Bharati Bhattacharjee et al. / Journal of Pharmacy Research 2016,10(11), Available online through

Transcription

1 Research Article ISSN: Available online through Terminalia arjuna aqueous bark extract protects against cadmium acetate-induced injury to rat liver and heart through antioxidant mechanisms: a dose response study Bharati Bhattacharjee 1, Arnab Kumar Ghosh 1, Sanatan Mishra 1, 2, Jayita Das 1, Aindrila Chattopadhyay 2, and Debasish Bandyopadhyay 1# 1 Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, 92, A.P.C. Road, Kolkata , India, # Principal Investigator, Centre with Potential for Excellence in a Particular Area (CPEPA), University of Calcutta, University College of Science and Technology, 92, APC Road, Kolkata , India 2 Department of Physiology, Vidyasagar College, 39, SankarGhosh Lane, Kolkata , India Received on: ; Revised on: ; Accepted on: ABSTRACT Background: Cadmium-acetate is a potent hepatotoxic and cardiotoxic heavy metal, which induces oxidative stress by disturbing a number of antioxidant enzymes in rat heart and liver. Terminalia arjuna is an effective antioxidant and free radical scavenger against cadmium acetate induced oxidative stress. The present study was designed to investigate the efficacy of Terminalia arjuna aqueous bark extract in protecting against the cadmium induced oxidative injury in rat heart and liver. Materials and method: The dose and time dependent changes were studied in heart and liver of male Wister rats following the subcutaneous administration of increasing concentrations of cadmium acetate (0.22, 0.44, and 0.88 mg/kg body weight), in every alternate day for a period of 5, 10, and 15 days respectively, to determine the maximum effective dose of cadmium acetate without any mortality. Pre-treatment of rats with aqueous bark extract of Terminalia arjuna (TA) was also studied by oral administration with the increasing doses (10, 20, and 40 mg/kg BW) against cadmium-acetate (0.44mg/kg BW, s.c.,) induced oxidative stress in rat heart and liver to determine the minimum effective dose of TA which can be protected these alterations in both the organs in a concentration dependent manner. The alterations in the activity of the different bio-markers of hepatic and cardiac damage, biomarkers of oxidative stress, and activities of the antioxidant enzymes were studied. Histo-pathological and histo-chemical alterations were also studied through H-E staining, PAS staining and Picrosirius red staining respectively. Structural integrity of hepatic and cardiac tissue were also studied through scanning electron microscopy. Results: The studies revealed that pre-treatment of aqueous bark extract of Terminalia arjuna protects the biomarkers of organ damage, oxidative stress, antioxidant enzymes, from getting altered in the rat heart and liver tissue following treatment with cadmium acetate. Conclusion: The results of the present study suggest that the treatment of aqueous bark extract of Terminalia arjuna might be beneficial to alleviate cadmium induced toxicity in rat heart and liver. KEYWORDS: Cadmium acetate (CH 3 COO) Cd. 2H O, Oxidative stress, Cardiac and hepato protection, Antioxidant efficacy, Terminalia 2 2 arjuna (TA) 1. INTRODUCTION: Cadmium (Cd) is a nonessential heavy metal which has received the attention of scientists during the last decade due to its high toxicity. It is a transition metal obtained as a byproduct of zinc ores. Exposure of humans to cadmium has been and continuous to be of major Corresponding author. Prof. Debasish Bandyopadhyay Oxidative stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta University College of Science and Technology, 92, APC Road, Kolkata , India. concern in the modern world [1, 2]. As cadmium is classified to be a human carcinogen by International Agency for Research on Cancer, it exerts multiple adverse effects on a variety of tissues and is linked with various acute and chronic diseases [3]. Humans are exposed to cadmium from cigarette smoke, food, contaminated water, industrial, occupational and environmental pollution [4]. Cadmium use has been increased dramatically during the last years because of the metal high corrosion and valuable electro chemical properties. Less than 5% of metal is recycled. So, environmental pollution is evident [5]. Cadmium poses a significant threat to the human population and

2 environment. Since cadmium cannot be degraded and biological halflife in humans is found to be more than 20 years, it has been recognized as one of the most toxic environmental heavy metal pollutant [6,7]. The mechanism of cadmium toxicity may be multifactorial. It has been suggested that cadmium acts as a catalyst in the oxidative reactions of biological macromolecules and, therefore, the toxicities associated with the metal might be due to oxidative tissue damage [8]. Cadmium being a redox-inactive metal cannot undergo redox cycling and unable to generate free radicle by itself. However, reports have indicated cadmium may cause an increase in the production of reactive oxygen species (ROS) such as hydroxyl radical (HO ), superoxide anion free radical (O 2- ), nitric oxide radical, or hydrogen peroxide (H 2 O 2 ) indirectly [9,10]. It has been shown that non-radical hydrogen peroxide which by itself became a significant source of free radicals, generated via the Fenton chemistry [11]. In addition other researchers have reported that cadmium can compete with some of the essential divalent elements for ligands.the displacement of essential elements may affect its transfer, storage as well as function at the active site of an enzyme; as a result it can change the conformation of the proteins or nucleic acids required for normal function [12]. It has been reported that cadmium could replace iron and copper from a number of cytoplasmic and membrane protein like ferritin, which in turn would release and increase the concentration of unbound iron and copper ions. These free ions mainly generate oxidative stress via the Fenton reactions [13,14]. Cadmium stimulates free radical production in different organs and tissues by acute and chronic exposure, resulting in oxidative deterioration of lipids, structural and functional proteins, and DNA, and also initiating various pathological condition in humans and animals. Consequently, it is suggested that cadmium induced oxidative stress in cells and tissues can be partially responsible for the toxic effect of the metal [15,16]. Once absorbed, cadmium is circulated in the blood, bound mainly to the blood cells and albumin, then rapidly cleared from the circulation and concentrates in various tissues. It primarily distributed to the liver after that redistributed progressively to the kidney as cadmium-metallothionein (Cd-MT) [17,18]. Since the numerous toxic effect of cadmium is mainly due to its high binding affinity to sulfhydryl groups, thus the metal may disturb the maintenance of thiol-disulfide balance through binding to reduced glutathione in cells, or may inhibit a number of enzymes containing essential thiols [1,19]. Besides the modification of thiol-containing proteins, Cd toxicity is also reported to be associated with lipid peroxidation, membrane protein degradation, inhibition of energy metabolism, membrane damage, and altered gene expression [20]. However, in cases of chronic exposure, when the metal is bound to metallothionein, chelation therapy is only weakly effective. In recent times, attention has been drawn to the healthpromoting activity of naturally occurring antioxidants which can be readily induced in our diet in specific amounts to combat stress in polluted areas [21]. In this context, we have chosen a very well known plant of medicinal significance, viz., Terminalia arjuna (Arjuna Tree) which is distributed throughout the world, in particular more in India than elsewhere.through screening of ethno-botanical and scientific literature revealed that Terminalia arjuna (Combretaceae) with its multiple phytoconstituents has great potential to provide several health benefits and also holds a reputed position in ayurvedic medicine since ancient times [22]. Bark of TA has been used in Indian system of medicine for the cure of number of diseases from thousands of years. TA is reported to contain many specific phyto-constituents including triterpene glycosides-arjunetin, argunoglucoside, arjunoside, arjunolitin, terminolitin; triterpine saponins-arjunic acid, arjunolic acid, arjungenin; falvonoidsarjunone, arjunolone, and also contain non-specific phytocompounds including phytosyerols i.e., β-sitosterol, proantho-cyanidines, and minerals-ca, Mg, Zn, and Cu [22,23]. Literature revealed that ancient Indian physician Vagbatta first advocated the use of this bark powder for the treatment of heart disease. TA has also been found to possess anti-carcinogenic antidyslipidaemic [24], hypocholesterolaemic [25], antibacterial [26], anti-carcinogenic [27], antiinflammatory and antioxidant activity [24]. Reactive oxygen species (ROS) is thought to play a critical role in the pathogenesis of various cardiovascular and other diseases, it may be presumed that the protective effect of TA might be exerted through its antioxidant mechanism(s). Removal of cadmium generated ROS by natural antioxidant like Terminalia arjuna can be quite a good approach to protect the cells from tissue damage. Therefore, our present study mainly concentrates on the determination of the maximum effective dose and duration of cadmium acetate in inducing toxicity in rat heart and liver and also focused on the therapeutic potential of Terminalia arjuna on combating such kind of toxicity in a dose dependent manner. 2. MATERIALS AND METHODS 2.1. Chemicals and reagents: Cadmium acetate (CH 3 COO) Cd. 2H O was purchased from Qualigens 2 2 Limited, Mumbai, India. Powder of bark of Terminaliaarjuna (TA) was purchased from Herby House, Kolkata, India. All the other chemicals used including the solvents, were of analytical grade obtained from Sisco Research Laboratories (SRL), Mumbai, India, Qualigens (India/Germany), SD fine chemicals (India), Merck Limited, Delhi, India.

3 2.2. Animals Male Wister rats, weighing g were handled as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA),Ministry of Environment and Forest, Government of India, with the approval of the Institutional Animal Ethics Committee (IAEC) of department of Physiology, University of Calcutta. Prof. P. K. Samanta, M.Sc. (Vet), Ph.D., CPCSEA nominee to Department of Physiology, University of Calcutta, acted as the advisor for animal care and also monitored animal experimentations Preparation of aqueous bark extract of Terminalia arjuna The method of preparation of aqueous bark extract of Terminalia arjuna (TA) was followed as according to Saha A et al., [28] with some modifications. The bark powder of TA was soaked for 4 hrs in double distilled water (1:4) (5 g per 20 ml), filtered through loin cloth (fine cotton cloth). The filtrate was centrifuged at 3000 rpm for 15 min (using a REMI cold-centrifuge). The supernatant, thus obtained, was filtered again through loin cloth, then the filtrate collected, concentrated and was stored in sterile polypropylene tubes at -20 C until further use. The yield of TA was approx. 10% (w/w) Experimental design Experiment 1: Cadmium acetate induced cardiac and hepatic toxicity: a time dependent study with the increasing doses of cadmium acetate Male Wister rats were randomly divided into four groups - A, B, C, and D. Each group of animals comprised of 5 rats. Rats from group A served as control. Cadmium acetate was administered subcutaneously to three groups at the doses of 0.22, 0.44, and 0.88mg/kg BW, In every alternate day for a period of 5, 10, 15days to group B, C and D respectively. Rats were sacrificed on 5 th, 10 th, and 15 th day after initiation of the experiment. The treatment of rats was carried out as per the schedule mentioned below: (i) (ii) (iii) (iv) Group A:The rats of the first group constituted the control group,where rats were treated with 0.9% normal saline (the vehicle control) for 5days, 10days and 15 days respectively. Group B:The rats of the second group were administered cadmium acetate subcutaneously at a dose of 0.22mg/kg body weight for 5days, 10 days and 15days respectively. Group C:The rats of the third group were administered cadmium acetate subcutaneously at a dose of 0.44mg/kg body weight for 5days, 10 days and 15days respectively. Group D: the rats of the fourth group were administered cadmium acetate subcutaneously at a dose of 0.88mg/kg body weight for 5 days, 10 days and 15days respectively Experiment 2: Cadmium acetate induced oxidative stress in vivo in rat heart and liver: Protected with different doses of Terminalia arjuna (TA) In this set of experiment the total no. of animal was 40 and they were divided into the following eight groups, with 5 rats in each group (n=5): (i) Group I: Control group (C), rats were treated with 0.9% normal saline (the vehicle control) (ii) Group II: Positive control group ( Terminalia arjuna(ta) administered orally at a dose of 10 mg/kg body weight, every day for a period of 15 days) (iii) Group III: Positive control group ( Terminalia arjuna (TA) administered orally at a dose of 20 mg/kg body weight, every day for a period of 15 days) (iv) Group IV: Positive control group ( Terminalia arjuna (TA) administered orally at a dose of 40 mg/kg body weight, every day for a period of 15 days) (v) Group V: Cadmium acetate treated group (Cd administered subcutaneously at a dose of 0.44 mg/kg body weight,in every alternate day for a period of 15 days.) (vi) Group VI: Cadmium acetate treated group (Cd administered subcutaneously at a dose of 0.44 mg/kg body weight) plus aqueous bark extract of Terminalia arjuna (10 mg/kg body weight, administered orally) (vii) Group VII: Cadmium acetate treated group (Cd at a dose of 0.44 mg/kg body weight to be administered subcutaneously) plus aqueous bark extract of Terminalia arjuna (20 mg/kg body weight, administered orally) (viii) Group VIII: Cadmium acetate treated group (Cd at a dose of 0.44 mg/kg body weight to be administered subcutaneously) plus aqueous bark extract of Terminalia arjuna (40 mg/kg body weight, administered orally) 2.5. Collection of blood and tissue samples At the end of the treatment period all animals were kept fasted overnight and were sacrificed through cervical dislocation after subjecting them to mild ether anesthesia. The chest cavity was opened first through a vertical incision and the blood was carefully collected through cardiac puncture for the preparation of serum. Thereafter, the abdomen was opened and the heart and liver were surgically extirpated, collected, rinsed well in saline and soaked properly with a piece of blotting paper and stored in sterile plastic vials at -20 C for further biochemical analyses. For histological studies, a suitable amount of the cardiac and hepatic tissue were placed immediately after removal in appropriate fixative Assessment of serum specific markers related to cardiac and hepatic damage Serum glutamate oxaloacetate transaminase (SGOT) and Serum glutamate pyruvate transaminase (SGPT) were measured by the

4 method of Reitman and Frankel. Values were expressed as IU/L [29]. The cardiac specific Type 1 isoform of Lactate Dehydrogenase (LDH1) activity was obtained by incubating the serum samples at 65 C for 30min which destroys all isoforms except LDH1 [30]. The enzyme activity was then determined by measuring the NADH oxidation. The enzyme activity was expressed as IU/L. Type 5 isoform of Lactate Dehydrogenase (LDH5) activities and the total serum activities of lactate dehydrogenase (LDH-T) were obtained by measuring the oxidation of NADH (0.1mM) to NAD+ at 340nm using 1.0mM sodium pyruvate as substrate. The samples for the measurement of total LDH were prepared by incubating the serum at 37 C for 30 min. Likewise; the samples for the measurement of LDH5 were prepared by incubating the serum samples at 57 C for 30min, which destroys the isoform LDH5. The resulting enzyme activity was then subtracted from the total serum LDH activity to obtain the activity of LDH 5 [31]. The enzyme activity was expressed as IU/L Preparation of heart and liver tissue homogenate, measurement of lipid peroxidation (LPO) level, reduced glutathione (GSH) content, and protein carbonyl (PCO) content The heart and liver tissue were homogenized (10%) in ice-cold 0.9% saline (ph 7.0) with a Potter Elvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA) for 30s and the lipid peroxides in the homogenate were determined as thiobarbituric acid reactive substances (TBARS) according to the method of Buegeand Aust, [32] with some modifications as adopted by Bandyopadhyay et al. [33]. The GSH content (as acid soluble sulfhydryl) of heart and liver tissue homogenate were estimated by its reaction with DTNB (Ellman s reagent) following the method of Sedlac and Lindsay, 1968 [34] with some modifications by Bandyopadhyay et al. [35]. The values were expressed as nmoles GSH/mg protein. Catalase activity was assayed by the method of Beers and Seizer [38] with some modifications as adopted by Chattopadhyay et al. [39]. The enzyme activity was expressed as µmoles of H2O2 consumed/ min/ mg tissue protein Determination of the activities of the enzymes of the glutathione metabolizing pathway Glutathione reductase (GR) assay was carried out according to the method of Krohne-Ehrich et al. [40]. The specific activity of the enzyme was calculated as Units/min/mg of tissue protein. Glutathione peroxidase (GPx) activity was measured according to the method of Paglia and Valentine [40] with some modifications [41]. The specific activity was expressed as Units/min/mg of tissue protein Estimation of protein Protein content was estimated by the method of Lowry et al. [42] using the bovine serum albumin as standard Histo-pathological and histo-chemical studies Studies using tissue sections stained with hematoxylineosin (H &E) A portion of the extirpated rat heart and liver were fixed immediately in 10% formalin and embedded in paraffin following routine histological procedure. Cardiac and hepatic tissue sections (5 μm thick) were prepared and stained with hematoxylin eosin (Sigma).The stained tissue sections were examined under Leica microscope and the images were captured with a digital camera attached to it [43]. Simultaneously, a small portion hepatic tissue was fixed in 10% natural buffered formalin acetic acid alcohol fixative and processed further for Per-iodic-Acid Schiff (PAS) staining for glycogen. Protein carbonyl (PCO) content of the above mentioned tissues were estimated by DNPH assay [35]. The absorbance was recorded at 370 nm using a UV / VIS spectrophotometer.the values were expressed as nmoles /mg protein Measurement of the activities of Copper-Zinc superoxide dismutase (Cu-Zn SOD or SOD1), Manganese superoxide dismutase (Mn-SOD or SOD2) and Catalase (CAT) Copper-zinc superoxide dismutase (Cu-Zn SOD or SOD1) activity was measured by the method of Martin et al., 1987 [36]. The enzyme activity was expressed as units/min/mg of tissue protein. Manganese superoxide dismutase (Mn-SOD or SOD2) activity was assayed by the method of Marklund and Marklund [37]. One unit of enzyme activity is 50% inhibition of the rate of autooxidation of pyrogallol as determined by change in absorbance/min at 420 nm. The enzyme activity was expressed as units/min/mg of tissue protein Quantification of fibrosis by Confocal Microscopy Another set of heart and liver tissue sections (5 μm thick) were fixed in 10% formalin and embedded in paraffin following routine procedure. The tissue sections were stained with Sirius red (Direct Red 80; Sigma Chemical Co, Louis, MO, USA) and imaged with a laser scanning confocal system (Zeiss LSM 510 META, Germany) and the stacked images through multiple slices were captured [44] Scanning electron microscopy (SEM) For scanning electron microscopy, small pieces of rat heart and liver tissue were fixed in 2.5% cold glutaraldehyde immediately after dissecting the animal for h. The specimens were then washed 3-4 times in phosphate buffer (PH 7.2), after that the tissue sections were dehydrated by ascending grades of alcohol (30, 50, 70, 90, and 100% for 10min).The dehydrated tissue sections were then embedded in pure tert-butyl alcohol and were then placed into a 4 0 C refrigerator

5 until the tert-butyl alcohol solidified. The frozen samples were dried by placing them into a vacuum bottle. The cardiac and liver tissue morphology were evaluated by scanning electron microscopy (SEM; Zeiss Evo 18 model EDS 8100) Statistical evaluation Each experiment was repeated at least three times with different rats. The data for various biochemical parameters were expressed as means ± S.E.M. The statistical significance of the data has been determined using one-way analysis of variance (ANOVA) after ascertaining the homogeneity of variances between the treatments and significant difference among treatment groups were evaluated by Scheffes test. The results were considered statistically significant at p < All statistical analyses were made using Microcal Origin version 7.0 for Windows. 3. RESULTS 3.1. Experiment 1: Dose response and time dependent study of cadmium acetate Biomarkers of cardiac and hepatic damage Figure.1 (A) reveals a significant increase in the level of activity of serum specific cardiac damage marker LDH1and figure. 1 (B, C) also depicts a significant increase in the level of hepatic damage markers LDH5 and total LDH respectively. All the biomarkers in both heart and liver tissue damage were found to be altered in a dose dependent and time dependent manner following the treatment of rats with cadmium acetate, at the doses of 0.22, 0.44 and 0.88 mg/kg body weight, s.c., in every alternative day for a period of 5 days, 10 days and 15 days respectively. The increased and decreased level of cardiac and hepatic damage markers in Cd-treated groups of rat compared to control groups (P< 0.001). Figure.1. Bar graphs represent damaging effect of cadmium acetate followed by the administration of the increasing doses of cadmium (0.22, 0.44, 0.88 mg/kg body weight, subcutaneously in male Wister rat and the results shows an increase in serum LDH-1 (A), LDH-5 (B) and total LDH (C). Serum lactate dehydrogenase-1 IU/L Serum lactate dehydrogenase-5 IU/L Serum lactate dehydrogenase activity (Total) IU/L The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA Biomarkers of oxidative damage Figure.2 (A), (B) and (C)showed a significant increase in the level of LPO, PCO content and decrease GSH content in cardiac tissue respectively. On the other hand, figure.2. (D), (E) and (F)also reveals a significant increase in the level of LPO, PCO content and decrease in GSH content in hepatic tissues respectively. All the biomarkers of oxidative stress in both cardiac and liver tissue were found to be dose and time dependently changed when the rats were treated with cadmium at the increasing doses of 0.22, 0.44 and 0.88 mg/kg body weight, s.c., in every alternative day for a period 5 days, 10 days and 15 days respectively. Furthermore, the level of increased and decreased oxidative damage biomarkers of Cd-treated groups were compared to respective control groups (P < 0.001).

6 Figure.2. Bar graphs represent damaging effect of cadmium acetate followed by the administration of the increasing doses of cadmium (0.22, 0.44, 0.88 mg/kg body weight, subcutaneously in male Wister rat heart and result shows an increase in the level of oxidative stress biomarker LPO (A), PCO (B) and decrease in endogenous antioxidant GSH (C), on the other hand, increase in the level of LPO (D), PCO (E) and also decrease in the concentration of GSH (F) in rat liver. nmoles TBARS/mg tissue protein Protein Carbonyl Content nmoles/mg tissue protein nmoles of GSH/mg tissue protein nmoles TBARS/mg tissue protein Protein Carbonyl Content nmoles/mg tissue protein nmoles of GSH/mg tissue protein The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA Status of the antioxidant enzymes Figure.3 (A) and (B) further showed a significant decrease in antioxidant enzyme activities of Mn-SOD and Cu-Zn SOD in heart tissue. On the other hand, figure.3(c) and (D) also demonstrate the significant increase inthe enzyme activities of Mn-SOD and Cu-Zn SOD in liver tissue. These parameters of antioxidant enzymes in both heart and liver tissue were found to be altered dose-dependently as

7 well as time-dependently following cadmium acetate treated groups of rat, where cadmium acetate administered at the doses of 0.22, 0.44 and 0.88 mg/kg body weight, s.c.,in every alternate day for a period of 5 days, 10 days and 15 days respectively. The level of increased and decreased antioxidant enzyme activities of Cd-treated groups were compared to control groups (P < 0.001). Figure. 3(E) reveals that the treatment of rats with cadmium acetate at the doses of 0.22, 0.44 and 0.88 mg/kg body weight, s.c., resulted in increase in the activity of catalase in cardiac tissue in a dose and time dependent manner as compared to control (P< 0.001) and figure. 3(F) also shows a significant decrease in the activity of catalase in liver tissue in a dose and time dependent fashion as compared to control (P< 0.001). Figure. 3(G) and (H) depict a dose and time dependent decrease in the enzyme activity of glutathione reductase (GR) in both heart and liver tissue respectively as compared to control (P< 0.001), and figure. 3(I) and 3(J) also demonstrate a significant increase in the activity of glutathione peroxidase (GPx) in both heart and liver tissue dose dependently with a time dependent manner following the treatment of rats with cadmium acetate (at the doses of 0.22, 0.44 and 0.88 mg/kg body weight) as compared to control (P< 0.001) which indicates myocardial and hepatic tissue damage. Figure. 3. shows the alteration in Mn SOD (A), Cu-Zn SOD (B) in heart and also shows alteration in Mn SOD (C), Cu-Zn SOD (D) in liver.cadmium treated group of rats show alteration in the activity of catalase in both heart and liver tissue in figure. 3. (E) and (F) respectively. Cadmium treated group of rats show alteration in the activity of GR and GPx in figure. 3. (G, H) and figure. 3. (I, J) respectively. The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA. Mn-SOD activity Units/mg tissue protein Cu-Zn SOD activity units/mg tissue protein Mn-SOD activity Units/min/mg tissue protein Cu-Zn SOD activity units/mg tissue protein

8 Catalase activity µm H 2 O 2 consumed/min/mg tissue protein Catalase activity µm H 2 O 2 consumed/min/mg tissue protein Glutathione reductase activity Units/min/ mg tissue protein Glutathione reductase activity Units/min/ mg tissue protein Glutathione Peroxidase activity Units/min/mg tissue protein Glutathione Peroxidase activity Units/min/mg tissue protein The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA Histological studies Figure 4(A) and (B) demonstrate that the treatment of rats with cadmium at the doses of 0.22, 0.44 and 0.88mg/kg body weight, s.c., caused damage to the cardiac and liver tissue respectively. There were clear signs of degenerative changes along with myocardial fiber necrosis, capillary dilatation, and vascular congestion in the cardiac tissue sections as well as mild dilatation of central vein, mild inflammatory cell infiltration in portal tract, congested portal vein, necrotic hepatocytes were observed in HE stained hepatic tissue following the treatment of cadmium.

9 Figure 4 shows routine H and E staining of the rat cardiac and hepatic tissue. Black arrows indicate the damaged portion of the cardiac and hepatic tissue in different concentration with the doses of 0.22, 0.44, and 0.88 mg/kg body weight, s.c, followed by the administration of cadmium acetate. H & E stained heart tissue, (400 x magnification) Scanning electron microscopy Figure 5(A) and(b) depict the topological changes on the surfaces of the cardiac and liver tissue were studied using scanning electron microscopy. The cardiac tissue sections of cadmium treated rats showed that the branching pattern of muscle fibers become irregular and degenerated, with the presence of necrotic tissue. Simultaneously, the architectural changes were also observed in hepatic tissue section of cadmium treated groups with dilated central vein, congested portal vein and also the arrangement of the hepatocytes were distorted with profound tissue necrosis. The purpose of the above mentioned experiments was to determine the most effective dose of cadmium acetate in inducing oxidative H& E stained liver tissue, (400 x magnification) stress in rat organs (i.e., liver and heart) without any mortality of the animals during the experimental period. In our experiment, cadmium acetate at a dose of 0.22 mg/kg body weight, s.c., did not provide significant damage compared to control. In contrast, there was no mortality of rats at a dose of 0.44 mg/kg body weight, s.c., of cadmium acetate during the entire period of treatment and showed maximum damage in case of 15 days but 40% mortality of rats were observed at a dose of 0.88 mg/kg body weight, s.c., of cadmium acetate although it showed maximum damage in case of 15 days of treatment. Therefore, the subsequent experiments were carried out with 0.44 mg/kg body weight, s.c.,dose of cadmium acetate in every alternate day for a period of 15 days.

10 Figure. 5 (A) and (B) also show the structural damages in heart and liver tissue examined through scanning electron microscopy in a dose and time dependent manner CH Cd0.22 Cd0.44 Cd0.88 CL Cd0.22 Cd0.44 Cd days 10 days 5 days Scanning electron microscopy of heart and liver tissue section 3.2. Experiment 2: Cadmium acetate induced oxidative stress in vivo in rat heart and liver: Protected with different doses of Terminalia arjuna (TA) Changes in serum specific cardiac and hepatic damage markers Figure. 6(A) and (B) demonstrate that the level of activity of serum specific cardiac damage markers, SGOT and LDH1, as well as hepatic damage markers SGPT, LDH-5 and T-LDH(figure. 6. C, D and E) were found to be significantly higher in Cd-treated (0.44mg/kg BW, s.c.,) group of rats compared to control (3.16 and 2.01 folds increases respectively in heart and 2.29, 2.11 and 1.82 folds increases respectively in liver tissue, P < vs. control). These activities of the enzymes were found to be significantly decreased in a dose dependent manner, when the animals were pre-treated with aqueous bark extract of TA at the doses of 10, 20, 40mg/kg body weight, fed orally, the maximum restoration being at a dose of 20mg/kg BW (Cd 0.44+TA 20) followed by cadmium acetate treatment (35.38% and 51.82% decreases respectively in heart tissue, and 51.55%, 47.99% and 56.95% decreases respectively in liver tissue, P < vs. Cd-treated group). However, the extract by itself did not possess any effect on the activities of these enzymes. The results indicate that the aqueous bark extract of TA do possess the capability to provide protection against Cd-induced cardiac and hepatic damage.

11 Figure. 6 (A) and (B) depict the protective effect of TA, administered orally to different group of rats in different doses, against cadmium induced alterations in SGOT(A), LDH-1(B), SGPT (C), LDH-5 (D), and T-LDH (E). Serum glutamate oxaloacetate transaminase activity (IU/L) Lactate dehydrogenase-1 activity IU/L Serum glutamate pyruvate transaminase activity (IU/L) Lactate dehydrogenase-5 activity IU/L Lactate dehydrogenase activity (total) IU/L Changes in biomarkers of oxidative damagein heart and liver tissue Figure. 7 (A, B) and (C, D) show that the significant increase in the level of LPO,and PCO content in heart and liver respectively, following Cd-treatment (0.44mg/kg BW, s.c.) of rats compared to control (1.67 and 1.69 folds increases respectively in heart, and 2.07 and 1.98 folds increases respectively in liver tissue, P < vs. control).these elevated level of lipid peroxidation products and protein carbonyl content were found to be decreased significantly in a dose dependent manner (60.60% and 67.90% decreases respectively in heart, and 51.49% and 57.41% decreases respectively in liver tissue from Cdtreated group, P < 0.001; reaching almost control level) when the rats were pre-treated increasing doses of aqueous bark extract of TA, where it observed that the maximum protection provided at a dose of 20 mg/kg BW, fed orally followed by the treatment with cadmium acetate(cd mg/kg BW). The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA; P<0.001 comparedto cadmium-induced values using ANOVA. On the other hand, figure. 7 (E) and (F) shows a significant decrease in the reduced glutathione content of heart and liver tissues following

12 treatment of rats with cadmium acetate (0.44mg/kg Bw, s.c.,) (48.58% and 34.55% decreases respectively, P < vs. control). These decreased level of reduced glutathione content were found to be increased significantly (1.97 and 2.64 folds increases respectively P< vs. cadmium treated groups) in a dose dependent manner when the rats were pre-treated with increasing doses of aqueous bark extract of TA and the maximum protection being provided at a dose of 20mg/kg BW. However, the aqueous bark extract of TA by itself (positive control) has no effect on these biomarkers. The results indicate the protective ability of the aqueous bark extract of TA against Cd-induced oxidative stress in rat heart and liver. Figure.7 Protective effect of TA, administered to different groups of rats in different doses, against cadmium induced alteration in LPO (A) and PCO (B) in heart, LPO (C), and PCO (D) in liver and also GSH (E)in heart and GSH (F) in liver respectively. Lipid Peroxidation nmoles TBARS/mg protein Protein Carbonyl Content nmoles of carbonyl/mg protein Lipid Peroxidation nmoles TBARS/mg protein Protein Carbonyl Content nmoles of carbonyl/mg protein The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA; P<0.001 compared to cadmium-induced values using ANOVA.

13 3.2.3.Changes in antioxidant enzyme activities of heart and liver Figure 8 (A, B and C) reveals a significant decrease in the activity of cytosolic Cu-Zn-SOD, mitochondrial MnSOD and increase in the activity of catalase in rat heart tissue following Cd-treatment (0.44mg/ kg BW, s.c.,) of rats compared to control (52.26% and 67.28%decreases and 1.30 fold increase respectively, P < vs. control). These alterations were found to be restored significantly (1.85 fold, 1.46 fold increases and %decrease respectively P<0.001 vs. cadmium treated groups) in a dose dependent manner when the rats were pre-treated with increasing doses of aqueous bark extract of TA and the best possible protection being provided at a dose of 20mg/kg BW. On the other hand, figure. 8( D, E and F) also shows a significant increase cytosolic Cu-Zn SOD, mitochondrial MnSOD and decrease in the activity of catalase in rat liver tissue following Cd-treatment (0.44mg/kg BW, s.c.,) of rats compared to control (1.36 fold and 1.90 fold increases and 62.64% decrease respectively, P < vs. control). These cadmium treated alterations in antioxidant enzymes were found to be restored significantly (75.34% and 53.32% decreases and 1.54 fold increase respectively P<0.001 vs. cadmium) in a dose dependent manner when the rats were pre-treated with increasing concentrations of aqueous bark extract of TA and the maximum protection being provided at a dose of 20mg/kg BW. The results indicate the protective ability of the aqueous bark extract of TA against Cd-induced oxidative stress in rat heart and liver. Figure.8 Protective effect of TA, administered to different groups of rats in different doses, against cadmium induced alteration in the activities of Cu-Zn SOD (A), Mn-SOD (B), and Catalase(C) in heart, and also Cu-Zn SOD (D), Mn-SOD (E), and Catalase(F) in liver respectively. Cu-Zn SOD activity Units/min/mg protein Mn-SOD activity Units/min/mg protein Catalase activity µm H 2 O 2 consumed/min/mg protein Cu-Zn SOD activity units/min/mg protein

14 Mn-SOD activity Units/min/mg protein Catalase activity µm H 2 O 2 consumed /min/mg tissue protein The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA; P<0.001 comparedto cadmium-induced values using ANOVA Status of the enzymes of glutathione metabolizing pathway Figure 9 (A) and (B) reveals that a significant reduction and elevation in the activity of glutathione reductase (GR) and glutathione peroxidase (GPx) respectively in cardiac tissue following treatment of rats with cadmium ( 77.96% decreased and 1.88 fold increased respectively, P < vs. control) (0.44 mg/kg BW, s.c.,). Pretreatment of the rats with increasing doses of aqueous bark extract of TA restored the activities of these enzymes significantly (1.26 fold increased and 57.35%decreased P<0.001 vs. cadmium treated groups)to those observed in the control rats in a dose dependent manner and the best possible protection being provided at a dose of 20 mg/kg BW. On the other hand, Figure. 9 (C) and (D) also reveals that a significant decrease in the activity of GR and increase in the activity of GPx in rat liver tissue following Cd-treatment (0.44mg/kg BW, s.c.,) of rats compared to control (53.55% decrease and 1.96 fold increase respectively, P < vs. control). These alterations were found to be restored significantly (1.85 fold increase and 54.83% decrease respectively P<0.001 vs. cadmium) in a dose dependent manner when the rats were pre-treated with increasing doses of aqueous bark extract of TA and the maximum protection being provided at a dose of 20mg/kg BW. However, the aqueous bark extract of TA by itself (positive control) has no effect on these enzymes of glutathione metabolizing pathway. The results indicate the protective ability of the aqueous bark extract of TA against Cd-induced oxidative stress in rat heart and liver. Figure. 9. Protective effect of TA, administered to different groups of rats in different doses, against cadmium induced alteration in the activities of GR (A), GPx (B) in heart, and GR(C), GPx (D) in liver respectively. Glutathione Reductase activity Units/min/mg protein Glutathione Peroxidase activity Units/min/mg protein

15 Glutathione Reductase activity Units/min/mg tissue protein Glutathione Peroxidase activity Units/min/mg tissue protein The values are expressed as Mean ± S.E.; P < 0.001; compared to control values using ANOVA; P<0.001 comparedto cadmiuminduced values using ANOVA Histo-pathological and histo-chemical studies Studies using tissue sections stained with hematoxylineosin (H & E) Figure.10 (A) (magnification 400X) demonstrates that there were clear sign of degenerative changes along with capillary dilatation, vascular congestion and myocardial fibre necrosis in the cardiac tissue sections and on the other hand, Figure. 11 (A), also shows marked congestion in portal vein, necrotic hepatocytes, dilated Figure. 10 (Panel A) represents the H and E stained cardiac tissue section. (Panel B) represents the picrisirius stained cardiac tissue section captured by light microscope. (Panel C) represent the acidsirius stained cardiac tissue captured by confocal microscope. Black and white arrows indicate the damaged portion of the cardiac tissue compared to control. All these changes were protected by the pre-treatment of TA at a dose of 20 mg/kg body weight, fed orally. The tissue sections marked with (a), (b), (c), (d), (e), (f), (g) and (h) in all the panel (A,B and C) mainly represent the groups such as, CH, TA10 mg/kg BW, TA20 mg/kg BW, TA40 mg/kg BW, Cd(0.44 mg/kg BW), Cd+TA10 mg/kg BW, Cd+TA20 mg/kg BW, Cd+TA40 mg/kg BW respectively. H & E stained heart tissue section, (400x maginfication) Picrisirius stained heart tissue section, Bright field image, (400 x magnification) Picrosirius stained heart tissue section, confocal microscopy image, (400x magnification)

16 Figure. 11 (Panel A) represents the H and E stained cardiac tissue section. (Panel B) represents the PAS stained liver tissue section captured by light microscope. (Panel C) represent the acidsirius stained liver tissue captured by light microscope and also Panel (D) represents the acidsirius stained liver tissue captured by confocal microscope. Black arrows indicate the damaged portion of the cardiac tissue compared to control.all these changes were protected by the pretreatment of TA at a dose of 20 mg/kg body weight, fed orally. The tissue section marked with (a), (b), (c), (d), (e), (f), (g) and (h) in all the panel (A,B and C) mainly represent the groups such as, CL, TA10 mg/kg BW, TA20 mg/kg BW, TA40 mg/kg BW, Cd(0.44 mg/kg BW), Cd+TA10 mg/kg BW, Cd+TA20 mg/kg BW, Cd+TA40 mg/kg BW respectively. H & E stained Liver tissue, (200x magnification) PAS stained liver tissue section, (200 x magnification) Picrosirius stained liver tissue section, bright field image, (200x magnification) Picrosirius stained liver tissue section, confocal microscopy image, (200x magnification) sinusoids,much enlarged nuclei and light stained cytoplasm with vacuoles, following the treatment of cadmium acetate (0.44 mg/kg body weight, s.c.,). The damage in heart and liver tissues were found to be protected when the rats were pre-treated with aqueous bark extract of TA in a dose dependent manner, the maximum protection being provided at a dose of 20mg/kg body weight. These results from HE stain indicate that TA has the ability to provide protection to the cardiac and liver tissue against cadmium-induced damage.

17 Studies using tissue sections stained with periodic acid Schiff (PAS) As shown in Figure 11 (B), treatment of cadmium acetate (0.44 mg/kg BW. s.c.) caused severe liver damage including congestion of vessels, vacuolization, polymorphic nuclei, and degenerative hepatocytes and marked glycogen depletion were also observed in PAS stained liver tissue. The damage in liver tissues were found to be protected when the rats were pre-treated with aqueous bark extract of TA in a dose dependent manner, the maximum protection being provided at a dose of 20mg/kg body weight. These results from PAS stain indicate that TA has the ability to provide protection to the liver tissue against cadmium-induced damage.however, there were no significant changes observed with the increasing concentration of aqueous bark extract of TA by itself (positive control) Quantification of fibrosis by Confocal Microscopy The figure 10 (C), (D) reveals that Picrosirius red stained cardiac tissue sections demonstrate a significant depletion of collagen from the intracellular space of cardiac muscle fiber and a deposition of collagen especially around the central vein of the hepatic lobule also in demonstrated in figure. 11 (C), (D), indicative of tissue fibrosis, following the treatment of cadmium acetate (0.44 mg/kg body weight, s.c.),. These alterations were found to be protected when the rats were pre-treated with aqueous bark extract of TA in a dose dependent manner, the best possible protection being provided at a dose of 20mg/kg body weight. The results indicate that TA has the ability to provide protection to the hepatic and cardiac tissue against cadmiuminduced damage Scanning electron microscopy Figure. 12 (A) shows the changes brought about to the cardiac and hepatic tissue section,following the treatment of cadmium (0.44 mg/kg BW. s.c.) and studied through scanning electron microscopy. The fibrillar collagen fibers of cardiac tissue found to cross-linked randomly to form a complicated matrix network and also showed that the branching pattern of muscle fibers become irregularfollowing the treatment of cadmium acetate. Simultaneously, the architectural changes were also observed in hepatic tissue section of cadmium-treated groups (Cd 0.44) with dilated central vein and also the arrangement of the hepatocytes were distorted with profound tissue necrosis (figure. 12.B). These cadmium-induced changes in the rat heart and liver were found to be signiûcantly prevented when the rats were pretreated with TA in a dose dependent manner, the maximum protection being provided at a dose of 20 mg/kg BW. Figure. 12 (A) and (B) represents the scanning electron microscopic images of the rat cardiac and liver tissue. Yellow arrows indicate the damaged portion of the cardiomyocytes and hepatocytes in cadmium treated rats (0.44 mg/kg BW). But no such changes were found in Cd+TA 20 group. CH TA 10 TA 20 TA 40 Cd 0.44 Cd + TA 10 Cd + TA 20 Cd + TA 40 CL TA 10 TA 20 TA 40 Cd 0.44 Cd + TA 10 Cd + TA 20 Cd + TA 40

18 4. DISCUSSION Heavy metal ions even at a very low level can cause serious health problems including humans and other mammals because it can accumulate in tissues, causing metabolic, histological and pathological changes [45]. Occupational exposure to Cd has been associated with occurrence of increased oxidative stress followed by the generation of reactive oxygen species [46]. Cadmium generated reactive oxygen species have the potential to affects the growth and development, cancer formation, damage to organs, nervous system damage and in severe cases it leads to death [45]. To our knowledge, this is the first report showing the protective role of aqueous bark extract of Terminalia arjuna against cadmium acetate induced structural and functional disturbances in heart and liver tissue of male Wister rats. In our present study, rats were injected subcutaneously with the increasing doses of cadmium acetate and we examined the responses in the heart and liver at 5, 10, and 15 days post-exposure. Cadmium induced toxicity, which increased with increasing concentration of cadmium and time from exposure, due to its strong oxidizing property in cells which causes membrane damage and mortality due to penetrating power of cadmium acetate into the tissue. Our results also determine the maximum effective dose of cadmium acetate (0.44mg/kg body weight, s.c.,) thrice at an interval of 15 days without any mortality of rats during the entire treatment period. The present study also emphasizes the specific role of aqueous bark extract of Terminalia arjuna (TA) against cadmium acetate induced oxidative stress. Here, we provide the evidence that aqueous bark extract of Terminalia arjuna ameliorate the damaging effect of cadmium acetate in experimental rats in a dose dependent manner mainly because of its antioxidant property. Literature suggests that cadmium accumulates in considerable amounts in rat heart and liver because these are highly perfused organ and contains huge amounts of metal binding useful substances like metallo-thionine. The accumulation of the metal in the organ may be a valid reason leading to damage and dysfunction [47], in the present study the oxidative stress induced by exposure to cadmium caused a significant increase in the activities of serum SGOT and LDH-1 in case of heart tissue as well as SGPT, LDH-5 and total LDH in liver tissue, were well known indicator of cardiac and hepatic injury respectively. In cases such as cardiac and liver damage with cardiac and hepatocellular lesion, these enzymes normally located in the cytosol of the organ were released into the blood stream [48,49]. Pretreatment with aqueous bark extract of TA significantly lowered the level of these enzymes in a dose dependent manner, the maximum protection being provided at a dose of 20 mg/kg BW, and the values were comparable with that of the control group of rats. This protection might have been exerted through some phytochemical(s)/ phytonutrient(s) present in the extract. Accumulation of cadmium in heart and liver tissue also caused a peroxidative disorder with a significantly increased level of membrane lipid peroxidation markers viz. TBARS and protein carbonyls, when compared to those in control group. Several studies demonstrate the ability of Cd to replace iron (Fe), a redox active metal, thereby increasing the availability of free Fe in the cells and hence induced oxidative stress. Free iron in turn produces highly damaging hydroxyl radicals via the Fenton reaction and hence increases the production of lipid peroxidation [50]. Membrane lipids are highly susceptible to free radical damage. Lipids when reacted with free radicals can undergo the highly damaging chain reaction of lipid peroxidation [51]. Protein carbonyl groups represent an irreversible protein modification, often leading to the inactivation of the proteins. Protein carbonyl content (PCO) is reported to be a sensitive and early marker of oxidative stress to tissue as compared with TBARS [52,53]. However, when the rats were pre-treated with aqueous bark extract of TA, a dose dependent protection was observed by decreasing the lipid peroxidation and the protein carbonyl content of heart and liver tissue. The results indicate that the aqueous bark extract of TA seems to possess anti-oxidative properties. As a thiol-affectionate metal, free Cd primarily targets the highly abundant cellular GSH, a ROS scavenger. Depletion of the GSH pool leads to poor scavenging of Cd, which thereafter results in disturbance of the cellular redox balance leading to oxidative stress [54]. In our present experiments, sub-chronic treatment of rats with cadmium acetate caused a significant reduction of the concentration of GSH in both heart and liver tissue, which may be due to the oxidative damage to the bio-molecules of the tissue. However, pretreatment of rats with aqueous bark extract of Terminalia arjuna dose dependently increases the concentration of GSH in both cardiac and hepatic tissue may be by increasing the level of endogenous antioxidant. With respect to the activity of SOD and catalase, a significant decrease was observed in case of SOD, with a concomitant increase in the activity of catalase in heart tissue, probably due to the breakdown of hydrogen peroxide by SOD and catalase also reduces the tissue injury by catalyzing the conversion of H 2 O 2 to H 2 O.Decrease in SOD activity and hence superoxide accumulation can have several deleterious effects on the cell such as, inactivation of a number of mitochondrial enzymes, lipid peroxidation and oxidation, production of reactive nitrogen species and so on [55]. On the other hand, hepatic tissue showed a significant increase and