Materials and Methods free essay sample

Technical grade Diazinon (DZN); O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate (98% purity) was donated from El-Helb, Pesticides and Chemicals, New Damietta, Egypt. It was diluted in corn oil for preparing the required concentrations. Dosing concentrations were freshly prepared during the administration period. Moringa seed oil (MSO) was purchased from Earths Moringa P.O. Box 39503, Los Angeles, CA 90039. Reduced glutathione (GSH), 1-chloro-2, 4-dinitrobenzene, nicotinamide adenine dinucleotide phosphate (NADPH), thiobarbituric acid (TBA), trichloroacetic acid (TCA), H2O2 (33%), ethylenediaminetetraacetic acid (EDTA), reduced glutathione (GSH), 5,5 dithiobis-(2-nitrobenzoic acid (DTNB), potassium fihydrogenphosphate (KH2PO4), butanol and sodium chloride (NaCl) of technical grade used in this study were purchased from Sigma Chemical Company (Saint Louis, USA). Other chemicals were supplied from Merck Led. SRL Pvt., Led., Mumbai, India.2.2. AnimalsTwenty male Albino rats Sprague–Dawley, weighing 180–200 g, were supplied from the Animal Breeding House of the Medical Research Institute, Alexandria University, Alexandria, Egypt. Animals were maintained at the animal care facility in the Faculty of Medicine, in plastic cages under controlled temperature (23  ± 2 oC), 12-h light/dark cycle and 50  ± 5% relative humidity. We will write a custom essay sample on Materials and Methods or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Water and food were available ad libitum. Rats were acclimatized to the laboratory environment for two weeks prior to the start of the experiments. Animal Care ; Experimental Committee, Alexandria University, Alexandria, Egypt, and all animal procedures were carried out in accordance with the Ethics Committee of the National Research Centre conformed to the Guide for the Care and Use of Laboratory [15]. During the experiments, maximum care was taken to minimize animal suffering and in addition, the number of rats used was kept at minimum.2. 3. Experimental designAfter two weeks of acclimatization, animals were divided into four (n = 5 rats per group) equal groups.†¢ Group 1: Control group; rats were given 1 mL/kg body weight (b,w)/day by gavage for 28 days;†¢ Group 2: DZN group; rats were given DZN 12.50 mg/kg b.w/day (1/100 LD50) by gavage for 28 days. The LD50 and the regime schedule were selected according to the previous study [16,17];†¢ Group 3: MSO group; MSO was given, 200 mg/kg b.w/day, by gavage for 28 days according to the previous study [18];†¢ Group 4: MSO + DZN group; rats were given first with (MSO (200 mg/kg b. w/day) by gavage and after 30 min were given DZN (12.50 mg/kg b.w/day (1/100 LD50) by gavage for 28 days.2.4. Sample collection and preparationThe animals were starved overnight for 12h before blood was collected. Rats were anaesthetized with rats were weighed and anesthetized with sodium pentobarbital (40 mg/kg i. p.), and venous blood samples were collected by direct heart puncture into sterilized vials. Blood samples were allowed set to clot at 4 oC and centrifuged at 2500 g for 10 min. Then 1000 ?l aliquots of serum were placed in microfuge tubes and frozen on dry ice. Labeled bags were placed into freezer at -20 oC until the time of the assay.Livers was removed from rats under anesthesia, after 28 days of treatment and washed with cold saline buffer to remove any clotted blood or tissue debris. Washed livers were immediately stored at – 80 oC. To obtain the enzymatic extract, tissues were homogenized in ice-cold 50 mM sodium phosphate buffer (pH 7.0) contains 0.1 mM ethylendiaminetetra-acetic acid (EDTA) to yield 10% (W/V) homogenate. The tissue homogenates were then centrifuged 1500 Xg for 20 minutes at 4  ºC. The supernatants were kept at – 80  ºC till the time of determination of oxidative/antioxidant parameters.2.5. Serum biomarkersAll serum parameters were determined using a commercial kit in accordance with manufacturers instructions using a spectrophotometer (Shimadzu UV-VIS Recording 2401 PC, Japan). Serum samples were analyzed for total protein by Lowry et al. [19]. Albumin concentration was determined by Doumas et al. [20]. Serum alanine aminotransferase (ALT; EC 2.6. 1.2) and aspartate aminotransferase (AST; EC 2.6.1.1) were determined using commercial kits obtained from Biodiagnostic kit (Cairo, Egypt). The principle reaction of the colorimetric determination of AST or ALT activity is based on the reaction of aspartate or alanine with ?-ketoglutarate to form oxaloacetate or pyruvate hydrazone formed with 2, 4-dinitrophenylhydrazine [21]. Serum alkaline phosphatase (ALP; EC 3. 1.3.1) activity was measured at 405 nm by the formation of para-nitrophenol from para-nitrophenylphosphate as a substrate [22] using commercial kits obtained from Biodiagnostic kit (Cairo, Egypt). Serum lactate dehydrogenase (LDH; EC 1.1.1.27) was determined according to the method of Friedman and Young [23], using kit obtained from Spinreact (Santa Coloma, Spain). Cholesterol and triglycerides was measured according to the method Carr et al. [24] using Biodiagnostic kit (Cairo, Egypt).2.6. Lipid peroxidation assayThe extent of LPO was estimated as the concentration of thiobarbituric acid reactive product malondialdehyde (MDA) by using the method of Ohkawa et al. [25]. MDA concentrations were determined using 1,1,3,3-tetraethoxypropane as standard and expressed as nmol/g liver tissue. 2.7. Antioxidant enzymesCatalase (CAT, EC. 1.11.1.6) activity was measured according to the method described by Aebi by assaying the hydrolysis of H2O2 and the resulting decrease in absorbance at 240 nm over a 3 min period at 25 oC [26]. The activity of CAT enzyme is expressed as U/gm tissue.Glutathione peroxidase (GPx; EC 1.11.1.9) activity was measured using H2O2 as substrate according to the method described by Paglia and Valentine [27]. The reaction was monitored indirectly as the oxidation rate of NADPH at 240 nm for 3 min. Enzyme activity was expressed as U/gm tissue. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined according to the method described by Marklund and Marklund by assaying the autooxidation and illumination of pyrogallol at 440 nm for 3 min [28].2.8. Reduced glutathione assayReduced GSH estimation was performed by Beutler et al. [29]. Livers were homogenized in 1 ml of 1.1% KCl cooled, then homogenate (100  µl) was mixed with 750  µL of precipitate solution (1.67 g glacial meta-phosphoric acid, 0.2 g EDTA and 30 g of NaCl in 100 ml D.W.) and 900  µl of D. W. Homogenate tissue was centrifuged at 2000g for 15 min to precipitate proteins. Protein-free supernatant (250  µl) was added to 1ml of Na2HPO4 (0.0 M) and the reaction was initiated by adding 125  µl of DTNB (6 mM) and the absorbance of 5-thio-nitrobenzoic acid (TNB) formed was measured at 412 nm. The level of GSH was obtained by standard curve and expressed as U/g tissue.2.9. Statistical analysisAll data were expressed as mean  ± standard deviation (SD) and then subjected to one-way analysis of variance followed by Tukeys multiple comparison tests. Values of p