Cheshchevik, V.T.

Dremza, I.K.

Lapshina, E.A.

Prokopchik, N.I.

Reiter, R.J.

Zabrodskaya, S.V.

Zavodnik, I.B.

Repository of Polessky State University (Репозиторий Полесского государственного университета)

IBBHBw Я m X > Г CENTRAL EUROPEAN INITIATIVE > Г ФАРМАЦЕВТИЧЕСКИЕ И П И Щ Е В Ы Е Б И О Т Е Х Н О Л О Г И И И С И С Т Е М А О Б Р А З О В А Н И Я : П Р А В О В Ы Е А С П Е К Т Ы P H A R M A C E U T I C A L A N D F O O D T E C H N O L O G I E S A N D T H E S Y S T E M O F E D U C A T I O N : L E G A L A S P E C T S НАУЧНЫХ СТАТЕЙ Гродненский Государственный университет имени Янки Купалы, Фармацевтические и пищевые б и о т е х н о л о г и и и с и с т е м а о б р а з о в а н и я : правовые аспекты Сборник научных статей 'V. Т. Cheshchevik, 'Е. A. Lapshina, 21. К. Dremza, 'S. V. Zabrodskaya, sR. J. Reiter, 2N. I. Prokopchik, Ч. B. Zavodnik 'Department of Biochemistry, Yanka Kupala Grodno State University, Grodno, Belarus 2Grodno State Medical University, Grodno, Belarus 3Department of Cellular and Structural Biology, University of Texas Health Science Center, USA e-mail: zavodnik_il@mail.ru RAT LIVER MITOCHONDRIAL PROTECTION BY MELATONIN AND FLAVONOIDS UNDER CARBON TETRACHLORIDE-INDUCED INTOXICATION We investigated the oxidative disorders of rat liver mitochondria during and intoxication and evaluated the possibility of corrections of mitochondrial disorders. After 30-day chronic rat intoxication we observed an increase (by 25 %, p < 0.01) of oxygen consumption rate V2 of liver mitochondria in the case of succinate (not glutamate) as a substrate. The ACR and ADP/O did not change. The level of mitochondrial glutathione as well as cytoplasmic mixed protein-glutathione disulfides enhanced considerably (by 60 %, p < 0.01 and by 30 %, p < 0.01, respectively) in the liver cells of chronically intoxicated animals. The activities of mitochondrial enzymes succinate dehydrogenase and glutathione peroxidase in liver cells inhibited markedly (by 15 %, p 0.05, 45 %, p < 0.001, respectively) Long-term melatonin administration (10 mg/kg, 30 days, daily) to chronically intoxicated rats diminished the toxii effects of CC14, reducing elevated plasma activities of alanine aminotrans ferase and aspartate aminotransferase and bilirubin concentration, prevented the enhancement of cellular and mitochondrial GSH contents during intoxica tion and accumulation of membrane lipid peroxidation products in rat livci The treatment of the animals by the complex of melatonin (10 mg/kg) plus succinate (50 mg/kg) plus cranberry flavonoids (7 mg/kg) was even more effective in prevention of toxic liver injury and liver mitochondria damage. Introduction Mitochondria play a key role in coordination of the main cellular func tions. They provide energy required for all cellular processes as well as pai 8 Фармацевтические и пищевые б и о т е х н о л о г и и и с и с т е м а о б р а з о в а н и я : правовые а с п е к т ы Сборник научных статей ticipate in cell signaling and in necrotic and apoptotic cell death. There is evidence that defective mitochondrial oxidative phosphorylation plays an important role in the pathogenesis of many diseases: Alzheimer's disease, diabetes, and ageing [1]. The mitochondrial function is particularly suscepti-ble to oxidative stress, leading to decreased mitochondrial ATP synthesis, induction of mitochondrial permeability transition, all of which predispose cells to necrosis or apoptosis [2]. The risk of toxic liver damage has markedly increased in recent years due to the exposure to environmental toxins, pesticides and chemotherapeu-tics. Many compounds, including useful drugs, can cause liver cell damage through their metabolic conversion to highly reactive substances and the gen-eration of free radicals [3]. It was suggested that reactive metabolite forma-tion, gluthatione depletion, outer mitochondrial membrane pore formation, intramembrane protein release, and the diminished capacity to ATP synthe-sis, are critical events in hepatotoxicity [4]. Mitochondrial dysfunction is an early manifestation of hepatotoxic agent effects. Prevention of the mitochon-drial oxidative damage is a therapeutic strategy in diabetes and intoxication and mitochondria-targeted antioxidants are to have a therapeutic potential under toxic liver damage. Correction of the mitochondrial functions is the basis of «mitochondrial medicine» [1]. The aim of the present work was to investigate the possible role of a specific functional damage in rat liver mitochondria under intoxication as well as evaluate the possibility of mitochondrial impairment corrections by antioxidants (melatonin, flavonoids) and succinate. Materials and methods Chemicals Melatonin, carbon tetrachloride (ССЦ), 5, 5'-dithiobis (2-nitrobenzoic acid) (Ellman's reagent), reduced glutathione (GSH), trichloroacetic acid (TCA) were from Sigma-Aldrich, St. Louis, USA. All other reagents were of analytical grade and were purchased from Reakhim, Moscow, Russia. All the solutions were made with water purified in the Milli-Q system. Animal models The investigations were performed using 80 male albino Wistar rats (150-180 g). A standard balanced diet and tap water were provided ad libi-tum. The animals were adapted to an intermittent 12-h light and dark phases cycle for 1 week. 9 Фармацевтические и пищевые б и о т е х н о л о г и и и с и с т е м а о б р а з о в а н и я : правовые аспекты Сборник научных статей Chronic rat intoxication with carbon tetrachloride. The rats were sub divided into 5 groups. The first group served as controls and received a sub cutaneous injection (s.c.) of olive oil (2.0 ml/kg b.w., 30 days, twice a week) and a physiologic solution containing 5 % ethanol (i.g. in the same volume as the melatonin solution). The second group received an injection of CC'U (1.6 g/kg b.w., 30 days, twice a week, as a 50 % solution in olive oil, s.c.) ul 9 h and a physiologic solution (i.g., 30 days, every day) at 20 h. The third, fourth and fifth groups were given melatonin (10 mg/kg b.w., 30 days, i.g.). melatonin and succinate (50 mg/kg b.w., 30 days, i.g.); melatonin, succinate and cranberry flavonoids (7 mg/kg b.w., 30 days, i.g.), respectively. The animals were sacrificed by decapitation 24 hours after last ССЦ injection Blood was collected and the activity of serum aminotransferases [(alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] as well as total, free and conjugated bilirubin levels in blood plasma were measured The animals were killed according to the rules defined by the European C011 vention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. Isolation of rat liver mitochondria Mitochondria were isolated by differential centrifugation from the liver 151. The protein concentration was determined by the method of Lowry et al. [6]. The respiration of mitochondria was measured using a laboratory-made oxygen Clark-type electrode and a hermetic polarographic cell (volume 1.25 ml) with constant gentle stirring [7]. The incubation medium contained 125 mM sucrose, 20 mM Tris-HCl, 50 mM KC1, 20 mM KH2P04, 5 niM MgS04, 1 mM EDTA, pH 7.5. The experiments were performed at 26.5°( using 5 mM succinate or 4 mM L-glutamate as substrates. Mitochondrial protein concentration in the probe was 1.0 mg/ml. The functional state ol mitochondria was determined by the acceptor control ratio (ACR, V3/V?), and the coefficient of phosphorylation (ADP/O). The rate of mitochondrial respiration corresponding to State 3 (V3) was registered after addition of IXO (iM ADP. State 1 corresponded to the respiration in the presence of endogc nous substrates (Vi), State 2 corresponded to the respiration in the presence of substrate (glutamate, or succinate) added (V2), and State 4 corresponded lo the respiration when the ADP added was exhausted (V4). Biochemical measurements 10 Фармацевтические и пищевые б и о т е х н о л о г и и и с и с т е м а о б р а з о в а н и я : правовые а с п е к т ы Сборник научных статей The concentration of non-protein thiols (predominantly, of reduced glu-tathione, GSH) in mitochondria was determined spectrophotometrically by the method of Ellman [8]. Statistical analysis Data for 8-10 rats in each group are presented as a mean ± SD for the normally distributed parameters or as a median and interquartile range for the data showing departures from normality. We used the standard Student t test for the comparison of the raw and transformed data showing no departures from normality (according to Shapiro-Wilk's test), and the non-parametric Mann-Whitney U test for the remaining variables. P<0.05 was taken to indi-cate statistical significance. Results Earlier we have shown that the experimental diabetes mellitus resulted in a considerable damage of respiratory activity in rat liver mitochondria [9]. The effects of diabetes and melatonin administration on mitochondrial func-tional activity we compared with those of ССЦ - induced rat intoxication and melatonin treatment. In the present work, we evaluated the parameters of mitochondrial physiology, metabolic state after chronic intoxication by car-bon tetrachloride and melatonin and cranberry flavonoids treatment in rats. Under chronic rat intoxication we observed some increase in the rate of endogenous oxygen consumption V, (by 20 %, p < 0.05), substrate-dependent respiration rate V2 (by 25 %, p < 0.05) and ADP-stimulated oxygen consumption rate V3 (by 15 %, p < 0.05) of succinate, but not glutamate, as respiratory substrate [10]. There was significant change in the redox state of mitochondria: the level of mitochondrial reduced glutathione (GSH) was enhanced considerably (by 60 %, p < 0.01) after 30 days of intoxication. Combined administration of succinate and melatonin or the treatment by the combination of melatonin plus succinate plus crude cranberry flavonoid extract for 30 days did not influence the GSH levels in mitochondria. Increased level of mixed protein - glutathione disulfides (GSSP) in mitochondria under intoxication was also noted (by 30 %, p < 0.01). We observed some changes of the activities of the mitochondrial enzymes under chronic intoxication: succinate dehydrogenase (complex II) activity and glutathione peroxidase activity decreased (by 15 %, P < 0.05, and by 45 %, p < 0.001, respectively). At the same time the activities of the main enzyme of the Krebs cycle, 2-oxoglutarate dehydrogenase, and the main detoxifying 11 Ф а р м а ц е в т и ч е с к и е и пищевые б и о т е х н о л о г и и и с и с т е м а о б р а з о в а н и я : правовые аспекты Сборник научных статей enzyme, glutathione transferase, in mitochondria did not change. Long-term melatonin administration (10 mg/kg, 30 days, daily) diminished many signs of liver damage (aminotransferase activities and bilirubin content in the blood plasma), prevented the enhancement of cellular and mitochondrial GSI1 contents and cellular lipid peroxidation products levels under chronic intoxication [11]. Much more effective in the prevention of the toxic liver injury and liver mitochondria impairments was the animal treatment by the complex melatonin (10 mg/kg) plus succinate (50 mg/kg) plus cranberry flavonoids (7 mg/kg) [11]. Discussion Melatonin (N-acetyl-5-methoxy-tryptamine) has been shown to be an effective antioxidant in a number of experimental models both in vitro and in vivo [12-13]. In our experimental animal models, the intoxication as well as diabetes resulted in impairment of mitochondrial state and respiration activity [9-1 I ]. The elevated content of GSH in mitochondria may serve as a factor of mitochondrial adaptation to constant effect of the toxic agent. Mitochon drial dysfunction may be the first sign of toxic and diabetic liver tissue dam age. Under intoxication melatonin administration decreased the level of oxi dative stress and, correspondingly, prevented a considerable elevation of eel lular and mitochondrial glutathione content. It has been shown that melatonin may recycle NADH by electron donation and by this way may improve the efficiency of NADH as an energy carrier and antioxidant [14]. The effects of melatonin might be due to both its radical scavenging properties, its meta bolic effects and specific interaction with complexes of the respiratory chain. The complex of melatonin plus succinate plus cranberry flavonoids was much more effective in preventing chronic liver damage. Literature 1. Fosslien, E. Mitochondrial medicine - molecular pathology of defective oxidii tive phosphorylation / E. Fosslien // Arm. Clin. Lab. Sci. - 2001. - Vol. 31. - P. 25-67. 2. James, A. M. How mitochondrial damage affects cell function / A. M. James, M. P. Murphy // J. Biomed. Sci. - 2002. - Vol. 9. - P. 475^187. 12 Фармацевтические и пищевые б и о т е х н о л о г и и и с и с т е м а о б р а з о в а н и я : правовые а с п е к т ы Сборник научных статей 3. Mahesh, A. Hepatocurative potential of sesquiterpene lactones of Taraxacum officinale on carbon tetrachloride induced liver toxicity in mice / A. Mahesh [et al.] // Acta Biol. Hung. -2010. - Vol. 61. - P. 175-190. 4. Jaeschke, H. Intracellular signaling mechanisms of acetaminophen-induced liver cell death / H. Jaeschke, M. L. Bajt // Toxicological Sciences. - 2006. - Vol. 89. -P. 31—41. 5. Johnson, D. Isolation of liver or kidney mitochondria / D. Johnson, H. A. Lardy // Methods in Enzymology / In: R. Estabrook, M. E. Pullmam (eds.). — Academic Press. -N.Y. , London. - 1967. - Vol. 10. - P. 94-96. 6. Lowry, O.H. Protein measurement with the Folin phenol reagent / O.H. Lowry [et al.] / /J. Biol. Chem. - 1951. - Vol. 193. - P. 265-275. 7. Dremza, I. K. Oxygen-related processes in red blood cells exposed to tert-butyl hydroperoxide /1. K. Dremza [et al. ] // Redox Rep. - 2006. - Vol. 11. - P. 185-192. 8. Ellman, G. Tissue sulfhydryl groups / G. Ellman // Arch. Biochem. Bio-phys. - 1959. - Vol. 82. - P. 70-77. 9. Zavodnik, I. B. Melatonin and succinate reduce rat liver mitochondrial dys-function in diabetes / I. B. Zavodnik [et al.] // J. Physiol. Pharmacol. - 2011. -Vol. 62. - P. 421—427. 10. Cheshchevik, V. Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats / V. Cheshchevik [et al.] // Cell Biochem. Funct. - 2011. - Vol. - 29. - P. 481 -488. 11. Cheshchevik, V. T. Rat liver mitochondrial damage under acute or chronic carbon tetrachloride-induced intoxication: protection by melatonin and cranberry fla-vonoids / V. T. Cheshchevik [et al.] // Toxicol, and Appl. Pharmacol. — 2012. — Vol. 261 . -P . 271-279. 12. Allegra, M. The chemistry of melatonin's interaction with reactive species / M. Allegra [et al.] // J. Pineal. Res. - 2003. - Vol. 34. - P. 1-10. 13. Sudnikovich, E. J. Melatonin attenuates metabolic disorders due to strepto-zotocin-induced diabetes in rats / E. J. Sudnikovich [et al.] // Eur. J. Pharmacol. -2007. - Vol. 569. - P. 180-187. 15. Tan, D. X. Interactions between melatonin and nicotinamide nucleotide: NADH preservation in cells and in cell-free systems by melatonin / D. X. Tan [et al.] // J. Pineal. Res. - 2005. - Vol. 39. - P. 185-194. 13 

Rat liver mitochondrial protection by melatonin and flavonoids under carbon tetrachloride-induced intoxication

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Rat liver mitochondrial protection by melatonin and flavonoids under carbon tetrachloride-induced intoxication

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