510 JOURNAL OF STUDIES ON ALCOHOL / JULY 2006 Protective Effect of Black Tea Against Ethanol-Induced Oxidative Modifications of Liver Proteins and Lipids* WOJCIECH LUCZAJ, PH.D., EWA SIEMIENIUK, PH.D., WIESÒAWA ROSZKOWSKA-JAKIMIEC, PH.D.,† AND ELZBIETA SKRZYDLEWSKA, PH.D.† Department of Analytical Chemistry, Medical University of Bia¬ystok, 15–230 Bia¬ystok 8, P.O. Box 14, Poland ABSTRACT. Objective: Black tea has been recently ascertained as a source of water-soluble antioxidants that may enhance cellular antioxidant abilities. The present study was designed to investigate the efficacy of the preventive effect of black tea on oxidative modifications of liver lipids and proteins of 2-month-old rats intoxicated chronically (28 days) with ethanol. Method: Lipid peroxidation was estimated by measurement of lipid hydroperoxides, malondialdehyde, and 4-hydroxynonenal by high-performance liquid chromatography (HPLC) and by spectrophotometric determination of conjugated dienes. The markers of protein oxidative modification products—bistyrosine and tryptophan— were quantified by spectrofluorimetry, whereas levels of amino, sulfhydryl, and carbonyl groups were estimated spectrophotometrically. Results: Ethanol intoxication caused changes in liver antioxidant abilities that led to the generation of oxidative stress and, consequently, to the significant increase in products of lipid and protein oxidative modification. Enhanced lipid peroxidation was confirmed by assessment of the concentration of lipid peroxidation products measured at all examined levels. Protein modifications were evidenced by increase in levels of bistyrosine and carbonyl groups and by decrease in concentration of tryptophan and levels of sulfhydryl and amino groups. The metabolic consequences of oxidative modifications of lipids and proteins were reduced by cathepsin B activity and translocation of this lysosomal protease into cytosol as well as markers of liver damage—alanine aminotransferase (ALT) and aspartate aminotransferase (AST)—into the blood serum. Administration of black tea to ethanol-intoxicated rats partially protected antioxidant parameters and, remarkably, prevented the significant increase in concentrations of all measured lipid peroxidation products. Moreover, the levels of markers of the protein-modification process were similar to those of the control group. Protection of biological membranes by black tea prevents changes in the permeability of these membranes and translocation of the examined enzymes. Conclusions: Our findings indicate that black tea protects proteins and lipids against oxidative modif ication induced by chronic ethanol intoxication, which preserves changes in redox and proteolytic homeostasis. (J. Stud. Alcohol 67: 510-518, 2006) S EVERAL XENOBIOTICS, SUCH AS ALCOHOLS, for example, are increasingly recognized as substances that disturb cellular metabolism, including redox balance. In most cases, it is connected with metabolism of these xenobiotics. Ethanol is metabolized into acetaldehyde and then into acetate mainly in the liver, and this process is accompanied by formation of free radicals. It has been shown that chronic ethanol intoxication enhances the generation of mainly superoxide radical and hydrogen peroxide (Kukielka and Cederbaum, 1994). Enhanced generation of superoxide radical is caused by an increase in the level of NADH (reduced form of the nicotinamide adenine dinucleotide [NAD]) produced during oxidation of ethanol as well as its metabolite (i.e., acetaldehyde) (Sochman, 1994). A decrease in the NAD/NADH ratio causes conversion of xanthine dehydro- genase into xanthine oxidase, an enzyme responsible for generation of superoxide radical (Kato et al., 1990). This transformation also occurs during acetaldehyde oxidation into acetate (Puntarulo and Cederbaum, 1989). The increase in NADH concentration is also responsible for enhanced release of iron (II) ions from ferritin (Shaw et al., 1988). An increase in free iron (II) ions, which catalyze free radical reactions, leads to an increase in the level of reactive oxygen species (ROS) observed in ethanol intoxication (Shaw et al., 1988). Chronic alcohol intoxication is also accompanied by a decrease in the activities of ethanol-metabolizing enzymes (i.e., alcohol and aldehyde dehydrogenases), and consequently, by an increase in acetaldehyde accumulation. In this situation, xanthine oxidase may also catalyze superoxide radical generation using acetaldehyde as a substrate (Kellogg and Fridovich, 1975). Moreover, increased generation of oxygen and ethanol-derived free radicals has been observed at the microsomal level, especially through the application of the ethanol-inducible cytochrome P450 isoform (Krikun et al., 1984). A decrease in antioxidant status during ethanol intoxication caused the generation of oxidative stress (Kurose, 1996). This oxidative stress results in enhanced lipid peroxidation and changes in the structure and function of other important cellular Received: June 24, 2005. Revision: March 22, 2006. *This research was supported by Medical University of Bia¬ystok, Poland, grant 3-02410F. †Correspondence may be sent to Elzbieta Skrzydlewska at the above address or via email at: email@example.com. Wies¬awa Roszkowska-Jakimiec is with the Department of Instrumental Analysis, Medical University of Bia¬ystok, Bia¬ystok, Poland. 510 LUCZAJ ET AL. components such as protein and DNA (Rouach et al., 1997; Wang et al., 1990). Therefore, scientists have searched for potent antioxidants, especially among natural products. One such potentially health-promoting beverage is tea. It was generally believed that only green tea prepared by dehydration of Camellia sinensis leaves, which contain monomeric polyphenols, possesses antioxidant properties (Graham, 1992). Recent investigations, however, indicate that black tea obtained by fermentation of tea leaves and containing only a small amount of monomeric polyphenols (catechins) and multimeric polyphenols, (theaflavins [TFs], and thearubigins)— whose biological activities are less documented but still extensively examined—also reveals antioxidant abilities (Frei and Higdon, 2003; Graham, 1992). As a result, black tea has been proved to protect against cancer progression and heart diseases (Dufresne and Farnworth, 2001; Yang et al., 2003; Rietveld and Wiseman, 2003). However, the biological activity of black tea as a source of antioxidants requires further investigation. The purpose of this study was to investigate potential protective effects of black tea on the consequences of ethanol-induced oxidative stress in the liver manifested by lipid and protein modifications. Method 511 • The control group was treated intragastrically with 1.8 ml of physiological saline every day for 4 weeks (n = 6). • The black-tea group was given black-tea solution ad libitum instead of water for 1 week. Then, the group was treated intragastrically with 1.8 ml of physiological saline and received black-tea solution ad libitum instead of water every day for 4 weeks (n = 6). • The ethanol group was treated intragastrically with 1.8 ml of ethanol in doses increased by ethanol concentration from 2.0 to 6.0 g/kg body weight every day for 4 weeks. The dose of ethanol was gradually increased by 0.5 g/kg body weight every 3 days (n = 6). • The ethanol-and-black-tea group was given black-tea solution ad libitum instead of water for 1 week. Next, it was treated intragastrically with 1.8 ml of ethanol in doses increased by ethanol concentration from 2.0 to 6.0 g/kg body weight and received black-tea solution ad libitum instead of water every day for 4 weeks. The animals were deprived of food 12 hours before being intubated and then received saline or ethanol solution intragastrically by gavage. The amount of black tea given to rats was an amount equivalent to 40-50 cups of tea daily (approximately 100 mg/kg body weight). However, this amount was necessary to achieve the therapeutic effect. Black tea Preparation of tissue Black tea—Camellia sinensis (Linnaeus) O. Kuntze (standard research blends—lyophilized extract)—was provided by TJ Lipton (Englewood Cliffs, NJ) and was dissolved in drinking water at concentration of 3 g/l. Tea was prepared three times per week and stored at 4∞ C until use. The content of drinking vessels was renewed every day. Blacktea extract contained catechins (epigallocatechin gallate [EGCG]: 14.53 mg/l; epigallocatechin [EGC]: 2.21 mg/l; and epicatechin [EC]: 2.83 mg/l) and TFs (theaflavin [TF1]; theaflavin 3-gallate [TF2A]; theaflavin 3’-gallate [TF2B]; and theaflavin 3, 3’-digallate [TF3] in the amount of 156.16 mg/g dried extract for all four TFs). The levels of catechins and TFs were determined by modified high-performance liquid chromatography (HPLC) methods of Mattila et al. (2000) and Lee et al. (2000). Animals Two-month-old male Wistar rats were used for the experiment. They were housed in groups with free access to a granular standard diet and water and were maintained under a normal light-dark cycle. All experiments were approved by the Local Ethics Committee in Bia¬ystok, Poland, referring to the Polish Act Protecting Animals of 1997. The animals were divided into the following groups: After the above procedure, the rats were sacrificed under ether anesthesia (six animals in each group). The livers 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 ice-cold 0.25 M sucrose and 0.15 M NaCl with the addition of 6 ml 250 mM butylated hydroxytoluene (BHT) in ethanol to prevent the formation of new peroxides during the assay. The homogenization procedure was performed under standardized conditions; 10% of the homogenate was centrifuged at 10.000 ¥ g for 15 minutes at 4∞ C, and the supernatant was kept on ice until assayed. To assay protein oxidation, liver samples were homogenized in 5 mM phosphate buffer (pH 7.5) with protease inhibitors (leupeptin: 0.5 mg/ml; aproteinin: 0.5 mg/ml; pepstatin: 0.7 mg/ml) and 0.1% Triton X-100. The homogenate was centrifuged at 7800 ¥ g for 20 minutes, and biochemical analysis was performed on the supernatant. To assay cathepsin B activity, liver samples were homogenized in a glass-Teflon Potter homogenizer in 0.25 M sucrose with and without 0.2% Triton X-100. The homogenates were centrifuged at 100,000 ¥ g (4∞ C) for 60 minutes to settle the organelles or their membranes. Supernatant obtained from homogenate prepared in sucrose was 512 JOURNAL OF STUDIES ON ALCOHOL / JULY 2006 termed cytosol, whereas supernatant obtained from homogenate prepared in sucrose with Triton X-100 was termed homogenate. The content of cathepsin B in lysosomes was calculated as the difference between the homogenate and cytosol activities. Biochemical assays Lipid peroxidation was assayed by HPLC measurement of lipid hydroperoxides (LOOH) (Tokumaru et al., 1995), malondialdehyde (MDA) as malondialdehyde-thiobarbituric acid adducts (Londero and Greco, 1996), 4-hydroxynonenal (4-HNE) as the fluorimetric derivatives (Yoshino et al., 1986), and by spectrophotometric measurement of conjugated dienes at 234 nm (Recknagel and Glende, 1984). Protein oxidative modifications were examined by assessing addition of carbonyl groups, bistyrosine, tryptophan, sulfhydryl groups, and amino groups. Presence of carbonyl groups was determined spectrophotometrically using 2,4dinitrophenylhydrazine (Levine et al., 1990). Bistyrosine content was estimated by fluorescence spectrophotometry at 325 nm excitation and 420 nm emission (Rice-Evans, 1991). Fluorescence emission at 338 nm and excitation at 288 nm were used as a reflection of tryptophan content (Rice-Evans, 1991). The spectrofluorimeter was calibrated using 0.1 µg/ml quinine sulphate in 0.1 M sulfuric acid, and fluorescence of this solution was accepted as 1 unit. The presence of free amino groups was assessed by reaction with ninhydrin (Devenyi and Gregely, 1968), whereas presence of sulfhydryl groups was determined by the Ellman reaction (Ellman, 1959). In liver cytosol and homogenate, the activity of cathepsin B was determined with Bz-DL-Arg-pNA (Sigma Chemical Co., St. Louis, MO) as a substrate, at pH 6.0, by measuring released p-nitroaniline at 405 nm during a 2hour incubation at 37° C (Towatari et al., 1979). The content of cathepsin B in lysosomes was calculated as the difference between the homogenate and cytosol activities. The protein concentration was determined according to the method of Lowry et al. (1951). Diagnostic Biomerieux tests were used for assessment of blood serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Glutathione peroxidase (EC.18.104.22.168) activity was measured spectrophotometrically using the method of Paglia and Valentine (1967). GSSG (oxidized glutathione) formation was assayed by measuring the conversion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to NADP (nicotinamide adenine dinucleotide phosphate). One activity unit was defined as the amount of enzyme catalyzing conversion of 1 µmol of NADPH/minute/mg protein at 25° C and pH 7.4. Glutathione reductase (EC.22.214.171.124) activity was measured spectrophotometrically using the method of Mize and Langdon (1962), which monitors the oxidation of NADPH at 340 nm. The enzyme activity was expressed in units per milligram of protein. Glutathione (GSH) concentration was measured using the Bioxytech GSH-400 test. The method is applied 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. In the second step, the substitution products obtained with GSH are specifically transformed into a chromophoric thione whose maximal absorbance wavelength is 400 nm. The liver levels of black-tea components—EC, EGC, EGCG, and TFs—were determined according to the methods of Lee et al. (2000). Statistical analysis The data obtained in this study are expressed as mean (SD). The data were analyzed using standard statistical analyses: one-way analysis of variance with Scheffe’s F test for multiple comparisons to determine significance between different groups. A value of p < .05 was considered significant. Pearson’s correlation coefficients were calculated using statistical package Statistica 6.0 (StatSoft, Tulsa, OK). Results Chronic ethanol intoxication enhanced lipid peroxidation; this is visible in the statistically significant increase in the level of lipid peroxidation products, that is, conjugated dienes (CD), LOOH, MDA, and 4-HNE, whereas black tea significantly decreased all these levels (Table 1). Comparison of two rat groups—one receiving ethanol and the other ethanol with black tea—showed that all levels of lipid peroxidation markers significantly decreased during consumption of black tea after alcohol administration. A small increase was observed in the levels of all measured markers in the group receiving ethanol with black tea in comparison with the control group. TABLE 1. The levels of lipid peroxidation products: conjugated dienes (CD), lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4hydroxynonenal (4-HNE) in the liver of rats receiving black tea, ethanol, and ethanol and black tea Groups of rats Mean (SD) Analyzed parameter CD, µmol/g tissue LOOH, µmol/g tissue MDA, nmol/g tissue 4-HNE, nmol/g tissue Control Black tea 1.27 (0.05) 119 (8) 6.9 (0.4) 1.51 (0.05) 1.09 (0.13)a 103 (9)a 5.5 (0.8)a 1.40 (0.05)a Ethanol Black tea + ethanol 1.35 (0.15)b 1.29 (0.07)b 199 (15)a,b 121 (13)b,c 11.9 (0.9)a,b 7.7 (0.5)b,c 1.92 (0.09)a,b 1.55 (0.05)b,c aSignificantly different from control (p < .05); bsignificantly different from black-tea group (p < .05); csignificantly different from ethanol group (p < .05). LUCZAJ ET AL. TABLE 2. The levels of protein modification process markers: carbonyl groups, bistyrosine, tryptophan, sulfhydryl groups, and amino groups in the liver of rats receiving black tea, ethanol, and ethanol and black tea Groups of rats Mean (SD) Analyzed parameter Control Black tea Ethanol Carbonyl groups, nmol/mg protein 0.91 (0.06) 0.83 (0.05)a 1.23 (0.08)a,b Bistyrosine, U/mg protein 0.38 (0.02) 0.33 (0.02)a 0.54 (0.04)a,b Tryptophan, U/mg protein 7.68 (0.41) 7.45 (0.47) 8.23 (0.58)b Sulfhydryl groups, nmol/mg protein 4.21 (0.29) 4.08 (0.30) 3.17 (0.30)a,b Amino groups, nmol tyrosine/ mg protein 45.8 (3.1) 46.7 (3.2) 36.1 (3.1)a,b Black tea + ethanol 1.05 (0.07)a,b,c 0.45 (0.04)a,b,c 7.91 (0.49) 3.95 (0.32)c 42.9 (3.3)c aSignificantly different from control (p < .05); bsignificantly different from black-tea group (p < .05); csignificantly different from ethanol group (p < .05). Table 2 shows that the levels of markers of the proteinmodification process in the rat liver—such as carbonyl groups, bistyrosine, and tryptophan—were significantly increased after ethanol intoxication, whereas sulfhydryl and amino groups were decreased in the same group. The content of almost all of the above markers (except amino groups) in a group of rats drinking black tea decreased in comparison with the control. Alcohol given with black tea increased the levels of carbonyl groups, bistyrosine, and 513 tryptophan but decreased the levels of sulfhydryl and amino groups compared with the control group. Ethanol administered together with black tea caused significantly smaller changes than when ingested alone in comparison with control group. Ethanol intoxication caused a statistically significant decrease in lysosomal as well as total activity of cathepsin B (Figure 1). Drinking black tea did not significantly influence the level of cathepsin B in comparison with the control group. Giving black tea together with alcohol caused changes in the activity of this enzyme in the same direction but with a smaller enhancement than in the alcohol group. Decrease in the activities of antioxidant enzymes during chronic ethanol intoxication was observed in the case of glutathione peroxidase (GSH-Px), whereas the activity of glutathione reductase (GSSG-R) was increased in the same group (Table 3) compared with the control group. However, drinking of black tea caused an increase in the activities of both enzymes. Alcohol ingested with black tea did not cause significant changes in the activity of GSSG-R in comparison with control group but caused a decrease in the activity of GSH-Px. The level of nonenzymatic antioxidant (GSH) in the liver was also decreased after ethanol administration compared with the control group. However, black tea caused a significant increase in the level of this parameter. Ethanol administered together with black tea caused significantly smaller changes than ethanol ingested alone in comparison with the control group. FIGURE 1. Activity of cathepsin B (pNA, mmol/g tissue) in the liver homogenate, cytosol, and lysosomes of rats received black tea, ethanol, and ethanol and black tea. aSignificantly different from control (p < .05); bsignificantly different from black-tea group (p < .05); csignificantly different from ethanol group (p < .05). 514 JOURNAL OF STUDIES ON ALCOHOL / JULY 2006 TABLE 3. Activities of glutathione peroxidase (GSH-Px) and reductase (GSSG-R) and level of glutathione reduced (GSH) in the liver of rats receiving black tea, ethanol, and ethanol and black tea TABLE 4. The activities of liver damage markers: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats receiving black tea, ethanol, and ethanol and black tea Groups of rats Mean (SD) Analyzed parameter Control Black tea Ethanol Groups of rats Mean (SD) Black tea + ethanol GSH-Px, U/ml 10.06 (0.35) 11.09 (0.25)a 9.47 (0.56) 9.66 (0.59)b GSSG-R, U/ml 0.37 (0.02) 0.40 (0.02) 0.53 (0.03)a 0.53 (0.03)a GSH, mmol/g tissue 0.82 (0.04) 0.98 (0.05)a 0.74 (0.04)a 0.89 (0.05)a,c Analyzed parameter Control ALT, U/l AST, U/l 37.1 (2.4) 156 (10) Black tea Ethanol Black tea + ethanol 32.6 (2.1)a 69.5 (5.9)a,b 44.8 (3.8)a,b,c 150 (10) 271 (19)a,b 198 (15)a,b,c aSignificantly aSignificantly different from control (p < .05); bsignificantly different from black-tea group (p < .05); csignificantly different from ethanol group (p < .05). Table 4 shows that the serum activities of the liver damage markers—ALT and AST—were also significantly changed during ethanol intoxication. An almost twofold increase in ALT and AST activities was observed in the ethanol group of rats in comparison with both control and black-tea groups. After ingestion of alcohol together with black tea, the activities of ALT and AST were much lower than after ethanol administration alone but higher than in the control and black-tea groups. Components of black tea—monomeric polyphenols (catechins) as well as multimeric polyphenols (TFs)—were found and amounts determined in the liver of rats receiving black tea and black tea and ethanol. It was observed that the levels of both catechins and TFs were diminished after ethanol administration (Figure 2). A correlation of the levels of black-tea components with the levels of lipids and markers of protein oxidative modifications was made (Table 5). Levels of lipid peroxidation products as well as the basic product of protein oxidative modifications, bistyrosine, were significantly and negatively correlated with the levels of TFs and EGC in the black-tea group. However, a significantly positive correlation was noticed between the levels of tryptophan and sulfhydryl groups and TFs as well as EGC. The most unanimously significant negative correlations were found between the levels of all lipid peroxidation products and the TFs as well as between the levels of LOOH and 4-HNE versus EGC level in the group of rats receiving black tea and alcohol. A negative correlation between the level of protein oxidative modification products—carbonyl groups and bistyrosine—and the different from control (p < .05); bsignificantly different from black-tea group (p < .05); csignificantly different from ethanol group (p < .05). FIGURE 2. The levels of black-tea components—catechins (epicatechin [EC], epigallocatechin [EGC], and epigallocatechin gallate [EGCG]) and theaflavins (TFs)—in the liver of rats received black tea and ethanol and black tea. aSignificantly different from black-tea group (p < .05). LUCZAJ ET AL. 515 TABLE 5. Pearson’s correlation coefficients between levels of lipid peroxidation products, protein oxidative modifications products, and measured levels of black-tea components (EC, EGC, EGCG, and TFs) Black-tea group (n = 6) Variable CD LOOH MDA 4-HNE Carbonyl groups Bistyrosine Tryptophan Sulfhydryl groups Amino groups Black-tea + ethanol group (n = 6) EC EGC EGCG TFs EC EGC EGCG TFs -.80 -.74 -.63 -.72 -.42 -.35 .62 .81 .32 -.43 -.94 -.88 -.95 -.43 -.42 .87 .90 .75 -.27 -.58 -.45 -.83 -.37 -.72 .43 .45 .41 -.45 -.97 -.92 -.89 -.56 -.89 .91 .88 .90 -.56 -.82 -.87 -.71 -.69 -.74 .55 .91 .89 -.61 -.89 -.67 -.86 -.91 -.83 -.27 .55 .19 -.83 -.63 -.77 -.64 -.64 -.52 .33 .82 .73 -.88 -.82 -.85 -.88 -.92 -.70 .09 .86 .58 Notes: Statistically significant correlation coefficients (for α = .05) are bolded. EC = epicatechin; EGC = epigallocatechin; EGCG = epigallocatechin gallate; TFs = theaflavins; CD = conjugated dienes; LOOH = lipid hydroperoxides; MDA = malondialdehyde; 4-HNE = 4-hydroxynonenal. level of TFs and EGC was also observed. However, a significantly positive correlation was found between the level of sulfhydryl groups and TFs as well as levels of EC and EGCG. Discussion It was revealed that, during ethanol intoxication, free radicals are generated (Lieber, 1997). This fact is very important in connection with a significant decrease in the antioxidant capacity of the liver after ethanol intoxication, which was manifested by the decrease in activity or concentration of basic cellular enzymatic and nonenzymatic antioxidants observed here and in other studies (Nordmann, 1994; Scott et al., 2000). As a result, cell components are exposed to enhanced action of free radicals. Proteins are known to be the major target as well as the first target for free radicals, which are formed in both the intra- and extracellular environment in vivo (Stadtman and Levine, 2003). All proteins are susceptible to attack by free radicals, though some of them are more vulnerable than others, and the most sensitive to oxidation are aromatic amino acids such as tryptophan and tyrosine (Stadtman and Levine, 2003). Our study has proved that during ethanol ingestion, the level of bistyrosine—the product of a reaction between free radicals and tyrosine—was increased in comparison with the control. Bistyrosine production appears to be a useful “marker” for protein modification, especially modification by hydroxyl radical (Giulivi et al., 2003). Cysteine is also extremely sensitive to free radicals. Most reports show that cysteine/cystine ratio of the proteins is altered under oxidizing conditions (Kalyanaraman, 1995). Other protein modifications are connected with the generation of free radicals at the alpha-carbon atom of the peptide bond and, consequently, the fragmentation of the polypeptide chain with formation of a new carbonyl group (Davies, 1987). The increase in the amount of protein carbonyl groups during ethanol intoxication was described in this article. During ethanol intoxication, protein structure can also be changed by reactions with other compounds generated during ethanol metabolism, such as lipid peroxidation products. Free radicals reacting with membrane phospholipids produce, at first, conjugated dienes and LOOH whose levels are enhanced during ethanol intoxication, as has been shown here. A variety of compounds, which can be produced by decomposition of these compounds, may exert more toxic effects on cells, but the most reactive are MDA and 4-HNE as well as other carbonyls (Aust et al., 1985; Esterbauer et al., 1991). These compounds are electrophilic and may form adducts with nucleophilic sulfhydryl, primary amino, and histydyl groups of proteins, which cause changes in protein structure and function (e.g., 4-HNE inhibits aldehyde dehydrogenase-mediated oxidation of ethanol to acetaldehyde) (Mitchell and Petersen, 1987). Aldehydes generated during lipid peroxidation also form couplings with GSH, the main cellular nonenzymatic antioxidant, leading to a significant decrease in the cellular concentration of GSH, which may result in the cytotoxicity of oxidative stress. Moreover, 4-HNE reacts with sulfhydryl compounds, causing rapid loss of protein sulfhydryl groups, which consequently leads to a decrease in cathepsin B lysosomal sulfhydryl protease activity (O’Neil et al., 1997). The diminution decrease in total activity of cathepsin B after ethanol intoxication was observed here. Another aldehyde, acetaldehyde, which can modify protein structure in ethanol intoxication, is a very reactive ethanol metabolite, which reacts with different cellular components including proteins (Tuma et al., 1996). Acetaldehyde reacts most readily with free amino and sulfhydryl groups mainly of lysine, cysteine, methionine, arginine, and tyrosine residues of proteins and, to a smaller degree, with other amino acid residues and peptide bonds (Braun et al., 1995; Kenyon et al., 1998). The occurrence of these types of reactions corresponds with the decrease in the number of amino and sulfhydryl groups observed in this study. The consequence of modification (by free radicals and/or acetaldehyde) of 516 JOURNAL OF STUDIES ON ALCOHOL / JULY 2006 sulfhydryl groups, which are localized in the active center of cathepsin B, is a decrease in this protease activity, which was observed in this study. The disturbances in protein structure described previously may be accompanied by changes in the biological functions of the proteins. Observed changes in the activities of GSH-Px and GSSG-R as well as a decrease in GSH concentration are especially important, because they may cause a breakdown of the antioxidant protection. Decreases in the activities of these enzymes after chronic alcohol consumption were also observed by others (Oh et al., 1998). However, the data concerning the increase in liver GSH-Px and GSSG-R activity were also presented (Bailey et al., 2001). A decrease in the antioxidant enzyme activity during chronic ethanol intoxication may be caused by many factors. In addition to the reactions described previously, this decrease may be due to inhibition of biosynthesis of protein molecules, which was earlier observed in ethanol intoxication (Bengtsson et al., 1984). Changes in lipid and protein structure caused by free radicals and acetaldehyde may be also the reason for disturbances in lipid-protein interaction necessary for biological membrane functions (Chen and Yu, 1994). First, the membrane fluidity is changed and an increase in permeability of the membranes is observed (Benedetti et al., 1980). The results of this study confirm that ethanol-induced oxidative stress leads to lysosomal fragility and release of cathepsin B into cytosol. The decrease in synthesis of adenosine triphosphate (ATP) also contributes to membrane destabilization during ethanol intoxication (Deaciuc et al., 1992). Furthermore, the increases in lipid peroxidation products and disruption of lysosomal membranes were confirmed by ultrastructure examinations (unpublished data). The increase in the quantity of autophagosomes containing fragments of disturbed organelles as well as in secondary lysosomes with differential density content was also examined. Proteolytic enzymes passing from the organelles into the cytosol may cause uncontrolled proteolysis in liver cells. The increase in the activities of AST and ALT, the markers of the liver destruction, in the blood indicates such processes. However, the reduction of proteolytic enzyme activity in the lysosomes may cause accumulation of modified proteins. Changes in cellular metabolism during ethanol intoxication are connected mainly with free radical actions. Therefore, it is necessary to find protective compounds. Ingestion of black tea seems to be useful because of the protective effect of its components, which possess antioxidative properties (Luczaj and Skrzydlewska, 2005; Yoshino et al., 1994). Black-tea components with proved antioxidant abilities are catechins (Rice-Evans et al., 1996, Ostrowska et al., 2004). During the last few years, it has been shown that the multimeric polyphenols of black tea (TFs and thearubigins formed during fermentation of tea leaves) possess even stronger antioxidant abilities than their precur- sors, catechins (Leung et al., 2001). Both catechins and TFs are absorbed from the gastrointestinal tract and are distributed into different tissues, including the liver (Mulder et al., 2001; Warden et al., 2001). In this study, determinable levels of these compounds were found both in the liver of rats drinking only black tea for 5 weeks and in the liver of rats receiving black tea and ethanol. As in other studies (Rechner, 2002), because of the administration of significantly higher doses of daily tea intake than average human consumption, the level of catechins that could elicit a therapeutic effect was obtained relatively quickly. It has been shown that TF3 inhibits the activity of xanthine oxidase, the enzyme that generates the main and first formed of all radicals—the superoxide anion (Lin et al., 2000). The main source of xanthine oxidase, especially during ethanol metabolism, is the liver. Moreover, TFs, to a larger degree TF3 and to a smaller degree TF2 and TF1, are able to scavenge superoxide anion, singlet oxygen, and hydroxyl radical, and the TFs react with these free radicals 10 times faster than the strongest antioxidant of all the catechins, EGCG (Jovanovic et al., 1997; Lin et al., 2000). Moreover, black-tea catechins as well as TFs, as a result of their chelating effects, may diminish pro-oxidative action of transition metal ions, whose amount is enhanced during ethanol intoxication. TFs also cause a decrease in iron absorption from the digestive tract (Hurrell et al., 1999). Moreover, it can be suggested that black tea changes ethanol metabolism. However, it is only known that catechins, a minor fraction of black-tea components, can inhibit the activity of cytochrome P450 2E1 (Goodin and Rosengren, 2003), which participates in ethanol metabolism, especially in chronic intoxication. Consequently, the above effects reduce the generation of free radicals and decrease the possibility of their reaction with integral cell components, especially proteins and lipids. Our results here indicate the protective action of black tea on liver proteins. This may explain the increase in GSH-Px, GSSG-R, and cathepsin B activities in the liver of rats drinking black tea and alcohol in comparison with the ethanol-only group in our study. In this article, it has been proved that giving black tea to rats intoxicated with ethanol prevents the enhancement of lipid peroxidation as well. Apart from the antioxidative effect of black-tea components, membrane protection may be connected to the fact that catechins, and to a largerer degree TFs, may show partially lipophilic character, and they can penetrate the interior of membranes, where they exert a membrane-stabilizing effect by modifying the lipid packing order (Arora et al., 2000; Nakayama et al., 1998). In conclusion, black tea protects liver lipids and proteins against ethanol-induced oxidative modifications and, consequently, prevents changes in their biological functions. Moreover, black tea prevents changes in protease activities and in protease distribution within hepatocytes, which may lead to disturbances in cellular protein catabolism and to LUCZAJ ET AL. destruction of the liver cells. Considering that the metabolism of ethanol and tea flavonoids is the same in rats as in humans, the results obtained here suggest that black tea may also protect human liver cells against the consequences of oxidative stress caused, for example, by ethanol intoxication. Acknowledgment The authors thank professor Joe Vinson (University of Scranton) for providing the TFs standards and to Unilever for providing black-tea extract. References ARORA, A., BYREM, T.M., NAIR M.G., AND STRASBURG, G.M. Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids. Arch. Biochem. Biophys. 373: 102-109, 2000. AUST, S.D., MOREHOUSE, L.A., AND THOMAS, C.E. Role of metals in oxygen radical reactions. J. Free Radic. Biol. Med. 1: 3-25, 1985. BAILEY, S.M., PATEL, V.B., YOUNG, T.A., ASAYAMA, K., AND CUNNINGHAM, C.C. Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver. Alcsm Clin. Exp. Res. 25: 726-733, 2001. BENEDETTI, A., COMPORTI, M., AND ESTERBAUER, H. Identification of 4hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim. Biophys. Acta 620: 281-296, 1980. BENGTSSON, G., SMITH-KIELLAND, A., AND MORLAND, J. Ethanol effects on protein synthesis in nonparenchymal liver cells, hepatocytes, and density populations of hepatocytes. Exp. Mol. Pathol. 41: 44-57, 1984. BRAUN, K.P., CODY, R.B., JR., JONES, D.R., AND PETERSON, C.M. A structural assignment for a stable acetaldehyde-lysine adduct. J. Biol. Chem. 270: 11263-11266, 1995. CHEN, J.J. AND YU, B.P. Alterations in mitochondrial membrane fluidity by lipid peroxidation products. Free Radic. Biol. Med. 17: 411-418, 1994. DAVIES, K.J.A. Protein damage and degradation by oxygen radicals: I. General aspects. J. Biol. Chem. 262: 9895-9901, 1987. DEACIUC, I.V., D’SOUZA, N.B., LANG, C.H., AND SPITZER, J.J. Effects of acute alcohol intoxication on gluconeogenesis and its hormonal responsiveness in isolated, perfused rat liver. Biochem. Pharmacol. 44: 1617-1624, 1992. DEVENYI, T. AND GREGELY, J. Analytische Methoden zur Untersuchung von Aminosauren: Peptiden und Proteinen, Budapest, Hungary: Akademie Kiado, 1968, pp. 70-71. DUFRESNE, C.J. AND FARNWORTH, E.R. A review of latest research findings on the health promotion properties of tea. J. Nutr. Biochem. 12: 404421, 2001. ELLMAN, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 7077, 1959. ESTERBAUER, H., SCHAUR, R.J., AND ZOLLNER, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11: 81-128, 1991. FREI, B. AND HIGDON, J.V. Antioxidant activity of tea polyphenols in vivo: Evidence from animal studies. J. Nutr. 133: 3275S-3284S, 2003. GIULIVI, C., TRAASETH, N.J., AND DAVIES, K.J. Tyrosine oxidation products: Analysis and biological relevance. Amino Acids 25: 227-232, 2003. GOODIN, M.G. AND ROSENGREN, R.J. Epigallocatechin gallate modulates CYP450 isoforms in the female Swiss-Webster mouse. Toxicol. Sci. 76: 262-270, 2003. GRAHAM, H.N. Green tea composition, consumption and polyphenol chemistry. Prev. Med. 21: 334-350, 1992. 517 HURRELL, R.F., REDDY, M., AND COOK, J.D. Inhibition of non-haem iron absorption in man by polyphenolic-containing beverages. Brit. J. Nutr. 81: 289-295, 1999. JOVANOVIC, S.V., HARA, Y., STEENKEN S., AND SIMIC, M.G. Antioxidant potential of theaflavins: A pulse radiolysis study. J. Amer. Chem. Soc. 119: 5337-5343, 1997. KALYANARAMAN, B. Radical intermediates during degradation of ligninmodel compounds and environmental pollutants: An electron spin resonance study. Xenobiotica 25: 667-675, 1995. KATO, S., KAWASE, T., ALDERMAN, J., INATOMI, N., AND LIEBER, C.S. Role of xanthine oxidase in ethanol-induced lipid peroxidation in rats. Gastroenterology 98: 203-210, 1990. KELLOGG, E.W., 3RD, AND FRIDOVICH, I. Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem. 250: 8812-8817, 1975. KENYON, S.H., NICOLAOU, A., AND GIBBONS, W.A. The effect of ethanol and its metabolites upon methionine synthase activity in vitro. Alcohol 15: 305-309, 1998. KRIKUN, G., LIEBER, C.S., AND CEDERBAUM, A.I. Increased microsomal oxidation of ethanol by cytochrome P-450 and hydroxyl radical-dependent pathways after chronic ethanol consumption. Biochem. Pharmacol. 33: 3306-3309, 1984. KUKIELKA, E. AND CEDERBAUM, A.I. DNA strand cleavage as a sensitive assay for the production of hydroxyl radicals by microsomes: Role of cytochrome P4502E1 in the increased activity after ethanol treatment. Biochem. J. 302 (Pt 3): 773-779, 1994. KUROSE, I., HIGUCHI, H., KATO, S., MIURA, S., AND ISHII, H. Ethanol-induced oxidative stress in the liver. Alcsm Clin. Exp. Res. 20 (1 Suppl.): 77A-85A, 1996. LEE, M.J., PRABHU, S., MENG, X., LI, C., AND YANG, C.S. An improved method for determination of green and black tea polyphenols in biomatrices by high-performance liquid chromatography with coulometric array detection. Analyt. Biochem. 279: 164-169, 2000. LEUNG, L.K., SU, Y., CHEN, R., ZHANG, Z., HUANG, Y., AND CHEN, Z.-Y. Theaflavins in black tea and catechins in green tea are equally effective antioxidants. J. Nutr. 131: 2248-2251, 2001. LEVINE, R.L., GARLAND, D., OLIVER, C.N., AMICI, A., CLIMENT, I., LENZ, A.G., AHN, B.W., SHALTIEL, S., AND STADTMAN, E.R. Determination of carbonyl content in oxidatively modified proteins. Meth. Enzymol. 186: 464-478, 1990. LIEBER, C.S. Role of oxidative stress and antioxidant therapy in alcoholic and nonalcoholic liver diseases. Adv. Pharmacol. 38: 601-628, 1997. LIN, J.K., CHEN, P.C., HO, C.T., AND LIN-SHIAU, S.Y. Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3’-digallate, (-)-epigallocatechin-3-gallate, and propyl gallate. J. Agric. Food Chem. 48: 2736-2743, 2000. LONDERO, D. AND LO GRECO, P. Automated high-performance liquid chromatographic separation with spectrofluorometric detection of a malondialdehyde-thiobarbituric acid adduct in plasma. J. Chromatogr. A 729: 207-210, 1996. LOWRY, O.H., ROSENBROUGH, N.J., FARR, A.L., AND RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265275, 1951. LUCZAJ, W. AND SKRZYDLEWSKA, E. Antioxidative properties of black tea. Prev. Med. 40: 910-918, 2005. MATTILA, P., ASTOLA, J., AND KUMPULAINEN, J. Determination of flavonoids in plant material by HPLC with diode-array and electro-array detections. J. Agric. Food Chem. 48: 5834-5841, 2000. MITCHELL, D.Y. AND PETERSEN D.R. The oxidation of alpha-beta unsaturated aldehydic products of lipid peroxidation by rat liver aldehyde dehydrogenases. Toxicol. Appl. Pharmacol. 87: 403-410, 1987. MIZE, C.E. AND LANGDON R.G. Hepatic glutathione reductase: I. Purification and general kinetic properties. J. Biol. Chem. 237: 1589-1595, 1962. MULDER, T.P., VAN PLATERINK, C.J., WIJNAND SCHUYL, P.J., AND VAN AMELSVOORT, J.M. Analysis of theaflavins in biological fluids using 518 JOURNAL OF STUDIES ON ALCOHOL / JULY 2006 liquid chromatography-electrospray mass spectrometry. J. Chromatogr. B Biomed. Sci. Applicat. 760: 271-279, 2001. NAKAYAMA, T., ONO, K., AND HASHIMOTO, K. Affinity of antioxidative polyphenols for lipid bilayers evaluated with liposome system. Biosci. Biotechnol. Biochem. 62: 1005-1007, 1998. NORDMANN, R. Alcohol and antioxidant systems. Alcohol Alcsm 29: 513522, 1994. OH, S.I., KIM, C.-I., CHUN, H.J., AND PARKS, S.C. Chronic ethanol consumption affects glutathione status in rat liver. J. Nutr. 128: 758-763, 1998. O’NEIL, J., HOPPE, G., SAYRE, L.M., AND HOFF, H.F. Inactivation of cathepsin B by oxidized LDL involves complex formation induced by binding of putative reactive sites exposed at low pH to thiols on the enzyme. Free Radic. Biol. Med. 23: 215-225, 1997. OSTROWSKA, J., LUCZAJ, W., KASACKA, I., ROZANSKI, A., AND SKRZYDLEWSKA, E. Green tea protects against ethanol-induced lipid peroxidation in rat organs. Alcohol 32: 25-32, 2004. PAGLIA, D.E. AND VALENTINE, W.N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70: 158-169, 1967. PUNTARULO, S. AND CEDERBAUM, A.I. Chemiluminescence from acetaldehyde oxidation by xanthine oxidase involves generation of and interactions with hydroxyl radicals. Alcsm Clin. Exp. Res. 13: 84-90, 1989. RECHNER, A.R., WAGNER, E., VAN BUREN, L., VAN DE PUT, F., WISEMAN, S., AND RICE-EVANS, C.A. Black tea represents a major source of dietary phenolics among regular tea drinkers. Free Radic. Res. 36: 1127-1135, 2002. RECKNAGEL, R.O. AND GLENDE, E.A., JR. Spectrophotometric detection of lipid conjugated dienes. Meth. Enzymol. 105: 331-337, 1984. RICE-EVANS, C.A., DIPLOCK, A.T., AND SYMONS, M.C.R. Techniques in free radical research. In: BURDON, R.H. AND VAN KNIPPENBERG, P.H. (Eds.) Laboratory Techniques in Biochemistry and Molecular Biology, New York: Elsevier, 1991, pp. 232-233. RICE-EVANS, C.A., MILLER, N.J., AND PAGANGA, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20: 933-956, 1996. RIETVELD, A. AND WISEMAN, S. Antioxidant effects of tea: Evidence from human clinical trials. J. Nutr. 133: 3285S-3292S, 2003. ROUACH, H., FATACCIOLI, V., GENTIL, M., FRENCH, S.W., MORIMOTO, M., AND N ORDMANN , R. Effect of chronic ethanol feeding on lipid peroxidation and protein oxidation in relation to liver pathology. Hepatology 25: 351-355, 1997. SCOTT, R.B., REDDY, K.S., HUSAIN, K., SCHLORFF, E.C., RYBAK, L.P., AND SOMANI, S.M. Dose response of ethanol on antioxidant defense system of liver, lung, and kidney in rat. Pathophysiology 7: 25-32, 2000. SHAW, S., JAYATILLEKE, E., AND LIEBER, C.S. Lipid peroxidation as a mechanism of alcoholic liver injury: Role of iron mobilization and microsomal induction. Alcohol 5: 135-140, 1988. SOCHMAN, J. Regional ischemic and reperfusion injury, free oxygen radicals and the possible role of n-acetylcysteine in acute myocardial infarction. Core Vasa 36: 269-279, 1994. STADTMAN, E.R. AND LEVINE, R.L. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25: 207-218, 2003. TOKUMARU, S., TSUKAMOTO, I., IGUCHI, H., AND KOJO, S. Specific and sensitive determination of lipid hydroperoxides with chemical derivatization into 1-naphthyldiphenylphosphine oxide and high-performance liquid chromatography. Analyt. Chim. Acta 307: 97-102, 1995. TOWATARI, T., KAWABATA, Y., AND KATUNUMA, N. Crystallization and properties of cathepsin B from rat liver. Europ. J. Biochem. 102: 279-289, 1979. TUMA, D.J., THIELE, G.M., XU, D., KLASSEN, L.W., AND SORRELL, M.F. Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration. Hepatology 23: 872-880, 1996. WANG, C.J., WANG, S.W., SHIAH, H.S., AND LIN, J.K. Effect of ethanol on hepatotoxicity and hepatic DNA-binding of aflatoxin B1 in rats. Biochem. Pharmacol. 40: 715-721, 1990. W ARDEN, B.A., S MITH , L.S., B EECHER, G.R., BALENTINE, D.A., AND CLEVIDENCE, B.A. Catechins are bioavailable in men and women drinking black tea throughout the day. J. Nutr. 131: 1731-1737, 2001. YANG, C.S., CHUNG, J.Y., YANG, G.Y., LI, C., MENG, X., AND LEE, M.J. Mechanisms of inhibition of carcinogenesis by tea. Biofactors 13 (14): 73-79, 2000. YOSHINO, K., HARA, Y., SANO, M., AND TOMITA, I. Antioxidative effects of black tea theaflavins and thearubigin on lipid peroxidation of rat liver homogenates induced by tert-butyl hydroperoxide. Biol. Pharmaceut. Bull. 17: 146-149, 1994. YOSHINO, K., MATSUURA, T., SANO, M., SAITO, S., AND TOMITA, I. Fluorometric liquid chromatographic determination of aliphatic aldehydes arising from lipid peroxides. Chem. Pharmaceut. Bull. 34: 1694-1700, 1986.