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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Protective effect of chemically modified SOD on lipid peroxidation and antioxidant status in diabetic rats

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Banu Mansuro˘glu a,∗ , Serap Derman b , Aylin Yaba c , Kadriye Kızılbey d Department of Molecular Biology and Genetics, Faculty of Science and Letters, Yildiz Technical University, 34220 Esenler, I˙ stanbul, Turkey Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University, 34220 Esenler, I˙ stanbul, Turkey c Department of Histology and Embryology, Faculty of Medicine, I˙ stanbul Bilim University, 34394 I˙ stanbul, Turkey d Science and Technology Application and Research Center, Yildiz Technical University, Bes¸iktas¸, I˙ stanbul, Turkey a

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Article history: Received 26 February 2014 Received in revised form 14 July 2014 Accepted 15 July 2014 Available online xxx

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Keywords: Superoxide dismutase Chemical modification Lipid peroxidation Antioxidant status

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1. Introduction

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Reactive oxygen species mediated oxidative stress play an important role on the injury of tissue damage and increased attention has been focused on the role of free radicals in diabetes mellitus (DM). In the present study firstly superoxide dismutase (SOD) enzyme was chemically modified with two different polymer and physicochemical properties of these conjugates clearly analyzed. Then, the stability of carboxymethylcellulose–SOD (CMC–SOD) and poly methyl vinyl ether-co-maleic anhydride–SOD (PMVE/MA–SOD) conjugates was investigated against temperature and externally added H2 O2 . Moreover, we investigated the effect of chemically modified SOD enzyme on lipid peroxidation and antioxidant status in streptozotocin (STZ)-induced diabetic rats. PMVE/MA–SOD conjugate treatment significantly reduced MDA level compared with the control groups, native and CMC–SOD conjugate treated groups in brain, kidney and liver tissue. GSH and SOD enzyme activity in diabetic groups was significantly increased by treatment of CMC–SOD and PMVE/MA–SOD conjugates. The protective effects on degenerative changes in diabetic rats were also further confirmed by histopathological examination. This study provides the preventative activity of SOD–polymer conjugates against complication of oxidative stress in experimentally induced diabetic rats. These results suggest that chemically modified SOD is effective on the oxidative stress-associated disease and offer a therapeutic advantage in clinical use. © 2014 Elsevier B.V. All rights reserved.

Diabetes mellitus (DM) is defined as a chronic metabolic disorder. DM affects more than 346 million people worldwide and these numbers are expected to be double by 2030 [1,2]. Several studies have shown that generation of reactive oxygen species and free radicals increase and/or decrease antioxidant defense potential in patients with diabetes mellitus [2,3]. Studies have displayed that DM is related to oxidative stress, leading to an increased production of reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide radicals (O2 •− ), and hydroxyl radical (• OH) or reduction of antioxidant defense system [2,4]. The balance between radical generating and radical scavenging systems is extremely important in DM [5]. SOD, Cu,Zn type [EC 1.15.1.1] catalyzes the dismutation of highly reactive superoxide anion radical (O2 •− ), to molecular oxygen

∗ Corresponding author. Tel.: +90 212 383 44 66; fax: +90 212 383 43 64. E-mail address: [email protected] (B. Mansuro˘glu).

(O2 ) and hydrogen peroxide (H2 O2 ), which are then converted to molecular oxygen and water by catalase [6,7]. SOD has long been recognized that high levels of excessive reactive oxygen species play an important role in cancer [8], diabetes and inflammatory diseases, various cardiovascular diseases [8,9]. SOD is essential enzyme eliminating superoxide radicals and hence is helping to protect cells against the toxic byproducts of aerobics metabolism. However, SOD has short plasma half-life in vivo (less than 5 min) currently limits clinical applications of this enzyme [10–12]. In addition, SOD is rapidly inactivated by its own reactive products H2 O2 , yielding highly reactive oxidant species [10,13]. Various strategies have been developed to increase the intravascular half-life of SOD [6,8,10–12,14–18]. Due to its poor pharmacokinetic profile [11,12,19], and short half-life in biological system [20] new controlled delivery strategies are investigated. Chemical transformations of its protein surface with synthetic water soluble polymers have been reported as a useful approach for improving the pharmacological and pharmacokinetic properties of SOD [18]. Various natural and synthetic polymers have been used as agents for chemically modifying proteins [21] and antioxidant enzymes

http://dx.doi.org/10.1016/j.ijbiomac.2014.07.039 0141-8130/© 2014 Elsevier B.V. All rights reserved.

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[22–25]. Several Cu,Zn–SOD conjugates were developed including poly(vinlypyrrolidone) (PVP) [11], polyethylene glycol (PEG) [10,17,26–28], carboxymethylcellulose (CMC) [16], CM-dextran [10,29], DIVEMA (divinly ether and maleic anhyride) [11,20], poly[N-(2hydroxypropyl)methacrylamide) (PHPMA) [11,30,31] in literature. However, the effect of chemically modification of SOD with poly methyl vinyl ether-co-maleic anhydride (PMVE/MA) and CMC polymers has not been reported against streptozotocin-induced diabetic rats. PMVE/MA has a great potential in biomedical application due to its biodegradable and biocompatible properties [32]. Similarly, CMC is a natural biodegradable and biocompatible anionic polymer [33] and has several potential applications in biomedical science [34]. To our knowledge, there is no study published about chemical modification of SOD with PMVE/MA polymer and the role of chemically modified SOD with CMC and PMVE/MA on oxidative stress in DM. Hence, the primary aim of the present study is to investigate the usefulness of the CMC–SOD and PMVE/MA–SOD conjugates for the first time as a protein modifier for improving the functional stability of SOD enzyme. Secondarily, it is aimed to investigate the protective effect of CMC–SOD and PMVE/MA–SOD conjugates on lipid peroxidation and antioxidant status in different tissue on STZ-induced diabetic rats using both biochemical and histological methods.

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2. Materials and methods

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2.1. Chemicals

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Carboxymethylcellulose (CMC) sodium salt with 90 kDa Mw was purchased from Sigma–Aldrich. Poly methyl vinyl ether-comaleic anhydride (PMVE/MA) with 216 kDa Mw was obtained from Sigma–Aldrich. SOD from bovine erythrocytes (3231 U/mg) was purchased from Biochemika (Fluka BioChemika, Switzerland). 1Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), activator of carboxylate groups of CMC and PMVE/MA, was obtained from the Sigma Chemical, St. Louis, MO. In addition to Xanthine sodium salt, xanthine oxidase from bovine milk, sodium carbonate, nitrotetrazolium blue, bovine serum albumin and ethylenediaminetetraacetic acid sodium salt purchased from the Sigma Chemical, St. Louis, MO. NaH2 PO4 , Na2 HPO4 ·7H2 O, NaCl, NaOH were obtained from Fluka and NaN3 was from Applichem (Applichem GmbH, Darmstadt, Germany). Ultra-pure water, which used in preparation of the solutions and chromatographic analysis, was obtained from Millipore MilliQ Gradient system.

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2.2. Chemically modification of SOD enzyme

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SOD enzyme was chemically modified with CMC and PMVE/MA polymers by covalent binding procedure which involves watersoluble carbodiimide [35,36]. CMC and PMVE/MA were dissolved in water, stirred at 4 ◦ C and pH values of mixture adjusted to pH 5 with NaOH. SOD enzyme was dissolved in water, stirred at 4 ◦ C. SOD enzyme solution was added into CMC and PMVE/MA polymer solutions at pH 5 and mixture stirred at 4 ◦ C. After 1 h, carboxyl groups of CMC and PMVE/MA polymers were activated with EDC. Dry EDC was added to mixture and stirred at 4 ◦ C. Then, pH values of mixtures were adjusted to pH 7.0 by NaOH. The conjugate was separated from the unreacted polymer, low molecular weight compounds (SOD and O-acylisourea intermediate) by dialyzes using a dialysis membrane with the cut-off molecular weight of 100 000 Da (Spectrum medical Industries Inc., Los Angeles, CA). After 2 days dialysis at 4 ◦ C, the sample was lyophilized. The lyophilized

conjugates were dissolved in 50 mM phosphate-buffered saline (PBS) at 4 ◦ C for the chromatographic analysis.

2.3. Characterization of chemically modified SOD–polymer conjugates 2.3.1. Size exclusion chromatography (SEC) with a triple detection system SOD, CMC, PMVE/MA, CMC–SOD and, PMVE/MA–SOD conjugates were analyzed using size exclusion chromatography (SEC) with a triple detection system. Viscotek TDA 302 detector system with refractive index (RI), right angle light scattering (RALS) detectors was used for on-line SEC signal detection. A separate UV detector obtained from Viscotek was connected to this detector system. OmniSEC4.1 software program was used for the acquisition and analysis of SEC data and calculation of molecular weight of CMC and PMVE/MA conjugates in the solution. Viscotek triple detector arrays were calibrated with BSA monomer peak in a mobile phase of PBS at 1.0 ml/min flow rate. 0.185 were used as dn/dc value and extinction coefficient of BSA, respectively [37]. All size exclusion chromatography experiments were repeated three times, and chromatograms are depicted using the mean values of replicate analyses. A Shim-Pack Diol 300 column (50 cm × 0.79 cm) was used for separation at room temperature. PBS (pH 7.1) was used for the mobile phase and flow rate was 1.0 ml/min.

2.4. Determination of protein content and SOD activity in conjugates Native SOD activity and SOD activity in the conjugates (CMC–SOD and PMVE/MA–SOD) were measured by the cytochrome c reduction assay using xanthine and xanthine oxidase as an O2 • generation system as described by McCord JM and Fridovich I [38]. Protein concentration in the CMC–SOD and PMVE/MA–SOD conjugates was estimated as described in the study of Lowry et al. [39] using bovine serum albumin as standard. The conjugation reaction yield was estimated using both protein content analysis and GPC analysis results. Maximum conjugation yield was obtained in CMC–SOD2 (55%) and PMVE/MA–SOD1 (75%) according to our results, these conjugates (CMC–SOD2 and PMVE/MA–SOD1) were used in-vitro animal studies.

2.5. Evaluation of thermostability of SOD and its conjugates The stability of the native and chemically modified SOD (5 ␮g/ml of native SOD) at different temperatures between 70 and 95 ◦ C was estimated by measuring the residual activities after the treatments. The thermostability of SOD was tested by treating the enzyme at 40 ◦ C, 50 ◦ C, 75 ◦ C, 85 ◦ C, and 95 ◦ C for 5 min in 0.15 M NaCl. After incubation, the samples were cooled and the residual SOD activities were measured by the method as described above.

2.6. Stability against externally added hydrogen peroxide The SOD inhibitor H2 O2 was used to determine the effect of native and SOD–polymer conjugates (5 ␮g/ml of native SOD) that were incubated with 0.4 mM H2 O2 in 25 mM sodium phosphate buffer (pH 7.0) at 37 ◦ C for 3 h. Aliquots were removed after 3 h, cooled in a cold bath and tested for residual SOD activity. The enzyme was treated with distilled water in the same way used as the control.

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2.7. Animals

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Sprague Dawley rats (12 weeks old; 350–380 g) were obtained from Animal Laboratory of Bezmialem Vakif University. They were housed in clean, sterile, polypropylene cages, under controlled photoperiod (12 h light/dark cycle) and humidity (55–60%). The animals were fed with standard rat pellet chow and tap water ad libitum. All animals were acclimatized for seven days before the study. All of the experimental protocols were performed according to the guidelines for the ethical treatment of experimental animals. The study was approved by the appropriate Animals Ethical committee of Bezmialem Vakif University.

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2.8. Experimental design

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The rats were randomly divided into 5 groups with six animals in each group. Experimental diabetes (Groups II, III, IV and V) was induced in 12 h fasted rats by single i.p. injection of streptozotocin (STZ, 60 mg/kg b.w.) dissolved in cold citrate buffer (0.1 M, pH 4.5). STZ-injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemia. The normal control group (Group I) received only the serum physiological. STZ-induced animals exhibited hyperglycemia within a few days. Glucose measurements were carried out using Gluco-check Active (Roche Diagnostics, Germany), after injection with STZ in 72 h. Animals with blood glucose levels above 350 mg/dl were selected for the experiment. Group I, non-diabetic (Normal Control) rats; Group II, Diabetic Control rats; Group III, native SOD treated diabetic rats; Group IV, CMC–SOD conjugate treated diabetic rats; Group V, SOD-PMVE/MA conjugate treated diabetic rats.

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Both native SOD and SOD–polymer conjugates administered intraperitoneally (i.p.) two times with a dose of 5560 U/rat and 2280 U/rat with one week intervals. After 4 weeks SOD–CMC and SOD-PMVE/MA conjugates treated rats were anaesthetized with an intramuscular injection of 50 mg kg−1 ketamine hydrochloride [40] (Ketalar, Eczacibasi, I˙ stanbul, Turkey). All animals were sacrificed under anesthesia and their brain, kidney and liver were carefully removed. The specimens were stored at −20 C until biochemical assays. Brain, liver, and kidney tissues were dissected and fixed in 10% buffered formalin for morphological evaluation.

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2.9. Lipid peroxidation measurement

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For biochemical evaluations, brain, kidney and liver biopsy samples were washed in cold (+4 ◦ C), 1.15% potassium chloride (KCl) and dried with blotting paper. The tissues were homogenized in KCl at 1600 rpm for 2 min. Lipid peroxidation (LP) in terms of thiobarbituric acid reactive substance (TBARS) formation was determined by the method of Esterbauer and Cheeseman [41]. Tissue homogenates were mixed with 2 volumes of cold 10% trichloroacetic acid and 2 volumes of cold 10% thiobarbituric acid then incubated for 1 h at 100 ◦ C. After cooling, the precipitate was removed by centrifugation. The absorbance of reaction mixtures was measured at 535 nm using a blank containing all the reagents except the tissue homogenates. Results were expressed as nmol MDA/g tissue.

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2.10. Antioxidant defenses

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2.10.1. Determination of reduced glutathione (GSH) activity For biochemical evaluations, brain, kidney and liver biopsy samples were washed in 1 M cold (+4 ◦ C) PBS and dried with blotting paper. The tissues were homogenized in metaphosphoric acid

Fig. 1. GPC chromatograms of native SOD (5), PMVE/MA (4), and PMVE/MA–SOD conjugates has molecular weight ratio of polymer/SOD 16 (3), 24 (2), 32 (1) obtained from RI (A), UV (B) and RALS (C) detectors.

(0.5 M) at 1600 rpm for 2 min. The homogenate was centrifuged at 3500 rpm for 10 min at +4 ◦ C. Reduced glutathione (GSH) tissue content (supernatant) was determined after reaction with 5,5 dithiobis-(2-nitrobenzoicacid) and the absorbance was measured at 412 nm [42]. A standard UV curve prepared with cysteine was

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used to calculate the content of GSH in tissue samples, expressed as mg GSH/g tissue.

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2.10.2. Determination of SOD activity For biochemical evaluations, brain, kidney and liver biopsy samples were washed in 1 M cold (+4 ◦ C) phosphate buffer saline (PBS) and dried with blotting paper. The tissues were homogenized in phosphate buffer saline (0.15 M) at 1600 rpm for 2 min. The homogenate was centrifuged at 7000 rpm for 30 min at +4 ◦ C. SOD activity was spectrophotometrically quantified in tissues using the method as described below [38]. SOD activity was also expressed as units per milligram protein.

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2.11. Morphological and histological investigation

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Tissues of brain, liver, and kidney were collected, and immediately fixed with 10% buffered formalin. After deparaffinization in xylene and rehydration in a graded series of ethanols and finally cleared with xylene. The samples were then embedded in paraffin. Paraffin-embedded samples were cut into 5-␮m sections and deparaffinized in xylene and rehydrated in a decreasing ethanol series. Afterwards sections were stained with hematoxylin and eosin (DAKO Corporation, Carpinteria, CA, USA) for histological analysis and they were evaluated by light microscopy and photographs were taken.

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Data were expressed as mean ± standard deviation. All statistical analyses were performed using SPSS 15.0 [43]. Non parametric analysis with Mann–Whitney U-test was carried out on the data of the biochemical variables to examine differences between the groups. p value less than 0.05 (p < 0.05) was accepted as significant.

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3. Results and discussion

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The present study was designed to compare the effect of native and chemically modified SOD with polymer on lipid peroxidation and antioxidant status in STZ-induced diabetic rats. 3.1. Characterization of SOD–CMC and SOD-PMVE/MA conjugates with size exclusion chromatography A carbodiimide conjugation procedure was followed to obtain SOD–polymer conjugates as described previously. SOD-PMVE/MA or SOD–CMC were then conjugated at the different weight ratios of polymer/SOD16, 24, 32 using water-soluble EDC as a zero-length cross-linker. In the first hand, all conjugates were characterized with SEC in order to identify if the conjugation occurred successfully. Detailed information about physicochemical properties of SOD–polymer conjugates were obtained from SEC, RI and RALS detectors that are sensitive to concentration and molar mass, respectively, whereas UV absorbance detector is sensitive to protein content of samples [44]. SEC chromatograms of free SOD and SOD–polymer conjugates with PMVE/MA (Fig. 1) and CMC (Fig. 2) shown where SOD concentrations were equal to 2.5 mg/ml in all solutions. In Fig. 1, SOD-PMVE/MA conjugates (1, 2 or 3) and free SOD in system (5) were labeled at the SEC chromatograms. As it shown in Fig. 1, all SOD chromatograms obtained from different detectors, the peak eluting at 18.27 ml was attributed to SOD. When conjugation reaction occurring, there was a new peak emerged (1, 2 or 3) that were eluted between 10 and 17.5 ml and detected by UV detector (Fig. 1B). This indicates that conjugation between carboxyl group of PMVE/MA and amino group of SOD enzyme was occurred successfully.

Fig. 2. GPC chromatograms of native SOD (5), CMC (4), and CMC–SOD conjugates has molecular weight ratio of polymer/SOD 16 (3), 24 (2), 32 (1) obtained from RI (A), UV (B) and RALS (C) detectors.

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Table 1 Physicochemical parameters and enzymatic activity results of SOD–polymer conjugates.

Native SOD CMC CMC–SOD 1 CMC–SOD 2 CMC–SOD 3 PMVE/MA PMVE/MA–SOD 1 PMVE/MA–SOD 2 PMVE/MA–SOD 3

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Weight ratios of polymer/SOD

Mw (kDA)

Mn (kDa)

PDI (Mw /Mn )

Specific activity (U/mg)

– – 16 24 32 – 16 24 32

27.370 160.96 291.83 317.89 524.03 54.74 212.30 194.99 208.08

27.2 52.64 141.29 154.26 208.58 17.83 47.19 47.66 49.30

1 3.05 2.06 2.06 2.50 2.9 4.49 4.09 4.22

3231 – 2744 2615 2951 – 2660 2815 2744

In Fig. 2 the same results for CMC–SOD conjugation was obtained from SEC analysis. As a result of conjugation reaction, larger molecules (SOD–CMC conjugates) are synthesized and these molecules eluted column earlier than both free CMC and SOD molecules as seen in Fig. 2B. According to Figs. 1 and 2, the value of peak area obtained by UV, RI and RALS detectors clearly increased with the increase in molecular weight ratio of polymer/SOD (16, 24, 32). RI signal of all molecules is directly proportional to dn/dc and concentration. Polymers dn/dc values are generally related to their chemical composition. Change in RI area of conjugates can be correlated to the dn/dc value. In RALS chromatograms peak areas of conjugates increased with the increase in polymer concentration used in conjugation reaction, as seen in Figs. 1C and 2C. Light scattering signal is related to the molecular weight and concentration of the molecules and considerable increase in the peak area of RALS chromatograms represents the chemical modification of SOD with PMVE/MA or CMC. Molecular weight of the native SOD and SOD–polymer conjugates (for different ratio of polymers) were determined from molecular mass-sensitive-light scattering (RALS) detectors and exhibited in Table 1. Although weight-average molecular weights of CMC–SOD conjugates increased linearly with the increase in the weight ratio of polymer/SOD, weight-average molecular weight of PMVE/MA–SOD conjugates increased non-linearly. When evaluating the changes in chromatograms and increases in molecular weight of all components we can say that SOD and polymers (both PMVE/MA and CMC) were covalently conjugated successfully. Furthermore, we examined the effect of chemically modification of SOD on the specific activity using xanthine and xanthine oxidase methods and obtained results were given in Table 1. Concerning the specific activity results; it was observed that SOD–polymer conjugates have a less enzymatic activity than native SOD: SOD–PMVE/MA retained 87.12% of the native SOD activity, while SOD–CMCs retained 91.3% when the max. specific activity of conjugates is compared with native SOD. 3.2. Stability of SOD–CMC and SOD-PMVE/MA conjugates In this study the enzymatic stability of PMVE/MA–SOD and CMC–SOD conjugates against temperature or externally added H2 O2 were tested. The temperature stability of native and chemically modified SOD was determined from 40 to 95 ◦ C for 5 min (Fig. 3A). Both PMVE/MA–SOD and CMC–SOD conjugates showed better temperature stability than the native SOD. The stability against externally added 0.4 mM H2 O2 were shown Fig. 3B. Most of polymer–SOD conjugates became more stable against H2 O2 than native SOD; except of SOD–CMC conjugates which has molecular weight ratio 16 and 24. According to these results we can assume that PMVE/MA–SOD conjugates are more stable than SOD–CMC conjugates against externally added H2 O2 .

Table 2 Malondialdehyde (MDA) status of the brain, kidney and liver in normal control (I), diabetic control (II), native control (III) and chemically modified SOD (IV, V) treated diabetic rats. Groups

Group I Group II Group III Group IV Group V

MDA Levels (nmol/g tissue) Brain

Kidney

Liver

34.42 ± 4.43 40.47 ± 5.04 36.17 ± 4.22 31.69 ± 3.03 19.38 ± 3.29

10.95 ± 1.74 13.51 ± 1.74 10.45 ± 2.12 9.32 ± 1.44 8.20 ± 1.33

8.59 ± 1.23 10.75 ± 1.14 8.09 ± 1.30 6.29 ± 0.90 7.46 ± 0.23

I–V (0.010) II–V (0.010) III–V (0.010) IV–V (0.004)

I–V (0.016) II–IV (0.005) II–V (0.004)

II–IV (0.004) II–V (0.004)

p values

Values are mean ± SD. NS, no significant.

3.3. Effect of SOD–CMC and PMVEMA–SOD conjugates on MDA levels in brain, kidney and liver tissues As it can be seen from Table 2, the levels of MDA in brain, kidney and liver are increased in diabetic control rats (Group II) when compared with the normal control (Group I) and native SOD (Group III) or SOD conjugates (Groups IV and V) treated diabetic groups (p < 0.016). The concentration of lipid peroxidation products in the tissue, which is an indirect proof of excessive free radical production, increased in STZ-induced diabetic rats [2,45–48]. Subsequently increased MDA levels were in agreement with the results of previous studies in literature [46–48]. Thus, the increased MDA level in diabetes mellitus suggests that hyperglycemia induces the peroxidative reactions in lipids [46,48]. However, SOD-PMVE/MA conjugate treatment significantly (p < 0.01) reduced MDA level when compared with the both controls (Group I and II), native and CMC–SOD conjugate treated groups (Group III, IV) in brain tissue. Treatment with PMVE/MA–SOD conjugate has the best effect on MDA level STZ-induced diabetic rats in brain, kidney and liver when compared with diabetic control groups (Group II) (p < 0.01, p < 0.004 and p < 0.004 respectively). Therefore, we can say that the chemically modified SOD with PMVE/MA prevents the lipid peroxidation in STZ-induced diabetic rat in brain, kidney and liver. PMVE/MA–SOD conjugates could be promising candidate as a new therapeutic drug for lipid peroxidation injuries. 3.4. Effect of SOD–CMC and PMVE/MA–SOD conjugates on GSH levels in brain, kidney and liver tissues The levels of non-enzymatic antioxidants (GSH) in brain, kidney and liver were represented in Table 3. It was seen that GSH level was significantly decreased in STZ-induced diabetic rats when compared with normal control rats. Otherwise, GSH activities of chemically modified SOD treated groups were

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Fig. 3. Enzymatic stability results of native SOD and SOD polymer conjugates () against (A) temperature and (B) externally added H2 O2 .

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significantly higher than in both control groups (Groups I and II) (p < 0.004, p < 0.004) of diabetic rats. But, there is no significant effect of PMVE/MA–SOD or CMC–SOD conjugates in brain tissue. The increased GSH activity may be an adaptive response to the increased oxidative stress in STZ-induced diabetic rats [49]. On administration of SOD–polymer conjugates the MDA levels have decreased and GSH levels have increased. This indicates that in the presence of chemically modified SOD has protective effect in the development of the complication of oxidative stress.

3.5. Effect of SOD–CMC and PMVE/MA–SOD conjugates on SOD levels in brain, kidney and liver tissues Table 4 represents the activities of antioxidant enzyme (SOD) in the in STZ-induced diabetic rat in brain, kidney and liver. In diabetic control group (Group II) the brain SOD activities level significantly reduced when compared to normal control group (p < 0.004), whereas the liver SOD activities level increased. Native SOD treated diabetic rats (Group III), kidney and liver SOD activities were significantly higher than both control groups (p < 0.004,

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Fig. 4. Histopathologically examination tissue sections of kidney (A–E), liver (F–J), and brain (K–O). Group I, non-diabetic (Normal Control) rats (A, F and K); GR2, Diabetic Control rats; (B, G and L); GR3, native SOD treated diabetic rats (C, H and M); GR4, CMC–SOD conjugate treated diabetic rats (D, I and N); GR5, PMVE/MA–SOD conjugate treated diabetic rats (E, J and O), objectives A–J: 20×; K–O: 10×.

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both). Also, treatment with SOD–CMC conjugates (Group IV) has the best effect on SOD level, when compared to diabetic control groups (Group II) (p < 0.01, p < 0.004 and p < 0.004 respectively) in STZ-induced diabetic rats. SOD is an important protective enzyme that accelerates the dismutation of superoxide anion radicals to hydrogen peroxide acts as a primary defense [5]. But there is no agreement in the level of antioxidant enzyme of many organs in diabetic rats [46]. Although some studies reported the reduction on SOD enzyme activities in diabetes mellitus [50,51], some other studies showed increases in the activity of this enzyme with STZ-induced diabetic rats [52,53]. The balance between oxidant and antioxidant systems is extremely important in the pathogenesis of diabetes mellitus and in the progression of tissue injury. 3.6. Morphological and histological investigation Tissue sections of kidney, liver and brain were examined histopathologically. Examined kidney sections present normal glomerular and tubular morphology in non-diabetic (normal control) rats (Group I) (Fig. 4A). Histological examination of liver in normal control rats showed normal hepatic cells with

well-preserved cytoplasm prominent nucleus that most of the cells contain a central rounded nucleus. The blood sinusoids are organized between the cords. The sinusoidal endothelium is formed of endothelial lining cells and the phagocytic kupffer cells (Fig. 4F). Normal cortical neuronal structure and vessels were observed in non-diabetic control group rats (Fig. 4K). In microscopic examination slight, degenerative changes were observed in glomerular interstitial and renal tubular epithelial cells of the kidney in STZ-induced diabetic rats (Group II, Fig. 4B). Focal interstitial fibrosis was observed with glomerular lesions. The livers of STZ rats showed increased number of kupffer cells and moderate sinusoidal congestion and dilation (Group II, Fig. 4G). Pathological changes in vessels and neural cells were detected in brain cortex of STZ-induced diabetic rats (Group II, Fig. 4L). Histopathological observation of the liver sections of native (Group III), CMC–SOD (Group VI) and PMVE/MA–SOD (Group V) conjugate treated groups diabetic rats showed similar morphology with control group (Group I) (Fig. 4C, D and E, respectively). Treatment of diabetic rats with native SOD and SOD–polymer conjugates treatment improve the impairment of degenerative changes in STZinduced diabetic rats in kidney (C, H and M), liver (D, I and N) and brain (E, J and O) in Fig. 4.

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Table 3 Antioxidant status (GSH) of the brain, kidney and liver in normal control (I), diabetic control (II), native control (III) and chemically modified SOD (IV, V) treated diabetic rats. Groups

Group I Group II Group III Group IV Group V

GSH Levels (mgr/g tissue) Brain

Kidney

Liver

2.11 ± 0.15 2.10 ± 0.15 2.17 ± 0.18 2.72 ± 0.22 2.14 ± 0.18

1.31 ± 0.07 1.20 ± 0.06 2.02 ± 0.15 2.22 ± 0.11 1.93 ± 0.18

2.74 ± 0.10 2.42 ± 0.19 3.33 ± 0.21 4.17 ± 0.58 3.84 ± 0.19

p values NS

I–III (0.004) I–IV (0.004) I–V (0.004) II–III (0.004) II–IV (0.004) II–IV (0.004)

I–III (0.004) I–IV (0.004) I–V (0.004) II–III (0.004) II–IV (0.004) II–V (0.004)

Values are mean ± SD. NS, no significant.

Table 4 Antioxidant status (SOD) of the brain, kidney and liver in normal control (I), diabetic control (II), native control (III) and chemically modified SOD (IV, V) treated diabetic rats. Groups

Group I Group II Group III Group IV Group V

SOD Levels (U/mg protein) Brain

Kidney

Liver

63.54 ± 2.8 53.57 ± 4.43 54.57 ± 2.86 65.24 ± 3.31 42.01 ± 1.34

69.47 ± 1.02 66.32 ± 0.03 70.57 ± 0.87 61.26 ± 2.11 65.73 ± 2.01

64.30 ± 0.01 84.52 ± 0.95 103.85 ± 0.10 107.85 ± 1.23 96.18 ± 2.31

I–II (0.004) I–III (0.004) I–V (0.004) II–IV (0.004) III–IV (0.004) III–V (0.004) IV–V (0.004)

I–IV (0.004) II–III (0.004) II–IV (0.004) III–IV (0.004) III–V (0.004)

I–II (0.004) I–III (0.004) I–IV (0.004) I–V (0.004) II–III (0.004) II–IV (0.004) III–IV (0.004) III–V (0.004) IV–V (0.004)

p values

Values are mean ± SD. NS, no significant.

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4. Conclusion Diabetes mellitus in rats is a useful and successful model for observing the protective effects of investigated agents rapidly on diabetes-induced damage. It has been clearly described that the levels of lipid peroxidation and antioxidant stress increased in diabetes mellitus [46]. This is the first study in which SOD enzyme conjugated PMVE/MA polymer and physicochemical properties of these conjugates clearly characterized. In addition, for the first time SOD–CMC and SOD–PMVE/MA conjugates were used against the complication of oxidative stress in diabetes mellitus. The results have shown that chemical modification increased resistance the enzyme against to temperature and oxidative inactivation by H2 O2 . The treatment with chemically modified SOD reduced the level of lipid peroxidation and increased the antioxidant status in STZ-induced diabetic rats. Furthermore, histopathological results demonstrates that prophylactic administration of chemically modified SOD protect brain, kidney and liver tissues from oxidative stress damage. Due to its rapid degradation in biological systems, it can be concluded that chemically modified SOD treatment is more effective and more prominent than uncoupled SOD.

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Protective effect of chemically modified SOD on lipid peroxidation and antioxidant status in diabetic rats.

Reactive oxygen species mediated oxidative stress play an important role on the injury of tissue damage and increased attention has been focused on th...
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