Group I represented the control and consisted of fish intraperitoneally (IP) injected with 0.7% NaCl. Group II was the experimental group, and the fish were IP injected with a dose of 2 mg/kg QDs (prepared in 0.7% MAPK Inhibitor Library research buy NaCl) per body weight. No food was supplied to the fish during the experimental period, and no obvious changes in fish body weight were recorded. After 1, 3, and 7 days from QDs injection, six fish from each group were sacrificed by trans-spinal dissection and the liver was quickly removed. Organs were immediately frozen

in liquid nitrogen and stored at -80°C until biochemical analyses were performed. Preparation of tissue homogenates and total protein measurements Liver was homogenized (1:10 w/v) using a Mixer Mill MM 301 homogenizer (Retsch, Haan, Germany) in ice-cold buffer (0.1 M Tris-HCl, 5 mM ethylenediaminetetraacetic see more acid (EDTA), pH 7.4), containing a few crystals of phenylmethylsulfonyl fluoride as protease inhibitor. The resulting homogenate was centrifuged at 8,000×g for 30 min, at 4°C. The supernatant was decanted, aliquoted, and stored at -80°C until needed. Protein concentration was determined using Lowry’s method with bovine serum albumin as standard [40] and was expressed as mg/mL. Oxidative stress markers Lipid peroxidation Lipid peroxidation was determined by measuring MDA content according to the fluorimetric method of Del Rio [41]. Briefly, 700 μL of 0.1 M HCl and

200 μL of a sample with a total protein concentration of 4 mg/mL were incubated for 20 min at room temperature. Then, 900 μL of 0.025 M thiobarbituric acid was added, and the mixture Etomidate was incubated for 65 min at 37°C. Finally, 400 μL of Tris-EDTA protein extraction buffer was added. The fluorescence of MDA was recorded using a Jasco

FP750 spectrofluorometer (Tokyo, Japan) with a 520/549 (excitation/emission) filter. MDA content was calculated based on a 1,1,3,3-tetramethoxy propane standard curve with concentrations up to 10 μM. The results were expressed as nanomoles of MDA per milligram of protein. Protein sulfhydryl groups assay The protein thiols were assayed using 4,4′-dithiodipyridine (DTDP) according to the method of Riener [42]. A volume of 100 μL of total protein extract was mixed with 100 μL of 20% trichloracetic acid (TCA) and thoroughly homogenized. After 10 min on ice, the samples were centrifuged at 10,000×g for 10 min. The pellet was rendered soluble in 20 μL 1 M NaOH and mixed with 730 μL 0.4 M Tris-HCl buffer (pH 9). Then, 20 μL of 4 mM DTDP were supplemented, and after 5-min incubation at room temperature (in the dark), the absorbance at 324 nm was measured. The concentration of PSH was quantified using a N-acetylcysteine standard curve with concentrations up to 80 μM. The values were expressed as nanomoles per milligram of protein. Carbonyl derivates of proteins CP were quantified using the reaction with 2,4-dinitrophenylhydrazine (DNPH) according to the method described by Levine [43].