The experiment was conducted during August 2011 to December 2014. Methodology involved with this experiment discussed below.
Fish: C. punctatus weighing 50 g to 60 g were used and maintained in normal water with ambient temperature (25.0 ± 1oC). They are very strong and survive in the environment for prolonged period. In the day of experiment, exposure of mercuric chloride and calcium carbonate were given to the different groups of fish in small plastic pots for 1h period with full aeration and with free access of water. After the treatment, fish were quickly decapitated and liver was sampled carefully and taken weight by digital balance (Chyo, JL - 180, China) and kept at – 20oC. Control fish were similarly used for sampling of tissue except giving mercuric chloride or calcium carbonate exposure.
Mercury treatment: To examine the role of heavy toxic element on the regulation of metabolic activity involving the amount of protein, triglyceride and cholesterol in liver, groups of fish were used with different concentrations of mercuric chloride (1 and 10 μM) (HgCl2, BDH Chemical Ltd.) in water (600 mL) for 1h. After the treatment, fish were quickly decapitated and liver was sampled carefully and taken weight by digital balance. Cholesterol, triglyceride and protein contents in extracts of liver of fish treated with HgCl2 were determined.
Calcium carbonate treatment: Calcium carbonate (100 μM and 1 mM) was prepared with water, dissolved with concentrated HCl and was made pH 7.0 with diluted NaOH. The following groups of fish treated with CaCO3 (100 μM and 1 mM) (600 mL) for 1h were used to examine the role of calcium carbonate on metabolic regulation in liver induced by HgCl2: a) Control b) HgCl2 (1 μM) c) HgCl2 (10 μM) d) CaCO3 (100 μM) e) CaCO3 (1 mM) f) HgCl2 (1 μM) + CaCO3 (100 μM) g) HgCl2 (10 μM) + CaCO3 (1 mM). The groups of fish were treated with HgCl2 and CaCO3 in ambient temperature for determination of different parameters. The liver was sampled after the treatment similarly as mentioned above and cholesterol, triglyceride and protein contents in each liver extract we re determined.
Assay of tissue cholesterol, triglyceride and protein content: Cholesterol content in liver was determined by using the method of Liebermann-Barchard reaction (Kenny, 1952). For assay of cholesterol, 0.5 mL of crud e extract was taken to test tubes and 10 mL of ethanol-ether mixture (3:1) were added. The test tubes were shaken vigorously and the contents were taken to centrifuge tubes and were centrifuged for 15 min at 8000 rpm. The supernatants were transferred to new glass tubes and evaporated to dryness in a water bath. After evaporation, 5 mL of chloroform were added to dissolve the residue and 2 mL of acetic anhydride - H2SO4 mixture (20 mL of acetic anhydride and 1 mL of concentrated H2SO4) were given, mixed an d allowed to stand in dark at 25oC for 20 min to develop the color. The spectrophotometer reading was taken at 680 nm against the blank. Cholesterol content was measured with the help of standard solution of cholesterol (20 mg/100 mL in chloroform) where 2.5 mL of standard solution was taken in test tubes and 2.5 mL of chloroform were mixed and followed the same procedure. For blank, only 5 mL of chloroform and 2 mL of acetic anhydride - H2SO4 mixture were used. The amount of cholesterol was expressed as mg/100 g of tissue weight. Triglyceride content in liver of different groups of fish was measured quantitatively by LABKIT (Triglycerides kits), Crest Biosystems, Bambolim Complex Post Office, Goa - 403 202, INDIA. For assay of triglyceride, 100 µL of crude liver sample were used. Tissues were homogenized with pre-cooled water and were centrifuged at 8000 rpm for 10 min. The supernatants from each tissue homogenate were used as crude extract for assay of protein by using 50 µL extract. The protein content in tissue was determined by the procedure of Lowry et al. (1951). Briefly, alkaline solution was prepared by mixing 50 mL of alkaline Na2CO3 solution (2% Na2CO3 in 0.1N NaOH) and 1.0 mL of copper-sodium potassium tartarate solution (1 g sodium potassium tartarate and 0.5 g CuSO 4 . 5H2O were dissolved in 100 mL distilled water). Fifty micro liters of tissue extract was taken to the test tube and made up to 1 mL with distilled water. For blank, 1 ml water was used in place of tissue extract. Five milliliters of alkaline solution was added to each tube and mixed well. The tubes were allowed to stand for 10 min at room temperature and 0.5 mL of diluted FCR (Commercial FCR was diluted with equal volume of water) was added and mixed well. After 30 min, the absorbance was taken at 650 nm against the blank. The protein content in each tissue was calculated from the standard graph of bovine albumin (1 mg mL-1) and is expressed as mg/100 g of tissue weight.
Statistical analysis: Results of the experiments were expressed as mean and standard error of different groups. The differences between the mean values were evaluated by ANOVA followed by paired t - test using SPSS software.