J Basic Clin Physiol Pharmacol 2015; 26(3): 237–251
Reham Z. Hamza, Nahla S. El-Shenawy* and Hayat A.A. Ismail
Protective effects of blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes in the hepatic, renal, testis and brain tissue of male rat Abstract Background: Sodium fluoride (NaF) intoxication is associated with oxidative stress and altered antioxidant defense mechanism. The present study was carried out to evaluate the potential protective role of blackberry and quercetin (Q) against NaF-induced oxidative stress and histological changes in liver, kidney, testis and brain tissues of rats. Methods: The rats were allocated evenly to seven groups. The first group was maintained as the control, whereas groups 2, 3, 4, 5, 6 and 7 were administered blackberry juice (BBJ), Q, NaF, BBJ+NaF, Q+NaF and BBJ+Q+NaF, respectively, for a period of 30 days. Results and conclusions: NaF caused an elevation in lipid peroxidation level paralleled with significant decline in glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase activities as well as the total antioxidant activity in liver, kidney, testes and brain. Some histopathological changes were detected in all tested tissues of the NaF treated group. Q and BBJ had successfully maintained normal histological architecture and mitigated the induction of oxidative stress caused by NaF. Q effectively reduced the elevation in thiobarbituric acid reactive substances level and restored the activities of antioxidant enzymes in liver, kidney, testis and brain. Less histopathological changes were observed in Q+NaF and BBJ+NaF treated groups. As a result, BBJ and Q significantly reduced NaF-induced oxidative and histological changes in rats. In the combination of BBJ and Q against NaF toxicity, the effects were more severe than from separate exposure, thus indicating that these flavonoids exhibited synergistic effects on all antioxidant and histological parameters. *Corresponding author: Nahla S. El-Shenawy, Faculty of Science, Zoology Department, Suez Canal University, Ismailia, 41522, Egypt, E-mail: [email protected] Reham Z. Hamza: Faculty of Science, Zoology Department, Zagazig University, Zagazig, Egypt Hayat A.A. Ismail: Faculty of Science, Biology Department, King Abdel Aziz University, Jeddah, Saudi Arabia
Keywords: blackberry; brain; histology; kidney; liver; oxidative/antioxidant parameters; quercetin; testis. DOI 10.1515/jbcpp-2014-0065 Received June 10, 2014; accepted August 24, 2014; previously published online October 28, 2014
Introduction Fluorosis is a serious public health problem in many parts of the world. As in the case of many chronic degenerative diseases, increased production of reactive oxygen species (ROS) has been considered to play an important role, even in the pathogenesis of chronic sodium fluoride (NaF) toxicity [1]. Studies revealed that fluoride (F) induces excessive production of ROS and might cause depletion in biological activities of some antioxidant enzymes [1–3]. Toxic effects of F on various biochemical parameters are known [2]. Increasing free radical generation and lipid peroxidation (LPO) levels is proposed to mediate the toxic effects of F on soft tissues [4]. Increased LPO and disturbed antioxidant defense systems in brain, erythrocytes and liver of rats exposed to F had been reported [5–7]. Because F has been found to penetrate readily into the brain and to accumulate in the brain tissues [8], daily intake of it for several days is likely to produce neurotoxic effects. Toxic effects in the central nervous system occur after its concentration reaches an adequate level in the brain. Chronic administration of NaF (1 ppm) in rats with drinking water resulted in damage of the hippocampus and Purkinje cells and formation of beta-amyloid plaques (the classic brain abnormality) [9]. Microscopic study showed neurodegeneration in the hippocampus of mice chronically exposed to NaF (100 ppm) [10]. A marked increase in oxidative stress, LPO and a decrease in the activity of antioxidant enzymes were found in the brain regions of rats chronically exposed to 100 ppm of F through drinking water [11]. Chronic F exposure (100 and 200 ppm) has been found to increase LPO and to decrease
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238 Hamza et al.: Blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes the activity of antioxidant enzymes in three generations. Thus, an induction of oxidative stress has been suggested by these investigators as one of the mediatory factors in the toxicity of F in the brain [3]. Fluoride was determined to cause adverse effects in mice on erythrocyte and liver tissue malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Hassan and Abdel-Aziz [12] indicated that NaF-induced oxidative stress as evidenced by elevated levels of LPO and nitric oxide in red blood cells, kidney, testis and brain tissues. Moreover, significantly decreased glutathione (GSH) level, total antioxidant capacity and SOD activity were observed in the previous tissues. NaF-induced blood oxidative stress in mice had been reported [13]. Flavonoids are a major class of phytochemicals found ubiquitously in fruits and vegetables, they are rarely found to be toxic and are highly efficient against ROS-mediated injury [14]. One of the most important natural diets with antioxidant properties is blackberries. Berry fruits, wild or cultivated, have been proven to be a traditional and rich source of bioactive compounds, possessing important biological activities such as flavonoids (anthocyanin), some minerals (Na, K, Ca, Se, Zn and P), vitamins (vitamin A, B complex, C and E), phenolic acids (galic, p-coumaric, caffeic, ferulic) and phenolic polymers (ellagic acids) [15]. The antioxidant capacity of these berries was related to their constituents, particularly total phenolics and anthocyanins [16]. These contents have antioxidants and can improve immunity, playing an antagonistic role of protective agent against toxic substances [17]. On the other hand, the induced oxidative stress and the alterations in antioxidant systems were normalized by the oral administration of blackberry juice (BBJ). Therefore, it can be concluded that blackberry administration could minimize the toxic effects of F indicating its free radical-scavenging and potent antioxidant activities [12]. Quercetin (Q) is a plant-derived flavonoid widely found in vegetables, fruits and cereals. It is the aglycone form of some flavonoid glycosides, such as quercitrin and rutin. Q is characterized by a phenyl benzo(c)pyronederived structure and is mainly found in different foods and food products such as onions and black tea [18]. It exhibits its antioxidant properties against many diseases, including ischemic heart disease, atherosclerosis, liver fibrosis, renal injury and biliary obstruction [19, 20]. Nabavi et al. [21] suggested that Q protects rat liver from NaF-induced oxidative stress, probably via its antioxidant activity. It also protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead [22].
In our previous study, BBJ and Q successfully restored liver functions and normalized kidney functions as well as improved the sex hormone reduction induced by NaF. These two antioxidants also alleviated hematotoxicity as compared with NaF. However, Q exhibited a more pronounced protection toward liver function and hematotoxicity than BBJ. Concurrent intake of BBJ or Q with NaF can mitigate its toxicity. Still, further investigations should be done to estimate the mechanism of phytochemicals to protect different tissues in rats [23]. To the best of our knowledge, there is no scientific report about the protective effect of Q against NaF-induced renal, testis and brain oxidative stress but only on hepatic tissue. Moreover, there are few reports about the uses of BBJ in alleviating adverse effects on NaF. Therefore, it seems of interest to evaluate the antioxidant activity of Q and BBJ separately or in combination as well as their scavenging capacity toward fluoride-induced free radicals in rats. The extent of this effect of BBJ or Q or both of them was estimated by measuring the level of LPO, activities of the antioxidant enzymes (namely, SOD and CAT), the GSH metabolic enzymes activities [GPx, glutathione reductase (GRx) and glutathione S-transferase (GST)] and total antioxidant levels.
Materials and methods Chemicals NaF was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The tested dose of NaF (10.3 mg/kg body weight) was chosen based on the previous studies of Zabulyte et al. [24]. Q was purchased from Sigma Chemical Co. (St. Louis, MO, USA). It was given in a dose of 75 mg/kg according to the previous study by Seivac et al. [25]. Fresh blackberry fruits were obtained from the local market (Zagazig, Egypt) and then washed and homogenized, and its juice was freshly prepared daily. The dose of blackberry (1.6 g/kg body weight equal to 9 mL/kg body weight, which contains 5 mg active constituent, anthocyanine) used in this study was according to the previous studies of Sauebin et al. [26] and Siriwoharn et al. [27]. All other chemicals used in the experiment were analytical grade and were procured from Merck Ltd., SRL Pvt., Ltd., Mumbai, India.
Experimental animals Seventy adult male Wistar albino rats (Rattus rattus) weighing 170–180 g were purchased from King Fahed Medical Research Center in Jeddah (Kingdom of Saudi Arabia). The animals were housed in metal cages and bedded with wood shavings and kept under standard laboratory conditions of aeration and room temperature at about 25 °C with 12/12 light and dark cycle. The animals were allowed to free access of standard diet and water ad libtum throughout the
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/10/15 11:49 AM
Hamza et al.: Blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes 239 experimental period. We have followed the European Community Directive (86/609/EEC) and national rules on animal care that were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals 8th edition. The animals were acclimated to the laboratory conditions for 2 weeks before being experimented on; one group served as controls, and six as the treated groups.
Experimental design After 2 weeks of acclimation, animals were divided into seven groups, ten rats each. All the groups were treated interaperitoneally (i.p.) for 30 successive days. Group 1 served as untreated controls and received 1.0 mL/kg of distilled water i.p. daily. Group 2 was given i.p. BBJ at a dose of 1.6 g/kg body weight containing 5 mg anthocyanine. Group 3 was given Q at a dose of 75 mg/kg body weight. Group 4 was treated with NaF at 10.3 mg/kg body weight. Group 5 was given i.p. BBJ followed by NaF at the same doses. Group 6 was given Q followed by NaF, and finally group 7 was treated with BBJ and Q then followed by NaF at mentioned previous doses.
Preparation of tissues for measurement of oxidative/ antioxidant parameters The tissues of liver, kidney, testis and brain were used for the analysis of oxidative stress and antioxidant parameters. Prior to dissection, tissue was perfused with a 50 mM (sodium phosphate buffer saline (100 mM Na2HPO4/NaH2PO4) (pH 7.4) and 0.1 mM ethylenediaminetetra acetic acid (EDTA) to remove any red blood cells and clots. Then tissues were homogenized in 5 mL cold buffer per gram tissue by a Potter-Elvehjem type homogenizer. The homogenate was centrifuged at 10,000 × g for 20 min at 4 °C, and the resultant supernatant transferred into Eppendorf tubes and preserved in a deep freezer until used. The supernatant was used for the determination of some biochemical parameters of liver, kidney, testis and brain tissues.
Lipid peroxidation assay The extent of LPO was estimated as the concentration of thiobarbit uric acid reactive product MDA by using the method of Ohkawa et al. [28]. MDA concentrations were determined using 1,1,3,3-tetraethoxypropane as standard and expressed as micromoles per gram of tissue.
Antioxidant enzymes SOD activity was measured according to the method described by Marklund and Marklund [29] in which pyrogallol underwent autoxidation at 440 nm for 3 min. One unit of SOD activity was calculated as the amount of protein that caused 50% pyrogallol autoxidation inhibition. The SOD activity was expressed as units per gram tissue. Before determination of the CAT activity, samples were diluted 1:9 with 1% Triton X-100 (v/v). CAT activity was measured according to the method described by Aebi [30]. The hydrolysis of H2O2 and the resulting decrease in absorbance at 240 nm over a 3-min period at 25 °C was measured. CAT activity was expressed as units per gram tissue.
Measurement of enzymes involved in glutathione metabolism The GPx activity was measured by the method of Hafeman et al. [31]. The reaction mixture contained 0.5 mL of 0.4 M sodium phosphate buffer (pH 7.0) and 0.4 mM EDTA, supplemented with 0.25 mL of sodium azide (1 mM), 0.5 mL of GSH (2 mM) and 0.25 mL of distal water. About 0.5 mL of homogenate was added and allowed to equilibrate for 5 min at 37 °C. The reaction was initiated by adding 0.5 mL of H2O2 (1.25 mM). Absorbance at 340 nm was recorded at 1, 3, and 6 min. The activity of GPx was expressed in terms of nanomoles GSH consumed per minute per gram of tissue (U/g). The GRx activity was measured by the method of Hafeman et al. [31]. The reaction mixture contained 1.2 mL of 67 mM sodium phosphate buffer (pH 7.0), 0.2 mL of sodium azide (1 mM) and 0.1 mL of oxidized glutathione (7.5 mM). Homogenate (0.5 mL) was added and allowed to equilibrate for 5 min at 37 °C. Reaction was initiated by adding 0.25 mL of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (2 mM). Absorbance at 340 nm was recorded at 1, 2 and 3 min. The activity of GRx was expressed in terms of micromolesmol GSH produced per minute per gram of tissue (U/g). GST was determined using spectrophotometric assay by Alin et al. [32]. It uses 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substance that binds to GSH with the participation of the enzyme and forms a colored GSH-substrate complex. This method measures the total GST activity (cytosolic and microsomal) by measuring the conjugation of CDNB with reduced glutathione; the conjugation is accompanied by an increase in absorbance at 340 nm; the rate of increase is directly proportional to the GST activity in the sample. The activity of GST was expressed as units per gram tissue (1 unit is the amount of enzyme that conjugates 1 nmol of CDNB with GSH/min). The extinct ion coefficient 9.6 mM–1 cm–1 CDNB was used for the calculations.
Total antioxidant capacity Total antioxidant capacity levels of tested tissues were determined using ferric reducing antioxidant power assay (FRAP). The reagent [300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tri(Z-pyridy)-S(riazine, 99%] in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10:1:1 was prepared. FRAP reagent (1.5 mL) was added to 50 μL of homogenated liver, kidney, testis or brain and incubated at 37 °C for exactly 5 min. The change in absorbance was measured at 593 nm due to the formation of a blue-colored FeII-tripyridyltriazine complex from the colorless oxidized FeIII form by the action of electron-donating antioxidants. The absorbance of the sample was read against reagent blank (1.5 mL FRAP reagent and 50 μL distilled water) at 593 nm [33].
Histological evaluations Histological examination of the tissues was conducted after removal of liver, kidney, testis and brain tissues from rats. The tissues were gently rinsed with a physiological saline solution (0.9% NaCl) to remove blood and adhering debris. They were then fixed in 5% formalin for 24 h, and the fixative was removed by washing overnight with running tap water. After dehydration through a graded series of alcohols, the tissues were cleared in methyl benzoate and embedded
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240 Hamza et al.: Blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes
in paraffin. Sections were cut by a microtome at 6 μm thickness and stained with hematoxylin staining as described by Gabe [34] and counterstained with eosin dissolved in 95% ethanol (H&E). After dehydration and clearing, sections were mounted with digital picture exchange and observed under a microscope.
Statistical analysis The SPSS 11.0 statistical software package program for Windows was used for statistical calculations. Data were given in the form of arithmetical mean values and ± standard error. Differences between groups were evaluated by one-way analysis of variance according to p
Reham Z. Hamza, Nahla S. El-Shenawy* and Hayat A.A. Ismail
Protective effects of blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes in the hepatic, renal, testis and brain tissue of male rat Abstract Background: Sodium fluoride (NaF) intoxication is associated with oxidative stress and altered antioxidant defense mechanism. The present study was carried out to evaluate the potential protective role of blackberry and quercetin (Q) against NaF-induced oxidative stress and histological changes in liver, kidney, testis and brain tissues of rats. Methods: The rats were allocated evenly to seven groups. The first group was maintained as the control, whereas groups 2, 3, 4, 5, 6 and 7 were administered blackberry juice (BBJ), Q, NaF, BBJ+NaF, Q+NaF and BBJ+Q+NaF, respectively, for a period of 30 days. Results and conclusions: NaF caused an elevation in lipid peroxidation level paralleled with significant decline in glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase activities as well as the total antioxidant activity in liver, kidney, testes and brain. Some histopathological changes were detected in all tested tissues of the NaF treated group. Q and BBJ had successfully maintained normal histological architecture and mitigated the induction of oxidative stress caused by NaF. Q effectively reduced the elevation in thiobarbituric acid reactive substances level and restored the activities of antioxidant enzymes in liver, kidney, testis and brain. Less histopathological changes were observed in Q+NaF and BBJ+NaF treated groups. As a result, BBJ and Q significantly reduced NaF-induced oxidative and histological changes in rats. In the combination of BBJ and Q against NaF toxicity, the effects were more severe than from separate exposure, thus indicating that these flavonoids exhibited synergistic effects on all antioxidant and histological parameters. *Corresponding author: Nahla S. El-Shenawy, Faculty of Science, Zoology Department, Suez Canal University, Ismailia, 41522, Egypt, E-mail: [email protected] Reham Z. Hamza: Faculty of Science, Zoology Department, Zagazig University, Zagazig, Egypt Hayat A.A. Ismail: Faculty of Science, Biology Department, King Abdel Aziz University, Jeddah, Saudi Arabia
Keywords: blackberry; brain; histology; kidney; liver; oxidative/antioxidant parameters; quercetin; testis. DOI 10.1515/jbcpp-2014-0065 Received June 10, 2014; accepted August 24, 2014; previously published online October 28, 2014
Introduction Fluorosis is a serious public health problem in many parts of the world. As in the case of many chronic degenerative diseases, increased production of reactive oxygen species (ROS) has been considered to play an important role, even in the pathogenesis of chronic sodium fluoride (NaF) toxicity [1]. Studies revealed that fluoride (F) induces excessive production of ROS and might cause depletion in biological activities of some antioxidant enzymes [1–3]. Toxic effects of F on various biochemical parameters are known [2]. Increasing free radical generation and lipid peroxidation (LPO) levels is proposed to mediate the toxic effects of F on soft tissues [4]. Increased LPO and disturbed antioxidant defense systems in brain, erythrocytes and liver of rats exposed to F had been reported [5–7]. Because F has been found to penetrate readily into the brain and to accumulate in the brain tissues [8], daily intake of it for several days is likely to produce neurotoxic effects. Toxic effects in the central nervous system occur after its concentration reaches an adequate level in the brain. Chronic administration of NaF (1 ppm) in rats with drinking water resulted in damage of the hippocampus and Purkinje cells and formation of beta-amyloid plaques (the classic brain abnormality) [9]. Microscopic study showed neurodegeneration in the hippocampus of mice chronically exposed to NaF (100 ppm) [10]. A marked increase in oxidative stress, LPO and a decrease in the activity of antioxidant enzymes were found in the brain regions of rats chronically exposed to 100 ppm of F through drinking water [11]. Chronic F exposure (100 and 200 ppm) has been found to increase LPO and to decrease
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238 Hamza et al.: Blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes the activity of antioxidant enzymes in three generations. Thus, an induction of oxidative stress has been suggested by these investigators as one of the mediatory factors in the toxicity of F in the brain [3]. Fluoride was determined to cause adverse effects in mice on erythrocyte and liver tissue malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Hassan and Abdel-Aziz [12] indicated that NaF-induced oxidative stress as evidenced by elevated levels of LPO and nitric oxide in red blood cells, kidney, testis and brain tissues. Moreover, significantly decreased glutathione (GSH) level, total antioxidant capacity and SOD activity were observed in the previous tissues. NaF-induced blood oxidative stress in mice had been reported [13]. Flavonoids are a major class of phytochemicals found ubiquitously in fruits and vegetables, they are rarely found to be toxic and are highly efficient against ROS-mediated injury [14]. One of the most important natural diets with antioxidant properties is blackberries. Berry fruits, wild or cultivated, have been proven to be a traditional and rich source of bioactive compounds, possessing important biological activities such as flavonoids (anthocyanin), some minerals (Na, K, Ca, Se, Zn and P), vitamins (vitamin A, B complex, C and E), phenolic acids (galic, p-coumaric, caffeic, ferulic) and phenolic polymers (ellagic acids) [15]. The antioxidant capacity of these berries was related to their constituents, particularly total phenolics and anthocyanins [16]. These contents have antioxidants and can improve immunity, playing an antagonistic role of protective agent against toxic substances [17]. On the other hand, the induced oxidative stress and the alterations in antioxidant systems were normalized by the oral administration of blackberry juice (BBJ). Therefore, it can be concluded that blackberry administration could minimize the toxic effects of F indicating its free radical-scavenging and potent antioxidant activities [12]. Quercetin (Q) is a plant-derived flavonoid widely found in vegetables, fruits and cereals. It is the aglycone form of some flavonoid glycosides, such as quercitrin and rutin. Q is characterized by a phenyl benzo(c)pyronederived structure and is mainly found in different foods and food products such as onions and black tea [18]. It exhibits its antioxidant properties against many diseases, including ischemic heart disease, atherosclerosis, liver fibrosis, renal injury and biliary obstruction [19, 20]. Nabavi et al. [21] suggested that Q protects rat liver from NaF-induced oxidative stress, probably via its antioxidant activity. It also protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead [22].
In our previous study, BBJ and Q successfully restored liver functions and normalized kidney functions as well as improved the sex hormone reduction induced by NaF. These two antioxidants also alleviated hematotoxicity as compared with NaF. However, Q exhibited a more pronounced protection toward liver function and hematotoxicity than BBJ. Concurrent intake of BBJ or Q with NaF can mitigate its toxicity. Still, further investigations should be done to estimate the mechanism of phytochemicals to protect different tissues in rats [23]. To the best of our knowledge, there is no scientific report about the protective effect of Q against NaF-induced renal, testis and brain oxidative stress but only on hepatic tissue. Moreover, there are few reports about the uses of BBJ in alleviating adverse effects on NaF. Therefore, it seems of interest to evaluate the antioxidant activity of Q and BBJ separately or in combination as well as their scavenging capacity toward fluoride-induced free radicals in rats. The extent of this effect of BBJ or Q or both of them was estimated by measuring the level of LPO, activities of the antioxidant enzymes (namely, SOD and CAT), the GSH metabolic enzymes activities [GPx, glutathione reductase (GRx) and glutathione S-transferase (GST)] and total antioxidant levels.
Materials and methods Chemicals NaF was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The tested dose of NaF (10.3 mg/kg body weight) was chosen based on the previous studies of Zabulyte et al. [24]. Q was purchased from Sigma Chemical Co. (St. Louis, MO, USA). It was given in a dose of 75 mg/kg according to the previous study by Seivac et al. [25]. Fresh blackberry fruits were obtained from the local market (Zagazig, Egypt) and then washed and homogenized, and its juice was freshly prepared daily. The dose of blackberry (1.6 g/kg body weight equal to 9 mL/kg body weight, which contains 5 mg active constituent, anthocyanine) used in this study was according to the previous studies of Sauebin et al. [26] and Siriwoharn et al. [27]. All other chemicals used in the experiment were analytical grade and were procured from Merck Ltd., SRL Pvt., Ltd., Mumbai, India.
Experimental animals Seventy adult male Wistar albino rats (Rattus rattus) weighing 170–180 g were purchased from King Fahed Medical Research Center in Jeddah (Kingdom of Saudi Arabia). The animals were housed in metal cages and bedded with wood shavings and kept under standard laboratory conditions of aeration and room temperature at about 25 °C with 12/12 light and dark cycle. The animals were allowed to free access of standard diet and water ad libtum throughout the
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/10/15 11:49 AM
Hamza et al.: Blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes 239 experimental period. We have followed the European Community Directive (86/609/EEC) and national rules on animal care that were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals 8th edition. The animals were acclimated to the laboratory conditions for 2 weeks before being experimented on; one group served as controls, and six as the treated groups.
Experimental design After 2 weeks of acclimation, animals were divided into seven groups, ten rats each. All the groups were treated interaperitoneally (i.p.) for 30 successive days. Group 1 served as untreated controls and received 1.0 mL/kg of distilled water i.p. daily. Group 2 was given i.p. BBJ at a dose of 1.6 g/kg body weight containing 5 mg anthocyanine. Group 3 was given Q at a dose of 75 mg/kg body weight. Group 4 was treated with NaF at 10.3 mg/kg body weight. Group 5 was given i.p. BBJ followed by NaF at the same doses. Group 6 was given Q followed by NaF, and finally group 7 was treated with BBJ and Q then followed by NaF at mentioned previous doses.
Preparation of tissues for measurement of oxidative/ antioxidant parameters The tissues of liver, kidney, testis and brain were used for the analysis of oxidative stress and antioxidant parameters. Prior to dissection, tissue was perfused with a 50 mM (sodium phosphate buffer saline (100 mM Na2HPO4/NaH2PO4) (pH 7.4) and 0.1 mM ethylenediaminetetra acetic acid (EDTA) to remove any red blood cells and clots. Then tissues were homogenized in 5 mL cold buffer per gram tissue by a Potter-Elvehjem type homogenizer. The homogenate was centrifuged at 10,000 × g for 20 min at 4 °C, and the resultant supernatant transferred into Eppendorf tubes and preserved in a deep freezer until used. The supernatant was used for the determination of some biochemical parameters of liver, kidney, testis and brain tissues.
Lipid peroxidation assay The extent of LPO was estimated as the concentration of thiobarbit uric acid reactive product MDA by using the method of Ohkawa et al. [28]. MDA concentrations were determined using 1,1,3,3-tetraethoxypropane as standard and expressed as micromoles per gram of tissue.
Antioxidant enzymes SOD activity was measured according to the method described by Marklund and Marklund [29] in which pyrogallol underwent autoxidation at 440 nm for 3 min. One unit of SOD activity was calculated as the amount of protein that caused 50% pyrogallol autoxidation inhibition. The SOD activity was expressed as units per gram tissue. Before determination of the CAT activity, samples were diluted 1:9 with 1% Triton X-100 (v/v). CAT activity was measured according to the method described by Aebi [30]. The hydrolysis of H2O2 and the resulting decrease in absorbance at 240 nm over a 3-min period at 25 °C was measured. CAT activity was expressed as units per gram tissue.
Measurement of enzymes involved in glutathione metabolism The GPx activity was measured by the method of Hafeman et al. [31]. The reaction mixture contained 0.5 mL of 0.4 M sodium phosphate buffer (pH 7.0) and 0.4 mM EDTA, supplemented with 0.25 mL of sodium azide (1 mM), 0.5 mL of GSH (2 mM) and 0.25 mL of distal water. About 0.5 mL of homogenate was added and allowed to equilibrate for 5 min at 37 °C. The reaction was initiated by adding 0.5 mL of H2O2 (1.25 mM). Absorbance at 340 nm was recorded at 1, 3, and 6 min. The activity of GPx was expressed in terms of nanomoles GSH consumed per minute per gram of tissue (U/g). The GRx activity was measured by the method of Hafeman et al. [31]. The reaction mixture contained 1.2 mL of 67 mM sodium phosphate buffer (pH 7.0), 0.2 mL of sodium azide (1 mM) and 0.1 mL of oxidized glutathione (7.5 mM). Homogenate (0.5 mL) was added and allowed to equilibrate for 5 min at 37 °C. Reaction was initiated by adding 0.25 mL of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (2 mM). Absorbance at 340 nm was recorded at 1, 2 and 3 min. The activity of GRx was expressed in terms of micromolesmol GSH produced per minute per gram of tissue (U/g). GST was determined using spectrophotometric assay by Alin et al. [32]. It uses 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substance that binds to GSH with the participation of the enzyme and forms a colored GSH-substrate complex. This method measures the total GST activity (cytosolic and microsomal) by measuring the conjugation of CDNB with reduced glutathione; the conjugation is accompanied by an increase in absorbance at 340 nm; the rate of increase is directly proportional to the GST activity in the sample. The activity of GST was expressed as units per gram tissue (1 unit is the amount of enzyme that conjugates 1 nmol of CDNB with GSH/min). The extinct ion coefficient 9.6 mM–1 cm–1 CDNB was used for the calculations.
Total antioxidant capacity Total antioxidant capacity levels of tested tissues were determined using ferric reducing antioxidant power assay (FRAP). The reagent [300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tri(Z-pyridy)-S(riazine, 99%] in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10:1:1 was prepared. FRAP reagent (1.5 mL) was added to 50 μL of homogenated liver, kidney, testis or brain and incubated at 37 °C for exactly 5 min. The change in absorbance was measured at 593 nm due to the formation of a blue-colored FeII-tripyridyltriazine complex from the colorless oxidized FeIII form by the action of electron-donating antioxidants. The absorbance of the sample was read against reagent blank (1.5 mL FRAP reagent and 50 μL distilled water) at 593 nm [33].
Histological evaluations Histological examination of the tissues was conducted after removal of liver, kidney, testis and brain tissues from rats. The tissues were gently rinsed with a physiological saline solution (0.9% NaCl) to remove blood and adhering debris. They were then fixed in 5% formalin for 24 h, and the fixative was removed by washing overnight with running tap water. After dehydration through a graded series of alcohols, the tissues were cleared in methyl benzoate and embedded
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/10/15 11:49 AM
240 Hamza et al.: Blackberry and quercetin on sodium fluoride-induced oxidative stress and histological changes
in paraffin. Sections were cut by a microtome at 6 μm thickness and stained with hematoxylin staining as described by Gabe [34] and counterstained with eosin dissolved in 95% ethanol (H&E). After dehydration and clearing, sections were mounted with digital picture exchange and observed under a microscope.
Statistical analysis The SPSS 11.0 statistical software package program for Windows was used for statistical calculations. Data were given in the form of arithmetical mean values and ± standard error. Differences between groups were evaluated by one-way analysis of variance according to p
