Cadmium induced hepatotoxicity in chickens (Gallus domesticus) and ameliorative effect by selenium

Ecotoxicology and Environmental Safety 96 (2013) 103–109

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Cadmium induced hepatotoxicity in chickens (Gallus domesticus) and ameliorative effect by selenium Jin-Long Li 1, Cheng-Yu Jiang 1, Shu Li, Shi-Wen Xu n College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People′s Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 29 May 2012 Received in revised form 29 June 2013 Accepted 4 July 2013 Available online 29 July 2013

Cadmium (Cd) is one of the most toxic metal compounds released into the environment. It was well known that Cd induced hepatotoxicity in animal models. However, little is known about the negative effects of Cd toxicity in the liver of birds. To investigate the Cd hepatotoxicity in birds and the protective effects of selenium (Se) against subchronic exposure to dietary Cd, 100-day-old cocks received either Se (as 10 mg Na2SeO3 per kg of diet), Cd (as 150 mg CdCl2 per kg of diet) or Cd+Se in their diets for 60 days. Histological and ultrastructural changes in the liver, the concentrations of Cd and Se, the lipid peroxidation (LPO) and nitric oxide (NO) production, the activities of the antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx), nitric oxide synthase (NOS) activities and apoptosis were determined. Exposure to Cd significantly reduced SOD and GPx activity, Se content in the liver tissue. It increased the LPO and NO production, the numbers of apoptotic cells and Cd concentration and caused obvious histopathological changes in the liver. Concurrent treatment with Se reduced the Cd-induced liver histopathological changes, oxidative stress, overexpression of NO and apoptosis, suggesting that the toxic effects of Cd on the liver is partly ameliorated by inorganic Se. Se supplementation also modified the distribution of Cd in the liver. & 2013 Elsevier Inc. All rights reserved.

Keywords: Cadmium Selenium Oxidative stress Apoptosis Chicken liver

1. Introduction Cadmium (Cd), a non essential heavy metal is a demonstrated environmental pollutant, arising primarily from battery, electroplating, pigment, plastic and fertilizer industries. It occurs in almost all soils, surface waters and plants, and it is readily mobilized by human activities. Cd contamination of environment is a subject of serious international concern since the metal is known to enter the food chain and can undergo bioaccumulation, endangering animal health (Roccheri et al., 2004; Lucia et al., 2009). Once the metal is absorbed, it is retained within the body with little excretion and thus, even in uncontaminated environment or very low concentration, there is an accumulation of the metal within the vital organs, causing hepatotoxicity, nephrotoxicity, and cardiomyopathy (Rikans and Yamano, 2000; Thévenod, 2003). The liver is the target organ in which Cd primarily accumulates and exerts its several deleterious effects (Bernard, 2004). Most in vivo and in vitro studies of the Cd toxicity in many mammalian organs have shown that Cd preferentially localized in hepatocytes and causes various adverse effects, mainly the accumulation,

n

Corresponding author. E-mail address: [email protected] (S.-W. Xu). 1 These two authors contributed equally to this work.

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.07.007

histopathological and cellular changes, enhancement of lipid peroxidation, influence on mitochondrial function and DNA chain break (Toman et al., 2005; Berzina et al., 2007). Cd toxicity has also been linked to declines in body condition in birds (Wayland et al., 2002; Anteau et al., 2007; Pollock and Machin, 2009). Several studies have shown that the liver and kidney are organs which accumulate Cd the most in chickens (Marettová et al., 2012). In fowl, the attention was paid to residual Cd in the organs and its transfer into eggs (Sato et al., 1997). Investigations by our lab have found that dietary exposure to Cd caused histopathological changes, oxidative stress, endocrine disorder and apoptosis in cock testes (Li et al., 2010a). Cd inhibits the viability of the chicken splenic lymphocyte and induces the oxidative stress and subsequently DNA damage and apoptosis (Li et al., 2010b). Cd exposure altered the activities of antioxidant enzymes of erythrocytes and produce oxidative stress by disturbing the oxidative and antioxidative balance of the adult poultry birds (Kant et al., 2011). Mitigation of Cd induced toxicity is of primary importance in the field of toxicological research and public health. Various mechanisms have been reported to explain the Cd-induced oxidative liver injury, including increased lipid peroxidation, depleting GSH or by inhibition of antioxidant enzymes and interaction with membrane components. Selenium (Se) has been found and accepted as an essential micronutrient and a potent chemopreventive agent. Several studies have indicated that the treatment with Se protects various organs and

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tissues against the toxicity of Cd (El-Sharaky et al., 2007; Messaoudi et al., 2009). Some studies found a protective effect of Se against the Cd hepatotoxicity in rodents and humans (Newairy et al., 2007; Ognjanović et al., 2008; Jihen et al., 2009; Lazarus et al., 2009). Other studies showed that organic Se administered could protect broilers from Cd toxicity (Pappas et al., 2011; Al-Waeli et al., 2013). Although there are several deleterious effects of Cd exposure on the liver in humans and rodents, there are no studies focusing on the hepatotoxicity in birds. Also the exact mechanism of toxic effects in birds is not clear. As liver being the primary target organ for Cd induced toxicity, in the study herein, the present work investigated the liver injury in chickens after a sub-chronic exposure to Cd and the simultaneous administration of Cd and Se by a dietary route. We evaluated histopathology, oxidative stress and apoptosis in the liver of chickens.

2. Materials and methods 2.1. Birds and experimental design All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. We determined that oral 50 percent lethal dose (LD50) of Cd for chicken was 218.44 mg/kg B W. To exacerbate the Cd hepatotoxicity and reveal the Se detoxification effects, the dose levels of Cd and Se was 150 mg/kg CdCl2 and 10 mg/kg Na2SeO3 (super-nutritional Se but not Se toxicity) respectively. The dose and duration of this study have been described previously (Li et al., 2010a). In brief, twenty-four 100-day-old Isa Brown male chickens were divided randomly into four groups (n¼ 6 per group). Group I (Control) were fed a basal diet; group II (Se-treated) were fed with the basic diet supplemented with 10 mg/kg Na2SeO3; group III (Cd/Se-treated group) were fed with the basic diet supplemented with 150 mg/kg CdCl2+10 mg/kg Na2SeO3 and group IV (Cd-treated group) were fed the basal diet supplemented with 150 mg/kg CdCl2. Birds were maintained in the Laboratory Animal Center, College of Veterinary Medicine, Northeast Agricultural University, China, kept under a 16/8 h light/dark cycle and given free access to standard food and water. On the 60th day of the experiment, all of the chickens were fasted overnight. Following euthanasia, the livers were immediately excised, blotted and then rinsed with ice-cold 0.9 percent NaCl solution. They were dried with filter paper and weighed. Fractions of livers (500 mg) were homogenized using glass Teflon homogenizer in cold 0.9 percent NaCl solution. The homogenates were centrifuged at 1000g for 10 min at 4 1C. The supernatant was collected and used for the experiments.

2.2. Estimation of hepatic Cd and Se concentrations The content of Cd in the livers was detected using flame atomic absorption spectrometry (FAAS). The optimal operating conditions were: wavelength λ 228.8 nm, slit 0.2 nm, burner height 5.0 mm, light current I 2.0 mA, acetylene discharge 1.5 L/min, air discharge 6.0 L/min. The wet tissue samples (1.0 g) were cut into small pieces with a stainless-steel knife and were transferred into beakers. For digestion, 25 mL of concentrated HNO3/HCl (4:1) was added to each beaker and warmed on a low temperature electric hot plate to solution transparence. The samples were metered volume to 10 mL by 0.5 percent HNO3 and measured using FAAS. The content of Cd was calculated from a standard curve. The content of Se in the livers was estimated by the method described by Hasunuma et al. (1982). The assay is based on the principle that Se in samples following acid digestion is converted to selenous acid. The reaction between selenous acid and aromatic-o-diamines, such as 2,3-diamino-naphthalene (DAN), leads to the formation of 4,5-benzopiazselenol, which displays a brilliant limegreen fluorescence when excited at 366 nm in cyclohexane. Fluorescence emission in extracted cyclohexane was read using a spectrophotometer using 366 nm as the excitation wavelength and 520 nm as the emission wavelength. The content of Se was calculated from a standard curve.

2.3. Light microscopic examination Parts of liver tissue obtained from each chickens were fixed in 10 percent buffered neutral formalin, dehydrated in ascending grades of alcohol and embedded in paraffin. Sections of about 5 μm thickness were taken, stained with hematoxylin and eosin (H&E) and examined under light microscope, by a pathologist unaware of the treatment protocol, to detect degeneration, vacuolization and necrosis of hepatocytes.

2.4. Electron microscopic examination For electron microscopy, small pieces of liver tissue (about 1 mm3) were rapidly fixed with 2.5 percent glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 1C, washed in the same buffer for 1 h at 4 1C and postfixed with 1 percent osmium tetroxide in sodium phosphate buffer for 1 h at 4 1C. The tissues were then dehydrated in graded series of ethanol, starting at 50 percent each step for 10 min, after two changes in propylene oxide. The tissue specimens were embedded in Araldite. Ultrathin sections were stained with Mg-uranyl acetate and lead citrate for transmission electron microscope (TEM) evaluation. 2.5. In situ apoptosis detection Cleavage of genomic DNA during apoptosis yields single strand breaks (nicks) in high molecular weight DNA. Apoptotic nuclei in liver tissue sections were identified using the in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) biotin nick-end labeling (TUNEL) technique that identifies DNA strand breaks by labeling their free 3′-OH termini. The present work used in situ cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany). The method distinguishes apoptotic cells from those undergoing necrosis, because damaged DNA in the former leads to a different distribution of staining and nuclear morphology. Paraffin wax-embedded tissue sections were treated with proteinase K and the endogenous peroxidase activity was blocked with hydrogen peroxide. The sections were incubated at 37 1C with the terminal TdT/nucleotide mixture for 1 h. Then, the reaction was stopped and the slides were rinsed with phosphate buffered saline. Nuclear labeling was developed with horseradish peroxidase and diaminobenzidine. Hematoxylin was used for counterstaining. Quantitative evaluation of the apoptotic index was performed by manual counting of positively stained nuclei at 400  magnification. Apoptosis was determined in five testes from each group of chickens by counting at least 1000 cells from 5–6 sections of each testis. The results are expressed as the percentage of TUNEL-positive cells among the total number of cells counted. 2.6. Determination of protein content Protein determinations were made using the dye-binding method of Bradford (1976). Bovine serum albumin (BSA) was used to construct the standard curve. 2.7. Lipid peroxidation Lipid peroxidation (LPO) was assessed by measuring malondialdehyde (MDA) formation, using the thiobarbituric acid assay (Esterbauer and Cheeseman, 1990). The content of LPO in livers was carried out with the MDA detection kit (A003-1, Nanjing Jiancheng Bioengineering Institute, P. R. China) according to the manufacturer′s protocol. MDA was determined using the thiobarbituric acid (TBA) method based on its reaction with TBA to form thiobarbituric acid-reactive substances (TBARS). 2.8. Antioxidant assays The activity levels of the antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in liver homogenates of all experimental chickens. The GPx activity assay was performed with GPx detection kits according to the manufacturer′s protocol (A005-1, Nanjing Jiancheng Bioengineering Institute, P. R. China). The GPx activity was detected by the oxidizing speed of the GSH, which can be expressed by the GSH reduction in a certain time. One unit of GPx activity was defined as 1 mmol/L GSH oxidized to GSSG (glutathione disulphide) per milligram of protein per minute. The SOD activity was measured using SOD detection kit (A001-1, Nanjing Jiancheng Bioengineering Institute, P. R. China) according to the manufacturer′s protocol. The SOD activity was determined by hydroxylamine assay-developed from xanthine oxidase assay, which was presented as units per milligram of protein. 2.9. Nitric oxide and nitric oxide synthase activity assay The liver homogenates of all experimental chickens were used for nitric oxide (NO) and total nitric oxide synthase (NOS) activity assay. The activities of NOS were spectrophotometrically measured with commercial-available kits (A014, Nanjing Jiancheng Bioengineering Institute, P. R. China) based on the oxidation of oxyhaemoglobin to methaemoglobin by nitric oxide. The concentration of nitrite was measured to reflect the production of NO commercial-available kits (A012, Nanjing Jiancheng Bioengineering Institute, P. R. China), according to the manufacturer′s protocol. In brief, the supernatant was mixed with the Griess reagent (1 percent sulfanilamide, 0.1 percent N-l-naphathyletylenediamine dihydrochloride and 2.5 percent phosphoric acid) at room temperature for 10 min. Nitrite products in the supernatants were determined by

J.-L. Li et al. / Ecotoxicology and Environmental Safety 96 (2013) 103–109 measuring absorbance at 550 nm with NaNO2 being used for a standard curve. The results were expressed as μmol/g protein.

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Statistical analysis of all data was performed using SPSS for Windows (version 13; SPSS Inc., Chicago, IL, USA). When a significant value (Po 0.05) was obtained by one-way analysis of variance, further analysis was carried out. All data showed a normal distribution and passed equal variance testing. Differences between means were assessed using Tukey′s honestly significant difference test for post hoc multiple comparisons. Data are expressed as the mean 7 standard deviation.

margination (Fig. 3c1 and c3), formation of multiple folds and pores in the nuclear membrane (Fig. 3c2 and c3), loss of rough endoplasmic reticulum (Fig. 3c1 and c2), and swollen mitochondria with distorted cristae (Fig. 3c1 and c4). Se co-treatment restored the normal electron microscopic appearance of liver tissue, similar to that observed in the control group. Slightly dilated cisternae of the rough endoplasmic reticulum and swollen mitochondria were observed in the hepatocyte (Fig. 3b1 and b3). Cytoplasmic vacuolation and abnormal chromatin distribution of the liver cells were not evident.

3. Results

3.3. Apoptosis in the liver

3.1. Cd and Se concentrations

The number of apoptotic cells in the livers shown by TUNEL assay was significantly increased in the Cd-treated group compared with the control and Se groups (Fig. 2d–f). Se supplementation significantly reduced the number of apoptotic cells in the Cd/Se-treated group compared with the Cd group (P o0.01; Fig. 4). The numbers of apoptotic cells were similar in the control and Se groups (Fig. 4).

2.10. Statistical analysis

After 60 d exposure to Cd in the feed, there were significant differences in Cd and Se concentrations between groups (Fig. 1). A significant (P o0.01) accumulation of Cd in the liver compared with the values in the control chickens was showed in Fig. 1. Cotreatment of Cd with Se also induced a significant (P o0.01) increase in the level of Cd accumulated in the liver compared with the level of the Cd group. Se in the diet reduced the level of Cd in the liver tissue and significantly increased the Se content of the liver tissue in the Cd+Se group (P o0.01). Se content in the liver tissue of the Se-treated group was significantly higher than the other groups (Po 0.05 or P o0.01), but Cd concentration was lower than the control group. 3.2. Histopathology and ultrastructure

Cd content

14

Se content

1.4

# a** # b** #

12 10

1

#b#

8

0.8 #b#

6 4 2 0

1.2

** **

Control

0.6 # a**

0.2

# a**

Se

0.4

Cd+Se

Cd

0

Se content in the liver tissuse (ug/g)

Cd content in the liver tissuse (ug/g)

Histology of the livers in control and Se-treated cocks showed the normal architecture of hepatic cords and their radiation from the central vein (CV) and blood sinusoids with phagocytic Kupffer cells. Chickens treated with Cd showed marked damage of hepatocytes in the form of ballooning degeneration, cytoplasmic vacuolation, coagulative necrosis with focal areas of macroscopic peliosis hepatis (Fig. 2a). Se treatment markedly attenuated the Cd-induced liver tissue injury and restored the same histopathological picture observed with the control group (Fig. 2b and c). Electron microscopy showed normal hepatic ultrastructure in the control and Se groups (Fig. 3a1 and a2). The control hepatocyte showed the normal appearance of polygonal hepatocyte and smooth rounded nucleus with abundant normally distributed chromatin and prominent nucleolus (Fig. 3a1), normal endothelial cell (Fig. 3a2), rough endoplasmic reticulum, intact mitochondria with normal cristae (Fig. 3a1). Cd treatment alone caused extensive hepatocellular injury as evidenced by nuclear chromatin condensation and

Fig. 1. Cd and Se contents in the liver tissue of the normal and experimental chickens. Each value is the mean 7S.D. Statistically significant differences: # Po 0.05 compared with control group; ## P o0.01 compared with the control group; *Po 0.05 compared with the Cd+Se group; ** values differ significantly from the Cd+Se group (P o0.01).

3.4. Lipid peroxidation As shown in Table 1, the LPO levels of liver tissue in the Cd group were significantly higher than in the control and Se groups (P o0.01), but the levels were not significantly different between the control and Se groups (P 40.05). Se supplementation significantly reduced the LPO levels in the Cd/Se-treated group compared with the Cd group (Po 0.01). 3.5. Antioxidant activity The SOD and GPx activity levels in the liver tissue of the experimental chickens are shown in Table 1. Cd administration significantly reduced the activities compared with the control and Se groups (Po 0.01). Thus, Se addition in the diet of birds fed Cd (Se+Cd group) improved the level of antioxidant enzymes compared to Cd alone group but did not restore it to the levels of control. 3.6. NO levels and NOS activity in the liver As shown in Fig. 5, the NO levels and NOS activity in the liver tissue of the Cd-treated chickens was increased significantly. However, Cd/Se co-treatment was able to partly reverse this. The NO levels and NOS activity showed no statistically significant differences between the control and Se groups (Fig. 5).

4. Discussion Cd is one of the most toxic metal compounds released into the environment and is one of the important environment pollutant that can be transferred among various levels of the food chain (Larison et al., 2000). Elevated levels of Cd have been reported in birds and mammals caused by long-range transport of pollution (Berzina et al., 2007; Kumar et al., 2007; Pan et al., 2008; Roodbergen et al., 2008; Lucia et al., 2009; Li et al., 2010a, 2010b; Kant et al., 2011; Marettová et al., 2012). Although Cd is widely distributed in the body, it preferentially localizes in the liver and has varying degrees of toxicity. It was well known that Cd induced hepatotoxicity in humans, farm and laboratory animals (Casalino et al., 2002; Arnold et al., 2006; Koyu et al., 2006; Jihen et al., 2008, 2009; El-Sokkary et al., 2010; Renugadevi and Prabu, 2010; Pari and Shagirtha, 2012).

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Fig. 2. Histology ((a)–(c), hematoxylin and eosin staining,  400) and TUNEL staining ((d)–(f), counterstained with hematoxylin,  400) of the liver section in different groups. (a) Liver section from the Cd-treated group showing ballooning degeneration, cytoplasmic vacuolization, coagulative necrosis of hepatocytes, focal areas of macroscopic peliosis hepatis and loss of definition of liver plates. (b) Liver section from the Cd/Se-treated group showing preserved hepatic architecture without ballooning or coagulative necrosis of hepatocytes nor cytoplasmic vacuolation, and intact liver plates. (c) Liver section from the Cd-treated group. (d) Liver section from the Cd-treated group showing increased numbers of apoptotic cells (arrows). (e) Liver section from the Cd/Se-treated group showing a low level of apoptosis. (f) Liver section from the Cd-treated group.

Liver is one of the major sites of Cd accumulation in the organism and target organs for this metal under both chronic and acute exposure (Renugadevi and Prabu, 2010). Liver changes in response to Cd toxicity have been seen in a variety of animal models. Cd can cause, even in very low Cd concentrations, distinct pathological changes to the liver (Koyu et al., 2006). In fact, several morphological changes in the hepatic tissue was observed after exposure to Cd (Mitsumori et al., 1998; Jihen et al., 2008; El-Sokkary et al., 2010). Zonal necrosis demonstrated in liver after acute Cd intoxication in rats (Tzirogiannis et al., 2004). Exposure to Cd resulted in its accumulation and severe histological changes in the liver in male rats (Jihen et al., 2008). Hepatic histoarchitecture of the Cd-treated rats resulted in severe damage of parenchyma with necrosis, lymphatic infiltration, dilation of sinusoids, cellular degeneration and intracellular vacuolation and pyknotic nuclei (Pari and Shagirtha, 2012). Cd exposure caused hepatic cell death mainly via the necrotic pathway (Sinha et al., 2009). Similarly severe macro and micro-morphological changes were detected in the liver of the rats administered with Cd (El-Sokkary et al., 2010). The administration of dietary Cd into birds (hens, cocks, broilers and ducks) reflected in a high increase of Cd level in the livers (Lucia et al., 2009; Marettová et al., 2012; Pappas et al., 2011; Al-Waeli et al., 2013). Through the present study, this work investigated some effects of dietary Cd on its accumulation in the liver and on their histological structure in the chickens. The dietary Cd induced similar histopathological changes in the livers (ballooning degeneration, cytoplasmic vacuolation, coagulative necrosis with focal areas of macroscopic peliosis hepatis) as was observed following Cd administration in rodents (Jihen et al., 2008; Sinha et al., 2009; El-Sokkary et al., 2010; Pari and Shagirtha, 2012). The results confirmed the involvement of apoptosis and necrosis in the pathways of Cd hepatotoxicity. The character of the pathological changes caused by Cd in the liver is

in accordance with findings of other authors (Murugavel and Pari, 2007; Jihen et al., 2008; Renugadevi and Prabu, 2010). Mitochondria are the proverbial powerhouses of the cell, running the fundamental biochemical processes that produce energy from nutrients using oxygen. They are among the key intracellular targets for different stressors including Cd (Sanni et al., 2008; Cannino et al., 2009). In rat liver the metal modifies mitochondrial function, inhibiting oxidative phosphorylation: in particular low concentrations stimulate resting state respiration, while high concentrations depress basal respiration (Belyaeva and Korotkov, 2003). It has been proposed that Cd initially binds to protein thiols in the mitochondrial membrane, affects the mitochondrial permeability transition, inhibits the respiratory chain reaction and that this generates ROS (Dorta et al., 2003). These effects on mitochondrial electron transfer are the major source of Cd-generated ROS not only in mammalian cells but also in plants (Heyno et al., 2008). In this study, markedly swollen mitochondria with degenerated or missing cristae were observed in hepatocytes by electron microscopy. These results are in agreement with previous studies (Fouad et al., 2009; Lucia et al., 2009). Cd is redox stable metal, therefore, most in vivo and in vitro studies seemed to point to a direct and major role for oxidative stress as an inducer of hepatic toxicity. The metal is known to disturb the oxidative balance of the organism and the free radical processes have been recognized to be involved in the mechanisms of health effects of intoxication with these substances. Cd induces oxidative damage by increasing the production of ROS (Chen et al., 2008; Liu et al., 2008) and decreasing the biological activities of some antioxidants, such as SOD and GPx (El-Sharaky et al., 2007; Jihen et al., 2008; Renugadevi and Prabu, 2010), which play an important role in antioxidant defense and in the elimination of free radicals. Researches on cock testes and chicken splenic lymphocytes in our laboratory have shown the potential for Cd

J.-L. Li et al. / Ecotoxicology and Environmental Safety 96 (2013) 103–109

107

NU

MI

NU MI

NU

NU MI

MI

ER NU

NU

MI

ER

MI NU

MI

ER

MI MI

NU MI

NU

NU

ER

Fig. 3. Transmission electron microscopy of liver tissues. Liver of chicken from the control and Se-treated group ((a1)  30,000; (a2)  10,000) showing the normal appearance of hepatocyte and smooth rounded nucleus with abundant normally distributed chromatin and prominent nucleolus, rough endoplasmic reticulum and intact mitochondria with normal cristae. Liver of chicken from the Cd/Se-treated group ((b1)  8000; (b2) and (b3)  30,000) showing slightly dilated cisternae of the rough endoplasmic reticulum and swollen mitochondria. Hepatocytes in the Cd-treated group ((c1)  10,000) displayed morphological characteristics of apoptosis, including chromatin condensation showing as apoptotic bodies (black arrows). Liver of chicken from the control and Se-treated group ((c2) and (c3)  12,000; (c4)  50,000) showing severe liver damage in the form of loss the normal hepatocyte architecture and irregular folded pyknotic nuclei with dense clumped marginal chromatin, widespread vacuolization, severe dilated cisternae of the rough endoplasmic reticulum, multiple pores in the nuclear membrane and swollen mitochondria with distorted cristae. Key: SER, smooth endoplasmic reticulum; MI, mitochondria; NU, nucleus.

8

Apoptosis (%)

7 6 5 4 3 2 1 0

control

Se

Cd+Se

Cd

Fig. 4. Effects of Se treatment on liver apoptosis in Cd-treated chickens. Each value is the mean 7 S.D. Statistically significant differences: # Po 0.05 compared with control group; ## Po 0.01 compared with the control group; * P o0.05 compared with the Cd+Se group; ** values differ significantly from the Cd+Se group (Po 0.01).

to significantly affect the antioxidant enzymes: SOD and GPx and to increase lipid peroxide concentration (MDA) and ROS (Li et al.,

2010a, 2010b). In the current study, the results of the present work are in agreement with previous studies, which clearly demonstrated that Cd exposure increases LPO and suppresses the antioxidant defense mechanisms in liver tissue with significant morphological changes (Murugavel and Pari, 2007; Rana, 2008; Jihen et al., 2009; Templeton and Liu, 2010). The oxidative stress could induce many kinds of negative effects including membrane peroxidation and apoptosis. Apoptosis is an essential mechanism for eliminating critically damaged cells in the liver of Cd-intoxicated rats (Tzirogiannis et al., 2003). Cd intoxication has been reported to induce dose-dependent apoptosis in the mouse liver (Habeebu et al., 1998). In this study, the microscopic examination of the liver in chicken exposed to Cd revealed morphological changes typical of apoptosis such as chromatin condensation showing as apoptotic bodies. In accordance with these morphological changes, apoptosis was further confirmed by TUNEL assay. The number of apoptotic chicken hepatocytes induced by Cd was significantly increased in the Cd-treated group compared with the control. Cd-induced

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Table 1 Effects of Se treatment on LPO levels and antioxidant activities in the liver tissue of Cd-treated chickens. Parameters

Control

Se

Cd+Se

Cd

LPO (nmol/mg protein) SOD (NU/mg protein) GPx (U/mg protein)

0.6047 0.107c 11.3527 1.562d 140.781 7 12.867c

0.553 7 0.058c 13.6477 1.548d 170.3427 19.452a,d

0.929 7 0.166b 5.3387 0.425b 114.755 7 12.401b

1.6677 0.157b,d 1.263 7 0.094b,d 97.360 7 9.233b,d

Values are expressed as the mean 7 SD, for six cocks in each group. a

Values differ significantly from the control group (Po 0.05). Values differ significantly from the control group (Po 0.01). Values differ significantly from the Cd+Se group (P o0.05). d Values differ significantly from the Cd+Se group (P o0.01). b c

NOS activity 7 # b** #

5 #b#

4 3 2

** *

*

# b** #

5 4

# a

3 *

2

1 0

6

1

Control

Se

Cd+Se

Cd

0

The activity of NOS(U/mg.protein)

The level of NO(nmol/mg.protein)

NO level 6

Fig. 5. Effects of Se treatment on the NO levels and NOS activity of liver tissues in Cd-treated chickens. Each value is the mean 7S.D. Statistically significant differences: # P o0.05 compared with the control group; ## Po 0.01 compared with the control group; *P o 0.05 compared with the Cd+Se group; ** values differ significantly from the Cd+Se group (Po 0.01).

oxidative stress is known to play a major role in its potential to induce apoptosis. The attention of the present work also was focused on the relationship between oxidative damage and excessive NO. It was shown that excessive NO, which might be regulated by NO synthase, contributed to cytotoxicity and induced LPO. Cd may induce NO generation to form a peroxynitrite anion. The accumulated peroxynitrite anion may result in LPO and damage of the cell membrane. In this study, the levels of NO and the acticities of NOS in chicken livers exposed to Cd were significantly increased. Overexpression of NO in the livers of birds is one of the mechanisms of oxidative damage caused by Cd. However, it is well known that endogenous NO protects against liver cell necrosis and apoptosis. This is probably due to conservation of cellular bioenergetics for maintenance of tissue homeostasis (Taniai et al., 2004). Also, it was found that the NO donor, O2-vinyl 1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate (V-PYRRO-NO), protects against Cdinduced hepatotoxicity in mice (Liu et al., 2004). Thus, the accurate relation between Cd hepatotoxicity and NO needs to be elucidated in further study. As oxidative stress is one of the important mechanisms of Cdinduced liver damages, it can be expected that administration of some antioxidants should be an important therapeutic approach. Se has a paradoxical position in animal nutrition because it is well established both as a natural toxicant and as an essential micronutrient. Some studies with concurrent Cd and organic Se addition in feed showed organic Se can help against the negative effects of Cd, but cannot counteract all of its negative effects (Pappas et al., 2011; Al-Waeli et al., 2013). In this study, concurrent treatment with inorganic Se and Cd reduced the Cd-induced hepatic histopathological changes, oxidative stress and apoptosis. Together, this evidence suggests that Se treatment partly counteracts the toxic effect of Cd on the bird liver. This element is a well-established antioxidant and can prevent or decrease the harmful effects of oxidants

and ROS in various tissues. This upregulation of GPx production induced by Se may explain why the GPx and SOD activity levels in the liver tissues of the Cd+Se group were higher than in the Cd group in the present work. Dietary Se also decreased the MDA concentration in the Cd+Se group. These results can be explained by the important role of Se in preventing LPO and in protecting the integrity and functioning of liver tissues. However, some have reported that Se exerts its protective effect by significantly decreasing Cd accumulation in organs or by inducing a redistribution of Cd in rodents (Ognjanović et al., 2008; Lazarus et al., 2009). It was found here that Se supplementation reduced the distribution of Cd to the liver tissues and decreased Cd concentrations in the testis with previous reports (Li et al., 2010a). Cd forms an equimolar complex with Se ingested as selenite, in the form of selenide in plasma. This complex binds to selenoprotein P as a high molecular weight protein complex and changes the metabolism of Cd (Sasakura and Suzuki, 1998). Selenoprotein P is the most common Se-binding protein and is important for the supply of Se to organs, especially the liver, testis and kidney. It has been implicated in Se transport, Se detoxification and antioxidant defense systems (Schomburg et al., 2003). Furthermore, the activity of Se within animal systems is mainly elicited via selenoproteins (Stadtman, 2000). Hence, The present work hypothesizes that selenoproteins, especially selenoprotein P, might play an important role in the detoxification and interactions between Se and Cd.

5. Conclusions The present work demonstrated that dietary exposure to Cd caused histopathological changes, oxidative stress, overexpression of NO and apoptosis in chicken liver. There were significant reductions in the antioxidant enzyme capacity, as well as increases of the LPO and NO production and apoptosis. The oxidative stress and apoptosis induced by Cd is the important mechanisms of the Cd hepatotoxicity to bird. Moreover, dietary Se ameliorated these effects by enhancing antioxidant systems and by decreasing Cd accumulation in the liver. However, the experimental design of the present work could not give information about the relationship between Cd retention and selenoprotein synthesis, so further investigations are needed to clarify this.

Acknowledgments This work was supported by Bureau of Education of Heilongjiang Province (No. 10551038). References Al-Waeli, A., Zoidis, E., Pappas, A.C., Demiris, N., Zervas, G., Fegeros, K., 2013. The role of organic selenium in cadmium toxicity: effects on broiler performance and health status. Animal 7, 386–393.

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