Effect of climate change on hematotoxicity/hepatoxicity oxidative stress, Oncorhynchus mykiss, under controlled conditions

sizing of blood cells by electrical impedance method [21]. Changes in electrical resistance pro- duced by a particle (WBC and RBC) passing through the aperture of a sensor are measured. Since blood cells are non-conductive, the sample blood is diluted in a conductive liquid (lysis solution). A counting pool and sensing circuit counts the WBC and another counting pool and sensing circuit counts the RBC. The instrument’s microprocessor calculates and analyzes the cells (WBC and RBC) and then provides histograms. For HGB measurement, lysis is added to the blood, rapidly breaking down the red blood cell and releasing hemoglobin. Hemoglobin and lysis form a new mixture that can absorb a wavelength of 540 nm. Based on the equations, the device determines other hematological parameters (HCT, MCH and MCHC). b. Determination of antioxidant enzyme activity. In order to evaluate oxidative parame- ters, the fish were stunned by neck crushing. Blood samples were taken and placed in a test tube, and after 2 mL of physiological (0.9% NaCl) was added, they were centrifuged for 15 minutes in a refrigerated centrifuge at 3000 rpm at +4˚C. After the formed plasma was discarded, an equal amount of saline (0.9% NaCl) was added to the remaining volume, and the erythrocytes were washed. Physiological saline added tubes were centrifuged at 2000 rpm for 8 minutes at +4 ˚C (this step was repeated three times). Then, after blasting the erythrocytes with cold distilled water, the obtained erythrocyte package (-20˚C) was kept in deep freeze [22]. The obtained supernatants were used to deter-

mine antioxidant analyses (SOD, CAT, GPx, GSH, ROS, TOS, TAS, OSI, MPO, MDA). Tissue homogenate preparation. Liver and muscle samples of fish were homogenized by adding 3 times their weight of buffer solution (KH2PO4). The tissues were thoroughly crushed with a glass baguette and homogenized for 3 minutes in an Ultrasonic Processor homogenizer. The extract was centrifuged at 9500 rpm for 30 minutes at +4 ˚C [23]. Clear supernatants obtained from liver and muscle tissue were used for enzyme assays (SOD, CAT, GPx, GSH, ROS, TOS, TAS, OSI, MPO, and MDA) determined in the targeted parameter supernatants. The protein concentration of each sample was determined spectrophotometrically at 595 nm according to Bradford method [24]. Measurement of superoxide dismutase (SOD) enzyme activity. SOD activity was determined spectrophotometer (560 nm) according to the method of NBT (nitro blue tetrazolium chlo- ride) reduction by O-2 under light [25]. Measurement of catalase (CAT) activity. Catalase converts hydrogen peroxide into water and molecular oxygen with its catalytic activity. For the determination of catalase, the decreas- ing absorbance of hydrogen peroxide, which gives maximum absorbance at a wavelength of 240 mn, was measured [26]. Measurement of glutathione peroxidase (GPx) activity. GPx activity was calculated by a change in absorbance at 340 nm (decrease in values read over 3 min) with reference to Beutler [27]. GSH amount was determined via a carefully optimized enzymatic cyclic method using

glutathione reductase. The sulfhydryl group of GSH reacts with DTNB (5,5’-dithio-bis2-(nitro- benzoic acid), Ellman’s reagent) to produce yellow 5-thio-2-nitrobenzoic acid (TNB). The for- mation of 5-thio-2-nitrobenzoic acid, which was proportional to the GSH concentration, was observed at 412 nm and 25 ˚C (against reactive controls). Fish tissues’ ROS levels were obtained by modifying the Gupta et al. [28] method. The 2’,7’- dichlorofluorescein diacetate kit (DCFDA; ABCAM, DCFDA Cellular ROS Detection Assay Kit, ab113851) was used for this purpose. The sample tissues were homogenized with Tris-HCl buffer (50 mM, pH = 7.4) at a 1:10 w/v rate inside an ice bath. Homogenate samples (100 μL) of fish tissues were mixed with 1 mL of the same buffer and 5 μL of DCFDA (10 μM). The resulting mixtures were incubated for 30 min at 37˚C (Lab. Companion SI-600 incubator shaker, Jelio Tech., Korea). After the incubation, the absorbance changes were measured at Ex 485 nm/Em 525 nm with a fluorescent spectrophotometer (LS55, PerkinElmer, USA). PLOS ONE Effect of temperature on fish physiology and biochemistry PLOS ONE | https://doi.org/10.1371/journal.pone.0294656 November 30, 2023 4 / 13