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Effects of insulin and clonazepam on DNA damage in diabetic rats submitted to the forced swimming test
Mutation Research 703 (2010) 187–190
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Effects of insulin and clonazepam on DNA damage in diabetic rats submitted to the forced swimming test Carlos Alberto Yasin Wayhs a,b,∗ , Vanusa Manfredini a,b , Angela Sitta b,c , Marion Deon b,c , Graziela S. Ribas a,b , Camila S. Vanzin b , Giovana B. Biancini b , Maurício S. Nin d , Helena M.T. Barros d , Carmen Regla Vargas a,b,c,∗ a
Programa de Pós-Graduac¸ão em Ciências Farmacêuticas, Porto Alegre, RS, Brazil Servic¸o de Genética Médica, HCPA, Porto Alegre, RS, Brazil c Programa de Pós-Graduac¸ão em Ciências Biológicas: Bioquímica, UFRGS, Porto Alegre, RS, Brazil d Departamento de Farmacologia, UFCSPA, Porto Alegre, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 24 May 2010 Received in revised form 18 August 2010 Accepted 26 August 2010 Available online 9 September 2010 Keywords: Comet assay DNA damage Diabetes Depression Insulin Clonazepam Streptozotocin
a b s t r a c t Diabetes mellitus (DM) is a chronic hyperglycemic state. DM may be associated with moderate cognitive deficits and neurophysiologic/structural changes in the brain (diabetic encephalopathy). Psychiatric manifestations seem to accompany this encephalopathy, since the prevalence of depression in diabetic patients is much higher than in the general population, and clonazepam is being used to treat this complication. The excessive production of oxygen free radicals that may occur in diabetes induces a variety of lesions in macromolecules, including DNA. In this work, we analyzed DNA damage in leukocytes from streptozotocin-induced diabetic rats submitted to the forced swimming test. The DNA damage index was significantly elevated (DI = 61.00 ± 4.95) in the diabetic group compared to the control group (34.00 ± 1.26). Significant reductions of the damage index were observed in diabetic animals treated with insulin (45.00 ± 1.82), clonazepam (52.00 ± 1.22), or both agents (39.00 ± 5.83, not significantly different from control levels). Insulin plus clonazepam can protect against DNA damage in stressed diabetic rats. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by a hyperglycemic chronic state that can lead to complications affecting the retina, kidney, muscle, blood vessels, and also the central nervous system (CNS). DM is regarded as one of the major metabolic diseases of the 21st C. [1,2]. Cognitive dysfunction is developed by some diabetic individuals [3]. A wealth of studies have described neuropsychological and neurobehavioral changes in both type 1 and type 2 diabetic subjects, suggesting that diabetic encephalopathy should be recognized as a complication of diabetes [4,5]. Psychiatric manifestations may accompany this encephalopathy, since the prevalence of depression in diabetic patients is much higher than in the general population [6–9].
∗ Corresponding authors at: Servic¸o de Genética Médica, HCPA, Rua Ramiro Barcelos, 2350 CEP 90.035-903, Porto Alegre, RS, Brazil. Tel.: +55 51 3359 8011; fax: +55 51 3359 8010. E-mail addresses: [email protected] (C.A.Y. Wayhs), [email protected] (C.R. Vargas). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.08.017
Diabetic encephalopathy can be modeled in experimental animals; diabetic mice and rats exhibit depressive-like behavior when submitted to the forced swimming test (FST) [10–12]. Some studies have demonstrated that insulin administration can prevent neuronal damage in the cortex of streptozotocin (STZ)-induced diabetic rats and also produce behavioral changes [11,13]. Insulin also affects synaptosomal ␥-aminobutyric acid (GABA) and glutamate transport under oxidative stress conditions [14] and clonazepam (CNZ), a positive GABAA receptor modulator, shows an antidepressant effect in these animals [10,15]. Oxidative stress may play a role in the development of diabetic complications [16], since hyperglycemia generates abnormally high levels of free radicals via autoxidation of glucose and protein glycation [17]. These free radicals induce a variety of lesions in DNA, including oxidized bases, abasic sites, strand breaks, and formation of DNA–protein cross-links. Hydroxyl radical, produced by the Fenton reaction in the presence of transition metal ions, is responsible for this DNA damage [18]. Using the comet assay, we have investigated DNA damage in peripheral whole blood leukocytes from streptozotocin-induced diabetic rats submitted to the FST, and we have evaluated the effects of acute treatment with insulin and/or clonazepam upon this process.
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C.A.Y. Wayhs et al. / Mutation Research 703 (2010) 187–190
Fig. 1. Time-line chart of the experiment.
2. Materials and methods 2.1. Animals Male Wistar rats (250 ± 50 g) were obtained from the Animal House of Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). The animals were housed in groups of four per polypropylene cage. Food and water were available ad libitum, except where otherwise stated, and the animals were maintained in a temperature-controlled room (22 ± 2 ◦ C) under a light–dark cycle (7:00 a.m.–7:00 p.m.). The animals were divided into five groups: control (nondiabetic); diabetic (STZ); diabetic plus insulin (STZ–INS); diabetic plus clonazepam (STZ–CNZ); and diabetic plus insulin and clonazepam (STZ–INS–CNZ). All in vivo experiments followed the guidelines of the International Council for Laboratory Animal Science (ICLAS) and were approved by the Ethical Committee for Animal Experimentation of UFCSPA (08404). All efforts were made to minimize animal suffering and to use only the number of animals necessary to generate reliable data. 2.2. Drugs Insulin (dose, 4 IU/mL) was administered intraperitoneally (i.p.) (Humulin® , Lilly, USA). CNZ (0.25 mg/mL; Rivotril® , Roche, Brazil) was prepared in saline with Tween 0.05% (v/v) and streptozotocin (60 mg/mL; Sigma, St. Louis, MO, USA) was prepared in citrate buffer (pH 4.3). All solutions were prepared immediately before i.p. administration. 2.3. Diabetes induction Diabetes was induced by a single i.p. dose of STZ, 60 mg/kg, as already described [10]. Increased blood glucose levels (≥250 mg/dL) of STZ-rats (blood collected from tail) were confirmed with a glucometer (AccuChek Aviva® , Roche, Germany) after 72 h. Nondiabetic control rats received i.p. injections of saline (1 mL/kg) and were also submitted to blood glucose measurement. 2.4. Forced swimming test (FST) The design of the experiments is summarized in Fig. 1. After 21 days of diabetes induction, animals were submitted to the FST [10,19]. On the first day of the experiment, 24 h before the FST, the animals were placed in the aquarium for 15 min (22 cm × 22 cm × 35 cm) with water of height 27 cm (temperature, 24–26 ◦ C). Soon after, the rats were dried with towels and the first drug dose was administered (insulin (4 IU/kg i.p.), clonazepam (0.25 mg/kg i.p.), insulin + clonazepam or saline (1 mL/kg i.p.)). 5 h and 1 h before being submitted to the FST, the animals were again dosed with the indicated treatment. The FST session was recorded on videotape for subsequent analysis. 2.5. Blood sample Animals were sacrificed by decapitation 30 min after the FST. Whole blood was collected aseptically in heparinized vials and stored at 4 ◦ C until analysis. 2.6. Single cell gel electrophoresis (comet assay) The alkaline comet assay was performed as described by Singh et al. [20] in accordance with general guidelines for use of the assay [21,22]. Isolated rat leukocytes
Fig. 2. Glycemia from streptozotocin-induced diabetic rats not treated (STZ) or treated with insulin (STZ–INS), clonazepam (STZ–CNZ), or insulin + clonazepam (STZ–INS–CNZ) and submitted to the forced swimming test (n = 12–13), and controls (n = 8). Data represent mean ± S.D. *p < 0.05 compared to the control; # p < 0.05 compared to the diabetic group (ANOVA followed by the Duncan test). were suspended in agarose and spread onto a glass microscope slide pre-coated with agarose. Agarose was allowed to set at 4 ◦ C for 5 min. Slides were incubated in icecold lysis solution [12.35% NaCl (2.5 M), 3.15% EDTA (100 mM), 0.10% TRIS (10 mM), 0.68% NaOH (30 mM), 0.84% sodium sarcosinate, 82.88% distilled water] to remove cell proteins, leaving DNA as “nucleoids”. After the lysis procedure, slides were placed on a horizontal electrophoresis unit and covered with fresh buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min at 4 ◦ C to allow DNA unwinding and the expression of alkali-labile-sites. Electrophoresis was performed for 20 min (25 V; 300 mA; 0.9 V/cm). The cells were then neutralized, washed in double-distilled water, and stained according to a silver-staining protocol [23]. After drying at room temperature overnight, gels were analyzed using an optical microscope. One hundred cells (50 cells from each of the two replicate slides) were selected and analyzed. Cells were visually scored according to tail length and received scores from 0 (no migration) to 4 (maximal migration) according to tail intensity. Therefore, the damage index (DI) for cells ranged from 0 (all cells with no migration) to 400 (all cells with maximal migration). The slides were analyzed under blind conditions by at least two different individuals. 2.7. Statistical analyses Blood glucose measurement and comet assay data were expressed as mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA), followed by the Duncan multiple range test when the F value was significant. A p value <0.05 was considered significant. The Pearson correlation test was used to evaluate the correlation between the variables. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software.
3. Results Table 1 shows the DI values and the number of cells found in each damage class for each of the five groups of animals. Fig. 2
C.A.Y. Wayhs et al. / Mutation Research 703 (2010) 187–190 Table 1 DI values and number of cells found in each damage class in the different groups. Groups
Control
Number of rats
9
DI (mean ± S.D.)
Damage class 0
1
2
3
4
608
272
17
2
0
487
316
75 20
2
661
405
32
2
0
556
394
35 15
0
752
429
18
0
35.22 ± 3.80
STZ
9
59.22 ± 14.8
STZ–INS
11
STZ–CNZ
10
50.90 ± 3.87
STZ–INS–CNZ
12
39.00 ± 5.83
43.18 ± 6.06
1
DI: damage index.
shows the glycemia status of the animals, 30 min post-FST (before sacrifice). Acute treatment with insulin or insulin plus clonazepam significantly decreased glycemia (p < 0.05). Fig. 3 shows the effect of treatment with insulin and/or CNZ on DNA damage in peripheral blood leukocytes of STZ-induced diabetic rats and controls submitted to FST. We verified that the damage index was significantly increased in STZ group (DI = 59.22 ± 14.86) when compared to the control group (35.22 ± 3.80). Additionally, STZ animals treated with insulin (43.18 ± 6.06) or CNZ (50.90 ± 3.87) presented a significant decrease in damage index, when compared to untreated rats. Furthermore, the damage index (39.00 ± 5.83) was reduced to control levels in STZ-rats treated with insulin plus CNZ. A significant positive correlation between glycemia and damage index (r = 0.4460; p < 0.05) was observed in treated and untreated STZ-rats submitted to the FST. 4. Discussion Oxidative stress has been suggested to be a contributor to the development of DM complications. Growing evidence indicates that oxidative stress is increased in diabetes, due to the overproduction of reactive oxygen species (ROS) and decreased efficiency of antioxidant defenses as a result of hyperglycemia [24]. Oxidation of lipids, proteins, and other macromolecules, such as DNA, occurs during the development of DM and its complications [25].
Fig. 3. DNA damage (comet assay) of peripheral blood leukocytes from streptozotocin-induced diabetic rats not treated (STZ) or treated with insulin (STZ–INS), clonazepam (STZ–CNZ), or insulin + clonazepam (STZ–INS–CNZ) and submitted to the forced swimming test (n = 9–12), and controls (n = 9). Data represent mean ± S.D. *p < 0.05 compared to the control; # p < 0.05 compared to STZ group; a p < 0.05 compared to STZ–INS. b p < 0.05 compared to STZ–CNZ (ANOVA followed by the Duncan test).
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Some studies have demonstrated a significantly higher DNA damage in lymphocytes from STZ-induced diabetic rats, as measured by percentage of tail DNA in the comet assay, when compared to nondiabetic rats [26,27]. Tail moment, defined as the product of the tail length and the fraction of DNA in the tail, was also elevated in diabetic rats [28]. Studies with the comet assay have shown increased levels of oxidative DNA breakage in peripheral blood lymphocytes of diabetic patients with poor glycemia control, but not in patients with normal glycemia [29,30]. Dandona et al. [31] reported that, in both type 1 and type 2 DM patients, oxidative DNA damage was higher than in a healthy control group. Type 1 male and female DM patients were investigated by Dinc¸er et al. [32], who verified that DNA damage in mononuclear leukocytes, analyzed by the comet assay, was significantly increased when compared to controls. Considering that: (1) the risk of developing depression is greater in patients with diabetes than in the general population [33]; (2) insulin modulates GABA and/or glutamate transport, thus having a neuroprotective role under oxidizing and/or diabetic conditions [14]; (3) oxidative stress occurs in diabetic rats submitted to an experimental depression model; and (4) CNZ exerts a protective effect against oxidative stress, decreasing immobility in the FST of diabetic rats [12], our goal was to evaluate the effect of acute treatment with insulin and/or CNZ on DNA damage in leukocytes from streptozotocin-induced diabetic rats submitted to the FST. We determined DNA damage in leukocytes via the alkaline comet assay and confirmed increased levels of DNA migration, and thus DNA damage, in leukocytes from diabetic group, when compared to the control group, in agreement with the literature [26–28]. STZ animals treated with insulin or clonazepam presented a significant decrease in DNA damage index and treatment with insulin plus clonazepam reduced DNA migration to control levels. A significant positive correlation was observed between glycemia and DNA damage index in STZ-induced diabetic rats. Our results confirm previous studies showing that DNA damage in diabetes can be provoked by an oxidative event, since hyperglycemia leads to high free radicals production, attacking essential molecules such as DNA. Comparing the distribution of damage classes in the groups studied, we found that the differences were caused primarily by an increased number of cells in damage class 1, reflecting a homogeneously distributed increase in the number of slightly damaged cells, rather than a few highly damaged cells. On the other hand, a significant number of cells presented damage classes 2 and 3 in diabetic rats, when compared to the other groups studied. Only one animal of the diabetic group presented cells with damage class 4. Combination of the two treatments (insulin plus CNZ) gave better results, in terms of DNA damage, compared to either treatment alone, since the distributions of cells in damage classes 3 and 4 were very similar to the control group. Our findings show that DNA damage index is increased in this diabetes/depression animal model and treatment with insulin and CNZ can protect against DNA damage. The effect of insulin can be explained by the decrease of hyperglycemia, since this pathological chronic state leads to oxidative stress, increased production of free radicals, and reduction in antioxidant potential [24]. Oxidative stress produces DNA base modifications, strand breakage, and other DNA damages [34]. Acute treatment with insulin may be acting as an antioxidant scavenger, since Kocic et al. [35] demonstrated that the application of an intensive insulin treatment regimen significantly improves total antioxidant plasma capacity, as compared to the application of conventional insulin therapy regimen. In addiction, Monnier et al. [36] showed an independent inhibitory effect of insulin therapy on oxidative stress in patients with type 1 and type 2 diabetes.
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In a previous study [12], it was demonstrated that CNZ treatment reduced immobility time in diabetic rats submitted to the FST, suggesting that this drug might be clinically useful for depressed diabetic patients. CNZ exerts an antioxidant effect in STZ-induced diabetic rats submitted to the FST, protecting against the action of free radicals which can contribute for the development of longterm diabetes complications, such as oxidative DNA damage. This could be related to the reduced GABA levels, through destruction of GABAergic neurons in diabetic encephalopathy. CNZ may significantly decrease glucose tolerance, insulin secretion, and glucose effectiveness at basal insulin levels in volunteers [37]. Diabetic animals present low levels of GABA in the CNS [38], as well as a reduction of dopamine and serotonin. Diabetic patients have high titers of anti-glutamic acid decarboxylase (the enzyme responsible for the synthesis of GABA in CNS) autoantibodies (antiGAD) in serum and cerebrospinal fluid [39]. Oxidative stress could lead to destruction of GABAergic neurons, which could be involved in the pathophysiology of depression in diabetes; CNZ plus insulin could have a protect action on this process. In conclusion, this work provides evidence that DNA damage is increased in streptozotocin-induced diabetic rats submitted to forced swimming test and that treatment with insulin plus clonazepam can protect against oxidative DNA damage. Conflict of interest
[14]
[15]
[16]
[17]
[18] [19] [20]
[21]
[22]
[23] [24] [25]
The authors declare that there are no conflicts of interest. Acknowledgements
[26]
This work was supported in part by grants from FAPERGS, CAPES, CNPq and FIPE/HCPA-Brazil.
[27]
References
[28]
[1] J. Apelqvist, K. Bakker, W.H. Van Houtum, N.C. Schaper, Practical guidelines on the management and prevention of the diabetic foot: based upon the International Consensus on the Diabetic Foot (2007) Prepared by the International Working Group on the Diabetic Foot, Diabetes Metab. Res. Rev. 24 (2008) 116–118. [2] L.L. Bellush, S.G. Reid, D. North, The functional significance of biochemical alterations in streptozotocin-induced diabetes, Physiol. Behav. 50 (1991) 973–981. [3] W.R. Miles, H.F. Root, Psychologic tests applied to diabetic patients, Arch. Int. Med. 30 (1922) 767–777. [4] G.J. Biessels, L.P. Van Der Heide, A. Kamal, R.L. Bleys, W.H. Gispen, Aging and diabetes: implications for brain function, Eur. J. Pharmacol. 441 (2002) 1–14. [5] A.A. Sima, H. Kamiya, Z.G. Li, Insulin C-peptide, hyperglycemia, and central nervous system complications in diabetes, Eur. J. Pharmacol. 490 (2004) 187–197. [6] J.A. Gavard, P.J. Lustman, R.E. Clouse, Prevalence of depression in adults with diabetes, Diabetes Care 16 (1993) 1167–1178. [7] J.F. Téllez-Zentero, M.H. Cardiel, Risk factors associated with depression in patients with type 2 diabetes mellitus, Arch. Med. Res. 33 (2002) 53–60. [8] P.J. Lustman, L.S. Griffith, J.A. Gavard, R.E. Clouse, Depression in adults with diabetes, Diabetes Care 15 (1992) 1631–1639. [9] A. Shehatah, M.A. Rabie, A. Al-Shahry, Prevalence and correlates of depressive disorders in elderly with type 2 diabetes in primary health care settings, J. Affect. Disord. 123 (2010) 197–201. [10] R. Gomez, H.M.T. Barros, Ethopharmacology of the antidepressant effect of clonazepam in diabetic rats, Pharmacol. Biochem. Behav. 66 (2000) 329–335. [11] L.A. Hilakivi-Clarke, K.M. Wozniak, M.J. Durcan, M. Linnoila, Behavior of streptozotocin-diabetic mice in tests of exploration, locomotion, anxiety, depression and aggression, Physiol. Behav. 48 (1990) 429–433. [12] A.S. Haeser, A. Sitta, A.G. Barschak, M. Deon, A.T. Barden, G.O. Schmitt, S. Landgraff, R. Gomez, H.M. Barros, C.R. Vargas, Oxidative stress parameters in diabetic rats submitted to forced swimming test: the clonazepam effect, Brain Res. 1154 (2007) 335–338. [13] L.L. Guyot, F.G. Diaz, M.H. O’regan, D. Song, J.W. Phillis, The effect of streptozotocin-induced diabetes on the release of excitotoxic and other amino
[29]
[30]
[31] [32]
[33] [34]
[35]
[36]
[37]
[38] [39]
acids from the ischemic rat cerebral cortex, Neurosurgery 48 (2001) 385– 391. A.I. Duarte, M.S. Santos, R. Seica, C.R. Oliveira, Oxidative stress affects synaptosomal gamma-aminobutyric acid and glutamate transport in diabetic rats: the role of insuline, Diabetes 53 (2004) 2110–2116. G. Rajtar, D. Zolkowska, Z. Keinrok, Effect of diazepam and clonazepam on the function of isolated rat platelet and neutrophils, Intern. Med. J. Exp. Clin. Res. 8 (2002) 37–44. V. Imaeda, T. Aoki, Y. Kondo, M. Hori, M. Ogata, H. Obayashi, G. Hasegawa, N. Nakamura, K. Tokuda, H. Nishino, T. Yoshikawa, M. Kondo, Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis, Free Radic. Res. 34 (2001) 33–44. D. Bonnefont-Rousselot, J.L. Beaudeux, P. Therond, J. Peynet, A. Legrand, J. Delattre, Diabetes mellitus, oxidative stress and advanced glycation endproducts, Ann. Pharm. Fr. 62 (2004) 147–157. B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd ed., Oxford University Press, New York, 2007. R.D. Porsolt, M. Le Pichon, M. Jalfre, Depression: a new model sensitive to antidepressant treatment, Nature 266 (1977) 730–732. N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantification of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184–191. R.R. Tice, D. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35 (2000) 206–221. A. Hartmann, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A. Collins, A. Smith, G. Speit, V. Thybaud, R.R. Tice, Recommendations for conducting the in vivo alkaline comet assay, Mutagenesis 18 (2003) 45–51. S. Nadin, L. Vargas-Roig, D. Ciocca, A silver staining method for single-cell gel assay, J. Histochem. Cytochem. 49 (2001) 1183–1186. N.F. Wiernsperger, Oxidative stress as a therapeutic target in diabetes: revisiting the controversy, Diabetes Metab. 29 (2003) 579–585. R. Stetina, J. Varvarovska, Z. Rusavy, R. Pomahacova, J. Racek, S. Lacigov, L. Trefil, K. Siala, F. Stozicky, Oxidative stress DNA damage and DNA repair capacity in children with type 1 diabetes mellitus, Toxicol. Lett. 164 (2006) S134. H. Wu, H. Guo, R. Zhao, Effect of Lycium barbarum polysaccharide on the improvement of antioxidant ability and DNA damage in NIDDM rats, Yakugaku Zasshi 126 (2006) 365–371. A. Fidan, Y. Dündar, The effects of Yucca schidigera and Quillaja saponaria on DNA damage, protein oxidation, lipid peroxidation, and some biochemical parameters in streptozotocin-induced diabetic rats, J. Diabetes Complications 22 (2008) 348–356. B.S. Balkis, R.N. Fadilah, A. Ezlan, O. Khairul, A.B. Mokhtar, M. Jamaludin, Oxidative stress and DNA damage in streptozotocin-induced diabetic rats: effects of alpha lipoic acid, J. Biol. Sci. 8 (2008) 622–627. S.H. Saydah, E.A. Platz, N. Rifai, M.N. Pollak, F.L. Brancati, K.J. Helzlsouer, Association of markers of insulin and glucose control with subsequent colorectal cancer risk, Cancer Epidemiol. Biomarkers Prev. 12 (2003) 412–418. D. Anderson, T.W. Yu, J. Wright, C. Ioannides, An examination of DNA strand breakage in the comet assay and antioxidant capacity in diabetic patients, Mutat. Res. 398 (1998) 151–161. P. Dandona, K. Thuru, S. Cook, B. Snyder, J. Makowski, D. Armstrong, T. Nicotera, Oxidative damage to DNA in diabetes mellitus, Lancet 347 (1996) 444–445. Y. Dinc¸er, T. Akc¸ay, H. I˙ lkova, Z. Alademir, G. Ozbay, DNA damage and antioxidant defense in peripheral leukocytes of patients with Type I diabetes mellitus, Mutat. Res. 527 (2003) 49–55. E.E. Leonard, Effects of depression on work loss and disability bed days in individuals with diabetes, Diabetes Care 27 (2004) 1751–1753. A. Imaeda, T. Kaneko, T. Aoki, Y. Kondo, H. Nagase, DNA damage and the effect of antioxidants in streptozotocin-treated mice, Food Chem. Toxicol. 40 (2002) 979–987. R. Kocic, D. Pavlovic, G. Kocic, Impact of intensive insulin treatment on the development and consequences of oxidative stress in insulin-dependent diabetes mellitus, Vojnosanit Pregled. 64 (2007) 623–628. L. Monnier, C. Colette, E. Mas, F. Michel, J.P. Cristol, C. Boegner, D.R. Owens, Regulation of oxidative stress by glycaemic control: evidence for an independent inhibitory effect of insulin therapy, Diabetologia 53 (2010) 562–571. H. Chevassus, I. Mourand, N. Molinier, B. Lacarelle, J.F. Brun, P. Petit, Assessment of single-dose benzodiazepines on insulin secretion, insulin sensitivity and glucose effectiveness in healthy volunteers: a double-bind, placebo-controlled, randomized cross-over trial, BMC Clin. Pharmacol. 4 (2004) 4: 3. D.A. Pearce, M. Atkinson, D.A. Tagle, Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders, Neurology 63 (2004) 2001–2005. T. Yoshimoto, M. Doi, N. Fukai, H. Izumiyama, T. Wago, I. Minami, I. Uchimura, Y. Hirata, Type 1 diabetes mellitus and drug-resistant epilepsy: presence of high titer of anti-glutamic acid decarboxylase autoantibodies in serum and cerebrospinal fluid, Intern. Med. 44 (2005) 1174–1177.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Effects of insulin and clonazepam on DNA damage in diabetic rats submitted to the forced swimming test Carlos Alberto Yasin Wayhs a,b,∗ , Vanusa Manfredini a,b , Angela Sitta b,c , Marion Deon b,c , Graziela S. Ribas a,b , Camila S. Vanzin b , Giovana B. Biancini b , Maurício S. Nin d , Helena M.T. Barros d , Carmen Regla Vargas a,b,c,∗ a
Programa de Pós-Graduac¸ão em Ciências Farmacêuticas, Porto Alegre, RS, Brazil Servic¸o de Genética Médica, HCPA, Porto Alegre, RS, Brazil c Programa de Pós-Graduac¸ão em Ciências Biológicas: Bioquímica, UFRGS, Porto Alegre, RS, Brazil d Departamento de Farmacologia, UFCSPA, Porto Alegre, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 24 May 2010 Received in revised form 18 August 2010 Accepted 26 August 2010 Available online 9 September 2010 Keywords: Comet assay DNA damage Diabetes Depression Insulin Clonazepam Streptozotocin
a b s t r a c t Diabetes mellitus (DM) is a chronic hyperglycemic state. DM may be associated with moderate cognitive deficits and neurophysiologic/structural changes in the brain (diabetic encephalopathy). Psychiatric manifestations seem to accompany this encephalopathy, since the prevalence of depression in diabetic patients is much higher than in the general population, and clonazepam is being used to treat this complication. The excessive production of oxygen free radicals that may occur in diabetes induces a variety of lesions in macromolecules, including DNA. In this work, we analyzed DNA damage in leukocytes from streptozotocin-induced diabetic rats submitted to the forced swimming test. The DNA damage index was significantly elevated (DI = 61.00 ± 4.95) in the diabetic group compared to the control group (34.00 ± 1.26). Significant reductions of the damage index were observed in diabetic animals treated with insulin (45.00 ± 1.82), clonazepam (52.00 ± 1.22), or both agents (39.00 ± 5.83, not significantly different from control levels). Insulin plus clonazepam can protect against DNA damage in stressed diabetic rats. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a metabolic disorder characterized by a hyperglycemic chronic state that can lead to complications affecting the retina, kidney, muscle, blood vessels, and also the central nervous system (CNS). DM is regarded as one of the major metabolic diseases of the 21st C. [1,2]. Cognitive dysfunction is developed by some diabetic individuals [3]. A wealth of studies have described neuropsychological and neurobehavioral changes in both type 1 and type 2 diabetic subjects, suggesting that diabetic encephalopathy should be recognized as a complication of diabetes [4,5]. Psychiatric manifestations may accompany this encephalopathy, since the prevalence of depression in diabetic patients is much higher than in the general population [6–9].
∗ Corresponding authors at: Servic¸o de Genética Médica, HCPA, Rua Ramiro Barcelos, 2350 CEP 90.035-903, Porto Alegre, RS, Brazil. Tel.: +55 51 3359 8011; fax: +55 51 3359 8010. E-mail addresses: [email protected] (C.A.Y. Wayhs), [email protected] (C.R. Vargas). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.08.017
Diabetic encephalopathy can be modeled in experimental animals; diabetic mice and rats exhibit depressive-like behavior when submitted to the forced swimming test (FST) [10–12]. Some studies have demonstrated that insulin administration can prevent neuronal damage in the cortex of streptozotocin (STZ)-induced diabetic rats and also produce behavioral changes [11,13]. Insulin also affects synaptosomal ␥-aminobutyric acid (GABA) and glutamate transport under oxidative stress conditions [14] and clonazepam (CNZ), a positive GABAA receptor modulator, shows an antidepressant effect in these animals [10,15]. Oxidative stress may play a role in the development of diabetic complications [16], since hyperglycemia generates abnormally high levels of free radicals via autoxidation of glucose and protein glycation [17]. These free radicals induce a variety of lesions in DNA, including oxidized bases, abasic sites, strand breaks, and formation of DNA–protein cross-links. Hydroxyl radical, produced by the Fenton reaction in the presence of transition metal ions, is responsible for this DNA damage [18]. Using the comet assay, we have investigated DNA damage in peripheral whole blood leukocytes from streptozotocin-induced diabetic rats submitted to the FST, and we have evaluated the effects of acute treatment with insulin and/or clonazepam upon this process.
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Fig. 1. Time-line chart of the experiment.
2. Materials and methods 2.1. Animals Male Wistar rats (250 ± 50 g) were obtained from the Animal House of Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). The animals were housed in groups of four per polypropylene cage. Food and water were available ad libitum, except where otherwise stated, and the animals were maintained in a temperature-controlled room (22 ± 2 ◦ C) under a light–dark cycle (7:00 a.m.–7:00 p.m.). The animals were divided into five groups: control (nondiabetic); diabetic (STZ); diabetic plus insulin (STZ–INS); diabetic plus clonazepam (STZ–CNZ); and diabetic plus insulin and clonazepam (STZ–INS–CNZ). All in vivo experiments followed the guidelines of the International Council for Laboratory Animal Science (ICLAS) and were approved by the Ethical Committee for Animal Experimentation of UFCSPA (08404). All efforts were made to minimize animal suffering and to use only the number of animals necessary to generate reliable data. 2.2. Drugs Insulin (dose, 4 IU/mL) was administered intraperitoneally (i.p.) (Humulin® , Lilly, USA). CNZ (0.25 mg/mL; Rivotril® , Roche, Brazil) was prepared in saline with Tween 0.05% (v/v) and streptozotocin (60 mg/mL; Sigma, St. Louis, MO, USA) was prepared in citrate buffer (pH 4.3). All solutions were prepared immediately before i.p. administration. 2.3. Diabetes induction Diabetes was induced by a single i.p. dose of STZ, 60 mg/kg, as already described [10]. Increased blood glucose levels (≥250 mg/dL) of STZ-rats (blood collected from tail) were confirmed with a glucometer (AccuChek Aviva® , Roche, Germany) after 72 h. Nondiabetic control rats received i.p. injections of saline (1 mL/kg) and were also submitted to blood glucose measurement. 2.4. Forced swimming test (FST) The design of the experiments is summarized in Fig. 1. After 21 days of diabetes induction, animals were submitted to the FST [10,19]. On the first day of the experiment, 24 h before the FST, the animals were placed in the aquarium for 15 min (22 cm × 22 cm × 35 cm) with water of height 27 cm (temperature, 24–26 ◦ C). Soon after, the rats were dried with towels and the first drug dose was administered (insulin (4 IU/kg i.p.), clonazepam (0.25 mg/kg i.p.), insulin + clonazepam or saline (1 mL/kg i.p.)). 5 h and 1 h before being submitted to the FST, the animals were again dosed with the indicated treatment. The FST session was recorded on videotape for subsequent analysis. 2.5. Blood sample Animals were sacrificed by decapitation 30 min after the FST. Whole blood was collected aseptically in heparinized vials and stored at 4 ◦ C until analysis. 2.6. Single cell gel electrophoresis (comet assay) The alkaline comet assay was performed as described by Singh et al. [20] in accordance with general guidelines for use of the assay [21,22]. Isolated rat leukocytes
Fig. 2. Glycemia from streptozotocin-induced diabetic rats not treated (STZ) or treated with insulin (STZ–INS), clonazepam (STZ–CNZ), or insulin + clonazepam (STZ–INS–CNZ) and submitted to the forced swimming test (n = 12–13), and controls (n = 8). Data represent mean ± S.D. *p < 0.05 compared to the control; # p < 0.05 compared to the diabetic group (ANOVA followed by the Duncan test). were suspended in agarose and spread onto a glass microscope slide pre-coated with agarose. Agarose was allowed to set at 4 ◦ C for 5 min. Slides were incubated in icecold lysis solution [12.35% NaCl (2.5 M), 3.15% EDTA (100 mM), 0.10% TRIS (10 mM), 0.68% NaOH (30 mM), 0.84% sodium sarcosinate, 82.88% distilled water] to remove cell proteins, leaving DNA as “nucleoids”. After the lysis procedure, slides were placed on a horizontal electrophoresis unit and covered with fresh buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min at 4 ◦ C to allow DNA unwinding and the expression of alkali-labile-sites. Electrophoresis was performed for 20 min (25 V; 300 mA; 0.9 V/cm). The cells were then neutralized, washed in double-distilled water, and stained according to a silver-staining protocol [23]. After drying at room temperature overnight, gels were analyzed using an optical microscope. One hundred cells (50 cells from each of the two replicate slides) were selected and analyzed. Cells were visually scored according to tail length and received scores from 0 (no migration) to 4 (maximal migration) according to tail intensity. Therefore, the damage index (DI) for cells ranged from 0 (all cells with no migration) to 400 (all cells with maximal migration). The slides were analyzed under blind conditions by at least two different individuals. 2.7. Statistical analyses Blood glucose measurement and comet assay data were expressed as mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA), followed by the Duncan multiple range test when the F value was significant. A p value <0.05 was considered significant. The Pearson correlation test was used to evaluate the correlation between the variables. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software.
3. Results Table 1 shows the DI values and the number of cells found in each damage class for each of the five groups of animals. Fig. 2
C.A.Y. Wayhs et al. / Mutation Research 703 (2010) 187–190 Table 1 DI values and number of cells found in each damage class in the different groups. Groups
Control
Number of rats
9
DI (mean ± S.D.)
Damage class 0
1
2
3
4
608
272
17
2
0
487
316
75 20
2
661
405
32
2
0
556
394
35 15
0
752
429
18
0
35.22 ± 3.80
STZ
9
59.22 ± 14.8
STZ–INS
11
STZ–CNZ
10
50.90 ± 3.87
STZ–INS–CNZ
12
39.00 ± 5.83
43.18 ± 6.06
1
DI: damage index.
shows the glycemia status of the animals, 30 min post-FST (before sacrifice). Acute treatment with insulin or insulin plus clonazepam significantly decreased glycemia (p < 0.05). Fig. 3 shows the effect of treatment with insulin and/or CNZ on DNA damage in peripheral blood leukocytes of STZ-induced diabetic rats and controls submitted to FST. We verified that the damage index was significantly increased in STZ group (DI = 59.22 ± 14.86) when compared to the control group (35.22 ± 3.80). Additionally, STZ animals treated with insulin (43.18 ± 6.06) or CNZ (50.90 ± 3.87) presented a significant decrease in damage index, when compared to untreated rats. Furthermore, the damage index (39.00 ± 5.83) was reduced to control levels in STZ-rats treated with insulin plus CNZ. A significant positive correlation between glycemia and damage index (r = 0.4460; p < 0.05) was observed in treated and untreated STZ-rats submitted to the FST. 4. Discussion Oxidative stress has been suggested to be a contributor to the development of DM complications. Growing evidence indicates that oxidative stress is increased in diabetes, due to the overproduction of reactive oxygen species (ROS) and decreased efficiency of antioxidant defenses as a result of hyperglycemia [24]. Oxidation of lipids, proteins, and other macromolecules, such as DNA, occurs during the development of DM and its complications [25].
Fig. 3. DNA damage (comet assay) of peripheral blood leukocytes from streptozotocin-induced diabetic rats not treated (STZ) or treated with insulin (STZ–INS), clonazepam (STZ–CNZ), or insulin + clonazepam (STZ–INS–CNZ) and submitted to the forced swimming test (n = 9–12), and controls (n = 9). Data represent mean ± S.D. *p < 0.05 compared to the control; # p < 0.05 compared to STZ group; a p < 0.05 compared to STZ–INS. b p < 0.05 compared to STZ–CNZ (ANOVA followed by the Duncan test).
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Some studies have demonstrated a significantly higher DNA damage in lymphocytes from STZ-induced diabetic rats, as measured by percentage of tail DNA in the comet assay, when compared to nondiabetic rats [26,27]. Tail moment, defined as the product of the tail length and the fraction of DNA in the tail, was also elevated in diabetic rats [28]. Studies with the comet assay have shown increased levels of oxidative DNA breakage in peripheral blood lymphocytes of diabetic patients with poor glycemia control, but not in patients with normal glycemia [29,30]. Dandona et al. [31] reported that, in both type 1 and type 2 DM patients, oxidative DNA damage was higher than in a healthy control group. Type 1 male and female DM patients were investigated by Dinc¸er et al. [32], who verified that DNA damage in mononuclear leukocytes, analyzed by the comet assay, was significantly increased when compared to controls. Considering that: (1) the risk of developing depression is greater in patients with diabetes than in the general population [33]; (2) insulin modulates GABA and/or glutamate transport, thus having a neuroprotective role under oxidizing and/or diabetic conditions [14]; (3) oxidative stress occurs in diabetic rats submitted to an experimental depression model; and (4) CNZ exerts a protective effect against oxidative stress, decreasing immobility in the FST of diabetic rats [12], our goal was to evaluate the effect of acute treatment with insulin and/or CNZ on DNA damage in leukocytes from streptozotocin-induced diabetic rats submitted to the FST. We determined DNA damage in leukocytes via the alkaline comet assay and confirmed increased levels of DNA migration, and thus DNA damage, in leukocytes from diabetic group, when compared to the control group, in agreement with the literature [26–28]. STZ animals treated with insulin or clonazepam presented a significant decrease in DNA damage index and treatment with insulin plus clonazepam reduced DNA migration to control levels. A significant positive correlation was observed between glycemia and DNA damage index in STZ-induced diabetic rats. Our results confirm previous studies showing that DNA damage in diabetes can be provoked by an oxidative event, since hyperglycemia leads to high free radicals production, attacking essential molecules such as DNA. Comparing the distribution of damage classes in the groups studied, we found that the differences were caused primarily by an increased number of cells in damage class 1, reflecting a homogeneously distributed increase in the number of slightly damaged cells, rather than a few highly damaged cells. On the other hand, a significant number of cells presented damage classes 2 and 3 in diabetic rats, when compared to the other groups studied. Only one animal of the diabetic group presented cells with damage class 4. Combination of the two treatments (insulin plus CNZ) gave better results, in terms of DNA damage, compared to either treatment alone, since the distributions of cells in damage classes 3 and 4 were very similar to the control group. Our findings show that DNA damage index is increased in this diabetes/depression animal model and treatment with insulin and CNZ can protect against DNA damage. The effect of insulin can be explained by the decrease of hyperglycemia, since this pathological chronic state leads to oxidative stress, increased production of free radicals, and reduction in antioxidant potential [24]. Oxidative stress produces DNA base modifications, strand breakage, and other DNA damages [34]. Acute treatment with insulin may be acting as an antioxidant scavenger, since Kocic et al. [35] demonstrated that the application of an intensive insulin treatment regimen significantly improves total antioxidant plasma capacity, as compared to the application of conventional insulin therapy regimen. In addiction, Monnier et al. [36] showed an independent inhibitory effect of insulin therapy on oxidative stress in patients with type 1 and type 2 diabetes.
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In a previous study [12], it was demonstrated that CNZ treatment reduced immobility time in diabetic rats submitted to the FST, suggesting that this drug might be clinically useful for depressed diabetic patients. CNZ exerts an antioxidant effect in STZ-induced diabetic rats submitted to the FST, protecting against the action of free radicals which can contribute for the development of longterm diabetes complications, such as oxidative DNA damage. This could be related to the reduced GABA levels, through destruction of GABAergic neurons in diabetic encephalopathy. CNZ may significantly decrease glucose tolerance, insulin secretion, and glucose effectiveness at basal insulin levels in volunteers [37]. Diabetic animals present low levels of GABA in the CNS [38], as well as a reduction of dopamine and serotonin. Diabetic patients have high titers of anti-glutamic acid decarboxylase (the enzyme responsible for the synthesis of GABA in CNS) autoantibodies (antiGAD) in serum and cerebrospinal fluid [39]. Oxidative stress could lead to destruction of GABAergic neurons, which could be involved in the pathophysiology of depression in diabetes; CNZ plus insulin could have a protect action on this process. In conclusion, this work provides evidence that DNA damage is increased in streptozotocin-induced diabetic rats submitted to forced swimming test and that treatment with insulin plus clonazepam can protect against oxidative DNA damage. Conflict of interest
[14]
[15]
[16]
[17]
[18] [19] [20]
[21]
[22]
[23] [24] [25]
The authors declare that there are no conflicts of interest. Acknowledgements
[26]
This work was supported in part by grants from FAPERGS, CAPES, CNPq and FIPE/HCPA-Brazil.
[27]
References
[28]
[1] J. Apelqvist, K. Bakker, W.H. Van Houtum, N.C. Schaper, Practical guidelines on the management and prevention of the diabetic foot: based upon the International Consensus on the Diabetic Foot (2007) Prepared by the International Working Group on the Diabetic Foot, Diabetes Metab. Res. Rev. 24 (2008) 116–118. [2] L.L. Bellush, S.G. Reid, D. North, The functional significance of biochemical alterations in streptozotocin-induced diabetes, Physiol. Behav. 50 (1991) 973–981. [3] W.R. Miles, H.F. Root, Psychologic tests applied to diabetic patients, Arch. Int. Med. 30 (1922) 767–777. [4] G.J. Biessels, L.P. Van Der Heide, A. Kamal, R.L. Bleys, W.H. Gispen, Aging and diabetes: implications for brain function, Eur. J. Pharmacol. 441 (2002) 1–14. [5] A.A. Sima, H. Kamiya, Z.G. Li, Insulin C-peptide, hyperglycemia, and central nervous system complications in diabetes, Eur. J. Pharmacol. 490 (2004) 187–197. [6] J.A. Gavard, P.J. Lustman, R.E. Clouse, Prevalence of depression in adults with diabetes, Diabetes Care 16 (1993) 1167–1178. [7] J.F. Téllez-Zentero, M.H. Cardiel, Risk factors associated with depression in patients with type 2 diabetes mellitus, Arch. Med. Res. 33 (2002) 53–60. [8] P.J. Lustman, L.S. Griffith, J.A. Gavard, R.E. Clouse, Depression in adults with diabetes, Diabetes Care 15 (1992) 1631–1639. [9] A. Shehatah, M.A. Rabie, A. Al-Shahry, Prevalence and correlates of depressive disorders in elderly with type 2 diabetes in primary health care settings, J. Affect. Disord. 123 (2010) 197–201. [10] R. Gomez, H.M.T. Barros, Ethopharmacology of the antidepressant effect of clonazepam in diabetic rats, Pharmacol. Biochem. Behav. 66 (2000) 329–335. [11] L.A. Hilakivi-Clarke, K.M. Wozniak, M.J. Durcan, M. Linnoila, Behavior of streptozotocin-diabetic mice in tests of exploration, locomotion, anxiety, depression and aggression, Physiol. Behav. 48 (1990) 429–433. [12] A.S. Haeser, A. Sitta, A.G. Barschak, M. Deon, A.T. Barden, G.O. Schmitt, S. Landgraff, R. Gomez, H.M. Barros, C.R. Vargas, Oxidative stress parameters in diabetic rats submitted to forced swimming test: the clonazepam effect, Brain Res. 1154 (2007) 335–338. [13] L.L. Guyot, F.G. Diaz, M.H. O’regan, D. Song, J.W. Phillis, The effect of streptozotocin-induced diabetes on the release of excitotoxic and other amino
[29]
[30]
[31] [32]
[33] [34]
[35]
[36]
[37]
[38] [39]
acids from the ischemic rat cerebral cortex, Neurosurgery 48 (2001) 385– 391. A.I. Duarte, M.S. Santos, R. Seica, C.R. Oliveira, Oxidative stress affects synaptosomal gamma-aminobutyric acid and glutamate transport in diabetic rats: the role of insuline, Diabetes 53 (2004) 2110–2116. G. Rajtar, D. Zolkowska, Z. Keinrok, Effect of diazepam and clonazepam on the function of isolated rat platelet and neutrophils, Intern. Med. J. Exp. Clin. Res. 8 (2002) 37–44. V. Imaeda, T. Aoki, Y. Kondo, M. Hori, M. Ogata, H. Obayashi, G. Hasegawa, N. Nakamura, K. Tokuda, H. Nishino, T. Yoshikawa, M. Kondo, Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis, Free Radic. Res. 34 (2001) 33–44. D. Bonnefont-Rousselot, J.L. Beaudeux, P. Therond, J. Peynet, A. Legrand, J. Delattre, Diabetes mellitus, oxidative stress and advanced glycation endproducts, Ann. Pharm. Fr. 62 (2004) 147–157. B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd ed., Oxford University Press, New York, 2007. R.D. Porsolt, M. Le Pichon, M. Jalfre, Depression: a new model sensitive to antidepressant treatment, Nature 266 (1977) 730–732. N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantification of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184–191. R.R. Tice, D. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35 (2000) 206–221. A. Hartmann, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A. Collins, A. Smith, G. Speit, V. Thybaud, R.R. Tice, Recommendations for conducting the in vivo alkaline comet assay, Mutagenesis 18 (2003) 45–51. S. Nadin, L. Vargas-Roig, D. Ciocca, A silver staining method for single-cell gel assay, J. Histochem. Cytochem. 49 (2001) 1183–1186. N.F. Wiernsperger, Oxidative stress as a therapeutic target in diabetes: revisiting the controversy, Diabetes Metab. 29 (2003) 579–585. R. Stetina, J. Varvarovska, Z. Rusavy, R. Pomahacova, J. Racek, S. Lacigov, L. Trefil, K. Siala, F. Stozicky, Oxidative stress DNA damage and DNA repair capacity in children with type 1 diabetes mellitus, Toxicol. Lett. 164 (2006) S134. H. Wu, H. Guo, R. Zhao, Effect of Lycium barbarum polysaccharide on the improvement of antioxidant ability and DNA damage in NIDDM rats, Yakugaku Zasshi 126 (2006) 365–371. A. Fidan, Y. Dündar, The effects of Yucca schidigera and Quillaja saponaria on DNA damage, protein oxidation, lipid peroxidation, and some biochemical parameters in streptozotocin-induced diabetic rats, J. Diabetes Complications 22 (2008) 348–356. B.S. Balkis, R.N. Fadilah, A. Ezlan, O. Khairul, A.B. Mokhtar, M. Jamaludin, Oxidative stress and DNA damage in streptozotocin-induced diabetic rats: effects of alpha lipoic acid, J. Biol. Sci. 8 (2008) 622–627. S.H. Saydah, E.A. Platz, N. Rifai, M.N. Pollak, F.L. Brancati, K.J. Helzlsouer, Association of markers of insulin and glucose control with subsequent colorectal cancer risk, Cancer Epidemiol. Biomarkers Prev. 12 (2003) 412–418. D. Anderson, T.W. Yu, J. Wright, C. Ioannides, An examination of DNA strand breakage in the comet assay and antioxidant capacity in diabetic patients, Mutat. Res. 398 (1998) 151–161. P. Dandona, K. Thuru, S. Cook, B. Snyder, J. Makowski, D. Armstrong, T. Nicotera, Oxidative damage to DNA in diabetes mellitus, Lancet 347 (1996) 444–445. Y. Dinc¸er, T. Akc¸ay, H. I˙ lkova, Z. Alademir, G. Ozbay, DNA damage and antioxidant defense in peripheral leukocytes of patients with Type I diabetes mellitus, Mutat. Res. 527 (2003) 49–55. E.E. Leonard, Effects of depression on work loss and disability bed days in individuals with diabetes, Diabetes Care 27 (2004) 1751–1753. A. Imaeda, T. Kaneko, T. Aoki, Y. Kondo, H. Nagase, DNA damage and the effect of antioxidants in streptozotocin-treated mice, Food Chem. Toxicol. 40 (2002) 979–987. R. Kocic, D. Pavlovic, G. Kocic, Impact of intensive insulin treatment on the development and consequences of oxidative stress in insulin-dependent diabetes mellitus, Vojnosanit Pregled. 64 (2007) 623–628. L. Monnier, C. Colette, E. Mas, F. Michel, J.P. Cristol, C. Boegner, D.R. Owens, Regulation of oxidative stress by glycaemic control: evidence for an independent inhibitory effect of insulin therapy, Diabetologia 53 (2010) 562–571. H. Chevassus, I. Mourand, N. Molinier, B. Lacarelle, J.F. Brun, P. Petit, Assessment of single-dose benzodiazepines on insulin secretion, insulin sensitivity and glucose effectiveness in healthy volunteers: a double-bind, placebo-controlled, randomized cross-over trial, BMC Clin. Pharmacol. 4 (2004) 4: 3. D.A. Pearce, M. Atkinson, D.A. Tagle, Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders, Neurology 63 (2004) 2001–2005. T. Yoshimoto, M. Doi, N. Fukai, H. Izumiyama, T. Wago, I. Minami, I. Uchimura, Y. Hirata, Type 1 diabetes mellitus and drug-resistant epilepsy: presence of high titer of anti-glutamic acid decarboxylase autoantibodies in serum and cerebrospinal fluid, Intern. Med. 44 (2005) 1174–1177.