DNA protective effects of melatonin on oxidative stress in streptozotocin - induced diabetic rats. 11. International Comet Assay Workshop Selim Sekkin, PhD Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey. 1
Layout Introduction Diabetes Melatonin Materials & Methods Animals Induction of diabetes Parameters Statistical analysis Results Discussion & Conclusion 2
Introduction 3
Diabetes mellitus (DM) is a chronic disease characterized by elevated blood sugar levels. During diabetes, persistent hyperglycemia causes an increased production of free radicals. Melatonin is a compound synthesized primarily by the pineal gland. The aim of this study was to research the effects of melatonin on oxidative stress and DNA protective effects in streptozotocin-induced diabetic rats. 4
Diabetes atlas 2014 http://www.idf.org/diabetesatlas 6th Edition. 2015 5
Prevalence of diabetes mellitus type 2. http://www.idf.org/diabetesatlas 2014 7
Oxidative stress pathways in diabetes mellitus. Lazo-de-la-Vega-Monroy M-L, Fernández-Mejía C. Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions 2013 http://dx.doi.org/10.5772/51788 9
The ominous octet of hyperglycaemia in diabetes mellitus type 2. DeFronzo RA, Ferrannini E, Groop L, Henry RR, Herman WH, Holst JJ, et al. Type 2 diabetes mellitus. Nature Reviews Disease Primers. 2015:15019. 10
Mechanisms modulating oxidant/antioxidant balance in obesity Savini I, Catani M, Evangelista D, Gasperi V, Avigliano L. Obesity-Associated Oxidative Stress: Strategies Finalized to Improve Redox State. International Journal of Molecular Sciences. 2013;14(5):10497. 11
Major endo- and exogenous antioxidants Endogenous antioxidants Enzymatic antioxidants: -Superoxide-dismutase (SOD) dependent on manganese, zinc and copper -Catalase (CAT) dependent on iron -Glutathione peroxidase (GPx)-dependent on selenium -Glutathione reductase (GR) -Thioredoxin reductase (TrxR) - dependent on selenium Non-enzymatic antioxidants: -glutathione (GSH) -melatonin -uric acid -coenzyme Q -lipoic acid -transferrin -albumin lactoferrin bilirubin -ceruloplasmin -nicotinamide adenine dinucleotide phosphate (NADPH) Exogenous antioxidants Mainly dietary antioxidants from plant based healthy foods: -Vitamins: Vitamin A, C, E -Trace elements: zinc, selenium (as parts of enzymes) -Carotenoids: ß-carotene, lycopene, lutein, zeaxanthin -Phenolic acids: chlorogenic acids, gallic acid, caffeic acid and others -Flavonols: quercetin, kaempferol, myricetin proanthocyanidins, catechins -Anthocyanidins: cyanidin, pelargonidin -Isoflavones: genistein, daidzein, glycitein Malwina S Munik, Ekmekçİoğlu C. Pro-oxidant effects of melatonin: a brief review. Turk J Biol 2015 (in press). 12
Antioxidant defenses in the organism. Lazo-de-la-Vega-Monroy M-L, Fernández-Mejía C. Oxidative Stress in Diabetes Mellitus and the Role Of Vitamins with Antioxidant Actions 2013 http://dx.doi.org/10.5772/51788 13
Dates correspond to the first research findings related to melatonin s clinical uses according to the PubMed database. Modfied from V. Srinivasan et al. (eds.), Melatonin and Melatonergic Drugs in Clinical Practice, DOI 10.1007/978-81-322-0825-9_3 Springer India 2014 14
Schematic representation of the pathway of synthesis, secretion, and catabolism of meatonin (1/2) Watson RR. Melatonin in the promotion of health, 2nd ed. Boca Raton, FL: CRC Press; 2012. xvii, 566 p. p. 15
Schematic representation of the pathway of synthesis, secretion, and catabolism of meatonin (2/2) Watson RR. Melatonin in the promotion of health, 2nd ed. Boca Raton, FL: CRC Press; 2012. xvii, 566 p. p. 16
Melatonin s antioxidant cascade and mechanisms of protection against oxidative damage Watson RR. Melatonin in the promotion of health, 2nd ed. Boca Raton, FL: CRC Press; 2012. xvii, 566 p. p. 17
Materials and Methods 22
Animals study design A total of 32 Wistar albino healthy male rats weighing 430-460 g were used. They were suspended screen bottomed stainless steel cages at 22-24ºC in a room with a 12/12 h light/dark cycle. The study was conducted at Adnan Menderes University, Faculty of Medicine, Aydin, Turkey. The rats received a commercial rodent diet and free access to tap water. After 21 days of acclimatization, rats were included in the study. The experimental protocol was approved by the Animal Ethic Committee of University of Adnan Menderes (64583101/2015/052). 23
Aſter overnight fasting, diabetes was induced in the rats by a single intraperitoneal (i.p.) injection of streptozotocin (STZ) at a dose of 60 mg/kg. Plasma glucose levels were measured 72 h after with a Contour TS strip test in a glucometer (Bayer). The rats whose blood glucose levels were 200 mg/ dl were considered as diabetic. Melatonin (M5250, Sigma Aldrich, St Louis, Missouri, USA) was injected (i.p.) daily 10 mg/kg body weight during 6 weeks. Groups were established as Control (n=8), Melatonin (n=8), Diabetic (n=8), and Diabetic + Melatonin (n=8). Plasma glucose levels and the body weights of the animals were measured weekly during the study. 24
Evaluated parameters Body weight Glucose (plasma) Glycosylated haemoglobin (HbA1c, plasma)* Malondialdehyde (MDA, liver, renal, brain and pancreas tissues) Glutathione (GSH, liver, renal, brain and pancreas tissues) Catalase (CAT, liver, renal, brain and pancreas tissues) Superoxide dismutase (SOD, liver, renal, brain and pancreas tissues) % DNA in Tail (lymphocytes) Mean tail moment (lymphocytes) *: Plasma samples were analysed with auto-analyser (Abbott, Architect C 8000). 25
Determination of SOD, CAT, GSH, and MDA levels in liver, renal, brain and pancreas tissues Superoxide dismutase (SOD) activity was measured at 560 nm by a spectrophotometer, and the results are shown as U/mg tissue protein (1) The activity of catalase (CAT) was measured spectrophotometrically by following the rate of hydrogen peroxide (H2O2) decomposition at 240 nm (2) GSH (reduced glutathione) level was spectrophotometrically determined at 412 nm and expressed as mg/g tissue protein (3). Malondialdehyde (MDA) levels were measured according to the method of Ohkawa, et al. (1979) (4). (1) Sun Y, Oberley LW, Li Y (1988). A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497-500 (2) Aebi H (1984). Catalase in vitro assay methods. Methods Enzymol 105:121-126 (3) Tietze F (1969). Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27:502-522 (4) Ohkawa H, Ohishi N, Yagi K (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351-358 26
Comet assay Lymphocytes were isolated from the fresh blood samples in accordance with a modified procedure described by Collins et al. (1997). The DNA strand breaks were detected with comet assay. The alkaline comet assay was applied with several modifications as previously described by Singh et al. (1988) and Collins et al. (1997). DNA of lymphocytes were stained with 4',6-diamidino-2-phenylindole (DAPI). Measurements of DNA in tail intensity and mean tail moment of comets were made by a computer-based image analysis system (Comet Assay IV, Perceptive Instruments, UK) for 100 (2 gel X 50 ) randomly selected cells from each sample. Singh NP, McCoy MT, Tice RR, Schneider EL (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184-191 Collins A, Dušinská M, Franklin M, Somorovská M, Petrovská H, Duthie S, Fillion L, Panayiotidis M, Rašlová K, Vaughan N (1997). Comet assay in human biomonitoring studies: Reliability, validation, and applications. Environ Mol Mutagen 30:139-146 27
Statistical analysis The data were compared among groups using Kruskal-Wallis analysis of variance (ANOVA) or one-way ANOVA. Post hoc multiple comparisons were performed using the Mann-Whitney U test with Bonferroni corrected or Duncan's test (SPSS 21.0 version). Differences were considered statistically significant if P<0.05. All data were expressed as the mean and standard error. Differences were considered statistically significant if P<0.05, P<0.01 and P<0.001. 28
Results 29
Diabetic rats weight were lesser than the Control and Melatonin group rats during the 6 week (P<0.001). Melatonin administration did not changed the body weight of the rats (P>0.05) During the experiment melatonin did not changed the blood glucose levels (P>0.05) Melatonin administration was not able to decrease the serum %HbA1c levels (P>0.05). 30
Effects of melatonin administration on body weight and glucose levels in streptozotocin induced diabetic rats (n=8). */ 31
SOD (U/mg protein) 25 20 *** a b c b 15 10 5 0 *** a b c b ** a b b b a b c b,c Liver Renal Brain Pancreas Effects of melatonin administration on the superoxide dismutase (SOD) activities in tissues. ** NS: Not significant *: P<0.05 **: P<0.01 ***: P<0.001 Groups: Control Melatonin Diabetic Melatonin+Diabetic 32
CAT (kg/mg protein) *** 30 25 20 15 10 5 0 b a d c * a a b a,b NS NS Liver Renal Brain Pancreas Effects of melatonin administration on the catalase (CAT) activities in tissues. NS: Not significant *: P<0.05 **: P<0.01 ***: P<0.001 Groups: Control Melatonin Diabetic Melatonin+Diabetic 33
GSH (mg/g protein) 60 50 * a a,b b b,c 40 30 20 10 0 *** ** ** a b c c a a,b c b a a b b Liver Renal Brain Pancreas Effects of melatonin administration on the reduced glutathione (GSH) levels in tissues. NS: Not significant *: P<0.05 **: P<0.01 ***: P<0.001 Groups: Control Melatonin Diabetic Melatonin+Diabetic 34
MDA (nmol/mg protein) 200 180 160 140 *** c c a b *** b,c c a b ** b b a b ** b b a a 120 100 80 60 40 20 0 Liver Renal Brain Pancreas Effects of melatonin administration on the malondialdehyde MDA levels in tissues. NS: Not significant *: P<0.05 **: P<0.01 ***: P<0.001 Groups: Control Melatonin Diabetic Melatonin+Diabetic 35
Effects of melatonin administration on % Tail DNA and Mean Tail Moment parameters in streptozotocin induced diabetic rats (n=8). Parameters Groups % Tail DNA Mean Tail Moment Control 12.99 ± 1.43 c 2.29 ± 0.23 c Melatonin 18.26 ± 1.13 b 5.52 ± 0.55 b Diabetic 41.55 ± 2.03 a 17.98 ± 1.46 a Diabetic+Melatonin 17.68 ± 1.00 b,c 5.65 ± 0.56 b P *** *** 36
Discussion & Conclusion 37
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To the best of our knowledge, no information is currently available regarding the DNA protective effects of melatonin with a Comet assay against STZ - induced diabetic rats. The i.p. administration of daily 10 mg/kg of melatonin over 6 weeks might ameliorate oxidative stress parameters against diabetes. Therefore, the limitations of the present study suggested that melatonin might play an important role in preventing oxidative DNA damage in hyperglycemic conditions, such as DM. 42
Thank you for your attention. Research team: Selim Sekkin*, Eda Duygu Ipek**, Murat Boyacioglu*, Cavit Kum*, Umit Karademir*, Hande Sultan Yalınkılınc*, Mehmet Onur Ak*, Hulki Basaloglu** *:Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey. **:Department of Anatomy, Faculty of Medicine, Adnan Menderes University, Aydın, Turkey. Acknowledgments A part of this study was supported by Adnan Menderes University, Scientific Research Projects 43