Investigation on the Protective Effect of Grape Seed and Linseed Oils Against Cyclophosphamide Induced Genotoxicity in Mice
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Global Veterinaria 3 (5): 377-382, 2009 ISSN 1992-6197 © IDOSI Publications, 2009 Investigation on the Protective Effect of Grape Seed and Linseed Oils Against Cyclophosphamide Induced Genotoxicity in Mice Abeer H. Abd El-Rahim and Naglaa A. Hafiz Department of Cell Biology, National Research Center, Dokki, Giza, Egypt Abstract: The protection conferred by grape seed oil and linseed oil against cyclophosphamide induced bone marrow chromosomal aberrations and sperm abnormalities as well as DNA fragmentation had been evaluated in adult Swiss albino mice. Cyclophosphamide induced genotoxicity indicated by increased number of aberrant cells and different types of structural chromosomal aberrations (gap, break, fragment and deletion) and numerical aberrations (hypoploidy and hyperploidy). In sperm morphology cyclophosphamide induced sperm abnormalities for both head and tail abnormalities. Pretreatment with grape seed oil (0.1ml/kg b.w. /day) or linseed oil (0.1ml/kg b.w. /day) for 14 days prior to an interperitoneal dose of cyclophosphamide (25mg/kg b.w) reduced (P<0.05) the number of chromosomal aberrations and sperm abnormalities and also reduced the percentage of DNA fragmentation caused by cyclophosphamide. It could be concluded that each of grape seed oil and linseed oil acts as a potent antioxidant that prevented genotoxicity of bone marrow cells and sperm abnormalities as well as DNA fragmentation. Key words: Grape seed oil Linseed oil Chromosomal aberrations DNA fragmentation Sperm morphology INTRODUCTION useful essential fatty acids and tocopherols (vitamin E) [8]. Moreover, it is a highly concentrated source (76%) of Exposure to various environmental factors can lead linoleic acid [9]. Some studies suggested the use of grape to free radical formation. The most common form of free seed oil as a chemopreventive and cytoprotective agent radicals is oxygen. When an oxygen molecule (O2) [10]. It was found that grape seed extract can protect bone becomes electrically charged, it tries to steal electrons marrow chromosomes against genotoxic substances by from other molecules, causing damage to the DNA and reducing the total number of aberrant cells and different other molecules and therefore cause mutagenesis. A types of structural chromosomal aberrations [3], other critical factor in mutagenesis is cell division [1]. When the studies demonstrated no toxicity and mutagenecity for cell divides, an unrepaired DNA lesion can give rise to a this substance [11]. Also, grape seed extracts preventing mutation. To protect against oxidative damage, animals damage to human liver cells caused by chemotherapy have many different types of antioxidants defenses, medications [12]. these antioxidants decrease mutagenesis and thus Linseed oil is derived from the dried ripe seeds carcinogenesis, in two ways: by decreasing oxidative of the flax plant (Linum usitatissimum, Linaceae). DNA damage and by decreasing cell division [2]. Regular Linseed oil contains between 52 and 63 % alpha Grape seed oil and linseed oil were reported to have linolenic acid [13]. The linolenic acid has beneficial antioxidant activities [3, 4]. Grape seed oil is a vegetable effect in reducing inflammation leading to atherosclerosis. oil pressed from the seeds of various varieties of vitis Linseed oil may reduce cardiovascular risk through vinifera grapes. Grape seeds contain antioxidants platelet function and inflammation [14], has positive (polyphenols, including proanthocyanidins) [5], effect on femur bone mineral content, bone mineral sufficiently high amounts of resveratrol which can be density and lumbar vertebrae [15]. Linseed oil extracted commercially [6]. Resveratrol (3,5,4- supplementation was found to achieve a greater trihydroxystilbene) a polyphenolic phytoalexin, primarily reduction in lung and total metastases [16]. High doses of found in the skin in muscadine grapes as well as in the linseed oil could also delay in the growth of mammary seeds [7]. Also, grape seed oil contains nutritionally cancers [17]. Corresponding Author: Dr. Abeer H. Abd El-Rahim, Department of Cell Biology, National Research Center, Dokki, Giza, Egypt 377 Global Veterinaria, 3 (5): 377-382, 2009 In this study, the modulatory effects of grape seed cell suspension were spread onto slides that had just and linseed oils were examined through monitoring the been removed from the freezer. Five to seven slides were incidence of cyclophosphamide (CP) induced genotoxicity prepared for each mouse. Slides were stained with 10% in mice bone marrow, sperm abnormalities as well as DNA Giemsa in phosphate buffer ph 6.8. Scanning slides fragmentation. for mitotic metaphase spreads was conveniently accomplished with a 25x magnification objective and MATERIALS AND METHODS analyzed with a 100 X oil objective. Ten mice were taken for each treatment and 100 well spread metaphases were Experimental Animals and Treatments: Ninety male adult analyzed per mouse. Swiss albino mice weighing between 20-25 grams were used in this study. These animals were obtained from the Sperm Morphology and Count: The method of Wyrobek Animal House of the National Research Center. All and Bruce [22] was used for investigating sperm animals were fed a common diet and water ad libitium. morphology and count. Animals were killed 35 days after Animals were divided into six separated groups. Each the last injection. Sperm were sampled from the caudae group contains 15 mice. Animals of each group were epididymis. Both epidiymes from each mouse were minced divided into two subgroups; 10 animals for the together with small scissors in physiological saline chromosomal aberrations analysis and DNA (0.9%NaCl), pipetted up and down and then filtered in a fragmentation and 5 animals to be sacrificed at the day 35 small test tube. The volume is made up of 2 ml. smears from the last treatment or injection and then examined for were prepared on clean dry slides. Five mice were taken sperm shape and count. for each treatment and at least five slides were prepared Group 1 was the untreated control. Animals of group for each mouse to study sperm abnormalities. The slides 2 were intraperitoneal injected with single dose of were stained with eosin nefrosin stain. Sperm were cyclophosphamide (25 mg/kg body weight) according to examined under microscope for each group of animals Shukla and Taneja [18] and Seehy [19] and considered as more than 1000 sperm were examined for morphological positive control. abnormalities of the sperm shape. Some drops of the Animals of group 3 orally administered grape seed oil sperm suspension were put on haemocytometer to count (0.1 ml/animal/day) based on Al-Attar [20] and group 4 the sperm. received linseed oil (0.1ml/animal/day) based on Bhatia [4] for 14 consecutive days. DNA Fragmentation: The method of DNA fragmentation Animals of group 5 and 6 orally administered grape was carried out according to Perandones [23]. A bout seed oil and linseed oil for 14 consecutive days prior to 0.25g of the liver tissues were mechanically dissociated in interaperitoneal (i.p) injection with cyclophosphamide 400 µl hypotonic lysis buffer (10mM tris, 1mM EDTA and (25mg/kg body weight), respectively. Animals then were 0.2% triton X-100, ph 8.0). scarified after 48 hour. The cell lysate was centrifuged at 12.000 Xg for 15 min. the supernatant containing small DNA fragments Materials: Grape seed oil and linseed oil were obtained was immediately separated as well as the pellet containing from the Unit of Squeeze and Extraction of Natural Oils in large pieces of DNA, were used for the diphenylamine National Research Center, Dokki, Egypt. (DPA) assay. The pellet was resuspended in 400 µl of hypotonic lysis buffer. 400µl 10% trichloroacetic acid Chromosomal Aberrations for Bone Marrow Cells: The (TCA) was added to both the supernatant and the method of Yosida and Amano [21] was used with some resuspended pellet and incubated at room temperature modifications. Mice were injected intraperitonealy with for10 min. The tubes were centrifuged at 2000 rpm for colchicine at a final concentration of 3 mg/kg b.wt. Mice 15 min. at 4°C. After discarding the supernatant, the were anesthetized with ether 2h. after colchicine treatment precipitate was resuspended in 400 µl 5% TCA, incubated and the bone marrow from both femurs was collected in a at 80°C for 30 min. and then allowed to cool at room centrifuge tube containing 0.075 M Kcl. cell suspension temperature. After centrifugation, one volume of the were incubated for about 30 min. at 37°C then centrifuged extracted DNA was added to two volumes of colorimetric at 1500 rpm for 10 min. the cells were fixed in 3:1 methanol: solution (0.088 M diphenylamine (DPA), 98% V/V glacial glacial acetic acid which was freshly prepared. The fixative acetic acid, 1.5%V/V sulphoric acid and 0.5% V/V 1.6% was added gently to the cells. Fixation was done twice, Acetaldehyde solution). The samples were stored at 378 Global Veterinaria, 3 (5): 377-382, 2009 4°C for 48h. The colorimetric reaction was quantities linseed oil groups the frequencies of total aberrations spectrophotometrically at 578 nm. The percentage of DNA were slightly and insignificantly higher (16.8±1.35) and fragmentation was expressed by the formula: (14.8±1.35) than the control. In addition there was a significant (P<0.05) decrease in the mean percentage of O.D supernatant total aberrations of the two combined groups (grape DNA fragmentation percentage = ------------------------------------- X 100 O.D supernatant+O.D pellet seed oil + cyclophosphamide) and (linseed oil+cyclophosphamide) (68.4±1.720) and (40.0±1.549), Statistical Analysis: The experiment followed respectively as compared with cyclophosphamide group complete randomized (C R D). The obtained data were (112.4±2.4). subjected to analysis of variance (ANOVA) according to These results suggested that the two oils had Snedecor and Cochran [24]. Least significant differences protective effect against cyclophosphamide as monitored (L S D) were used to compare between means of by decreasing the incidence of aberrations.