Biol Trace Elem Res DOI 10.1007/s12011-014-9968-9
Protective Effects of Onion Extract on Cadmium-Induced Oxidative Stress, Histological Damage, and Apoptosis in Rat Heart Seref Alpsoy & Mehmet Kanter & Cevat Aktas & Mustafa Erboga & Aydın Akyuz & Dursun Cayan Akkoyun & Mustafa Oran
Received: 7 February 2014 / Accepted: 2 April 2014 # Springer Science+Business Media New York 2014
Abstract To date, there is no available information on the protective effect of onion (Allium cepa) extract (AcE) on cadmium (Cd)-induced cardiotoxicity. The present study was performed to assess the possible antioxidant and antiapoptotic roles of AcE in Cd-induced cardiotoxicity in rats. A Cd group was injected subcutaneously with CdCl2 dissolved in saline at a dose of 2 ml/kg/day for 30 days, resulting in a dosage of 1 mg/kg Cd. The rats in the AcE-treated group were given 1 ml of AcE via intragastric intubation for 30 days. The rats intoxicated with Cd for 30 days showed increased tissue malondialdehyde (MDA) levels and decreased levels of the enzymatic antioxidants superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in cardiac tissue. AcE attenuated these adverse effects of Cd. After Cd exposure, histological abnormalities were observed, including myofibrillar loss, vacuolization of cytoplasm and irregularity of myofibrils. These histological alterations were effectively attenuated by the treatment with AcE. Furthermore, our data indicate a significant reduction of apoptosis in the cardiomyocytes of the Cd group treated with AcE therapy. Animal studies show antioxidant effects of AcE. But to date, S. Alpsoy : A. Akyuz : D. C. Akkoyun Department of Cardiology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey M. Kanter (*) Department of Histology and Embryology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul, Turkey e-mail: [email protected] C. Aktas : M. Erboga Department of Histology and Embryology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey M. Oran Department of Internal Medicine, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey
no study reported the effect of AcE on biochemical and histopathological changes due to Cd induced on rat heart. Our study showed that AcE therapy reduced Cd-induced oxidative stress and apoptosis, possibly through its antioxidant and anti-apoptotic activity. Keywords Cadmium . Cardiotoxicity . Allium cepa . TUNEL . Oxidative stress
Introduction Cadmium (Cd) causes a number of clinical complications, including liver and kidney injury, disruption of the endocrine system, anemia, diabetes, respiratory diseases, neurological disorders, skeletal system damage, and reproductive system damage [1, 2]. Heart disease is a leading cause of death worldwide. There are a number of risk factors associated with different types of heart diseases. There is an increasing body of evidence indicating an association between Cd exposure and an augmented risk of cardiovascular disease [3]. Studies in animals have implicated Cd in the etiology and pathogenesis of hypertension and cardiotoxicity [4, 5]. Several experimental studies have shown that Cd induces oxidative stress in rats [6, 7]. Onions (Allium cepa) belong to the genus Allium, and they are one of the most widely and largely consumed vegetables. Studies have documented the antioxidant value of A. cepa extract (AcE) [8]. According to Hertog et al. [9], onion consumption is related to low rates of coronary heart disease. The antioxidant effect of AcE has been associated with reduced malondialdehyde (MDA) and increased superoxide dismutase (SOD) and catalase (CAT) levels [8]. The therapeutic and medicinal values of AcE have been the subjects of many studies. Several studies have shown their functional health
Alpsoy et al.
benefits in the reduction of cardiovascular disease risk by lowering serum cholesterol and blood pressure, in addition to their antioxidant, anticarcinogenic, antimutagenic, antidiabetic, anti-asthmatic, immunomodulatory, antimicrobial, and prebiotic effects [10]. These studies suggest that AcE may play a beneficial role during Cd-induced cardiotoxicity. To our knowledge, the effect of AcE on Cd-induced cardiac tissue impairment has not been reported yet. Taking into account all the above observations, this study was designed to investigate the protective effects of AcE against Cd-induced biochemical, histopathological, and apoptotic impairment in rats.
Material and Methods Animals Twenty-four healthy male Sprague-Dawley rats, weighing 250–300 g and averaging 16 weeks old, were utilized in our study. The animals were purchased from Trakya University Animal Care and Research Unit. Rats were fed on a standard rat chow and tap water ad libitum. In the windowless animal quarter, automatic temperature (22±2 °C) and lighting controls (light on at 7 a.m. and off at 9 p.m.: 14 h light/10 h dark cycle) was performed. Humidity ranged from 50 to 55 %. All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. The study was approved by the institutional animal ethical committee of Trakya University, Edirne, Turkey (protocol number: TÜHDYEK-2012/07). Experimental Design and Samples Collection A total of 24 male Sprague-Dawley rats were allotted into three groups as follows: control, Cd, and Cd treated with AcE; 8 animals of each. AcE extract was prepared following a method described by Nelson et al. [11]. The onions were washed with clean sterile distilled water and allowed to air dry for 1 h. The outer covering of the onion were manually peeled off. Exactly 200 g of fresh onion bulbs were blended and the raw juice was extracted after standing in a clean glass contained for 24 h. Juice was then filtrated and squeezed out of it. The extract was stored bellow 4 °C until given to rats. The rats in AcE-treated group were given 1 ml AcE (freshly prepared) by using intragastric (i.g.) intubation for 30 days starting at the same time first Cd injection. Control group was given the same volume of saline. This application continued daily for a total of 30 days. To induce cardiotoxicity, the Cd group was injected subcutaneously with CdCl2 dissolved in saline at a dose of 2 ml/kg/day for 30 days, resulting in a
dosage of 1 mg/kg Cd (Sigma, St. Louis, MO, USA). At the end of the experiment, all animals were anesthetized by intraperitoneal administration of 90 mg/kg ketamine and 10 mg/kg xylazine. The anesthetized rats were sacrificed and the cardiac tissue was evaluated for LPO products, antioxidant enzymes, and morphological appearance.
Chemical Content Analysis of Ace An onion extract weight gain control composition includes from 20 to 40 % by weight of polyphenols belonging to the flavonol family, the onion extract having the following composition characteristics, expressed in weight based on the dry extract total weight: from 60 to 90 % by weight of the total flavonols in the extract consist in glycosylated polyphenols, from 85 to 98 % by weight of the total flavonols in the extract consist in quercetin in a free form (aglycone) or in a glycosylated form, from 5 to 20 % by weight of the total flavonols in the extract consist in quercetin-3,4′-diglucoside, from 30 to 70 % by weight of the total flavonols in the extract consist in quercetin-3-monoglucoside and quercetin-4′-monoglucoside, and from 20 to 30 % by weight of the total flavonols in the extract consist in quercetin (Table 1) [12].
Biochemical Analysis Preparation of Tissue Samples All tissues were weighed and homogenized with 0.15 M KCl solution and 10 % homogenates (w/v) of these tissues were prepared. Tissue homogenates were centrifuged for 10 min at 4 °C cold centrifuge 600×g. Supernatant was centrifuged for 20 min at 10,000×g so postmitochondrial fraction was obtained. MDA level of tissues were determined in tissue homogenates; SOD and GSH-Px activities were examined in postmitochondrial fraction of these homogenates. The bicinchoninic acid method was used for determining the amount of protein in samples [13].
Table 1 Chemical content analysis of AcE Molecules
Weight of total flavonols (%)
Polyphenols Glycosylated polyphenols Quercetin (aglycone or glycosylated) Quercetin-3,4′-diglucoside Quercetin 3-monoglucoside and quercetin-4′-monoglucoside Quercetin
20 to 40 60 to 90 85 to 98 5 to 20 30 to 70 20 to 30
Protective Effects of Onion on Cadmium-Induced Heart Damage
Lipid Peroxidation Determination MDA, as an endpoint of lipid peroxidation (LPO), was calculated by detecting absorbance of thiobarbituric acid reactive substances at 532 nm [14]. MDA levels were expressed as MDA nmol/mg protein. Superoxide Dismutase Determination SOD activity was measured by principle of increasing the ability of photooxidation rate in o-dianisidin sensitived with riboflavin [15]. Colored product was measured spectrophotometrically at 460 nm, and the results IU/mg protein, as specified. Catalase Determination Catalase (CAT) activity was determined according to Aebi’s method [16]. The principle of the method was based on the determination of the rate constant (s−1, k) of the H2O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) per mg protein. Glutathione Peroxidase Determination GSH-Px activity was measured according to the protocol of Lawrence et al. [17]. The results were calculated using NADPH in extinction coefficient and nmol NADPH/mg protein/min were expressed. Histopathologic Examination Cardiac tissues were harvested from the sacrificed animals, and the tissues were fixed in 10 % neutral formalin and embedded in paraffin blocks. Sections of 5 μm were obtained, deparaffinized, and stained with hematoxylin and eosin (H&E). The cardiac tissue was examined, evaluated, and photographed in random order under blindfold conditions with standard light microscopy (Nikon Optiphot 2, Tokyo). The severity of changes was quantitated none (−) to severe (+++) based on the degree of cytoplasmic vacuolization, myocardial disorganization, and myofibrillar loss. The scoring system was as follows: (−) no damage, (+) mild, (++) moderate, and (+++) severe. Evaluation of Cardiomyocytes Apoptosis Cardiomyocytes apoptosis were evaluated by the TUNEL assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was employed using an apoptosis detection kit (TdT-Fragel™ DNA Fragmentation Detection Kit, cat. no. QIA33, Calbiochem, USA). All reagents listed below are from the
kit and were prepared following the manufacturer’s instructions. Five-micrometer-thick cardiac tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series as described previously. The sections were then incubated with 20 mg/ml proteinase K for 20 min and rinsed in PBS. Endogenous peroxidase activity was inhibited by incubation with 3 % hydrogen peroxide. Sections were then incubated with equilibration buffer for 10–30 s and then TdTenzyme, in a humidified atmosphere at 37 °C, for 90 min. Sections were subsequently put into prewarmed working strength stop/wash buffer at room temperature for 10 min and incubated with blocking buffer for 30 min. Each step was separated by thorough washes in PBS. Labeling was revealed using DAB, counterstaining was performed using methyl green, and sections were dehydrated, cleared, and mounted. TUNEL positive cells were counted, and the TUNEL positive cells/100 cardiomyocyte percentage was used as the index of apoptosis. This percentage represented the apoptotic index of the sample and was compared between groups. Statistical Analysis All statistical analyses were performed using PASWR Statistics 18 for Windows. All data were presented in mean (±) standard error of the mean (SEM). Differences in measured parameters among the three groups were analyzed with a nonparametric test (Kruskal–Wallis). Dual comparisons between groups exhibiting significant values were evaluated with a Mann–Whitney U test. These differences were considered significant when probability was less than 0.05.
Results Biochemical Findings Lipid Peroxidation Levels of Cardiac Tissues Cd-induced oxidative stress resulted in significant increases in the cardiac level of MDA (a marker of LPO) when compared to the control group (P
Protective Effects of Onion Extract on Cadmium-Induced Oxidative Stress, Histological Damage, and Apoptosis in Rat Heart Seref Alpsoy & Mehmet Kanter & Cevat Aktas & Mustafa Erboga & Aydın Akyuz & Dursun Cayan Akkoyun & Mustafa Oran
Received: 7 February 2014 / Accepted: 2 April 2014 # Springer Science+Business Media New York 2014
Abstract To date, there is no available information on the protective effect of onion (Allium cepa) extract (AcE) on cadmium (Cd)-induced cardiotoxicity. The present study was performed to assess the possible antioxidant and antiapoptotic roles of AcE in Cd-induced cardiotoxicity in rats. A Cd group was injected subcutaneously with CdCl2 dissolved in saline at a dose of 2 ml/kg/day for 30 days, resulting in a dosage of 1 mg/kg Cd. The rats in the AcE-treated group were given 1 ml of AcE via intragastric intubation for 30 days. The rats intoxicated with Cd for 30 days showed increased tissue malondialdehyde (MDA) levels and decreased levels of the enzymatic antioxidants superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in cardiac tissue. AcE attenuated these adverse effects of Cd. After Cd exposure, histological abnormalities were observed, including myofibrillar loss, vacuolization of cytoplasm and irregularity of myofibrils. These histological alterations were effectively attenuated by the treatment with AcE. Furthermore, our data indicate a significant reduction of apoptosis in the cardiomyocytes of the Cd group treated with AcE therapy. Animal studies show antioxidant effects of AcE. But to date, S. Alpsoy : A. Akyuz : D. C. Akkoyun Department of Cardiology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey M. Kanter (*) Department of Histology and Embryology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul, Turkey e-mail: [email protected] C. Aktas : M. Erboga Department of Histology and Embryology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey M. Oran Department of Internal Medicine, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey
no study reported the effect of AcE on biochemical and histopathological changes due to Cd induced on rat heart. Our study showed that AcE therapy reduced Cd-induced oxidative stress and apoptosis, possibly through its antioxidant and anti-apoptotic activity. Keywords Cadmium . Cardiotoxicity . Allium cepa . TUNEL . Oxidative stress
Introduction Cadmium (Cd) causes a number of clinical complications, including liver and kidney injury, disruption of the endocrine system, anemia, diabetes, respiratory diseases, neurological disorders, skeletal system damage, and reproductive system damage [1, 2]. Heart disease is a leading cause of death worldwide. There are a number of risk factors associated with different types of heart diseases. There is an increasing body of evidence indicating an association between Cd exposure and an augmented risk of cardiovascular disease [3]. Studies in animals have implicated Cd in the etiology and pathogenesis of hypertension and cardiotoxicity [4, 5]. Several experimental studies have shown that Cd induces oxidative stress in rats [6, 7]. Onions (Allium cepa) belong to the genus Allium, and they are one of the most widely and largely consumed vegetables. Studies have documented the antioxidant value of A. cepa extract (AcE) [8]. According to Hertog et al. [9], onion consumption is related to low rates of coronary heart disease. The antioxidant effect of AcE has been associated with reduced malondialdehyde (MDA) and increased superoxide dismutase (SOD) and catalase (CAT) levels [8]. The therapeutic and medicinal values of AcE have been the subjects of many studies. Several studies have shown their functional health
Alpsoy et al.
benefits in the reduction of cardiovascular disease risk by lowering serum cholesterol and blood pressure, in addition to their antioxidant, anticarcinogenic, antimutagenic, antidiabetic, anti-asthmatic, immunomodulatory, antimicrobial, and prebiotic effects [10]. These studies suggest that AcE may play a beneficial role during Cd-induced cardiotoxicity. To our knowledge, the effect of AcE on Cd-induced cardiac tissue impairment has not been reported yet. Taking into account all the above observations, this study was designed to investigate the protective effects of AcE against Cd-induced biochemical, histopathological, and apoptotic impairment in rats.
Material and Methods Animals Twenty-four healthy male Sprague-Dawley rats, weighing 250–300 g and averaging 16 weeks old, were utilized in our study. The animals were purchased from Trakya University Animal Care and Research Unit. Rats were fed on a standard rat chow and tap water ad libitum. In the windowless animal quarter, automatic temperature (22±2 °C) and lighting controls (light on at 7 a.m. and off at 9 p.m.: 14 h light/10 h dark cycle) was performed. Humidity ranged from 50 to 55 %. All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. The study was approved by the institutional animal ethical committee of Trakya University, Edirne, Turkey (protocol number: TÜHDYEK-2012/07). Experimental Design and Samples Collection A total of 24 male Sprague-Dawley rats were allotted into three groups as follows: control, Cd, and Cd treated with AcE; 8 animals of each. AcE extract was prepared following a method described by Nelson et al. [11]. The onions were washed with clean sterile distilled water and allowed to air dry for 1 h. The outer covering of the onion were manually peeled off. Exactly 200 g of fresh onion bulbs were blended and the raw juice was extracted after standing in a clean glass contained for 24 h. Juice was then filtrated and squeezed out of it. The extract was stored bellow 4 °C until given to rats. The rats in AcE-treated group were given 1 ml AcE (freshly prepared) by using intragastric (i.g.) intubation for 30 days starting at the same time first Cd injection. Control group was given the same volume of saline. This application continued daily for a total of 30 days. To induce cardiotoxicity, the Cd group was injected subcutaneously with CdCl2 dissolved in saline at a dose of 2 ml/kg/day for 30 days, resulting in a
dosage of 1 mg/kg Cd (Sigma, St. Louis, MO, USA). At the end of the experiment, all animals were anesthetized by intraperitoneal administration of 90 mg/kg ketamine and 10 mg/kg xylazine. The anesthetized rats were sacrificed and the cardiac tissue was evaluated for LPO products, antioxidant enzymes, and morphological appearance.
Chemical Content Analysis of Ace An onion extract weight gain control composition includes from 20 to 40 % by weight of polyphenols belonging to the flavonol family, the onion extract having the following composition characteristics, expressed in weight based on the dry extract total weight: from 60 to 90 % by weight of the total flavonols in the extract consist in glycosylated polyphenols, from 85 to 98 % by weight of the total flavonols in the extract consist in quercetin in a free form (aglycone) or in a glycosylated form, from 5 to 20 % by weight of the total flavonols in the extract consist in quercetin-3,4′-diglucoside, from 30 to 70 % by weight of the total flavonols in the extract consist in quercetin-3-monoglucoside and quercetin-4′-monoglucoside, and from 20 to 30 % by weight of the total flavonols in the extract consist in quercetin (Table 1) [12].
Biochemical Analysis Preparation of Tissue Samples All tissues were weighed and homogenized with 0.15 M KCl solution and 10 % homogenates (w/v) of these tissues were prepared. Tissue homogenates were centrifuged for 10 min at 4 °C cold centrifuge 600×g. Supernatant was centrifuged for 20 min at 10,000×g so postmitochondrial fraction was obtained. MDA level of tissues were determined in tissue homogenates; SOD and GSH-Px activities were examined in postmitochondrial fraction of these homogenates. The bicinchoninic acid method was used for determining the amount of protein in samples [13].
Table 1 Chemical content analysis of AcE Molecules
Weight of total flavonols (%)
Polyphenols Glycosylated polyphenols Quercetin (aglycone or glycosylated) Quercetin-3,4′-diglucoside Quercetin 3-monoglucoside and quercetin-4′-monoglucoside Quercetin
20 to 40 60 to 90 85 to 98 5 to 20 30 to 70 20 to 30
Protective Effects of Onion on Cadmium-Induced Heart Damage
Lipid Peroxidation Determination MDA, as an endpoint of lipid peroxidation (LPO), was calculated by detecting absorbance of thiobarbituric acid reactive substances at 532 nm [14]. MDA levels were expressed as MDA nmol/mg protein. Superoxide Dismutase Determination SOD activity was measured by principle of increasing the ability of photooxidation rate in o-dianisidin sensitived with riboflavin [15]. Colored product was measured spectrophotometrically at 460 nm, and the results IU/mg protein, as specified. Catalase Determination Catalase (CAT) activity was determined according to Aebi’s method [16]. The principle of the method was based on the determination of the rate constant (s−1, k) of the H2O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) per mg protein. Glutathione Peroxidase Determination GSH-Px activity was measured according to the protocol of Lawrence et al. [17]. The results were calculated using NADPH in extinction coefficient and nmol NADPH/mg protein/min were expressed. Histopathologic Examination Cardiac tissues were harvested from the sacrificed animals, and the tissues were fixed in 10 % neutral formalin and embedded in paraffin blocks. Sections of 5 μm were obtained, deparaffinized, and stained with hematoxylin and eosin (H&E). The cardiac tissue was examined, evaluated, and photographed in random order under blindfold conditions with standard light microscopy (Nikon Optiphot 2, Tokyo). The severity of changes was quantitated none (−) to severe (+++) based on the degree of cytoplasmic vacuolization, myocardial disorganization, and myofibrillar loss. The scoring system was as follows: (−) no damage, (+) mild, (++) moderate, and (+++) severe. Evaluation of Cardiomyocytes Apoptosis Cardiomyocytes apoptosis were evaluated by the TUNEL assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was employed using an apoptosis detection kit (TdT-Fragel™ DNA Fragmentation Detection Kit, cat. no. QIA33, Calbiochem, USA). All reagents listed below are from the
kit and were prepared following the manufacturer’s instructions. Five-micrometer-thick cardiac tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series as described previously. The sections were then incubated with 20 mg/ml proteinase K for 20 min and rinsed in PBS. Endogenous peroxidase activity was inhibited by incubation with 3 % hydrogen peroxide. Sections were then incubated with equilibration buffer for 10–30 s and then TdTenzyme, in a humidified atmosphere at 37 °C, for 90 min. Sections were subsequently put into prewarmed working strength stop/wash buffer at room temperature for 10 min and incubated with blocking buffer for 30 min. Each step was separated by thorough washes in PBS. Labeling was revealed using DAB, counterstaining was performed using methyl green, and sections were dehydrated, cleared, and mounted. TUNEL positive cells were counted, and the TUNEL positive cells/100 cardiomyocyte percentage was used as the index of apoptosis. This percentage represented the apoptotic index of the sample and was compared between groups. Statistical Analysis All statistical analyses were performed using PASWR Statistics 18 for Windows. All data were presented in mean (±) standard error of the mean (SEM). Differences in measured parameters among the three groups were analyzed with a nonparametric test (Kruskal–Wallis). Dual comparisons between groups exhibiting significant values were evaluated with a Mann–Whitney U test. These differences were considered significant when probability was less than 0.05.
Results Biochemical Findings Lipid Peroxidation Levels of Cardiac Tissues Cd-induced oxidative stress resulted in significant increases in the cardiac level of MDA (a marker of LPO) when compared to the control group (P
