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Biochemistry
Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats Mohamed E. El-Boshy a,b,∗∗ , Engy F. Risha b , Fatma M. Abdelhamid b , Mohammad S. Mubarak c , Taibi Ben Hadda d,∗ a
Laboratory Medicine Department, Faculty of Applied Medical Science, Umm Al-Qura University, PB 7296, Makkah 21955, Saudi Arabia Clinical Pathology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt c Department of Chemistry, The University of Jordan, Amman 11942, Jordan d Laboratoire de Chimie des Matériaux (LCM), Faculté des Sciences, Université Mohammed Premier, Oujda 60000, Morocco b
a r t i c l e
i n f o
Article history: Received 25 March 2014 Accepted 26 May 2014 Keywords: Selenium Cadmium Immunosuppressive Antioxidant Rats
a b s t r a c t Cadmium is a non-essential toxic metal used in industrial process, causes severe risk to human health. Selenium (Se) is an essential trace mineral of fundamental importance for human health. Selenium has antioxidant enzymes roles and is needed for the proper function of the immune system. In this study, the protective effects of selenium against cadmium intoxication in rats have been investigated by monitoring some selective cytokines (IL-1, TNF ␣, IL-6, IL-10 and IFN-␥), antioxidant enzymes reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation malondialdehyde (MDA) as well as some selective biochemical markers of liver and kidney functions. Thirty-two rats were divided into four equal groups; the first group was used as a control. Groups 2–4 were treated with selenium (Se; 0.1 mg/kg BW), cadmium (Cd; 40 mg/L drinking water) and selenium plus cadmium, respectively. Rats were orally administered their relevant doses daily for 30 days. Blood samples were collected from heart puncture at the end of the experiment (30 days) for complete blood picture (CBC) and serum was separated to evaluate the different immunological parameters and biochemical parameters, as well as liver specimens for Cd and Se estimation. Rats in the Cd treated group have a significantly higher hepatic concentration of Cd than in other treated groups. Results revealed that cadmium significantly increased IL-1, TNF ␣, IL-6 and IL-10, beside peripheral neutrophils count, while the IFN-␥ and lymphocytes were decreased in rat sera. In addition, GSH level, CAT, SOD and GPx activities were significantly decreased while lipid peroxidation (MDA) was increased. Regarding, liver and renal markers, they were significantly increased in the activities of aminotransferases (AST, ALT), urea and creatinine, while total plasma proteins and albumin were significantly decreased. On the other hand, selenium treated group, showed significantly increased IFN-␥, GSH level, CAT, and GPx activities, as well as lymphocyte count while IL-10 was decreased. Selenium in combination with cadmium, significantly improved the elevation of serum IL-1, IL-6, TNF ␣, IL-10 and malondialdehyde in addition to enhancing the antioxidant enzyme activities of GSH, CAT, GPx and SOD. Moreover, selenium has ameliorated the cadmium-induced liver and kidney damage by improving hepatic and renal markers. The results of this investigation demonstrated that selenium has the potential to countermeasure the immunosuppressive as well as hepatic and renal oxidative damage induced by cadmium in rats; selenium has shown promising effects against Cd toxicity. © 2014 Elsevier GmbH. All rights reserved.
Introduction ∗ Corresponding author. Tel.: +212 0666134178; fax: +212 05 36 50 06 03. ∗∗ Co-corresponding author at: Laboratory Medicine Department, Faculty of Applied Medical Science, Umm Al-Qura University, PB 7296, Makkah 21955, Saudi Arabia. Tel.: +966 0563025297. E-mail addresses: dr [email protected] (M.E. El-Boshy), [email protected], [email protected] (T.B. Hadda).
Adverse immune effects of chemicals, defined as immunotoxicity, has been used as a sensitive biomarker for assessing health effect of environmental pollution. Immunotoxic effects of nonessential toxic metal, as a typical environmental agent and their mechanism are areas of interest. Cadmium (Cd) is one of the most
http://dx.doi.org/10.1016/j.jtemb.2014.05.009 0946-672X/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: El-Boshy ME, et al. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.05.009
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toxic non-essential toxic metal, an environmental and occupational pollutant endangering human and animal health [1,2]. It has been suggested that Cd may be an environmental risk factor for osteoporosis and fertility as well as nephrotoxicity, hepatotoxicity, and immunosuppressiveness [3–7]. The immunotoxic effects of Cd on the development of immune organs, the differentiation of immune cells, and specific and non-specific immune responses have been reported by Koller [8]. This indicates an impact on the immune system as a whole, which can lead to a significant decrease in host resistance. Moreover, some of the specific changes that lead to tissue damage and death from chronic exposure to Cd have been related to oxidative stress [10]. The associated toxic effects of cadmium attributed to suppression of free radical scavenging function and the enhancement of ROS contribute to Cd induced oxidative stress and lipid peroxidation [10–12]. Selenium (Se) is an essential trace element for both animal and human begins. It has been detected that selenium function through participates in formation of selenoproteins, this element contributes in various biological processes such as antioxidant defense, thyroid hormone production, and immune responses [13,14]. Some reports indicate that a deficiency in selenium may be flat to certain diseases [15]. It is generally recognized to be of great importance for human health, protecting the cells from the harmful effects of free radical production [16]. Adverse health effects following selenium overexposure, although very rare, have been found in animals and people [15]. Selenium interaction with cadmium has been reported; this interaction may reduce cadmium accumulation and subsequent reduce its toxicity in the body [15]. Although many of the studies that have been carried out until now reported on the different adverse health effects of Cd, such as its nephrotoxicity, hepatotoxicity, and immunosuppressiveness, however, none has dealt with the association between cadmium and selenium. Therefore, the present study was conducted to shed some light on the relationship between the immunosuppressive, renal and hepatic damage, hematological disturbances as well as oxidative stress effects of cadmium and the putative protective effects of selenium in rats. Materials and methods Experimental animals The experiment was conducted in accordance with animal welfare and under ethical protocols by the Faculty of Veterinary medicine, Mansoura university, Egypt. Forty rat weight (160 ± 10 g), housed in a plastic cages 50 cm × 30 cm × 10 cm and placed in the room for 10 days for adaptation. The room temperature was maintained at 24 ± 2 ◦ C, the air of the room was changed continuously by using ventilated vacuum and with light\dark cycle of 12:12 h per day. The animal was fed on a pellet diet. Chemicals Sodium selenite (Na2 SeO3 ) and cadmium chloride anhydrous (CdCl2 ), which were analytical grade (≥99%) and were obtained from the Sigma Aldrich Chemical Co. (St. Louis, Missouri, USA). All chemicals and reagents used in this study were of analytical reagent grade.
used according to published literature procedures [17]. Group 4 was orally administered Cd at doses of 40 mg/L and Se at a dose of 0.1 mg/kg BW.
Samples collection and parameters measured The blood samples were collected in test tubes contained disodium salt of ethylene diamine tetra acetic acid (EDTA) anticoagulant and used for determination of red blood cell counts (RBCs), hemoglobin (Hb) concentration, Hematocrit (HCT) and erythrocyte indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), in addition, white blood cells (WBCs) and platelets (PLTs). Three blood films were made for each blood sample. Blood films were stained with Giemsa stain and differential leukocyte count was done by standard hematological techniques. The second blood samples were collected in test tubes without anticoagulant. The samples were centrifuged at 3000 rpm for 10 min and the clear serum was carefully separated from all samples. Selective humoral immunological parameters (Tumor necrosis factor – ␣ (TNF ␣), Interleukin 1 (IL-1), IL-6, IL-10 and gamma interferon (IFN-␥) were determined by Enzyme Amplified Sensitivity Immunoassay (EASIA, R & D Systems, Minneapolis, MN, USA) using microplates according to enclosed pamphlets (Aushon Searchlight Biosystem (Billerica, MA). The lower detection limits are 1.5 pg/mL for IL-1, 6.3 pg/mL for IL-6, 5.4 pg/mL for IL-10, 3.1 pg/mL for TNF␣, and 6.2 pg/mL for IFN-␥ and data are presented as pg cytokine/mL serum. Antioxidant markers, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), oxidized glutathione, (GPx) and malondialdehyde (MDA), were determined from undiluted serum samples using commercially available ELISA Kits (Cayman Chemical Co.). The plates were read at 450 nm and a correction wavelength of 550 nm on a computerized automated microplate ELISA reader. ALT, AST, alkaline phosphatase (ALP) activities were assayed by using commercial kits (Human, Diagnostic Co. Germany), while total protein, albumin, uric acid, urea and creatinine (Crescent Diagnostic Co. KSA) were estimated spectrophotometrically (BM Co. Germany, 5010) according to enclosed pamphlets.
Cadmium and selenium measurement The concentration of Cd in the liver was determined by atomic absorption spectrophotometry (Perkin-Elmer A.A. Model 800) with Zeeman-effect for background correction. Briefly, 0.5 g of each sample homogenized and digested with ultra-pure concentrated nitric acid: Perchloric acid at a ratio of (6:1), then complete dryness by using a hot plate. The samples were analyzed for cadmium using atomic absorption spectrophotometry after dilution with diluted with deionized water [18]. For selenium analyses, dried homogenized liver samples (0.5 g) were digested with ultra-pure nitric acid and hydrogen peroxide in the microwave digestion system. Selenium content was determined in digestion, samples by atomic absorption spectrometry [19].
Experimental design
Statistical analysis
Thirty two male albino rats were involved in this study. Rats were randomly divided into four groups of eight rats each; the first group was used as a control. Groups 2 and 3 were treated orally with Se (0.1 mg/kg BW), and cadmium 40 mg/L in drinking water, respectively, for 30 days. The cadmium dose used in the present study
Data were analyzed by means of one way (ANOVA) using the SPSS software statistical program (SPSS for windows, ver.15.00, USA) followed by least significant difference (LSD). Data are expressed as the mean ± SD, and P < 0.05 was considered statistically significant [20].
Please cite this article in press as: El-Boshy ME, et al. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.05.009
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Table 1 Cadmium (Cd) and selenium (Se) concentrations (mean ± SE), in liver of controls and rats treated with cadmium and selenium for 30 days. Parameters (g/g liver)
Experiment groups
Cd Se
Control
Se
Cd
Cd + Se
0.112 ± 0.04c 0.65 ± 0.06c
0.082 ± 0.02c 1.55 ± 0.12a
44.64 ± 4.62a 0.51 ± 0.08c
29.14 ± 2.12b 1.01 ± 0.10b
Cd
Cd + Se
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
Table 2 Effects of cadmium and selenium on cytokines markers (mean ± SE), 30 days post treatment. Parameters (pg/mL)
Experiment groups Control
TNF ␣ IL-1 IL-6 IFN-␥ IL-10
24.41 32.62 21.92 42.32 9.19
± ± ± ± ±
Se 1.2b 3.4b 1.2b 1.4b 0.42b
24.25 31.14 22.44 48.24 6.34
± ± ± ± ±
1.3b 2.9b 1.4b 1.6a 0.25c
34.34 48.16 35.85 34.71 14.67
± ± ± ± ±
1.8a 3.4a 1.2a 1.3c 1.01a
22.66 48.24 23.94 40.38 9.94
± ± ± ± ±
0.9b 2.1a 1.1b 1.8b 0.62b
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
Results
Hematological results
Metals concentrations
Cadmium treated group showed a significant decrease (P < 0.05) in RBCs, Hb, HCT, PLTs and lymphocyte count when compared with the control group, while neutrophils count were significantly increased, in addition, WBCs, MCV, MCH, MCHC, monocytes, esinophils and basophils were non-significant changed in comparison with control rats. There was a significantly increased in lymphocyte count in the Se treated group as compared to the control group. In Cd + Se treated group, there were a significantly increased in RBCs, Hb, HCT, PLTs and the lymphocytes count when compared with Cd treated rats, while neutrophils count was significantly reduced as compared to the cadmium treated group (Table 3).
The concentration of cadmium (Cd) in the liver was significantly increased in animals exposed to cadmium when compared with other treated rats, while the Cd concentration was significantly lower in Cd + Se treated group when compared with Cd treated group alone. The selenium liver concentration was significantly higher in selenium treated group in comparison with other treated groups, while selenium liver concentration significantly increased in Cd + Se treated group as compared with Cd treated group (Table 1). Cytokines parameters Results from the present study indicate that the serum cytokines IL-1, TNF ␣, IL-6 and IL-10 were significantly higher in cadmiumtreated group, while the serum level of IFN-␥, significantly decreased in the cadmium administrated group as compared with the control. On the other hand, the Se treated rats showed a significant decrease in IL-10 while IFN-␥ significantly increased when compared with control rats, while other cytokines (IL-1, TNF ␣, IL-6) showed no significant changes. Additionally, no significant changes were observed in the aforementioned cytokine serum level in Cd + Se treated groups when compared with the control group as shown in Table 2.
Antioxidant system and lipid peroxidation Results obtained showed a significant decrease (P < 0.05) in antioxidant enzymes, GSH, catalase, SOD and GPx were significantly decreased in the cadmium-treated group when compared to the control group (Table 4). In addition, peroxidation MDA was significantly higher in cadmium-treated group when compared with the control group. On the other hand, treatment with Se alone caused a significant increase in GSH, CAT and GPx as compared to the control group. The antioxidant markers, GSH, CAT, SOD, GPx and lipid peroxidation (MDA), in Cd + Se treated group did not
Table 3 Hematological picture (mean ± SE), 30 days post treatment with cadmium and seleniums. Parameters
Unit
RBCs Hb HCT MCV MCH MCHC PLTs WBCs Neutrophils Esinophils Basophils Lymphocytes Monocytes
106 /L g/dL % fL pg % 103 /L 103 /L 103 /L 103 /L 103 /L 103 /L 103 /L
Experiment groups Control
Se
Cd
Cd + Se
6.48 ± 0.46a 13.34 ± 0.42a 44.48 ± 1.62a 68.58 ± 2.38a 20.61 ± 0.48a 33.35 ± 0.92a 120.24 ± 5.84a 10.42 ± 0.68a 1.92 ± 0.14c 0.24 ± 0.10a 0.0 7.82 ± 0.18b 0.42 ± 0.06a
6.98 ± 0.66a 13.94 ± 0.92a 46.12 ± 2.56a 66.27 ± 2.92a 20.01 ± 0.52a 33.18 ± 0.91a 124.28 ± 5.46a 11.32 ± 0.76a 1.76 ± 0.10c 0.22 ± 0.12a 0.014 ± 0.014 8.98 ± 0.14a 0.34 ± 0.08a
4.94 ± 0.21b 10.21 ± 0.32b 34.28 ± 1.96b 69.32 ± 2.81a 20.68 ± 0.58a 33.62 ± 0.85a 91.68 ± 4.64b 10.86 ± 0.91a 3.64 ± 0.24a 0.32 ± 0.16a 0.0 6.42 ± 0.09c 0.44 ± 0.10a
5.98 ± 0.36a 12.12 ± 0.61a 42.01 ± 2.26a 70.21 ± 2.98a 20.29 ± 0.44a 34.62 ± 0.88a 116.24 ± 6.21a 10.98 ± 0.54a 2.52 ± 0.16b 0.34 ± 0.14a 0.0 7.61 ± 0.12b 0.46 ± 0.09a
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
Please cite this article in press as: El-Boshy ME, et al. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.05.009
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Table 4 Effects of cadmium and selenium on antioxidant and oxidative stress markers (mean ± SE), 30 days post treatment. Parameters
Experiment groups Control
GSH (mol/mL) GPx (mol/mL) CAT (U/mL) SOD (U/mL) MDA (mol/mL)
18.42 21.51 31.18 2.52 8.85
± ± ± ± ±
Se 0.45b 0.69b 0.98b 0.10a 0.42b
24.51 29.92 39.89 2.62 8.1
Cd ± ± ± ± ±
0.26a 0.88a 0.95a 0.18a 0.41b
14.14 15.47 21.41 1.69 12.52
Cd + Se ± ± ± ± ±
0.12c 0.41c 0.72c 0.14b 0.39a
16.92 19.14 29.08 2.39 9.01
± ± ± ± ±
0.64b 0.75b 0.96b 0.19a 0.41b
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
significantly differ from those of the control group as displayed in Tables 2 and 3. Hepatic markers The serum biochemical parameters in our work revealed variations in hepatic markers in cadmium administrated groups when compared with the control group. Results presented in Table 5 showed a significant increase in the ALT and AST serum activities, significant decrease in total protein and albumin, and a nonsignificant change in alkaline phosphatase in cadmium-treated group when compared with the control group. All hepatic markers were none significantly changed in Se treated group alone, when compared with the control group. In the cadmium and selenium treated group, non-significant changes in ALT and AST serum levels were observed when compared with control. On the other hand, the total plasma protein did not significantly change in the Cd + Se treated groups when compared with the control group, while the albumin serum level was significantly increased in cadmium and selenium treated groups when compared with the cadmium treated groups as shown in Table 5. Renal markers Displayed in Table 5, are results pertaining to the renal markers. Results revealed that urea and creatinine levels were higher in groups intoxicated with cadmium in comparison with the control group. Uric acid, however, did not significantly change in the cadmium treated group when compared with the control group. There were none significant changed in renal markers in Se treated group alone, as compared to the control group. Results also indicated that urea, creatinine, and uric acid levels in Cd + Se treated groups, did not significantly change when compared with the control group. Discussion Environmental toxicants, such as cadmium, may produce a variety of clinical manifestations. In man and animals, several organ systems, including the renal, hepatic and immune system may get affected [21].
In the current study, a significantly increased accumulation of Cd in liver was observed in rats treated with Cd. The liver is the primary organ for cadmium toxicity, where most of the absorbed Cd is trapped in the liver and gradually redistributed to other organs, moreover, Cd accumulation in the body depends on the route, dose and duration of the exposure [13,22]. The increased Se concentration in the liver could be explained by its redistribution from other tissues to forming Cd–Se protein complexes [23]. Several studies have documented the immunosuppressive effects of cadmium on the immune system [4,8,24]. In the present study, the cadmium administrated group showed elevation in the serum cytokines IL-1, IL-6, TNF ␣ and IFN-␥, compared with those of the control group. IL-1, IFN-␥ and TNF-␣ cytokines contribute to the inflammatory process by inducing the expression of adhesion molecules on endothelial cells, causing leukocytes in circulation to adhere to the endothelium [24]. Marit et al. [25] reported that cadmium has induced elevation in pro inflammatory cytokines IL-1 and IL-6 in human fibroblasts after exposure to 0.098 g/L Cd2+ for 7 h. Cadmium also induced the release of TNF-␣, IL-6 and IL-8, which may indicate some potential to induce deleterious effects through this pathway [5,22,26]. Similarly, treatment of albino rats with CdCl2 for 2 weeks, resulted in a significant increase in TNF-alpha and IL-1 [27]. Additionally, an elevation in IFN-gamma level was observed after incubation of cultivated peripheral blood mononuclear cell with high concentrations of 0.14 g/L of cadmium chloride [28]. They concluded, elevation of cytokines from monocytes exposed cells through signal transduction induced by cadmium. The immunomodulatory response of selenium was evaluated by the significant increase IFN-␥ and decrease IL-10 in Se treated groups. Cytokines immunomodulatory has been reported as IFN-␥ activates macrophages, while IL-10 prevents macrophage activation [24]. Regarding to the immune protective effects of Se, non-significant changes in the cytokine serum level in Cd + Se treated group when compared with the control group. With respect to the immunomodulatory effects of selenium, the immune response in albino rats dietary supplemented with selenium 8 and 12 mg/L for 14 days was enhanced by an increasing in antibody response to sheep red blood cells of the supplemented groups [29]. The in vivo immune-enhancement activity of
Table 5 Liver and kidney function biomarkers (mean ± SE), 30 days post treatment with cadmium and selenium. Parameters
Experiment groups Control
ALT (IU/L) AST (IU/L) ALP (IU/L) T. Protein (g/dL) Albumin (g/dL) Urea (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL)
41.2 52.8 118.2 7.56 3.72 40.1 0.46 0.92
± ± ± ± ± ± ± ±
Se 3.29b 3.56b 6.21a 0.49a 0.14a 2.45b 0.07b 0.08a
42.5 51.1 122.4 7.65 3.74 41.9 0.42 1.12
Cd ± ± ± ± ± ± ± ±
2.41b 3.28b 4.14a 0.38a 0.15a 2.15b 0.06b 0.09a
62.4 76.1 127.1 6.12 2.32 59.1 0.82 1.18
Cd + Se ± ± ± ± ± ± ± ±
3.32a 5.22a 6.16a 0.21b 0.10b 3.29a 0.09a 0.10a
45.1 55 121.2 7.25 3.01 44.6 0.48 1.10
± ± ± ± ± ± ± ±
3.22b 3.12b 6.15a 0.29a 0.12c 2.26b 0.08b 0.11a
Means in the same raw not followed by the same letter differ significantly (P < 0).
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selenium-exopolysaccharide (seeps) in immunosuppressed mice was researched. Se-EPS treatments increased macrophage phagocytosis, spleen and thymus indices and hemolytic complement activity [30]. Moreover, the dietary supplementation of selenium in a dose of 10−3 g Se/kg for the period of 10 days, enhanced neutrophils phagocytosis function [31]. In addition, IL-1 activity was unaffected by selenium administered to female Sprague-Dawley rats for 10 weeks at 0.5 and 2.0 mg/L [32]. The immunomodulatory of Se may be associated with a number of mechanisms, including the increased activity of natural killer (NK) cells, the proliferation of T-lymphocytes, increased production of IFN-␥ and increased IL-2 receptors, stimulation [14]. Hematopoietic system is one of the most sensitive system to assess the toxicity of environmental toxins and drugs in humans and animals [7]. Regarding to the leukogram in the present work, Cd exposure led to absolute neutrophilia with lymphopenia but total leukocytes, monocytes, esinophils and basophils not change (Table 3). In the agreement, it has been approved that Cd induces neutrophilia and lymphopenia in rats intoxicated with cadmium [5,22]. Neutrophilia and lymphopenia could be as a result of elevation of both IL-6 and TN-␣ induced bone marrow neutrophils release and mobilization as well as redistribution from tissues (marginal pool) into circulating neutrophils under base and inflammatory conditions [5,22]. Increase the absolute lymphocyte count in Se treated group, could be accredited to enhance lymphocyte proliferation under the stimulatory effect of IFN-␥ [14]. Regarding to the erythrogram, this study indicated that the Cd exposure can cause decrease RBCs, Hb and HCT with normal MCV, MCH, MCHC, indicates that Cd exposure can cause normocytic normochromic anemia, but not in Cd + Se treated group (Table 3). In, accordance with other results, anemia has been reported in rats exposed to the Cd [6,7]. Cadmium induced anemia could be as a result of the non-essential toxic metal accumulation in kidney, spleen and liver might suppress the activity of these hematopoietic tissues [6,33]. Moreover, Cd led to anemia as a result of accelerated erythrocyte destruction because of the altered erythrocyte membrane permeability, increased mechanical fragility, and/or failure of the intestinal uptake of Fe because of mucosal lesions [7]. The protective effects of Se on the hematological changes in Cd exposed rats could be attributed to modulator inflammatory cytokines and/or redistribution of cadmium in different organs. The enzymatic antioxidant defense system includes mainly SOD, CAT, GPx, and GSH; this system protects cells against reactive oxygen species (ROS) toxicity and lipid peroxidation. SOD converts the superoxide anion radical to hydrogen peroxide and CAT cleaves this hydrogen peroxide into water and oxygen [34]. Se in the form of amino acid selenocysteine incorporated in synthesis selenoproteins in all mammalian [14]. GPx is a selenoenzyme that catalyses the oxidation of GSH to GSSG, and thereby scavenges the H2 O2 [35]. Moreover, GPx is the most abundant selenoproteins in mammals [14]. The present results showed that treatment with Se alone caused a significant increase in GSH, SOD, CAT, and GPx activity content in rat serum. The antioxidant activity of Se as well as the enhance natural antioxidant body system has been documented by El-Demerdash and Nasr [16]. Our results show a significant decrease in antioxidant enzymes, GSH, CAT, and SOD and Gpx in cadmium treated groups, along with a significant increase in lipid peroxidation MDA. Cd is a toxic metal that is widely used in different industries. It promotes an early oxidative stress and afterwards contributes to the development of serious pathological conditions [27]. Cadmium has been shown to decrease the antioxidant markers activities such as GSH, CAT, SOD, nitric oxide, and to significantly increase malondialdehyde in rats which received cadmium chloride (5 mg/kg BW) for 28 days and (1.5 mg/4 mL/kg) daily for 5 days, respectively [36,37]. Moreover, our findings are consistent with those reported by other researchers, who discovered that
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antioxidant marker concentrations decreased during Cd intoxication [38,39]. Cadmium has been documented to impact the body system damage through inhibited antioxidant markers and induced oxidative damage with ROS generation which destroy proteins, lipids and DNA by oxidation. Lipid peroxidation involves a broad spectrum of alterations in cells and the consequent degeneration of cell membranes. Free radicals and intermediate products of peroxidation are capable of damaging the integrity and altering the functions of bio-membranes, which can lead to the development of many pathological processes [36]. Cd interferes with the intracellular signaling network and gene regulation at multiple levels. Some of the specific changes that lead to tissue damage and death associated with exposure to Cd have been related to oxidative stress and thiol depletion [9]. In addition, in vitro Cd2+ exposure caused cell death, ROS generation, activation, apoptosis, and finally inhibitors of cellular signaling pathways [40]. Studies with Cd revealed that the primary route for Cd toxicity is the depletion of GSH and binding of Cd to SH groups of proteins. The depletion of cellular sulfhydryl reserves seems to be an important indirect mechanism for oxidative stress induced by cadmium [41]. Suppression of free radical scavenging function and the enhancement of ROS, contributes to Cd induced oxidative stress, lipid peroxidation and its associated toxic effects [6,42,11]. On the other hand, lipid peroxidation is thought to be an important mechanism of cell membrane injury and malondialdehyde (MDA) is one of its end-products, which is generated during the oxidative degradation of lipids [43]. The enhanced lipid peroxidation (LPO) may result from the reduction in the activities of CAT, SOD and GSH observed in our work, since these antioxidant enzymes provide protection via elimination of reactive oxygen species (ROS). Cd induced liver damage is associated with increased lipid peroxidation [44,45]. Results from the present study, revealed insignificant changes in GSH, CAT, GPx, SOD antioxidant markers and malondialdehyde levels in cadmium and selenium treated groups when compared with the control group. Selenium (Se) supplementation may prevent the formation of free radicals and the process of lipid peroxidation [46]. It has been detected that Se act as a substrate for various enzymes such as GPx, and is important in sulphur amino acid metabolism that protects the body against several diseases through their antioxidant role [16]. The protective effect of selenium against cadmium induced tissue damage could be attributed to it is own antioxidant activity and enhancement the cellular antioxidant enzymes [47]. Cadmium produces a variety of health hazards in humans and animals due to its ability to induce severe pathological conditions in various organs because of its long retention in some tissues [27,36]. Meanwhile, liver and kidneys are considered as the main targets of Cd induced toxicity [37,48]. The hepato-toxicity of cadmium was demonstrated by alteration of serum hepatic markers in cadmium administrated groups. There was a significant increase in ALT and AST serum activities and a significant decrease in total protein and albumin. However, non-significant changes in alkaline phosphatase in cadmium treated groups compared with that of the control group were observed. Similar results were obtained by other researchers who reported a significant increase in hepatic markers, ALT, AST, alkaline phosphatase, total bilirubin in rats treated with cadmium chloride [6,22,27,36]. Similarly, ALT and AST were elevated in rats intraperitoneally injected with cadmium at a dose of 2 mg/kg/day for 8 days [49]. This elevation could be attributed to liver damage which included swelling and ruptured parenchymal cell leukocyte infiltration, and focal necrosis. The tissue damage, could be attributed to the following: During Cd exposure, most of the antioxidant enzymes become inactive due to their binding to the active sites of the enzyme containing SH groups, which leads to enhancement of reactive oxygen species and or direct damage effect of cadmium, where the liver is the primary organ for cadmium toxicity [50]. On the other hand, Obianime and
Please cite this article in press as: El-Boshy ME, et al. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.05.009
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Roberts [51] reported a decrease in serum alkaline phosphatase in rats after 4 h of cadmium administration at a dose of 40 mg/kg BW. The hypoproteinemia and hypoalbuminemia in the present work could be attributed to liver damage and proteinuria as a renal dysfunction in laboratory animals which are common in cadmium toxicity [48]. In the present work, selenium ameliorates cadmium hepatotoxicity in cadmium and selenium treated group. Several mechanisms could be operating in the protective action of selenium, which could result, for example, in changed absorption of the cadmium or in a change in their action and distribution in the organism and within target organs. The protective mechanisms of selenium compound and selanoproteins are well known for their ability to scavenge ROS and enhancement antioxidant system in cadmium induced tissue damage [47,49]. The kidney is generally recognized as the most critical organ affected by chronic exposure to cadmium. Renal dysfunction in laboratory animals is commonly reported in cadmium toxicity. Cadmium reaches the kidney in the form of cadmium-metallothionein that is filtrated in the glomerulus, and subsequently reabsorbed in the proximal tubules. It then, remains in the tubule cells and results in tubular damage [52]. As for the renal markers, urea and creatinine blood level were higher in groups intoxicated with cadmium. This is consistent with what other researchers observed; elevated serum levels of urea and creatinine were reported in rats [51]. Additionally, proteinuria and renal histopathologic damage have been observed in cadmium intoxicated rats [48]. Moreover, chronic environmental oral exposure to cadmium in humans leads to renal failure, characterized by proteinuria due to renal tubular dysfunction [21]. The urea and creatinine levels in the cadmium and selenium treated groups did not significantly change when compared with the control group. The evidence that selenium ameliorated the renal damage in cadmium intoxicated rats by reducing the urea and creatinine serum levels was reported in the literature [51]. The protective effect of selenium against cadmium toxicity could be attributed to it is antioxidant activity. Interactions between non-toxic levels of dietary selenium and relatively high levels of cadmium apparently resulted in partial amelioration of cadmium toxicity in different systems [47]. Conclusions In conclusion, results from this study demonstrated that cadmium is capable of causing marked oxidative stress in addition to inhibiting the activities of antioxidant enzymes. Treatment with selenium could significantly attenuate the cadmium induced immunosuppressive oxidative stress as well as hepatotoxicity and renal damage. Conflict of interest None declared. References [1] Jarup L. Cadmium overload and toxicity. Nephrol Dial Transplant 2002;17:35–9. [2] Satarug S, Baker JR, Urbenjapol S, Haswell-Elkins M, Reilly PE, Williams DJ, et al. Global perspective on cadmium pollution and toxicity in non-occupationally exposed population. Toxicol Lett 2003;137:65–83. [3] Małgorzata M, Brzo S, Janina MJ. Low-level exposure to cadmium during the lifetime increases the risk of osteoporosis and fractures of the lumbar spine in the elderly: studies on a rat model of human environmental exposure. Toxicol Sci 2004;82:468–77. [4] Stephane P, Michele D, Jacques B, Jean-Marie B, Michel F, Daniel GC. Immunomodulatory effects of estradiol and cadmium in adult female rats. Toxicol Sci 2006;92:423–32. [5] Kataranovski M, Jankovi S, Kataranovski J, Bogojevi D. Gender differences in acute cadmium induced systemic inflammation in rats. Biomed Environ Sci 2009;21:1–7.
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Biochemistry
Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats Mohamed E. El-Boshy a,b,∗∗ , Engy F. Risha b , Fatma M. Abdelhamid b , Mohammad S. Mubarak c , Taibi Ben Hadda d,∗ a
Laboratory Medicine Department, Faculty of Applied Medical Science, Umm Al-Qura University, PB 7296, Makkah 21955, Saudi Arabia Clinical Pathology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt c Department of Chemistry, The University of Jordan, Amman 11942, Jordan d Laboratoire de Chimie des Matériaux (LCM), Faculté des Sciences, Université Mohammed Premier, Oujda 60000, Morocco b
a r t i c l e
i n f o
Article history: Received 25 March 2014 Accepted 26 May 2014 Keywords: Selenium Cadmium Immunosuppressive Antioxidant Rats
a b s t r a c t Cadmium is a non-essential toxic metal used in industrial process, causes severe risk to human health. Selenium (Se) is an essential trace mineral of fundamental importance for human health. Selenium has antioxidant enzymes roles and is needed for the proper function of the immune system. In this study, the protective effects of selenium against cadmium intoxication in rats have been investigated by monitoring some selective cytokines (IL-1, TNF ␣, IL-6, IL-10 and IFN-␥), antioxidant enzymes reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation malondialdehyde (MDA) as well as some selective biochemical markers of liver and kidney functions. Thirty-two rats were divided into four equal groups; the first group was used as a control. Groups 2–4 were treated with selenium (Se; 0.1 mg/kg BW), cadmium (Cd; 40 mg/L drinking water) and selenium plus cadmium, respectively. Rats were orally administered their relevant doses daily for 30 days. Blood samples were collected from heart puncture at the end of the experiment (30 days) for complete blood picture (CBC) and serum was separated to evaluate the different immunological parameters and biochemical parameters, as well as liver specimens for Cd and Se estimation. Rats in the Cd treated group have a significantly higher hepatic concentration of Cd than in other treated groups. Results revealed that cadmium significantly increased IL-1, TNF ␣, IL-6 and IL-10, beside peripheral neutrophils count, while the IFN-␥ and lymphocytes were decreased in rat sera. In addition, GSH level, CAT, SOD and GPx activities were significantly decreased while lipid peroxidation (MDA) was increased. Regarding, liver and renal markers, they were significantly increased in the activities of aminotransferases (AST, ALT), urea and creatinine, while total plasma proteins and albumin were significantly decreased. On the other hand, selenium treated group, showed significantly increased IFN-␥, GSH level, CAT, and GPx activities, as well as lymphocyte count while IL-10 was decreased. Selenium in combination with cadmium, significantly improved the elevation of serum IL-1, IL-6, TNF ␣, IL-10 and malondialdehyde in addition to enhancing the antioxidant enzyme activities of GSH, CAT, GPx and SOD. Moreover, selenium has ameliorated the cadmium-induced liver and kidney damage by improving hepatic and renal markers. The results of this investigation demonstrated that selenium has the potential to countermeasure the immunosuppressive as well as hepatic and renal oxidative damage induced by cadmium in rats; selenium has shown promising effects against Cd toxicity. © 2014 Elsevier GmbH. All rights reserved.
Introduction ∗ Corresponding author. Tel.: +212 0666134178; fax: +212 05 36 50 06 03. ∗∗ Co-corresponding author at: Laboratory Medicine Department, Faculty of Applied Medical Science, Umm Al-Qura University, PB 7296, Makkah 21955, Saudi Arabia. Tel.: +966 0563025297. E-mail addresses: dr [email protected] (M.E. El-Boshy), [email protected], [email protected] (T.B. Hadda).
Adverse immune effects of chemicals, defined as immunotoxicity, has been used as a sensitive biomarker for assessing health effect of environmental pollution. Immunotoxic effects of nonessential toxic metal, as a typical environmental agent and their mechanism are areas of interest. Cadmium (Cd) is one of the most
http://dx.doi.org/10.1016/j.jtemb.2014.05.009 0946-672X/© 2014 Elsevier GmbH. All rights reserved.
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toxic non-essential toxic metal, an environmental and occupational pollutant endangering human and animal health [1,2]. It has been suggested that Cd may be an environmental risk factor for osteoporosis and fertility as well as nephrotoxicity, hepatotoxicity, and immunosuppressiveness [3–7]. The immunotoxic effects of Cd on the development of immune organs, the differentiation of immune cells, and specific and non-specific immune responses have been reported by Koller [8]. This indicates an impact on the immune system as a whole, which can lead to a significant decrease in host resistance. Moreover, some of the specific changes that lead to tissue damage and death from chronic exposure to Cd have been related to oxidative stress [10]. The associated toxic effects of cadmium attributed to suppression of free radical scavenging function and the enhancement of ROS contribute to Cd induced oxidative stress and lipid peroxidation [10–12]. Selenium (Se) is an essential trace element for both animal and human begins. It has been detected that selenium function through participates in formation of selenoproteins, this element contributes in various biological processes such as antioxidant defense, thyroid hormone production, and immune responses [13,14]. Some reports indicate that a deficiency in selenium may be flat to certain diseases [15]. It is generally recognized to be of great importance for human health, protecting the cells from the harmful effects of free radical production [16]. Adverse health effects following selenium overexposure, although very rare, have been found in animals and people [15]. Selenium interaction with cadmium has been reported; this interaction may reduce cadmium accumulation and subsequent reduce its toxicity in the body [15]. Although many of the studies that have been carried out until now reported on the different adverse health effects of Cd, such as its nephrotoxicity, hepatotoxicity, and immunosuppressiveness, however, none has dealt with the association between cadmium and selenium. Therefore, the present study was conducted to shed some light on the relationship between the immunosuppressive, renal and hepatic damage, hematological disturbances as well as oxidative stress effects of cadmium and the putative protective effects of selenium in rats. Materials and methods Experimental animals The experiment was conducted in accordance with animal welfare and under ethical protocols by the Faculty of Veterinary medicine, Mansoura university, Egypt. Forty rat weight (160 ± 10 g), housed in a plastic cages 50 cm × 30 cm × 10 cm and placed in the room for 10 days for adaptation. The room temperature was maintained at 24 ± 2 ◦ C, the air of the room was changed continuously by using ventilated vacuum and with light\dark cycle of 12:12 h per day. The animal was fed on a pellet diet. Chemicals Sodium selenite (Na2 SeO3 ) and cadmium chloride anhydrous (CdCl2 ), which were analytical grade (≥99%) and were obtained from the Sigma Aldrich Chemical Co. (St. Louis, Missouri, USA). All chemicals and reagents used in this study were of analytical reagent grade.
used according to published literature procedures [17]. Group 4 was orally administered Cd at doses of 40 mg/L and Se at a dose of 0.1 mg/kg BW.
Samples collection and parameters measured The blood samples were collected in test tubes contained disodium salt of ethylene diamine tetra acetic acid (EDTA) anticoagulant and used for determination of red blood cell counts (RBCs), hemoglobin (Hb) concentration, Hematocrit (HCT) and erythrocyte indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), in addition, white blood cells (WBCs) and platelets (PLTs). Three blood films were made for each blood sample. Blood films were stained with Giemsa stain and differential leukocyte count was done by standard hematological techniques. The second blood samples were collected in test tubes without anticoagulant. The samples were centrifuged at 3000 rpm for 10 min and the clear serum was carefully separated from all samples. Selective humoral immunological parameters (Tumor necrosis factor – ␣ (TNF ␣), Interleukin 1 (IL-1), IL-6, IL-10 and gamma interferon (IFN-␥) were determined by Enzyme Amplified Sensitivity Immunoassay (EASIA, R & D Systems, Minneapolis, MN, USA) using microplates according to enclosed pamphlets (Aushon Searchlight Biosystem (Billerica, MA). The lower detection limits are 1.5 pg/mL for IL-1, 6.3 pg/mL for IL-6, 5.4 pg/mL for IL-10, 3.1 pg/mL for TNF␣, and 6.2 pg/mL for IFN-␥ and data are presented as pg cytokine/mL serum. Antioxidant markers, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), oxidized glutathione, (GPx) and malondialdehyde (MDA), were determined from undiluted serum samples using commercially available ELISA Kits (Cayman Chemical Co.). The plates were read at 450 nm and a correction wavelength of 550 nm on a computerized automated microplate ELISA reader. ALT, AST, alkaline phosphatase (ALP) activities were assayed by using commercial kits (Human, Diagnostic Co. Germany), while total protein, albumin, uric acid, urea and creatinine (Crescent Diagnostic Co. KSA) were estimated spectrophotometrically (BM Co. Germany, 5010) according to enclosed pamphlets.
Cadmium and selenium measurement The concentration of Cd in the liver was determined by atomic absorption spectrophotometry (Perkin-Elmer A.A. Model 800) with Zeeman-effect for background correction. Briefly, 0.5 g of each sample homogenized and digested with ultra-pure concentrated nitric acid: Perchloric acid at a ratio of (6:1), then complete dryness by using a hot plate. The samples were analyzed for cadmium using atomic absorption spectrophotometry after dilution with diluted with deionized water [18]. For selenium analyses, dried homogenized liver samples (0.5 g) were digested with ultra-pure nitric acid and hydrogen peroxide in the microwave digestion system. Selenium content was determined in digestion, samples by atomic absorption spectrometry [19].
Experimental design
Statistical analysis
Thirty two male albino rats were involved in this study. Rats were randomly divided into four groups of eight rats each; the first group was used as a control. Groups 2 and 3 were treated orally with Se (0.1 mg/kg BW), and cadmium 40 mg/L in drinking water, respectively, for 30 days. The cadmium dose used in the present study
Data were analyzed by means of one way (ANOVA) using the SPSS software statistical program (SPSS for windows, ver.15.00, USA) followed by least significant difference (LSD). Data are expressed as the mean ± SD, and P < 0.05 was considered statistically significant [20].
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Table 1 Cadmium (Cd) and selenium (Se) concentrations (mean ± SE), in liver of controls and rats treated with cadmium and selenium for 30 days. Parameters (g/g liver)
Experiment groups
Cd Se
Control
Se
Cd
Cd + Se
0.112 ± 0.04c 0.65 ± 0.06c
0.082 ± 0.02c 1.55 ± 0.12a
44.64 ± 4.62a 0.51 ± 0.08c
29.14 ± 2.12b 1.01 ± 0.10b
Cd
Cd + Se
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
Table 2 Effects of cadmium and selenium on cytokines markers (mean ± SE), 30 days post treatment. Parameters (pg/mL)
Experiment groups Control
TNF ␣ IL-1 IL-6 IFN-␥ IL-10
24.41 32.62 21.92 42.32 9.19
± ± ± ± ±
Se 1.2b 3.4b 1.2b 1.4b 0.42b
24.25 31.14 22.44 48.24 6.34
± ± ± ± ±
1.3b 2.9b 1.4b 1.6a 0.25c
34.34 48.16 35.85 34.71 14.67
± ± ± ± ±
1.8a 3.4a 1.2a 1.3c 1.01a
22.66 48.24 23.94 40.38 9.94
± ± ± ± ±
0.9b 2.1a 1.1b 1.8b 0.62b
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
Results
Hematological results
Metals concentrations
Cadmium treated group showed a significant decrease (P < 0.05) in RBCs, Hb, HCT, PLTs and lymphocyte count when compared with the control group, while neutrophils count were significantly increased, in addition, WBCs, MCV, MCH, MCHC, monocytes, esinophils and basophils were non-significant changed in comparison with control rats. There was a significantly increased in lymphocyte count in the Se treated group as compared to the control group. In Cd + Se treated group, there were a significantly increased in RBCs, Hb, HCT, PLTs and the lymphocytes count when compared with Cd treated rats, while neutrophils count was significantly reduced as compared to the cadmium treated group (Table 3).
The concentration of cadmium (Cd) in the liver was significantly increased in animals exposed to cadmium when compared with other treated rats, while the Cd concentration was significantly lower in Cd + Se treated group when compared with Cd treated group alone. The selenium liver concentration was significantly higher in selenium treated group in comparison with other treated groups, while selenium liver concentration significantly increased in Cd + Se treated group as compared with Cd treated group (Table 1). Cytokines parameters Results from the present study indicate that the serum cytokines IL-1, TNF ␣, IL-6 and IL-10 were significantly higher in cadmiumtreated group, while the serum level of IFN-␥, significantly decreased in the cadmium administrated group as compared with the control. On the other hand, the Se treated rats showed a significant decrease in IL-10 while IFN-␥ significantly increased when compared with control rats, while other cytokines (IL-1, TNF ␣, IL-6) showed no significant changes. Additionally, no significant changes were observed in the aforementioned cytokine serum level in Cd + Se treated groups when compared with the control group as shown in Table 2.
Antioxidant system and lipid peroxidation Results obtained showed a significant decrease (P < 0.05) in antioxidant enzymes, GSH, catalase, SOD and GPx were significantly decreased in the cadmium-treated group when compared to the control group (Table 4). In addition, peroxidation MDA was significantly higher in cadmium-treated group when compared with the control group. On the other hand, treatment with Se alone caused a significant increase in GSH, CAT and GPx as compared to the control group. The antioxidant markers, GSH, CAT, SOD, GPx and lipid peroxidation (MDA), in Cd + Se treated group did not
Table 3 Hematological picture (mean ± SE), 30 days post treatment with cadmium and seleniums. Parameters
Unit
RBCs Hb HCT MCV MCH MCHC PLTs WBCs Neutrophils Esinophils Basophils Lymphocytes Monocytes
106 /L g/dL % fL pg % 103 /L 103 /L 103 /L 103 /L 103 /L 103 /L 103 /L
Experiment groups Control
Se
Cd
Cd + Se
6.48 ± 0.46a 13.34 ± 0.42a 44.48 ± 1.62a 68.58 ± 2.38a 20.61 ± 0.48a 33.35 ± 0.92a 120.24 ± 5.84a 10.42 ± 0.68a 1.92 ± 0.14c 0.24 ± 0.10a 0.0 7.82 ± 0.18b 0.42 ± 0.06a
6.98 ± 0.66a 13.94 ± 0.92a 46.12 ± 2.56a 66.27 ± 2.92a 20.01 ± 0.52a 33.18 ± 0.91a 124.28 ± 5.46a 11.32 ± 0.76a 1.76 ± 0.10c 0.22 ± 0.12a 0.014 ± 0.014 8.98 ± 0.14a 0.34 ± 0.08a
4.94 ± 0.21b 10.21 ± 0.32b 34.28 ± 1.96b 69.32 ± 2.81a 20.68 ± 0.58a 33.62 ± 0.85a 91.68 ± 4.64b 10.86 ± 0.91a 3.64 ± 0.24a 0.32 ± 0.16a 0.0 6.42 ± 0.09c 0.44 ± 0.10a
5.98 ± 0.36a 12.12 ± 0.61a 42.01 ± 2.26a 70.21 ± 2.98a 20.29 ± 0.44a 34.62 ± 0.88a 116.24 ± 6.21a 10.98 ± 0.54a 2.52 ± 0.16b 0.34 ± 0.14a 0.0 7.61 ± 0.12b 0.46 ± 0.09a
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
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Table 4 Effects of cadmium and selenium on antioxidant and oxidative stress markers (mean ± SE), 30 days post treatment. Parameters
Experiment groups Control
GSH (mol/mL) GPx (mol/mL) CAT (U/mL) SOD (U/mL) MDA (mol/mL)
18.42 21.51 31.18 2.52 8.85
± ± ± ± ±
Se 0.45b 0.69b 0.98b 0.10a 0.42b
24.51 29.92 39.89 2.62 8.1
Cd ± ± ± ± ±
0.26a 0.88a 0.95a 0.18a 0.41b
14.14 15.47 21.41 1.69 12.52
Cd + Se ± ± ± ± ±
0.12c 0.41c 0.72c 0.14b 0.39a
16.92 19.14 29.08 2.39 9.01
± ± ± ± ±
0.64b 0.75b 0.96b 0.19a 0.41b
Means in the same raw not followed by the same letter differ significantly (P < 0.05).
significantly differ from those of the control group as displayed in Tables 2 and 3. Hepatic markers The serum biochemical parameters in our work revealed variations in hepatic markers in cadmium administrated groups when compared with the control group. Results presented in Table 5 showed a significant increase in the ALT and AST serum activities, significant decrease in total protein and albumin, and a nonsignificant change in alkaline phosphatase in cadmium-treated group when compared with the control group. All hepatic markers were none significantly changed in Se treated group alone, when compared with the control group. In the cadmium and selenium treated group, non-significant changes in ALT and AST serum levels were observed when compared with control. On the other hand, the total plasma protein did not significantly change in the Cd + Se treated groups when compared with the control group, while the albumin serum level was significantly increased in cadmium and selenium treated groups when compared with the cadmium treated groups as shown in Table 5. Renal markers Displayed in Table 5, are results pertaining to the renal markers. Results revealed that urea and creatinine levels were higher in groups intoxicated with cadmium in comparison with the control group. Uric acid, however, did not significantly change in the cadmium treated group when compared with the control group. There were none significant changed in renal markers in Se treated group alone, as compared to the control group. Results also indicated that urea, creatinine, and uric acid levels in Cd + Se treated groups, did not significantly change when compared with the control group. Discussion Environmental toxicants, such as cadmium, may produce a variety of clinical manifestations. In man and animals, several organ systems, including the renal, hepatic and immune system may get affected [21].
In the current study, a significantly increased accumulation of Cd in liver was observed in rats treated with Cd. The liver is the primary organ for cadmium toxicity, where most of the absorbed Cd is trapped in the liver and gradually redistributed to other organs, moreover, Cd accumulation in the body depends on the route, dose and duration of the exposure [13,22]. The increased Se concentration in the liver could be explained by its redistribution from other tissues to forming Cd–Se protein complexes [23]. Several studies have documented the immunosuppressive effects of cadmium on the immune system [4,8,24]. In the present study, the cadmium administrated group showed elevation in the serum cytokines IL-1, IL-6, TNF ␣ and IFN-␥, compared with those of the control group. IL-1, IFN-␥ and TNF-␣ cytokines contribute to the inflammatory process by inducing the expression of adhesion molecules on endothelial cells, causing leukocytes in circulation to adhere to the endothelium [24]. Marit et al. [25] reported that cadmium has induced elevation in pro inflammatory cytokines IL-1 and IL-6 in human fibroblasts after exposure to 0.098 g/L Cd2+ for 7 h. Cadmium also induced the release of TNF-␣, IL-6 and IL-8, which may indicate some potential to induce deleterious effects through this pathway [5,22,26]. Similarly, treatment of albino rats with CdCl2 for 2 weeks, resulted in a significant increase in TNF-alpha and IL-1 [27]. Additionally, an elevation in IFN-gamma level was observed after incubation of cultivated peripheral blood mononuclear cell with high concentrations of 0.14 g/L of cadmium chloride [28]. They concluded, elevation of cytokines from monocytes exposed cells through signal transduction induced by cadmium. The immunomodulatory response of selenium was evaluated by the significant increase IFN-␥ and decrease IL-10 in Se treated groups. Cytokines immunomodulatory has been reported as IFN-␥ activates macrophages, while IL-10 prevents macrophage activation [24]. Regarding to the immune protective effects of Se, non-significant changes in the cytokine serum level in Cd + Se treated group when compared with the control group. With respect to the immunomodulatory effects of selenium, the immune response in albino rats dietary supplemented with selenium 8 and 12 mg/L for 14 days was enhanced by an increasing in antibody response to sheep red blood cells of the supplemented groups [29]. The in vivo immune-enhancement activity of
Table 5 Liver and kidney function biomarkers (mean ± SE), 30 days post treatment with cadmium and selenium. Parameters
Experiment groups Control
ALT (IU/L) AST (IU/L) ALP (IU/L) T. Protein (g/dL) Albumin (g/dL) Urea (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL)
41.2 52.8 118.2 7.56 3.72 40.1 0.46 0.92
± ± ± ± ± ± ± ±
Se 3.29b 3.56b 6.21a 0.49a 0.14a 2.45b 0.07b 0.08a
42.5 51.1 122.4 7.65 3.74 41.9 0.42 1.12
Cd ± ± ± ± ± ± ± ±
2.41b 3.28b 4.14a 0.38a 0.15a 2.15b 0.06b 0.09a
62.4 76.1 127.1 6.12 2.32 59.1 0.82 1.18
Cd + Se ± ± ± ± ± ± ± ±
3.32a 5.22a 6.16a 0.21b 0.10b 3.29a 0.09a 0.10a
45.1 55 121.2 7.25 3.01 44.6 0.48 1.10
± ± ± ± ± ± ± ±
3.22b 3.12b 6.15a 0.29a 0.12c 2.26b 0.08b 0.11a
Means in the same raw not followed by the same letter differ significantly (P < 0).
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selenium-exopolysaccharide (seeps) in immunosuppressed mice was researched. Se-EPS treatments increased macrophage phagocytosis, spleen and thymus indices and hemolytic complement activity [30]. Moreover, the dietary supplementation of selenium in a dose of 10−3 g Se/kg for the period of 10 days, enhanced neutrophils phagocytosis function [31]. In addition, IL-1 activity was unaffected by selenium administered to female Sprague-Dawley rats for 10 weeks at 0.5 and 2.0 mg/L [32]. The immunomodulatory of Se may be associated with a number of mechanisms, including the increased activity of natural killer (NK) cells, the proliferation of T-lymphocytes, increased production of IFN-␥ and increased IL-2 receptors, stimulation [14]. Hematopoietic system is one of the most sensitive system to assess the toxicity of environmental toxins and drugs in humans and animals [7]. Regarding to the leukogram in the present work, Cd exposure led to absolute neutrophilia with lymphopenia but total leukocytes, monocytes, esinophils and basophils not change (Table 3). In the agreement, it has been approved that Cd induces neutrophilia and lymphopenia in rats intoxicated with cadmium [5,22]. Neutrophilia and lymphopenia could be as a result of elevation of both IL-6 and TN-␣ induced bone marrow neutrophils release and mobilization as well as redistribution from tissues (marginal pool) into circulating neutrophils under base and inflammatory conditions [5,22]. Increase the absolute lymphocyte count in Se treated group, could be accredited to enhance lymphocyte proliferation under the stimulatory effect of IFN-␥ [14]. Regarding to the erythrogram, this study indicated that the Cd exposure can cause decrease RBCs, Hb and HCT with normal MCV, MCH, MCHC, indicates that Cd exposure can cause normocytic normochromic anemia, but not in Cd + Se treated group (Table 3). In, accordance with other results, anemia has been reported in rats exposed to the Cd [6,7]. Cadmium induced anemia could be as a result of the non-essential toxic metal accumulation in kidney, spleen and liver might suppress the activity of these hematopoietic tissues [6,33]. Moreover, Cd led to anemia as a result of accelerated erythrocyte destruction because of the altered erythrocyte membrane permeability, increased mechanical fragility, and/or failure of the intestinal uptake of Fe because of mucosal lesions [7]. The protective effects of Se on the hematological changes in Cd exposed rats could be attributed to modulator inflammatory cytokines and/or redistribution of cadmium in different organs. The enzymatic antioxidant defense system includes mainly SOD, CAT, GPx, and GSH; this system protects cells against reactive oxygen species (ROS) toxicity and lipid peroxidation. SOD converts the superoxide anion radical to hydrogen peroxide and CAT cleaves this hydrogen peroxide into water and oxygen [34]. Se in the form of amino acid selenocysteine incorporated in synthesis selenoproteins in all mammalian [14]. GPx is a selenoenzyme that catalyses the oxidation of GSH to GSSG, and thereby scavenges the H2 O2 [35]. Moreover, GPx is the most abundant selenoproteins in mammals [14]. The present results showed that treatment with Se alone caused a significant increase in GSH, SOD, CAT, and GPx activity content in rat serum. The antioxidant activity of Se as well as the enhance natural antioxidant body system has been documented by El-Demerdash and Nasr [16]. Our results show a significant decrease in antioxidant enzymes, GSH, CAT, and SOD and Gpx in cadmium treated groups, along with a significant increase in lipid peroxidation MDA. Cd is a toxic metal that is widely used in different industries. It promotes an early oxidative stress and afterwards contributes to the development of serious pathological conditions [27]. Cadmium has been shown to decrease the antioxidant markers activities such as GSH, CAT, SOD, nitric oxide, and to significantly increase malondialdehyde in rats which received cadmium chloride (5 mg/kg BW) for 28 days and (1.5 mg/4 mL/kg) daily for 5 days, respectively [36,37]. Moreover, our findings are consistent with those reported by other researchers, who discovered that
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antioxidant marker concentrations decreased during Cd intoxication [38,39]. Cadmium has been documented to impact the body system damage through inhibited antioxidant markers and induced oxidative damage with ROS generation which destroy proteins, lipids and DNA by oxidation. Lipid peroxidation involves a broad spectrum of alterations in cells and the consequent degeneration of cell membranes. Free radicals and intermediate products of peroxidation are capable of damaging the integrity and altering the functions of bio-membranes, which can lead to the development of many pathological processes [36]. Cd interferes with the intracellular signaling network and gene regulation at multiple levels. Some of the specific changes that lead to tissue damage and death associated with exposure to Cd have been related to oxidative stress and thiol depletion [9]. In addition, in vitro Cd2+ exposure caused cell death, ROS generation, activation, apoptosis, and finally inhibitors of cellular signaling pathways [40]. Studies with Cd revealed that the primary route for Cd toxicity is the depletion of GSH and binding of Cd to SH groups of proteins. The depletion of cellular sulfhydryl reserves seems to be an important indirect mechanism for oxidative stress induced by cadmium [41]. Suppression of free radical scavenging function and the enhancement of ROS, contributes to Cd induced oxidative stress, lipid peroxidation and its associated toxic effects [6,42,11]. On the other hand, lipid peroxidation is thought to be an important mechanism of cell membrane injury and malondialdehyde (MDA) is one of its end-products, which is generated during the oxidative degradation of lipids [43]. The enhanced lipid peroxidation (LPO) may result from the reduction in the activities of CAT, SOD and GSH observed in our work, since these antioxidant enzymes provide protection via elimination of reactive oxygen species (ROS). Cd induced liver damage is associated with increased lipid peroxidation [44,45]. Results from the present study, revealed insignificant changes in GSH, CAT, GPx, SOD antioxidant markers and malondialdehyde levels in cadmium and selenium treated groups when compared with the control group. Selenium (Se) supplementation may prevent the formation of free radicals and the process of lipid peroxidation [46]. It has been detected that Se act as a substrate for various enzymes such as GPx, and is important in sulphur amino acid metabolism that protects the body against several diseases through their antioxidant role [16]. The protective effect of selenium against cadmium induced tissue damage could be attributed to it is own antioxidant activity and enhancement the cellular antioxidant enzymes [47]. Cadmium produces a variety of health hazards in humans and animals due to its ability to induce severe pathological conditions in various organs because of its long retention in some tissues [27,36]. Meanwhile, liver and kidneys are considered as the main targets of Cd induced toxicity [37,48]. The hepato-toxicity of cadmium was demonstrated by alteration of serum hepatic markers in cadmium administrated groups. There was a significant increase in ALT and AST serum activities and a significant decrease in total protein and albumin. However, non-significant changes in alkaline phosphatase in cadmium treated groups compared with that of the control group were observed. Similar results were obtained by other researchers who reported a significant increase in hepatic markers, ALT, AST, alkaline phosphatase, total bilirubin in rats treated with cadmium chloride [6,22,27,36]. Similarly, ALT and AST were elevated in rats intraperitoneally injected with cadmium at a dose of 2 mg/kg/day for 8 days [49]. This elevation could be attributed to liver damage which included swelling and ruptured parenchymal cell leukocyte infiltration, and focal necrosis. The tissue damage, could be attributed to the following: During Cd exposure, most of the antioxidant enzymes become inactive due to their binding to the active sites of the enzyme containing SH groups, which leads to enhancement of reactive oxygen species and or direct damage effect of cadmium, where the liver is the primary organ for cadmium toxicity [50]. On the other hand, Obianime and
Please cite this article in press as: El-Boshy ME, et al. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.05.009
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Roberts [51] reported a decrease in serum alkaline phosphatase in rats after 4 h of cadmium administration at a dose of 40 mg/kg BW. The hypoproteinemia and hypoalbuminemia in the present work could be attributed to liver damage and proteinuria as a renal dysfunction in laboratory animals which are common in cadmium toxicity [48]. In the present work, selenium ameliorates cadmium hepatotoxicity in cadmium and selenium treated group. Several mechanisms could be operating in the protective action of selenium, which could result, for example, in changed absorption of the cadmium or in a change in their action and distribution in the organism and within target organs. The protective mechanisms of selenium compound and selanoproteins are well known for their ability to scavenge ROS and enhancement antioxidant system in cadmium induced tissue damage [47,49]. The kidney is generally recognized as the most critical organ affected by chronic exposure to cadmium. Renal dysfunction in laboratory animals is commonly reported in cadmium toxicity. Cadmium reaches the kidney in the form of cadmium-metallothionein that is filtrated in the glomerulus, and subsequently reabsorbed in the proximal tubules. It then, remains in the tubule cells and results in tubular damage [52]. As for the renal markers, urea and creatinine blood level were higher in groups intoxicated with cadmium. This is consistent with what other researchers observed; elevated serum levels of urea and creatinine were reported in rats [51]. Additionally, proteinuria and renal histopathologic damage have been observed in cadmium intoxicated rats [48]. Moreover, chronic environmental oral exposure to cadmium in humans leads to renal failure, characterized by proteinuria due to renal tubular dysfunction [21]. The urea and creatinine levels in the cadmium and selenium treated groups did not significantly change when compared with the control group. The evidence that selenium ameliorated the renal damage in cadmium intoxicated rats by reducing the urea and creatinine serum levels was reported in the literature [51]. The protective effect of selenium against cadmium toxicity could be attributed to it is antioxidant activity. Interactions between non-toxic levels of dietary selenium and relatively high levels of cadmium apparently resulted in partial amelioration of cadmium toxicity in different systems [47]. Conclusions In conclusion, results from this study demonstrated that cadmium is capable of causing marked oxidative stress in addition to inhibiting the activities of antioxidant enzymes. Treatment with selenium could significantly attenuate the cadmium induced immunosuppressive oxidative stress as well as hepatotoxicity and renal damage. Conflict of interest None declared. References [1] Jarup L. Cadmium overload and toxicity. Nephrol Dial Transplant 2002;17:35–9. [2] Satarug S, Baker JR, Urbenjapol S, Haswell-Elkins M, Reilly PE, Williams DJ, et al. Global perspective on cadmium pollution and toxicity in non-occupationally exposed population. Toxicol Lett 2003;137:65–83. [3] Małgorzata M, Brzo S, Janina MJ. Low-level exposure to cadmium during the lifetime increases the risk of osteoporosis and fractures of the lumbar spine in the elderly: studies on a rat model of human environmental exposure. Toxicol Sci 2004;82:468–77. [4] Stephane P, Michele D, Jacques B, Jean-Marie B, Michel F, Daniel GC. Immunomodulatory effects of estradiol and cadmium in adult female rats. Toxicol Sci 2006;92:423–32. [5] Kataranovski M, Jankovi S, Kataranovski J, Bogojevi D. Gender differences in acute cadmium induced systemic inflammation in rats. Biomed Environ Sci 2009;21:1–7.
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