Ipid peroxidation in hippocampal homogenatesThe effects of Cd and/or DMS on lipid peroxidation levels in

Ipid peroxidation in hippocampal homogenatesThe effects of Cd and/or DMS on lipid peroxidation levels in control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) were assessed by measuring malondialdehyde (MDA) formation using the Bioxytech MDA586 kit (Oxis Research, Portland, OR, USA). Briefly, bilateral hippocampi were collected using a surgical blade and the left part was homogenized in 20 mM PBS (pH 7.4) containing 5 mM butylated hydroxytoluene. After centrifugation of the homogenates at 3000 ?g for 10 min at 4 , the supernatants were collected. For each reaction, 10 L of probucol and 640 L of diluted R1 reagent (1:3 of methanol:N-methyl-2-phenylindole) were added and mixed with 150 L of 12 N HCl. Each reaction was incubated at 45 for 60 min and buy Ro4402257 centrifuged at 10,000 ?g for 10 min. The supernatant was collected and MDA formation was determined by measuring the absorbance at 586 nm. MDA data were normalized to the protein concentration of each sample.Protein carbonyl levelsriboflavin in 0.36 mM potassium phosphate buffer (pH 7.8). The gel was exposed to a fluorescent light source until the bands showed maximum resolution. CAT activity was assayed at 25 by determining the rate of H2O2 degradation in 10 mM of potassium phosphate buffer (pH 7.0) according to Aebi’s method [21]. An extinction coefficient of 43.6 mM/cm was used for the calculations. One unit is defined as 1 pmol of H2O2 consumed per min and the specific activity is reported as units per mg of protein. GPx activity was assayed by measuring nicotinamide adenine dinucleotide phosphate (NADPH) oxidation with t-butyl-hydroperoxide as a substrate according to the method of Maral et al. [22]. Briefly, the reaction was carried out at 25 in 600 L of a solution containing 100 mM potassium phosphate buffer (pH 7.7), 1 mM EDTA, 0.4 mM sodium azide, 2 mM glutathione, 0.1 mM NADPH, 0.62 U of glutathione reductase, and 50 L of homogenate.Measurement of glutathione-related enzymes in hippocampal homogenatesTo elucidate the effects of Cd and/or DMS on protein modification, carbonylation of proteins as a result of oxidative stress was determined in control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) by the Levine method [18]. The color intensity of the supernatant was measured at 370 nm using a spectrophotometer against 2 M HCl. Carbonyl content was calculated using the molar extinction coefficient (21 ?103 L/mol cm), and results were expressed as nmol/mg of protein.Measurement of antioxidant activity in hippocampal homogenatesThe effects of Cd and/or DMS on glutathione-related enzymes in the hippocampus of control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) were investigated using the tissue samples obtained for the measurement of protein carbonyl levels. Glutathione-S-transferase (GST) activity was determined spectrophotometrically using 1-chloro-2,4-dinitrobenzene as a substrate [23]. Glutathione reductase (GR), which has been shown to utilize NADPH to convert oxidized glutathione (GSSG) to the reduced form (GSH), was assayed by the method of Horn and Burns [24].Statistical analysisTo elucidate the effects of Cd and/or DMS on PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 Cu, Znsuperoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase (GPx), the activity of these enzymes was measured in control, DMS-, Cd-, and CdDMS-treated rats (n = 7 in each group). Briefly, the right part of the hippocampus (matching the right part used for measurement of lipid peroxidation) was homogenized.