Experimental organism
The experimental animal used in this research was the African catfish, Clarias gariepinus. A total of 30 juvenile C. gariepinus (mean weight: 2.8 ± 0.13 g) were used in the study. The fish were purchased from a commercial farm within Akure metropolis, and were transported in open head plastic containers to the Research Laboratory, Department of Biology, Federal University of Technology, Akure. The fish were allowed to acclimatize to laboratory conditions in chlorine-free tap water for one week prior to commencement of experiments. The fish were fed with commercial fish pellets twice daily during the period of the acclimatization and experiment.
Pre-exposure of fish to selenium
The laboratory acclimatized fish were allocated into six tanks (n = 5 per tank); three of which were maintained in clean water for 14 days while the remaining tanks were exposed to 50 µg/L selenium (prepared from the stock solution of selenium) for 14 days. After the 14 days of selenium pre-exposure, one tank each from the unexposed (control) and selenium-exposed (Sel) were collected and the fish in those tanks were sacrificed, the liver excised, homogenized in ice-cold phosphate buffer (50 mM, pH 7.4), and the homogenates was used for the determination of oxidative stress parameters; levels of reduced glutathione, lipid peroxidation, and activities of catalase, superoxide dismutase, and glutathione peroxidase. The blood was collected into EDTA bottle, centrifuged at 10,000 ×g for 20 min at 4 °C to obtain the plasma and the plasma levels of nitric oxide and respiratory burst activity were determined.
Experimental challenge of fish with S. marcescens
Fish in the remaining tanks were challenged with 5 × 103 CFU/mL inoculums of S. marcescens via oral gavage for either 24 h or 48 h. The tanks were designated as: unexposed fish for 14 days but challenged with bacteria for 24 h or 48 h (Bact) and pre-exposed to selenium for 14 days and challenged with bacteria for 24 or 48 h (Sel + Bact). The fish in each group were collected at 24 and 48 h post-inoculation periods, sacrificed, the liver excised for the determination of oxidative stress parameters while the blood was collected for the determination blood plasma levels of nitric oxide and respiratory burst activity.
Plasma level of nitric oxide
Nitric oxide levels of the blood plasma were determined according to the method described by Guevara-Guzman et al. (1994), with a slight modification. Aliquot of 100 μL of the plasma was mixed with 100 μL Griess reagent and incubated for 10 min at room temperature. The absorbance was read by a spectrophotometer at 540 nm. The nitrite concentration was measured with the nitrite standard curve and expressed as µmol/mL.
Respiratory burst assay
The level of respiratory burst was determined by the method of Anderson and Siwicki (1995). A 100 µL of blood plasma was mixed with 100 µL of 0.2 % nitroblue tetrazolium (NBT) solution which was homogenized and incubated for 30 min at 25 °C. The NBT solution was prepared in phosphate buffered saline (PBS), prepared with NaCl (0.137 M), KCl (2.7 mM), KH2PO4 (1.5 mM), Na2HPO4 (8.1 mM), CaCl2 (0.9 mM), MgCl2 (0.49 mM) in distilled water Milli-Q qsp 1 litre), pH 7.4. 50 µl from the mixture was added to 1 ml of N, N-dimethyl formamide in a glass tube. The solution was properly homogenized and afterwards centrifuged at 3000 × g for 5 min. The optical density of the supernatant was measured at 540 nm on a UV/visible spectrophotometer against a blank with same components except the blood was exchanged with distilled water.
Liver lipid peroxidation levels
Lipid peroxidation was determined by the thiorbarbituric acid reactive substances assay which quantifies the levels of malondialdehyde, the final product of lipid peroxidation. This method was based on the colorimetric determination of malondialdehyde (MDA) according to the method of Esterbauer and Cheeseman (1990). Homogenized suspension of liver was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid (TCA) to precipitate the protein. The precipitate was pelleted by centrifugation, and an aliquot of the supernatant was added to an equal volume of 0.67% (w/v) thiobarbituric acid and the mixture placed in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm. The concentration of MDA was expressed as nmol MDA mg protein-1 using a molar extinction coefficient of 153,000 M-1cm-1.
Liver reduced glutathione levels (GSH)
The levels of reduced glutathione in the liver homogenate was estimated by the method of Ellman (1959). Briefly, 0.5 mL of tissue homogenate was precipitated with 2.0 mL of 5% TCA and centrifuged. To 1.0 mL of the supernatant, 3.0 mL phosphate buffer and 0.5 mL Ellman’s reagent were added and incubated for 15 min at room temperature. The intensity of yellow color formed was measured at 420 nm. A series of standards (10– 50 μg GSH) were treated in a similar manner along with the blank containing 1.0 mL distilled water. The amount of GSH was expressed as nmol/mg protein.
Liver glutathione peroxidase activity
Glutathione peroxidase activity was determined according to the method of Hafeman et al. (1974). The tissue homogenate (approximately 0.5 mg protein) was incubated with 0.1 mL of 5 mM GSH, 0.1 mL of 1. 25 mM H2O2, 0.1 mL of 25 mM NaN3 and phosphate buffer (0.05 mM, pH 7) in a total volume of 2.5 mL at 37 °C for 10 min. The reaction was stopped by adding 2 mL of 1.65 % HPO32- and the reaction mixture was centrifuged at 1500 rpm for 10 min. 2 mL of the supernatant was mixed with 2 mL 0.4 M Na2HPO4 and 1 mL of 1 mM DTNB. The absorbance of the yellow colored complex was measured at 412 nm after incubation for 10 min at 37 °C against distilled water. A sample without the tissue homogenate processed in the same way was kept as non-enzymatic reaction.
Liver superoxide dismutase activity
Superoxide dismutase (SOD) activity was determined according to the method of Beauchamp and Fridovich (1971). The reaction mixture comprised 0.01 mL of the tissue homogenate, 0.2 mL of 0.1 M EDTA (containing 0.0015% NaCN), 0.1 ml of 1.5 mM NBT and phosphate buffer (67 mM, pH 7.8) in a total volume of 2.6 mL. Later, 0.05 mL of riboflavin was added to the mixture, and incubated in the dark for 1 h after which the absorbance was measured at 560 nm using a UV/visible spectrophotometer. The tissue homogenate was replaced with distilled water to get the reading for the blank. SOD activity was expressed as µmoles/min/mg protein.
Liver catalase (CAT) activity
The catalase activity was determined according to the method described by Aebi (1984). Aliquot of 25 μL of tissue homogenate was added to 1.0 mL of phosphate buffer, and the enzyme reaction was started by the addition of 250 μL of H2O2 solution. The decrease in absorbance was measured at 240 nm at 30 seconds intervals for 3 min. The enzyme blank was run simultaneously with 250 μL of distilled water instead of hydrogen peroxide. The enzyme activity is expressed as nmoles of H2O2 decomposed/min/mg protein.
Protein determination
The protein levels in the tissue homogenate was determined according to the method described by Bradford (1976), using bovine serum albumen as the standard.
Statistical analyses
Data obtained were expressed as mean ± standard deviation. The data on GSH, MDA, CAT activity, SOD activity, GPx activity, nitric oxide, and respiratory burst were subjected to two-way analysis of variance with selenium pre-exposure (pre-exposed to selenium for 14 days versus not pre-exposed to selenium) and post-infection time (24 h versus 48 h) as factors. The means were separated using Tukey’s multiple comparison tests. Statistical analyses were performed using GraphPad Prism software (version 5), and statistical significance was assumed at p < 0.05.