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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: skrzydle@amb.edu.pl. 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.1.11.1.6) 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.1.6.4.2) 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.
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