Faculty of Veterinary Medicine, Mustafa Kemal University,. Antakya-Hatay, Turkey; and 4Department of Cardiology,. Faculty of Medicine, Firat University, Elazig, ...
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Effect of Black Cumin (Nigella sativa) on Heart Rate, Some Hematological Values, and Pancreatic β-Cell Damage in Cadmium-Treated Rats HALIT DEMIR,1 MEHMET KANTER,*,2 OMER COSKUN,2 YESIM HULYA UZ,2 AHMET KOC,3 AND ABDULMELIK YILDIZ 4 1
Department of Chemistry, Faculty of Art and Science, Yuzuncu Yil University, Van, Turkey; 2Department of Histology–Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey; 3Department of Histology–Embryology, Faculty of Veterinary Medicine, Mustafa Kemal University, Antakya-Hatay, Turkey; and 4Department of Cardiology, Faculty of Medicine, Firat University, Elazig, Turkey Received April 5, 2005; Received June 28, 2005; Accepted July 21, 2005
ABSTRACT This study was designed to investigate the effect of Nigella sativa (NS) on the heart rate, some hematological values, and pancreatic β-cell damage in cadmium (Cd)-treated rats. The rats were randomly grouped into one of three experimental groups: Control, Cd treated, and Cd + NS treated. Each group contained 10 animals. The Cd-treated and Cd + NS-treated groups were injected subcutaneously daily with CdCl2 dissolved in isotonic NaCl in the amount of 2 mL/kg for 30 d, resulting in a dosage of 0.49 mg Cd/kg/d. The control group was injected with only isotonic NaCl (2 mL/kg/d) throughout the experiment (for 30 d). Three days prior to administration of CdCl2, the Cd + NS-treated group received the daily intraperitoneal (ip) injection of 2 mL/kg NS until the end of the study; animals in all three groups were fasted for 12 h and blood samples were taken for the determination of the glucose and insulin levels, red blood cell (RBC) and white blood cell (WBC) counts, packet cell volume (PCV), and hemoglobin (Hb) concentration. The heart rates of rats were also measured by a * Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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Demir et al. direct writing electrocardiograph before the blood withdrawals. It was found that NS treatment increased the lowered insulin levels, RBC and WBC counts, PCV, and neutrophil percentage in Cd-treated rats. However, the WBC count of Cd-treated rats with NS treatment was still lower than those of control values. NS treatment also decreased the elevated heart rate and glucose concentration of Cd-treated rats. Pancreatic tissues were also harvested from the sacrificed animals for morphological and immunohistochemical examinations. Cd exposure alone caused a degeneration, necrosis, and weak degranulation in the β-cells of the pancreatic islets. In Cd + NS-treated rats, increased staining of insulin and preservation of islet cells were apparent. It is concluded that NS treatment might decrease the Cdtreated disturbances on heart rate, some hematological values, and pancreatic β-cell. Index Entries: Nigella sativa; cadmium; heart rate; hematological values; pancreatic β-cell; rat.
INTRODUCTION This study was part of our investigations of the responses of Cdtreated oxidative stress in the blood of rats to a volatile oil of Nigella sativa (NS) (1). The heavy metal cadmium (Cd) is an industrial and environmental pollutant that is present in the soil, water, air, food, and cigaret smoke. Cd causes poisoning in various tissues of humans and animals (2,3). After the intake and resorption, Cd enters the blood, where it binds to the erythrocyte membranes and plasma albumin (4). In the blood and tissues, Cd stimulates the formation of metallothioneins (5) and reactive oxygen species (ROS), thus causing oxidative damage in erythrocytes and in various tissues, which results in a loss of membrane functions (6). Cd causes the destruction of erythrocytes, decreases the hematocrit value and hemoglobin concentration, and leads to anemia. Cd also influences the differential blood count (7,8). It has long been recognized, on the other hand, that Cd disturbs glucose homeostasis and induces hyperglycemia in experimental animals (9–11). Decreased insulin release, activation of gluconeogenic enzymes, and reduced insulin receptors have been implicated in Cd-treated hyperglycemia (9,11,12). Angiotensin-converting enzyme activity, blood pressure, and heart rate were all significantly higher in chronic Cd-poisoned rats (13). The heart rate was reduced in the rats exposed to the higher dose of Cd, but no electrocardiographic alteration was demonstrated (14). The effects of acute Cd at three different time-points increased the blood pressure and heart rate of rats (15). Nigella sativa belongs to the plant family Ranunculaceae, with a height of 20–30 cm. The black seed is composed of fixed and volatile oil and saponins. NS contains >30 of a fixed oil and 0.40–0.45 w/w of a volatile oil. The volatile oil has been shown to contain 18.4–24% thymoquinone and 46% several monoterpenes such as p-cymene, and a-pinene (16). Clinical and animal studies have shown that the extracts of the black seeds have Biological Trace Element Research
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many therapeutic effects such as antibacterial (17), immunomodulative (18), anti-inflammatory (19), antitumor (20), antidiabetic (21–24), and antiulcerogenic (25). The aim of this study was to investigate a possible protective effects of NS treatment on the heart rate, some hematological values, and pancreatic β-cell damage in Cd-treated rats.
MATERIAL AND METHODS Plant Material and Extraction Procedure The NS seeds were purchased from a local herb store (Zonguldak, Turkey). Voucher specimens have been kept at the Department of Histology–Embryology, Zonguldak Karaelmas University, Zonguldak, Turkey for the future reference. The seeds of NS were powdered in a mixer. Then 20 g of the powdered seeds were added to 400 mL of distilled water and the extraction was carried out by steam distillation. The process of distillation was continued until about 200 mL of the distillate were collected. The distillate was extracted three times with chloroform. Moisture was removed by anhydrous sodium sulfate and the resultant extract was evaporated using a water bath (40°C); this led to the appearance of the volatile oil. The products of various extractions were pooled together, giving an average yield of 0.2%. Five hundred milligrams of the volatile oil were dissolved by the initial addition of 1 mL of dimethyl sulfoxide (DMSO), followed by the addition of 9 mL of normal saline to yield a concentration of 50 mg volatile oil per 1-mL solution (26).
Treatment of Rats Thirty healthy male Wistar albino rats, weighing 200–250 g (aged 4 mo) and averaging 16 wk old were supplied from The Center of Medical Investigations of Yuzuncu Yil University. The animals were given standard rat pellets (Murat Animal Food Product Co., Ankara, Turkey) and tap water ad libitum. The rats were housed singly in temperature-controlled (20–25°C) cages. They were housed in Macrolon cages under standard laboratory conditions (light period 7:00 AM to 7:00 PM, 21±2°C, relative humidity 55%). The rats were randomly divided in three experimental groups: Control, Cd treated, and Cd + NS treated. Each group contained 10 animals. The Cd-treated and Cd + NS-treated groups were injected subcutaneously daily with CdCl2 dissolved in isotonic NaCl in the amount of 2 mL/kg for 30 d, resulting in a dosage of 0.49 mg Cd/kg/d (27). The control group was injected with only isotonic NaCl (2 mL/kg/d) throughout the experiment (for 30 d). Three days prior to administration of CdCl2, the Cd + NS-treated group received a daily intraperitoneal (ip) injection of 2 mL/kg NS until the end of the study. The dose of NS volatile oil was chosen on the basis of a previous study (24). At the end of the experiment, rats Biological Trace Element Research
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in all groups were starved overnight for 12 h and sacrificed under chloralhydrate (6 mL of 7% chloralhydrate/kg; Sigma, St. Louis, MO, USA) anesthesia. Blood samples were collected by cardiac puncture using a heparinized syringe. 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.
Hematological Analysis At the end of the experimental period (30 d), blood samples of animals in all three groups were taken for the determination of the glucose and insulin levels, red blood cell (RBC) and white blood cell (WBC) counts, packet cell volume (PCV), and hemoglobin (Hb) concentration. Serum glucose concentration was measured immediately by the glucose oxidized method (28), and insulin was determined by a double-antibody radioimmunoassay kit (Amersham Radiochemical Centre, Bucks, UK). The PCV was measured by a microhematocrit centrifuge. Hb concentration was determined by the cyanmethemoglobin technique. The RBC and WBC counting methods were based on the dilution of obtained blood with diluting fluids (Hayem and Turk) in RBC and WBC counting pipets. Individual cells were then counted in the counting chamber (hemocytometer). Giemsa’s staining method was used for the differential count of RBC. Heart rates of rats were measured by a direct writing electrocardiograph (Cardiofax 02611; Nihon Kohden, Tokyo) before the blood withdrawals.
Histopathological Procedures Pancreatic tissues were harvested from the animals, and tissue fragments were fixed in 10% neutral formaline solution, embedded in paraffin, and then stained with hematoxylin and eosin (H&E). The preparations were evaluated by means of a bright-field microscope and photographed (Optiphot 2; Nikon, Tokyo, Japan).
Immunohistochemical Procedures Harvested pancreatic tissues were fixed in 10% neutral buffered formaline, embedded in paraffin, and sectioned at 5-µm thickness. Immunocytochemical reactions were performed according to the ABC technique described by Hsu et al. (29). Specific monoclonal mouse antisera against human insulin protein (18-0066; Zymed, San Francisco, CA) were applied at a dilution of 1 : 50. The procedure involved the following steps: (1) Endogenous peroxidase activity was inhibited by 3% H2O2 in distilled water for 30 min; (2) the sections were washed in tap water for 30 min and in distilled water for 10 min; (3) nonspecific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907; Carpinteria, CA) with phosphate-buffered saline (PBS), diluted 1 : 4; (4) sections were Biological Trace Element Research
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incubated with monoclonal mouse antisera against human insulin protein, diluted 1 : 400 for 2 h, and then at room temperature; (5) sections were washed in PBS three times for 3 min each; (6) sections were incubated with biotinylated anti-mouse IgG (DAKO LSAB 2 Kit; (7) sections were washed in PBS three times for 3 min each; (8) sections were incubated with ABC complex (DAKO LSAB 2 Kit); (9) sections were washed in PBS three times for 3 min each; (10) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories); (11) sections were washed in tap water for 10 min and then dehydrated; (12) nuclei were stained with hematoxylin; and (13) sections were mounted in DAKO paramount.
Image Analysis The system we used consisted of a PC with hardware and software (Image-Pro Plus 5.0; Media Cybernetics, Silver Spring, MD) for image acquisition and analysis, a Spot Insight QE (Diagnostic Instruments, Silver Spring, MD) camera, and an optical microscope. The method requires preliminary software procedures involving spatial calibration (on a micron scale) and setting of color segmentation for quantitative color analysis. Ten Langerhans islets from each rat (100 islets for each group) were chosen randomly. The intensity of staining with anti-insulin antibodies of β-cells in pancreatic islets of control, Cd-treated, and Cd + NS treated groups was scored as 0 (absent), 1 (weak), 2 (moderate), 3 (strong), or 4 (very strong) in the image analysis system. Then the percentage of the insulin-immunoreactive β-cell area in the Langerhans islets (100 islets for each group) was estimated. The percentage of the insulin-immunoreactive β-cells was calculated according to these results. The investigator who obtained these measurements was unaware of the experiment being performed.
Statistical Analysis The data were expressed as the mean ± standard deviation (SD) and analyzed by repeated measures of variance. A Tukey test was used to test for differences among means when an analysis of variance (ANOVA) indicated a significant (p
