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E- ISSN: 2249 –1929

JCBPS; Section B; February 2016-April 2016; Vol. 6; No. 2; 274-290.

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online at www.jcbsc.org Section B: Biological Sciences

CODEN (USA): JCBPAT

Research Article

Protective Effects of Flaxseed Oil against Oxidative Injury Induced By Gamma-Irradiation and Carbon Tetrachloride in Rat Liver Amel F. M. Ismail1, Asmaa A. M. Salem2, Mamdouh M. T. Eassawy2, Fatma S. M. Moawed3 1

Drug Radiation Research Department, National Center for Radiation Research and Technology (NCRRT). P.O. Box 29, Atomic Energy Authority, Nasr City, Cairo, Egypt. 2

Regional Center for Food and Feed (RCFF), Agriculture Research Center, Giza, Egypt.

3

Health Radiation Research Department, National Center for Radiation Research and Technology (NCRRT). P.O. Box 29, Atomic Energy Authority, Nasr City, Cairo, Egypt. Received: 10 January 2016; Revised: 28 January 2016; Accepted: 05 February 2016

Abstract: The aim of this study was to investigate the protective effect of flaxseed oil (FSO) against acute liver injury induced by carbon tetrachloride (CCl 4) in γ-irradiated rats (7 Gray). Rats treated with CCl4 exhibited the elevations in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Further, the levels of malondialdehyde (MDA), nitric oxide (NO), and the relative ratio of the gene expression of cytochromeP450-2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-κB) and Caspase-3 were significantly increased, accompanied by the decrease of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione transferase (GST) activities as well as the reduced glutathione (GSH) content. Furthermore, the alterations of the trace elements; iron, calcium, copper, manganese, magnesium and zinc were observed. These effects were augmented after injection of CCl4 in γ-irradiated rats. However, the oral treatment of FSO ameliorated these parameters. FSO exhibit protective effects on the oxidative stress induced by CCl4 or CCl4/γ-irradiation in rats that could be attributed to its potent antioxidant, antiinflammatory and anti-apoptotic activities. The antioxidant enzymes activation by FSO 274

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol. 6; No. 2; 274-290.

Protective

Amel F. M. Ismail et al.

could be attributed to the down-regulation of the CYP2E1 and iNOS gene expression, which are responsible for the reactive species generation. The inhibition of the inflammatory response could be attributed to the down-regulation of NF-κB that might be mediated by the amelioration of the trace elements' levels in the liver tissues of the intoxicated groups. The down-regulation of Caspase-3 is responsible for the antiapoptotic activity of FSO. Keywords: Flaxseed Oil, carbon tetrachloride, γ-irradiation, nuclear factor kappa B, cytochrome P2E1 (CYP2E1) gene expression. INTRODUCTION Flaxseed oil (FSO), flax oil or linseed oil is derived from the seeds of the plant Linium usitatissimum (41 g oil /100 g flaxseed)1. Flaxseed has several nutritional properties and health benefits that gained worldwide awareness in research and in the food industry2. The flaxseed health benefits, including cardioprotective1, anti-diabetic3, anti-inflammatory4, immune-modulatory5 and anticancer6 activities are mainly attributed to its constituents of omega-3 fatty acids and the phenolic lignans7. FSO is rich in the polyunsaturated fats, which constitute about 75% of its weight1; approximately 40 to 60% are alphalinolenic acid (18:3, n-3 omega-3 fatty acid) 8 with lower quantities of linoleic acid and oleic acid (each about 15%). Alpha-linoleic acid is a precursor to eicosapentanoic and docosahexanoic acid, and it may have beneficial effects on health and in control of chronic diseases 9. In addition, flaxseed oil is the richest plant source that contains approximately 1-4% of the phenolic compound lignan, secoisolariciresinol diglycoside (SDG)10. SDG is a lignan precursor, is converted by the bacterial flora of the human colon to two major mammalian lignans, enterodiol and enterolactone 11. Other smaller quantities of matairesinol, isolariciresinol, lariciresinol, demethoxysecoisolariciresinol and pinoresinol are present in FSO12-14. The significant antioxidant activity of these lignans is attributed to their high levels of free radical scavenger properties15,16. Radiation is used for medical and occupational purposes in the diagnosis and the therapy of cancer. The destructive effect of radiation destroys the tumor cells, likewise extends to damage the healthy tissues in the areas which are being treated. The exposure to ionizing radiation increases the production of the reactive oxygen species (ROS) and this can lead the irradiated cells into a state of oxidative stress, or imbalance between ROS production and the detoxification process by the biological system17. Carbon tetrachloride (CCl4) is a well-known hepatotoxin that is widely used to induce acute toxic liver injury in a large range of laboratory animals 18. Free radicals - such as trichloromethyl (CCl3• and/or CCl3OO•) and oxygen centered lipid radicals (LO• and/or LOO•) - are pivotal in CCl4-induced hepatotoxicity, which are generated during CCl4 metabolism by hepatic cellular cytochrome P45019. In addition, the activation of Kupffer cells also contributes to the liver injury through releasing both direct toxic products and cytokines which promote the inflammatory response20. Accordingly, the present study was carried out to investigate the protective effect of FSO against γradiation and CCl4-induced acute liver damage in female albino rats. MATERIAL AND METHODS Chemicals: The FSO was obtained from Imtenan, herbal drugstore, Nasr City, Cairo, Egypt. Carbon tetrachloride (CCl4) was obtained from Merck. All other chemicals and reagents used in this study were of analytical grade. 275

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol. 6; No. 2; 274-290.

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Amel F. M. Ismail et al.

Irradiation Facility: Whole-body gamma-irradiation was performed at the National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt, using Canadian Gamma Cell-40 biological irradiator (137Cesium), manufactured by the Atomic Energy of Canada Limited, Ontario, Canada. The radiation dose rate was 0.456 Gray (Gy) /min at the time of exposure. The total radiation dose was 7 Gy as a single dose of the whole body. Animals were not anesthetized before irradiation. Animals: Female Wistar rats (weighing 100–120 g) were obtained from the Nile Pharmaceutical Co., Cairo, Egypt. They were housed at the animal facility at the National Center for Radiation Research and Technology. Upon arrival, the animals were allowed to acclimatize for one week before starting the experiment. The animals were kept under standard laboratory conditions of light/dark cycle (12/12 h), a temperature of 25 ± 2 °C and humidity of 60 ± 5%. The rats were housed in cages with free access to food and drinking water ad libitum. They were provided with a nutritionally adequate standard laboratory (pellet) diet. The study was conducted in accordance with international guidelines for animal experiments and approved by the Ethical Committee of the National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt. Experimental design: The rats were divided into the following groups, (6 animals / group): Control group (C): rats administered water orally by gastric intubation. Irradiated group (R): Rats were exposed to a single dose of 7 Gy of the whole body γ-irradiation21, then, they were administered water orally for 7 days. CCl4 treated group (CCl4): rats were orally administered water, then they were intraperitoneally (IP) injected with acute single dose, 2 ml /kg body weight of CCl 4 (prepared with olive oil 1:1 v/v) after 7 days from the beginning of the experiment22. Irradiated/CCl4 treated group (R / CCl4): Rats were exposed to a single dose of 7 Gy of the whole body γ-irradiation. Irradiated rats were orally administered water by gastric intubation for seven days, and then they were administered intra- peritoneal (IP) with an acute single dose, 2 ml /kg body weight of CCl4. FSO treated group (FSO): Rats were orally administered 500 mg/kg body weight of FSO23, till the end of the experimental period. Irradiated / FSO treated group (R / FSO): Rats were exposed to a single dose of 7 Gy of the whole body γ-irradiation21, 30 min later was orally administered FSO, till the end of the experimental period. FSO/CCl4 treated group (FSO / CCl4): rats were orally administered with FSO, for seven days. Then they were administered IP with acute single dose, 2 ml /kg body weight of CCl4. Irradiated / FSO / CCl4 treated group (R / FSO / CCl4): rats were exposed to a single dose of 7 Gy of the whole body γ-irradiation; 30 min later was orally administered with FSO for seven days. Then they were administered IP with acute single dose, 2 ml /kg body weight of CCl4. After 16 h of CCl4 administration22, including an overnight fasting, with free access to drinking water, blood samples were collected from the retro-orbital venous plexus of the eye of each animal using a glass capillary tube under light ether anesthesia. The blood was collected in glass-tubes free of anticoagulant, allowed to clot, centrifuged at 1200 g using universal centrifuge (16R, Germany) and sera were separated. 276

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol. 6; No. 2; 274-290.

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Amel F. M. Ismail et al.

The liver of each animal was excised immediately and washed with physiological saline and stored at 80oC. Determination of liver transaminases: ALT and AST were determined in serum by a biochemical blood analyzer (ALFA WASSERMANN DIGNOSTIC TECHNOLOGYIES, LLC, ACE, Alera, USA). Determination of the Oxidative Stress Parameters and the Antioxidant enzymes in the Liver homogenate: Part of the liver was weighed and homogenized (10%) in chilled 50 mmol/L phosphate buffered saline (pH 7.4), centrifuged at 1200 g, at 4 °C for 15 min by universal centrifuge (16R, Germany), then the supernatants were used for the determination of the following parameters: Lipid peroxides, in terms of malondialdehyde (MDA) were measured according to the method of Satoh 24, using 1, 1, 3, 3-tetraethoxypropane as a standard. MDA concentration was expressed as nmol/g wet tissue. Nitric oxide (NO) was determined as nitrite concentration. The method used depends on Griess reaction, which converts nitrite into a deep purple azo-compound which photometrically measured at 540 nm according to the method of Montgomery and Dymock25. NO concentration was expressed as μmol/g wet tissue. GSH concentration was measured according to Beutler et al.26, using DTNB and was expressed as mg/g wet tissue. Superoxide dismutase (SOD) activity was determined according to Nishikimi and Yogi27. The assay relies on the ability of the enzyme to inhibit the phenazine methosulphate-mediated reduction of nitroblue tetrazolium (NBT) dye, which was followed photometrically at 560 nm. Catalase (CAT) activity was assessed according to Aebi28. Catalase reacts with a known quantity of hydrogen peroxide (H2O2) in the presence of horseradish peroxidase (HRP), remaining H2O2 reacts with 3, 5dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP) to form a chromophore with a color intensity measured at 510 nm, which is inversely proportional to the amount of catalase in the original sample. The enzyme activity was expressed as U/g wet tissue. Glutathione-peroxidase (GPx) activity was measured according to Rotruck et al.29 that based on indirect determination of GPx, whereas GPx reacted with known amount of glutathione (GSH), then the residual GSH reacted with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The color developed was read at 412 nm. The enzyme activity was expressed as μM of GSH oxidized/g wet tissue/min. Glutathione-S-transferase (GST) activity was measured according to Habig and Jakoby30, by measuring the conjugation of 1- chloro- 2,4dinitrobenzene (CDNB) with reduced glutathione. The conjugation is accompanied by an increase in absorbance at 340 nm. The enzyme activity was expressed as U/g wet tissue Cytokines Assays: IL-6 and TNF-α were determined in serum, using ELISA kits for rat (Glory Science Co., Ltd, USA). The measurements were done according to the catalogue-instruction guidelines. Detection of relative gene expression ratio of Cytochrome P2E1, inducible nitric oxide, nuclear factor kappa B and Caspase-3 in the liver tissues by Quantitative Real Time PCR (qRT-PCR): 2.9.1 RNA Isolation and Reverse Transcription: Briefly, liver tissue (100 mg) was homogenized in 1 ml ice-cold TRIzol reagent (Invitrogen, U.S.A.) using a Polytron homogenizer and subsequently incubated for 10 min at room temperature. Samples were mixed with chloroform (0.2 ml) and incubated for 3 min at room temperature, followed by centrifugation (12,000 g, 15 min). The top aqueous phase was isolated and 0.5 ml isopropanol was added, samples were re-centrifuged (12,000 g, 10 min) and the resulting RNA pellet was washed with 75% ethanol and centrifuged again (7500 g, 5 min). The supernatants were discarded and the RNA pellets were air-dried (10 min), then dissolved in diethyl pyrocarbonate (DEPC), then incubated at 55–60 °C for 10 min. The concentration of isolated nucleic acids was determined spectrophotometrically by measuring the absorbance at 260 nm. All samples were 277

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol. 6; No. 2; 274-290.

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stored at − 80 °C until the analysis of cDNA synthesis reaction that was performed using the Reverse Transcription System (Promega, Leiden, The Netherlands). Total RNA was incubated at 70 °C for 10 min to prevent secondary structures. The RNA was supplemented with MgCl2 (25 mM), RTease buffer (10 ×), dNTP mixture (10 mM), oligo d(t) primers, RNase inhibitor (20 U) and AMV reverse transcriptase (20 U/μl). This mixture was incubated at 42 °C for 1 h. Quantitative Real Time PCR: QRT-PCR was performed using an optical 96-well plate with an ABI PRISM 7500 fast sequence detection system (Applied Biosystems, Carlsbad, California) and universal thermal cycling conditions ( 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 60 s). Each 10 μl reaction contained 5 μl SYBR Green Master Mix (Applied Biosystems), 0.3 μl gene-specific forward and reverse primers (10 μM), 2.5 μl cDNA and 1.9 μl nuclease-free water. The sequences of PCR primer pairs used for each gene as follows, CYP2E1 was carried out in liver tissues with the oligonucleotide 5'-ACT TCT ACC TGC TGA GCA C-3' (forward) and 5’ TTC AGG TCT CAT GAA CGG G-3' (reverse)31, iNOS: Forward 5'-GGG CCA CCT TTA TGT TTG TG-3', reverse: 5'-CCG GTG GGT TTC TTCTTC TTG AA-3' 32. NF-κB Forward 5'-AAC ACT GCC GAG CTC AAG AT-3', reverse: 5'-CAT CGG CTT GAG AAA AGG AG-3' 33, Caspase-3 Forward 5'-CCA CTC CCA GTC ATT CCT TTA GTG-3', reverse: 5'-ATG GAC AAC AAC GAA ACC TCC GTG-3' 34, and GAPDH: Forward 5'-ATG GGA GTT GCT GTT GAA GTC A-3’, reverse: 5'-CCG AGG GCC CAC TAA AGG-3' 35. Gel Electrophoresis: 10 μl of PCR product was analyzed on 2% agarose gel with ethidium bromide staining and the product was visualized on ultraviolet transilluminator, then gel documentation was performed. PCR products were semi-quantified by using a gel documentation system (Bio Doc Analyze) supplied by Biometra, Germany. Then, the data were analyzed with the ABI Prism sequence detection system software and quantified using the v1·7 Sequence Detection Software from PE Biosystems (Foster City, CA). The relative expression of the studied genes was calculated using the comparative threshold cycle method. All values were normalized to the GAPDH genes36. Determination of trace elements in the liver tissues: The liver tissues of different groups were digested in a mixture of concentrated nitric acid (HNO3) and hydrogen peroxide (H2O2) (5:1 v/v) until complete digestion of the organic materials using Milestone MLS-1200 Mega, High Performance Microwave Digester Unit, Italy. Calcium (Ca), iron (Fe), copper (Cu) manganese (Mn) and zinc (Zn) concentration levels were estimated in the prepared liver samples using Atomic Absorption spectrophotometer (Thermo Scientific, iCE 3000, England), at NCRRT. Statistical Analysis: All statistical analyses were conducted by using the statistical package for Windows Version 15.0 (SPSS Software, Chicago, IL). The results for continuous variables were expressed as mean + standard error. Values were compared by one-way analysis of variance (ANOVA). Post -hoc testing was performed for inter-group comparisons using the least significant difference (LSD) test, and significance of p values < 0.05 was considered statistically significant. RESULTS AND DISCUSSION This study aimed at examining the protective effect of flaxseed oil (FSO) on CCl 4-induced rat liver injury in γ-irradiated rats by mitigating the oxidative stress and inhibiting the inflammatory response. The activity of the hepatic enzymes (ALT and AST): The activity of the hepatic enzymes ALT and AST showed significant elevations (p0.05). (Fig.1). The FSO administration promoted a significant decrease in the serum activities of ALT and AST as compared to the CCl4 intoxicated groups (Fig.1). 3500

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Fig.1. The activities of serum AST and ALT of different experimental groups. Rats were treated with 500 mg/kg b. wt. FSO, γ-irradiation (7 Gy) and 2 ml/kg b. wt. CCl4. Data are presented as the mean ± SE of six rats. a: Statistical non-significant difference at p>0.05 vs. control, b: Statistical significant difference at p