Cadmium induced pathophysiology: Prophylactic role of edible jute (Corchorus olitorius) leaves with special emphasis on oxidative stress and mitochondrial involvement

Food and Chemical Toxicology 60 (2013) 188–198

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Cadmium induced pathophysiology: Prophylactic role of edible jute (Corchorus olitorius) leaves with special emphasis on oxidative stress and mitochondrial involvement Saikat Dewanjee a,⇑, Moumita Gangopadhyay a,b, Ranabir Sahu a, Sarmila Karmakar a a b

Advanced Pharmacognosy Research Laboratory, Department of Pharmaceutical Technology, Jadavpur University, Raja S C Mullick Road, Kolkata 700 032, India Division of Biophysics and Structural Genomics, Saha Institute of Nuclear Physics, Bidhannagar, Kolkata 700 064, India

a r t i c l e

i n f o

Article history: Received 22 May 2013 Accepted 16 July 2013 Available online 25 July 2013 Keywords: Antioxidant CdCl2 Cadmium toxicity Corchorus olitorius Oxidative stress

a b s t r a c t The present study was undertaken to evaluate the protective effect of aqueous extract of Corchorus olitorius leaves (AECO) against CdCl2 intoxication. In vitro bioassay on isolated mice hepatocytes confirmed dose dependent cytoprotective effect of AECO. The CdCl2 (30 lM) exhibited a significantly increased levels of lipid peroxidation, protein carbonylation along with the reduction of antioxidant enzymes and reduced glutathione levels in hepatocytes. AECO (200 and 400 lg/ml) + CdCl2 (30 lM) could significantly restore the aforementioned oxidation parameters in hepatocytes. Beside this, AECO could significantly reduce Cd-induced increase in Bad/Bcl-2 ratio and the over-expression of NF-jB, caspase 3 and caspase 9. In in vivo assay, CdCl2 (4 mg/kg body weight, for 6 days) treated rats exhibited a significantly increased intracellular Cd accumulation, oxidative stress and DNA fragmentation in the organs. In addition, the haematological parameters were significantly altered in the CdCl2 treated rats. Simultaneous administration of AECO (50 and 100 mg/kg body weight), could significantly restore the biochemical, antioxidant and haematological parameters near to the normal status. Histological studies of the organs supported the protective role of jute leaves. Presence of substantial quantity of phenolic compounds and flavonoids in extract may be responsible for overall protective effect. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cadmium (Cd) is a toxic metal emanating from both industrial and agricultural sources with biological half-life in the range of 10–30 years (Xiang et al., 2012). Human body may be exposed to Cd mainly through air, water or even through foods. After absorption from the alimentary tract, Cd forms durable combinations with the apoprotein thionein, forming metallothionein, the holoprotein that plays an important role in further metabolism of this metal. In chronic exposure, Cd has been considered to be a multitarget toxicant, and it causes damage of vital organs namely liver, kidney, brain, pancreas, intestine and heart (Al-Saleh et al., 2008; Oymak et al., 2009; Mortensen et al., 2011; Kossowska et al., 2013). However, the mechanism of Cd toxicity remains fairly unclear but Cd related augmented oxidative stress may be one of the critical features of Cd induced toxicity of critical tissues. Cd itself is unable to generate free radicals directly, since it has only one

⇑ Corresponding author. Address: Advanced Pharmacognosy Research Laboratory (3rd Floor), Department of Pharmaceutical Technology, Jadavpur University, Raja S C Mullick Road, Kolkata 700 032, India. Tel.: +91 33 24146666x2043; fax: +91 33 28371078. E-mail address: [email protected] (S. Dewanjee). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.07.043

oxidation state. However, indirect generation of various radicals involving the superoxide radical, hydroxyl radical and nitric oxide has been reported (Galan et al., 2001; Ghosh et al., 2010). It has been reported that Cd increases the free iron (Fe) concentration possibly by its replacement in various proteins and hence increases the cellular amount of free redox-active metals (Cuypers et al., 2010). Free redox-active metals directly enhance the generation of hydroxyl radicals through the Fenton reaction (Watanabe et al., 2003). Reduction of the oxidised metal ion can be achieved by the Haber-Weiss reaction (Karihtala and Soini, 2007) with superoxide radicals as a substrate, but also other reducing agents, such as ascorbate can catalyse this reaction (Winterbourn, 1979). Cd also exhibits other ways to induce oxidative stress. As a thiolaffectionate metal, Cd targets the highly abundant cellular GSH, thiol antioxidant (Nair et al., 2013). Thiol affinity of Cd is several times greater than its affinity for phosphate, chloride, carboxyl, or amino groups (Rikans and Yamano, 2000). Depletion of the GSH results impairment of the cellular redox balance leading to oxidative stress. Mitochondria are the primary cellular targets of Cd (Muller, 1986). Mitochondria are a major cellular site of electron-transfer-chain-dependent production of oxidative free radicals during Cd-intoxication. Later resulted decrease in mitochondrial membrane potential, which further leads to the

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activation of caspases and consequently cell death by apoptosis. In addition, it is also known that Bcl-2 family proteins are upstream regulator of mitochondrial events and play critical roles in mitochondrial-mediated cell death. Cd-intoxication up-regulates proapoptotic Bad and down-regulates anti-apoptotic Bcl-2 proteins regulated by NF-jB (Pal et al., 2011). So, it is clear that oxidative stress related responses versus signalling scenario are affected during Cd toxicity. Considering the relationship between Cd exposure and oxidative stress, attention has been focused on compounds having antioxidant properties to combat against Cd-induced toxicity. Tossa jute, Corchorus olitorius Linn. (Family: Malvaceae) is a commercially important fibre crop with a production in excess of 2 million tons annually. Jute fibre is 100% bio-degradable and thus environmentally friendly. Jute fibre has high tensile strength, low extensibility, and ensures better breathability of fabrics. It is extensively used as packaging material for the variety of substances. Therefore, it occupies the second to cotton in economic importance (Maiti and Chakravarty, 1977). Jute plants produce edible leaves that are used as a vegetable and food ingredient common to the people of Eastern Asia and Africa (Zeid, 2002). Tossa jute has been used in folk medicine against different ailments including inflammation, dysentery, gastroenteritis, diabetes and tumors (Yan et al., 2013). Besides this jute leaves possess significant antioxidant activity (Oboh et al., 2009) due to presence of significant quantity of polyphenolics, ascorbic acid and a-tocopherol (Azuma et al., 1999). Polyphenolics enriched jute leaves have been reported to regulate the obesity in mice fed with high-fat diets through reduction of oxidative stress and enhancing b-oxidantion (Wang et al., 2011). Earlier studies by our group revealed that C. Olitorius leaves possess significant protective role against augmented oxidative stress associated with arsenic and lead intoxication (Das et al., 2011; Dewanjee et al., 2013). The present study was undertaken to determine the protective role of C. olitorius leaves in Cd intoxication through in vitro and in vivo preclinical bioassays. The cytoprotective role of jute leaf was evaluated in isolated mouse hepatocytes. Intracellular antioxidant markers viz. antioxidant enzymes, reduced glutathione, lipid peroxidation and protein carbonylation were estimated in vitro. In addition, mechanistic aspect of Cd toxicity in hepatocytes and its protection by jute leaf extract was evaluated by immunoblotting. Finally the effect of Cd-intoxication in critical organs was estimated by suitable in vivo model in Wistar rats. Haematological, biochemical, antioxidant and histopathological markers were estimated to observe protective role of jute leaf against Cd toxicity. 2. Materials and methods 2.1. Chemicals Ammonium sulphate, 1-chloro-2,4-dinitrobenzene, 2,4-dinitrophenylhydrazine, 5,5-dithiobis(2-nitrobenzoic acid), ethylene diamine tetraacetic acid, N-ethylmaleimide, reduced nicotinamide adenine dinucleotide, nitro blue tetrazolium, phenazine methosulphate, potassium dihydrogen phosphate, reduced glutathione, sodium azide, sodium pyrophosphate, trichloro acetic acid, thiobarbituric acid and 5-thio-2-nitrobenzoic acid were purchased from Sisco Research Laboratory, Mumbai, India. Cadmium chloride (CdCl2), bovine serum albumin and Bradford reagent were procured from Sigma–Aldrich Chemical Company, St. Louis, USA. All antibodies for immunoblotting were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO), USA. HPLC grade solvents viz. acetonitrile, acetic acid and formic acid were procured from Merck, Mumbai, India.

2.2. Preparation of extract Mature leaves of C. olitorius were collected in the month of June, 2008 from the villages of Hoogly, India. The plant was authenticated by the Taxonomists, Central Research Institute for Jute and Allied Fibers, India. A voucher specimen JU/PT/PC/05/ 08 was deposited at our laboratory for future reference. The leaves were dried under shade, powdered and macerated for 48 h at 25 ± 2 °C with double distilled water

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containing few drops of chloroform with continuous stirring. The extract was filtered to remove particulate matters and lyophilised (Heto FD 3 Drywinner) to obtain the powdered crude extract (yield 14.4%, w/w). The testing sample was prepared by dissolving lyophilised powder (AECO) with double distilled water.

2.3. Phytochemical analysis Qualitative analysis of phytoconstituents within AECO was performed by preliminary phytochemical analysis. The content of total phenolic compounds and total flavonoid was determined spectrophotometrically (Das AK et al., 2011; Das J et al., 2011; Dewanjee et al., 2013). The identification of individual flavonoids and phenolic compounds determined in reverse phase HPLC system (Dionex, Germany) (Dewanjee et al., 2013).

2.4. Animals Healthy adult male Swiss albino mice (25 ± 5 g) and male Wistar rats (150 ± 20 g) were used for the in vitro and in vivo studies respectively. Animals were maintained under standard laboratory conditions of temperature (25 ± 2 °C), relative humidity (50 ± 15%), 12 h light-dark cycle, standard diet and water ad libitum. The principles of Laboratory Animals care (PHS, 1986) and the instructions given by our institutional animal ethical committee (Reg No. 0367/01/C/CPCSEA) were followed throughout the experiment.

2.5. In vitro bio-assay 2.5.1. Hepatocyte isolation The animals were sacrificed by cervical dislocation under light ether anaesthesia and the livers were collected. Hepatocytes were isolated from mouse liver by the method of Sarkar et al. (2009) with little modifications to obtain 2  106 cells/ml.

2.5.2. Assessment of cytoprotective role of AECO against Cd-induced cytopathophysiology Cell viability assessment has been carried out to determine cytoprotective role of AECO against Cd-induced cytopathophysiology. Briefly, six different sets of hepatocytes (each containing 2  106 cells) were incubated with CdCl2 (30 lM) and AECO (50, 100, 200 and 400 lg/ml) for different times (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h and 3 h). The cell viability was determined as per the method developed by Pal et al. (2011). One set without CdCl2 and one set with CdCl2 (30 lM) were kept as control and toxic control sets respectively.

2.5.3. Assay of antioxidant markers All experiments were performed with different sets of hepatocytes containing 1 ml of suspension (2  106 cells) in each. The combined effect of AECO and CdCl2 was studied by incubating hepatocytes with AECO (200 and 400 lg/ml) and CdCl2 (30 lM) together for 2 h at 37 °C (Pal et al., 2011). The toxin control was prepared by incubating the hepatocytes with CdCl2 (30 lM) for 2 h. One set without CdCl2 was kept as normal control. The extent of lipid peroxidation i.e. thiobarbituric acid reactive substances (TBARS) were measured by the method described by Ohkawa et al. (1979). The protein carbonylation was determined according to the method of Uchida and Stadtman (1993). The antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-6-phosphate dehydrogenase (G6PD) activity was measured according to the method described by Ghosh et al. (2010). CAT unit, ‘U’, is defined as lmoles of H2O2 consumed per minute. SOD unit, ‘U’, is defined as the lmoles inhibition of nitro blue tetrazolium chloride reduction per minute. Non-enzymatic antioxidant, reduced glutathione (GSH) level were assayed by the method of Hissin and Hilf (1973).

2.5.4. Immunoblotting of signalling proteins Samples containing 50 lg proteins were subjected to 10% SDS–PAGE and transferred to a nitrocellulose membrane (Ghosh et al., 2010; Das AK et al., 2011; Das J et al., 2011). Membranes were blocked at room temperature for 2 h in blocking buffer containing 5% non-fat dry milk to prevent nonspecific binding and then incubated with primary antibodies viz. anti-caspase 3 (1:1000 dilution), anti-NF-jB (1:250 dilution), anti-Bad (1:1000 dilution), anti-Bcl-2 (1:1000 dilution) at 4 °C overnight. An antibody against b-actin, was used to ensure equal protein loading and electrophoretic transfer. The membranes were washed in Tris-Buffered Saline with 0.1% Tween 20 (pH 7.6) for 30 min, incubated with appropriate HRP conjugated secondary antibody (1:2000 dilution) for 2 h at room temperature and developed by the HRP substrate 3,30 -diaminobenzidine tetrahydrochloride system (Bangalore genei, India). Protein bands were scanned and the band intensities quantified using a densitometer imaging system and the normal control band was given an arbitrary value of 1.

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2.6. In vivo bioassay 2.6.1. Experimental design Thirty Wistar rats were divided into five groups (six rats/group) and they were treated as suggested by Manna et al. (2008) and Sinha et al. (2009): Group I: Normal control (animals received only double distilled water as vehicle). Group II: Toxic control (animals received CdCl2 through double distilled water at a dose of 4 mg/kg body weight, p.o. for 6 days, once daily). Group III: Animals were treated with AECO (50 mg/kg body weight, p.o.) for 5 days followed by CdCl2 (4 mg/kg body weight, p.o., once daily) intoxication for next 6 days. Group IV: Animals were treated with AECO (100 mg/kg body weight, p.o.) for 5 days followed by CdCl2 (4 mg/kg body weight, p.o., once daily) intoxication for next 6 days. The dose for CdCl2 was decided on the basis of experiments conducted in the laboratory and the concentration of CdCl2 used in the experiment was 1/20 of LD50. The plant doses were decided on the basis of experiments conducted in our own laboratory and on the basis of other published report (Dewanjee et al., 2013). After 11 days, the animals were sacrificed by cervical dislocation under light ether anaesthesia. Before sacrificing the animals, blood samples were collected from retro-orbital venous plexus in eppendorf tubes rinsed with anticoagulant for haematological assays. The organs (liver, kidney, brain and heart) were excised, cleaned and washed with ice cold saline (pH 7.4). The organs were homogenised in 0.1 M Tris–HCl-0.001 M EDTA buffer (pH 7.4) and centrifuged at 12,000g for 30 min at 4 °C. The supernatant were collected and used for assaying biochemical parameters. 2.6.2. Haematological parameters Total erythrocyte count and haemoglobin content were determined by using standard laboratory procedures. Serum biochemical parameters viz. alanine aminotransferase (ALT) and aspartate aminotransferase (AST), urea, cholesterol and triglycerides were estimated by standard kits (Span Diagnostic Limited, India). 2.6.3. Assessment of antioxidant markers related to organ dysfunction Distribution of Cd in tissues has been measured by flame atomic absorption spectroscopy. The extent of DNA fragmentation in the selected tissues was determined by the method as described by Lin et al. (1997). The TBARS level, antioxidant enzymes, non-enzymatic antioxidant were assayed by the standard protocols mentioned earlier. Co-enzymes Q (Q9 and Q10) were isolated and estimated according to the method of Zhang et al. (1995). Briefly, One ml of 30% tissue homogenate (in 0.25 M sucrose) was put in a tube containing 50 ll butylated hydroxyl toluene (1 mg/ml), to which 1 ml, 0.1 mM sodium dodecyl sulfate (SDS) was added, vortexed for 30 s, sonicated for 15 s, chilled in ice–water and vortexed for 15 s. Two ml of ethanol were added and again vortexed for 30 s, sonicated for 15 s, chilled in ice–water and vortexed for 15 s. Then, 2 ml of hexane were added. The tube was vortexed and subsequently centrifuged at 2000 rpm for 3 min. Hexane layer (1.75 ml) was transferred to another tube and evaporated under gentle nitrogen stream. Dionex Ultimate 3000 HPLC system (Dionex, Germany), using a reverse phase C-18 column (250  4.6 mm, particle size 5l) and UV detector was employed for estimation og co-enzymes Q. The samples were dissolved in 100 ll of the mobile phase consisting of HPLC grade methanol and ethanol (70:30), filtered through cellulose nylon membrane filter (0.45 lm) (PALL, Life Sciences) and injected to HPLC. The aliquots of the filtrate were eluted with isocratic solvent mixture comprising methanol:ethanol (70:30). 2.6.4. Histological studies The organs from the normal and experimental rats were fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections of about 5 lm thickness were stained with haematoxylin and eosin to study the histology of organs of all experimental rats. 2.7. Statistical analysis Data were statistically analysed by utilising one way ANOVA and expressed as mean ± SE followed by Dunnett’s t-test using computerised GraphPad InStat version 3.05, Graph pad software, USA. The values were considered significant when p < 0.05.

3. Results 3.1. Effect of AECO in Cd toxicity in isolated hepatocytes 3.1.1. Dose and time dependent effect of AECO against CdCl2 induced cellular damage Results of the dose and time dependent protective effect of AECO in CdCl2 induced cellular damage in isolated mouse hepatocytes

was shown in Fig. 1. CdCl2 (30 lM) exposure caused time dependent reduction (p < 0.01, compared with control hepatocytes) in cell viability up to 3 h. Simultaneous treatment of hepatocytes with AECO at different concentrations viz. 50, 100, 200 and 400 lg/ml with Cd (30 lM) prevented the reduction in cell death for 3 h in a concentration dependent manner. However, AECO at the dose of 200 and 400 lg/ml show significantly (p < 0.05–0.01) restore cell viability despite Cd (30 lM) intoxication. 3.1.2. Effect of AECO against CdCl2 induced alteration of antioxidant markers in mouse hepatocytes Table 1 represents the effect of AECO against CdCl2 induced alteration of antioxidant markers in isolated in mouse hepatocytes. The extent of lipid peroxidation (TBARS) and protein carbonylation in isolated mouse hepatocytes was significantly increased (p < 0.01) with Cd (30 lM) intoxication. Simultaneous treatment with AECO at the doses of 200 and 400 lg/ml could significantly reduced, p < 0.05 and p < 0.01, respectively, the extent of lipid peroxidation (TBARS) and protein carbonylation in a dose dependent manner. The levels of CAT, SOD, GST, GPx, GR, G6PD, GR and GSH were significantly decreased (p < 0.01) in CdCl2 (30 lM) intoxicated mouse hepatocytes. Simultaneous treatment with AECO at the doses of 200 and 400 lg/ml could significantly improved (p < 0.01) SOD, GPx, G6PD levels in mouse hepatocytes in a dose dependant manner. The levels of CAT, GR and GSH were significantly improved (p < 0.05) at the dose of 400 lg/ml of AECO. While, no significant alteration of GST level was recorded at any of the selected doses of AECO. 3.1.3. Effect of AECO against CdCl2 induced alteration of signalling proteins Mitochondrial damage is a hallmark of cellular damage. We, therefore, studied the effect of AECO against CdCl2 induced mitochondrial events (Fig. 2A–D). It is known that Bcl-2 family proteins are upstream regulator of mitochondrial events and play important roles in mitochondria mediated cellular functions. Therefore Bad/ Bcl-2 ratio is an index of mitochondria mediated apoptosis. In the present study, we observed that CdCl2 (30 lM) caused a marked decrease in Bcl-2 expression in hepatocytes and increased the expression of Bad which led to increased Bad/Bcl-2 ratio (Fig. 2A). Simultaneous treatment of AECO (400 lg/ml) with CdCl2

Control Cd

100

AECO 50 µg/ml + Cd

Cell viability (% control)

190

*

AECO 100 µg/ml + Cd

*

**

**

80

AECO 200 µg/ml + Cd AECO 400 µg/ml + Cd

*

60

*

#

#

40

#

# #

20 0

0.5

1

1.5

2

2.5

3

Time (hours) Fig. 1. A time and dose-dependent effect on cell viability in absence (CdCl2) and presence of AECO (AECO + CdCl2) in hepatocytes. Values are expressed as mean ± SE (n = 3). Values are expressed as percent over control. Values are expressed as mean ± SE (n = 3). #Values differs significantly (p < 0.01) from normal control. * Values differs significantly (p < 0.05) from toxic control. **Values differs significantly (p < 0.01) from toxic control.

GSH (mg/g of tissue)

92.12 ± 4.54 60.36 ± 5.31# 76.58 ± 5.81

81.79 ± 4.74*

121.12 ± 6.67 72.03 ± 5.94# 104.24 ± 7.78**

105.19 ± 6.60**

191

(30 lM) significantly (p < 0.05) reduced Bad/Bcl-2 ratio and reciprocate regulation of Bad and Bcl-2. NF-jB usually gets activated in response to oxidative stress. In the present study, CdCl2 (30 lM) exposure caused a significant increase in the expression of NF-jB in mouse hepatocytes (Fig. 2B). However, Cd-induced increase in NF-jB activation was significantly (p < 0.01) prevented by co-treatment with AECO (400 lg/ml). Apoptotic nature of hepatocytes in Cd-induced cytotoxicity was also studied in terms of caspase 9 and caspase 3 activation. Exposure of hepatocytes with CdCl2 (30 lM) resulted in a marked increase in the levels of cleaved caspase 9 and caspase 3. Simultaneous incubation with AECO (200 and 400 lg/ml) could significantly (p < 0.01) inhibit this effect (Fig. 2C and D).

3.2.1. Effect on haematological and serum biochemical parameters The effects of different treatments on haematological and serum biochemical parameters were shown in Table 2. The haematological parameters showed a significant (p < 0.01) reduction in total erythrocyte counts and haemoglobin content in CdCl2 treated animals (group II). AECO supplementation could revert the total erythrocyte counts and haemoglobin content significantly in a dose dependant manner. Animals treated with CdCl2 showed a significant (p < 0.01) increase in ALT, AST, urea, total cholesterol and triglyceride levels, whereas, the level of HDL cholesterol was found to decrease significantly (p < 0.01). Animals received the combined treatment of AECO and CdCl2 showed significant improvements in all the biochemical parameters tested. This improvement was pronounced in the groups treated with AECO at the doses of 100 mg/kg body weight. 3.2.2. Effect on Cd bioaccumulation The effect of different treatments on intracellular Cd accumulation was shown in Table 3. CdCl2 exposure in rats significantly (p < 0.01) increased the bioaccumulation of Cd in liver, kidney, heart and brain, when compared with untreated animals (group I). Intracellular Cd content was found to be highest in kidney followed by heart, liver and brain. AECO supplementation at the doses of 50 and 100 mg/kg body weight significantly prevented intracellular Cd burden in aforementioned tissues, in comparison to CdCl2 treated animals (group II).

Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05). ** Values differ significantly from CdCl2 control (p < 0.01).

74.84 ± 7.75* 82.01 ± 4.86** 0.96 ± 0.05 71.53 ± 3.90** 42.71 ± 3.96** 4.92 ± 0.32**

247.04 ± 12.82*

81.05 ± 5.43 49.76 ± 4.13# 70.24 ± 7.11 84.32 ± 3.45 56.79 ± 4.35# 77.59 ± 4.05** 0.98 ± 0.06 0.75 ± 0.10# 0.93 ± 0.06 84.52 ± 3.01 36.81 ± 2.70# 65.73 ± 4.46** 40.21 ± 3.33 65.45 ± 4.35# 49.65 ± 3.78*

Control CdCl2 (30 lM) AECO (200 lM) + CdCl2 (30 lM) AECO (400 lM) + CdCl2 (30 lM)

3.76 ± 0.31 6.59 ± 0.42# 5.08 ± 0.24*

276.54 ± 15.32 174.19 ± 19.51# 230.02 ± 17.33

GPx (nmol/min/mg of protein) SOD (U/mg of protein) Protein carbonyl (nmol/mg of protein)

CAT (U/mg of protein)

GST (lmol/h/mg protein)

3.2. Effect of AECO against CdCl2 intoxication in Wistar rats

TBARS (lg/g of tissue)

Parameters Groups

Table 1 Effect on antioxidant parameters in absence (CdCl2) and presence of AECO (AECO + CdCl2) in isolated mice hepatocytes.

GR (nmol/min/mg of protein)

G6PD (nmol/min/mg of protein)

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3.2.3. Effect on DNA fragmentation The percentage of DNA fragmentation in liver, kidney, brain and heart tissues of the experimental rats was represented in Fig. 3. Daily administration of CdCl2 caused significant increase (p < 0.01) in DNA fragmentation in the renal, hepatic, cardiac and cerebral tissues amounting 141%, 137%, 132% and 112% respectively. The extent of DNA fragmentation was found to be highest in renal tissues. Animals received the combined treatment of AECO and CdCl2 exhibited significant inhibition (p < 0.05) in DNA fragmentation in hepatic and renal tissues at the dose of 50 mg/kg, while significant inhibition (p < 0.05) in DNA fragmentation was observed in all the selected tissues at the dose of 100 mg/kg. 3.2.4. Effect on co-enzymes Q The effects of different treatments on mitochondrial co-enzymes Q (Q9 and Q10) in liver, kidney, brain and heart were shown in Table 4, respectively. Total co-enzyme Q9 level was reduced significantly in CdCl2 treated rats (group II). Treatment with AECO could significantly (p < 0.05) elevate Q9 level in liver, heart and brain at the dose of 100 mg/kg, whereas, no significant alteration of co-enzyme Q9 level was observed in renal tissues. A significant decrease in total co-enzyme Q10 in the tissues was observed in

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B

A

D

C

Fig. 2. Respective western blot analysis of Bad (A), Bcl-2 (A), NF-jB (B), caspase 3 (C) and caspase 9 (D) to investigate the mitochondria-dependent pathway in absence (CdCl2) and presence of AECO (AECO + CdCl2) in hepatocytes followed by densitometric analysis of the respective protein levels and the normal control band was given an arbitrary value of 1. b-actin was used as an internal control. Values are expressed as mean ± SE (n = 3). #Values differs significantly (p < 0.01) from normal control. *Values differs significantly (p < 0.05) from toxic control. **Values differs significantly (p < 0.01) from toxic control. Table 2 Effect on haematological and serum biochemical parameters in (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Groups

Parameters Total erythrocyte count (106/mm3)

Haemoglobin (g/dl)

I

6.04 ± 0.24

8.67 ± 0.65

76.54 ± 3.65

42.16 ± 3.43

45.56 ± 2.98

102.15 ± 4.78

32.46 ± 2.02

86.51 ± 3.67

II

3.82 ± 0.25#

5.85 ± 0.42#

106.33 ± 4.75#

74.43 ± 4.87#

72.52 ± 4.21#

144.67 ± 5.64#

18.76 ± 1.55#

134.44 ± 5.05#

III

4.82 ± 0.33*

6.92 ± 0.52*

88.21 ± 4.12*

58.65 ± 5.02

58.61 ± 4.54

127.12 ± 4.15*

25.87 ± 1.98*

116.54 ± 4.92*

*

114.45 ± 4.62*

IV

5.14 ± 0.28

**

7.52 ± 0.67

*

ALT (IU/l)

AST (IU/l)

Urea (mg/dl)

Cholesterol (mg/dl) Total

84.05 ± 4.56

**

53.33 ± 4.76

*

*

57.92 ± 3.98

Triglyceride (mg/dl)

HDL

**

122.56 ± 3.78

27.15 ± 1.82

Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05). ** Values differ significantly from CdCl2 control (p < 0.01).

Table 3 Effect on intracellular Cd burden in blood, liver, kidney, heart and brain in absence (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Groups

Cadmium burden Blood (lg/ml)

Liver (ppm of wet tissue)

Kidney (ppm of wet tissue)

Heart (ppm of wet tissue)

Brain (ppm of wet tissue)

I

0.04 ± 0.01

0.06 ± 0.03

0.07 ± 0.04

0.07 ± 0.04

0.04 ± 0.02

II

1.07 ± 0.16#

16.26 ± 0.77#

27.59 ± 1.52#

19.84 ± 1.87#

8.44 ± 0.76#

III

0.81 ± 0.09

9.15 ± 0.57**

16.49 ± 1.68**

14.83 ± 1.04*

6.75 ± 0.65

**

**

13.78 ± 0.95**

5.56 ± 0.54**

IV

*

0.67 ± 0.09

7.21 ± 0.58

14.06 ± 1.12

Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05). ** Values differ significantly from CdCl2 control (p < 0.01).

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DNA fragmentation (% over control)

200

Liver

Kidney

Brain

Heart

3.2.5. Effect on lipid peroxidation and protein carbonylation The protective role of AECO on lipid peroxidation in liver, kidney, brain and heart was measured by estimating TBARS level of all experimental animals (Table 5). Toxic control rats (group II) exhibited a significant raise (p < 0.01) in TBARS level, when compared with untreated animals (group I). The maximum extent of lipid peroxidation was found in kidney followed by liver, brain and heart. AECO (50 mg/kg) could significantly prevent (p < 0.05) that increase in TBARS level in kidney and brain of experimental rats. However, AECO (100 mg/kg) could significantly prevent (p < 0.05) that increase in TBARS level in all the selected tissues. Protein carbonylation in selected tissues was also shown in Table 5. CdCl2 intoxication caused significant (p < 0.01) increase of protein carbonyl content compared to normal rats (group I). Co-administration of AECO (50 mg/kg) with CdCl2 significantly prevented that enhancement in the levels of protein carbonyl content hepatic (p < 0.05), renal (p < 0.01) and cardiac (p < 0.05) tissues. Treatment with AECO (100 mg/kg) along with CdCl2 significantly reduced the levels of protein carbonyl content hepatic (p < 0.01), renal (p < 0.01), cardiac (p < 0.01) and cerebral (p < 0.05) tissues.

#

150 *

*

*

* *

*

100

50

0 Group I

Group II

Group III

Group IV

Fig. 3. Effect on DNA fragmentation (% over normal control) in liver, kidney, heart and brain in absence (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/ kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. #Values differs significantly (p < 0.01) from normal control. *Values differs significantly (p < 0.05) from toxic control.

3.2.6. Effect on antioxidant enzymes The effect of AECO on antioxidant enzymes in liver, kidney, brain and heart of experimental rats was shown in Table 6. The levels of CAT and SOD levels were significantly (p < 0.01) reduced in hepatic, renal, cardiac and cerebral tissues of CdCl2 treated rats (group II) as compared with normal rats (group I). Concurrent treatment of AECO with CdCl2 could significantly improve the levels of aforementioned enzymes. CAT levels in liver (p < 0.05-0.01), kidney (p < 0.05) and brain (p < 0.05) tissues were improved significantly in both the selected doses, while a significant improvement

liver, kidney and heart in toxic control animals (group II). Simultaneous administration of AECO with CdCl2 could significantly (p < 0.05) elevate co-enzyme Q10 level in hepatic and renal tissues at both the selected doses of AECO. However, no significant improvement of Q10 level was observed in cardiac and cerebral tissues of experimental rats (group III and IV).

Table 4 Effect on co-enzymes Q in liver, kidney, heart and brain in absence (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Parameters

Groups

Liver

Kidney

Heart

Brain

Total coenzyme Q9 (nmol/g of wet tissue)

I II III IV

182.54 ± 9.41 133.24 ± 6.53# 161.34 ± 7.14 165.43 ± 8.43*

155.43 ± 7.53 127.65 ± 5.79$ 135.67 ± 6.28 138.34 ± 6.32

65.32 ± 4.53 44.54 ± 3.59# 57.68 ± 4.12 59.98 ± 4.37*

17.24 ± 1.72 7.65 ± 0.65# 12.34 ± 1.13 13.24 ± 1.52*

Total coenzyme Q10 (nmol/g of wet tissue)

I II III IV

32.12 ± 2.31 16.33 ± 1.02# 24.32 ± 2.12* 25.14 ± 1.92*

27.54 ± 1.74 14.55 ± 0.58# 21.32 ± 1.78* 21.89 ± 1.92*

28.24 ± 2.04 19.97 ± 1.45$ 25.32 ± 1.89 26.34 ± 1.92

5.41 ± 0.24 3.31 ± 0.17 3.87 ± 0.26 4.02 ± 0.25

Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). $ Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05).

Table 5 Effect on lipid peroxidantion and protein carbonylation in liver, kidney, heart and brain in absence (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Parameters

Groups

Heart

Brain

Lipid peroxidation (TBARS level in lg/g of tissue)

I II III IV

Liver 4.57 ± 0.67 8.11 ± 1.02# 5.98 ± 0.56 5.24 ± 0.48*

Kidney 5.56 ± 0.46 8.67 ± 0.78# 6.67 ± 0.52* 6.58 ± 0.45*

3.90 ± 0.26 6.36 ± 0.58# 5.39 ± 0.42 4.92 ± 0.35*

4.46 ± 0.28 7.04 ± 0.78# 5.24 ± 0.35* 5.15 ± 0.42*

Protein cabonylation (nmol/mg of protein)

I II III IV

14.23 ± 0.78 22.34 ± 1.51# 16.12 ± 1.56* 14.32 ± 1.38**

9.87 ± 0.67 22.34 ± 1.65# 15.21 ± 1.36** 13.21 ± 1.28**

2.45 ± 0.18 5.32 ± 0.45# 3.98 ± 0.41* 3.21 ± 0.33**

1.98 ± 0.11 3.21 ± 0.35# 2.45 ± 0.18 2.32 ± 0.21*

Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05). ** Values differ significantly from CdCl2 control (p < 0.01).

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Table 6 Effect on antioxidant enzymes in liver, kidney, heart and brain in absence (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Parameters

Groups

Liver

Kidney

Heart

Brain

CAT (U/mg of protein)

I II III IV

273.17 ± 24.21 140.83 ± 13.55# 221.05 ± 17.34* 233.25 ± 18.47**

244.93 ± 14.64 129.90 ± 11.54# 170.03 ± 6.94* 172.14 ± 9.73*

292.25 ± 14.04 157.40 ± 13.24# 184.43 ± 9.42 195.21 ± 12.93*

107.13 ± 8.86 54.52 ± 6.59# 82.82 ± 7.80* 85.86 ± 7.11*

SOD (U/mg of protein)

I II III IV

178.45 ± 15.90 74.08 ± 9.02# 135.55 ± 12.70** 141.78 ± 13.54**

138.51 ± 13.36 51.12 ± 7.23# 91.25 ± 9.28* 98.12 ± 9.55*

154.07 ± 11.17 84.31 ± 10.70# 124.36 ± 9.96* 138.41 ± 11.17**

95.93 ± 9.87 53.50 ± 7.99# 68.87 ± 8.25 78.65 ± 4.70*

GST (lmol/h/mg protein)

I II III IV

1.18 ± 0.11 0.65 ± 0.08# 0.89 ± 0.17* 0.95 ± 0.12*

0.74 ± 0.06 0.41 ± 0.05# 0.62 ± 0.07 0.65 ± 0.09

1.88 ± 0.12 1.02 ± 0.09# 1.58 ± 0.16* 1.61 ± 0.15*

0.57 ± 0.05 0.34 ± 0.04# 0.42 ± 0.06 0.45 ± 0.04

GPx (nmol/min/mg protein)

I II III IV

166.85 ± 8.95 104.73 ± 6.52# 134.30 ± 8.85 147.05 ± 9.28**

72.12 ± 6.73 43.18 ± 3.78# 56.40 ± 4.62* 61.52 ± 5.58*

152.07 ± 7.35 104.52 ± 9.70# 128.02 ± 6.76 139.68 ± 8.94*

71.78 ± 4.62 45.23 ± 3.72# 64.68 ± 4.98* 69.91 ± 4.41**

GR (nmol/min/mg protein)

I II III IV

108.23 ± 7.65 61.27 ± 4.54# 84.36 ± 5.67* 88.85 ± 7.33*

72.96 ± 4.21 51.20 ± 3.67# 64.85 ± 4.78 68.56 ± 4.55*

98.36 ± 6.24 55.13 ± 4.52# 80.10 ± 5.16* 89.21 ± 5.67**

62.11 ± 3.98 32.59 ± 2.782# 49.97 ± 3.24** 54.82 ± 4.02**

G6PD (nmol/min/mg protein)

I II III IV

212.34 ± 9.87 126.45 ± 6.12# 182.65 ± 7.65** 194.56 ± 8.76**

142.56 ± 6.21 103.34 ± 5.24# 126.35 ± 5.67* 131.46 ± 6.98*

104.32 ± 5.33 54.34 ± 4.05# 75.26 ± 4.77* 81.23 ± 4.25**

34.12 ± 2.45 17.34 ± 1.58# 23.12 ± 1.76 25.32 ± 2.33*

Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05). ** Values differ significantly from CdCl2 control (p < 0.01).

Table 7 Effect on GSH in liver, kidney, heart and brain in absence (CdCl2) and presence of AECO (AECO + CdCl2) in rats. Parameters

Groups

Liver

Kidney

Heart

Brain

GSH (mg/g tissue)

I II III IV

78.60 ± 4.56 50.98 ± 3.57# 62.95 ± 3.92 67.86 ± 4.21*

26.34 ± 1.45 12.38 ± 0.98# 17.67 ± 1.35 19.86 ± 1.42*

21.51 ± 1.61 10.36 ± 0.73# 12.14 ± 1.04 14.56 ± 1.13

18.68 ± 1.67 7.62 ± 0.75# 11.13 ± 1.04 13.26 ± 1.45*

Group I: normal control, Group II: toxic control (CdCl2, 4 mg/kg), Group III: AECO (50 mg/kg) + CdCl2 (4 mg/kg), Group IV: AECO (100 mg/kg) + CdCl2 (4 mg/kg). Values are expressed as mean ± SE, for six animals in each group. # Values differ significantly from normal control (p < 0.01). * Values differ significantly from CdCl2 control (p < 0.05).

(p < 0.05) of CAT activity was observed in cardiac tissues only at the dose of 100 mg/kg. Concurrent treatment of AECO with CdCl2 could significantly improve SOD levels in hepatic (p < 0.01), renal (p < 0.05) and cardiac (p < 0.05-0.01) tissues at the doses of 50 and 100 mg/kg. However, a significant improvement (p < 0.05) of SOD activity was observed in cerebral tissues only at the dose of 100 mg/kg. CdCl2 intoxication significantly (p < 0.01) inhibited glutathione enzyme systems viz. GST, GPx, GR and G6PD. Simultaneous administration of AECO could significantly (p < 0.05) improve GST level only in hepatic and cardiac tissues at all the selected doses. However, no significant improvement was observed in renal and cerebral tissues. The levels of GPx were significantly improved (p < 0.05–0.01) in the selected tissues of experimental rats by co-administration of AECO (100 mg/kg) along with CdCl2. Concurrent treatment of AECO (50 mg/kg) along with CdCl2 exhibited significant improvement in GR levels in hepatic (p < 0.05), cardiac (p < 0.05) and cerebral (p < 0.01) tissues. However, the effect is more pronounced at the dose of 100 mg/kg, which showed significant improvement of GR levels in hepatic (p < 0.05), renal (p < 0.05), cardiac (p < 0.01) and cerebral (p < 0.01) tissues of experimental rats. Treatment with AECO (100 mg/kg) caused significant improvement (p < 0.05–0.01) of G6PD levels in all the selected tissues.

3.2.7. Effect on reduced glutathione Table 7 showed the GSH levels in the organs of experimental rats. Oral administration of CdCl2 caused significant decrease of GSH levels in the selected organs compared to normal control. Treatment with AECO (100 mg/kg) significantly increased (p < 0.05) the GSH levels in liver, kidney and brain. However, an insignificant raise in GSH level was observed in cardiac tissues of AECO treated groups. 3.2.8. Effect on histology of organs The histological results revealed that the liver of the untreated rats (group I) showed normal hepatocytes and central vein (Fig. 4A). Rats treated with CdCl2 alone (group II) exhibited dilated portal tract with inflamed hepatocytes cells and hepatocytes focal necrosis (Fig. 4B). AECO supplementation reduced such abnormal changes and showed prominent improvement in hepatocytes (Fig. 4C–D). The histological examination of the kidney tissues of the control animals showed normal cyto-architecture of glomerulus structure (Fig. 5A). Rats treated with CdCl2 alone showed glomerular hypercellularity and cellular necrosis (Fig. 5B). Rats treated with CdCl2 plus AECO restored more or less normal glomerular structures and renal tubules in a dose dependent quality (Fig. 5C–D). Fig. 6A–D showed the histological assessments of heart

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Fig. 4. Histological sections (X 20) of livers of normal rats (A), CdCl2-treated rats (B), rat pretreated with AECO (50 mg/kg) followed by CdCl2 administration (C), and rat pretreated with AECO (100 mg/kg) followed by CdCl2 administration (D).

Fig. 5. Histological sections (X 20) of kidneys of normal rats (A), CdCl2-treated rats (B), rat pretreated with AECO (50 mg/kg) followed by CdCl2 administration (C), and rat pretreated with AECO (100 mg/kg) followed by CdCl2 administration (D).

of experimental rats. Cd-intoxication caused extensive degeneration in cardiac muscle and interstitial fibrosis (Fig. 6B) compared with normal rats (Fig. 6A). Treatment with AECO could prevent the disorganisation of cell plates and restore cardiac cytoarchitecture more or less comparable to that of normal rats (Fig. 6C–D). The histopathological assessments of brain of Cd-intoxicated rats exhibited vacuolated area within degenerated tissues, diffused edema and encephalomalacia (Fig. 7B) as compared to normal rats (Fig. 7A). Simultaneous treatment of AECO along with CdCl2 how-

195

Fig. 6. Histological sections (X 20) of hearts of normal rats (A), CdCl2-treated rats (B), rat pretreated with AECO (50 mg/kg) followed by CdCl2 administration (C), and rat pretreated with AECO (100 mg/kg) followed by CdCl2 administration (D).

Fig. 7. Histological sections (X 20) of brain of normal rats (A), CdCl2-treated rats (B), rat pretreated with AECO (50 mg/kg) followed by CdCl2 administration (C), and rat pretreated with AECO (100 mg/kg) followed by CdCl2 administration (D).

ever, could, reduce the incidence of cellular necrosis and showed almost normal architecture of brain (Fig. 7C–D).

4. Discussion The present study describes the protective role of aqueous extract of C. olitorius leaves in overall CdCl2 intoxication with the help of suitable in vitro and in vivo models. It has been observed that

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CdCl2 administration caused a significant oxidative stress and death in hepatocytes. Simultaneous treatment of the hepatocytes with AECO prevented Cd induced cellular toxicity. Oxidative free radicals are known to play a major role in Cd induced cellular damage in a variety of pathophysiological conditions and are responsible for oxidative injury of enzymes, lipid membranes and DNA in living cells and tissues. This protective action of AECO might arise due to the potent free radical scavenging activity and antioxidant property due to presence of phenolic compounds and flavonoids in extract. In vivo study showed that Cd was accumulated most in kidney and heart followed by liver and minimum quantity in brain. Extract treatment prior to Cd administration significantly lowered the intracellular Cd accumulation in selected tissues of experimental rats and contributed lowering Cd induced toxic manifestations. The metal chelating ability of flavonoids and phenolic compounds would potentiate the clearance of Cd from body (Heim et al., 2002). The alterations in haematological parameters serve as the earliest indicators of toxic effects on tissues. Following oral ingestion of CdCl2, a considerable reduction in total erythrocyte count and haemoglobin content was observed, what is in accordance to the observation by Sinha et al. (2008). AECO treatment could significantly increase the total erythrocyte count and haemoglobin content near to the normal status. An increase in ALT and AST levels in blood may indicate degenerative and necrotic changes in liver and kidneys due to Cd intoxication. The results also exhibited that Cd-intoxication significantly increased serum level of urea, which may be associated with protein catabolism and kidney dysfunction. The association between Cd exposure and high serum lipids levels is multifaceted and could be due to either increased synthesis or decreased removal of lipoproteins. Simultaneous administration of AECO, however, could significantly restore serum biochemical parameters near to the normal status. The reversal of haematotoxic effect of CdCl2 is not very clear. However, the antioxidative role of AECO may be suggested to be implicated (Dewanjee et al., 2009). Lipid peroxidation and protein carbonylation are the indicators of oxidative stress. In present study, the levels of TBARS and protein carbonylation have been significantly increased with Cd intoxication. Experimental findings suggested that oxidative stress plays an important role in Cd intoxication. Co-administration of AECO could prevent the Cd induced alteration in the levels of lipid peroxidation and protein carbonylation probably by quenching and detoxifying several reactive intermediates namely hypochlorous acid generated by myeloperoxidase, nitric oxide, hydrogen peroxide and hydroxyl radical (Sinha et al., 2008). Free radical scavenging property of phenolic compounds and flavonoids present within AECO could attenuate the lipid peroxidation and protein carbonylation (Dewanjee et al., 2009). The metalloenzyme SOD acts by quenching the superoxide radical resulting with the formation of H2O2 and O2 (O’Sullivan et al., 2011). The reduction in its activity due to Cd-intoxication may be attributed to dysfunctional conformational change which may be due to the replacement of zinc present in SOD by Cd leading to loss of enzymatic activity (Confod et al., 1991; Casalino et al., 2002). CAT, a hemeprotein, is localised in the peroxisomes or the microperoxisomes. At physiological pH of 7 (the pH at which CAT was assayed) the nitrogen atom of imidazole ring in His-74 is deprotonated and thus interacts with the Cd2+ ion causing reduction in CAT activity (Casalino et al., 2002). GPx, a selenoenzyme, is present in the cytosol and mitochondria. Simultaneous action of CAT and GPx accelerate the dismutation and reduction of H2O2 respectively (Wang et al., 2008). The enzyme GST plays an important role in the detoxification of lipid hydroperoxide and thus contributing to the protection of membrane integrity (Coruh et al., 2007). The intracellular redox status has been main-

tained by the coordinate activities of another two enzymes GR and G6PD (Sinha et al., 2009). Cd intoxication caused significant inhibition in the activities of GPx, GST, GR and G6PD by either the direct binding to the active sites of the enzymes containing thio groups or by the displacement of the metal cofactors from the active sites (Casalino et al., 2000; Pichardo and Pflugmacher, 2011). Co-administration of AECO, however, could significantly restore the activity of antioxidant enzymes through eliminating the toxin from the body or encapsulated Cd+2 and could prevent the toxin for further oxidative injury (Yu et al., 2002). Metal chelating property of phenolic acids viz. gallic acid, chlorogenic acid, p-cumaric acid, ferulic acid, and ellagic acid in AECO could promote the clearance of Cd from the body (Psotová et al., 2003; Dewanjee et al., 2013). Thiol-based antioxidant system, GSH, helps to protect cells from reactive oxygen species and consequently converted to its oxidised form, GSSG (García-Nebot et al., 2011). Cd has a strong affinity for thiol groups (Fotakis et al., 2006) and depletes GSH, resulting in enhanced production of reactive oxygen species thus causing damage consistent with oxidative stress. AECO treatment could significantly prevent the Cd induced alteration of non-enzymatic antioxidant marker probably due to its free radical scavenging activity. Ubiquinones (co-enzymes Q) function as important cellular electron carriers distributed in intracellular major organelles principally in mitochondria. Co-enzymes act as antioxidants (inhibiting lipid peroxidation and/or scavenging free radicals). The experimental results suggested that the coenzyme Q9 and Q10 were significantly decreased in the tissues of Cd-intoxicated rats. These results may be attributed to the raised level of Cd in these tissues that may disrupt the activity of the enzymes responsible for the reduction of ubiquinone to ubiquinol (Kishi et al., 1999). AECO could exert its effect on ubiquinones levels through promoting the clearance of Cd from tissues and organs. In the present study, it was observed that Cd intoxication induced over production of intracellular free radicals which caused fragmentation of genomic DNA. DNA fragmentation act as a mediator of cell death, suggesting Cd induced cell damage via necrotic pathway (Sinha et al., 2009). However, treatment with AECO prior to Cd intoxication significantly reduced the DNA fragmentation. Mitochondria participates a vital role in regulating apoptosis. The prime executers of the apoptotic pathways are some proand anti-apoptotic proteins and cysteinyl aspartic acid-specific proteases (caspases). Bcl-2 is anti-apoptotic member, which regulate apoptosis via intrinsic pathway by modulating the release of cytochrome c from mitochondria to cytosol. Bad is pro-apoptotic in nature and can modulate the pro-apoptotic processes by inhibiting the contributions of anti-apoptotic Bcl-2 members. To confirm Cd induced apoptosis in the hepatocytes used in the present study, we performed immunoblot analyses of Bad and Bcl-2. It was observed that CdCl2 intoxication up-regulated pro-apoptotic Bad and down-regulated anti-apoptotic Bcl-2 proteins resulting a higher Bad/Bcl-2 ratio. AECO treatment, however, could significantly reduce Bad/Bcl-2 ratio. Initiation of the caspase cascade required for apoptotic cellular demise. To confirm Cd, indeed, induced apoptosis in the hepatocytes used in the present study, we performed immunoblot analyses of caspase 3 and caspase 9. Up-regulation of caspase 3 and caspase 9 confirmed the apoptotic hepatocytes damage due to CdCl2 intoxication. AECO treatment, however, could significantly reduce Cd induced up-regulation of caspase 3 and caspase 9. Oxidative stress perturbs cellular redox status which causes oxidative damage to cellular molecules and alters gene expression mainly via post-translational modification of redox sensitive transcription factor viz. NF-jB. However, activation of NF-jB is associated with carcinogenesis through the regulation of genes involved in cell transformation, proliferation and angiogenesis. In our study, we observed that CdCl2 exposure increased

S. Dewanjee et al. / Food and Chemical Toxicology 60 (2013) 188–198

Cd++

Free radicals

197

NFBad

, Bcl-2

O2 • Caspase9 , Caspase3

AECO

H2 O2

OH• Oxidative stress

Activation of mitochondrial pathways

Lipid peroxidation & DNA fragmentation

Apoptosis

Fig. 8. Schematic diagram of the CdCl2 induced toxicity and its protective mechanism of AECO. The bold arrows represent the possible mechanism of Cd toxicity. The dotted lines represent the possible targets of AECO in the toxic pathway.

the expression of NF-jB. AECO treatment, however, could prevent this Cd-induced alteration in the expressions of NF-jB. The early generation of reactive oxidative species during Cdintoxication is closely associated with NADPH oxidases (NOXs) activation, which is coincident with an increment in p47Phox phosphorylation (Flores et al., 2013). NOXs function as multi-component enzymes, and use electrons derived from intracellular ´ et al., 2012). The NADPH to generate O 2 from O2 (Radosavljevic NOX family consists of seven members that participate in important cellular processes, related to signalling, cell proliferation and apoptosis. Chen et al. (2011) reported that Cd induced generation of oxidative free radicals is caused by up-regulation of the expression of NOX-2 gene and its regulatory proteins. NADPH oxidases play an important role in apoptosis via activation of gene transcription of the mitogen activated protein kinases (MAPKs) family viz. cJun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase ERK1/2 (Ghosh et al., 2010). Cd-induced cellular stress disturbs the proper folding of membranes and secreted proteins in the endoplasmic reticulum (ER) and triggers unfolded protein response (Kitamura and Hiramatsu, 2010). Reactive oxygen species may operate up- or down-stream regulation of the ER stress (Xu et al., 2012). The SOD, but not H2O2, appears to trigger selectively activation of the pro-apoptotic branches of the unfolded protein response induced by Cd. However, we did not evaluate the effect of AECO on NOXs and ER stress but overall protective role of AECO could suggest that it would have some ameliorative role on NOXs and ER stress. Histopathological assessment of liver, kidney, brain and heart tissues revealed that Cd caused abnormal ultrastructural changes in the tissues. However, pretreatment with AECO could prevent the changes and could also maintain the ultrastructure similar to that of normal control. In present investigation, it was found that the administration of C. olitorius leaves induced a reduction of the toxic manifestation of Cd. The mechanism of Cd intoxication and multimodal protective role of C. olitorius leaves were confirmed in this study (Fig. 8). The results of the present study suggest that AECO treatment provides protection against Cd induced toxicity through its oxidative free radical scavenging, promoting Cd clearance via metal chelating and anti-oxidant properties. Free radical mediated up-regulation of mitochondrial signalling proteins namely Bad, NF-jB, caspase 3, and casapase 9 and down-regulation of Bcl-2 involves

in apoptotic pathway. AECO also acts on those signalling molecules by down-regulating the activation of Bad, NF-jB, caspase 3 and casapase 9 as well as by up-regulating the expression of Bcl-2. In other words, AECO treatment could block the apoptotic signalling cascade and ameliorates Cd induced pathophysiology. Earlier studies revealed presence of rutin, quercetin, gallic acid, chlorogenic acid, p-cumaric acid, ferulic acid and ellagic acid in AECO (Dewanjee et al., 2013). It also consists antioxidant vitamins viz. ascorbic acid and a-tocopherol. Subtantial quantity of aforementioned Phyto-antioxidants would be responsible for the overall protective role. In conclusion, the protective role of C. olitorius represents a promising approach for the protection against Cd-intoxication. Conflict of Interest The authors declare that there is no conflict of interest. Acknowledgements Authors are thankful to University Grants Commission, New Delhi, India for providing financial support. Authors are also grateful to Jadavpur University, Kolkata, India and Ramakrishna Mission Vivekananda University, Narendrapur, India for providing necessary facilities for this study. The authors would like to thank the reviewers for their time and valuable comments. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2013.07.043. References Al-Saleh, I., Coskun, S., Mashhour, A., Shinwari, N., El-Doush, I., Billedo, G., Jaroudi, K., Al-Shahrani, A., Al-Kabra, M., Mohamed, G.E.D., 2008. Exposure to heavy metals (lead, cadmium and mercury) and its effect on the outcome of in-vitro fertilization treatment. Int. J. Hyg. Environ. Health 211, 560–579. Azuma, K., Nakayama, M., Koshioka, M., Ippoushi, K., Yamaguchi, Y., Kohata, K., Yamauchi, Y., Ito, H., Higashio, H., 1999. Phenolic antioxidants from the leaves of Corchorus olitorius L.. J. Agric. Food Chem. 47, 3963–3966. Casalino, E., Calzaretti, G., Sblano, L., Landriscina, C., 2000. Cadmium dependent enzyme activity alteration is not imputable to lipid peroxidation. Arch. Biochem. Biophys. 383, 288–295.

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