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The potential role of combined antioxidant treatment on pancreas of STZ-diabetic mice
Experimental and Toxicologic Pathology 65 (2013) 255–262
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Review
The potential role of combined antioxidant treatment on pancreas of STZ-diabetic mice Ayse Karatug ∗ , Sehnaz Bolkent Istanbul University, Faculty of Science, Department of Biology, 34134 Vezneciler, Istanbul, Turkey
a r t i c l e
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Article history: Received 10 December 2010 Accepted 30 August 2011 Keywords: Diabetes Oxidative stress Mitochondrial and cytosolic antioxidative systems Pancreas Mice
a b s t r a c t In diabetes, cells and tissues are damaged due to the imbalance between production of free radicals and removal of them. The effective biologic antioxidants for oxidative stress such as ␣-lipoic acid, vitamin E and selenium are effective in diminishing oxidative damage such as membrane lipid peroxidation. The experiment aimed to investigate the oxidative stress occurring in mitochondrial and cytoplasmic fraction of pancreatic tissues in streptozotocin-diabetic mice and the possible effects of ␣-lipoic acid + vitamin E + selenium combination on oxidative damage and antioxidative system by using microscopic and biochemical methods. The mice were divided into five groups. These groups were treated by citrate buffer, the solvents of the antioxidants, combined the antioxidants [␣-lipoic acid (50 mg/kg), vitamin E (100 mg/kg), selenium (0.25 mg/kg)], streptozotocin (40 mg/kg × 5), combined the antioxidants and streptozotocin. The mice were sacrificed by cervical dislocation. In the experimental group given combined antioxidants following results were observed compared to diabetic group: increased percent insulin-positive cell area; decreased blood glucose levels; increased manganase superoxide dismutase activities and unsignificantly increased superoxide dismutase activities; unsignificantly decreased lipid peroxidase levels in both of fraction; unsignificantly decreased in mitochondrial fraction and unsignificantly increased in cytosolic fraction for catalase levels; not any alteration glutathione levels; not any activity in both of fraction for glutathione peroxidase. We can say that by taking the blood glucose levels and immunohistochemical results into account, the combination of triple antioxidants has a partly positive effect on diabetes. This positive effect could increase when trying different doses of combined antioxidant treatment. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Diabetes is a disease characterized with the change of carbohydrate, lipid and protein metabolism due to the decreasing insulin level (Leach, 2007; Modak et al., 2007). Diabetes is related with antioxidative defense system which is accepted to be important in the increases of reactive oxygen species (ROS) and free radical species that cause development and progress of diabetic complications (Barbosa et al., 2008; Di Leo et al., 2004; West, 2000; Yi and Maeda, 2006). Streptozotocin (STZ) causes pancreatic beta cell damage with the decrease of insulin level and evidence of hyperglycemia (Wada and Yagihashi, 2004). One of the first effect of STZ on pancreatic beta cells is the oxidative damage caused by the generation of free radicals (Seven et al., 2004; Yi and Maeda, 2006). Production of ROS in various tissues of diabetic individuals has a role in early and late period complications in diabetes (Kaneto et al.,
∗ Corresponding author. Tel.: +90 212 4555700; fax: +90 212 5280527. E-mail address: [email protected] (A. Karatug). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.08.012
1999). Production of most ROS and free radicals during physiological and pathological metabolism generates the defense mechanism against oxidative stress. However, the decay of balance between producing and scavenging free radicals due to an increased oxidative stress or decreased antioxidative defence causes cell and tissue damage in diabetes (Dincer et al., 2002). Both internal and external cell defense of endogeneous and exogeneous antioxidative molecules render inactive damage caused by oxidant molecules (Altan et al., 2006). ␣-Lipoic acid (ALA) is characterized as a powerful antioxidant due to the ability of scavenging ROS (Foster, 2007). Therefore, ALA is a biological antioxidant and a powerful scavenger of free radicals, also an essential cofactor for mitochondrial enzymes (Somani et al., 2000). STZ-damaged beta cells may partially be improved and islet cell functions may be obtained again by using ALA in rodents. It is suggested that giving exogeneous ALA to mice, beta cell is protected aganist hyperglycemia (Yi and Maeda, 2006). Vitamin E (Vit E) is one of the most effective biological antioxidants that supplies protection against damage that occurred in the end of oxidative stress in animals. Giving additional Vit E to the animal organizes cytotoxic effects of
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quickly removed for immunohistochemical and biochemical analyses.
2.2. Immunohistochemical assay
Fig. 1. Diagram of control and experimental groups.
agents producing free radicals (Slonim et al., 1983). Selenium (Se) is an important component for the glutathione peroxidase (GSHPx) enzyme and a functional antioxidant along with its feature of reducing lipid peroxidation (LPO) (Eskew et al., 1985). Se plays an important role in increasing the scavenger effects of oxygen-free radicals and thus, there is a protective effect of Se in early period of diabetes or progress of diabetic complications (Mukherjee et al., 1998). It is suggested that ALA-Vit E and Vit E-Se couples have a synergistic effect. ALA provides recycling of Vit E with increased coenzyme Q level. Vit E plays a role for preventing the formation and removal of hydroperoxide by interacting with Se-dependent systems (Hoekstra, 1975). In this study, we aimed to investigate synergistic effects of ALA + Vit E + Se combination on the alterations which occurred by increased oxidative stress and mitochondrial defense mechanism in an important chronic disease such as diabetes. 2. Materials and methods
Pancreatic tissue was fixed in 10% buffered formalin for immunohistochemical assay. After dehydration in a series of ethanol, pancreatic tissues were cleared in xylene and embedded in paraffin. Paraffin-embedded tissues were cut as 4-m sections and stuck on microscope slide coated with poly-l-lysine. Sections were kept in toluene to remove paraffin. After hydration, sections were treated with 3% hydrogen peroxide (H2 O2 ) prepared in distilled water to quench the endogeneous peroxidase activity. Histostain Plus Broad Spectrum Kit (Zymed, 85-9743) and insulin antibody (Ab-6, MS-1379-P, Neomarkers, Fremond, CA, dilution 1:400, 30 min at room temperature) were used for insulin labeling by using streptavidin–biotin–peroxidase technique. Immunoreactivity was revealed by using 3-amino-9-ethylcarbazole. Sections were counter-stained in Mayer’s haematoxylin and mounted in glycerol-vinyl alcohol. Microscopic analysis was performed by using 40× objective and 10× ocular system of Olympus CX-45 microscope. The image was transferred to computer by Olympus DP-71 imaging system and the measurements were done with Olympus Analysis Computer Programme. Thirty islets were evaluated for all groups. Insulin cell area % was calculated by measuring the evaluated pancreatic islets and labeling the insulin area.
2.3. Preparation of cellular fractions Pancreatic tissue was homogenized in cold 0.15 M KCl with a glass homogenizer for 10% homogenate. First, the homogenates were centrifuged at +4 ◦ C and 2000 × g for 10 min and the supernatants were centrifuged again at +4 ◦ C and 10,000 × g for 10 min. Supernatants were used as cytosolic fraction. Mitochondrial fractions were obtained by suspending the mitochondrial pellets with 0.15 M KCl and supernatants were stored at −86 ◦ C until analyzed and used for measuring the enzyme activities.
2.1. Animals and in vivo treatment In the study, 2–2.5-month-old Balb/c mice were provided by Institute’s Animal Care and Use Committee of Istanbul University. All mice were housed in a temperature-controlled clean room with a 12 h–12 h light–dark cycle and fed with standard chow and tap water ad libitum. The mice were selected randomly and divided into five groups. Group I: the control group given citrate buffer (n = 8), Group II: the control group given the solvents of the antioxidants (n = 8), Group III: the control group given combined the antioxidants (n = 8), Group IV: the experimental group given STZ (n = 10), and Group V: the experimental group given combined the antioxidants and STZ (n = 10) (Fig. 1). The mice were treated by intraperitoneal injections of multiple low-dose streptozotocin as five consecutive daily doses of 40 mg/kg to experimental groups. STZ was dissolved in a freshly prepared 0.01 M pH 4.5 citrate buffer. ALA (50 mg/kg), Vit E (100 mg/kg) and Se (0.25 mg/kg) were given by gavage technique for five consecutive days to the control group and before STZ injections to the experimental group. The antioxidants were dissolved in distilled water including a trace of NaOH for ALA, flower oil for Vit E and distilled water for Se. The mice were sacrificed by cervical dislocation on day 30, starting from the administration of the following: Group I, first citrate buffer, Group II, last antioxidant, Group III, solvent of the last antioxidant, Group IV, first dose of STZ, and Group V, first dose of STZ after receiving the antioxidants. After the mice were sacrificed, the pancreatic tissue was
2.4. Biochemical assays Mice fasting blood glucose levels (after 12-h period of fasting) were determined with an Accu Check Go Glucometer with a small drop of blood from tail of each mouse on the glucometer strip. Spectrophotometric methods were used in the determinations of glutathione (GSH) levels by Beutler’s method (Beutler, 1975) and of LPO by Buege and Aust method (Buege and Aust, 1978) and of the activities of the enzymes, namely, catalase (CAT) by Aebi’s method (Aebi, 1984) and GSH-Px by Paglia–Valentine method (Paglia and Valentine, 1967) for cytosolic and mitochondrial fraction of the pancreatic tissue. McCord and Fridovich method (McCord and Fridovich, 1969), modified by Flohe et al. (1989) for manganese superoxide dismutase (MnSOD) in mitochondrial fraction and the method of Sun et al. (1988) for superoxide dismutase (SOD) in cytosolic fraction were used.
2.5. Statistical analysis The immunohistochemical and biochemical results were analyzed by two-way ANOVA and Mann–Whitney U tests by using GraphPad Prism version 4.0 computer package. Results were reported as mean ± SE. P values less than 0.05 were considered to be significant.
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Table 1 Fasting blood glucose levels and body weights for all groups.
BGL (mg/dl) BW (g)
Citrate buffer (CB)
Solvents of antioxidants (AS)
Combined antioxidants (A)
Diabetes (D)
A+D
86.875 ± 5.37 25.625 ± 0.52
97.125 ± 6.90 27.563 ± 0.62
100.63 ± 4.90 29.375 ± 0.86
183.50 ± 13.29a 24.725 ± 0.62
134.00 ± 8.37b 25.938 ± 1.17
Mean ± SE. BGL: blood glucose level; BW: body weight. a P < 0.001 versus to CB group. b P < 0.01 versus to D group.
antioxidants, there was not a significant decrease compared to the diabetic groups (P > 0.05; Fig. 5B).
Fig. 2. Insulin positive cell area. a P < 0.001 versus to CB group; b P < 0.001 versus to D group. Groups that given citrate buffer (CB) and STZ (D).
3. Results 3.1. Immunohistochemical results Insulin-positive cell area in diabetic mice was significantly decreased compared to the control group given citrate buffer (P < 0.001). The decreasing of insulin-positive cell area in antioxidant-treated diabetic group was prevented by combined antioxidant treatment (P < 0.001; Figs. 2 and 3). 3.2. Biochemical results 3.2.1. Blood glucose levels and body weight Fasting blood glucose levels and body weights of mice are presented in Table 1. Fasting blood glucose levels of diabetic mice were significantly increased compared to control group given citrate buffer (P < 0.001). The increase of fasting blood glucose levels in diabetic mice were significantly decreased by combined antioxidative treatment (P < 0.01). Body weights of the diabetic mice (P < 0.001) were decreased compared to the control groups and body weights of the antioxidant-treated diabetic group were not significantly increased compared to the diabetic group (P > 0.05). 3.2.2. Oxidative stress in mitochondrial and cytosolic fractions of the pancreas GSH and LPO levels of mice in mitochondrial and cytosolic fractions of the pancreas were presented in Figs. 4A, B and 5A, B. Mitochondrial and cytosolic GSH levels for all groups were not significantly different (P > 0.05; Fig. 4A and B). But, mitochondrial LPO levels in diabetic mice were not significantly increased compared to control group given citrate buffer (P > 0.05). Mitochondrial LPO levels in diabetic group given combined the antioxidants did not cause a significant decrease compared to the diabetic mice (P > 0.05; Fig. 5A). Furthermore, cytosolic LPO levels in the diabetic group were not significantly increased compared to the control group given citrate buffer (P > 0.05). On the other hand, cytosolic LPO levels in the experimental group treated with combined the
3.2.3. Antioxidative activities in mitochondrial and cytosolic fractions of the pancreas Antioxidative enzyme activities of mice in mitochondrial and cytosolic fractions of the pancreas are presented in Figs. 6A, B and 7A, B. MnSOD activities in diabetic mice were significantly decreased compared to the control group given citrate buffer (P < 0.05). Decrease of MnSOD activities in antioxidant-treated diabetic group was prevented by combined antioxidants. Namely, MnSOD activities in antioxidant-treated diabetic group were significantly increased compared to the diabetic group (P < 0.05; Fig. 6A). While cytosolic SOD activities in diabetic mice were not significantly decreased compared to the control group given citrate buffer (P > 0.05), the results of the experimental group given combined antioxidants were not significantly increased compared to the diabetic group (P > 0.05; Fig. 6B). There was not a significant difference of mitochondrial CAT activity for all groups (P > 0.05; Fig. 7A). While the diabetic group was not significantly increased compared to the control group given citrate buffer, experimental group given combined antioxidant was not significantly decreased compared to diabetic group. Cytosolic CAT activities in diabetic mice were significatly decreased compared to control group given citrate buffer (P < 0.01). But, there was not a significant increase in the experimental group given combined antioxidants compared to the diabetic mice (P > 0.05; Fig. 7B). GSH-Px activities in both mitochondrial and cytosolic fractions of the pancreas could not be observed. 4. Discussion Diabetes is a metabolic defect characterized with developed hyperglycemia after the insufficiency of insulin release from the pancreas, increased oxidative stress, non-enzymatic glycolization, LPO and changed antioxidative defence system after being exposed to free radicals (Dincer et al., 2002; Jang et al., 2000; Kaneto et al., 2005; Raza et al., 2004). STZ is a broad-spectrum antibiotic obtained from Streptomyces achromogenes, an agent causing diabetes through damaging pancreatic beta cells (Aughsteen, 2000; Srinivasan and Ramarao, 2007). We did not encounter any experimental study on mice involving the combined administration of ALA, Vit E and Se for prevention and treatment of diabetes. It was determined that administration of exogeneous ALA did not decrease the blood glucose levels (Dincer et al., 2002; Khamaisi et al., 1999; Maritim et al., 2003; Obrosova et al., 2003) while it was observed an inhibitor effect on the increase of blood glucose level in long time experiments in diabetic rats (Kojima et al., 2007; Lateef et al., 2005;). In some studies, a significant difference was not observed in blood glucose levels of diabetic mice treated with Vit E and Se (Barbosa et al., 2008; Gocmen et al., 2000; Mukherjee et al., 1998), there is another study showing a significant decrease of blood glucose level (Shirpoor et al., 2007). In our study, when the results of the experimental group given triple antioxidants were
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Fig. 3. Pancreas Langerhans islet (L) and insulin positive cells (→) in control sections that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A) and in sections of experimental groups that given STZ (D) and antioxidant + STZ (A + D), 540×.
compared to the diabetic group, we found a significant decrease in fasting blood glucose levels (Table 1). Immunohistochemical studies related with diabetes showed that the density of insulin-positive reaction area (Clark et al., 2001; Shao et al., 2006), quantity of beta cells (Koyuturk et al., 2005; Yanardag et al., 2003;) and percentage of beta cells (Kawasaki et al., 2005) in pancreatic islets of diabetic individuals were few compared to nondiabetic individuals. In our study, the percentage of insulin positive cell in the experimental group given antioxidant
and STZ was excessive compared to the experimental group given STZ, so this result showed that there is a protective effect of triple antioxidative application on pancreatic beta cells (Fig. 2). Oxidative stress is characterized with increased LPO and/or changed enzymatic or non-enzymatic antioxidative system (Kanbagli et al., 2002). Mitochondrial GSH is shown to be a reliable parameter of oxidative stress. The decrease of GSH shows a decrease of mitochondrial function (Kowluru et al., 2006). While the cytosolic fraction of pancreatic tissue of diabetic individuals
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Fig. 4. Mitochondrial (A) and cytosolic (B) GSH levels for all group. P > 0.05 for all groups. Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
Fig. 5. Mitochondrial (A) and cytosolic (B) LPO levels for all group. P > 0.05 for all groups. Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
showed decreased GSH levels (Bastar et al., 1998), there are also some studies with significantly increased (Dincer et al., 2002) or unchanged GSH levels (Raza et al., 2004; Wohaieb and Godin, 1987). It is reported that Se prevented the decrease of GSH levels which important for decrease of superoxide formation during diabetes (Mukherjee et al., 1998). Busse et al. (1992) showed that ALA increased the intracellular GSH levels. In our study, we did not observe significantly different GSH levels among all groups of both fractions (Fig. 4A and B). Kanbagli et al. (2002) reported that when mitochondria did not synthesize GSH, mitochondrial GSH is provided from cytoplasmic pool. But, in this work, GSH, an indicator of oxidative damage, there was not a significant difference among all groups of both fractions, so we considered that the damage was not too much or given antioxidants did not contribute much to GSH synthesis. MDA, which seems to be connected to various diseases, is used as an indicator of oxidative damage and is a product of multiple unsaturated fatty acid peroxidation (Berryman et al., 2004; Kannan and Jain, 2000; Zhang et al., 2008). It is known that LPO increased after increasing the oxidative damage in hyperglycemia (Bastar et al., 1998; Berryman et al., 2004; Kannan and Jain, 2000), on the other hand no change was reported by Rungby et al. (1992). In the diabetes group of this study, we observed not significantly increased LPO levels in both fractions (Fig. 5A and B). In addition to the control group given citrate buffer, we determined significantly increased LPO levels compared to the control groups receiving the solvents of antioxidants and antioxidants in the mitochondrial fraction (Fig. 5A). We considered that this can occur due to giving citrate buffer for five consecutive days. Since the citrate buffer pH was 4.5 and it was given for five consecutive days, the mitochondrial fraction of this study can allow more excessive and quick
development of free radicals which occurred after damage. Our opinion was verified by the fact that we did not find a similar result in cytosolic fraction. A survey of literature did not show any work about LPO levels in mitochondrial fraction of diabetic individuals induced by multiple low-dose STZ administration. In vitro studies showed that antioxidative vitamins could prevent LPO (Ozkan et al., 2005) Cytotoxic effects of agents producing free radicals decrease with the administration of Vit E to animals, whereas it was shown that membrane LPO increase the tendency in animals fed with a diet not including Vit E and Se (Slonim et al., 1983). In our study, we observed an unsignificant decrease in LPO levels in the experimental group given antioxidant and STZ compared to the experimental group receiving STZ in both fractions (Fig. 5A and B) and we think that these antioxidants can prevent only a slight oxidative damage. Some key enzymes like GSH-Px are present at further low levels in the pancreatic tissue according to the other tissues in diabetes for defending toward ROS and islet cells could be more sensitive and defenseless to damage from oxidative stress in these conditions (Ho and Bray, 1999; Jang et al., 2000; Kaneto et al., 2005). In a study which investigates the mitochondrial fraction of pancreatic tissue of single-dose STZ-induced rats was observed to be more excessive GSH-Px acitivity in diabetic group according to control group (Jang et al., 2000). In our study, GSH-Px activity was not observed both in mitochondrial and cytosolic fraction of pancreatic tissue (data not shown). We thought that diabetes induced by multiple lowdose STZ administration could not cause major damage and these conditions could be the cause of not observing GSH-Px activity in mitochondrial and cytosolic fractions of this study. SOD, which catalyzes the transformation between molecular O2 and H2 O2 of superoxide anion radicals, is an important intracellular antioxidative enzyme (Shirpoor et al., 2007). Different
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Fig. 6. Mitochondrial MnSOD levels for all group. a P < 0.05 versus to CB group; b P < 0.05 versus to D group (A). Cytosolic SOD levels for all group (B). Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
Fig. 7. Mitochondrial CAT levels for all group. P > 0.05 for all groups (A). Cytosolic CAT levels for all group. a P < 0.01 versus to CB group (B). Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
forms of SOD are represented both in cytosol and mitochondria. When we searched the literature for the SOD activity, which is important for the determination of oxidative damage, we found that while mice continue living with mutations in the gene encoding Cu/ZnSOD, which is the cytosolic form of the enzyme (Reaume et al., 1996) the mice died shortly after birth with mutations in the gene encoding MnSOD, which is the mitochondrial form of the enzyme (Lebovitz et al., 1996; Li et al., 1995). Increased MnSOD activitiy has an important role for mitochondrial sensitivity (Kanbagli et al., 2002). SOD activities were increased in cytosolic fraction of pancreas and kidney tissues of diabetic rats because of production of increased superoxide anions when the cell function was destroyed (Coskun et al., 2005; Dincer et al., 2002). In mitochondrial fraction of pancreas tissue showed by Jang et al. (2000) that MnSOD activity of diabetic individuals were more excessive according to control individuals. In our study, SOD activity was significantly decreased in mitochondrial fractions (Fig. 6A) and not significantly decreased in cytosolic fraction (Fig. 6B) in the diabetic group according to control group. Also previously as explained, increased oxidative stress with diabetes was decreased to activity of this enzyme and given antioxidants have a protective role aganist this damage so that MnSOD activity rised with the reduction of oxidative stress (Fig. 6A). There was not a significant increase after given antioxidants in cytosolic fraction (Fig. 6B). Futhermore, Papaccio et al. (2000) demostrated that while SOD activities were decreased with given multiple low dose STZ by day 11, SOD activities were transiently increased with given a single high dose STZ (24 h from the administration), then SOD activities are decreased in isolated islets. Our experiment model as associate with STZ did not have much excessive damage such as seen in the other parameter. We thought that given antioxidants focus on mitochondrial damage and repair to this damage (Lebovitz et al., 1996; Li et al., 1995; Reaume et al., 1996). Maybe if we were to change the amount of given antioxidants, we could have obtained important results in the cytosolic fraction. CAT is an important free radical scavenger enzyme that breaks down H2 O2 into H2 O and O2 (Mukherjee et al., 1998; Shirpoor et al., 2007). The deficiency of this enzyme in beta cells causes increased oxidative stress and damaged beta cells (Heales, 2001). While beta cells are poor on amount of CAT, they are rich in mitochondria which is the resource for developing superoxide and H2 O2 . Thus, oxidative stress in beta cells due to deficiency of CAT and developing diabetes occur with slow accumulation of low-level peroxides for years (Góth and Eaton, 2000). In related studies with diabetes, CAT enzyme activities were observed to decrease (Gezginci Oktayoglu and Bolkent, 2009) and it has also been reported that CAT activities returned to normal or increased with separate administration of ALA, Vit E and Se (Barbosa et al., 2008; Reddi and Bollineni, 2001; Sadi et al., 2008; Shirpoor et al., 2007). On the contrary, another study reported an increase of CAT activity in diabetic individuals. In this study, CAT activity was determined to increase due to increasing H2 O2 and oxygen radicals (Bhor et al., 2004). However, we have not come across any study which determines the CAT activity in mitochondrial fraction of pancreatic tissue in diabetes. In our study, we could not find a significant result for CAT activity in mitochondrial fraction (Fig. 7A). This suggested that there was not an excessive amount of H2 O2 in this fraction. In cytosolic fraction, while CAT activity decreased significantly in the diabetic group, it was not a significant change in the experimental group given combined antioxidants (Fig. 7B) and we thought that these conditions could be due to the insufficiency of the given antioxidants. When STZ is given multiple low dose, islet beta cells are destroyed within a few days (Like and Rossini, 1976). However, when STZ is given a single high dose, it rapidly destroys islet beta cells (Yamamoto et al., 1981). Futhermore, the mortality rate is lower in mice receiving multiple low doses of STZ than in those
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receiving single high dose (Jin et al., 2009). Namely, STZ causes more damage with single high dose in the beta cells. We are considered that the beta cells are damaged after a long period of time by given multiple low dose STZ and this damage may be reduced because of the antioxidant treatment. In our study, combined antioxidants were given as a protective in the first 5 day. For this reason, in conclusion, the diabetes model induced by low dose STZ administration did not cause an excessive damage. The different doses of the combined antioxidants could try for the exact removal of the damage that has occurred. We can consider that by taking into account the blood glucose levels and immunohistochemical results, the combination of triple antioxidants has a positive effect on diabetes. This positive effect could increase when trying different doses of combined antioxidant treatment. Acknowledgments This study was supported by the Research Fund of Istanbul University. Project Nos. T-891 and UDP-3975. References Aebi H. Catalase in vitro. Methods in Enzymology 1984;105:121–6. Altan N, Sepici Dinc¸el A, Koca C. Diabetes mellitus ve oksidatif stres. Turkish Journal of Biochemistry 2006;31(2):51–6. Aughsteen AA. An ultrastructural study on the effect of streptozotocin on the islets of Langerhans in mice. Journal of Electron Microscopy (Tokyo) 2000;49(5):681–90. Barbosa NB, Rocha JB, Soares JC, Wondracek DC, Goncalves JF, et al. Dietary diphenyl diselenide reduces the STZ-induced toxicity. Food and Chemical Toxicology 2008;46(1):186–94. Bastar I, Seckin S, Uysal M, Aykac Toker G. Effect of streptozotocin on glutathione and lipid peroxide levels in various tissues of rats. Research Communications in Molecular Pathology & Pharmacology 1998;102(3):265–72. Berryman AM, Maritim AC, Sanders RA, Watkins 3rd JB. Influence of treatment of diabetic rats with combinations of pycnogenol, beta-carotene, and alpha-lipoic acid on parameters of oxidative stress. Journal of Biochemical and Molecular Toxicology 2004;18(6):345–52. Beutler E. Glutathione in red blood cell metabolism, a manual of biochemical methods, vol. 36, second ed. New York: Grune and Stratton; 1975. p. 112–4. Bhor VM, Raghuram N, Sivakami S. Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats. The International Journal of Biochemistry & Cell Biology 2004;36(1):89–97. Ho E, Bray TM. Antioxidants, NFkappaB activation, and diabetogenesis. Proceedings of the Society for Experimental Biology and Medicine 1999;222(3):205–13. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods in Enzymology 1978;52:302–10. Busse E, Zimmer G, Schopohl B, Kornhuber B. Influence of alpha-lipoic acid on intracellular glutathione in vitro and in vivo. Arzneimittelforschung 1992;42(6):829–31. Clark A, Jones LC, De Koning E, Hansen BC, Matthews DR. Decreased insulin secretion in type 2 diabetes: a problem of cellular mass or function? Diabetes 2001;50(1):S169–71. Coskun O, Kanter M, Korkmaz A, Oter S. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and -cell damage in rat pancreas. Pharmacological Research 2005;51(2):117–23. Di Leo MA, Santini SA, Silveri NG, Giardina B, Franconi F, Ghirlanda G. Long-term taurine supplementation reduces mortality rate in streptozotocin-induced diabetic rats. Amino Acids 2004;27(2):187–91. Dincer Y, Telci A, Kayali R, Yilmaz IA, Cakatay U, Akcay T. Effect of alpha-lipoic acid on lipid peroxidation and anti-oxidant enzyme activities in diabetic rats. Clinical and Experimental Pharmacology and Physiology 2002;29(4):281–4. Eskew ML, Scholz RW, Reddy CC, Todhunter DA, Zarkower A. Effects of vitamin E and selenium deficiencies on rat immune function. Immunology 1985;54(1):173–80. Flohe L, Becker R, Brigelius R, Lengfelder E, Otting F. CRC handbook of free radicals and antioxidants in biomedicine, convenient assays for superoxide dismutase, vol. 3. Boca Raton, FL: CRC Press, Inc; 1989. p. 287–93. Foster TS. Efficacy and safety of alpha-lipoic acid supplementation in the treatment of symptomatic diabetic neuropathy. The Diabetes Educator 2007;33(1):111–7. Gezginci Oktayoglu S, Bolkent S. Exendin-4 exerts its effects through the NGF/p75NTR system in diabetic mouse pancreas. Biochemistry and Cell Biology 2009;87(4):641–51. Gocmen C, Secilmis A, Kumcu EK, Ertug PU, Onder S, Dikmen A, et al. Effects of vitamin E and sodium selenate on neurogenic and endothelial relaxation of corpus cavernosum in the diabetic mouse. European Journal of Pharmacology 2000;398(1):93–8. Góth L, Eaton JW. Hereditary catalase deficiencies and increased risk of diabetes. The Lancet 2000;356(9244):1820–1. Heales SJ. Catalase deficiency, diabetes, and mitochondrial function. The Lancet 2001;357(9252):314.
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Review
The potential role of combined antioxidant treatment on pancreas of STZ-diabetic mice Ayse Karatug ∗ , Sehnaz Bolkent Istanbul University, Faculty of Science, Department of Biology, 34134 Vezneciler, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 10 December 2010 Accepted 30 August 2011 Keywords: Diabetes Oxidative stress Mitochondrial and cytosolic antioxidative systems Pancreas Mice
a b s t r a c t In diabetes, cells and tissues are damaged due to the imbalance between production of free radicals and removal of them. The effective biologic antioxidants for oxidative stress such as ␣-lipoic acid, vitamin E and selenium are effective in diminishing oxidative damage such as membrane lipid peroxidation. The experiment aimed to investigate the oxidative stress occurring in mitochondrial and cytoplasmic fraction of pancreatic tissues in streptozotocin-diabetic mice and the possible effects of ␣-lipoic acid + vitamin E + selenium combination on oxidative damage and antioxidative system by using microscopic and biochemical methods. The mice were divided into five groups. These groups were treated by citrate buffer, the solvents of the antioxidants, combined the antioxidants [␣-lipoic acid (50 mg/kg), vitamin E (100 mg/kg), selenium (0.25 mg/kg)], streptozotocin (40 mg/kg × 5), combined the antioxidants and streptozotocin. The mice were sacrificed by cervical dislocation. In the experimental group given combined antioxidants following results were observed compared to diabetic group: increased percent insulin-positive cell area; decreased blood glucose levels; increased manganase superoxide dismutase activities and unsignificantly increased superoxide dismutase activities; unsignificantly decreased lipid peroxidase levels in both of fraction; unsignificantly decreased in mitochondrial fraction and unsignificantly increased in cytosolic fraction for catalase levels; not any alteration glutathione levels; not any activity in both of fraction for glutathione peroxidase. We can say that by taking the blood glucose levels and immunohistochemical results into account, the combination of triple antioxidants has a partly positive effect on diabetes. This positive effect could increase when trying different doses of combined antioxidant treatment. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Diabetes is a disease characterized with the change of carbohydrate, lipid and protein metabolism due to the decreasing insulin level (Leach, 2007; Modak et al., 2007). Diabetes is related with antioxidative defense system which is accepted to be important in the increases of reactive oxygen species (ROS) and free radical species that cause development and progress of diabetic complications (Barbosa et al., 2008; Di Leo et al., 2004; West, 2000; Yi and Maeda, 2006). Streptozotocin (STZ) causes pancreatic beta cell damage with the decrease of insulin level and evidence of hyperglycemia (Wada and Yagihashi, 2004). One of the first effect of STZ on pancreatic beta cells is the oxidative damage caused by the generation of free radicals (Seven et al., 2004; Yi and Maeda, 2006). Production of ROS in various tissues of diabetic individuals has a role in early and late period complications in diabetes (Kaneto et al.,
∗ Corresponding author. Tel.: +90 212 4555700; fax: +90 212 5280527. E-mail address: [email protected] (A. Karatug). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.08.012
1999). Production of most ROS and free radicals during physiological and pathological metabolism generates the defense mechanism against oxidative stress. However, the decay of balance between producing and scavenging free radicals due to an increased oxidative stress or decreased antioxidative defence causes cell and tissue damage in diabetes (Dincer et al., 2002). Both internal and external cell defense of endogeneous and exogeneous antioxidative molecules render inactive damage caused by oxidant molecules (Altan et al., 2006). ␣-Lipoic acid (ALA) is characterized as a powerful antioxidant due to the ability of scavenging ROS (Foster, 2007). Therefore, ALA is a biological antioxidant and a powerful scavenger of free radicals, also an essential cofactor for mitochondrial enzymes (Somani et al., 2000). STZ-damaged beta cells may partially be improved and islet cell functions may be obtained again by using ALA in rodents. It is suggested that giving exogeneous ALA to mice, beta cell is protected aganist hyperglycemia (Yi and Maeda, 2006). Vitamin E (Vit E) is one of the most effective biological antioxidants that supplies protection against damage that occurred in the end of oxidative stress in animals. Giving additional Vit E to the animal organizes cytotoxic effects of
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quickly removed for immunohistochemical and biochemical analyses.
2.2. Immunohistochemical assay
Fig. 1. Diagram of control and experimental groups.
agents producing free radicals (Slonim et al., 1983). Selenium (Se) is an important component for the glutathione peroxidase (GSHPx) enzyme and a functional antioxidant along with its feature of reducing lipid peroxidation (LPO) (Eskew et al., 1985). Se plays an important role in increasing the scavenger effects of oxygen-free radicals and thus, there is a protective effect of Se in early period of diabetes or progress of diabetic complications (Mukherjee et al., 1998). It is suggested that ALA-Vit E and Vit E-Se couples have a synergistic effect. ALA provides recycling of Vit E with increased coenzyme Q level. Vit E plays a role for preventing the formation and removal of hydroperoxide by interacting with Se-dependent systems (Hoekstra, 1975). In this study, we aimed to investigate synergistic effects of ALA + Vit E + Se combination on the alterations which occurred by increased oxidative stress and mitochondrial defense mechanism in an important chronic disease such as diabetes. 2. Materials and methods
Pancreatic tissue was fixed in 10% buffered formalin for immunohistochemical assay. After dehydration in a series of ethanol, pancreatic tissues were cleared in xylene and embedded in paraffin. Paraffin-embedded tissues were cut as 4-m sections and stuck on microscope slide coated with poly-l-lysine. Sections were kept in toluene to remove paraffin. After hydration, sections were treated with 3% hydrogen peroxide (H2 O2 ) prepared in distilled water to quench the endogeneous peroxidase activity. Histostain Plus Broad Spectrum Kit (Zymed, 85-9743) and insulin antibody (Ab-6, MS-1379-P, Neomarkers, Fremond, CA, dilution 1:400, 30 min at room temperature) were used for insulin labeling by using streptavidin–biotin–peroxidase technique. Immunoreactivity was revealed by using 3-amino-9-ethylcarbazole. Sections were counter-stained in Mayer’s haematoxylin and mounted in glycerol-vinyl alcohol. Microscopic analysis was performed by using 40× objective and 10× ocular system of Olympus CX-45 microscope. The image was transferred to computer by Olympus DP-71 imaging system and the measurements were done with Olympus Analysis Computer Programme. Thirty islets were evaluated for all groups. Insulin cell area % was calculated by measuring the evaluated pancreatic islets and labeling the insulin area.
2.3. Preparation of cellular fractions Pancreatic tissue was homogenized in cold 0.15 M KCl with a glass homogenizer for 10% homogenate. First, the homogenates were centrifuged at +4 ◦ C and 2000 × g for 10 min and the supernatants were centrifuged again at +4 ◦ C and 10,000 × g for 10 min. Supernatants were used as cytosolic fraction. Mitochondrial fractions were obtained by suspending the mitochondrial pellets with 0.15 M KCl and supernatants were stored at −86 ◦ C until analyzed and used for measuring the enzyme activities.
2.1. Animals and in vivo treatment In the study, 2–2.5-month-old Balb/c mice were provided by Institute’s Animal Care and Use Committee of Istanbul University. All mice were housed in a temperature-controlled clean room with a 12 h–12 h light–dark cycle and fed with standard chow and tap water ad libitum. The mice were selected randomly and divided into five groups. Group I: the control group given citrate buffer (n = 8), Group II: the control group given the solvents of the antioxidants (n = 8), Group III: the control group given combined the antioxidants (n = 8), Group IV: the experimental group given STZ (n = 10), and Group V: the experimental group given combined the antioxidants and STZ (n = 10) (Fig. 1). The mice were treated by intraperitoneal injections of multiple low-dose streptozotocin as five consecutive daily doses of 40 mg/kg to experimental groups. STZ was dissolved in a freshly prepared 0.01 M pH 4.5 citrate buffer. ALA (50 mg/kg), Vit E (100 mg/kg) and Se (0.25 mg/kg) were given by gavage technique for five consecutive days to the control group and before STZ injections to the experimental group. The antioxidants were dissolved in distilled water including a trace of NaOH for ALA, flower oil for Vit E and distilled water for Se. The mice were sacrificed by cervical dislocation on day 30, starting from the administration of the following: Group I, first citrate buffer, Group II, last antioxidant, Group III, solvent of the last antioxidant, Group IV, first dose of STZ, and Group V, first dose of STZ after receiving the antioxidants. After the mice were sacrificed, the pancreatic tissue was
2.4. Biochemical assays Mice fasting blood glucose levels (after 12-h period of fasting) were determined with an Accu Check Go Glucometer with a small drop of blood from tail of each mouse on the glucometer strip. Spectrophotometric methods were used in the determinations of glutathione (GSH) levels by Beutler’s method (Beutler, 1975) and of LPO by Buege and Aust method (Buege and Aust, 1978) and of the activities of the enzymes, namely, catalase (CAT) by Aebi’s method (Aebi, 1984) and GSH-Px by Paglia–Valentine method (Paglia and Valentine, 1967) for cytosolic and mitochondrial fraction of the pancreatic tissue. McCord and Fridovich method (McCord and Fridovich, 1969), modified by Flohe et al. (1989) for manganese superoxide dismutase (MnSOD) in mitochondrial fraction and the method of Sun et al. (1988) for superoxide dismutase (SOD) in cytosolic fraction were used.
2.5. Statistical analysis The immunohistochemical and biochemical results were analyzed by two-way ANOVA and Mann–Whitney U tests by using GraphPad Prism version 4.0 computer package. Results were reported as mean ± SE. P values less than 0.05 were considered to be significant.
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Table 1 Fasting blood glucose levels and body weights for all groups.
BGL (mg/dl) BW (g)
Citrate buffer (CB)
Solvents of antioxidants (AS)
Combined antioxidants (A)
Diabetes (D)
A+D
86.875 ± 5.37 25.625 ± 0.52
97.125 ± 6.90 27.563 ± 0.62
100.63 ± 4.90 29.375 ± 0.86
183.50 ± 13.29a 24.725 ± 0.62
134.00 ± 8.37b 25.938 ± 1.17
Mean ± SE. BGL: blood glucose level; BW: body weight. a P < 0.001 versus to CB group. b P < 0.01 versus to D group.
antioxidants, there was not a significant decrease compared to the diabetic groups (P > 0.05; Fig. 5B).
Fig. 2. Insulin positive cell area. a P < 0.001 versus to CB group; b P < 0.001 versus to D group. Groups that given citrate buffer (CB) and STZ (D).
3. Results 3.1. Immunohistochemical results Insulin-positive cell area in diabetic mice was significantly decreased compared to the control group given citrate buffer (P < 0.001). The decreasing of insulin-positive cell area in antioxidant-treated diabetic group was prevented by combined antioxidant treatment (P < 0.001; Figs. 2 and 3). 3.2. Biochemical results 3.2.1. Blood glucose levels and body weight Fasting blood glucose levels and body weights of mice are presented in Table 1. Fasting blood glucose levels of diabetic mice were significantly increased compared to control group given citrate buffer (P < 0.001). The increase of fasting blood glucose levels in diabetic mice were significantly decreased by combined antioxidative treatment (P < 0.01). Body weights of the diabetic mice (P < 0.001) were decreased compared to the control groups and body weights of the antioxidant-treated diabetic group were not significantly increased compared to the diabetic group (P > 0.05). 3.2.2. Oxidative stress in mitochondrial and cytosolic fractions of the pancreas GSH and LPO levels of mice in mitochondrial and cytosolic fractions of the pancreas were presented in Figs. 4A, B and 5A, B. Mitochondrial and cytosolic GSH levels for all groups were not significantly different (P > 0.05; Fig. 4A and B). But, mitochondrial LPO levels in diabetic mice were not significantly increased compared to control group given citrate buffer (P > 0.05). Mitochondrial LPO levels in diabetic group given combined the antioxidants did not cause a significant decrease compared to the diabetic mice (P > 0.05; Fig. 5A). Furthermore, cytosolic LPO levels in the diabetic group were not significantly increased compared to the control group given citrate buffer (P > 0.05). On the other hand, cytosolic LPO levels in the experimental group treated with combined the
3.2.3. Antioxidative activities in mitochondrial and cytosolic fractions of the pancreas Antioxidative enzyme activities of mice in mitochondrial and cytosolic fractions of the pancreas are presented in Figs. 6A, B and 7A, B. MnSOD activities in diabetic mice were significantly decreased compared to the control group given citrate buffer (P < 0.05). Decrease of MnSOD activities in antioxidant-treated diabetic group was prevented by combined antioxidants. Namely, MnSOD activities in antioxidant-treated diabetic group were significantly increased compared to the diabetic group (P < 0.05; Fig. 6A). While cytosolic SOD activities in diabetic mice were not significantly decreased compared to the control group given citrate buffer (P > 0.05), the results of the experimental group given combined antioxidants were not significantly increased compared to the diabetic group (P > 0.05; Fig. 6B). There was not a significant difference of mitochondrial CAT activity for all groups (P > 0.05; Fig. 7A). While the diabetic group was not significantly increased compared to the control group given citrate buffer, experimental group given combined antioxidant was not significantly decreased compared to diabetic group. Cytosolic CAT activities in diabetic mice were significatly decreased compared to control group given citrate buffer (P < 0.01). But, there was not a significant increase in the experimental group given combined antioxidants compared to the diabetic mice (P > 0.05; Fig. 7B). GSH-Px activities in both mitochondrial and cytosolic fractions of the pancreas could not be observed. 4. Discussion Diabetes is a metabolic defect characterized with developed hyperglycemia after the insufficiency of insulin release from the pancreas, increased oxidative stress, non-enzymatic glycolization, LPO and changed antioxidative defence system after being exposed to free radicals (Dincer et al., 2002; Jang et al., 2000; Kaneto et al., 2005; Raza et al., 2004). STZ is a broad-spectrum antibiotic obtained from Streptomyces achromogenes, an agent causing diabetes through damaging pancreatic beta cells (Aughsteen, 2000; Srinivasan and Ramarao, 2007). We did not encounter any experimental study on mice involving the combined administration of ALA, Vit E and Se for prevention and treatment of diabetes. It was determined that administration of exogeneous ALA did not decrease the blood glucose levels (Dincer et al., 2002; Khamaisi et al., 1999; Maritim et al., 2003; Obrosova et al., 2003) while it was observed an inhibitor effect on the increase of blood glucose level in long time experiments in diabetic rats (Kojima et al., 2007; Lateef et al., 2005;). In some studies, a significant difference was not observed in blood glucose levels of diabetic mice treated with Vit E and Se (Barbosa et al., 2008; Gocmen et al., 2000; Mukherjee et al., 1998), there is another study showing a significant decrease of blood glucose level (Shirpoor et al., 2007). In our study, when the results of the experimental group given triple antioxidants were
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Fig. 3. Pancreas Langerhans islet (L) and insulin positive cells (→) in control sections that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A) and in sections of experimental groups that given STZ (D) and antioxidant + STZ (A + D), 540×.
compared to the diabetic group, we found a significant decrease in fasting blood glucose levels (Table 1). Immunohistochemical studies related with diabetes showed that the density of insulin-positive reaction area (Clark et al., 2001; Shao et al., 2006), quantity of beta cells (Koyuturk et al., 2005; Yanardag et al., 2003;) and percentage of beta cells (Kawasaki et al., 2005) in pancreatic islets of diabetic individuals were few compared to nondiabetic individuals. In our study, the percentage of insulin positive cell in the experimental group given antioxidant
and STZ was excessive compared to the experimental group given STZ, so this result showed that there is a protective effect of triple antioxidative application on pancreatic beta cells (Fig. 2). Oxidative stress is characterized with increased LPO and/or changed enzymatic or non-enzymatic antioxidative system (Kanbagli et al., 2002). Mitochondrial GSH is shown to be a reliable parameter of oxidative stress. The decrease of GSH shows a decrease of mitochondrial function (Kowluru et al., 2006). While the cytosolic fraction of pancreatic tissue of diabetic individuals
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Fig. 4. Mitochondrial (A) and cytosolic (B) GSH levels for all group. P > 0.05 for all groups. Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
Fig. 5. Mitochondrial (A) and cytosolic (B) LPO levels for all group. P > 0.05 for all groups. Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
showed decreased GSH levels (Bastar et al., 1998), there are also some studies with significantly increased (Dincer et al., 2002) or unchanged GSH levels (Raza et al., 2004; Wohaieb and Godin, 1987). It is reported that Se prevented the decrease of GSH levels which important for decrease of superoxide formation during diabetes (Mukherjee et al., 1998). Busse et al. (1992) showed that ALA increased the intracellular GSH levels. In our study, we did not observe significantly different GSH levels among all groups of both fractions (Fig. 4A and B). Kanbagli et al. (2002) reported that when mitochondria did not synthesize GSH, mitochondrial GSH is provided from cytoplasmic pool. But, in this work, GSH, an indicator of oxidative damage, there was not a significant difference among all groups of both fractions, so we considered that the damage was not too much or given antioxidants did not contribute much to GSH synthesis. MDA, which seems to be connected to various diseases, is used as an indicator of oxidative damage and is a product of multiple unsaturated fatty acid peroxidation (Berryman et al., 2004; Kannan and Jain, 2000; Zhang et al., 2008). It is known that LPO increased after increasing the oxidative damage in hyperglycemia (Bastar et al., 1998; Berryman et al., 2004; Kannan and Jain, 2000), on the other hand no change was reported by Rungby et al. (1992). In the diabetes group of this study, we observed not significantly increased LPO levels in both fractions (Fig. 5A and B). In addition to the control group given citrate buffer, we determined significantly increased LPO levels compared to the control groups receiving the solvents of antioxidants and antioxidants in the mitochondrial fraction (Fig. 5A). We considered that this can occur due to giving citrate buffer for five consecutive days. Since the citrate buffer pH was 4.5 and it was given for five consecutive days, the mitochondrial fraction of this study can allow more excessive and quick
development of free radicals which occurred after damage. Our opinion was verified by the fact that we did not find a similar result in cytosolic fraction. A survey of literature did not show any work about LPO levels in mitochondrial fraction of diabetic individuals induced by multiple low-dose STZ administration. In vitro studies showed that antioxidative vitamins could prevent LPO (Ozkan et al., 2005) Cytotoxic effects of agents producing free radicals decrease with the administration of Vit E to animals, whereas it was shown that membrane LPO increase the tendency in animals fed with a diet not including Vit E and Se (Slonim et al., 1983). In our study, we observed an unsignificant decrease in LPO levels in the experimental group given antioxidant and STZ compared to the experimental group receiving STZ in both fractions (Fig. 5A and B) and we think that these antioxidants can prevent only a slight oxidative damage. Some key enzymes like GSH-Px are present at further low levels in the pancreatic tissue according to the other tissues in diabetes for defending toward ROS and islet cells could be more sensitive and defenseless to damage from oxidative stress in these conditions (Ho and Bray, 1999; Jang et al., 2000; Kaneto et al., 2005). In a study which investigates the mitochondrial fraction of pancreatic tissue of single-dose STZ-induced rats was observed to be more excessive GSH-Px acitivity in diabetic group according to control group (Jang et al., 2000). In our study, GSH-Px activity was not observed both in mitochondrial and cytosolic fraction of pancreatic tissue (data not shown). We thought that diabetes induced by multiple lowdose STZ administration could not cause major damage and these conditions could be the cause of not observing GSH-Px activity in mitochondrial and cytosolic fractions of this study. SOD, which catalyzes the transformation between molecular O2 and H2 O2 of superoxide anion radicals, is an important intracellular antioxidative enzyme (Shirpoor et al., 2007). Different
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Fig. 6. Mitochondrial MnSOD levels for all group. a P < 0.05 versus to CB group; b P < 0.05 versus to D group (A). Cytosolic SOD levels for all group (B). Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
Fig. 7. Mitochondrial CAT levels for all group. P > 0.05 for all groups (A). Cytosolic CAT levels for all group. a P < 0.01 versus to CB group (B). Groups that given citrate buffer (CB), solvents of antioxidants (AS), antioxidants (A), STZ (D) and antioxidant + STZ (A + D).
forms of SOD are represented both in cytosol and mitochondria. When we searched the literature for the SOD activity, which is important for the determination of oxidative damage, we found that while mice continue living with mutations in the gene encoding Cu/ZnSOD, which is the cytosolic form of the enzyme (Reaume et al., 1996) the mice died shortly after birth with mutations in the gene encoding MnSOD, which is the mitochondrial form of the enzyme (Lebovitz et al., 1996; Li et al., 1995). Increased MnSOD activitiy has an important role for mitochondrial sensitivity (Kanbagli et al., 2002). SOD activities were increased in cytosolic fraction of pancreas and kidney tissues of diabetic rats because of production of increased superoxide anions when the cell function was destroyed (Coskun et al., 2005; Dincer et al., 2002). In mitochondrial fraction of pancreas tissue showed by Jang et al. (2000) that MnSOD activity of diabetic individuals were more excessive according to control individuals. In our study, SOD activity was significantly decreased in mitochondrial fractions (Fig. 6A) and not significantly decreased in cytosolic fraction (Fig. 6B) in the diabetic group according to control group. Also previously as explained, increased oxidative stress with diabetes was decreased to activity of this enzyme and given antioxidants have a protective role aganist this damage so that MnSOD activity rised with the reduction of oxidative stress (Fig. 6A). There was not a significant increase after given antioxidants in cytosolic fraction (Fig. 6B). Futhermore, Papaccio et al. (2000) demostrated that while SOD activities were decreased with given multiple low dose STZ by day 11, SOD activities were transiently increased with given a single high dose STZ (24 h from the administration), then SOD activities are decreased in isolated islets. Our experiment model as associate with STZ did not have much excessive damage such as seen in the other parameter. We thought that given antioxidants focus on mitochondrial damage and repair to this damage (Lebovitz et al., 1996; Li et al., 1995; Reaume et al., 1996). Maybe if we were to change the amount of given antioxidants, we could have obtained important results in the cytosolic fraction. CAT is an important free radical scavenger enzyme that breaks down H2 O2 into H2 O and O2 (Mukherjee et al., 1998; Shirpoor et al., 2007). The deficiency of this enzyme in beta cells causes increased oxidative stress and damaged beta cells (Heales, 2001). While beta cells are poor on amount of CAT, they are rich in mitochondria which is the resource for developing superoxide and H2 O2 . Thus, oxidative stress in beta cells due to deficiency of CAT and developing diabetes occur with slow accumulation of low-level peroxides for years (Góth and Eaton, 2000). In related studies with diabetes, CAT enzyme activities were observed to decrease (Gezginci Oktayoglu and Bolkent, 2009) and it has also been reported that CAT activities returned to normal or increased with separate administration of ALA, Vit E and Se (Barbosa et al., 2008; Reddi and Bollineni, 2001; Sadi et al., 2008; Shirpoor et al., 2007). On the contrary, another study reported an increase of CAT activity in diabetic individuals. In this study, CAT activity was determined to increase due to increasing H2 O2 and oxygen radicals (Bhor et al., 2004). However, we have not come across any study which determines the CAT activity in mitochondrial fraction of pancreatic tissue in diabetes. In our study, we could not find a significant result for CAT activity in mitochondrial fraction (Fig. 7A). This suggested that there was not an excessive amount of H2 O2 in this fraction. In cytosolic fraction, while CAT activity decreased significantly in the diabetic group, it was not a significant change in the experimental group given combined antioxidants (Fig. 7B) and we thought that these conditions could be due to the insufficiency of the given antioxidants. When STZ is given multiple low dose, islet beta cells are destroyed within a few days (Like and Rossini, 1976). However, when STZ is given a single high dose, it rapidly destroys islet beta cells (Yamamoto et al., 1981). Futhermore, the mortality rate is lower in mice receiving multiple low doses of STZ than in those
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