Hychem Experiments

Materials and methods:
1. Materials:
Urethane was purchased form Hychem Laboratories. AST, ALT, and ALP was purchased from ----------. All the others reagents use are of high purity and of analytical grade.
2. Animals:
BALB/C mice, 15-20 g were housed in colony rooms with 12/12 hr light/dark cycle at 21±2°C and had free access to food and water. All animal experiments were carried out in accordance with CPCSEA guidelines, Ethical committee Acts.
3. Experimental protocol:
Animals were acclimatized for a period of 1 week prior to the experiment. Later they were divided in to five groups containing six animals each. For induction of lung carninogen BALB/c mice were given Urethane in all the group except normal control, twice a week for a period of 4 weeks by intraperitonial injection[1].
Group I serves as normal control receive normal saline.
Group II serves as Disease control receive Urethane at a dose

MDA level was determined by thiobarbituric acid reactive substances (TBARS) in serum, based on the reaction between MDA and TBARS. Standard Malondialdehyde solution in 5 mL of volume was processed along with test samples. 1.5 mL of 0.8% of TBA was added to 1 mL of the serum sample. Then 0.4 mL of 8.1% sodium dodecyl sulphate and 1.5 mL of acetic acid was added. The mixture was finally made upto 5 mL with distilled water and placed in hot water bath at 95ºC for 1 h. After cooling, 1 mL of distilled water and 5 mL of the mixture of n-butanol and pyridine (15:1, v/v) was added. The mixture was vortexed and after centrifugation at 4000 rpm for 10 minutes, the absorbance of the organic layer (upper layer) was measured in UV-Vis spectrophotometer (Shimatzu) at 532 nm against blank using distilled water. TBA when allowed to react with MDA aerobically formed a colored complex [MDA-(TBA) 2 complex] which was measured with spectrophotometer. MDA concentration (measured as TBARS) was calculated as