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Green tea protects against ethanol-induced lipid peroxidation in rat organs
Alcohol 32 (2004) 25–32
Green tea protects against ethanol-induced lipid peroxidation in rat organs Justyna Ostrowskaa, Wojciech Łuczaja, Irena Kasackab, Andrzej Ro´z˙an´skic, Elz˙bieta Skrzydlewskaa,* a
Department of Analytical Chemistry, Medical Academy of Bialystok, P.O. Box 14, 15-230 Bialystok, Poland b Department of Histology and Embryology, Medical Academy of Bialystok, 15-089 Bialystok, Poland c Department of Organic Chemistry, Medical Academy of Bialystok, 15-230 Bialystok, Poland Received 9 August 2003; received in revised form 25 October 2003; accepted 4 November 2003
Abstract Ethanol metabolism is accompanied by generation of free radicals, which stimulates lipid peroxidation. Natural antioxidants are particularly useful in such a situation. The current study was designed to investigate the efficacy of green tea, as a source of watersoluble antioxidants (catechins), on lipid peroxidation in liver, brain, and blood induced by chronic (4 weeks) ethanol intoxication in rats. Feeding of ethanol led to a significant increase in lipid peroxidation, as measured by increased concentrations of lipid hydroperoxides, 4hydroxynonenal, and malondialdehyde. Feeding of ethanol also changed the glutathione-dependent lipid hydroperoxide decomposition system, resulting in a decrease in both reduced glutathione concentration and activity of glutathione peroxidase. Observed changes were statistically significant in all examined tissues. Enhancement in lipid peroxidation was associated with disruption of hepatocyte cell membranes, as observed through electron microscopic evaluation. Green tea protects phospholipids from enhanced peroxidation and prevents changes in biochemical parameters and morphologic changes observed after ethanol consumption. These results support the suggestion that green tea protects membranes from peroxidation of lipids associated with ethanol consumption. 쑖 2004 Elsevier Inc. All rights reserved. Keywords: Green tea; Ethanol; Liver; Brain; Lipid peroxidation products
1. Introduction Ethanol manifests its harmful effects either through direct generation of reactive metabolites, including free radical species that react with most of the cell components, changing their structures and functions, or by contributing to other mechanisms that finally promote enhanced oxidative damage (Kato et al., 1990; Nordmann, 1994). The liver is the major target of ethanol toxicity, and the role of oxidative stress in the pathogenesis of alcohol-related disease, particularly in the liver, has been repeatedly confirmed (Lieber, 1997). Experimentally, chronic ethanol feeding leads to a decrease in the major antioxidant factors in the liver, including enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase (Polavarapu et al., 1998; Schisler & Singh, 1989), and nonenzymatic antioxidants, such as reduced glutathione and vitamins C and E (Hagen et al., 1989; Loguercio
* Corresponding author. Tel.: ⫹48-85-7485707; fax: ⫹48-85-7485707. E-mail address: [email protected] (E. Skrzydlewska). Editor: T.R. Jerrells 0741-8329/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2003.11.001
et al., 1996). These changes lead to (among other effects) an enhanced lipid peroxidation (Barclay, 1993). It has also been demonstrated that chronic ethanol exposure leads to an increased susceptibility of brain microsomes to iron-induced lipid peroxidation, without modification of the glutathione content or the activity of glutathione peroxidase or reductase of the whole brain (Omodeo-Sale et al., 1997). This finding is in agreement with results of another report, which demonstrated that chronic alcohol treatment reduced vitamin E levels, but did not influence glutathione peroxidase activity, in rat cerebellum (Rouach et al., 1997). Lipid peroxidation products are constantly involved in some of the pathophysiologic effects associated with oxidative stress in cells and tissues. Unlike reactive free radicals, aldehydes can produce lipid peroxidation products, which are rather long lived and can therefore diffuse from the site of their origin, reaching and attacking intracellular and extracellular targets (Esterbauer et al., 1991). They disrupt the various important structural and protective functions associated with biomembranes in various in vivo pathologic events and are implicated as a result of this oxidation (Barclay, 1993).
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The deleterious consequences of membrane peroxidation have stimulated investigations on the efficacy and mechanisms of action of biologically relevant antioxidants, particularly naturally occurring ones, including those used as foods and beverages (i.e., green tea). The main components of green tea are catechins, which have a polyphenol structure. Because of this characteristic, they are considered effective scavengers of superoxide, hydroxyl, and peroxyl radicals (Guo et al., 1996; Jovanovic et al., 1995; Khan et al., 1992). A free radical–scavenging mechanism has generally been described as the basis of their antioxidative activity. In addition, catechins are known to chelate metal ions, another possible contributory mechanism in their protective action against the destructive effect of free radicals on the components of the cells (Guo et al., 1996). Results from other studies have demonstrated that catechins suppress the formation of lipid peroxidation products in biologic tissues and subcellular fractions (Terao et al., 1994). It has been demonstrated that the chemically induced lipid peroxidation in rat liver and kidney could be inhibited by the intake of tea catechins (Sano et al., 1995). In addition, catechins incorporated in cell membrane were shown to prevent or reduce the morphologic and biochemical alteration of hepatocytes induced by hepatotoxic agents (Varga & Buris, 1990). In the current study, we evaluated the influence of green tea, as a source of water-soluble antioxidants, on lipid peroxidation in liver and brain, as well as on the concentration of lipid peroxidation products in blood, associated with chronic ethanol consumption.
2. Materials and methods 2.1. Animals Two-month-old, male, Wistar rats (each weighing, initially, between 210 and 230 g) were used for the study. All experiments were approved by the Local Ethic Committee in Bialystok (Poland) according to the Polish Act Protecting Animals of 1997. Rats were housed in individual cages and pair-fed with either a liquid Lieber–DeCarli diet—containing 47% of total energy as carbohydrate, 18% protein, and 35% lipid—or an identical diet with ethanol substituted isocalorically for carbohydrate (36% of total energy) (Lieber & DeCarli, 1982). Liquid diet (control and ethanol), containing 7 g of green tea extract per liter of diet, was also prepared. Green tea—Camellia sinensis (Linnaeus) O. Kuntze (standard research blends - lyophilized extract)—was provided by TJ Lipton (Englewood Cliffs, NJ). The amount of catechins in this diet, measured by high-performance liquid chromatography [(HPLC); Khokhar et al. (1997)], was equal to the amount of these compounds in 3 g of green tea extract per liter of water, which is frequently used for human consumption. The content of components in diet with green tea
was as follows: epigallocatechin gallate (337 mg/l), epigallocatechin (268 mg/l), epicatechin (90 mg/l), epicatechin gallate (60 mg/l), and coffeic acid (35 mg/l). The animals were divided into four groups, designated as control, green tea, ethanol, and ethanol ⫹ green tea. The control group (n ⫽ 6) was fed a control liquid Lieber– DeCarli diet for 5 weeks. Animals in the green tea group (n ⫽ 6) were fed a control liquid Lieber–DeCarli diet containing green tea (7 g/l) for 5 weeks. Animals in the ethanol group (n ⫽ 6) were fed a control liquid Lieber–DeCarli diet for 1 week, and for the next 4 weeks they received a liquid Lieber–DeCarli diet containing ethanol. Animals in the ethanol ⫹ green tea group (n ⫽ 6) were fed a control liquid Lieber–DeCarli diet containing green tea (7 g/l) for 1 week, and for the next 4 weeks they received a liquid Lieber– DeCarli diet containing ethanol as well as green tea (7 g/l). 2.2. Preparation of tissues After 5 weeks of study, all rats (six animals in each of four groups) were killed under ether anesthesia by exsanguination. Animals were anesthetized with ether 10 min before blood and tissues were obtained. This level of anesthesia was sufficient for cardiac puncture and euthanasia as we did not observe any pain reflexes elicited by paw pinch. Blood was taken by cardiac puncture. Livers and brains were removed quickly and placed in iced 0.15 M NaCl solution, perfused with the same solution to remove blood cells, blotted on filter paper, weighed, and homogenized in 9 ml of ice-cold 0.15 M NaCl or 0.25 M sucrose solution with the addition of 6 µl of 250 µM butylated hydroxytoluene in ethanol to prevent the formation of new peroxides during the assay. The homogenization procedure was performed under standardized conditions. Homogenates (10%) were centrifuged at 10,000g for 15 min at 4ºC, and the supernatant was kept on ice until assayed. 2.3. Biochemical assays Epigallocatechin gallate, epigallocatechin, and epicatechin concentrations in blood (serum samples) and liver were determined by HPLC (Maiani et al., 1997; Wan et al., 2001). Lipid peroxidation was assayed by HPLC by measuring the concentrations of lipid hydroperoxides (Tokumaru et al., 1995); malondialdehyde, as a malondialdehyde–thiobarbituric acid adduct (Londero & Lo Greco, 1996); and 4-hydroxynonenal, as a fluorimetric derivative (Yoshino et al., 1986). Reduced glutathione concentration was measured by using Bioxytech GSH-400 test. The method proceeds in two steps. The first step leads to the formation of substitution products between a patented reagent and all mercaptans (RSH), which are present in the sample. The second step specifically transforms the substitution products obtained with glutathione into a chromophoric thione, whose maximal absorbance wavelength is 400 nm.
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Glutathione peroxidase (EC.1.11.1.6) activity was measured specrophotometrically by using a technique based on the method of Paglia and Valentine (1967), whereas glutathione formation was assayed by measuring the conversion of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP). The enzyme activity was expressed in units per milligram of protein. Protein was determined by the method of Lowry et al. (1951). 2.4. Ultrastructural analysis of livers Sections (1 mm3) of livers, five from each animal, were collected for ultrastructural examinations. They were immediately fixed in 3.6% glutaraldehyde, then in 2% osmium tetroxide, to be embedded finally in Epon 812 after dehydration in a series of alcohol concentrations and propylene oxide. Ultrathin sections were contrasted with lead citrate and uranyl acetate and evaluated under a transmission electron microscope (OPTON 900 PC). 2.5. Statistical analysis Data obtained in the current study are expressed as mean ⫾ S.D. These data were analyzed by using standard statistical analyses, one-way analysis of variance (ANOVA) with Tukey test for multiple comparisons, to determine significance between different groups. A P value of ⬍.05 was considered significant.
3. Results Catechins were not detectable in liver and blood (serum samples) of rats in either the control or the ethanol group. Epicatechin, epigallocatechin, and epigallocatechin gallate concentrations in liver and blood of rats in the ethanol ⫹ green tea group were less than those in liver and blood of rats in the green tea group (Fig. 1). Table 1 displays data obtained for liver concentrations of lipid peroxidation products (i.e., lipid hydroperoxides, malondialdehyde, and 4-hydroxynonenal), as well as glutathione peroxidase activity and reduced glutathione concentration. After consumption of ethanol (i.e., in the ethanol group), the concentration of lipid hydroperoxides was about threefold higher than that in the control group, whereas consumption of green tea led to a significant decrease in this parameter. Consumption of ethanol together with green tea led to a significantly smaller increase in the concentration of lipid hydroperoxides in comparison with findings for the control group. Similarly, malondialdehyde and 4hydroxynonenal concentrations were increased in the ethanol group and decreased in the green tea group. The concentrations for these parameters in the liver of rats that received ethanol with green tea were not changed in comparison with findings for the control group. The glutathione
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peroxidase activity and concentration of reduced glutathione were decreased in the liver of rats in the ethanol group. Consumption of green tea led to an increase in the activity of enzyme and the concentration of reduced glutathione. After consumption of ethanol with green tea, the glutathione peroxidase activity and concentration of reduced glutathione were similar to findings for these parameters in the control group. The brain concentrations of lipid hydroperoxides and their metabolites (malondialdehyde and 4-hydroxynonenal) after ethanol consumption were significantly different from those of the control group (Table 2). However, the group provided diet containing green tea had a significant decrease in these parameters. After consumption of ethanol together with green tea, the concentrations of lipid hydroperoxides, malondialdehyde, and 4-hydroxynonenal were all significantly lower than those recorded with consumption of ethanol alone. Malondialdehyde concentration in the ethanol ⫹ green tea group was significantly lower than that of the control group. The glutathione peroxidase activity and the concentration of reduced glutathione were significantly decreased in the brain of rats receiving ethanol. In rats that consumed diet containing green tea, the levels of antioxidants were significantly increased compared with findings for the control group. After consumption of ethanol with green tea, the concentrations of antioxidants were significantly higher than those of the ethanol group. The direction of changes in the concentration of lipid peroxidation products in blood was similar to that in the liver and brain of the examined rats (Table 3). Consumption of ethanol, with or without green tea added, resulted in a significantly lower level of glutathione peroxidase activity compared with findings for the control group. Consumption of green tea modestly reversed the changes in glutathione peroxidase concentrations observed after ethanol consumption. Changes suggestive of impairment of the biologic membranes were found on electron microscopic evaluation of parenchymous cells of liver obtained from rats intoxicated with ethanol. These changes extended to subcellular structures (lesion of internal structure and, occasionally, blurring of membranes surrounding cellular organelle) as well as plasmolemma. In comparison with findings obtained by evaluation of the liver of control rats (presented in Fig. 2), liver from ethanol-fed rats (Fig. 3) showed pronounced changes in the cell membrane on the hepatocyte surface directed to the Disse’s spaces, where irregular decomposition or total atrophy of microvilluses was observed. In addition, markedly widened perisinusoid space was seen to be filled with varied contents (Fig. 3). Feeding of ethanol with green tea resulted in more orderly arrangement of subcellular structures of hepatocytes, and pathologic changes of cell membrane were slight (Fig. 4). The vascular surface of liver cells obtained from the group provided green tea was covered by regularly distributed microvilluses found in the Disse’s spaces, which are separated from the sinusoid by endothelial
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Fig. 1. Concentrations of green tea catechins [epicatechin (EC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG)] in blood (A) and livers (B) of two groups of rats. Green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks, and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6. *Significantly different in comparison with values for green tea group (P ⬍ .05).
cells. Therefore, no effect of ethanol was noted in the group provided green tea in the diet. A demonstrated decrease in the toxic effect of ethanol on liver cells in the group of animals provided green tea was observed in the ultrastructural examination of parenchymal cells.
4. Discussion Proper composition of cellular membranes is of great importance to their biologic functions. A wide range of membrane functions could be modulated by ethanol through an alcohol-associated increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells and resulting increase in the membrane fluidity
(Zloch, 1994). Results from the current study support the suggestion that chronic ethanol consumption leads to important changes in membrane lipid organization in liver and brain, evidenced by an increase in lipid peroxidation products, such as lipid hydroperoxides and their metabolites (malondialdehyde and 4-hydroxynonenal), and, as a consequence, to damage of liver cell membrane, as observed on electron microscopic evaluation. It is known that, during ethanol intoxication by the liver alcohol and aldehyde dehydrogenases, an increase in the reduced form of nicotinamide adenine dinucleotide (NADH) and acetaldehyde leads to manifestation of xanthine oxidase activity, the main source of superoxide anions (Nordmann, 1994). After chronic ethanol intoxication, metabolism by microsomal cytochrome P450 enzymes is enhanced and is
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Table 1 Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE)] and reduced glutathione (GSH), as well as activity of glutathione peroxidase (GSH-Px), in the liver of four groups of rats Analyzed parameters
Control group
Green tea group
Ethanol group
Ethanol ⫹ green tea group
LOOH (µmol/g tissue) MDA (nmol/g tissue) 4-HNE (nmol/g tissue) GSH ((µmol/g tissue) GSH-Px (U/mg protein)
115 ⫾ 6 7.42 ⫾ 0.51 1.43 ⫾ 0.11 1.91 ⫾ 0.11 135 ⫾ 7
92 ⫾ 9* 4.79 ⫾ 0.40* 1.32 ⫾ 0.08 2.16 ⫾ 0.13* 185 ⫾ 12*
296 ⫾ 14* 10.63 ⫾ 0.93* 1.74 ⫾ 0.14* 1.72 ⫾ 0.15* 123 ⫾ 7*
131 ⫾ 8*,**,*** 7.27 ⫾ 0.43**,*** 1.50 ⫾ 0.13**,*** 2.01 ⫾ 0.14** 142 ⫾ 10**,***
Control group was fed a control Lieber–DeCarli liquid diet for 5 weeks; green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks; and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6 (*P ⬍ .05 in comparison with values for control group; **P ⬍ .05 in comparison with values for ethanol group; ***P ⬍ .05 in comparison with values for green tea group).
accompanied by free radicals as well. Moreover, ethanol metabolism leads to the release of iron ions (Schisler & Singh, 1989; Shaw & Jayatilleke, 1990). These ions, therefore, become more reactive in liver and brain (Double et al., 1998). Free iron ions released from iron-containing proteins participate in the generation of hydroxyl radicals. Hydroxyl radicals are the most reactive of the reactive oxygen species, and they react readily with most cellular components, including organic molecules. In particular, their reactions with polyunsaturated fatty acids of membrane phospholipids and with lipid hydroperoxides are characterized by extremely high rate constants. The reduction and the degradation of phospholipid hydroperoxides are also carried out by glutathione peroxidase, and this is suggested to be a pathway of cytoprotection against the deleterious effects of phospholipid hydroperoxides (Kinter & Roberts, 1996). Diminution in this enzyme activity during ethanol intoxication, observed in the current study, would decrease the efficiency of this protective mechanism. Moreover, at the same time, a decrease in co-substrate of glutathione peroxidase–reduced glutathione has been observed. In such a situation, lipid peroxidation products may increase to toxic levels. The transition metal ions are also able to break down lipid hydroperoxides to trigger free radical–mediated lipid
peroxidation products. A variety of compounds could be produced from such decomposition that may exert more toxic effects on cells, but the most reactive are malondialdehyde, 4-hydroxynonenal, and acrolein as well as other carbonyls (Aust et al., 1985; Esterbauer et al., 1991). Therefore, the oxidative breakdown of the membrane polyunsaturated fatty acids is known to be accompanied by the formation of secondary products. These compounds are highly reactive and may act as “secondary toxic messengers” of the primary free radical event. The property of these biogenic aldehydes can be attributed to their α,β-unsaturated configuration, which gives them strong electrophilic properties, which is reflected in the ability to form adducts with nucelophilic sulfhydryl, primary amino and histydyl groups of proteins changing proteins structure and function (e.g., 4-hydroxynonenal inhibits aldehyde dehydrogenase–mediated oxidation of ethanol to acetaldehyde) (Mitchell & Petersen, 1987). Slowing the rate of acetaldehyde oxidation may lead to formation of acetaldehyde–protein adducts or malondialdehyde–acetaldehyde–protein adducts and, as a consequence, may increase the probability of cell injury (Tuma et al., 1987). Aldehydes generated during lipid peroxidation also form a conjugate with glutathione, leading to a significant decrease in cellular concentration of glutathione, which may result in the cytotoxicity of oxidative stress. In addition,
Table 2 Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE)] and reduced glutathione (GSH), as well as activity of glutathione peroxidase (GSH-Px), in the brain of four groups of rats Analyzed parameters
Control group
Green tea group
Ethanol group
Ethanol ⫹ green tea group
LOOH (µmol/g tissue) MDA (nmol/g tissue) 4-HNE (nmol/g tissue) GSH (µmol/g tissue) GSH-Px (U/mg protein)
103 ⫾ 9 30.8 ⫾ 1.7 3.94 ⫾ 0.18 0.82 ⫾ 0.06 51.0 ⫾ 2.3
92 ⫾ 6* 13.5 ⫾ 1.0* 3.05 ⫾ 0.19* 0.92 ⫾ 0.07* 54.3 ⫾ 1.7*
231 ⫾ 15* 40.1 ⫾ 2.0* 5.16 ⫾ 0.24* 0.67 ⫾ 0.06* 34.6 ⫾ 1.9*
93 ⫾ 7** 24.0 ⫾ 1.7*,**,*** 4.41 ⫾ 0.21*,**,*** 0.80 ⫾ 0.07**,*** 47.8 ⫾ 1.4*,**,***
Control group was fed a control Lieber–DeCarli liquid diet for 5 weeks; green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks; and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6 (*P ⬍ .05 in comparison with values for control group; **P ⬍ .05 in comparison with values for ethanol group; ***P ⬍ .05 in comparison with values for green tea group).
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Table 3 Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE)] and reduced glutathione (GSH), as well as activity of glutathione peroxidase (GSH-Px), in blood obtained from four groups of rats Analyzed parameters
Control group
Green tea group
Ethanol group
Ethanol ⫹ green tea group
LOOH (µmol/ml) MDA (nmol/ml) 4-HNE (nmol/ml) GSH (µmol/ml) GSH-Px (U/mg protein)
11.3 ⫾ 0.5 0.42 ⫾ 0.02 0.29 ⫾ 0.01 16.2 ⫾ 0.9 10.51 ⫾ 0.64
10.5 ⫾ 0.6* 0.36 ⫾ 0.02* 0.25 ⫾ 0.01* 17.3 ⫾ 0.9 10.24 ⫾ 0.41
19.7 ⫾ 0.9* 0.49 ⫾ 0.02* 0.38 ⫾ 0.03* 13.4 ⫾ 1.1* 8.49 ⫾ 0.82*
12.6 ⫾ 0.5*,**,*** 0.45 ⫾ 0.02*,**,*** 0.32 ⫾ 0.02*,**,*** 15.4 ⫾ 1.2**,*** 8.91 ⫾ 0.52*,***
Control group was fed a control Lieber–DeCarli liquid diet for 5 weeks; green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks; and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6 (*P ⬍ .05 in comparison with values for control group; **P ⬍ .05 in comparison with values for ethanol group; ***P ⬍ .05 in comparison with values for green tea group).
the turnover of glutathione in the brain is considered to be slower than in the liver. The above aldehydes may also react with the selenocysteine residue of glutathione peroxidase (Kinter & Roberts, 1996), which reduces its activity, so that lipid hydroperoxide degradation decreases. The brain contains particularly large amounts of polyunsaturated fatty acids and a high content of catalytically active metal ions, especially in the striatum and hippocampus. Thus, the brain tissue is particularly vulnerable to membrane lipid peroxidation that disturbs fundamental functions of the brain. Findings from numerous studies have shown that lipid peroxidation (an outcome of free radical generation) may be implicated in the irreversible loss of neuronal tissue after brain or spinal cord injury as well as in degenerative neurologic disorders. The damage of nerve endings by peroxidation products may lead to large changes in neurotransmitter transport, resulting in an alteration of the function of the
Fig. 2. Transmission electron micrograph of hepatocytes obtained from liver of control rat, which was fed a control Lieber–DeCarli liquid diet for 5 weeks. D ⫽ Disse’s spaces; H ⫽ vascular surface of hepatocytes; M ⫽ mitochondria. (Magnification, ×4,400.)
CNS (Rafałowska et al., 1989). This process is regulated as a passive event ultimately because of ATP depletion, leading to failure of Na⫹/K⫹ ion pumps, secondary cell swelling, as well as lysis of intracellular components into surrounding tissue and a low concentration of ATP (below 15%), which can cause cell death (Siems et al., 1996). In the current study, giving green tea simultaneously with ethanol to rats resulted in normalization of lipid peroxidation process as well as glutathione concentration and glutathione peroxidase activity in liver and brain. Compounds of green tea scavenge a wide range of free radicals, including the most active hydoxyl radical, which may initiate lipid peroxidation. Therefore, catechins may decrease the concentration of lipid free radicals and terminate initiation and propagation of
Fig. 3. Transmission electron micrograph of hepatocytes obtained from livers of ethanol group of rats, which were fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks (n ⫽ 6). D ⫽ Disse’s spaces; H ⫽ vascular surface of hepatocytes; M ⫽ mitochondria. (Magnification, ×12,000.)
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Fig. 4. Transmission electron micrograph of hepatocytes obtained from livers of ethanol ⫹ green tea group of rats, which were fed a control Lieber– DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks (n ⫽ 6). D ⫽ Disse’s spaces; H ⫽ vascular surface of hepatocytes; Lu ⫽ vascular endothelial cell; M ⫽ mitochondria. (Magnification, ×4,400.)
lipid peroxidation. Catechins may chelate metal ions, especially iron and copper, which, in turn, inhibit the generation of hydroxyl radicals and degradation of lipid hydroperoxides, which causes reactive aldehydes formation. Furthermore, the green tea polyphenols have been demonstrated to inhibit iron-induced oxidation of synaptosomes by scavenging hydroxyl radicals generated in the lecithin/lipoxidase system (Guo et al., 1996). The chelating effect of green tea results in a reduction of the free form of iron (Guo et al., 1996). Catechins, which are water-soluble antioxidants, could reduce the mobility of the free radicals into the lipid bilayer as well. Flavonoids preferentially enter the hydrophobic core of the membrane where they exert a membrane-stabilizing effect by modifying the lipid packing order (Arora et al., 2000). They can penetrate the lipid bilayer, decreasing free radicals concentration or influencing antioxidant capability in biomembranes (Saija et al., 1995). Moreover, catechins can also interact with phospholipid head groups, particularly with those containing hydroxyl groups, so they could decrease the fluidity in the polar surface of phospholipid bilayer (Chen et al., 2002). In addition, catechins prevent the loss of the lipophilic antioxidant α-tocopherol, by repairing tocopheryl radicals, and protection of the hydrophilic antioxidant ascorbate, which also repairs this radical (Rice-Evans et al., 1996; Saija et al., 1995; Skrzydlewska et al., 2002; Zhu et al., 1999). In such a way, they decrease the lipid peroxidation when membrane phospholipids are exposed to oxygen radicals
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from the aqueous phase. The oxidative attack from the aqueous phase seems to be an important reaction for initiating membrane lipid peroxidation. Perhydroxyl radicals, which are produced by the promotion of superoxide and whose concentration is enhanced during ethanol intoxication, are regarded as one of the most likely radicals for initiating lipid peroxidation in vivo. Consequently, the slower pace of free radical reactions leads to inhibition of lipid peroxidation and, as a consequence, to a decrease in membrane fluidity (Tsuchiya, 1999). The effect of green tea catechins on lipid peroxidation was most evident in the ethanol-fed group of rats because alcohol oxidation promotes such lipid peroxidation reactions. However, it has also been observed that green tea affects lipid peroxidation in control rats, especially in liver and brain. It is important to suggest that green tea may decrease lipid peroxidation in healthy individuals and may prevent disorganization and disruption of cell membranes and, as a consequence, may prevent metabolic changes in these organs. In conclusion, ethanol significantly impairs the antioxidant defense system and, as a consequence, leads to a significant increase in the lipid peroxidation processes, both in liver and brain. Consumption of ethanol simultaneously with green tea in rats leads to improvement in the antioxidant system and diminution in the amounts of lipid peroxidation products. Taking into consideration that metabolism of ethanol and catechins is the same in rats as in human beings, results obtained from the current study support the suggestion that green tea may also protect liver and brain cells of human beings against the sequelae of oxidative stress caused by ethanol intoxication.
Acknowledgments This work was supported by a grant from the Polish Committee of Scientific Research, No. 6 PO5F 017 20.
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and impaired antioxidant enzyme function is associated with pathological liver injury in experimental alcoholic liver disease in rats diets high in corn oil and fish oil. Hepatology 27, 1317–1323. Rafałowska, U., Liu, G. J., & Floyd, R. A. (1989). Peroxidation induced changes in synaptosomal transport of dopamine and gamma-aminobutyric acid. Free Radic Biol Med 6, 485–492. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 20, 933–956. Rouach, H., Houze´, P., Gentil, M., Orfanelli, M.-T., & Nordmann, R. (1997). Changes in some pro- and antioxidants in rat cerebellum after chronic alcohol intake. Biochem Pharmacol 53, 539–545. Saija, A., Scalese, M., Lanza, M., Marzullo, D., Bonina, F., & Castelli, F. (1995). Flavonoids as antioxidant agents: importance of their interaction with biomembranes. Free Radic Biol Med 19, 481–486. Sano, M., Takahashi, Y., Yoshino, K., Shimoi, K., Nakamura, Y., Tomita, I., Oguni, I., & Konomoto, H. (1995). Effect of tea (Camellia sinensis L.) on lipid peroxidation in rat liver and kidney: a comparison of green and black tea feeding. Biol Pharm Bull 18, 1006–1008. Schisler, N. J., & Singh, S. M. (1989). Effect of ethanol in vivo on enzymes which detoxify oxygen free radicals. Free Radic Biol Med 7, 117–123. Shaw, S., & Jayatilleke, E. (1990). The role of aldehyde oxidase in ethanolinduced hepatic lipid peroxidation in the rat. Biochem J 268, 579–583. Siems, W. G., Hapner, S. J., & van Kuijk, F. J. G. M. (1996). 4-Hydroxynonenal inhibits Na⫹-K⫹-ATPase. Free Radic Biol Med 20, 215–223. Skrzydlewska, E., Ostrowska, J., Stankiewicz, A., & Farbiszewski, R. (2002). Green tea as a potent antioxidant in alcohol intoxication. Addict Biol 7, 307–314. Terao, J., Piskula, M., & Yao, Q. (1994). Protective effect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers. Arch Biochem Biophys 308, 278–284. Tokumaru, S., Tsukamoto, I., Iguchi, H., & Kojo, S. (1995). Specific and sensitive determination of lipid hydroperoxides with chemical derivatization into 1-naphtyldiphenylphosphine oxide and HPLC. Anal Chim Acta 307, 97–102. Tsuchiya, H. (1999). Effects of green tea catechins on membrane fluidity. Pharmacology 59, 34–44. Tuma, D. J., Newman, M. R., Donohue, T. M. Jr., & Sorrell, M. F. (1987). Covalent binding of acetaldehyde to proteins: participation of lysine residues. Alcohol Clin Exp Res 11, 579–584. Varga, M., & Buris, L. (1990). Quantitative ultrastructural analysis of hepatoprotective effects of (⫹)-cyanidanol-3 on alcoholic liver damage. Exp Mol Pathol 52, 249–257. Wan, Y., Vinson, J. A., Etherton, T. D., Proch, J., Lazarus, S. A., & KrisEtherton, P. M. (2001). Effects of cocoa powder and dark chocolate and LDL oxidative susceptibility and prostaglandin concentrations in humans. Am J Clin Nutr 74, 596–602. Yoshino, K., Matsuura, T., Sano, M., Saito, S., & Tomita, I. (1986). Fluorometric liquid chromatographic determination of aliphatic aldehydes arising from lipid peroxides. Chem Pharm Bull (Tokyo) 34, 1694–1700. Zhu, Q. Y., Huang, Y., Tsang, D., & Chen, Z.-Y. (1999). Regeneration of α-tocopherol in human low-density lipoprotein by green tea catechin. J Agric Food Chem 47, 2020–2025. Zloch, Z. (1994). Temporal changes of the lipid peroxidation in rats after acute intoxication by ethanol. Z Naturforsch [C] 49, 359–362.
Green tea protects against ethanol-induced lipid peroxidation in rat organs Justyna Ostrowskaa, Wojciech Łuczaja, Irena Kasackab, Andrzej Ro´z˙an´skic, Elz˙bieta Skrzydlewskaa,* a
Department of Analytical Chemistry, Medical Academy of Bialystok, P.O. Box 14, 15-230 Bialystok, Poland b Department of Histology and Embryology, Medical Academy of Bialystok, 15-089 Bialystok, Poland c Department of Organic Chemistry, Medical Academy of Bialystok, 15-230 Bialystok, Poland Received 9 August 2003; received in revised form 25 October 2003; accepted 4 November 2003
Abstract Ethanol metabolism is accompanied by generation of free radicals, which stimulates lipid peroxidation. Natural antioxidants are particularly useful in such a situation. The current study was designed to investigate the efficacy of green tea, as a source of watersoluble antioxidants (catechins), on lipid peroxidation in liver, brain, and blood induced by chronic (4 weeks) ethanol intoxication in rats. Feeding of ethanol led to a significant increase in lipid peroxidation, as measured by increased concentrations of lipid hydroperoxides, 4hydroxynonenal, and malondialdehyde. Feeding of ethanol also changed the glutathione-dependent lipid hydroperoxide decomposition system, resulting in a decrease in both reduced glutathione concentration and activity of glutathione peroxidase. Observed changes were statistically significant in all examined tissues. Enhancement in lipid peroxidation was associated with disruption of hepatocyte cell membranes, as observed through electron microscopic evaluation. Green tea protects phospholipids from enhanced peroxidation and prevents changes in biochemical parameters and morphologic changes observed after ethanol consumption. These results support the suggestion that green tea protects membranes from peroxidation of lipids associated with ethanol consumption. 쑖 2004 Elsevier Inc. All rights reserved. Keywords: Green tea; Ethanol; Liver; Brain; Lipid peroxidation products
1. Introduction Ethanol manifests its harmful effects either through direct generation of reactive metabolites, including free radical species that react with most of the cell components, changing their structures and functions, or by contributing to other mechanisms that finally promote enhanced oxidative damage (Kato et al., 1990; Nordmann, 1994). The liver is the major target of ethanol toxicity, and the role of oxidative stress in the pathogenesis of alcohol-related disease, particularly in the liver, has been repeatedly confirmed (Lieber, 1997). Experimentally, chronic ethanol feeding leads to a decrease in the major antioxidant factors in the liver, including enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase (Polavarapu et al., 1998; Schisler & Singh, 1989), and nonenzymatic antioxidants, such as reduced glutathione and vitamins C and E (Hagen et al., 1989; Loguercio
* Corresponding author. Tel.: ⫹48-85-7485707; fax: ⫹48-85-7485707. E-mail address: [email protected] (E. Skrzydlewska). Editor: T.R. Jerrells 0741-8329/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2003.11.001
et al., 1996). These changes lead to (among other effects) an enhanced lipid peroxidation (Barclay, 1993). It has also been demonstrated that chronic ethanol exposure leads to an increased susceptibility of brain microsomes to iron-induced lipid peroxidation, without modification of the glutathione content or the activity of glutathione peroxidase or reductase of the whole brain (Omodeo-Sale et al., 1997). This finding is in agreement with results of another report, which demonstrated that chronic alcohol treatment reduced vitamin E levels, but did not influence glutathione peroxidase activity, in rat cerebellum (Rouach et al., 1997). Lipid peroxidation products are constantly involved in some of the pathophysiologic effects associated with oxidative stress in cells and tissues. Unlike reactive free radicals, aldehydes can produce lipid peroxidation products, which are rather long lived and can therefore diffuse from the site of their origin, reaching and attacking intracellular and extracellular targets (Esterbauer et al., 1991). They disrupt the various important structural and protective functions associated with biomembranes in various in vivo pathologic events and are implicated as a result of this oxidation (Barclay, 1993).
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The deleterious consequences of membrane peroxidation have stimulated investigations on the efficacy and mechanisms of action of biologically relevant antioxidants, particularly naturally occurring ones, including those used as foods and beverages (i.e., green tea). The main components of green tea are catechins, which have a polyphenol structure. Because of this characteristic, they are considered effective scavengers of superoxide, hydroxyl, and peroxyl radicals (Guo et al., 1996; Jovanovic et al., 1995; Khan et al., 1992). A free radical–scavenging mechanism has generally been described as the basis of their antioxidative activity. In addition, catechins are known to chelate metal ions, another possible contributory mechanism in their protective action against the destructive effect of free radicals on the components of the cells (Guo et al., 1996). Results from other studies have demonstrated that catechins suppress the formation of lipid peroxidation products in biologic tissues and subcellular fractions (Terao et al., 1994). It has been demonstrated that the chemically induced lipid peroxidation in rat liver and kidney could be inhibited by the intake of tea catechins (Sano et al., 1995). In addition, catechins incorporated in cell membrane were shown to prevent or reduce the morphologic and biochemical alteration of hepatocytes induced by hepatotoxic agents (Varga & Buris, 1990). In the current study, we evaluated the influence of green tea, as a source of water-soluble antioxidants, on lipid peroxidation in liver and brain, as well as on the concentration of lipid peroxidation products in blood, associated with chronic ethanol consumption.
2. Materials and methods 2.1. Animals Two-month-old, male, Wistar rats (each weighing, initially, between 210 and 230 g) were used for the study. All experiments were approved by the Local Ethic Committee in Bialystok (Poland) according to the Polish Act Protecting Animals of 1997. Rats were housed in individual cages and pair-fed with either a liquid Lieber–DeCarli diet—containing 47% of total energy as carbohydrate, 18% protein, and 35% lipid—or an identical diet with ethanol substituted isocalorically for carbohydrate (36% of total energy) (Lieber & DeCarli, 1982). Liquid diet (control and ethanol), containing 7 g of green tea extract per liter of diet, was also prepared. Green tea—Camellia sinensis (Linnaeus) O. Kuntze (standard research blends - lyophilized extract)—was provided by TJ Lipton (Englewood Cliffs, NJ). The amount of catechins in this diet, measured by high-performance liquid chromatography [(HPLC); Khokhar et al. (1997)], was equal to the amount of these compounds in 3 g of green tea extract per liter of water, which is frequently used for human consumption. The content of components in diet with green tea
was as follows: epigallocatechin gallate (337 mg/l), epigallocatechin (268 mg/l), epicatechin (90 mg/l), epicatechin gallate (60 mg/l), and coffeic acid (35 mg/l). The animals were divided into four groups, designated as control, green tea, ethanol, and ethanol ⫹ green tea. The control group (n ⫽ 6) was fed a control liquid Lieber– DeCarli diet for 5 weeks. Animals in the green tea group (n ⫽ 6) were fed a control liquid Lieber–DeCarli diet containing green tea (7 g/l) for 5 weeks. Animals in the ethanol group (n ⫽ 6) were fed a control liquid Lieber–DeCarli diet for 1 week, and for the next 4 weeks they received a liquid Lieber–DeCarli diet containing ethanol. Animals in the ethanol ⫹ green tea group (n ⫽ 6) were fed a control liquid Lieber–DeCarli diet containing green tea (7 g/l) for 1 week, and for the next 4 weeks they received a liquid Lieber– DeCarli diet containing ethanol as well as green tea (7 g/l). 2.2. Preparation of tissues After 5 weeks of study, all rats (six animals in each of four groups) were killed under ether anesthesia by exsanguination. Animals were anesthetized with ether 10 min before blood and tissues were obtained. This level of anesthesia was sufficient for cardiac puncture and euthanasia as we did not observe any pain reflexes elicited by paw pinch. Blood was taken by cardiac puncture. Livers and brains were removed quickly and placed in iced 0.15 M NaCl solution, perfused with the same solution to remove blood cells, blotted on filter paper, weighed, and homogenized in 9 ml of ice-cold 0.15 M NaCl or 0.25 M sucrose solution with the addition of 6 µl of 250 µM butylated hydroxytoluene in ethanol to prevent the formation of new peroxides during the assay. The homogenization procedure was performed under standardized conditions. Homogenates (10%) were centrifuged at 10,000g for 15 min at 4ºC, and the supernatant was kept on ice until assayed. 2.3. Biochemical assays Epigallocatechin gallate, epigallocatechin, and epicatechin concentrations in blood (serum samples) and liver were determined by HPLC (Maiani et al., 1997; Wan et al., 2001). Lipid peroxidation was assayed by HPLC by measuring the concentrations of lipid hydroperoxides (Tokumaru et al., 1995); malondialdehyde, as a malondialdehyde–thiobarbituric acid adduct (Londero & Lo Greco, 1996); and 4-hydroxynonenal, as a fluorimetric derivative (Yoshino et al., 1986). Reduced glutathione concentration was measured by using Bioxytech GSH-400 test. The method proceeds in two steps. The first step leads to the formation of substitution products between a patented reagent and all mercaptans (RSH), which are present in the sample. The second step specifically transforms the substitution products obtained with glutathione into a chromophoric thione, whose maximal absorbance wavelength is 400 nm.
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Glutathione peroxidase (EC.1.11.1.6) activity was measured specrophotometrically by using a technique based on the method of Paglia and Valentine (1967), whereas glutathione formation was assayed by measuring the conversion of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP). The enzyme activity was expressed in units per milligram of protein. Protein was determined by the method of Lowry et al. (1951). 2.4. Ultrastructural analysis of livers Sections (1 mm3) of livers, five from each animal, were collected for ultrastructural examinations. They were immediately fixed in 3.6% glutaraldehyde, then in 2% osmium tetroxide, to be embedded finally in Epon 812 after dehydration in a series of alcohol concentrations and propylene oxide. Ultrathin sections were contrasted with lead citrate and uranyl acetate and evaluated under a transmission electron microscope (OPTON 900 PC). 2.5. Statistical analysis Data obtained in the current study are expressed as mean ⫾ S.D. These data were analyzed by using standard statistical analyses, one-way analysis of variance (ANOVA) with Tukey test for multiple comparisons, to determine significance between different groups. A P value of ⬍.05 was considered significant.
3. Results Catechins were not detectable in liver and blood (serum samples) of rats in either the control or the ethanol group. Epicatechin, epigallocatechin, and epigallocatechin gallate concentrations in liver and blood of rats in the ethanol ⫹ green tea group were less than those in liver and blood of rats in the green tea group (Fig. 1). Table 1 displays data obtained for liver concentrations of lipid peroxidation products (i.e., lipid hydroperoxides, malondialdehyde, and 4-hydroxynonenal), as well as glutathione peroxidase activity and reduced glutathione concentration. After consumption of ethanol (i.e., in the ethanol group), the concentration of lipid hydroperoxides was about threefold higher than that in the control group, whereas consumption of green tea led to a significant decrease in this parameter. Consumption of ethanol together with green tea led to a significantly smaller increase in the concentration of lipid hydroperoxides in comparison with findings for the control group. Similarly, malondialdehyde and 4hydroxynonenal concentrations were increased in the ethanol group and decreased in the green tea group. The concentrations for these parameters in the liver of rats that received ethanol with green tea were not changed in comparison with findings for the control group. The glutathione
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peroxidase activity and concentration of reduced glutathione were decreased in the liver of rats in the ethanol group. Consumption of green tea led to an increase in the activity of enzyme and the concentration of reduced glutathione. After consumption of ethanol with green tea, the glutathione peroxidase activity and concentration of reduced glutathione were similar to findings for these parameters in the control group. The brain concentrations of lipid hydroperoxides and their metabolites (malondialdehyde and 4-hydroxynonenal) after ethanol consumption were significantly different from those of the control group (Table 2). However, the group provided diet containing green tea had a significant decrease in these parameters. After consumption of ethanol together with green tea, the concentrations of lipid hydroperoxides, malondialdehyde, and 4-hydroxynonenal were all significantly lower than those recorded with consumption of ethanol alone. Malondialdehyde concentration in the ethanol ⫹ green tea group was significantly lower than that of the control group. The glutathione peroxidase activity and the concentration of reduced glutathione were significantly decreased in the brain of rats receiving ethanol. In rats that consumed diet containing green tea, the levels of antioxidants were significantly increased compared with findings for the control group. After consumption of ethanol with green tea, the concentrations of antioxidants were significantly higher than those of the ethanol group. The direction of changes in the concentration of lipid peroxidation products in blood was similar to that in the liver and brain of the examined rats (Table 3). Consumption of ethanol, with or without green tea added, resulted in a significantly lower level of glutathione peroxidase activity compared with findings for the control group. Consumption of green tea modestly reversed the changes in glutathione peroxidase concentrations observed after ethanol consumption. Changes suggestive of impairment of the biologic membranes were found on electron microscopic evaluation of parenchymous cells of liver obtained from rats intoxicated with ethanol. These changes extended to subcellular structures (lesion of internal structure and, occasionally, blurring of membranes surrounding cellular organelle) as well as plasmolemma. In comparison with findings obtained by evaluation of the liver of control rats (presented in Fig. 2), liver from ethanol-fed rats (Fig. 3) showed pronounced changes in the cell membrane on the hepatocyte surface directed to the Disse’s spaces, where irregular decomposition or total atrophy of microvilluses was observed. In addition, markedly widened perisinusoid space was seen to be filled with varied contents (Fig. 3). Feeding of ethanol with green tea resulted in more orderly arrangement of subcellular structures of hepatocytes, and pathologic changes of cell membrane were slight (Fig. 4). The vascular surface of liver cells obtained from the group provided green tea was covered by regularly distributed microvilluses found in the Disse’s spaces, which are separated from the sinusoid by endothelial
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Fig. 1. Concentrations of green tea catechins [epicatechin (EC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG)] in blood (A) and livers (B) of two groups of rats. Green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks, and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6. *Significantly different in comparison with values for green tea group (P ⬍ .05).
cells. Therefore, no effect of ethanol was noted in the group provided green tea in the diet. A demonstrated decrease in the toxic effect of ethanol on liver cells in the group of animals provided green tea was observed in the ultrastructural examination of parenchymal cells.
4. Discussion Proper composition of cellular membranes is of great importance to their biologic functions. A wide range of membrane functions could be modulated by ethanol through an alcohol-associated increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells and resulting increase in the membrane fluidity
(Zloch, 1994). Results from the current study support the suggestion that chronic ethanol consumption leads to important changes in membrane lipid organization in liver and brain, evidenced by an increase in lipid peroxidation products, such as lipid hydroperoxides and their metabolites (malondialdehyde and 4-hydroxynonenal), and, as a consequence, to damage of liver cell membrane, as observed on electron microscopic evaluation. It is known that, during ethanol intoxication by the liver alcohol and aldehyde dehydrogenases, an increase in the reduced form of nicotinamide adenine dinucleotide (NADH) and acetaldehyde leads to manifestation of xanthine oxidase activity, the main source of superoxide anions (Nordmann, 1994). After chronic ethanol intoxication, metabolism by microsomal cytochrome P450 enzymes is enhanced and is
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Table 1 Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE)] and reduced glutathione (GSH), as well as activity of glutathione peroxidase (GSH-Px), in the liver of four groups of rats Analyzed parameters
Control group
Green tea group
Ethanol group
Ethanol ⫹ green tea group
LOOH (µmol/g tissue) MDA (nmol/g tissue) 4-HNE (nmol/g tissue) GSH ((µmol/g tissue) GSH-Px (U/mg protein)
115 ⫾ 6 7.42 ⫾ 0.51 1.43 ⫾ 0.11 1.91 ⫾ 0.11 135 ⫾ 7
92 ⫾ 9* 4.79 ⫾ 0.40* 1.32 ⫾ 0.08 2.16 ⫾ 0.13* 185 ⫾ 12*
296 ⫾ 14* 10.63 ⫾ 0.93* 1.74 ⫾ 0.14* 1.72 ⫾ 0.15* 123 ⫾ 7*
131 ⫾ 8*,**,*** 7.27 ⫾ 0.43**,*** 1.50 ⫾ 0.13**,*** 2.01 ⫾ 0.14** 142 ⫾ 10**,***
Control group was fed a control Lieber–DeCarli liquid diet for 5 weeks; green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks; and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6 (*P ⬍ .05 in comparison with values for control group; **P ⬍ .05 in comparison with values for ethanol group; ***P ⬍ .05 in comparison with values for green tea group).
accompanied by free radicals as well. Moreover, ethanol metabolism leads to the release of iron ions (Schisler & Singh, 1989; Shaw & Jayatilleke, 1990). These ions, therefore, become more reactive in liver and brain (Double et al., 1998). Free iron ions released from iron-containing proteins participate in the generation of hydroxyl radicals. Hydroxyl radicals are the most reactive of the reactive oxygen species, and they react readily with most cellular components, including organic molecules. In particular, their reactions with polyunsaturated fatty acids of membrane phospholipids and with lipid hydroperoxides are characterized by extremely high rate constants. The reduction and the degradation of phospholipid hydroperoxides are also carried out by glutathione peroxidase, and this is suggested to be a pathway of cytoprotection against the deleterious effects of phospholipid hydroperoxides (Kinter & Roberts, 1996). Diminution in this enzyme activity during ethanol intoxication, observed in the current study, would decrease the efficiency of this protective mechanism. Moreover, at the same time, a decrease in co-substrate of glutathione peroxidase–reduced glutathione has been observed. In such a situation, lipid peroxidation products may increase to toxic levels. The transition metal ions are also able to break down lipid hydroperoxides to trigger free radical–mediated lipid
peroxidation products. A variety of compounds could be produced from such decomposition that may exert more toxic effects on cells, but the most reactive are malondialdehyde, 4-hydroxynonenal, and acrolein as well as other carbonyls (Aust et al., 1985; Esterbauer et al., 1991). Therefore, the oxidative breakdown of the membrane polyunsaturated fatty acids is known to be accompanied by the formation of secondary products. These compounds are highly reactive and may act as “secondary toxic messengers” of the primary free radical event. The property of these biogenic aldehydes can be attributed to their α,β-unsaturated configuration, which gives them strong electrophilic properties, which is reflected in the ability to form adducts with nucelophilic sulfhydryl, primary amino and histydyl groups of proteins changing proteins structure and function (e.g., 4-hydroxynonenal inhibits aldehyde dehydrogenase–mediated oxidation of ethanol to acetaldehyde) (Mitchell & Petersen, 1987). Slowing the rate of acetaldehyde oxidation may lead to formation of acetaldehyde–protein adducts or malondialdehyde–acetaldehyde–protein adducts and, as a consequence, may increase the probability of cell injury (Tuma et al., 1987). Aldehydes generated during lipid peroxidation also form a conjugate with glutathione, leading to a significant decrease in cellular concentration of glutathione, which may result in the cytotoxicity of oxidative stress. In addition,
Table 2 Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE)] and reduced glutathione (GSH), as well as activity of glutathione peroxidase (GSH-Px), in the brain of four groups of rats Analyzed parameters
Control group
Green tea group
Ethanol group
Ethanol ⫹ green tea group
LOOH (µmol/g tissue) MDA (nmol/g tissue) 4-HNE (nmol/g tissue) GSH (µmol/g tissue) GSH-Px (U/mg protein)
103 ⫾ 9 30.8 ⫾ 1.7 3.94 ⫾ 0.18 0.82 ⫾ 0.06 51.0 ⫾ 2.3
92 ⫾ 6* 13.5 ⫾ 1.0* 3.05 ⫾ 0.19* 0.92 ⫾ 0.07* 54.3 ⫾ 1.7*
231 ⫾ 15* 40.1 ⫾ 2.0* 5.16 ⫾ 0.24* 0.67 ⫾ 0.06* 34.6 ⫾ 1.9*
93 ⫾ 7** 24.0 ⫾ 1.7*,**,*** 4.41 ⫾ 0.21*,**,*** 0.80 ⫾ 0.07**,*** 47.8 ⫾ 1.4*,**,***
Control group was fed a control Lieber–DeCarli liquid diet for 5 weeks; green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks; and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6 (*P ⬍ .05 in comparison with values for control group; **P ⬍ .05 in comparison with values for ethanol group; ***P ⬍ .05 in comparison with values for green tea group).
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Table 3 Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE)] and reduced glutathione (GSH), as well as activity of glutathione peroxidase (GSH-Px), in blood obtained from four groups of rats Analyzed parameters
Control group
Green tea group
Ethanol group
Ethanol ⫹ green tea group
LOOH (µmol/ml) MDA (nmol/ml) 4-HNE (nmol/ml) GSH (µmol/ml) GSH-Px (U/mg protein)
11.3 ⫾ 0.5 0.42 ⫾ 0.02 0.29 ⫾ 0.01 16.2 ⫾ 0.9 10.51 ⫾ 0.64
10.5 ⫾ 0.6* 0.36 ⫾ 0.02* 0.25 ⫾ 0.01* 17.3 ⫾ 0.9 10.24 ⫾ 0.41
19.7 ⫾ 0.9* 0.49 ⫾ 0.02* 0.38 ⫾ 0.03* 13.4 ⫾ 1.1* 8.49 ⫾ 0.82*
12.6 ⫾ 0.5*,**,*** 0.45 ⫾ 0.02*,**,*** 0.32 ⫾ 0.02*,**,*** 15.4 ⫾ 1.2**,*** 8.91 ⫾ 0.52*,***
Control group was fed a control Lieber–DeCarli liquid diet for 5 weeks; green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 5 weeks; ethanol group was fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks; and ethanol ⫹ green tea group was fed a control Lieber–DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean ⫾ S.D.; n ⫽ 6 (*P ⬍ .05 in comparison with values for control group; **P ⬍ .05 in comparison with values for ethanol group; ***P ⬍ .05 in comparison with values for green tea group).
the turnover of glutathione in the brain is considered to be slower than in the liver. The above aldehydes may also react with the selenocysteine residue of glutathione peroxidase (Kinter & Roberts, 1996), which reduces its activity, so that lipid hydroperoxide degradation decreases. The brain contains particularly large amounts of polyunsaturated fatty acids and a high content of catalytically active metal ions, especially in the striatum and hippocampus. Thus, the brain tissue is particularly vulnerable to membrane lipid peroxidation that disturbs fundamental functions of the brain. Findings from numerous studies have shown that lipid peroxidation (an outcome of free radical generation) may be implicated in the irreversible loss of neuronal tissue after brain or spinal cord injury as well as in degenerative neurologic disorders. The damage of nerve endings by peroxidation products may lead to large changes in neurotransmitter transport, resulting in an alteration of the function of the
Fig. 2. Transmission electron micrograph of hepatocytes obtained from liver of control rat, which was fed a control Lieber–DeCarli liquid diet for 5 weeks. D ⫽ Disse’s spaces; H ⫽ vascular surface of hepatocytes; M ⫽ mitochondria. (Magnification, ×4,400.)
CNS (Rafałowska et al., 1989). This process is regulated as a passive event ultimately because of ATP depletion, leading to failure of Na⫹/K⫹ ion pumps, secondary cell swelling, as well as lysis of intracellular components into surrounding tissue and a low concentration of ATP (below 15%), which can cause cell death (Siems et al., 1996). In the current study, giving green tea simultaneously with ethanol to rats resulted in normalization of lipid peroxidation process as well as glutathione concentration and glutathione peroxidase activity in liver and brain. Compounds of green tea scavenge a wide range of free radicals, including the most active hydoxyl radical, which may initiate lipid peroxidation. Therefore, catechins may decrease the concentration of lipid free radicals and terminate initiation and propagation of
Fig. 3. Transmission electron micrograph of hepatocytes obtained from livers of ethanol group of rats, which were fed a control Lieber–DeCarli liquid diet for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol for the next 4 weeks (n ⫽ 6). D ⫽ Disse’s spaces; H ⫽ vascular surface of hepatocytes; M ⫽ mitochondria. (Magnification, ×12,000.)
J. Ostrowska et al. / Alcohol 32 (2004) 25–32
Fig. 4. Transmission electron micrograph of hepatocytes obtained from livers of ethanol ⫹ green tea group of rats, which were fed a control Lieber– DeCarli liquid diet containing green tea (7 g/l) for 1 week, followed by feeding of a Lieber–DeCarli liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks (n ⫽ 6). D ⫽ Disse’s spaces; H ⫽ vascular surface of hepatocytes; Lu ⫽ vascular endothelial cell; M ⫽ mitochondria. (Magnification, ×4,400.)
lipid peroxidation. Catechins may chelate metal ions, especially iron and copper, which, in turn, inhibit the generation of hydroxyl radicals and degradation of lipid hydroperoxides, which causes reactive aldehydes formation. Furthermore, the green tea polyphenols have been demonstrated to inhibit iron-induced oxidation of synaptosomes by scavenging hydroxyl radicals generated in the lecithin/lipoxidase system (Guo et al., 1996). The chelating effect of green tea results in a reduction of the free form of iron (Guo et al., 1996). Catechins, which are water-soluble antioxidants, could reduce the mobility of the free radicals into the lipid bilayer as well. Flavonoids preferentially enter the hydrophobic core of the membrane where they exert a membrane-stabilizing effect by modifying the lipid packing order (Arora et al., 2000). They can penetrate the lipid bilayer, decreasing free radicals concentration or influencing antioxidant capability in biomembranes (Saija et al., 1995). Moreover, catechins can also interact with phospholipid head groups, particularly with those containing hydroxyl groups, so they could decrease the fluidity in the polar surface of phospholipid bilayer (Chen et al., 2002). In addition, catechins prevent the loss of the lipophilic antioxidant α-tocopherol, by repairing tocopheryl radicals, and protection of the hydrophilic antioxidant ascorbate, which also repairs this radical (Rice-Evans et al., 1996; Saija et al., 1995; Skrzydlewska et al., 2002; Zhu et al., 1999). In such a way, they decrease the lipid peroxidation when membrane phospholipids are exposed to oxygen radicals
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from the aqueous phase. The oxidative attack from the aqueous phase seems to be an important reaction for initiating membrane lipid peroxidation. Perhydroxyl radicals, which are produced by the promotion of superoxide and whose concentration is enhanced during ethanol intoxication, are regarded as one of the most likely radicals for initiating lipid peroxidation in vivo. Consequently, the slower pace of free radical reactions leads to inhibition of lipid peroxidation and, as a consequence, to a decrease in membrane fluidity (Tsuchiya, 1999). The effect of green tea catechins on lipid peroxidation was most evident in the ethanol-fed group of rats because alcohol oxidation promotes such lipid peroxidation reactions. However, it has also been observed that green tea affects lipid peroxidation in control rats, especially in liver and brain. It is important to suggest that green tea may decrease lipid peroxidation in healthy individuals and may prevent disorganization and disruption of cell membranes and, as a consequence, may prevent metabolic changes in these organs. In conclusion, ethanol significantly impairs the antioxidant defense system and, as a consequence, leads to a significant increase in the lipid peroxidation processes, both in liver and brain. Consumption of ethanol simultaneously with green tea in rats leads to improvement in the antioxidant system and diminution in the amounts of lipid peroxidation products. Taking into consideration that metabolism of ethanol and catechins is the same in rats as in human beings, results obtained from the current study support the suggestion that green tea may also protect liver and brain cells of human beings against the sequelae of oxidative stress caused by ethanol intoxication.
Acknowledgments This work was supported by a grant from the Polish Committee of Scientific Research, No. 6 PO5F 017 20.
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