Toxicology Mechanisms and Methods
ISSN: 1537-6516 (Print) 1537-6524 (Online) Journal homepage: http://www.tandfonline.com/loi/itxm20
Modulatory Effects of Quercetin on Liver Histopathological, Biochemical, Hematological, Oxidative Stress and DNA Alterations in Rats Exposed To Graded Doses of Score 250 Sabrine Kasmi, Intithar Bkhairia, Bahira Harrabi, Hela Mnif, Rim Marrakchi, Hanen Ghozzi, Choumous Kallel, Moncef Nasri, Khaled Zeghal, Kamel Jamoussi & Ahmed Hakim To cite this article: Sabrine Kasmi, Intithar Bkhairia, Bahira Harrabi, Hela Mnif, Rim Marrakchi, Hanen Ghozzi, Choumous Kallel, Moncef Nasri, Khaled Zeghal, Kamel Jamoussi & Ahmed Hakim (2017): Modulatory Effects of Quercetin on Liver Histopathological, Biochemical, Hematological, Oxidative Stress and DNA Alterations in Rats Exposed To Graded Doses of Score 250, Toxicology Mechanisms and Methods, DOI: 10.1080/15376516.2017.1351507 To link to this article: http://dx.doi.org/10.1080/15376516.2017.1351507
Accepted author version posted online: 05 Jul 2017.
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Date: 06 July 2017, At: 02:17
Modulatory Effects of Quercetin on Liver Histopathological, Biochemical, Hematological, Oxidative Stress and DNA Alterations in Rats Exposed To Graded Doses of Score 250 Running title: Modulatory Effects of Quercetin Sabrine KASMI1, Intithar BKHAIRIA2, Bahira HARRABI1, Hela MNIF3, Rim MARRAKCHI4, Hanen
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GHOZZI1, Choumous KALLEL5, Moncef NASRI2, Khaled ZEGHAL1, Kamel JAMOUSSI4, Ahmed HAKIM1
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Laboratory of Pharmacology, UR/12 ES-13, Faculty of Medicine of Sfax, Sfax University,
Laboratory of Enzyme engineering and Microbiology, National Engineering School of Sfax,
Sfax University, B.P. 1173, 38 Sfax, Tunisia.
Laboratories of Histology and Embryology, Faculty of Medicine of Sfax, SfaxUniversity,
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Biochemistry Department, Hedi Chaker Hospital, Sfax University , Tunisia.
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Sfax, Tunisia.
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2
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Sfax, Tunisia.
Hematology Laboratory, Habib Bourguiba University Hospital, 3029 Sfax, Sfax University,
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Tunisia. *
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Corresponding author: E-mail:
[email protected], Laboratory of Pharmacology,
Faculty of Medicine of Sfax, Sfax University, Tunisia, Avenue Majida Boulila, 3029 Sfax, Tunisia.
Abstract This study investigated the morphological, biochemical and molecular aspects of liver injury in rats after the exposure to difenoconazole and the protective effects of quercetin against hepatotoxicity and genotoxicity induced by this fungicide. Rats were given graded doses of difenoconazole associated or not to quercetin daily for 20 days. Our results showed a significant increase in PLT (Platelets) and WBC (white blood cells) in rats treated with higher doses of difenoconazole (1/38 and 1/9 of LD50). However, a significant decrease in Hb
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(Hemoglobin) rate and RBC (Red blood cells) number in rats treated with higher doses of difenoconazole (1/38 and 1/9 of LD50) was obtained. Besides, difenoconazole treatment
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caused an increase in hepatic enzyme activities of alanine transaminase (ALT), aspartate
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transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH).
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Difenoconazole increased the levels of malondialdehyde (MDA) and advanced oxidation protein products (AOPP), and decreased superoxide dismutase (SOD), catalase (CAT),
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glutathione peroxidase (GPx) activities and vitamin C levels in liver tissues compared to the
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control group. We also noted a degradation of nucleic acids, testifying difenoconazole genotoxicity. Changes in hepatic tissues were confirmed by histological findings. Co-
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administration of quercetin (20 mg/ kg) improved hematological and biochemical parameters
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and showed a significant liver protective effect by decreasing malondialdehyde levels and producing advanced oxidation protein, along with increased antioxidative enzyme activities
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and vitamin C levels. Results were confirmed by the improvement of histological impairments. Thus, it appears that quercetin was effective in preventing acute liver injury induced by exposure to difenoconazole. Keywords Quercetin; difenoconazole; liver; oxidative stress; biochemical parameters; hematological parameters.
Introduction Difenoconazole, 1-[2-[2-chloro-4-(4-chlorophenoxy) phenyl]-4-methyl-1, 3-dioxolan2-ylmeth-yl]-1H-1, 2, 4-triazole, is a fungicide with a broad spectrum, for the control of fungal disease on vegetables, fruit, cereals and other field crops (Vawdrey et al., 2008; Horsfield et al., 2010). Like other triazole fungicides, difenoconazole inhibits fungal lanosterol-14α-demethylase activity (CYP51) in the biosynthesis pathway of ergosterol. In
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consequence, cell membrane integrity, function and cell growth are affected (Ragsdale, 1977;
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Vanden Bossche et al., 1990; Buchnauer et al., 1995; Ghannoum and Rice, 1999; Sheehan et
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al., 1999; Hamada et al., 2011). This fundamental mechanism is the base of the use of
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antifungal triazoles in agriculture (Zarn et al., 2003). Difenoconazole is widely used in agriculture. Hence, farm workers can be exposed during the application. Besides, consumers
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can be exposed to the residues of this fungicide in water and food (Danuta et al., 2001).
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Incorrect application and the absence of the protective gears during pesticide application are responsible for skin injuries, dermal sensitization and mucous membranes (James et al.,
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2006).
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Pesticides, including triazoles, are shown to increase the generation of the reactive oxygen species (ROS) which initiate oxidative stress in diverse tissues (Mehta et al., 2009).
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ROS may interact with cellular lipids, proteins and DNA, resulting in alterations in cell
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function. Organism cells contribute to the elimination of harmful effects of ROS. They integrate antioxidant systems that include enzymes (Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) and non-enzymatic antioxidants (vitamin E, vitamin C, flavonoïdes) (Scott et al., 2000; Karaoz et al., 2002; Kalender et al., 2007; Uzunhisarcikli et al., 2007; Ogutcu et al., 2008 ). In fact, the antioxidant enzymes (SOD, GPx, and CAT) are the first line of defense of the cells and help to protect them from excessive generation of oxidative stress.
The liver has a vital role in xenobiotics transformation; thus it is highly vulnerable to injury. In fact, it is the major site of detoxification and xenobiotic metabolism. It can accumulate huge concentrations of metabolites (Sultatos et al., 1985; Sultatos, 1987). The function of liver is defined through serum markers such as AST, ALT, ALP, and LDH (Padma et al; 2012). In order to cope with the enhanced production of ROS, antioxidant intake has been shown to inhibit the ROS generation (Durak et al., 2010). Antioxidants, particularly flavonoids, have been reported to prevent lipid damage by integrating in lipid bilayer, to
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scavenge ROS and lipid peroxyl radicals (Chander et al., 2003; Zal et al., 2007). Quercetin is
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the most abundant polyphenolic flavonoid in human diet including broccoli, onions, tomatoes, berries and apples, and it is one of the most potent flavonoids. Quercetin has been reported to
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display hepatoprotective effect against lindane (insecticide) induced hepatotoxicity and
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against the cadmium-induced toxicity by increasing the expression of endothelial nitric oxide synthase (eNOS) and increasing the methyl transferase (MT) (Filipe et al., 2004; Morales et
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al., 2006; Padma et al., 2012). In addition, quercetin has been reported to display anti-
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inflammatory, antiviral, anticarcinogenic and antibacterial effects (Formica and Regelson, 1995; Di Carlo et al., 1999; Harborne and williams, 2000; Anjaneyulu and chopra, 2003). thus used as a powerful antioxidant due to its efficiency particularly for
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oxidative damage.
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Quercetin is
Therefore, the purpose of this study was to investigate the protective effect of
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quercetin against hematological perturbations and liver damage induced by difenoconazole, a widely used triazole fungicide.
Materials and methods Animals Forty eight adult and healthy male Wistar rats weighing 230±10 g, were received from the breeding centre of Tunisia (SIFAT) and housed per group (six per cage) in eight plexiglass cages under controlled conditions of light (12h light /dark cycle), temperature (23±2°C) and humidity (50±10%). Animals were allowed free access to commercial pellet diet supplied
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from the Industrial Society of Nutrients (SICO, Sfax, Tunisia) and distilled water. All
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experimental protocols were carried out in accordance with the guidelines for the care and use
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of laboratory animals with the consent of the national ethical committee (University of Sfax,
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Tunisia; Protocol no. 94-1939).
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Chemicals
The commercial product of score 250 was purchased from Syngenta Ltd, Tunisia .It contains,
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as an active ingredient, difenoconazole (EC 25%), surfactant (25%) and water (10%). Dimethyl sulfoxide (DMSO) was provided by Merck (Germany), and quercetin by Sigma-
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Experimental design
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Aldrich (USA). All other reagents were purchased from Sigma-Aldrich.
Group 1 (control group) received by oral gavage 2 ml of distilled water, served as
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Animals were divided into seven groups of six rats each. They were treated as follows:
negative control;
Group 2 (quercetin, Q group) was given via oral gavage quercetin at a dose of 20mg/kg (n=6), served as positive control;
Group 3 (D1 group) received by oral gavage difenoconazole at a dose of 24 mg/kg b.w/day (D1 (low dose)≈1/60 LD50);
Group 4 (D2 group) received by oral gavage difenoconazole at a dose of 48 mg/kg b.w/day (D2 (medium dose)≈1/38 LD50) (n=6);
Group 5 (D3 group) was given daily via oral gavage difenoconazole at a dose of 160 mg/kg b.w/day (D3 (high dose)≈1/9 LD50) (n=6);
Group 6 (D1+Q) received by oral gavage difenoconazole (D1≈1/60 LD50) and quercetin (20 mg/kg b.w/day) (n=6).
Group 7 (D2+Q) was given via oral gavage difenoconazole (D2≈1/38 LD50) and
Group 8 (D3+Q) received by oral gavage difenoconazole (D3≈1/9 LD50) and
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quercetin (20 mg/kg b.w/day) (n=6).
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quercetin (20 mg/kg b.w/day) (n=6).
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In a preliminary-study, we fixed the lethal dose LD50 (the median lethal dose is the dose required to kill half the rats of a tested population after a specified test duration
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(3days)) at 1453 mg/kg and we assessed different doses of difenoconazole. The doses of
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difenoconazole used in this study represented 1/60, 1/38, 1/9 of lethal dose (LD50= 1453mg/kg): no toxic effects were observed (no mortality) in rats treated with difenoconazole
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at doses under 1/60 LD50. With this dose, toxic effects and oxidative stress were identified in
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male rats without lethal effects. But, with doses over 1/9 LD50, difenoconazole provoked mortality. The rationale behind choosing 1/60, 1/38, 1/9 LD50 doses was to compare the toxic
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effects of low (1/60 LD50); medium (1/38 LD50) and a high (1/9 LD50) doses. The fungicide and quercetin were administered once a day during 20 days via gavage. For quercetin and difenoconazole treatment, rats received quercetin dissolved in DMSO (0.5%) at a dose of 20 mg/kg b.w/day). After 30 minutes, animals were given difenoconazole (3 doses). The use of the single dose of quercetin was selected according to previous researches (Uzun et al., 2013). In fact, this dose (20 mg/kg) showed a potent protective role against oxidative stress via the
oral gavage method (Uzun et al., 2013). We chose the oral gavage for treatment with difenoconazole and quercetin because it is the most reliable and accurate method for administering substances into the gastro-intestinal tract, as it eliminates variability in intake between animals which may arise when substances are administered through delivery in food and/or water. In order to get a more stable internal dose of difenoconazole, the oral gavage route of exposure was used in an acute model (for 20 days). At the end of treatment, all male rats were sacrificed after receiving intramuscular injections of ketamine (50 mg/ml) (200 µl)
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and midazolam 5 mg/ml (20 µl) to avoid stress.
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Hematological parameters
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Blood samples were obtained by cardiac puncture in EDTA treated tubes and analyzed for
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hematological parameters: white blood cell (WBC) counts, red blood cell (RBC) counts, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),
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platelets (PLT) and mean corpuscular hemoglobin concentration (MCHC) using a hematology
Biochemical parameters
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analyzer.
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Other blood samples of the male rats were taken into sterile tubes and centrifuged at 3500 × g
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for 20min.Serum was separated and was stored at –20°C until analysis for liver biochemical markers (AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline
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phosphatase) and LDH (lactate dehydrogenase)). Samples preparation
Livers were quickly dissected out and washed. About 0.3 g of the liver samples were homogenized (10Mm Tris HCl; PH8.5) with an Ultra Turrax homogenizer in ice-cold buffer. Homogenates were then centrifuged at 10,000 × g for 15 min at 4°C. Some samples were quickly fixed in 10% formalin solution to assess histological changes.
Biochemical assays Protein quantification. Liver protein contents were measured according to the method described by Lowry et al. (1951) using bovine serum albumin (BSA) as standard.
Determination of malondialdehyde levels
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Malondialdehyde (MDA) concentrations in liver were used as an index of lipid peroxidation
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and determined spectrophotometrically according to the method described by Draper and
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Hadley (1990). Thiobarbituric acid (TBA)-MDA complex absorbance was measured at 532
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nm. Malondialdehyde values were determined using 1,1,3,3-tetraethoxypropane as standard
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and the results were expressed as nanomoles of malondialdehyde per gram of protein.
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Determination of AOPP levels
Levels of advanced oxidation of protein products (AOPP) in liver were estimated according
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to the method of Kayali et al. (2006). Briefly, about 0.4 mL of liver extract supernatant was treated with 0.8 mL phosphate buffer (0.1 M; pH 7.4).After 2 min, 0.1 mL of potassium
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iodide (1.16 M) was added and followed by 0.2 mL of acetic acid. The absorbance of the
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reaction mixture was immediately recorded at 340 nm. AOPP concentration for each liver
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sample was estimated by using the extinction coefficient (261 cm−1 mM−1). Results were expressed in terms of AOPP nanomoles per gram of protein. Determination of antioxidant enzyme activities Catalase (CAT) activity CAT activity in liver was determined by the method of Aebi (1984). An aliquot of 20 μl of supernatant of liver homogenate was added to the substrate (H2O2, 0.5 M) in a medium
containing phosphate buffer (100 mM, PH 7.4). The absorbance was measured at 240 nm. CAT activity was expressed as micromoles of H2O2 per min per mg of protein. Superoxide dismutase (SOD) SOD activity in liver was assayed by the method of Beauchamp and Fridovich (1971). The reaction mixture contained liver homogenates in 50 mM potassium phosphate buffer (pH 7.8), 13 mM L-methionine, 0.1 M EDTA, 2 mM riboflavin, 75 mM nitro blue tetrazolium
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(NBT). The blue color developed in this reaction was recorded at 560 nm. The enzyme
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activity was expressed in terms of units per milligram of protein. Units of SOD activity were
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defined as the amount of enzyme required to inhibit the reduction of nitro blue tetrazolium
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(NBT) by 50%.
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Glutathione peroxidase (GPx)
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GPx activity in liver was recorded as described by Flohe and Gunzler (1984). Liver GPx activity was estimated as nanomoles of GSH oxidized per minute per milligram of protein.
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Measurement of vitamin C levels
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Vitamin C levels in livers were determined according to the method of Jacques-Silva et al.
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(2001). Protein was precipitated in 10 v of a cold 4% TCA solution. 300 μl of supernatant were adjusted with distilled H2O to 1 ml (final volume) and was incubated for 3h at 38°C;
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then, 1 ml of sulfuric acid 65% (v/v) was added to the medium. The resulting product was determined by using a color reagent with 0.075 mg/ml CuSO4 and 4.5 mg/ml dinitrophenylhydrazin. The reaction absorbance of the mixture was measured at 540 nm. Results were expressed as micromoles of ascorbic acid per milligram of protein.
DNA fragmentation analysis
100 mg of liver tissues were used to isolate total DNA according to the method of Chamczynski and Sacchi (2006). DNA fragmentation from kidney was done in accordance with Kanno et al. (2004). Then, the DNA fragmentation assay was analysed by electrophoresis on agarose gel treated with ethydium bromide, following the procedure described by Sellins and Cohen (1995). Band intensities were measured using Quantity One analysis software. All experiments were carried out in triplicate.
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Histological studies
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Hepatic tissues were taken to assess histological changes by light microscope examination.
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Tissue samples were fixed for 48 h in 10% neutral formalin; then, liver samples were processed in ascending graded series of ethanol solutions and embedded in paraffin. Paraffin
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sections (5-6μm) were cut using a rotary microtome. They were then stained with hematoxylin and eosin for microscope examination. Slides were prepared from each liver (6
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slides). All liver sections were examined for the degree of liver injury.
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Statistical analysis
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The data were analyzed using SPSS software (version 20.0; SPSS inc., Chicago, IL, USA).
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Statistical analysis between all groups was performed using one way analysis of variance (ANOVA) followed by the Tukey post hoc test. For each parameter, values were expressed as
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the means±standard deviation (SD). Results were considered significant if P
