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Consumption of green tea protects rats from exercise-induced oxidative stress in kidney and liver
Nutrition Research 22 (2002) 1177–1188 www.elsevier.com/locate/nutres
Consumption of green tea protects rats from exerciseinduced oxidative stress in kidney and liver Helaine M. Alessioa,*, Ann E. Hagermanb, Mary Romanelloc, Stephane Carandob, Melinda S. Threlkeldb, J. Rogersc, Yoana Dimitrovab, Subiquah Muhammedb, Ronald L. Wileyc a
Department Physical Education, Health & Sport Studies, Miami University, Oxford, OH 45056, USA b Department Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA c Department Zoology, Miami University, Oxford, OH 45056, USA Received 1 November 2001; received in revised form 31 May 2002; accepted 3 June 2002
Abstract The effects of green tea on biomarkers of exercise-induced oxidative status were measured in young male Sprague-Dawley rats. Rats (n ⫽ 12) drank green tea or water ad lib for 6.5 weeks. Half of each group was sacrificed at rest, and the other half ran 25 m/min at 0% grade for approximately 30 min immediately before sacrifice. Green tea had no effect on resting heart rate, blood pressure, body weight, cholesterol, or triglycerides. Tea consumption had a mild influence on total plasma antioxidants, heart glutathione, and plasma ascorbic acid. Exercise had a major impact on malonaldehyde (MDA) equivalents in kidney (⫹290%, p ⫽ 0.0001), and to a lesser extent, liver (⫹81%, p ⫽ 0.18) in rats that drank water. In contrast, kidney MDA equivalents were unchanged by exercise in rats that drank green tea. Green tea may have selective protective effects within the body, especially on the kidney. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Tannins; Polyphenols; Antioxidants; Prooxidants; Physical activity
1. Introduction Epidemiological studies of cancer and cardiovascular disease suggest that consumption of fruits, vegetables, and plant-derived beverages is correlated with reduced risk of chronic
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disease [1,2]. The benefits of plant-based foods may be a consequence of bioactive phytochemicals found in these foods. Phytochemicals include a wide variety of non-nutritive plant constituents that have diverse biochemical activities, including antioxidant properties. One of the most widely distributed classes of phytochemicals is the polyphenolic compounds, a group that includes flavonoids and tannins [3]. The common feature of all these compounds is their phenolic structure, and it is a consequence of this characteristic that makes polyphenolic compounds potent antioxidants [4]. Consumption of fruits, vegetables, and plantderived beverages can provide protection from some diseases by providing polyphenolic compounds that may supplement or enhance activities of known dietary and endogenous antioxidants. For example, the health benefits associated with consumption of green tea [5,6] may be derived from its principal flavonoid constituent, epigallocatechin gallate (EGCG), which acts as a potent reducing agent [7] and as an antioxidant in vivo [8]. Beverages containing EGCG have recently been marketed for health and sports performance. Yet, short term ingestion of EGCG as a health or ergogenic aid has not been studied. Green tea thus represents a useful way to study the potential of dietary polyphenolic compounds, including EGCG, to act as beneficial antioxidants that may affect health and performance. Although oxidative stress generally seems to contribute to chronic diseases via a lifetime accumulation of oxidative events, systems for studying the role of dietary antioxidants in vivo generally include imposition of an acute oxidative stress so that responses can be studied in a reasonably short time frame. Acute exercise is a well-accepted model to induce oxidative stress. Decades of research on exercise and oxidative stress have contributed to our understanding of mechanisms that underlie the health benefits of chronic exercise and the potential dangers of strenuous acute exercise [9 –12]. A single bout of exercise can alter tissue antioxidant enzyme activity [13]. Chronic exercise enhances some antioxidants [14 –16], reduces leakage and uncoupling in the mitochondrial membrane so that fewer oxygencentered radicals are produced, and may signal transcription factors that regulate cellular redox reactions [17]. Most dietary interventions have focused on nutritive factors such as vitamin antioxidants, or drugs that mediate exercise-induced oxidative stress [18 –22]. The present study is the first to investigate the effects of a non-nutritive substance, green tea, on oxidative stress indicators with acute exercise. We hypothesize that ingestion of EGCG via green tea will protect tissues in experimental animals from exercise-induced oxidative stress.
2. Methods and materials Male Sprague Dawley rats (11 weeks old) were pair weighted and equally divided into groups of six: 1) water only, 2) water ⫹ aerobic exercise (AE), 3) tea (EGCG) only, 4) EGCG ⫹ AE. ECGC was administered by substituting decaffeinated green tea for drinking water. All groups consumed rat chow throughout the 6.5 week experiment. One rat from
* Corresponding author. Tel.: ⫹1-513-529-2707; fax: ⫹1-513-529-5006. E-mail address: [email protected] (H. Alessio).
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group 3 died part way through the experiment, so the final n for that group was 5. Autopsy showed that the death was unrelated to treatment. Rats were housed in pairs in climatecontrolled rooms with 12 h light/dark cycle. The study was conducted in accordance with ethical procedures and policies approved by the Institutional Animal Care and Use Committee of Miami University. Tea was prepared daily by steeping 3 decaffeinated green tea bags (Celestial Seasonings, Boulder, CO) in 1 L of boiling distilled water for 3 min. Tea was cooled before presenting it to the rats in water bottles. Each day when fresh tea or water was provided, the volume of liquid consumed during the previous 24 h was recorded. All rats were removed from their cages twice per week for 30 min periods for mild physical activity in a large box (0.9 m ⫻ 0.6 m). Rats were placed in the box in groups of 4, and were monitored during this time to ensure that all animals were normally active and not aggressive. Blood pressure and heart rate were measured weekly during the last four weeks of the study with a tail cuff connected to a sphygmomanometer, IITC amplifier, and Grass Polygraph (Model 7 D, Quincy, Massachusetts), using a multi-channel pen recorder. Resting heart rate, systolic blood pressure, mean arterial pressure, and calculated diastolic blood pressure were averaged over three samplings for each rat. Rats were sacrificed in random order between 8 am and noon on two successive days by decapitation and exsanguination, in an unfasted state. Rats from groups 1 and 3 were sacrificed in a rested state whereas rats from groups 2 and 4 were exercised for approximately 30 minutes on a treadmill (Columbus Instrument, Columbus, Ohio) at an average speed of 25 m/min on a 0% grade immediately prior to sacrifice. When a rat no longer continued running despite receiving a low-level electrical current to its tail, it was removed from the treadmill and immediately sacrificed by decapitation and exsanguination. Within one minute, blood was collected into tubes containing EDTA, kept on ice, and centrifuged to recover plasma. Within five minutes, heart, lung, kidney, liver, and red and white portions of vastus lateralis muscle were harvested and immediately frozen in liquid nitrogen. Subsamples of plasma were removed for ascorbic acid analysis on the day of sample collection. Whole blood was used to assess blood lipids and glucose immediately after collection. The remaining samples were kept frozen at ⫺40oC over a period of up to four months. Biomarkers of oxidative stress included a lipid peroxidation marker, malonaldehyde (MDA) equivalents, protein carbonyls, glutathione (GSH), ascorbic acid, nitric oxide metabolites, and total antioxidants (oxygen radical absorbance capacity, ORAC). Lipid peroxidation by-products were measured in tissue samples after reaction with thiobarbituric acid [23]. The method was standardized with MDA (bis(dimethyl acetyl) 1,1,3,3-tetramethoxypropane, Sigma Chemical Co., St. Louis, MO). The concentration of lipid peroxidation products was expressed as MDA equivalents per mg protein determined with the Bradford method (BioRad, Hercules, CA) standardized with bovine serum albumin. Total protein carbonyls were determined spectrophotometrically after conversion to the dinitrophenylhydrazine derivatives [24]. Absorbances were converted to molar quantities using the reported extinction coefficient (22,000 M⫺1 cm⫺1) [24]. Values were expressed per mg protein determined with the biocinchoninic acid method (Pierce Chemical Co., Rockford, IL) standardized with bovine serum albumin.
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GSH was determined fluorimetrically after reaction with ortho-phthalaldehyde [25] using commercial GSH (Sigma) as a standard. Values were normalized for protein determined with the Bradford method using bovine serum albumin as a standard. Nitric oxide metabolic products (nitrates and nitrites) were determined in plasma using Griess reagents and a protocol similar to that described by Green et al. [26] (Caymen Chemical Co., Ann Arbor, MI). The assay was standardized with the commercial nitrite standard provided by Caymen. Total antioxidant level was determined with the ORAC assay [27] using samples that were not de-proteinized. After adding the target (beta-phycoerythrin type P1286 from Sigma Chemical Co., St. Louis, MO) and the free radical generator (2,2'-azobis (2-amidinopropane) dihydrochloride, AAPH) to the sample, fluorescence was monitored at 38°C until the emission reached zero (approximately 45– 60 minutes). The area under the curve was determined arithmetically [27] and compared to that of the standard, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), a water-soluble vitamin E analog. Results are expressed as ORAC units, where 1 ORAC unit equals the net protection produced by 1 mM Trolox [27]. Blood lipids were determined immediately after sacrifice with a Cholestech LDX lipid profile plus glucose cartridge (Cholestech, Hayward, CA) using 35 l samples of whole blood. Total triglycerides, high density lipoproteins (HDL) and glucose were measured by enzymatic reactions which were assessed spectrophotometrically. Samples to be used for ascorbic acid determination were mixed with dithiothreitol and perchloric acid immediately after collection and were then prepared for immediate HPLC analysis [28]. The ascorbic acid peak was confirmed by incubation of identical samples with ascorbic acid oxidase (Sigma) before acid precipitation of the protein and and was quantitated with commercial ascorbic acid (Sigma) as the standard. Analysis of variance was used to compare means from these stress biomarkers. Sphericity was tested by the Greenhouse-Geisser method, and the Bonferroni correction within each response-variable comparison was made to decrease the chance of a Type I error [29]. Post hoc comparisons were made by comparison-contrast tests. A probability level of 0.05 initially was set for significance, but we divided the original probability level of 0.05 by the number of ANOVA’s [4] performed so that the 0.0125 level of significance had to be achieved before we recorded a change as significant.
3. Results Tea consumption had no effect on basic physiological parameters (Table 1). Liquid consumption and average body mass were the same for rats in all groups throughout the study. Rats in all groups had similar systolic blood pressures and heart rates (Table 1), indicating that tea consumption did not change these cardiovascular functions over time. There were no changes associated with tea consumption for blood triglycerides, HDL cholesterol, or glucose (Table 1). In all rats, total blood cholesterol was below the limit of detection (100 mg/dL) and therefore it was not possible to calculate LDL cholesterol.
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Table 1 Body weight, liquid intake, cardiovascular parameters and blood chemistry for resting animals consuming water or tea
Weight at 7 weeksb (g) Liquid intake per dayc (mL) Resting heart rateb (beats/min) Systolic blood pressureb (mm Hg) Blood triglyceridesd (mg/dL) Blood HDL cholesterold (mg/dL) Blood glucosed (mg/dL)
Control, no exercise (water)a (n ⫽ 6)
Tea, no exercisea (n ⫽ 5)
411 ⫾ 8 260 ⫾ 5 419 ⫾ 20 153 ⫾ 13 94 ⫾ 6 29 ⫾ 1 128 ⫾ 7
430 ⫾ 11 273 ⫾ 7 412 ⫾ 25 146 ⫾ 15 105 ⫾ 21 23 ⫾ 3 146 ⫾ 13
Values are means ⫾ SEM. Weights, heart rate, and blood pressure were obtained weekly. c Liquid intake was determined daily when fresh tea or water was provided. d Blood chemistry was determined at time of sacrifice. a
b
3.1. Main effect of green tea The main effect of green tea on plasma ORAC (F3,19 ⫽ 2.57, p ⫽ 0.080) failed to reach significance (Fig. 1a) and green tea had no effect on ORAC levels in other tissues measured. The main effect of green tea on plasma GSH (F3,19 ⫽ 4.10, p ⫽ 0.02) (Fig. 1b) and heart GSH (F3,18 ⫽ 2.72, p ⫽ 0.07) (Fig. 2) did not reach significance using the Bonferonni probability level of p ⫽ 0.0125. Despite 32% lower heart GSH levels in rats that consumed green tea compared to water, tea consumption did not significantly decrease heart GSH in rested animals (p ⫽ 0.03) (Fig. 2). Tea consumption had no effect on liver, kidney, white or red muscle GSH in rats (Fig. 2). Tea consumption had no significant effect on the levels of MDA equivalents in liver, heart, and red and white muscle (Fig. 3). Protein carbonyls were unaffected by tea consumption in liver and plasma (data not shown). Tea consumption did not significantly change plasma ascorbic acid or nitrite plus nitrates in resting rats (Fig. 1c, 1d). 3.2. Main effect of exercise The main effect of exercise occurred when comparing MDA equivalents in the kidney (F ⫽ 14.669, p ⫽ 0.0001) (Fig. 3). Acute exercise dramatically increased levels of lipid peroxidation by-products in the kidney. MDA equivalents in the water, exercise group were 290% greater than in the water, rest group; p ⫽ 0.001) (Fig. 3). In other tissues there were no significant changes in MDA equivalents due to exercise by the water-consuming animals (Fig. 3). Acute exercise did not influence levels of protein oxidation in either the plasma or the liver in the water-consuming animals (data not shown). Acute exercise was associated with elevated blood glucose levels in both tea (24% change, p ⫽ 0.04) and water (34% change, p ⫽ 0.010) groups, but only the water group reached statistical significance using the Bonferroni probability level of p ⫽ 0.0125. Blood triglyc-
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Fig. 1. Antioxidants in plasma of animals sacrificed at rest [■] or after aerobic exercise (䊐). Error bars indicate SEM. * indicates significant difference (p ⬍ 0.0125) from control (water, rest). (A) Total antioxidants. (B) Glutathione. (C) Ascorbic acid. (D) Nitrite plus nitrate.
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Fig. 2. Levels of GSH in various tissues of animals sacrificed at rest or after aerobic exercise. Error bars indicate SEM. * indicates significant difference (p ⬍ 0.0125) from control (water, rest).
erides and HDL cholesterol did not change with exercise in tea- or water-consuming groups. Running performance (i.e. time on treadmill) did not differ between water and tea groups. Plasma ORAC levels (F ⫽ 2.561, p ⫽ 0.126), GSH (F ⫽ 7.360, p ⫽ 0.138), ascorbic acid (F ⫽ 3.251, p ⫽ 0.0872) and total nitrites (F ⫽ 4.203, p ⫽ 0.06) did not significantly change following exercise in the water groups (Fig 1a– d). Tissue ORAC levels and tissue GSH (Fig. 2) were not significantly changed by exercise in the water-consuming animals.
3.3. Interaction of exercise and tea Using the Bonferroni level of significance (p ⱕ 0.0125), tea plus exercise did not significantly change plasma ORAC (p ⫽ 0.02), GSH (p ⫽ 0.04), or ascorbic acid (p ⫽ 0.03) (Fig. 1a– c). Levels of nitrite plus nitrate in the plasma of tea-consuming animals were not changed by acute exercise (Fig. 1d). Heart, liver, kidney, white, and red muscle GSH levels did not change following acute exercise in the tea consumption group (Fig. 2). Acute exercise did not change the level of protein oxidation in either the plasma or the liver in animals consuming tea. In animals consuming tea, exercise did not change levels of MDA equivalents in any tissue including kidney and liver (Fig. 3), implying protection against oxidative stress in these tissues. These results differed from those obtained with animals that consumed water, in which kidney MDA equivalents significantly increased after acute exercise.
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Fig. 3. Levels of lipid peroxidation products in various tissues of animals sacrificed at rest or after aerobic exercise. Lipid peroxidation products were measured as malonaldehyde equivalents (MDA). Error bars indicate SEM. * indicates significant difference (p ⬍ 0.0125) from control (water, rest).
4. Discussion Consumption of green tea had positive effects in some tissues and negative effects in others. Positive effects included slightly elevated (10%) plasma ORAC and plasma ascorbic acid (13%), and maintenance of MDA equivalents in kidney and to a lesser degree, in liver following acute exercise by tea-drinking animals. Exercise increased mean MDA equivalents 290% in the kidneys of rats that consumed water instead of green tea. In the heart, green tea was associated with a negative effect as evidenced by heart GSH levels that were approximately 20% lower on average than heart GSH in the water consumption group. Both the water and tea consumption groups maintained their heart GSH levels following exercise. Heart MDA equivalents were not elevated by exercise in either group despite the lower heart GSH levels in the tea consumption group. All rats consumed similar volumes of either tea or water throughout the study, suggesting that tea is an appropriate vehicle for administration of dietary polyphenolic compounds and is associated with neither taste aversion nor stimulation. Consumption of tea did not affect animal growth rate as indicated by similar body weights in all rats throughout the study. Measures of resting heart rate and blood pressure indicated normal cardiovascular and metabolic function in all rats. Short-term green tea ingestion did not affect the blood lipid profile or blood glucose in our study. Although Zeyuan et al. [30] found that providing green tea to mature rats for nearly 11 weeks diminished blood glucose 24% and blood triglycerides 33%, that study is difficult to evaluate since the reported values for blood glucose and blood
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triglycerides were unusually low. Our animals were sacrificed when they were only 17 weeks old, and it is therefore possible that age-related metabolic changes are responsible for the differences between our study and Zeyuan’s. ORAC levels, ascorbic acid, nitrite plus nitrate, and GSH in plasma and in tissues of animals in our study (Fig. 1a– d; Fig. 2) are similar to levels reported in other studies [11,19,31–36]. Furthermore, the general trend observed in many studies for GSH in various tissues (liver ⬎ heart ⬎ kidney ⬎ muscle) is followed in our results (Fig. 2). The levels of lipid peroxidation products we report for the control (water, rest) rats (Fig. 3) are comparable to values from the literature [37–39] although our value for white muscle is higher than the value reported by Ji et al. [11] (Fig. 3). Levels of protein carbonyls in plasma and in liver homogenates were similar to those reported in other studies [40,41]. The effects of acute exercise on antioxidant levels in our control (water-consuming) animals were similar to those reported in other studies [22,41,42], although the magnitude of the changes we report are in some cases relatively small. For example, in our study, the minor increase in ascorbic acid accompanying exercise was not sufficient to significantly elevate total antioxidant levels. Similarly, we found that acute exercise had only a slight tendency to decrease plasma nitrite plus nitrate compared to the decrease in serum nitrate reported in the early stages of exercise training in spontaneously hypertensive rats [43]. Our study is unique in several respects; we sacrificed the animals when they were still young (17 weeks old) and healthy; we used 30 min periods twice per week in the course of the 6.5 week study when rats were provided with moderate physical activity; and we used only 30 min of acute exercise before sacrifice. Changes in oxidative status and antioxidants during exercise vary with workload, time of exercise, and the health and age of the animal [11]. Many exercise studies use animals that are totally sedentary throughout the study and are then exhaustively exercised by as much as 60 min of running immediately before sacrifice [38]. At the time of sacrifice we noted that the mean blood glucose of our exercised rats was elevated, indicating that they were not exhausted [44]. The general trends in MDA equivalents we noted for animals drinking water and subjected to acute exercise before sacrifice (Fig. 3) correspond well with reports from the literature. Our data support the conclusion that there is little oxidative stress in white muscle during exercise [11,37]. Significant increases in the levels of lipid oxidation products in red muscle have been noted in some studies [11] but other studies [38,45], were similar to ours in reporting no accumulation in red muscle. A typical outcome of exercise is an increase in lipid peroxidation by-products in the liver [46,47] but in several studies including ours, the increase does not reach significance [38,45] (Fig. 3). Exercise significantly increased kidney lipid peroxidation as evidenced by a 290% increase in MDA equivalents in rats that consumed water only (p ⫽ 0.0001) (Fig. 3). Other studies employing exhaustive exercise [37] or dietary manipulation of lipids [48] have reported lipid peroxidation changes in kidney. It has been estimated that exercise decreases blood flow to the kidney about five-fold [37]. Reduced blood flow may diminish the ability of the kidney to accumulate antioxidants from the plasma [46] or may reduce excretion of oxidized waste products. Current data does not distinguish between these physiological mechanisms for buildup of lipid peroxidation products in the kidney after acute exercise. In our study, kidney tissue appeared to be protected from exercise-induced oxidative stress in
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the tea consumption group as evidenced by no change in MDA equivalents compared to a 290% increase in the kidneys of the water consumption group (Fig. 3). It is possible that a bioactive component found in tea was transported to the kidney for metabolic processing and excretion, and thus enhanced antioxidant protection specifically in that tissue. The ability of EGCG to serve as a potent antioxidant in vitro [4] and to provide protection from oxidative damage in vivo [49] suggest that EGCG from green tea is responsible for the protective effect, but the mode of action of tea has not been directly demonstrated. In humans, EGCG and other flavonoids from green tea are absorbed from the gastrointestinal tract and can be detected at micromolar levels in the plasma 30 minutes after consumption of the tea [50]. However, Kohri et al. [51]reported that in rats, most of the ECGC taken into the body is not absorbed intact, but rather is degraded in the intestine, and then absorbed and circulated to the kidney. In our study, rats freely drank the green tea for 6.5 weeks but EGCG levels in plasma remained below our detection limit (4 M), consistent with Kohri’s observations. We suggest that the ECGC and its byproducts are efficiently transported to the kidney for processing and excretion and provide protection from exercise-induced lipid oxidation in kidney. Our dietary treatment with green tea prevented exercise-induced accumulation of lipid peroxidation products in kidney and, to a lesser extent, in liver, but our study did not identify mechanisms of inhibition. Further studies of the absorption, transport and accumulation of EGCG and related compounds are required to determine the basis for the beneficial effects of tea drinking. Development of a more sensitive method for determining EGCG in plasma samples in small rodents, and extension of that method to tissue samples are mandated before further studies are undertaken.
Acknowledgments This work was funded by the National Cancer Institute and Miami University Faculty Research Committee. Funding for undergraduate students was provided by the Howard Hughes Research Institute.
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[34] Leeuwenburgh C, Hollander J, Leichtweis S, Griffiths M, Gore M, Ji LL. Adaptations of glutathione antioxdant system to endurance training are tissue and muscle fiber specific. Am J Physiol 1997;272:R363– R369. [35] Wang JS, Chow S-E, Chen J-K, Wong M-K. Effect of exercise training on oxidized LDL-mediated platelet function in rats. Thromb Haemost 2000;83:503–508. [36] Atalay M, Seene T, Hanninen O, Sen CK. Skeletal muscle and heart antioxidant defences in response to sprint training. Acta Physiol Scand 1996;158:129 –134. [37] Radak Z, Kaneko T, Tahara S, Nakamoto H, Ohno H, Sasvari M, Nyakas C, Goto S. The effect of exercise training on oxidative damage of lipids, proteins and DNA in rat sketetal muscle: evidence for beneficial outcomes. Free Rad Biol Med 1999;27:69 –74. [38] Ji LL, Stratman FW, Lardy HA. Enzymatic down regulation with exercise in rat skeletal muscle. Arch Biochem Biophys 1988;263:137–149. [39] Rathore N, John S, Kale M, Bhatnagar D. Lipid peroxidation and antioxidant enzymes in isoproterenol induced oxidative stress in rat tissues. Pharrm Res 1998;38:297–302. [40] Reznick AZ, Packer L. Oxidative damage to protein: spectrophotometric method for carbonyl assay. Methods Enzymol 1994;233:357–363. [41] Witt EH, Reznick AZ, Viguie CA, Starke-Reed P, Packer L. Exercise, oxidative damage, and effects of antioxidant manipulation. J Nutr; 1992;122:766 –773. [42] Child RB, Wilkinson DM, Fallowfield JL, Donnelly AE. Elevated serum antioxidant capacity and plasma malondialdehyde concentration in response to a simulated half-marathon run. Med Sci Sports Exerc 1998;30:1603–1607. [43] Jonsdottir IH, Jungersten L, Johansson C, Wennmalm A, Thoren P, Hoffmann P. Increase in nitric oxide formation after chronic voluntary exercise in spontaneously hypertensive rats. Acta Physiol Scan 1998; 162:149 –153. [44] Rennie MJ, Winder WW, Holloszy JO. A sparing effect of increased plasma fatty acids on muscles, and liver glycogen content in the exercising rat. Biochem J 1976;156:647– 655. [45] Leeuwenburgh C, Ji LL. Alteration of glutathione and antioxidant status with exercise in unfed and refed rats. J Nutr 1996;126:1833–1843. [46] Leeuwenburgh C, Ji LL. Glutathione depletion in rested and exercised mice: biochemical consequence and adaptation. Arch Biochem Biophys 1995;316:941–949. [47] Radak Z, Asano K, Inoue M, Kizaki T, Oh-Ishi S, Suzuki K, Tahniguchi N, Ohno H. Superoxide dismutase derivative prevents oxidative damage in liver and kidney of rats induced by exhausting exercise. Eur J Appl Physiol 1996;72:189 –194. [48] Draper HH, Polensek L, Hadley M, McGirr LG. Urinary malondialdehyde as an indicator of lipid peroxidation in the diet and in the tissues. Lipids 1984;19:836 – 843. [49] Nagasawa T, Hayashi H, Fujimaki N, Nishizawa N, Kitts DD. Induction of oxidatively modified proteins in skeletal muscle by electrical stimulation and its suppression by dietary supplementation of (-)-epigallocatechin gallate. Biosci Biotech Biochem 2000;64:1004 –1010. [50] Maiani G, Serafini M, Salucci M, Azzini E, Ferro-Luzzi A. Application of a new high-performance liquid chromatographic method for measuring selected polyphenols in human plasma. J Chrom B 1997;692:311– 317. [51] Kohri Toshiyuki, Matsumoto Natsuki, Yamakawa Mana, Suzuki Masayuki, Nanjo Fumio, Hara Yukihiko, Oku Naoto. Metabolic fate of (-)-[4-3H]epigallocatechin gallate in rats after oral administration. J Agric Food Chem 2001;49:4102– 4112.
Consumption of green tea protects rats from exerciseinduced oxidative stress in kidney and liver Helaine M. Alessioa,*, Ann E. Hagermanb, Mary Romanelloc, Stephane Carandob, Melinda S. Threlkeldb, J. Rogersc, Yoana Dimitrovab, Subiquah Muhammedb, Ronald L. Wileyc a
Department Physical Education, Health & Sport Studies, Miami University, Oxford, OH 45056, USA b Department Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA c Department Zoology, Miami University, Oxford, OH 45056, USA Received 1 November 2001; received in revised form 31 May 2002; accepted 3 June 2002
Abstract The effects of green tea on biomarkers of exercise-induced oxidative status were measured in young male Sprague-Dawley rats. Rats (n ⫽ 12) drank green tea or water ad lib for 6.5 weeks. Half of each group was sacrificed at rest, and the other half ran 25 m/min at 0% grade for approximately 30 min immediately before sacrifice. Green tea had no effect on resting heart rate, blood pressure, body weight, cholesterol, or triglycerides. Tea consumption had a mild influence on total plasma antioxidants, heart glutathione, and plasma ascorbic acid. Exercise had a major impact on malonaldehyde (MDA) equivalents in kidney (⫹290%, p ⫽ 0.0001), and to a lesser extent, liver (⫹81%, p ⫽ 0.18) in rats that drank water. In contrast, kidney MDA equivalents were unchanged by exercise in rats that drank green tea. Green tea may have selective protective effects within the body, especially on the kidney. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Tannins; Polyphenols; Antioxidants; Prooxidants; Physical activity
1. Introduction Epidemiological studies of cancer and cardiovascular disease suggest that consumption of fruits, vegetables, and plant-derived beverages is correlated with reduced risk of chronic
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disease [1,2]. The benefits of plant-based foods may be a consequence of bioactive phytochemicals found in these foods. Phytochemicals include a wide variety of non-nutritive plant constituents that have diverse biochemical activities, including antioxidant properties. One of the most widely distributed classes of phytochemicals is the polyphenolic compounds, a group that includes flavonoids and tannins [3]. The common feature of all these compounds is their phenolic structure, and it is a consequence of this characteristic that makes polyphenolic compounds potent antioxidants [4]. Consumption of fruits, vegetables, and plantderived beverages can provide protection from some diseases by providing polyphenolic compounds that may supplement or enhance activities of known dietary and endogenous antioxidants. For example, the health benefits associated with consumption of green tea [5,6] may be derived from its principal flavonoid constituent, epigallocatechin gallate (EGCG), which acts as a potent reducing agent [7] and as an antioxidant in vivo [8]. Beverages containing EGCG have recently been marketed for health and sports performance. Yet, short term ingestion of EGCG as a health or ergogenic aid has not been studied. Green tea thus represents a useful way to study the potential of dietary polyphenolic compounds, including EGCG, to act as beneficial antioxidants that may affect health and performance. Although oxidative stress generally seems to contribute to chronic diseases via a lifetime accumulation of oxidative events, systems for studying the role of dietary antioxidants in vivo generally include imposition of an acute oxidative stress so that responses can be studied in a reasonably short time frame. Acute exercise is a well-accepted model to induce oxidative stress. Decades of research on exercise and oxidative stress have contributed to our understanding of mechanisms that underlie the health benefits of chronic exercise and the potential dangers of strenuous acute exercise [9 –12]. A single bout of exercise can alter tissue antioxidant enzyme activity [13]. Chronic exercise enhances some antioxidants [14 –16], reduces leakage and uncoupling in the mitochondrial membrane so that fewer oxygencentered radicals are produced, and may signal transcription factors that regulate cellular redox reactions [17]. Most dietary interventions have focused on nutritive factors such as vitamin antioxidants, or drugs that mediate exercise-induced oxidative stress [18 –22]. The present study is the first to investigate the effects of a non-nutritive substance, green tea, on oxidative stress indicators with acute exercise. We hypothesize that ingestion of EGCG via green tea will protect tissues in experimental animals from exercise-induced oxidative stress.
2. Methods and materials Male Sprague Dawley rats (11 weeks old) were pair weighted and equally divided into groups of six: 1) water only, 2) water ⫹ aerobic exercise (AE), 3) tea (EGCG) only, 4) EGCG ⫹ AE. ECGC was administered by substituting decaffeinated green tea for drinking water. All groups consumed rat chow throughout the 6.5 week experiment. One rat from
* Corresponding author. Tel.: ⫹1-513-529-2707; fax: ⫹1-513-529-5006. E-mail address: [email protected] (H. Alessio).
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group 3 died part way through the experiment, so the final n for that group was 5. Autopsy showed that the death was unrelated to treatment. Rats were housed in pairs in climatecontrolled rooms with 12 h light/dark cycle. The study was conducted in accordance with ethical procedures and policies approved by the Institutional Animal Care and Use Committee of Miami University. Tea was prepared daily by steeping 3 decaffeinated green tea bags (Celestial Seasonings, Boulder, CO) in 1 L of boiling distilled water for 3 min. Tea was cooled before presenting it to the rats in water bottles. Each day when fresh tea or water was provided, the volume of liquid consumed during the previous 24 h was recorded. All rats were removed from their cages twice per week for 30 min periods for mild physical activity in a large box (0.9 m ⫻ 0.6 m). Rats were placed in the box in groups of 4, and were monitored during this time to ensure that all animals were normally active and not aggressive. Blood pressure and heart rate were measured weekly during the last four weeks of the study with a tail cuff connected to a sphygmomanometer, IITC amplifier, and Grass Polygraph (Model 7 D, Quincy, Massachusetts), using a multi-channel pen recorder. Resting heart rate, systolic blood pressure, mean arterial pressure, and calculated diastolic blood pressure were averaged over three samplings for each rat. Rats were sacrificed in random order between 8 am and noon on two successive days by decapitation and exsanguination, in an unfasted state. Rats from groups 1 and 3 were sacrificed in a rested state whereas rats from groups 2 and 4 were exercised for approximately 30 minutes on a treadmill (Columbus Instrument, Columbus, Ohio) at an average speed of 25 m/min on a 0% grade immediately prior to sacrifice. When a rat no longer continued running despite receiving a low-level electrical current to its tail, it was removed from the treadmill and immediately sacrificed by decapitation and exsanguination. Within one minute, blood was collected into tubes containing EDTA, kept on ice, and centrifuged to recover plasma. Within five minutes, heart, lung, kidney, liver, and red and white portions of vastus lateralis muscle were harvested and immediately frozen in liquid nitrogen. Subsamples of plasma were removed for ascorbic acid analysis on the day of sample collection. Whole blood was used to assess blood lipids and glucose immediately after collection. The remaining samples were kept frozen at ⫺40oC over a period of up to four months. Biomarkers of oxidative stress included a lipid peroxidation marker, malonaldehyde (MDA) equivalents, protein carbonyls, glutathione (GSH), ascorbic acid, nitric oxide metabolites, and total antioxidants (oxygen radical absorbance capacity, ORAC). Lipid peroxidation by-products were measured in tissue samples after reaction with thiobarbituric acid [23]. The method was standardized with MDA (bis(dimethyl acetyl) 1,1,3,3-tetramethoxypropane, Sigma Chemical Co., St. Louis, MO). The concentration of lipid peroxidation products was expressed as MDA equivalents per mg protein determined with the Bradford method (BioRad, Hercules, CA) standardized with bovine serum albumin. Total protein carbonyls were determined spectrophotometrically after conversion to the dinitrophenylhydrazine derivatives [24]. Absorbances were converted to molar quantities using the reported extinction coefficient (22,000 M⫺1 cm⫺1) [24]. Values were expressed per mg protein determined with the biocinchoninic acid method (Pierce Chemical Co., Rockford, IL) standardized with bovine serum albumin.
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GSH was determined fluorimetrically after reaction with ortho-phthalaldehyde [25] using commercial GSH (Sigma) as a standard. Values were normalized for protein determined with the Bradford method using bovine serum albumin as a standard. Nitric oxide metabolic products (nitrates and nitrites) were determined in plasma using Griess reagents and a protocol similar to that described by Green et al. [26] (Caymen Chemical Co., Ann Arbor, MI). The assay was standardized with the commercial nitrite standard provided by Caymen. Total antioxidant level was determined with the ORAC assay [27] using samples that were not de-proteinized. After adding the target (beta-phycoerythrin type P1286 from Sigma Chemical Co., St. Louis, MO) and the free radical generator (2,2'-azobis (2-amidinopropane) dihydrochloride, AAPH) to the sample, fluorescence was monitored at 38°C until the emission reached zero (approximately 45– 60 minutes). The area under the curve was determined arithmetically [27] and compared to that of the standard, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), a water-soluble vitamin E analog. Results are expressed as ORAC units, where 1 ORAC unit equals the net protection produced by 1 mM Trolox [27]. Blood lipids were determined immediately after sacrifice with a Cholestech LDX lipid profile plus glucose cartridge (Cholestech, Hayward, CA) using 35 l samples of whole blood. Total triglycerides, high density lipoproteins (HDL) and glucose were measured by enzymatic reactions which were assessed spectrophotometrically. Samples to be used for ascorbic acid determination were mixed with dithiothreitol and perchloric acid immediately after collection and were then prepared for immediate HPLC analysis [28]. The ascorbic acid peak was confirmed by incubation of identical samples with ascorbic acid oxidase (Sigma) before acid precipitation of the protein and and was quantitated with commercial ascorbic acid (Sigma) as the standard. Analysis of variance was used to compare means from these stress biomarkers. Sphericity was tested by the Greenhouse-Geisser method, and the Bonferroni correction within each response-variable comparison was made to decrease the chance of a Type I error [29]. Post hoc comparisons were made by comparison-contrast tests. A probability level of 0.05 initially was set for significance, but we divided the original probability level of 0.05 by the number of ANOVA’s [4] performed so that the 0.0125 level of significance had to be achieved before we recorded a change as significant.
3. Results Tea consumption had no effect on basic physiological parameters (Table 1). Liquid consumption and average body mass were the same for rats in all groups throughout the study. Rats in all groups had similar systolic blood pressures and heart rates (Table 1), indicating that tea consumption did not change these cardiovascular functions over time. There were no changes associated with tea consumption for blood triglycerides, HDL cholesterol, or glucose (Table 1). In all rats, total blood cholesterol was below the limit of detection (100 mg/dL) and therefore it was not possible to calculate LDL cholesterol.
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Table 1 Body weight, liquid intake, cardiovascular parameters and blood chemistry for resting animals consuming water or tea
Weight at 7 weeksb (g) Liquid intake per dayc (mL) Resting heart rateb (beats/min) Systolic blood pressureb (mm Hg) Blood triglyceridesd (mg/dL) Blood HDL cholesterold (mg/dL) Blood glucosed (mg/dL)
Control, no exercise (water)a (n ⫽ 6)
Tea, no exercisea (n ⫽ 5)
411 ⫾ 8 260 ⫾ 5 419 ⫾ 20 153 ⫾ 13 94 ⫾ 6 29 ⫾ 1 128 ⫾ 7
430 ⫾ 11 273 ⫾ 7 412 ⫾ 25 146 ⫾ 15 105 ⫾ 21 23 ⫾ 3 146 ⫾ 13
Values are means ⫾ SEM. Weights, heart rate, and blood pressure were obtained weekly. c Liquid intake was determined daily when fresh tea or water was provided. d Blood chemistry was determined at time of sacrifice. a
b
3.1. Main effect of green tea The main effect of green tea on plasma ORAC (F3,19 ⫽ 2.57, p ⫽ 0.080) failed to reach significance (Fig. 1a) and green tea had no effect on ORAC levels in other tissues measured. The main effect of green tea on plasma GSH (F3,19 ⫽ 4.10, p ⫽ 0.02) (Fig. 1b) and heart GSH (F3,18 ⫽ 2.72, p ⫽ 0.07) (Fig. 2) did not reach significance using the Bonferonni probability level of p ⫽ 0.0125. Despite 32% lower heart GSH levels in rats that consumed green tea compared to water, tea consumption did not significantly decrease heart GSH in rested animals (p ⫽ 0.03) (Fig. 2). Tea consumption had no effect on liver, kidney, white or red muscle GSH in rats (Fig. 2). Tea consumption had no significant effect on the levels of MDA equivalents in liver, heart, and red and white muscle (Fig. 3). Protein carbonyls were unaffected by tea consumption in liver and plasma (data not shown). Tea consumption did not significantly change plasma ascorbic acid or nitrite plus nitrates in resting rats (Fig. 1c, 1d). 3.2. Main effect of exercise The main effect of exercise occurred when comparing MDA equivalents in the kidney (F ⫽ 14.669, p ⫽ 0.0001) (Fig. 3). Acute exercise dramatically increased levels of lipid peroxidation by-products in the kidney. MDA equivalents in the water, exercise group were 290% greater than in the water, rest group; p ⫽ 0.001) (Fig. 3). In other tissues there were no significant changes in MDA equivalents due to exercise by the water-consuming animals (Fig. 3). Acute exercise did not influence levels of protein oxidation in either the plasma or the liver in the water-consuming animals (data not shown). Acute exercise was associated with elevated blood glucose levels in both tea (24% change, p ⫽ 0.04) and water (34% change, p ⫽ 0.010) groups, but only the water group reached statistical significance using the Bonferroni probability level of p ⫽ 0.0125. Blood triglyc-
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Fig. 1. Antioxidants in plasma of animals sacrificed at rest [■] or after aerobic exercise (䊐). Error bars indicate SEM. * indicates significant difference (p ⬍ 0.0125) from control (water, rest). (A) Total antioxidants. (B) Glutathione. (C) Ascorbic acid. (D) Nitrite plus nitrate.
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Fig. 2. Levels of GSH in various tissues of animals sacrificed at rest or after aerobic exercise. Error bars indicate SEM. * indicates significant difference (p ⬍ 0.0125) from control (water, rest).
erides and HDL cholesterol did not change with exercise in tea- or water-consuming groups. Running performance (i.e. time on treadmill) did not differ between water and tea groups. Plasma ORAC levels (F ⫽ 2.561, p ⫽ 0.126), GSH (F ⫽ 7.360, p ⫽ 0.138), ascorbic acid (F ⫽ 3.251, p ⫽ 0.0872) and total nitrites (F ⫽ 4.203, p ⫽ 0.06) did not significantly change following exercise in the water groups (Fig 1a– d). Tissue ORAC levels and tissue GSH (Fig. 2) were not significantly changed by exercise in the water-consuming animals.
3.3. Interaction of exercise and tea Using the Bonferroni level of significance (p ⱕ 0.0125), tea plus exercise did not significantly change plasma ORAC (p ⫽ 0.02), GSH (p ⫽ 0.04), or ascorbic acid (p ⫽ 0.03) (Fig. 1a– c). Levels of nitrite plus nitrate in the plasma of tea-consuming animals were not changed by acute exercise (Fig. 1d). Heart, liver, kidney, white, and red muscle GSH levels did not change following acute exercise in the tea consumption group (Fig. 2). Acute exercise did not change the level of protein oxidation in either the plasma or the liver in animals consuming tea. In animals consuming tea, exercise did not change levels of MDA equivalents in any tissue including kidney and liver (Fig. 3), implying protection against oxidative stress in these tissues. These results differed from those obtained with animals that consumed water, in which kidney MDA equivalents significantly increased after acute exercise.
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Fig. 3. Levels of lipid peroxidation products in various tissues of animals sacrificed at rest or after aerobic exercise. Lipid peroxidation products were measured as malonaldehyde equivalents (MDA). Error bars indicate SEM. * indicates significant difference (p ⬍ 0.0125) from control (water, rest).
4. Discussion Consumption of green tea had positive effects in some tissues and negative effects in others. Positive effects included slightly elevated (10%) plasma ORAC and plasma ascorbic acid (13%), and maintenance of MDA equivalents in kidney and to a lesser degree, in liver following acute exercise by tea-drinking animals. Exercise increased mean MDA equivalents 290% in the kidneys of rats that consumed water instead of green tea. In the heart, green tea was associated with a negative effect as evidenced by heart GSH levels that were approximately 20% lower on average than heart GSH in the water consumption group. Both the water and tea consumption groups maintained their heart GSH levels following exercise. Heart MDA equivalents were not elevated by exercise in either group despite the lower heart GSH levels in the tea consumption group. All rats consumed similar volumes of either tea or water throughout the study, suggesting that tea is an appropriate vehicle for administration of dietary polyphenolic compounds and is associated with neither taste aversion nor stimulation. Consumption of tea did not affect animal growth rate as indicated by similar body weights in all rats throughout the study. Measures of resting heart rate and blood pressure indicated normal cardiovascular and metabolic function in all rats. Short-term green tea ingestion did not affect the blood lipid profile or blood glucose in our study. Although Zeyuan et al. [30] found that providing green tea to mature rats for nearly 11 weeks diminished blood glucose 24% and blood triglycerides 33%, that study is difficult to evaluate since the reported values for blood glucose and blood
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triglycerides were unusually low. Our animals were sacrificed when they were only 17 weeks old, and it is therefore possible that age-related metabolic changes are responsible for the differences between our study and Zeyuan’s. ORAC levels, ascorbic acid, nitrite plus nitrate, and GSH in plasma and in tissues of animals in our study (Fig. 1a– d; Fig. 2) are similar to levels reported in other studies [11,19,31–36]. Furthermore, the general trend observed in many studies for GSH in various tissues (liver ⬎ heart ⬎ kidney ⬎ muscle) is followed in our results (Fig. 2). The levels of lipid peroxidation products we report for the control (water, rest) rats (Fig. 3) are comparable to values from the literature [37–39] although our value for white muscle is higher than the value reported by Ji et al. [11] (Fig. 3). Levels of protein carbonyls in plasma and in liver homogenates were similar to those reported in other studies [40,41]. The effects of acute exercise on antioxidant levels in our control (water-consuming) animals were similar to those reported in other studies [22,41,42], although the magnitude of the changes we report are in some cases relatively small. For example, in our study, the minor increase in ascorbic acid accompanying exercise was not sufficient to significantly elevate total antioxidant levels. Similarly, we found that acute exercise had only a slight tendency to decrease plasma nitrite plus nitrate compared to the decrease in serum nitrate reported in the early stages of exercise training in spontaneously hypertensive rats [43]. Our study is unique in several respects; we sacrificed the animals when they were still young (17 weeks old) and healthy; we used 30 min periods twice per week in the course of the 6.5 week study when rats were provided with moderate physical activity; and we used only 30 min of acute exercise before sacrifice. Changes in oxidative status and antioxidants during exercise vary with workload, time of exercise, and the health and age of the animal [11]. Many exercise studies use animals that are totally sedentary throughout the study and are then exhaustively exercised by as much as 60 min of running immediately before sacrifice [38]. At the time of sacrifice we noted that the mean blood glucose of our exercised rats was elevated, indicating that they were not exhausted [44]. The general trends in MDA equivalents we noted for animals drinking water and subjected to acute exercise before sacrifice (Fig. 3) correspond well with reports from the literature. Our data support the conclusion that there is little oxidative stress in white muscle during exercise [11,37]. Significant increases in the levels of lipid oxidation products in red muscle have been noted in some studies [11] but other studies [38,45], were similar to ours in reporting no accumulation in red muscle. A typical outcome of exercise is an increase in lipid peroxidation by-products in the liver [46,47] but in several studies including ours, the increase does not reach significance [38,45] (Fig. 3). Exercise significantly increased kidney lipid peroxidation as evidenced by a 290% increase in MDA equivalents in rats that consumed water only (p ⫽ 0.0001) (Fig. 3). Other studies employing exhaustive exercise [37] or dietary manipulation of lipids [48] have reported lipid peroxidation changes in kidney. It has been estimated that exercise decreases blood flow to the kidney about five-fold [37]. Reduced blood flow may diminish the ability of the kidney to accumulate antioxidants from the plasma [46] or may reduce excretion of oxidized waste products. Current data does not distinguish between these physiological mechanisms for buildup of lipid peroxidation products in the kidney after acute exercise. In our study, kidney tissue appeared to be protected from exercise-induced oxidative stress in
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the tea consumption group as evidenced by no change in MDA equivalents compared to a 290% increase in the kidneys of the water consumption group (Fig. 3). It is possible that a bioactive component found in tea was transported to the kidney for metabolic processing and excretion, and thus enhanced antioxidant protection specifically in that tissue. The ability of EGCG to serve as a potent antioxidant in vitro [4] and to provide protection from oxidative damage in vivo [49] suggest that EGCG from green tea is responsible for the protective effect, but the mode of action of tea has not been directly demonstrated. In humans, EGCG and other flavonoids from green tea are absorbed from the gastrointestinal tract and can be detected at micromolar levels in the plasma 30 minutes after consumption of the tea [50]. However, Kohri et al. [51]reported that in rats, most of the ECGC taken into the body is not absorbed intact, but rather is degraded in the intestine, and then absorbed and circulated to the kidney. In our study, rats freely drank the green tea for 6.5 weeks but EGCG levels in plasma remained below our detection limit (4 M), consistent with Kohri’s observations. We suggest that the ECGC and its byproducts are efficiently transported to the kidney for processing and excretion and provide protection from exercise-induced lipid oxidation in kidney. Our dietary treatment with green tea prevented exercise-induced accumulation of lipid peroxidation products in kidney and, to a lesser extent, in liver, but our study did not identify mechanisms of inhibition. Further studies of the absorption, transport and accumulation of EGCG and related compounds are required to determine the basis for the beneficial effects of tea drinking. Development of a more sensitive method for determining EGCG in plasma samples in small rodents, and extension of that method to tissue samples are mandated before further studies are undertaken.
Acknowledgments This work was funded by the National Cancer Institute and Miami University Faculty Research Committee. Funding for undergraduate students was provided by the Howard Hughes Research Institute.
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