distilled water.
Solution B: 2.1 g of citric acid was dissolved in 100 ml of distilled water.
88.5 ml of Solution A and 11.5 ml of Solution B was mixed and the pH was
adjusted to 7.0.

1.13.2. Glucose-1-phosphate (0.02 M)


37 mg of glucose-1-phosphate was dissolved in 5 ml of distilled water.

1.13.3. AMP (0.0025 M)


8.68 mg of AMP was dissolved in 10 ml of distilled water.

1.13.4. Sodium fluoride (0.075 M)


30 mg of sodium fluoride was dissolved in 10 ml of distilled water.

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Preparation of reagents

1.13.5. Glycogen (1 %)
50 mg of glycogen was dissolved in 5 ml with distilled water.

1.13.6. Aminonapthosulphonic acid (0.25 %)


Solution A: 3 g of sodium bisulphate was dissolved in 20 ml of distilled water.
Solution B: 4 g of sodium sulphite was dissolved in 20 ml of distilled water.
19.5 ml of Solution A and 0.5 ml of Solution B was mixed and 50 mg of
ANSA was dissolved in it.

1.13.7. Ammonium molybdate (2.5 %)


1.25 g of ammonium molybdate was dissolved in 50 ml of 10 N H2SO4.

1.14. Bouins fluid


Picric acid (75 ml), formalin (25 ml) and glacial acetic acid (5 ml) was mixed
and used as a fixative.

1.15. Glucose-6-Phosphatase

1.15.1. Citrate buffer (0.1 M, pH 6.0)


Solution A: 2.94 g of sodium citrate was dissolved in 100 ml of distilled water.
Solution B: 2.1 g of citric acid was dissolved in 100 ml of distilled water.
88.5 ml of Solution A and 11.5 ml of Solution B was mixed and the pH was
adjusted to 7.0.

1.15.2. Glucose-6-phosphate (0.02 M)


5.61 mg of glucose-6-phosphate was dissolved in 10 ml of distilled water.

1.16. Lysis buffer for immunoblot analyses

1.16.1. Lysis buffer for immunoblot analysis


0.606 g of Tris was dissolved in 100 ml of distilled water. To this, 0.877 g of
NaCl, 10 ml of glycerol and 1 ml of NP-40 were added and pH was adjusted to
7.4. For every 10 ml of buffer mixture, 50 l of sodium fluoride (200 mM), 50
l of sodium orthovanadate (200 mM), and 100 l of protease inhibitor
cocktail [PMSF (1mM), EDTA (1mM), bestatin (150 M), leupeptin (1 M)
and aprotinin (1 M)] were added.

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Preparation of reagents

1.17. Immunoblotting

1.17.1. Transfer buffer


6.06 g of Tris, 28.8 g of glycine and 2 g of SDS were dissolved in 1 L of
distilled. To this, 400 ml of methanol was added and made upto 2 L with
distlled water.

1.17.2. Ponceau S
500 mg of Ponceaus S, 7.5 g of TCA and 7.5 g of sulfosalicylic acid were
dissolved in and made up to 25 ml with distilled water.

1.17.3. Phosphate-buffered saline-Tris buffer


0.121 g of Tris was dissolved in distilled water. To this 0.138 g of sodium
dihydrogen phosphate and 0.876 g of NaCl were added and pH adjusted to 7.4.

1.17.4. Blocking buffer


5 g of skimmed milk powder was dissolved in 100 ml of PBS-Tris buffer
containing 1 % Tween 20.

1.18. Immunofluorescent staining

1.18.1. Phosphate-buffered saline buffer


0.138 g of sodium dihydrogen phosphate was dissolved in 100 ml of distilled
water. To this 0.876 g of NaCl was added and pH adjusted to 7.4.

1.18.2. 1% non-immune goat serum


0.1 ml of 10% non-immune goat serum was made upto 10 ml with PBS buffer.

1.19. Mitochondrial membrane potential

1.19.1. 0.25 % trypsin


25 mg of trypsin was made upto 10 ml with DMEM.

1.19.2. 0.2 % trypan blue


20 mg trypan blue was made upto 10 ml with PBS.

1.19.3. Rhodamine 123


Stock solution: 1 mg of Rhodamine 123 was dissolved in 1 ml of distilled
water.
Working solution: 1 l of the stock solution was made upto 1000 l with
distilled water.

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Preparation of reagents

1.20. TUNEL assay

1.20.1. 4% paraformaldehyde solution in PBS


4 ml of paraformaldehyde was dissolved in 100 ml of PBS.

1.20.2. 0.3 % H2O2


0.3 ml of H2O2 was made up to 100 ml with distilled water.

161