The Investigation of the Antioxidative Properties of the Novel Synthetic Organoselenium Compounds in Some Rat Tissues

Structure of Novel Synthetic Organoselenium Compounds.

Novel synthetic organoselenium compounds (SeI and SeII) (Fig. 1A and B) were synthesized in our laboratories by methods found in the literatures (15, 16). The novel compounds synthesized were characterized by ¹H-NMR (300 MHz), ¹³C-NMR (75.5 MHz), and FT-IR spectroscopic techniques and microanalysis.

Setting of Groups.

In this study, 35 healthy female Albino Wistar rats (body weight 180–200 g and 3 and 4 months old) were divided into five groups, each consisting of six to eight animals. Each rat was weighed just before the start of the study. All drugs were administered intra-peritoneally (ip). DMBA was dissolved in corn oil, and rats were injected with 50 mg/kg body weight. SeI and SeII were dissolved in corn oil, and rats were injected with 25 μmol/ kg. Animals in Group I were used as a control group. Animals in Group II received only the vehicle solution, (i.e., corn oil at 2-day intervals over 4 weeks). Animals in Group III were given a single dose of 50 mg/kg DMBA and were sacrificed 4 weeks later. Rats in Group IV also received DMBA as in Group III, but after 6 hrs of DMBA application, SeI at 25 μmol/kg was applied at 2-day intervals over 4 weeks. Animals in Group V were also treated exactly as Group IV animals, except that SeII was used instead of SeI.

The rules outlined in the “Guide for the Care and Use of Laboratory Animals” were followed throughout the study, which was closely monitored by the ethical commission of Medicine School of Inonu University.

Preparation of Tissues for Biochemical Analysis.

All animals were successively sacrificed after being anaesthetized with 75 mg/kg of sodium pentobarbital. The vena cava was also cut, and 30 ml of 0.9% NaCl was injected into the heart to rinse blood from the body. The heart and brain were removed and frozen in liquid nitrogen. Tissues were stored at −80°C until used.

The tissues were separated into two parts for determination of enzymatic activity and lipid peroxidation. The samples for enzymatic activity analysis were homogenized in phosphate-buffered saline (PBS) (pH 7.4) using PCV Kinematica Status Homogenizer (Germany). Homogenized samples were then sonicated for 1.5 mins (30-sec sonications interrupted with a 30-sec pause on ice). Samples were then centrifuged at 17,000 g for 15 mins, and supernatants, if not used for enzyme assays immediately, were kept in the deep freeze at −80°C.

The second part of tissues homogenate was used for lipid peroxidation analysis. Tissue was washed three times with ice-cold 0.9% NaCl solution and homogenized in 1.15% KCl. The homogenates were assayed for malone-dialdehyde (MDA), the product of lipid peroxidation.

Protein Assay.

The protein content of the supernatants for enzyme assays and MDA assay was determined using the colorometric method of Lowry et al. and using bovine serum albumin (BSA) as the standard (17). All analyses were performed in duplicates.

Enzyme assays.

The samples were determined spectrophotometrically for CAT (μmol/mg protein), SOD (ng/mg protein), Se-GSH-Px (nmol/mg protein), and GR (μmol/mg protein) activities in the supernatant fraction.

CAT Activity.

CAT activity was measured at 37°C by following the rate of disappearance of H₂O₂ at 240 nm (ε₂₄₀ =40 M⁻¹ cm⁻¹) (18). One unit of CAT activity was defined as the amount of the degradation of 1 μmol of H₂O₂/min at 37°C by enzyme catalyzing and as the specific activity corresponds to μmol transformation of substrate (H₂O₂) min⁻¹ per mg protein.

Cu, Zn-SOD Activity.

SOD activity in the supernatant fraction was measured by using the xanthine oxidase/ cytochrome c method (19), in which one unit of activity was the amount of enzyme needed to cause half-maximal inhibition of cytochrome c reduction. The amount of SOD in the extract was determined as ng of enzyme per mg protein, by utilizing a commercial SOD preparative as the standard.

Se-GSH-Px Activity.

Se-GSH-Px activity was determined though a coupled assay with GR and by measuring the rate of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidation at 340 nm and by using hydrogen peroxide as the substrate (20). Specific activity is given as nmol NADPH disappearing min⁻¹ per mg protein.

GR Activity.

GR is a ubiquitous enzyme, which catalyzes the reduction of oxidized glutathione (GSSG) to GSH. This assay is based on the oxidation of NADPH to nicotinamide adenine dinucleotide phosphate (NADP), which is catalyzed by limiting a concentration of GR. One GR activity unit is defined as the amount of enzyme catalyzing the reduction of 1 μmole of GSSG per minute at pH 7.6 and 25°C. One molecule of NADPH is consumed for each molecule of GSSG reduced. Therefore, the reduction of GSSG was determined indirectly by the measurement of the consumption of NADPH, demonstrating decreases in absorbance at 340 nm (A340) as a function of time (21).

GSH Assay.

The formation of 5-thio-2-nitrobenzoate (TNB) is followed spectrophotometrically at 412 nm (22). The amount of GSH in the extract was determined as nmol/ mg protein by utilizing a commercial GSH (Sigma, St. Louis, MO) as the standard.

Lipid Peroxidation Assay.

The analysis of lipid peroxidation was carried out as described by Beuge and Aust with a minor modification (23). The reaction mixture was prepared by adding 1 ml homogenate into 4 ml reaction solution (15% trichloroacetic acid, 0.375% thiobarbituric acid, 0.25 N NaOH; 1:1:1, w/v) and heated at 100°C for 10 mins. The mixture was cooled to room temperature, centrifuged (10,000 g for 10 mins), and the absorbance of the supernatant was recorded at 532 nm. MDA results were expressed as nmol/mg protein in the supernatant.

Statistical Analysis.

The data were analyzed with SPSS 9.0 for Windows (SPSS Inc., Chicago, IL) by using one-way analyses of variance (ANOVA). Differences between means were determined using Duncan’s multiple range test in which the significance level was defined as P < 0.05.