Antioxidant status in streptozotocin induced diabetic rats treated with vanadium complex

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1 Indian J. Anim. Res., 50 (1) 2016: Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Antioxidant status in streptozotocin induced diabetic rats treated with vanadium complex P. Kannan, A. Vijayaraj #, P. S. L. Sesh*, V. Narayanan #, A. Thangavel and V. Pandiyan Department of Veterinary Biochemistry, Madras Veterinary College, Chennai , India. Received: Accepted: DOI: /ijar.8564 ABSTRACT As lipid peroxidation and oxidative stress play a key role in the pathogenesis of diabetes, the antioxidant status of streptozotocin induced diabetic rats, treated with vanadium complex was explored in the present study. Diabetes was induced by single intraperitoneal injection of streptozotocin (STZ) at the dose rate of 45 mg per kg body weight. Diabetes was confirmed after 72 hours of STZ injection by estimating blood glucose level and those rats showing more than 250 mg/ dl were considered as diabetic. Vanadium complex at the dose rates of 5 and 10 mg / kg body weight was administered orally to normal control and STZ induced diabetic rats. Glimepiride was used as the positive control and was given orally at the dose rate of 800 µg / Kg body weight. The study on the hepatic, renal and pancreatic tissues showed that vanadium complex at both the predetermined dosages significantly increased the antioxidant activities of superoxide dismutase and glutathione peroxidase along with a significant increase in the level of glutathione and a significant decrease in the level of lipid peroxidation. The study also revealed that there is a significant reduction in the activity of catalase after treatment with vanadium complex at both the dosage levels. Key words: Antioxidants, Diabetes Mellitus, Hypoglycemia, Rats,Vanadium. INTRODUCTION Diabetes mellitus is a syndrome characterized by chronic hyperglycemia and disturbances of carbohydrate, fat and protein metabolism associated with absolute or relative deficiency in insulin secretion and action. Diabetes is widely recognized as one of the leading causes of death and disability worldwide. In 2011, according to the World Health Organization there were 346 million people affected with diabetes. WHO projects that diabetic death will be doubled between 2005 and Among domestic animals, diabetes is most commonly found in middle aged to older dogs and cats: particularly in female dogs and in male cats. In dogs, breeds like Terriers, Daschunds, Golden retriever, Labrador retriever, Poodles, Puli and Schnauzers are the most vulnerable to diabetes. It causes number of complications like retinopathy, neuropathy, and peripheral vascular insufficiencies (Chehade and Mooaradian, 2000). The pathogenesis of these complications is attributed largely to lipid peroxidation and oxidative stress (Ramadasan and Chako Sabu, 2009). The main alteration related to diabetes is hyperglycemia. Reactive oxygen species (ROS) have been implicated in the pathogenesis of diabetes leading to hyperglycemia (Baynes and Thorpe, 1991). ROS are produced due to autoxidation of plasma glucose, nonenzymatic protein glycation, activation of leukocytes, and increased transition metal bioavailability (Hunt et al., 1990). When the generation of ROS and other free radical overwhelms cellular defences, these unstable radicals react with essential molecules within the cell such as lipids, protein and DNA, leading to histological changes as well as functional abnormalities. The increase of free radical mediated toxicity, spread throughout the different tissues, is well documented in clinical diabetes and in STZ-diabetic rats. Degeneration of vital tissues leading to diabetic complications may be indirectly due to the increased oxidative stress of the diabetic condition (Rauscher et al., 2001). The combination of protective effect exerted by antioxidants and free radicals scavenging are of utmost importance in preventing or slowing down the complications of diabetes. The increased oxidative stress in diabetic conditions might be caused not only by an accelerated production of reactive oxygen species but also by a decreased removal of these molecules (Tashima et al., 2000). *Corresponding author s pslsesh@yahoo.com. #Department of Inorganic Chemistry, University of Madras, Chennai , India.

2 58 INDIAN JOURNAL OF ANIMAL RESEARCH Since the currently available oral hypoglycaemic drugs lack desired properties of an ideal drug, researches are aimed at to find out effective, safe and less expensive drugs.vanadium complexes have been demonstrated to exert various insulin-mimetic, anti-diabetic effects, improved lipid and protein metabolism and antioxidant properties without apparent signs of liver and kidney toxicity. The present study was undertaken to evaluate the biochemical effect of Vanadium complex, on enzymatic and non-enzymatic antioxidant levels in normal, streptozotocin (STZ) induced diabetic, Glimepiride and Vanadium complex treated diabetic rats (STZ induced). MATERIALS AND METHODS All chemicals used were of analytical grade. Streptozotocin (STZ) was purchased from Sigma Chemicals Co., St. Louis, USA. Glimepiride was obtained as gratis from Orchid Pharmaceuticals Limited, Chennai. Chemicals for antioxidant assay were purchased from M/s Merck Chemicals, Mumbai, India. Vanadium complex (9E)-N1-(2-((E) 2-(2- hydroxy-5-methylbenzylidene amino) ethylamino)ethyl)-n2- (2-hydroxy-5-methyl benzylidene) ethane-1,2-diamine VO(IV) was obtained as gratis from Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 25. Experimental Animals: Female albino Wistar rats, weighing about grams were obtained from Laboratory Animal Medicine Unit, Tamil Nadu Veterinary and Animal Sciences University, Chennai - 51, India. They were randomly divided into seven groups, each consisting of eight animals. Animals were identified by marking different parts of the body. The mean body weight variation was not exceeding ± 20%. The animals were maintained on standard rat feed supplied by M/s. Tetragon Chemie Private Limited, Bangalore, India. All animals were housed in cages with 12/12 hours light/dark cycle. The animals were fed ad libitum feed and water throughout the experimental period. The animals were acclimatized for one week prior to the start of the experiment. The animal experiments were carried out after prior approval of Institutional Animal Ethical Committee (IAEC), MVC, Chennai - 7. In the present study before administration of STZ injection, the blood glucose level was assessed to rule out spontaneous diabetes in the rats. Those animals, showing normal blood glucose levels of mg/dl, were selected for the study. Induction of diabetes mellitus: The animals were fasted overnight and a single intraperitoneal injection of a freshly prepared solution of STZ (45 mg/kg b.wt) in 0.1 M cold citrate buffer (ph 4.5) was given to induce diabetes. The animals were allowed to access to 5 per cent glucose solution overnight, to prevent total hypoglycaemia, induced by STZ by massive pancreatic insulin release. The animals were considered as diabetic, if their blood glucose values were above 250 mg/dl on the third day (72 hours) after STZ injection. The treatment was started on the fourth day after STZ injection and the day was considered as first day of treatment. The treatment was continued for 28 days. Experimental design: Group I served as normal control. Groups II and III were normal control treated with vanadium complex at the dose rate of 5 mg/kg b.wt and 10 mg/kg b.wt respectively for toxicity study. Group IV served as diabetic control. Groups V and VI were streptozotocin induced diabetic rats treated with vanadium complex at the dose rate of 5 mg/kg b.wt and 10 mg/kgb.wt respectively. Group VII was treated with standard drug Glimepiride at the dose rate of 800 g/kg b.wt for comparison. Groups Treatments Animals Group I Normal control 8 Group II Normal control + vanadium complex p/o@ 5 mg/kg b.wt 8 Group III Normal control + vanadium complex p/o@ 10 mg/kg b.wt 8 Group IV Diabetic control - Streptozotocin IP injection@ 45 mg/kg b.wt 8 Group V Diabetic control + vanadium complex p/o@ 5 mg/kg b.wt 8 Group VI Diabetic control + vanadium complex p/o@ 10 mg/kg b.wt 8 Group VII Diabetic control + standard hypoglycemic drug (Glimepiride 800 mg/kg b.wt p/o) 8 Total 56 Antioxidant assay: At the end of the twenty-eighth day after the start of the treatment, the animals were sacrificed by cervical dislocation and liver, kidney and pancreas were isolated from normal and experimental rats (i.e. from all the above groups) for assay of tissue antioxidants. The following assays were performed to evaluate the tissue antioxidant status in the control, diabetic and treated rats: I. Enzymatic antioxidants: 1. Activity of catalase (E.C ): Catalase was assayed according to the method of Sinha, (1972).The enzyme activity is expressed as µmoles of H 2 decomposed/minute/mg protein. 2. Activity of superoxide dismutase (SOD) (E.C ): Superoxide dismutase activity was assayed by the method of Misra and Fridovich, (1972).The enzyme activity is expressed as 50 per cent inhibition of epinephrine auto-oxidation/ minute/mg protein in tissue.

3 Volume 50 Issue 1 (2016) Activity of glutathione peroxidase (GPx) (E.C ): The activity of glutathione peroxidase was assayed by the method of Rotruck et al. (1972). Glutathione peroxidase activity is expressed as nmoles of glutathione oxidized/ minute/mg protein. II. Non enzymatic antioxidant Reduced glutathione (GSH): Reduced glutathione was assayed according to the method of Moron et al. (1979). The amount of glutathione in tissues is expressed as nmoles / mg of protein. III. Lipid peroxidation (LPO): Assay of lipid peroxidation was done by the method of Yagi et al., The level of lipid peroxides is expressed as nmoles of MDA released/mg of protein. Estimation of tissue protein: The protein contents of the tissues were assayed by the method of Lowry et al. (1951). Statistical analysis: The results were expressed as mean ± S.E. All the data were analyzed by one way analysis of variance followed by Duncan s test multiple comparison test (Snedecor and Cochran, 1994). A value of p< 0.05 was considered statistically significant. RESULTS AND DISCUSSION Two predetermined doses of vanadium i.e., 5mg and 10mg (Groups II and III) respectively were included in the experimental protocol as drug control, so as to find out the adverse effects, toxic effects, behavioural effects and other biochemical effects, if any. Incidentally, vanadium alone at these two doses did not have any impairment on any of the parameter under study and behaved in a similar manner as that of non-diabetic control group (group I). Hence, the control group (Group-I) alone was taken for comparison with the other experimental groups (Groups IV, V, VI and VII). The effect of Vanadium complex (at both the doses) and Glimepiride on the levels of endogenous antioxidants and TBARS are furnished below: Enzymatic antioxidants Catalase: The catalase activity in liver, kidney and pancreas increased significantly in diabetic rats. The values are presented in table 1.The values were reversed to almost normal levels in vanadium complex treated (at both the doses) and Glimepiride treated groups and there was no significant difference among the treatment and normal control groups. However they differed significantly from the diabetic control group. Catalase catalyses the reduction of hydrogen peroxides and protects tissues from highly reactive hydroxyl radicals. The increased activity of catalase may be a response to the increased production of H 2 and by the auto oxidation of glucose and non-enzymatic glycation (Yanardag et al., 2009). The increase in CAT activity in liver, kidney and pancreatic tissues in the present study is in accordance with the above report. This reflects a condition of higher oxidative stress and a consequent increase in endogenous hydrogen peroxide. Kurt et al. (2011) have found an increase in tissue catalase activity in diabetic rats due to the production of ROS which demands more antioxidant defense systems leading to the increased expression of antioxidant enzymes. Oral administration of vanadium complex to diabetic rats, reverse the elevated CAT activity. These findings are in accordance with the reports of Kurt et al. (2011) and Yanardag et al. (2009). On the contrary, Ravi et al. (2004) have found a decrease in catalase activity in diabetic rats. Superoxide dismutase: SOD activity in liver, kidney and pancreas decreased significantly in diabetic rats. The values are presented in Table 1. The values were reversed to normal in vanadium complex treated (at both the doses) and Glimepiride treated groups in liver and there was no significant difference between the treatment and normal control groups. However they differed significantly from the diabetic control group. In kidneys, the values were reversed to normal in vanadium complex treated (at both the doses) rats, whereas the Glimepiride treated group showed no significant difference between the vanadium complex treatment groups, normal control group as well as the diabetic control group. In pancreas, the values were reversed to normal in 10mg/kg b.wt vanadium complex treated group and the Glimepiride treated group, while the 5mg/kg b.wt vanadium complex treated group significantly differed from all the other groups except the diabetic control group. SOD scavenges the superoxide radical by converting it into H 2 and molecular oxygen. It is known that the SOD activity is low in diabetes mellitus (Damasceno et al., 2004 and Rajasekaran et al., 2005). This is in agreement with the findings of this study. In the present study, administration of vanadium complex increased the activity of SOD that may help to control free radicals in diabetic rats. This may be in part due to normalizing the blood glucose value. The findings are similar to Ramachandran et al. (2004) and Yanardag et al. (2009) who also reported an increased tissue SOD activity in vanadium treated diabetic rats. Glutathione peroxidase: GPx activity in liver, kidney and pancreas decreased significantly in diabetic rats.the values are presented in Table 1. In liver, the values were reversed to normal in vanadium complex treated (at both the doses) rats and the Glimepiride treated group; however the 10mg/ kg

4 60 INDIAN JOURNAL OF ANIMAL RESEARCH b.wt. vanadium treated group significantly differed from the other two treatment groups. The values were reversed to almost normal in vanadium complex treated (at both the doses) and Glimepiride treated groups in kidneys and there was no significant difference between the treatment and normal control groups. However they differed significantly from the diabetic control group. In pancreas, the values were reversed to normal in 10mg/ kg b.wt vanadium complex treated group and the Glimepiride treated group, while the 5mg/ kg b.wt vanadium complex treated group significantly differed from the other treatment groups and showed no significant difference with respect to the diabetic control and normal control groups. The reduced activity may be attributed to the unavailability of reduced GSH. Administration of vanadium increased the content of reduced GSH in the tissues of diabetic rats, which in turn would have increased the GPx activity. These observations are similar to the findings of Ramachandran et al. (2004) and Yanardag et al. (2009) who had reported that the reduced GPx activity in the tissues of diabetic rats, increased after the administration of vanadium. Non enzymatic antioxidant assay - reduced glutathione (GSH): The liver, kidney and pancreas GSH was significantly decreased in diabetic rats as compared to normal control Table 2. Administration of vanadium complex at both the doses and Glimepiride significantly increased the GSH level compared to the diabetic control group in all the three tissues. Similar findings have been reported by Koyutruk et al. (2005) in STZ-induced diabetic rats. Chronic hyperglycemia induced toxicity may also decrease the level of GSH in tissues and plasma (Rajasekaran et al., 2005). Decreased activity of glucose-6-phosphate dehydrogenase in diabetes results in reduced availability of NADPH and hence decreased level of GSH in tissue. Administration of MBOV complex increases the activity of glucose-6-phosphate dehydrogenase in diabetic rats which in turn enhances NADPH levels (Nanda et al.,1995). GSH is replenished by the administration of vanadium complex, which may in turn maintain the antioxidant status in the tissues (Ramachandran et al., 2004 and Yanardag et al., 2009). Lipid peroxidation: The amount of TBARS in liver, kidney and pancreas increased significantly in diabetic rats as compared to normal control Table 2. Administration of Vanadium complex at both the doses and Glimepiride significantly decreased the TBARS level compared to the diabetic control group in all the three tissues. Increased level of LPO observed in the present study in the diabetic control may be due to hyperglycemia followed by oxidative stress as suggested by Koyutruk et al.,(2005), Wolff and Dean (1987), Das et al.,(2000) and Klepac et al., (2005). Vanadyl ion may act as a scavenger of oxyradicals and prevent liver (Koyutruk et al., 2005) and heart dysfunctions (Matsubara et al., 1995). Sutradhar et al. (2011) found that administration of malonato complexes of oxidovanadium (IV) to STZ-induced diabetic rats increased the antioxidant status and lowered the MDA level in liver of vanadium treated diabetic rats. Decreased LPO level found in the present study in vanadium treated diabetic rats shows the protective effect of vanadium in diabetic groups by scavenging the oxyradicals produced due to oxidative stress. TABLE 1: Effect of vanadium complexon enzymatic antioxidants in liver, kidneys and pancreas of normal, control and experimental rats (n=8, Mean ± S.E.) Group / Treatment Group-I Normal control Group-II Normal control + 5mg of Vanadium Group-III Normal control + 10mg of Vanadium Group-IV Diabetic control Group-V Diabetic control + 5 mg of Vanadium Group-VI Diabetic control + 10mg of Vanadium Group-VII Diabetic control + 800µg Glimepiride Liver Kidney Pancreas CAT SOD GPx CAT SOD GPx CAT SOD GPx a 6.29 a 7.11 bc a b 9.11 b 4.22 a b 6.35 bc ± 7.17 ± 1.42 ± 0.14 ± 5.09 ± 2.11 ± 1.78 ± 0.84 ± 2.44 ± a 4.44 a 7.43 bc a b 8.44 ab 4.33 a b 6.76 bc ± 5.48 ± 1.23 ± 0.60 ± 6.29 ± 2.93 ± 0.79 ± 0.71 ± 2.14 ± a 6.79 a 7.11 bc a 8.77 ab b 7.29 a b 7.13 bc ± 5.60 ± 1.08 ± 0.62 ± 7.91 ± 2.64 ± 3.72 ± 0.76 ± 2.98 ± b 1.43 a b 4.50 a 2.69 a b 2.23 a 3.02 a b ± 8.19 ± 1.09 ± 0.27 ± ± 0.10 ± 0.65 ± 7.62 ± 0.88 ± a 6.44 a 5.63 b a b b a 6.65 a 4.33 ab ± 0.25 ± 1.05 ± 0.41 ± 9.49 ± 0.53 ± 1.41 ± 0.71 ± 1.53 ± a 8.15 a 8.74 c a b b 8.97 a b 8.99 c ± 4.85 ± 0.22 ± 1.74 ± 6.28 ± 0.65 ± 1.12 ± 1.34 ± 2.52 ± a 7.42 a 6.49 b a 8.52 ab 8.71 b 6.03 a b 7.08 bc ± 5.48 ± 0.82 ± 0.31 ± 5.32 ± 1.19 ± 1.08 ± 1.67 ± 5.72 ± 1.93 Units: Catalase (CAT) activity was expressed as µmoles of H 2 decomposed/minute/mg of protein. Superoxide dismutase (SOD) activity was expressed as 50% inhibition of epinephrine autooxidation / minute/ mg of protein. Glutathione peroxidase (GPx) was expressed as nmoles of glutathione (GSH) oxidized/minute/mg of protein.

5 Group / Treatment Group-I Normal control Group-II Normal control + 5mg of Vanadium Group-III Normal control + 10mg of Vanadium Group-IV Diabetic control Group-V Diabetic control + 5 mg of Vanadium Group-VI Diabetic control + 10mg of Vanadium Group-VII Diabetic control + 800µg Glimepiride Volume 50 Issue 1 (2016) 61 TABLE 2: Effect of vanadium complex on reduced glutathione and lipid peroxidation levels in liver, kidneys and pancreas of normal, control and experimental rats (n=8, Mean ± S.E.) Units: Reduced glutathione (GSH) was expressed as nmoles /mg of protein. Lipid peroxidation (LPO) was expressed as nmoles of MDA released / mg of protein. Liver Kidney Pancreas GSH LPO GSH LPO GSH LPO c 0.18 a c 0.18 a 9.68 c 0.08 a ± 7.75 ± 0.03 ± 4.38 ± 0.03 ± 1.97 ± c 0.19 a c 0.14 a 7.84 ab 0.09 a ± 7.44 ± 0.03 ± 1.78 ± 0.02 ± 4.23 ± c 0.15 a c 0.10 a c 0.06 a ± 1.70 ± 0.02 ± 5.60 ± 0.02 ± 1.77 ± a 0.46 b 5.15 a 0.24 b 1.99 a 0.47 b ± 1.38 ± 0.05 ± 2.64 ± 0.05 ± 1.62 ± b 0.24 a b 0.12 a 4.65 b 0.15 a ± 1.79 ± 0.01 ± 0.70 ± 0.02 ± 0.00 ± b 0.28 a b 0.16 a 5.55 b 0.21 a ± 2.88 ± 0.06 ± 2.47 ± 0.01 ± 1.30 ± b 0.26 a b 0.14 a 5.44 b 0.21 a ± 2.53 ± 0.07 ± 4.97 ± 0.02 ± 0.42 ± 0.03 In general, for all the parameters under study, the reversal of alterations back to normalcy, by the action of Vanadium complex / commercial Glimepiride was maximal in liver, followed by kidneys and at minimal in the pancreas; this can be attributed to the selective sensitivity of STZ on the target tissues as well as the regenerative capacity of these tissues for the oxidative stress caused by STZ-induced diabetes. In conclusion, it can be noted that Vanadium c o m p l e x ( 9 E ) - N 1 - ( 2 - ( ( E ) 2 - ( 2 - h yd r o x y methylbenzylidene amino) ethylamino)ethyl)-n2-(2- hydroxy-5-methyl benzylidene) ethane-1, 2-diamine VO(IV) used in this study, can serve as a potent candidate in the treatment and control of diabetes; however, further work is required to find out the optimum dosage to optimize the glycemic control. REFERENCES Baynes, J.W. and Thorpe, S.R. (1991). Role of oxidative stress in diabetic complications: A new perspective on an old paradigm. Diab.,48:1-9. Chehade, J.M. and Mooradian, A.D. (2000).A Rational Approach to Drug Therapy of Type 2 Diabetes Mellitus, Disease Management.Drugs,60: Damasceno, D.C., Volpato, G.T., Calderon, I.M.P., Aguilar, R. and Rudge, M.V.C.(2004). Effect of Bauhinia forficataextract in diabetic pregnant rats: maternal repercussions. Phytomedicine,11: Das, S., Vasisht,S., Snehalata, M., Das, N. and Srivastava, M. (2000). Correlation between total antioxidant status and lipid peroxidation in hypercholesterolemia. Curr. Sci.,78: 486. Hunt, J.V., Smith, C. C. and Wolff, S. P. (1990). Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose.diab.,39: Klepac, N., Rudes, Z. and Klepac,R.(2005). Effects of melatonin on plasma oxidative stress in rats with streptozotocin induced diabetes. Biomed.Pharmacother.,60: Koyuturk, M., SevimTunali, Sehnaz Bolkent and RefiyeYanardag.(2005). Effects of VanadylSulfate on Liver of Streptozotocin- Induced Diabetic Rats. Biol. Trace Elem. Res.,104: Kurt, O., Tugba Yilmaz Ozden, Nurten Ozsoy, SevimTunali, Ayse Can, Nuriye Akev and RefiyeYanardag.(2011). Influence of vanadium supplementation on oxidative stress factors in the muscle of STZ-diabetic rats.biometals,24: Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem.,193: Matsubara, T., Marcu, S.M., Misra, P.H. and Dhalla, N.S.(1995). Protective effect of vanadate on oxyradical-induced changes in isolated perfused heart.mol. Cell Biochem.,153: Misra, H.P. and Fridovich, I.(1972).The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase.j. Biol. Chem.,247:

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