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]

1078

Jabeen and Chaudhry

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

Cadmium Chloride and Sodium Selenite for Rats

1079

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

1080

Jabeen and Chaudhry

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

Cadmium Chloride and Sodium Selenite for Rats

1081

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 P<0.05. Tukey’s test was used to compare treatments means at P<0.05. TUNEL assay was assessed by recording visual observations of the photographic illustrations of relevant tissues.

Results The effects of Cd, Se and Cd+Se on body weight and relative weight of liver (liver weight/ body weight of rat×100) in male Sprague–Dawley rats are shown in Table 1. There was no significant difference in the body weight or relative weight of liver between different rat treatment groups (P>0.05). Figure 1 represents the photomicrographs of the comet assay of the liver tissues of all treatment groups. Table 2 shows the data on head length, tail length, head intensity, tail intensity and extent tail moment of comets in liver tissues of different rat treatment groups. The head length was significantly lower in Cd than other groups (P< 0.001). The tail length of the Cd group was significantly greater than the control and other treatment groups (P<0.001). While the tail length in Cd+Se group was 4.7 and 2.4 times greater than the control and Se respectively, it was 2.3 times less than the Cd group (P<

1082

Jabeen and Chaudhry

Table 1 Mean (±SD) initial and final body weight (BW) and mean liver weight as percent of BW of different treatment groups of male Sprague–Dawley rats Items

Treatment groups Control

SEM Cd

Se

Cd–Se

Initial BW (g)

86.2±4.4

86.6±6.5

86.2±6.4

83.6±4.7

2.8

Final BW (g)

217.6±4.7

229.4±8.8

225.0±6.9

217.8±12.2

4.3

3.9±0.1

3.4±0.1

4.1±0.2

4.3±0.6

0.1

Liver (% BW)

SD standard deviation, SEM standard error of means

0.01). The head intensity for the Cd group was significantly lower but its Tail intensity was significantly higher than other treatment groups (P<0.001). There was no difference in tail intensity between Cd+Se and Se group (P>0.05) but it was different from the control group (P<0.001). There was a significant difference between treatments for the tail extent moment (P<0.01). Figure 2 represents the photomicrograph of the rat livers representing different treatments. Here the control group showed normal histology of rat liver (Fig. 2a

Fig. 1 Comet assay of liver cells from different treatment groups of male Sprague–Dawley rats representing a control, b selenium, c cadmium and d Cd+Se treatment groups

Cadmium Chloride and Sodium Selenite for Rats

1083

Table 2 Mean (±SD) of head and tail lengths (micrometres), head and tail intensity (% DNA) and extent tail moment (arbitrary units=tail length×tail % DNA) of comets for liver cells from different treatment groups of male Sprague–Dawley rats Comet parameters

Treatment groups Control

Cd

Se

Cd+Se

Head length

19.5±2.6 b

17.1±4.4 a

21.0±3.9 b

21.0±3.4 b

Tail length

8.3±1.2 c

89.1±19.9 a

16.4±2.2 c

38.7±5.3 b

Head intensity

98.8±8.6 c

48.2±6.2 a

77.1±3.4 b

75.2±6.1 b

Tail intensity

1.2±0.3 c

51.8±7.2 a

22.9±3.4 b

24.8±3.1 b

10.1±2.1 d

4,746.7±214.6 a

374.5±53.9 c

972.5±94.2 b

Extent tail moment

Means with same letters in the same row did not differ significantly (P>0.05) 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<0.001). There was no significant difference between the Se and control groups of rats for the MDA levels (P> 0.05). The LHP levels were significantly higher in Cd than control, Cd+Se, and Se groups of rats (P<0.05). There was no significant difference between the Control, Cd+Se and Se groups of rats for their liver LHP (P>0.05). 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 (P<0.001). There was no difference in GSH levels between the control and Se groups (P>0.05). The catalase activities were significantly lower in the Cd, Se and Cd+Se groups than the control group (P<0.05).

Discussion This study investigated the effects of either cadmium chloride or sodium selenite alone or together to observe their effects on growth and the liver of male Sprague–Dawley rats. For this purpose the comet assay, the TUNEL assay, oxidative stress and histological

1084

Jabeen and Chaudhry

Fig. 2 The photomicrograph of rat liver exposed to different treatments. a, b Control group; c, d Cd group in which arrows represent the mitotic division of nuclei, 5-point stars represent the degenerating hepatocytes and vaculation, triangles represent the apoptotic bodies, and 4-point star represents the degenerating epithelium of the portal vein; e Se group in which downward arrow represents the degenerating line of the portal system, upward arrow represents the hepatocytes with pycnotic nuclei, 5-point star represents the degenerating hepatocytes and vaculation and triangle represents the apoptotic bodies; and f Cd–Se group where less degenerative changes were observed as compared with the Cd and Se group, upward arrow represents the hepatocytes with pycnotic nuclei, 5-point star represents the degenerating hepatocytes and vaculation, and in this group less degenerative changes were observed as compared with the Cd and Se group

Cadmium Chloride and Sodium Selenite for Rats

1085

Fig. 3 The photomicrographs of TUNEL assay of rat livers exposed to different treatments: a positive control, b negative control, c control group, d Cd group, e Se group and f Cd+Se group. Apoptotic bodies in positive control and treatment groups are shown with arrows

parameters were considered. The comet assay was used for its simplicity, speed, and sensitivity to assess the DNA damage and repair quantitatively as well as qualitatively in individual cell populations [36]. Although different parameters of comet like head length, tail length, head intensity, tail intensity and extent tail moment were recorded; the DNA

1086

Jabeen and Chaudhry

Table 3 Mean (±SD) levels of lipid peroxides (MDA as nanomolars per gramme of tissues), hydroperoxides (LHP as millimolars per gramme), reduced glutathione (GSH as micromolars per gramme) and catalase (units per millilitre of tissues) in liver tissues of different treatment groups of male Sprague–Dawley rats Parameters

Treatment groups

SEM

Control

Cd

MDA

462.0±21.9 b

1,726.2±129.4 a

522.9±29.8 b

701.6±41.8 c

LHP

8.0±0.3 b

9.1 ±0.2 a

7.7±0.4 b

8.2±0.3 b

0.1*

GSH

2,245±115 a

1,262±43.7 b

2,448±247.9 a

2,729±66.6 c

71.3***

2.5±0.2 c

1.9±0.03 b

2.1±0.1 b, a

Catalase

Se

Cd–Se

2.1±0.1 b, a

35.3***

0.1*

Means with same letters within a same row did not differ significantly (P>0.05) *P<0.05; ***P<0.001, significance

damage was expressed mostly on the basis of its tail intensity and extent tail moment. This is because the tail DNA and olive tail moment are known to give a good correlation with genotoxicity [37] even when these are used routinely. Tail intensity could be a better parameter since tail moment was reported as arbitrary units where different image analysis systems gave different values [37]. It appeared from the comet assay, TUNEL and histological analysis of this study that the subcutaneous administration of either cadmium chloride or sodium selenite alone at the dose of 1 mg/kg body weight was genotoxic and cytotoxic for rats. This response was expected because although the dose of 1 mg Se/kg BW was not fatal for rats of this study, it was in the high pharmacological range [29, 30]. In fact, these findings agreed well with the previous findings [38, 39] where separate intraperitoneal administration of Cd or Se at the doses of 5–20 μM/kg did cause DNA damage and apoptosis in the liver or hepatocytes of Sprague–Dawley rats. These findings were further supported by previous studies [40], where the DNA damage of human lymphocytes being induced by sodium selenate, sodium selenite, and selenous acid on their own was observed by the comet assay. Conversely, it was interesting to note the antigenotoxic, antagonistic and anticytotoxic effects of 1 mg Se/kg body weight when it was administered together with Cd in the Cd–Se group of rats. This is in line with the findings of previous studies [41, 42] where Se at certain doses could antagonize DNA damage, apoptosis, changes of cell cycle and DNA relative content being induced by Cd in rat hepatocytes in vivo. The DNA damage and apoptosis due to Se alone in this study may be correlated well with several in vivo, in vitro, and epidemiological studies where adverse effects of Se were observed. It was found that Se can induce DNA damage [16, 17], oxidative stress [18], lipid peroxidation [19] and no protection against adverse actions of other compounds such as arsenic [20] or sodium metavanadate [21]. However, many of these effects depended on the level and the chemical form of Se. The apparent cytotoxic role of 1 mg Se per kg body weight of this study agreed well with other studies where several seleno compounds (mainly sodium selenite but also SeMet, Se dioxide, and methyl seleninic acid) induced cell death in different mammalian cell lines [43–49]. Although the precise mechanisms of apoptosis induced by the Se compounds were not well understood [44]; it was believed that ROS may have played a crucial role in Se-decreased cell viability and Se-induced apoptosis [45]. In this study, Cd treated rats showed not only a significant increase in the MDA and LHP levels, but also a decrease in the activity of Catalase and glutathione and an increase in the production of oxygen reactive forms. These results support other reports where Cd was able to up-regulate oxidative stress marker such as

Cadmium Chloride and Sodium Selenite for Rats

1087

MDA and decrease the activity of antioxidants such as GSH and Catalase [11, 50]. The reduction in the lipid peroxidation as indicated by the normalization of MDA, LHP and liver antioxidants (GSH and CAT) in response to the co-administration of Se and Cd agreed well with other reports [51]. These researchers indicated that Cd might induce phagocytic cells for the production of ROS which might be involved in the initiation of lipid peroxidation and oxidative stress in different tissues. Increased MDA and LHP levels and depressed antioxidant status in the livers of rats receiving 1 mg Cd per kg body weight suggest that the cytotoxic effect was imposed by this oxidative insult. Peroxidation of cellular membranes leads to molecular disorganization of lipids resulting in increased membrane permeability and leakage of cellular enzymes into circulation [52]. The decreased level of MDA in the Cd+Se than the Cd group was also a strong evidence of the antagonistic role of sodium selenite. In this study the lower GSH in the liver extracts of Cd than the control group may have been due to its consumption by the scavenging free radicals generated by Cd [50, 53]. Moreover, the sulfhydryl group of cysteine moiety of glutathione has a high affinity for metals, forming thermo dynamically stable mercaptide complexes with several metals, e.g. Cd. These complexes are inert and excreted via bile, so decreased GSH might have been due to its consumption during the Cd detoxification [54]. The catalase activities were decreased in the liver extracts of the Cd than the control rats which agreed with the earlier studies [50]. Inhibition of catalase after Cd treatment may be due to the depletion of Se in Cd detoxification. This suggestion is supported by previous studies [55] where Se joins Cd to form an inert Cd–Se complex. In this study, inhibitions of catalase activities were recovered by Se treatment of rats during Cd administration. In contrast, Se supplementation may have increased the activities of selenoproteins, e.g. GPx and TrxR perhaps due to an increased incorporation of seleno cysteine to form selenoproteins [56]. These complexes can decrease free radical-mediated lipid peroxidation and regenerate the GSH [57]. It was exhibited that a significant increase in DNA damage, apoptosis and histological changes in liver cells occurred via ROS as it played a very important role in DNA damage and apoptosis induction under both physiological and pathological conditions. Production of GSH is considered to be the first line of defence against oxidative damage and free radical generation where GSH functions as a scavenger and a cofactor in metabolic detoxification of ROS [58]. So the rats treated with a combination of Cd and Se could directly react with lipid hydroperoxides and increase the levels of GSH and catalase which then help prevent the DNA damage and apoptosis of their livers. The fact that the Se supplementation ameliorated the liver damage in the Cd group, indicated that the prevention was attributed to the inhibition of DNA damage and apoptosis by the increased levels of catalase and GSH. To the best of our knowledge, this is the first study in which all the tested parameters gave similar or complementary responses as observed by the comet and TUNEL assays which were then verified by the histology and oxidative stress parameters. So in the future, we can utilise any one or two of the above techniques to study toxicology to save time and cost and yet obtain reliable estimates. As Cd is a ubiquitous toxin of the natural and occupational environment and a large number of smokers are exposed to this metal, this study suggested the genotoxic and cytotoxic roles of Cd (as cadmium chloride) in rats. Hence, Cd and its compounds should be regarded with concerns. While Se is an essential and functional component of the antioxidant system of living organisms, its high levels or long term use can also cause the disruption of endocrine system, DNA and oxidation. However, it appears that Se could be used at its appropriate levels to counter some negative impacts of Cd on rat health and production. Indeed similar studies can help to monitor the

1088

Jabeen and Chaudhry

optimum dose of Se for its potential protection against genetic damage and toxicity being caused by metals such as Cd in living organisms.

Conclusions This study showed the genotoxic and cytotoxic effects of either Cd (as cadmium chloride) or Se (as sodium selenite) alone at the dose of 1 mg/kg body weight on alternate days for 1 month. This study also showed the antagonistic effect of Se in Cd-induced toxicity. It appeared that the subcutaneous administration of Se as sodium selenite was able to curtail the Cd-induced toxic effects in male Sprague–Dawley rats. It appeared that techniques such as comet, TUNEL, histology and oxidative stress analysis were promising in assessing either the genotoxic and cytotoxic potential of cadmium chloride and sodium selenite alone or their interactive effects when used together in rats. Acknowledgements Thanks to the Islamic Development Bank Jeddah, Saudi Arabia for funding this postdoctoral research at Newcastle University and Professor Dr. Samina Jalali, Dr. Sarwat Jahan, Dr. Robina Shaheen, Riffat Gillani, Noshaba Memon and Aysha Ambreen for their help and support during the rat trials at Quaid-i-Azam University Islamabad, Pakistan. Conflicts of Interest Authors have no conflict of interest.

References 1. Gałaz yn-Sidorczuk M, Brzóska MM, Moniuszko-Jakoniuk J (2008) Estimation of Polish cigarettes contamination with cadmium and lead, and exposure to these metals via smoking. Environ Monit Assess 137:481–493 2. Vromman V, Saegerman C, Pussemier L et al (2008) Cadmium in the food chain near non-ferrous metal production sites. Food Addit Contam 25:293–301 3. Palus J, Rydzynski K, Dziubaltowska E et al (2003) Genotoxic effects of occupational exposure to lead and cadmium. Mutat Res 540:19–28 4. Waalkes MP (2003) Cadmium carcinogenesis. Mutat Res 533:107–120 5. Waisberg M, Joseph P, Hale B et al (2003) Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicol 192:95–117 6. Gennari A, Cortese E, Boveri M (2003) Sensitive endpoints for evaluating cadmium-induced acute toxicity in LLC-PK1 cells. Toxicol 183:211–220 7. López E, Figueroa S, Oset-Gasquem MJ, González MP (2003) Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture. Br J Pharmacol 138:901–911 8. Wätjen W, Beyersmann D (2004) Cadmium-induced apoptosis in C6 glioma cells: influence of oxidative stress. Biometals 17:65–78 9. Shih CM, Ko WC, Yang L et al (2005) Detection of apoptosis and necrosis in normal human lung cells using 1H NMR spectroscopy. Ann NY Acad Sci 1042:488–496 10. Potten C, Wilson J (2004) How to die. In: Apoptosis. The life and death of cells. Cambridge University Press, Cambridge, pp 15–60 11. Stohs SJ, Bagchi D, Hassoun E, Bagchi M (2001) Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Toxicol Oncol 20:77–88 12. Yiin SJ, Chern CL, Sheu JY et al (2000) Cadmium induced liver, heart, and spleen lipid peroxidation in rats and protection by selenium. Biol Trace Elem Res 78:219–230 13. Letavayová L, Vlasakova D, Spallholz JE et al (2008) Toxicity and mutagenicity of selenium compounds in Saccharomyces cerevisiae. Mutat Res 638:1–10

Cadmium Chloride and Sodium Selenite for Rats

1089

14. Wu Q, Huang K (2004) Effect of long-term Se deficiency on the antioxidant capacities of rat vascular tissue. Biol Trace Elem Res 98:73–84 15. Agay D, Sandre C, Ducros V et al (2005) Optimization of selenium status by a single intraperitoneal injection of Se in Se deficient rat: possible application to burned patient treatment. Free Radic Biol Med 39:762–768 16. Biswas S, Talukder G, Sharma A (2000) Chromosome damage induced by selenium salt in human peripheral lymphocytes. Toxicol In Vitro 14:405–408 17. Machado Mda S, Villela IV, Moura DJ et al (2009) 3′3-ditriXuoromethyldiphenyl diselenide: a new organoselenium compound with interesting antigenotoxic and antimutagenic activities. Mutat Res 673:133–140 18. Wycherly BJ, Moak MA, Christensen MJ (2004) High dietary intake of sodium selenite induces oxidative DNA damage in rat liver. Nutr Cancer 48:78–83 19. Colado-Megía MI, Sánchez-Sánchez V, Camarero-Jiménez J, O’Shea-Gaya E (2004) Effect of dietary selenium on MDMA (“ecstasy”)-induced neurotoxicity in brain mouse (in Spanish). Mapfre Med 15:53–62 20. Hasgekar N, Beck JP, Dunkelberg H et al (2006) Influence of antimonite, selenite, and mercury on the toxicity of arsenic in primary rat hepatocytes. Biol Trace Elem Res 111:167–183 21. Zwolak I, Zaporowska H (2009) Preliminary studies on the effect of zinc and selenium on vanadiuminduced cytotoxicity in vitro. Acta Biol Hung 60:55–56 22. Alaejos MS, Diaz Romero FJ, Diaz Romero C (2000) Selenium and cancer: some nutritional aspects. Nutrition 16:376–383 23. Ognjanović B, Žikić RV, Štajn A et al (1995) The effects of selenium on the antioxidant defense system in the liver of rats exposed to cadmium. Physiol Res 44:293–300 24. Žikić RV, Štajn AŠ, Ognjanović BI et al (1998) The effect of cadmium and selenium on the antioxidant enzyme activities in rat heart. J Environ Pathol Toxicol Oncol 17:259–264 25. Xiao P, Jia XD, Zhong WJ et al (2002) Restorative effects of zinc and selenium on cadmium induced kidney oxidative damage in rats. Biomed Environ Sci 15:67–74 26. Combs G, Gray WP (1998) Chemopreventive agents: selenium. Pharmacol Ther 79:179–192 27. Koyu A, Gokcimen A, Ozguner F, Bayram DS, Kocak A (2006) Evaluation of the effects of cadmium on rat liver. Mol Cell Biochem 284(1–2):81–85 28. Mitsumori K, Shibutani M, Sato S et al (1998) Relationship between the development of hepato-renal toxicity and cadmium accumulation in rats given minimum to large amounts of cadmium chloride in the long term: preliminary study. Arch Toxicol 72(9):545–552 29. Beer MH, Porter RS, Jones TV (2006) The Merck manual of diagnosis and therapy, 18th edn. Wiley, UK 30. Nuttall KL (2006) Review: evaluating selenium poisoning. Ann Clin Lab Sci 36:409–420 31. Singh NP, Mc Coy MT, Tice RR et al (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191 32. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358 33. Jiang ZY, Hunt JV, Wolff SD (1992) Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxides in low density lipoprotein. Anal Biochem 202:384–391 34. Sedlak J, Lindsay RH (1968) Estimation of total protein bound and non-protein sulfhydryl groups in tissue with ellmans reagent. Anal Biochem 25:293–298 35. Aebi H (1974) Catalase. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic, New York, pp 673–685 36. Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1(1):23–29 37. Kumaravel TS, Jha AN (2006) Reliable comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat Res 605(1–2):7–16 38. Yu RA, He LF, Chen XM (2007) Effects of cadmium on hepatocellular DNA damage, proto-oncogene expression and apoptosis in rats. Biomed Environ Sci 20(2):146–153 39. Yu RA, Yang CF, Chen XM (2006) DNA damage, apoptosis and C-myc, C-fos, and C-jun over expression induced by selenium in rat hepatocytes. Biomed Environ Sci 19(3):197–204 40. Cemeli E, Carder J, Anderson D et al (2003) Antigenotoxic properties of selenium compounds on potassium dichromate and hydrogen peroxide. Teratog Carcinog Mutagen 23:53–67 41. Yu RA, Chen XM (2004) Effects of selenium on rat hepatocellular DNA damage, apoptosis and changes of cell cycle induced by cadmium in vivo. Zhonghua Yu Fang Yi Xue Za Zhi 38(3):155–158 42. Cemeli E, Marcos R, Anderson D (2006) Genotoxic and antigenotoxic properties of selenium compounds in the in vitro micronucleus assay with human whole blood lymphocytes and TK6 lymphoblastoid cells. Sci World J 6:1202–1210

1090

Jabeen and Chaudhry

43. Xiang N, Zhao R, Zhong W (2009) Sodium selenite induces apoptosis by generation of superoxide via the mitochondrial-dependent pathway in human prostate cancer cells. Cancer Chemother Pharmacol 63:351–362 44. Philchenkov A, Zavelevich M, Khranovskaya N, Surai P (2007) Comparative analysis of apoptosis inductions by selenium compounds in human lymphoblastic leukaemia MT-4 cells. Exp Oncol 29:257– 261 45. Zou Y, Yang J, Liu X, Yuan J (2007) Relationship between reactive oxygen species and apoptosis in HepG2 cells induced by sodium selenite. Wei Sheng Yan Jiu 36:272–274 46. Goel A, Fuerst F, Hotchkiss E, Boland CR (2006) Selenomethionine induces p53 mediated cell cycle arrest and apoptosis in human colon cancer cells. Cancer Biol Ther 5:529–535 47. Last K, Maharaj L, Perry J (2006) The activity of methylated and non-methylated selenium species in lymphoma cell lines and primary tumours. Ann Oncol 7:773–779 48. Wang XH, Wei YM, Bai H et al (2004) Apoptosis and regulation of expressions of apoptosis-related gene Bcl-2 and p53 induced by selenium dioxide in three leukaemia cell lines. Di Yi Jun Yi Da Xue Xue Bao 24(10):1160–1163 49. Rooprai HK, Kyriazis I, Nuttall RK et al (2007) Inhibition of invasion and induction of apoptosis by selenium in human malignant brain tumour cells in vitro. Int J Oncol 30:1263–1271 50. Al-Hashem F, Dallak M, Bashir N et al (2009) Camel’s milk protects against cadmium chloride induced toxicity in white albino rats. Am J Pharmacol Toxicol 4(3):107–117 51. Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18:321–336 52. Mason RP, Walter MF, Mason PE (1997) Effect of oxidative stress on membrane structure: small angle X-ray diffraction analysis. Free Radic Biol Med 23:419–425 53. Koyuturk M, Yanardag R, Bulkent S, Tunali S (2006) Influence of combined antioxidants against cadmium induced testicular damage. Environ Toxicol Pharmacol 21:235–240 54. Mohanpuria P, Rana NK, Yadav SK (2007) Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis (L.) O. Kuntze. Environ Toxicol 22(4):368–374 55. Lazarus M, Orct T, Blanusa M (2006) Effect of selenium pre-treatment on cadmium content and enzymatic antioxidants in tissues of suckling rat. Toxicol Lett 164S1: S191 56. Saito Y, Takahashi K (2002) Characterization of selenoprotein P as a selenium supply protein. Eur J Biochem 269:5746–5751 57. Gan L, Liu Q, Xu HB et al (2002) Effects of selenium overexposure on glutathione peroxidase and Thioredoxin reductase gene expressions and activities. Biol Trace Elem Res 89:165–175 58. Tandon SK, Singh S, Prasad S et al (2003) Reversal of cadmium induced oxidative stress by chelating agent, antioxidant, or their combination in rat. Toxicol Lett 45:211–217