Effects of Cadmium Chloride and Sodium Selenite Alone or in Combination on the Liver of Male Sprague–Dawley Rats Assessed by Different Assays Farhat Jabeen & Abdul Shakoor Chaudhry
Biological Trace Element Research ISSN 0163-4984 Volume 143 Number 2 Biol Trace Elem Res (2011) 143:1077-1090 DOI 10.1007/s12011-010-8946-0
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Author's personal copy Biol Trace Elem Res (2011) 143:1077–1090 DOI 10.1007/s12011-010-8946-0
Effects of Cadmium Chloride and Sodium Selenite Alone or in Combination on the Liver of Male Sprague–Dawley Rats Assessed by Different Assays Farhat Jabeen & Abdul Shakoor Chaudhry
Received: 11 November 2010 / Accepted: 22 December 2010 / Published online: 7 January 2011 # Springer Science+Business Media, LLC 2011
Abstract This study assessed the impact of either cadmium chloride (Cd) or sodium selenite (Se) alone or in combination on male Sprague–Dawley rats. For this purpose, body and liver weights, comet and TUNEL assays, histological analysis and levels of lipid peroxidation and antioxidants in liver were determined in four groups of male Sprague– Dawley rats. The rats were given subcutaneous doses of 1 mg/kg body weight (BW) of either normal saline (control=Ct) or Cd or Se or Cd plus Se (Cd+Se) on alternate days for 4 weeks. The Cd group showed increased DNA damage, apoptosis and hepatic levels of lipid peroxidation and altered histology. Conversely, the antioxidant levels in this group were decreased as compared with the control group. The Se group also showed DNA damage, apoptosis and altered histology and reduced catalase activity, but it was less severe than the Cd group. In the Cd+Se group, ameliorating effects of Se on Cd-induced changes were observed. While the Se was able to curtail the toxic effect of Cd, the Cd or Se alone were genotoxic and cytotoxic for rats receiving a high pharmacological but non-fatal dose of 1 mg/kg BW. Keywords Sprague–Dawley rats . Lipid peroxidation . Cadmium . Selenium . Comet assay . TUNEL assay
Introduction Cadmium (Cd), a well-known environmental hazard, exerts a number of toxic effects on humans and animals. Tobacco smoke, food, environmental and industrial pollution are the
F. Jabeen (*) Department of Zoology, GC University Faisalabad, Faisalabad, Pakistan e-mail:
[email protected] F. Jabeen : A. S. Chaudhry School of Agriculture Food and Rural Development, Newcastle University, Newcastle upon Tyne, UK A. S. Chaudhry e-mail:
[email protected]
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main sources of Cd for their potential hazards for humans and animals [1–3]. Liver is a major target organ for showing toxic effects of Cd as a result of accumulation after both acute and chronic poisoning. This metal has been reported for its carcinogenic, mutagenic and teratogenic properties [4, 5]. Cd-induced apoptosis has been observed in several cell types at concentrations ranging from 1 to 250 μM [6–9]. The process of apoptosis is a strategic and organized mechanism of cell death where distinct morphological changes including membrane blebbing of cells can occur. Nuclear changes that accompany apoptosis include chromatin condensation and DNA fragmentation, and membrane changes involve the exposure of phosphatidylserine to external side of the cell membrane, which is necessary for the phagocytic removal of the apoptotic bodies [10]. Cd enhances production of reactive oxygen species (ROS), which results in increased lipid peroxidation, enhanced DNA and membrane damage, altered gene expression, apoptosis and cell proliferation [11, 12]. Intake of Cd results in the consumption of glutathione and protein binding sulfhydryl groups and subsequently the increased levels of free radicals such as hydrogen peroxide, hydroxide and superoxide. Selenium (Se) plays an important role in a number of biological processes for humans and animals. While low doses of selenium are needed to maintain animal and human health, its deficiency can induce coronary heart disease and liver necrosis [13–15]. Conversely the high Se levels can induce DNA damage [16, 17], oxidative stress [18], lipid peroxidation [19] and neurotoxicity and reduced protection against other compounds such as arsenic [20] or sodium metavanadate [21]. However, many of these effects depend on the level and chemical form of Se which at low concentrations could be antimutagenic but at high concentrations mutagenic and toxic [14, 22]. It has been suggested that Se could be protective against the toxic actions of Cd and other heavy metals [23–25]. This protection includes the capability of Se to alter Cd distribution in tissues by forming the Cd–Se complexes which in turn bind to proteins such as metallothioneins [26]. Therefore, it is essential to determine the optimum Se concentration which provides protection against Cdinduced genetic damage and toxicity. This study aimed to determine the appropriate dosage of Se that can counteract Cd toxicity by using rat liver as one of the most critical indicator of heavy metal toxicity. It can exhibit distinct histological [27] and morphological [28] changes in response to the Cd exposure. For this purpose, different biological assays were used in this study to examine the role of Se (sodium selenite) or Cd (cadmium chloride) to induce toxicity or the role of Se to counter the Cd-induced toxicity in male Sprague– Dawley rats that were exposed to the combined dose of Se and Cd.
Materials and Methods Chemicals All the chemicals and reagents were purchased from Sigma-Aldrich Co. Ltd. (UK) unless otherwise stated in the following sections. Animals and Treatments Following the approval by the Ethics Committee of the Quaid-iAzam University Islamabad, Pakistan, 20 post-weaning male Sprague–Dawley rats (28 days old) were housed at the animal unit of this University. The rats were acclimatized to their housing and feeding for 2 weeks before the commencement of this completely randomised study. The rats were housed in steel cages (38×23×10 cm) which were maintained in a room at 25±2°C with dark to light cycle of 14 to 10 h. All rats received the same commercial diet and fresh water throughout this study. The rats were weighed and distributed into four groups of five rats with similar initial mean body weight (BW) per
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group. Each rat received a subcutaneous injection of relevant treatment on alternate days over a 4-week period as follows: (I) control group, saline solution; (II) Cd group, CdCl2 in saline solution at a dose of 1 mg/kg BW; (III) Se group, saline solution of sodium selenite at a dose of 1 mg/kg BW and (IV) Cd–Se group, saline solution of CdCl2 plus sodium selenite each at a dose of 1 mg/kg BW. This dose was chosen because in our previous study these treatments at a lower dose of 0.5 mg/kg BW did not produce any effect on rats (data not shown). Although this chosen dose of Se was in the high pharmacological range [29], it was much lower than the fatal level of about 5–10 mg Se/kg BW [30]. All rats were weighed before their fasting for 12 h followed by their killing on 29th day. Liver tissues were isolated immediately, cleaned, weighed and washed with ice cold isotonic saline solution. A small piece of each fresh liver tissue was used for comet assay whereas 4–5 mm of each liver tissue was fixed in a fixative (60% ethanol+30% formalin+10% acetic acid) for histological observations. About 100 mg of each fresh liver tissue was homogenized in 0.1 M Tris–HCl buffer at pH 7.4 by using a Potter-Elvejham homogenizer at 4°C with a diluting factor of 4. The crude tissue homogenate was then centrifuged at 10,000 rpm for 15 min at 4°C to collect the supernatant which was kept at −20°C for the estimation of malondialdehyde (MDA), lipid hydroperoxides (LHP), reduced glutathione (GSH) and catalase activity (CAT). Assessment of DNA Damage The single-cell gel electrophoresis assay or comet assay was used for the detection of single-strand DNA breaks and reparation in individual cells [31]. Preparation of Liver Cell Suspensions Small pieces of relevant liver tissues were cut and minced individually in a cold phosphate-buffered saline (PBS) containing 20 mM ethylenediaminetetraacetic acid (EDTA) and 10% DMSO (dimethyl sulfoxide) which prevents lipid peroxidation. The mixture was allowed to settle followed by the aspiration of mincing solution. The fresh mincing solution was added again to mince the tissues into finer pieces. About 5–10 μl of each cell suspension was mixed with 75 μl of 0.5% low melting point agarose (LMPA) for further processing. Comet Assay Slides were prepared in triplicate per sample per experiment. Fully frosted slides were covered with 140 μl of 0.75% normal melting point agarose (NMPA) and allowed to polymerize at 4°C for 5 min. Then 20 μl of the liver suspension was mixed with 110 μl of 0.5% of LMPA, layered on the top of NMPA (stored at 37°C), covered with a cover slip and allowed to polymerize at 4°C for 10 min. The slides (without coverslips) were then immersed in freshly prepared, cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl pH 10, 1% N-lauroyl sarcosinate; 1% Triton X-100 and 10% DMSO were added just before use) in a Coplin jar for overnight at 4°C. After lysis, the slides were immersed in an alkaline buffer (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 25 min to allow unwinding of DNA. Electrophoresis of slides was conducted in dim light for 25 min at 25 V (0.66 V/cm) which was then adjusted to 300 mA. DNA fragments if any, due to DNA damage can migrate into the gel. The slides were then drained, placed on a tray, washed slowly with three changes for 5 min each in a neutralizing buffer (0.4 M Tris–HCl, pH 7.5), dehydrated in absolute methanol for 10 min and dried at room temperature. The slides were stained with 50 μl of SYBR Green staining solution (1 μl SYBR Green in 10 ml of TE buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA)), incubated at room temperature for 15 min in the dark and analysed by Leica TCS SP2 UV upright confocal system at ×20 by using AR488 nm filter. Images of 150 randomly selected cells (50 cells from each of three replicate slides) were analysed per sample using Comet Assay
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IV software from Perceptive Instruments Ltd. The cells with damaged DNA displayed the DNA migration from their nuclei towards anode. The damaged nucleoid formed a comet and the undamaged one formed a halo. The comet head and tail length were measured with a calibrated ocular micrometre disk. The quantification of the DNA damage was estimated as comet tail length, percent of tail DNA and extent tail moment which was calculated as follows: Extent tail moment ¼ Length of tail Tail DNA% Fixation, Staining and Histological Analysis For histological analysis the liver tissues with a diameter of 3–5 mm were fixed in sera (60% ethanol+30% formalin+10% acetic acid) for 3–4 h. The fixed samples were dehydrated at room temperature with ethanol and toluene series and embedded in paraffin. These paraffin embedded tissues were sectioned into thin slices of 4–5 μm by using a microtome (MICROM GmbH, HM 310, Ser.No. 6929, 69190 Walldorf, Germany), stretched in water and mounted on gelatin-coated marked glass slides. These sections were then stained with haematoxylin and eosin or used in the dUTP nick end-labelling (TUNEL) assay. The stained tissues were then examined under a light microscope (Vickers Ltd, England) and their images were captured by using a PC linked camera (Moticam 1000, Motic® China). TUNEL Assay Apoptosis in liver tissues was examined by the terminal deoxynucleotidyl transferase-mediated TUNEL assay by using the ApopTag® in situ apoptosis detection kit (Millipore, UK) according to the manufacturer’s guidelines. Here, the single- and doublestranded DNA breaks were detected by enzymatically labelling the free 3′-OH termini with modified nucleotides. The new DNA ends that were generated upon DNA fragmentation were typically localized in morphologically identifiable nuclei and apoptotic bodies. In contrast, normal or proliferative nuclei, which had relatively insignificant numbers of DNA 3′-OH ends, usually did not stain with the kit. After deparaffinization and rehydration the tissues were pretreated with freshly diluted proteinase K (20 μg/mL) for 15 min at room temperature in a coplin jar followed by 2 changes of dH2O for 2 min each. The tissues were quenched in 3% hydrogen peroxide in PBS for 5 min at about 20°C followed by washing in PBS for 5 min in a coplin jar. The tissue sections were shortly incubated with equilibration buffer at about 20°C followed by the addition of 55 μL/5 cm2 of working strength terminal deoxyribonucleotidyl transferase (TdT) and incubation in a humidified chamber at 37°C for 1 h. The tissues were incubated with working strength stop/wash buffer for 10 min followed by the addition of 65 μL/5 cm2 of Anti-Digoxigenin conjugate and incubation in a humidified chamber for 30 min at about 20°C. The tissue sections were stained with diaminobenzidine peroxidase substrate for 3 to 6 min at about 20°C in the dark. The slides were counterstained with haematoxylin and mounted under a glass cover slip in Canada balsam. The positive controls were prepared by treating the control tissues with DNase I and negative control included the omission of TdT enzyme from the labelling mixture. The slides were viewed under a light microscope (Vickers Ltd, England) and photographed by using the Moticam 1000 camera (Motic® China). Estimation of Lipid Peroxidation The concentration of lipid peroxidation end product (MDA) in the liver homogenate was determined by the method of Okhawa et al. [32]. Here, the reaction mixture contained 0.2 mL of 10% (w/v) tissue homogenate, 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid and 1.5 mL of 0.8% aqueous solution of
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thiobarbutaric acid. The pH of 20% acetic acid was pre-adjusted with 1 M NaOH to 3.5. The mixture was made up to 4 mL with distilled water, heated at 95°C for 1 h in a water bath by using antibumping granules. After cooling in tap water, 1 mL of distilled water and 5 mL of n-butanol and pyridine solution (15:1) were added and the mixture was vortex mixed (Bio Vortex; peQ Lab, UK). After centrifugation at 4,000 rpm for 10 min the absorbance of the upper organic layer was read at 532 nm. Tetramethoxypropane was used as an external standard, and the level of lipid peroxidation was expressed as nmol of MDA. The values of lipid peroxidation were expressed in nanomolars per gramme of tissues. Estimation of Lipid Hydroperoxides, Reduced Glutathione and Catalase The LHP were estimated by the method of Jiang et al. [33], in which 0.1 mL of 10% (w/v) tissue homogenate was treated with 0.9 mL of fox reagent (88 mg of butylated hydroxytoluene, 7.6 mg of xylenol orange and 9.8 mg of ammonium iron sulfate which were added to 90 mL methanol and 10 mL of 250 mM sulphuric acid) and incubated at 37°C for 30 min. The colour developed was then read at 560 nm and the lipid hydroperoxides were expressed as millimolars per gramme of tissues. The GSH content of the liver homogenate was measured at 412 nm by using the method of Sedlak and Lindsay [34]. The homogenate was precipitated with 50% trichloroacetic acid and then centrifuged at 1,000 rpm for 5 min. The reaction mixture contained 0.5 mL of supernatant, 2.0 mL of 0.2 M Tris–EDTA buffer (pH 8.9) and 0.1 mL of 0.01 M 5′5′- dithio-bis-2-nitrobenzoic acid. The solution was kept at about 20°C for 5 min and then read at 412 nm on the spectrophotometer. The values were expressed as μM/g of tissues. Catalase activity was assayed according to the method of Aebi [35]. About 50 μL of 10% (w/v) tissue homogenate (supernatant) were measured into a 3 mL cuvette containing 1.95 mL of 50 mM phosphate buffer (pH 7). About 1 mL of 30 mM hydrogen peroxide was added and the changes in absorbance were followed for 30 s at 240 nm at 15 s intervals. Catalase activity was expressed as unit released per mL of tissue homogenate. Statistical Analysis The data were statistically analysed by using ANOVA in Minitab software to determine the treatment effects on different parameters. The analysis compared the effect of the above mentioned treatments on body weight, relative weight of liver, lipid peroxidation, oxidative stress and comet assay parameters at P0.05) SD standard deviation
and b) whereas the Cd group showed many alterations including the mitotic division of nuclei, degenerating hepatocytes and vacuolation, the apoptotic bodies and the degenerating epithelium of the portal vein (Fig. 2c, d). Interestingly many changes in the rat liver structure were observed in the Se group which showed the degenerating line of the portal system, hepatocytes with pycnotic nuclei, the degenerating hepatocytes and vacuolation and the apoptotic bodies (Fig. 2e) although these changes were less severe than the Cd group. The liver histology of Cd+Se group showed less degenerative changes than the Cd and Se groups containing hepatocytes with pycnotic nuclei, the degenerating hepatocytes and vacuolation (Fig. 2f). Figure 3 represents the photomicrograph of TUNEL assay of rat livers from different treatments. Apoptotic bodies were found in positive control and different treatment groups whereas no apoptosis was found in the negative control and control groups. More apoptotic bodies were found in the Cd group than the Se and Cd+Se groups. It was interesting to note that the Cd+Se group showed less apoptotic bodies than the Se group. Table 3 presents the effects of Cd, Se and Cd+Se on lipid peroxidation end product (malondialdehyde, MDA), LHP, glutathione levels (GSH) and catalase activity in rat livers. The MDA levels were significantly higher in the Cd than the control and other rat groups (P 0.05). The LHP levels were significantly higher in Cd than control, Cd+Se, and Se groups of rats (P0.05). While the GSH levels were significantly lowered by the administration of Cd when compared with the control group, these were increased by the administration of Cd+Se than the Cd group (P0.05). The catalase activities were significantly lower in the Cd, Se and Cd+Se groups than the control group (P
