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Mechanism of protection by epicatechin against tert-butylhydroperoxide induced oxidative cell injury in isolated rat hepatocytes and calf thymus DNA
Process Biochemistry 37 (2002) 659– 664 www.elsevier.com/locate/procbio
Mechanism of protection by epicatechin against tert-butylhydroperoxide induced oxidative cell injury in isolated rat hepatocytes and calf thymus DNA V. Valls-Belle´s, P. Mun˜iz *, P. Gonza´lez, M.L. Gonza´lez-Sanjose´, S. Beltran Dpto Biotecnologia y Ciencia de los Alimentos, Facultad de Ciencias, Uni6ersidad de Burgos, 09001 Burgos, Spain Received 12 June 2001; received in revised form 12 June 2001; accepted 30 June 2001
Abstract The purpose of this investigation was to establish the antioxidant effect of flavonoid epicatechin (EC) a low concentration (0.75 mM). The relative antioxidant potentials were measured against radicals generated by tert-BOOH in isolated rat hepatocytes and calf thymus DNA. EC is an effective antioxidant, it completely prevents tert-BOOH induced oxidation and thereby prevents subsequent lipid peroxidation. It also plays an important role in conserving of endogenous antioxidants such as GSH and catalase. As it is a radical scavenger, it is a prominent antioxidant on calf thymus DNA exposed to tert-BOOH. Oxidative stress induced by tert-BOOH at different concentrations in these isolated cells led to lipid peroxidation and changes in endogenous antioxidant levels, which were prevented by EC at a dose of 0.75 mM. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Epicatechin; Antioxidant enzymes; Glutathione; Tert-butylhydroperoxide
1. Introduction Flavonoids are polyphenolic compounds widely distributed in foods and beverages extensively studied for their antioxidant and cytoprotective properties in various biological models [1 – 3]. As the constituents of an integral part of the human diet, they are considered to exert a wide range of beneficial effects on human health, including protection against cardiovascular diseases and certain forms of cancer [4]. They can protect against oxidative stress through their antioxidative activities involving free radical scavenging and also by metal ion-chelating [5]. ( −)-Epicatechin (EC) is a flavonoid belonging to the flavanol family and is commonly present in tea and red wine. The antioxidant effect of wine led to the hypothesis that the phenolic phytochemicals of wine could be beneficial against coronary heart disease by inhibition * Corresponding author. Address: Area Bioquimica y Biologı´a Molecular, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Ban˜uelos s/n, 09001 Burgos, Spain. Tel.: +34-6-947258800x8210; fax: +34-6-947-258831. E-mail address: [email protected] (P. Mun˜iz).
of low density lipoprotein oxidation in vivo [6]. However, factors effecting the scavenging activity of the catechin are still obscure. Catechins, ECs and isomers constitute the most abundant flavonoid class in wines. In studies on model systems, the catechins from tea have shown high activity in erythrocyte membranes and in rat liver microsomes with great protection from lipid oxidation being shown by epigalocatechin gallate and EC gallate, the latter being ten times more effective than vitamin E [7] Tert-BOOH is a well known inducer of oxidative stress in biological systems and it is believed that reactive free radical intermediates, including peroxyl-, alkoxyl- and carbon centred radicals, are mediators of peroxide-dependent injury [8,9]. In this study, we have investigated the effect of the flavonoid EC on cytotoxicity induced by tert-BOOH toxicity in isolated rat hepatocytes. In order to estimate the extent of oxidative stress, lipoperoxidation was evaluated as thiobarbituric acid reactant substances (TBARS). Lactate dehydrogenase (LDH) leakage and modifications in enzymic and non-enzymic antioxidants were also measured as indicators of cell damage. Re-
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sults strongly imply that EC possesses an ability to protect rat hepatocytes against free radicals induced by tert-BOOH. We also examined EC for its effect on DNA damage in vitro induced by tert-BOOH. The fact that DNA represents an important target molecule of the reactive species generated during oxidative stress prompted the present investigation. This study shows that the mechanism of action of EC could be free radical scavenging or iron chelatin mechanism. The results obtained indicate that the effects of EC are best explained by free radical scavenging.
2. Materials and methods
2.1. Materials Calf thymus DNA, tert-BOOH, EC, and enzymes used were purchased from Sigma Chemical Co. (St. Louis, MO). Reduced glutathione, ethanol, enzymes, coenzymes and other reagents were of the highest purity and were from Merck (Darmstadt, Germany).
2.2. Hepatocyte cells and treatment Rat hepatocytes were isolated from 3-month-old Wistar male animals by cannulating the portal vein and perfusing the liver with a collagenase solution as previously described [10,11]. Two milliliters of hepatocyte suspension cells in Krebs– Henseleit saline were incubated in a shaking water bath at 37 °C in 25-ml capacity conical flasks. Oxidative stress was induced by supplementation with tert-BOOH of different concentrations. EC was dissolved in water and added to hepatocytes at a concentration of 0.75 mM. Moreover, some cultures were maintained in the presence of tertBOOH or EC alone (control cultures). Reactions were stopped after 60 min with 0.4 ml 20% perchloric acid.
2.4. Measurement of lactate dehydrogenase leakage LDH leakage from isolated rat hepatocytes was measured in incubation mixtures by spectrophotometer method [13]. These were centrifuged at slow speed to spin down the cells and appropriate dilutions of the cell-free supernatants were used for determination of activity.
2.5. Measurement of antioxidant acti6ities The activity of the total SOD was measured by monitoring at 550 nm the rate of reduction of Cytochrome c by superoxide radicals (O2 − ) produced by the xanthine/xanthine oxidase system with the method developed by McCord and Fridovich [14]. The activity of catalase was determined by measurement of H2O2 concentration using UV absorbance at 240 nm according to the method of Clairbone [15]. Glutathione peroxidase activity was determined measuring the conversion of NADPH to NADP in the presence of reduced glutathione (GSH) and H2O2 spectrophotometrically at 340 nm according to the method described by Flohe and Gunzler [16] and GSH was analyzed spectrophotometrically by the glutathione-S-transferase assay [17].
2.6. Degradation of DNA. TBA-reacti6ity assay Reaction mixtures of 1 ml final volume contained in the following order: sodium phosphate buffer pH 7.4, calf thymus DNA (4 mg/ml) and tert-BOOH at the final concentration indicated in the study and where indicated 0.75 mM of EC. The release of TBA-reactivity as an indication of DNA degradation was measured following the procedure described by Quintlan and Gutteridge [18]. One milliliter of 1% in 50 mM thiobarbituric acid followed by 1 ml of 28% trichloroacetic acid (TCA) were added to each tube and mixed. The release of thiobarbituric acid reactive products was followed by spectrophotometer at 532 nm.
2.7. Statistical analysis 2.3. Determination of thiobarbituric acid reactant substances TBARS in hepatocytes The formation of lipid oxidation products was evaluated as MDA or TBARS [12] with 1,1,3,-tetramethoxypropane used as standard. After incubation, aliquots were treated with 1% phosphoric acid, 0.6% TBA. The mixtures were cooled, 4.0 ml of n-butanol was added for lipid extraction and they were then mechanically mixed for 10 min. Samples were subsequently centrifuged for 10 min at 4000× g. The lipid soluble phase was then extracted and absorbance was measured at 532 nm. MDA concentration was determined against a standard curve.
Experimental values are means9S.D. of the number of separate experiments indicated in the legends. Significance was assessed using the Student’s t-test. The minimal level of significance was PB 0.01.
3. Results The experimental model of isolated rat hepatocytes treated with tert-BOOH represents a useful tool to study the antioxidant capacity of EC. In hepatocytes treated with EC alone, LDH leakage was not significantly higher than the levels found in the
V. Valls-Belle´ s et al. / Process Biochemistry 37 (2002) 659–664
control group, showing that EC at the concentration used was not cytotoxic in the medium used (Fig. 1A). LDH leakage was increased in hepatocytes treated with 0.25 and 0.1 mM tert-BOOH during 60 min demonstrating it cytotoxic effect. When tert-BOOH was added with 0.75 mM EC, the levels of LDH released to the medium remained as low as in the control indicating a cytoprotective capacity of EC. In order to assess the antioxidant capacity of EC were studied the TBARS levels in hepatocytes treated with tert-BOOH and EC (Fig. 1B). In hepatocytes treated with EC alone, TBARS level were lower than control hepatocytes, showing that EC alone did not modify basal lipid peroxidation in hepatocytes. The hepatocytes treated with tert-BOOH showing a significative increase of TBARS levels. The addition of EC with tert-BOOH resulted in a decrease of peroxidative damage. This result showed that EC was able to counteract lipid peroxidation, which corresponded to an antioxidant effect of EC.
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The effect of EC on the endogenous enzymatic activities (SOD, catalase and GSH-PX) of isolated rat hepatocytes treated tert-BOOH were also evaluated (Fig. 2). In hepatocytes treated with EC alone, SOD and catalase activities were significantly (PB 0.05 and P B 0.005, respectively) lower than control values. No difference was observed between hepatocytes with and without EC treatment. These results show the antioxidant effect of EC in the absence of oxidative stress. As can observed, hepatocytes treated with tertBOOH alone, showed a decrease in SOD and GPX activities but in both cases the effect was not statistically significant. EC, therefore, does not have any effect on SOD and GSH-PX activity on hepatocytes treated with tert-BOOH, with the exception when they were incubated with 0.1 mM of tert-BOOH where SOD decreased significantly (PB 0.05). In contrast, catalase activity increased significantly (PB 0.005) when hepatocytes was incubated with tert-BOOH and the addition of EC allowed catalase to return to initial values.
Fig. 1. A – B. LDH leakage and MDA levels in isolated rat hepatocytes treated with tert-BOOH and 0.75 mM of EC. All values represent the mean9 S.D. from n =6 different hepatocyte populations. (A) *PB 0.05, **PB 0.005, T60 versus tert-BOOH and EC. (B) *PB 0.05, **PB 0.005, tert-BOOH versus tert-BOOH with EC.
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Fig. 2. SOD, Cat, GSH-PX activities in isolated rat hepatocytes treated with tert-BOOH and 0.75 mM EC. All values represent the mean 9 S.D. from n =6 different hepatocytes populations. **PB 0.005, control versus tert-BOOH and EC. **PB0.005, tert-BOOH versus (EC + tertBOOH).
The effect of EC on GSH levels was also investigated Fig. 3. GSH levels in the presence of tert-BOOH were 119 3 and 129 2 and in the presence of EC were of 32.69 10.5 and 35.399.7 mmol/g protein, levels similar to control group. So, GSH concentration was increase in presence of the antioxidant EC. The degradation of DNA in the presence of tertBOOH was substantially inhibited by the EC and the effect of increasing concentration of EC was investi-
gated. As illustrated in Table 1 treatment with tertBOOH was also highly toxic in calf thymus DNA resulting in a dose-dependent effect. The degradation of DNA, measured as release of TBA reactivity was most marked when the final tert-BOOH concentrations in the reaction mixture were 0.5 mM. Table 1 show that in the presence of EC the TBARS were significantly inhibited, with an inhibition of 40% in hepatocytes treated with 0.5 mM tert-BOOH and 1 mM EC.
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Importantly, EC alone was not cytotoxic at any of the concentrations tested. Showing that EC is an extremely effective antioxidant.
4. Discussion There is evidence that flavonoids exert an in vitro antioxidative effect [4,19]. This effect has attracted much attention in relation to the protective effect of the consumption of catechin-containing beverages such as wine [20] over different diseases. However, in contrast to these beneficial effects, cytotoxicity has also been observed with flavonoids. Potentially harmful effects have mostly been reported, this toxicity of flavonoids was thought to result from their pro-oxidant action [21]. The cytoprotective effect of EC was associated with a complete inhibition of LDH leakage and TBARS formation induced by tert-BOOH. Terao et al. [22] showed that flavonoids are localized near the surface of phospholipid bilayers suitable for scavenging oxygen radicals and thereby they prevent lipid peroxidation in phospholipid bilayers exposed to aqueous oxygen radicals. Tert-BOOH induced a decrease in GSH levels and these results are also consistent with those published by Bellomo et al. [23], who observed that the effect of tert-BOOH in intact hepatocytes led to a marked decrease in cellular GSH level. The decrease of GSH during oxidation produced by tert-BOOH was suppressed when rat hepatocytes were
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incubated with EC. The levels of catalase decreased in the presence of EC in isolated hepatocytes treated with tert-BOOH. This shows that the capacity of EC as antioxidant is also superior to catalase. All of this represents an original mechanism of cytoprotection by EC against oxidative stress. These actions may contribute to increase antioxidant capacity in vivo. It was reported that some antioxidative activity of EC still remains during the metabolic process, and it may positively affect the antioxidative defense of hepatocytes rats [24–26]. There is evidence that ingestion of tea in humans elevates the antioxidative capacity of human plasma [27] Inhibition of DNA oxidation by EC was in a dosedependent manner and was noted with different concentrations. Moreover, the percentage inhibition was more significant at high concentrations of tert-BOOH. These results indicate that the inhibitory effects of EC on DNA degradation caused by hydroperoxides can only be explained by a radical scavenging capacity. These results are not consistent with previously published work from other laboratories which indicates that DNA cleavage generated by tert-BOOH requires a source of iron [27,28] and is insensitive to radical antioxidants. In conclusion, the results presented in this study demonstrate a powerful antioxidant effect of EC in rat hepatocytes against lipid peroxidation. Furthermore, EC has been shown to conserve endogenous antioxidant as GSH and catalase. EC acts as a radical scavenger preventing the oxidation of calf thymus DNA exposed to tert-BOOH.
Fig. 3. GSH levels in isolated rat hepatocytes treated with tert-BOOH and 0.75 mM of EC. All values represent the mean 9 S.D. from n=6 different hepatocyte populations. **PB 0.005, control versus tert-BOOH and EC. **PB 0.005, tert-BOOH versus (EC + tert-BOOH).
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Table 1 Levels of TBARS in calf thymus DNA incubated with tBOOH and EC t-BOOH (mM) 0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5
EC (mM)
0.1 0.5 1 0.1 0.5 1 0.1 0.5 1
TBARS
Activation (%)
0.0990.013 0.0769 0.13 0.0759 0.011 0.0759 0.012 0.0979 0.007 0.0909 0.018 0.0809 0.012 0.0799 0.011 0.1189 0.013 0.1079 0.014 0.092 9 0.014 0.0899 0.007
23
Inhibition (%)
19 20 20 33 10 23 25 62 15 36 40
Acknowledgements This work was supported by a grant of the FEDER (1FD-97-1471/QUI).
References [1] Hermann K. Flavonols and flavones in food plants: a review. J Food Technol 1976;11:433 –48. [2] Hertog MGL, Hollman PCH, Katan MB, Krombout D. Estimation of daily intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutr Canc 1993;20:21 – 9. [3] Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease. Lancet 1993;342:1007 – 11. [4] Cook NC, Sammand S. Flavonoids-chemistry, metabolism cardioprotective effects, and dietary sources. J Nutr Biochem 1996;766 – 76. [5] Korkina LG, Afanas’ev IB. Antioxidant and chelantin properties of flavonoids. Adv Pharmacol 1997;38:151 – 63. [6] Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of human low-density lipoprotein by phenolic substances in red wine. Lancet 1993;341:454 – 7. [7] Namiki M. Antioxidants/antimutagens in food. Crit Rev Food Sci Nutr 1990;29:273 –300. [8] Iannone A, Bini A, Jin G, Vannini V, Tomasi A. tert-Butylhydroperoxide bioactivation to methyl radical in rat liver mitochondria and submitochondrial particles. Free Rad Res Comms 1993;19:141 – 7. [9] Sies H, Summer KHH. Hydropeoxide metabolizing systems in rat liver. Eur J Biochem 1975;57:503 –12. [10] Berry MN, Friend DS. High yield preparation of isolated rat liver parenchymal cells. J Cell Biol 1969;43:506 –20.
[11] Seglen PO. Preparation of rat liver cells. Effect of calcio and enzymatic dispersion of isolated perfused liver. Exptl C Res 1972;74:450 – 4. [12] Stacey N, Priestly BG. Lipid peroxidation in isolated rat hepatocytes: relationship to toxicity of diethyl maleate. Tos Appl Pharmacol 1978;45:41 – 8. [13] Bergmeyer HU, Bernt E. Lactate dehydrogenase, assay with pyruvate and NADH. In: Bergmeyer HU, editor. Methods of enzymatic analysis, 1974:574 – 9. [14] McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erytocuprein. Biol Chem 1969;244:6049 – 55. [15] Clairbone A. Catalasa activity. Handbook of methods for oxygen radical research. Boca Raton: CRC press, 1983:283 – 6. [16] Flohe L, Gunzler WA. Glutathione peroxidase. Methods Enzymol 1984;114 – 21. [17] Brigelius R, Muckel C, Akerboom TPM, Sies H. Identification and quantification of glutathione in hepatic protein mixed disulfides and its relation to glutathione disulfides. Biochem Pharmacol 1981;2529 – 34. [18] Quintlan GJ, Gutteridge JMC. Hydroxyl radical generation by the tetracyclin antibiotics with free radical damage to DNA, lipids and carbohydrates in the presence of iron and copper salts. Free Rad Biol Med 1988;5:341 – 8. [19] Negre-Salvayre A, Alomar Y, Troly M, Salvare R. Ultraviolettreated lipoproteins as a model system for the study of the biological effects of lipid peroxides of cultured cells III. The protective effect of antioxidants (probucol, catechim vitamin E) against the cytotoxicity of oxidized LDL ocurrc¸ s in two different ways. Biochim Biophys Acta 1991;1096:291 – 300. [20] Serafini M, Ghiselli A, Ferro-Luzi A. Red wine, tea and antioxidants. Lancet 1994;344:626. [21] Hippeli S, Elstner F. Transition metal ion-catalyzed oxygen activation during pathogenic process. 1999;443:1 – 7. [22] Terao J, Piskula M, Yao Q. Protective effect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers. Arch Biochem Biophys 1994;308:278 – 84. [23] Bellomo G, Jewell SA, Thor H, Orrenius S. Regulation of intracellular calcium compartmentation studies with isolated hepatocytes and tert-butylhydroperoxide. Proc Natl Acad Sci USA, 1982. [24] Salah N, Miller NJ, Paganga G, Tijburg L, Bolwell GP, RiceEvans C. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-brealing antioxidants. Arch Biochem Biophys 1995;322:2339 – 46. [25] Zhu Q-Y, Huang Y, Tsang D, Chen ZY. Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin. J Agri Food Chem 1990;47(5):2020 – 5. [26] Da Silva EL, Piskula M, Terao J. Enhancement of antioxidative ability of rat plasma by oral administration of ( − ) Epicatechin. Free Rad Biol Med 1998;24:1209 – 16. [27] Latour I, Demoulin JB, Buc-Calderon P. Oxidative DNA damage by tert-butylhydroperoxide causes DNA single strand breaks which in not linked to cell lysis. A mechanistic study in feshly isolated rat hepatocytes. FEBS Lett 1995;373:299 – 302. [28] Sestili P, Guidarelli A, Dacha M, Cantoni O. Quercetin prevents DNA single strand breakage and citotoxicity caused by tertbutylhydroperoxide: free radical scavenging versus iron chelating mechanism. Free Rad Biol Med 1998;25(2):196 – 200.
Mechanism of protection by epicatechin against tert-butylhydroperoxide induced oxidative cell injury in isolated rat hepatocytes and calf thymus DNA V. Valls-Belle´s, P. Mun˜iz *, P. Gonza´lez, M.L. Gonza´lez-Sanjose´, S. Beltran Dpto Biotecnologia y Ciencia de los Alimentos, Facultad de Ciencias, Uni6ersidad de Burgos, 09001 Burgos, Spain Received 12 June 2001; received in revised form 12 June 2001; accepted 30 June 2001
Abstract The purpose of this investigation was to establish the antioxidant effect of flavonoid epicatechin (EC) a low concentration (0.75 mM). The relative antioxidant potentials were measured against radicals generated by tert-BOOH in isolated rat hepatocytes and calf thymus DNA. EC is an effective antioxidant, it completely prevents tert-BOOH induced oxidation and thereby prevents subsequent lipid peroxidation. It also plays an important role in conserving of endogenous antioxidants such as GSH and catalase. As it is a radical scavenger, it is a prominent antioxidant on calf thymus DNA exposed to tert-BOOH. Oxidative stress induced by tert-BOOH at different concentrations in these isolated cells led to lipid peroxidation and changes in endogenous antioxidant levels, which were prevented by EC at a dose of 0.75 mM. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Epicatechin; Antioxidant enzymes; Glutathione; Tert-butylhydroperoxide
1. Introduction Flavonoids are polyphenolic compounds widely distributed in foods and beverages extensively studied for their antioxidant and cytoprotective properties in various biological models [1 – 3]. As the constituents of an integral part of the human diet, they are considered to exert a wide range of beneficial effects on human health, including protection against cardiovascular diseases and certain forms of cancer [4]. They can protect against oxidative stress through their antioxidative activities involving free radical scavenging and also by metal ion-chelating [5]. ( −)-Epicatechin (EC) is a flavonoid belonging to the flavanol family and is commonly present in tea and red wine. The antioxidant effect of wine led to the hypothesis that the phenolic phytochemicals of wine could be beneficial against coronary heart disease by inhibition * Corresponding author. Address: Area Bioquimica y Biologı´a Molecular, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Ban˜uelos s/n, 09001 Burgos, Spain. Tel.: +34-6-947258800x8210; fax: +34-6-947-258831. E-mail address: [email protected] (P. Mun˜iz).
of low density lipoprotein oxidation in vivo [6]. However, factors effecting the scavenging activity of the catechin are still obscure. Catechins, ECs and isomers constitute the most abundant flavonoid class in wines. In studies on model systems, the catechins from tea have shown high activity in erythrocyte membranes and in rat liver microsomes with great protection from lipid oxidation being shown by epigalocatechin gallate and EC gallate, the latter being ten times more effective than vitamin E [7] Tert-BOOH is a well known inducer of oxidative stress in biological systems and it is believed that reactive free radical intermediates, including peroxyl-, alkoxyl- and carbon centred radicals, are mediators of peroxide-dependent injury [8,9]. In this study, we have investigated the effect of the flavonoid EC on cytotoxicity induced by tert-BOOH toxicity in isolated rat hepatocytes. In order to estimate the extent of oxidative stress, lipoperoxidation was evaluated as thiobarbituric acid reactant substances (TBARS). Lactate dehydrogenase (LDH) leakage and modifications in enzymic and non-enzymic antioxidants were also measured as indicators of cell damage. Re-
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sults strongly imply that EC possesses an ability to protect rat hepatocytes against free radicals induced by tert-BOOH. We also examined EC for its effect on DNA damage in vitro induced by tert-BOOH. The fact that DNA represents an important target molecule of the reactive species generated during oxidative stress prompted the present investigation. This study shows that the mechanism of action of EC could be free radical scavenging or iron chelatin mechanism. The results obtained indicate that the effects of EC are best explained by free radical scavenging.
2. Materials and methods
2.1. Materials Calf thymus DNA, tert-BOOH, EC, and enzymes used were purchased from Sigma Chemical Co. (St. Louis, MO). Reduced glutathione, ethanol, enzymes, coenzymes and other reagents were of the highest purity and were from Merck (Darmstadt, Germany).
2.2. Hepatocyte cells and treatment Rat hepatocytes were isolated from 3-month-old Wistar male animals by cannulating the portal vein and perfusing the liver with a collagenase solution as previously described [10,11]. Two milliliters of hepatocyte suspension cells in Krebs– Henseleit saline were incubated in a shaking water bath at 37 °C in 25-ml capacity conical flasks. Oxidative stress was induced by supplementation with tert-BOOH of different concentrations. EC was dissolved in water and added to hepatocytes at a concentration of 0.75 mM. Moreover, some cultures were maintained in the presence of tertBOOH or EC alone (control cultures). Reactions were stopped after 60 min with 0.4 ml 20% perchloric acid.
2.4. Measurement of lactate dehydrogenase leakage LDH leakage from isolated rat hepatocytes was measured in incubation mixtures by spectrophotometer method [13]. These were centrifuged at slow speed to spin down the cells and appropriate dilutions of the cell-free supernatants were used for determination of activity.
2.5. Measurement of antioxidant acti6ities The activity of the total SOD was measured by monitoring at 550 nm the rate of reduction of Cytochrome c by superoxide radicals (O2 − ) produced by the xanthine/xanthine oxidase system with the method developed by McCord and Fridovich [14]. The activity of catalase was determined by measurement of H2O2 concentration using UV absorbance at 240 nm according to the method of Clairbone [15]. Glutathione peroxidase activity was determined measuring the conversion of NADPH to NADP in the presence of reduced glutathione (GSH) and H2O2 spectrophotometrically at 340 nm according to the method described by Flohe and Gunzler [16] and GSH was analyzed spectrophotometrically by the glutathione-S-transferase assay [17].
2.6. Degradation of DNA. TBA-reacti6ity assay Reaction mixtures of 1 ml final volume contained in the following order: sodium phosphate buffer pH 7.4, calf thymus DNA (4 mg/ml) and tert-BOOH at the final concentration indicated in the study and where indicated 0.75 mM of EC. The release of TBA-reactivity as an indication of DNA degradation was measured following the procedure described by Quintlan and Gutteridge [18]. One milliliter of 1% in 50 mM thiobarbituric acid followed by 1 ml of 28% trichloroacetic acid (TCA) were added to each tube and mixed. The release of thiobarbituric acid reactive products was followed by spectrophotometer at 532 nm.
2.7. Statistical analysis 2.3. Determination of thiobarbituric acid reactant substances TBARS in hepatocytes The formation of lipid oxidation products was evaluated as MDA or TBARS [12] with 1,1,3,-tetramethoxypropane used as standard. After incubation, aliquots were treated with 1% phosphoric acid, 0.6% TBA. The mixtures were cooled, 4.0 ml of n-butanol was added for lipid extraction and they were then mechanically mixed for 10 min. Samples were subsequently centrifuged for 10 min at 4000× g. The lipid soluble phase was then extracted and absorbance was measured at 532 nm. MDA concentration was determined against a standard curve.
Experimental values are means9S.D. of the number of separate experiments indicated in the legends. Significance was assessed using the Student’s t-test. The minimal level of significance was PB 0.01.
3. Results The experimental model of isolated rat hepatocytes treated with tert-BOOH represents a useful tool to study the antioxidant capacity of EC. In hepatocytes treated with EC alone, LDH leakage was not significantly higher than the levels found in the
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control group, showing that EC at the concentration used was not cytotoxic in the medium used (Fig. 1A). LDH leakage was increased in hepatocytes treated with 0.25 and 0.1 mM tert-BOOH during 60 min demonstrating it cytotoxic effect. When tert-BOOH was added with 0.75 mM EC, the levels of LDH released to the medium remained as low as in the control indicating a cytoprotective capacity of EC. In order to assess the antioxidant capacity of EC were studied the TBARS levels in hepatocytes treated with tert-BOOH and EC (Fig. 1B). In hepatocytes treated with EC alone, TBARS level were lower than control hepatocytes, showing that EC alone did not modify basal lipid peroxidation in hepatocytes. The hepatocytes treated with tert-BOOH showing a significative increase of TBARS levels. The addition of EC with tert-BOOH resulted in a decrease of peroxidative damage. This result showed that EC was able to counteract lipid peroxidation, which corresponded to an antioxidant effect of EC.
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The effect of EC on the endogenous enzymatic activities (SOD, catalase and GSH-PX) of isolated rat hepatocytes treated tert-BOOH were also evaluated (Fig. 2). In hepatocytes treated with EC alone, SOD and catalase activities were significantly (PB 0.05 and P B 0.005, respectively) lower than control values. No difference was observed between hepatocytes with and without EC treatment. These results show the antioxidant effect of EC in the absence of oxidative stress. As can observed, hepatocytes treated with tertBOOH alone, showed a decrease in SOD and GPX activities but in both cases the effect was not statistically significant. EC, therefore, does not have any effect on SOD and GSH-PX activity on hepatocytes treated with tert-BOOH, with the exception when they were incubated with 0.1 mM of tert-BOOH where SOD decreased significantly (PB 0.05). In contrast, catalase activity increased significantly (PB 0.005) when hepatocytes was incubated with tert-BOOH and the addition of EC allowed catalase to return to initial values.
Fig. 1. A – B. LDH leakage and MDA levels in isolated rat hepatocytes treated with tert-BOOH and 0.75 mM of EC. All values represent the mean9 S.D. from n =6 different hepatocyte populations. (A) *PB 0.05, **PB 0.005, T60 versus tert-BOOH and EC. (B) *PB 0.05, **PB 0.005, tert-BOOH versus tert-BOOH with EC.
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Fig. 2. SOD, Cat, GSH-PX activities in isolated rat hepatocytes treated with tert-BOOH and 0.75 mM EC. All values represent the mean 9 S.D. from n =6 different hepatocytes populations. **PB 0.005, control versus tert-BOOH and EC. **PB0.005, tert-BOOH versus (EC + tertBOOH).
The effect of EC on GSH levels was also investigated Fig. 3. GSH levels in the presence of tert-BOOH were 119 3 and 129 2 and in the presence of EC were of 32.69 10.5 and 35.399.7 mmol/g protein, levels similar to control group. So, GSH concentration was increase in presence of the antioxidant EC. The degradation of DNA in the presence of tertBOOH was substantially inhibited by the EC and the effect of increasing concentration of EC was investi-
gated. As illustrated in Table 1 treatment with tertBOOH was also highly toxic in calf thymus DNA resulting in a dose-dependent effect. The degradation of DNA, measured as release of TBA reactivity was most marked when the final tert-BOOH concentrations in the reaction mixture were 0.5 mM. Table 1 show that in the presence of EC the TBARS were significantly inhibited, with an inhibition of 40% in hepatocytes treated with 0.5 mM tert-BOOH and 1 mM EC.
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Importantly, EC alone was not cytotoxic at any of the concentrations tested. Showing that EC is an extremely effective antioxidant.
4. Discussion There is evidence that flavonoids exert an in vitro antioxidative effect [4,19]. This effect has attracted much attention in relation to the protective effect of the consumption of catechin-containing beverages such as wine [20] over different diseases. However, in contrast to these beneficial effects, cytotoxicity has also been observed with flavonoids. Potentially harmful effects have mostly been reported, this toxicity of flavonoids was thought to result from their pro-oxidant action [21]. The cytoprotective effect of EC was associated with a complete inhibition of LDH leakage and TBARS formation induced by tert-BOOH. Terao et al. [22] showed that flavonoids are localized near the surface of phospholipid bilayers suitable for scavenging oxygen radicals and thereby they prevent lipid peroxidation in phospholipid bilayers exposed to aqueous oxygen radicals. Tert-BOOH induced a decrease in GSH levels and these results are also consistent with those published by Bellomo et al. [23], who observed that the effect of tert-BOOH in intact hepatocytes led to a marked decrease in cellular GSH level. The decrease of GSH during oxidation produced by tert-BOOH was suppressed when rat hepatocytes were
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incubated with EC. The levels of catalase decreased in the presence of EC in isolated hepatocytes treated with tert-BOOH. This shows that the capacity of EC as antioxidant is also superior to catalase. All of this represents an original mechanism of cytoprotection by EC against oxidative stress. These actions may contribute to increase antioxidant capacity in vivo. It was reported that some antioxidative activity of EC still remains during the metabolic process, and it may positively affect the antioxidative defense of hepatocytes rats [24–26]. There is evidence that ingestion of tea in humans elevates the antioxidative capacity of human plasma [27] Inhibition of DNA oxidation by EC was in a dosedependent manner and was noted with different concentrations. Moreover, the percentage inhibition was more significant at high concentrations of tert-BOOH. These results indicate that the inhibitory effects of EC on DNA degradation caused by hydroperoxides can only be explained by a radical scavenging capacity. These results are not consistent with previously published work from other laboratories which indicates that DNA cleavage generated by tert-BOOH requires a source of iron [27,28] and is insensitive to radical antioxidants. In conclusion, the results presented in this study demonstrate a powerful antioxidant effect of EC in rat hepatocytes against lipid peroxidation. Furthermore, EC has been shown to conserve endogenous antioxidant as GSH and catalase. EC acts as a radical scavenger preventing the oxidation of calf thymus DNA exposed to tert-BOOH.
Fig. 3. GSH levels in isolated rat hepatocytes treated with tert-BOOH and 0.75 mM of EC. All values represent the mean 9 S.D. from n=6 different hepatocyte populations. **PB 0.005, control versus tert-BOOH and EC. **PB 0.005, tert-BOOH versus (EC + tert-BOOH).
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Table 1 Levels of TBARS in calf thymus DNA incubated with tBOOH and EC t-BOOH (mM) 0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5
EC (mM)
0.1 0.5 1 0.1 0.5 1 0.1 0.5 1
TBARS
Activation (%)
0.0990.013 0.0769 0.13 0.0759 0.011 0.0759 0.012 0.0979 0.007 0.0909 0.018 0.0809 0.012 0.0799 0.011 0.1189 0.013 0.1079 0.014 0.092 9 0.014 0.0899 0.007
23
Inhibition (%)
19 20 20 33 10 23 25 62 15 36 40
Acknowledgements This work was supported by a grant of the FEDER (1FD-97-1471/QUI).
References [1] Hermann K. Flavonols and flavones in food plants: a review. J Food Technol 1976;11:433 –48. [2] Hertog MGL, Hollman PCH, Katan MB, Krombout D. Estimation of daily intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutr Canc 1993;20:21 – 9. [3] Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease. Lancet 1993;342:1007 – 11. [4] Cook NC, Sammand S. Flavonoids-chemistry, metabolism cardioprotective effects, and dietary sources. J Nutr Biochem 1996;766 – 76. [5] Korkina LG, Afanas’ev IB. Antioxidant and chelantin properties of flavonoids. Adv Pharmacol 1997;38:151 – 63. [6] Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of human low-density lipoprotein by phenolic substances in red wine. Lancet 1993;341:454 – 7. [7] Namiki M. Antioxidants/antimutagens in food. Crit Rev Food Sci Nutr 1990;29:273 –300. [8] Iannone A, Bini A, Jin G, Vannini V, Tomasi A. tert-Butylhydroperoxide bioactivation to methyl radical in rat liver mitochondria and submitochondrial particles. Free Rad Res Comms 1993;19:141 – 7. [9] Sies H, Summer KHH. Hydropeoxide metabolizing systems in rat liver. Eur J Biochem 1975;57:503 –12. [10] Berry MN, Friend DS. High yield preparation of isolated rat liver parenchymal cells. J Cell Biol 1969;43:506 –20.
[11] Seglen PO. Preparation of rat liver cells. Effect of calcio and enzymatic dispersion of isolated perfused liver. Exptl C Res 1972;74:450 – 4. [12] Stacey N, Priestly BG. Lipid peroxidation in isolated rat hepatocytes: relationship to toxicity of diethyl maleate. Tos Appl Pharmacol 1978;45:41 – 8. [13] Bergmeyer HU, Bernt E. Lactate dehydrogenase, assay with pyruvate and NADH. In: Bergmeyer HU, editor. Methods of enzymatic analysis, 1974:574 – 9. [14] McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erytocuprein. Biol Chem 1969;244:6049 – 55. [15] Clairbone A. Catalasa activity. Handbook of methods for oxygen radical research. Boca Raton: CRC press, 1983:283 – 6. [16] Flohe L, Gunzler WA. Glutathione peroxidase. Methods Enzymol 1984;114 – 21. [17] Brigelius R, Muckel C, Akerboom TPM, Sies H. Identification and quantification of glutathione in hepatic protein mixed disulfides and its relation to glutathione disulfides. Biochem Pharmacol 1981;2529 – 34. [18] Quintlan GJ, Gutteridge JMC. Hydroxyl radical generation by the tetracyclin antibiotics with free radical damage to DNA, lipids and carbohydrates in the presence of iron and copper salts. Free Rad Biol Med 1988;5:341 – 8. [19] Negre-Salvayre A, Alomar Y, Troly M, Salvare R. Ultraviolettreated lipoproteins as a model system for the study of the biological effects of lipid peroxides of cultured cells III. The protective effect of antioxidants (probucol, catechim vitamin E) against the cytotoxicity of oxidized LDL ocurrc¸ s in two different ways. Biochim Biophys Acta 1991;1096:291 – 300. [20] Serafini M, Ghiselli A, Ferro-Luzi A. Red wine, tea and antioxidants. Lancet 1994;344:626. [21] Hippeli S, Elstner F. Transition metal ion-catalyzed oxygen activation during pathogenic process. 1999;443:1 – 7. [22] Terao J, Piskula M, Yao Q. Protective effect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers. Arch Biochem Biophys 1994;308:278 – 84. [23] Bellomo G, Jewell SA, Thor H, Orrenius S. Regulation of intracellular calcium compartmentation studies with isolated hepatocytes and tert-butylhydroperoxide. Proc Natl Acad Sci USA, 1982. [24] Salah N, Miller NJ, Paganga G, Tijburg L, Bolwell GP, RiceEvans C. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-brealing antioxidants. Arch Biochem Biophys 1995;322:2339 – 46. [25] Zhu Q-Y, Huang Y, Tsang D, Chen ZY. Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin. J Agri Food Chem 1990;47(5):2020 – 5. [26] Da Silva EL, Piskula M, Terao J. Enhancement of antioxidative ability of rat plasma by oral administration of ( − ) Epicatechin. Free Rad Biol Med 1998;24:1209 – 16. [27] Latour I, Demoulin JB, Buc-Calderon P. Oxidative DNA damage by tert-butylhydroperoxide causes DNA single strand breaks which in not linked to cell lysis. A mechanistic study in feshly isolated rat hepatocytes. FEBS Lett 1995;373:299 – 302. [28] Sestili P, Guidarelli A, Dacha M, Cantoni O. Quercetin prevents DNA single strand breakage and citotoxicity caused by tertbutylhydroperoxide: free radical scavenging versus iron chelating mechanism. Free Rad Biol Med 1998;25(2):196 – 200.