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Evaluation of the Protective Effect of Moringa (Moringa oleifera L) Seed Oil against Diazinon Induced Hepatotoxicity in Male RatsAqeel Khaleel Ibraheem1, 2; Tamara Shaker Mahmoud1; Ahmed M. Attia1, 3 1Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University2 Present address: Biology, Environmental Studies, Ministry of Education, Education Babylon, Iraq3Corresponding author:Ahmed M. AttiaProfessor of Environmental ToxicologyDepartment of Environmental StudiesInstitute of Graduate Studies ResearchAlexandria University163, Horrya Av. El-Shatbey, P.O. Box 832 # 21526Alexandria – EgyptEmail: [emailprotected]: +203 42 95 007; Fax: +203 42 85 792Abstract:Diazinon (DZN), is a commonly used organophosphorous (OP) pesticide to control a variety of insects in agriculture and in the environment. Moringa oleifera seed oil (MSO) is a pharmacologically active with documented antioxidant activity. The present study aims to investigate the hepatoprotective effect of DZN-induced liver damage. Twenty male Albino rats Sprague–Dawley were randomized into 4 groups. Group I: served as normal control given vehicle, 2) Group II: DZN group-rats received orally DZN at a dose 12. 50 mg/kg body weight (b.w)/day (1/100 LD50), Group III: MSO group-rats received orally 200 mg/kg b.w/day MSO, Group IV: MSO + DZN group DZN plus MSO was given. The administration of DZN increased (P 0.05) ALT, AST, ALP, lactic dehydrogenase, triglycerides and total cholesterol in serum, compared to control group, while protein and albumin were decreased. High levels of liver LPO and low antioxidant defenses, catalase (CAT), Superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities as well as glutathione (GSH) concentration in DZN-treated group. In rats supplemented with MSO as well as treated with DZN, hepatic specific marker enzymes were restored. Conclusions: Moringa oleifera seed oil may have hepatoprotective effect which is probably due to its antioxidant property. Keywords: Diazinon; Oxidative Stress; Antioxidants; Liver; Moringa Seed Oil 1. Introduction:Pesticides are occasionally used indiscriminately in large amounts causing environmental pollution and therefore, are a cause of concern. Organophosphorus insecticides (OPIs) are widespread use in agricultural and public health. However, most of applied agricultural pesticides never reach target organisms, but disperse through the environment [1,2]. Diazinon (O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate) (DZN), is a commonly used organophosphorus insecticide. It has been used since 1956 for the control of soil insects and pests, on ornamental plants, and on fruits, vegetables and field crops. Now it is used to control flies around animal facilities, greenhouses and other businesses and public places where food or animal wastes might be accumulated [3]. DZN can be highly toxic for animals and humankind [4,5]. The main mechanism of action of DZN is acetyl-cholinesterase enzyme inhibition [6]. However, DZN may induce imbalance in the free radical production/elimination processes with consequent induction of cellular damage [7-9].The importance of the naturally currently widespread botanicals that can be consumed in an individuals everyday diet because of their antioxidant and anti-inflammatory properties [10]. Nature has been a source of medicinal treatments and up to 40% of modern drugs may directly or indirectly be related to natural compounds [11].Moringa oleifera is called Miracle Vegetable because it is both a medical and a functional food [12]. All parts of the Moringa tree (leaves, seeds, roots and flowers) are suitable for human and animal consumption. Moringa oleifera seed consists of oil, protein, fiber, moisture and ash. Oil and protein contents were 34. 80, 31.65%, respectively, thus some people use it as a nutritional supplement or tonic [13]. Moringa oleifera seed kernels contain a oleic acid which is resistance to oxidative degradation [14]. Consequently, the aim of the present study was to investigate the antioxidant and the chemo protective effect of Moringa oleifera seed oil against diazinon-induced oxidative stress in rats.2. Materials and Methods2.1. ChemicalsTechnical 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. 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 biomarkers All serum biomarkers 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 the method of Doumas et al. [20]. Serum alanine aminotransferase (ALT; EC 2.6.1.2) and aspartate aminotransferase (AST; EC 2. 6.1.1) activities 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, 4dinitrophenylhydrazine [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 enzymes Catalase (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 the method of 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. Homogenated tissue were centrifuged at 2000g for 15 min to precipitate proteins. Protein-free supernatant (250  µl) was added to 1ml of Na2HPO4 (0.0 M) solution 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