Bioscience Methods 2024, Vol.15, No.4, 173-183 http://bioscipublisher.com/index.php/bm 178 in plastic containers of 150 × 80 × 50 mm, with twenty animals per container. The water used to maintain the control animals was free of Cr6+ contamination. The plastic containers were shielded with muslin cloth to allow for entree to oxygen but barred the animals from evasion during experiments. Control groups of periwinkles were also maintained under identical conditions without Cr6+ exposure. Following the exposure, representative animal samples were crushed, the soft tissues were removed, and the samples were frozen at −20 oC for later determination of activities of antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Likewise, the levels of reduced glutathione (GSH) and lipid peroxidation (LPO) were quantified. 5.3 Preparation of crude homogenate The soft bodies were homogenized by macerating in an ice-cold potassium phosphate buffer (0.05 M; pH 7.5) in a ceramic mortar. The homogenates were centrifuged at 10,000 ×g for 15 min at 4 °C. The obtained supernatants were kept at -20 °C for the determination of activities of antioxidant enzymes and the levels of reduced glutathione while the lipid peroxidation levels were determined using the whole homogenates. 5.4 Superoxide dismutase activity assay The Beauchamp and Fridovich (1971) method was used to measure the superoxide dismutase activity. This involved adding 0.25 mL of the supernatant to 1.5 mL of SOD reagent, which contained 1.17 mM ribofavin, 0.1 M methionine, 20 mM potassium thiocyanide, and 56 mM nitro blue tetrazolium. After that, the mixture was incubated at ambient temperature for one hour. A blank was also simultaneously prepared, which contained distilled water in the place of the supernatant. After the period of incubation, the absorbance was read at 560 nm, by means of a UV/Vis spectrophotometer (BK-UV 1900 model). The SOD activity was expressed as μmoles min-1mg protein-1. 5.5 Catalase activity assay Using Ellerby and Bredesen's (2000) approach, spectrophotometric quantification of catalase activity was conducted. The pace at which catalase in the supernatant breaks down hydrogen peroxide determines the course of the reaction. The amount of enzyme needed to break down one µmol of hydrogen peroxide in one minute at 25 ℃ is known as one unit of the enzyme. In brief, 0.3 mL of a 0.03 M hydrogen peroxide solution was introduced to 0.6 mL of potassium phosphate buffer (0.01 M; pH 7.5) in a cuvette. After that, 20 µL of the supernatant was added, and after a minute, the change in absorbance at 240 nm wavelength was measured. Catalase activity was expressed as µmol min-1 mg protein-1 using a molar extinction coefficient of 39.6 M-1 cm-1. 5.6 Glutathione peroxidase (GPx) activity Glutathione peroxidase activity was determined by the procedure of Paglia and Valentine (1967). First, 100 µL of the supernatant was added to 10 µL of 200 mM GSH in test tubes. Thereafter, 100 µL of H2O2 (2 mM) was then added to the mixture to initiate the reaction. The reaction was observed at 340 nm for 3 min using the UV-Visible spectrophotometer, and the activity of GPx was expressed as µmol NADPH min-1 mg-1 protein. 5.7 Reduced glutathione concentration The concentration of reduced glutathione in the supernatant was measured by adopting the method of Ellman (1959), with minor changes. Ellman’s reagent also known as 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was used as the substrate. The procedure involved mixing 10 µL of the supernatant with an equivalent volume of 10 mM DTNB in 0.1 M potassium phosphate (pH 7.5) that contained 17.5 mmol/L of EDTA. The samples were centrifuged at 2000 ×g for 10 minutes, and the resulting supernatants were added to cuvettes containing 10 µL of 0.5 U ml-1 glutathione reductase (GR) in 0.1 M potassium phosphate (pH 7.5). The cuvettes were then incubated at room temperature for one minute. The reaction assay was started by adding 220 nM of reduced nicotinamide adenine dinucleotide phosphate in 0.1 M potassium phosphate (pH 7.5) containing 5 mM EDTA in a concluding volume of 1 ml. The rate of reduction of DTNB was observed and recorded spectrophotometrically at a wavelength of 412 nm using a UV/Vis spectrophotometer. Using a standard curve generated from known concentrations of GSH in potassium phosphate buffer solution of pH 7.5, the total GSH concentration was determined.
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