Green tea supplementation was found to mitigate some of the negative effects of chronic ethanol consumption in the brains of rats of different ages. Ethanol consumption caused decreases in antioxidant enzyme activities and levels of antioxidants in the brain, along with increased oxidative damage to lipids and proteins, especially in older rats. Rats that received green tea supplementation alongside ethanol showed reduced oxidative stress and better protection of antioxidant abilities in the brain, particularly in young and adult rats, compared to rats that only received ethanol. The protective effects of green tea may result from its inhibition of free radicals generated by ethanol-induced oxidative stress and its ability to increase the brain's endogenous antioxidant defenses.

1. Green tea supplementation in rats of different ages mitigates
ethanol-induced changes in brain antioxidant abilities
Elz_bieta Skrzydlewska*, Agnieszka Augustyniak, Kamil Michalak, Ryszard Farbiszewski
Department of Analytical Chemistry, Medical University of Bialystok, 15-089 Bia1ystok, Poland
Received 5 August 2005; received in revised form 20 December 2005; accepted 21 December 2005
Abstract
Oxidative stress induced by chronic ethanol consumption, particularly in aging subjects, has been implicated in the pathophysiology of
many neurodegenerative diseases. Antioxidants with polyphenol structures, such as those contained in green tea, given alone for 5 weeks in
liquid Lieber de Carli diet followed by administration with ethanol for 4 weeks with ethanol have been investigated as potential therapeutic
antioxidant agents in the brain in rats of three ages (2, 12, and 24 months). Ethanol consumption caused age-dependent decreases in brain
superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase activities. In addition, ethanol consumption caused age-
dependent decreases in the levels of GSH, selenium, vitamins, E, A and C, and b-carotene and increases in the levels of oxidized glutathione
(GSSG). Changes in the brain’s antioxidative ability were accompanied by enhanced oxidative modification of lipids (increases in lipid
hydroperoxides, malondialdehyde, and 4-hydroxynonenal levels) and proteins (increases in carbonyl groups and bistyrosine). Reduced risk
of oxidative stress and protection of the central nervous system, particularly in young and adult rats, after green tea supplementation were
observed. Green tea partially prevented changes in antioxidant enzymatic as well as nonenzymatic parameters induced by ethanol and en-
hanced by aging. Administration of green tea significantly protects lipids and proteins against oxidative modifications in the brain tissue of
young and adult rats. The beneficial effect of green tea can result from the inhibition of free radical chain reactions generated during eth-
anol-induced oxidative stress and/or from green tea-induced increases in antioxidative abilities made possible by increases in the activity/
concentration of endogenous antioxidants. Ó 2005 Elsevier Inc. All rights reserved.
Keywords: Brain; Green tea; Ethanol; Aging; Antioxidants; Lipid peroxidation; Protein oxidation
1. Introduction
The mechanism underlying aging, an inevitable biolog-
ical process that affects most living organisms, is still an
area of significant controversy. It is known that aging is
a complex process involving many genetic and environ-
mental factors (Harman, 1998; Jazwinski, 1998; Samson
& Nelson, 2000). A large body of evidence indicates that
the antioxidative abilities decrease and in consequence ox-
idative damage of macromolecules such as DNA, protein,
and lipid occurs more frequently with age and in various
age-related diseases. Therefore, it has been suggested that
such an increase in the oxidative damage, which in turn
contributes to the aging process and the degeneration of
neurons, is the basic pathological change leading to neuro-
degenerative diseases probably associated with more slight
molecular changes in the plasticity of specific neurons and/
or synaptic efficacy (Morrison & Hof, 1997). Moreover,
several external factors could be implicated in the cell in-
jury, for example, chronic consumption of ethanol that in-
duces free radical production and in consequence
oxidative damage (Vallet et al., 1997). Free radicals could
contribute to the action of ethanol in the central nervous
system particularly in aging subjects.
Oxidation of ethanol in the central nervous system has
been revealed to be accompanied by enhanced free radical
formation with superoxide anion being generated as the first
one (Ponnappa & Rubin, 2000). Due to oxygen consump-
tion in metabolic reactions, central nervous system is influ-
enced by substantial amount of superoxide anion (Somani
et al., 1996). Increased generation of superoxide anion in
central nervous system, like in other tissues, is caused by
augmented conversion of xanthine dehydrogenase to xan-
thine (Kato et al., 1990). This results from an increase in
NADH concentration, which occurs during oxidation of
ethanol as well as that of acetaldehyde, the metabolite of
ethanol (Puntarulo & Cederbaum, 1989). Superoxide dis-
mutase metabolizes superoxide anion into hydrogen perox-
ide, which can activate some neurotransmitters responsible
* Corresponding author. Tel./fax: 148 085 7485707.
E-mail address: skrzydle@amb.edu.pl (E. Skrzydlewska).
0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi: 10.1016/j.alcohol.2005.12.003
Alcohol 37 (2005) 89–98

2. for control in rodents, for example, dopamine (Adachi
et al., 2001). Hydrogen peroxide is converted to highly re-
active hydroxyl radical under the influence of iron ions (II),
the content of which is very high in the central nervous sys-
tem (Gerlach et al., 1994). This radical easily reacts with all
cell components, with lipids in particular, causing peroxida-
tion. Due to a very high phospholipid content, the central
nervous system is extremely susceptible to hydroxyl radical
action (Halliwell, 1992; Porter, 1984) Moreover, reaction of
hydroxyl radical with acetaldehyde results in the formation
of acetyl radicals the level of which increases both in the
liver microsomes and in the central nervous system (Puntar-
ulo & Cederbaum, 1989). The hydroxyl radical can also re-
act with alcohol molecule forming 1-hydroxyethyl radical,
which due to relatively long half-life time contributes con-
siderably to cell damage. The level of hydroxyethyl radical
in spite of its minimal activity in the central nervous system
increases as a result of isoenzyme CYP2E1 induction (Sun
& Sun, 2001).
The nervous system cells of both humans and animals
are especially vulnerable to oxidative damage caused by
free radicals for a number of reasons. These include high
concentration of readily oxidazible substrate, in particular
membrane lipid polyunsaturated fatty acid, low level of
protective antioxidant enzymes (catalase and glutathione
peroxidase), high ratio of membrane surface area to cyto-
plasmic volume, and extended axonal morphology prone
to peripheral injury. In addition, some regions have high
nonheme iron concentrations. Thus, antioxidative defense
is critically important in nervous tissue protection. Growing
fundamental and clinical data indicate that the redox state
in neural structures plays a significant role in the pathogen-
esis of age-associated disorder observed in humans (Halli-
well, 2001).
If mild oxidative stress occurs, normal tissues often re-
spond with extra antioxidant defense. However, severe or
persistent oxidative stress can cause cellular component in-
jury, their degeneration, and finally brain cell death. In
addition, exposure of an aging organism to life threatening
conditions, for example, to chronic ethanol-induced oxida-
tion stress, aggravates the homeostatic level of the products
of the genes and gradually damages the cellular function of
the brain by inducing changes in the structure and function
of regulatory proteins (Cakatay et al., 2001).
Therefore, potent antioxidants especially those belong-
ing to natural products are investigated. One of such poten-
tially health-promoting beverages is green tea, whose
components mainly catechins and catechin derivates have
antioxidant properties (Guo et al., 1996; Rice-Evans et al.,
1996). Moreover, an important role of green tea in the im-
provement of defense system against ethanol-induced oxi-
dative stress was evidenced with reference to different
tissues, for example, liver and blood (Dobrzynska et al.,
2005; Luczaj et al., 2004; Ostrowska et al., 2004). The pro-
tective effects of the green tea extract on the central nervous
tissue expression in decreased level of lipid peroxidation
products were shown (Graham, 1992; Rice-Evans et al.,
1996).
Hence, the goal of the present study has been to investi-
gate the efficacy of green tea as a source of water-soluble
antioxidants in its action on the brain antioxidative poten-
tial of aged rats chronically exposed to an oxidative stress
induced by ethanol.
2. Materials and methods
All experiments were approved by the Local Ethic Com-
mittee in Bia1ystok (Poland) referring to Polish Act Protect-
ing Animals of 1997.
2.1. Animals (treatment)
Male Wistar rats of 2 (200–220 g b.w.), 12 (520–550 g
b.w.) and 24 months (750–780 g b.w.) were used for all ex-
periments. The rats were housed in individual cages and
pair-fed with either nutritional control or ethanol liquid
Lieber DeCarli diet for 5 weeks. Dietary intake was compa-
rable in all groups, with all rats demonstrating constant
weight gain throughout the 5-week feeding period. Etha-
nol-fed rats had only slightly decreased rates of weight gain,
consistent with the well-studied effects of isocaloric ethanol
feeding on intermediate metabolism. In Lieber DeCarli liq-
uid diet, ethanol provided 36% of total calories, 18% pro-
tein, 35% fat, and 11% carbohydrate (Lieber & De Carli,
1982). Pair-fed littermates consumed the same diet except
that carbohydrates replaced ethanol isocalorically. Liquid
diet (control and ethanol) containing 7 g green tea extract/
l diet was also prepared. Green tea, Camellia sinensis (Lin-
naeus) O. Kuntze (standard research blendsdlyophilized
extract), was provided by TJ Lipton (Englewood Cliffs,
NJ). Green tea extract contained epigallocatechin gallate
(554 mg/g dried extract), epigallocatechin (82 mg/g dried
extract), epicatechin (90 mg/g dried extract), epicatechin
gallate (86 mg/g dried extract), and coffeic acid (10 mg/g
dried extract), which have been determined by HPLC
(Maiani et al., 1997).
The animals from each age group were divided into the
following groups:
Control group was fed for 5 weeks on a control
Lieber DeCarli liquid diet (n 5 6).
Green tea group was fed for 5 weeks on a control
liquid Lieber DeCarli diet containing green tea
(7 g/l) (n 5 6).
Ethanol group was fed for 1 week on a control liq-
uid Lieber DeCarli diet and for the next 4 weeks on
ethanol Lieber DeCarli liquid diet (n 5 6).
Ethanol and green tea groups were fed for 1 week
on a control Lieber DeCarli liquid diet containing
green tea (7 g/l) and for the next 4 weeks with eth-
anol Lieber DeCarli liquid diet also containing
green tea (7 g/l) (n 5 6).
90 E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

3. 2.2. Preparation of tissues
After 5 weeks of experiment all rats were sacrificed un-
der ether anesthesia (six animals in each group). Brain was
removed quickly and placed in iced 0.15 M NaCl solution,
perfused with the same solution to remove blood cells, blot-
ted on filter paper, weighed, diluted into 10% w/v in ice-
cold 0.15 M NaCl or 0.25 M sucrose containing 167 mM
butylated hydroxytoluene in ethanol to prevent the forma-
tion of new peroxides during the assay. Homogenization
procedure was performed under standardized conditions.
Glass homogenizator with 1,500 rotatory speed of piston/
min was used and three shifts downwards and upwards
were done. Homogenates were centrifuged at 10,000 3 g
for 15 min at 4
C. The supernatants were used for bio-
chemical analysis.
To assay protein oxidation, brain samples were homog-
enized in 5 mM phosphate buffer (pH 7.5) with protease in-
hibitors (leupeptin 0.5 mg/ml, aproteinin 0.5 mg/ml, and
pepstatin 0.7 mg/ml) and 0.1% Triton X-100. The homoge-
nate was centrifuged at 7,800 3 g for 20 min, and the bio-
chemical analysis of the supernatant was performed.
2.3. Biochemical assays
Superoxide dismutase (Cu,Zn-SODdEC.1.15.1.1) ac-
tivity was determined by the method of Misra and Frido-
vich (1972) as modified by Sykes et al. (1978), and this
measures the activity of cytosolic SOD. Mn-SOD of the
brain mitochondria is known to be destroyed during this
procedure. A standard curve for SOD activity was con-
structed using SOD from bovine erythrocytes (Sigma Bio-
chemicals, St. Louis, MO). One unit of SOD was defined
as the amount of the enzyme inhibiting epinephrine oxida-
tion to adrenochrome by 50%. The enzyme specific activity
was expressed in units per milligram of protein.
Catalase (CATdEC.1.11.1.9) activity was determined
after a 30-min preincubation of the postmitochondrial frac-
tion of the brain homogenate with 1% Triton X-100 by
measuring the decrease in absorbance of hydrogen peroxide
at 240 nm (Aebi, 1984). The rates were determined at 25
C
using 10 mM hydrogen peroxide, and the activity was ex-
pressed in units per milligram of protein. One unit of
CAT was defined as the amount of the enzyme required
to catalyze 1 mmol H2O2 during 1 min. The enzyme specific
activity was expressed in units per milligram of protein.
Glutathione peroxidase (GSH–PxdEC.1.11.1.6) activ-
ity was assessed in the brain spectrophotometrically using
a technique based on Paglia and Valentine (1967). Applying
this technique, GSH formation was assayed by measuring
the conversion of nicotinamide adenine dinucleotide phos-
phate (NADPH) to NADP. The final concentration of GSH
was 0.2 mM and of H2O2 was 0.3 mM. The activity was ex-
pressed in universal units. One unit of activity was defined
as the amount of enzyme catalyzing the oxidation of
1 mmol of NADPH/min at 25
C and pH 7.4. The enzyme
specific activity was expressed in units per milligram of
protein.
Glutathione reductase (GSSG–RdEC.1.6.4.2) activity
was measured by the method of Mize and Langdon
(1962) by monitoring the oxidation of NADPH at
340 nm. The reaction mixture contained 0.2 mM KCl,
1 mM EDTA, and 1 mM oxidized GSH (GSSG) in 0.1 M
potassium phosphate buffer, pH 7.1. The enzyme activity
was expressed in units per milligram of protein. One unit
of GSSG-R oxidized 1 mmol of NADPH/min at 25
C
and pH 7.4. The enzyme specific activity was expressed
in units per milligram of protein.
Total glutathione was determined by HPLC technique
(Cereser et al., 2001). This measurement was performed by
reducing the disulfide group of oxidized glutathione with di-
thiothreitol. The main step of total glutathione determination
is based on the derivatization of GSH with o-phthalaldehyde,
which reacts with both the sulfhydryl and the primary amino
group of glutathione to form a highly fluorescent product.
Oxidized glutathione was measured by HPLC after elim-
ination of GSH with N-ethylmaleimide followed by reduc-
tion of disulfides with dithiothreitol and derivatization with
o-phthalaldehyde (Neuschwander-Tetri Roll, 1989).
The GSH concentration was obtained by subtraction of
the value of GSSG from the total glutathione value.
Selenium was determined with graphite furnace atomic
absorption spectrometry (Model Z-5000, Hitachi) (Hansson
et al., 1989).
HPLC methods were used to determine the levels of vi-
tamin C (Ivanovic et al., 1999), vitamins A and E (De
Leenher et al., 1979), and b-carotene (Elinder Walldius,
1992). For ascorbic acid determination, 300 ml of brain ho-
mogenate was mixed with an equal volume of metaphos-
phoric acid (100 g/l). Before analysis, the samples were
centrifuged (3,500 3 g, 4 min) to remove precipitated pro-
tein after which they were immediately assayed. The vita-
mins A and E were extracted from brain homogenate
with hexane containing 0.025% butylated hydroxytoluene.
The hexane phase was removed and dried with sodium sul-
fate, and 50 ml of the hexane extract was injected on the
column.
Lipid peroxidation was estimated by measuring lipid hy-
droperoxides (LOOH) (Tokumaru et al., 1995), malondial-
dehyde (Londero Greco, 1996), and 4-hydroxynonenal
(4–HNE) (Yoshino et al., 1986). Lipid hydroperoxides were
determined as an oxide 1-naephtylodiphenylophosphine
formed in reaction with 1-naephtylodiphenylophosphine
and determined by HPLC with DAD detector at 292 nm.
Malondialdehyde was determined as malondialdehyde–
thiobarbituric acid adducts separated by HPLC with spec-
trofluorometric quantification at 532 nm excitation and
553 nm emission. 4-Hydroxynonenal was determined as
one of the fluorescent decahydroacridine derivatives formed
in reaction with 1,3-cyclohexanodione. It was determined
by HPLC with fluorescence detector with excitation at
380 nm and emission at 445 nm.
91E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

4. Oxidative modifications of proteins were determined by
examination of bistyrosine and carbonyl groups. Bistyrosine
was spectrofluorometrically determined (Prutz et al., 1983).
Signal intensity was calibrated against 0.1 mg/ml quinine
sulfate solution in sulfuric acid the fluorescence of which
was assumed as a unit. Fluorescence emission at 420 nm
(325 nm excitation) was used as a reflection of bistyrosine
content. Carbonyl groups were determined using 2,4-dinitro-
phenylhydrazine (Levine et al., 1990). Homogenate proteins
were precipitated with 20% trichloroacetic acid and centri-
fuged at 11,000 3 g. The pellet was then used for the 2,4-di-
nitrophenylhydrazine assay.
Protein in brain homogenates was determined by the
method of Lowry et al. (1951).
Epicatechin, epigallocatechin, and epigallocatechin gal-
late in brain homogenates were determined as free forms
generated in reaction between their conjugated forms and
b-glucuronidase and sulfatase. Free forms of catechins
were determined by HPLC with amperometric detection
at 254 mV (Lee et al., 1995).
2.4. Statistical analysis
Data obtained in the current study are expressed as
mean 6 S.D. These data were analyzed by using standard
statistical analyses, one-way analysis of variance with Tu-
key test for multiple comparisons, to determine significant
differences between different groups. The data were ana-
lyzed separately for treatment and age groups. A p value
of !0.05 was considered significant.
3. Results
Changes in the activity of the antioxidant enzymes in the
brain of aged rats, which received green tea, ethanol, and
ethanol with tea, are shown in Table 1. Cu,Zn-SOD activity
in the brain of healthy rats clearly decreased with age from
74.6 U/mg protein in 2-month-old rats to 52.6 U/mg protein
in 12-month-old rats and to 39.8 U/mg protein in 24-month-
old rats. The differences in the activity of Cu,Zn-SOD in the
brain between age groups were statistically significant
( p ! 0.05). In animals treated with green tea, the activity
of this enzyme decreased with age as well. Chronic ethanol
ingestion caused a significant decrease in Cu,Zn-SOD activ-
ity in young as well as in aged rats. The activity values
changed from 26.9 U/mg protein in 2-month-old rats,
through 23.9 U/mg protein in 12-month-old rats to 15.4 U/
mg protein in 24-month-old rats. However, in 2- and 12-
month-old rats given ethanol with green tea, a significant in-
crease in Cu,Zn-SOD activity was observed in comparison
with those given green tea as well as the control diet.
CAT activities in the rats’ brains showed markedly dif-
ferential pattern. In the healthy rats’ brains this enzyme ac-
tivity amounted to 0.36 U/mg protein in 2-month-old rats,
0.47 U/mg protein in 12-month-old rats, and 0.41 U/mg
protein in 24-month-old rats. After alcohol intake, CAT
activity was decreased to 0.29, 0.39, and 0.30 U/mg protein,
respectively, in each of the age groups. After administration
of green tea and ethanol, CAT activity was similar to that in
the control groups in the 2- and 12-month-old rats and
slightly decreased to that in the 24-month-old rats com-
pared with the green tea group where this activity was
diminished.
Direction and intensity of changes in the activity of
GSH-Px were similar to changes in Cu,Zn-SOD activity
in the control groups. The brains from elderly control rats
showed significantly lower GSH-Px activity than the brains
from younger rats and the activity dropped to 13.8 U/mg
protein in comparison with 16.1 U/mg protein and
17.4 U/mg protein (for 12- and 24-month-old rats). Green
tea given to 2- and 12-month-old rats caused significant de-
crease in GSH-Px activity whereas it did not cause change
in the activity of this enzyme when given to 24-month-old
rats in comparison with control group. Significant decrease
in the activity of the examined enzyme in all age groups
was due to ethanol ingestion. Green tea given with ethanol
did not cause significant changes in GSH-Px activity in
comparison either with green tea or ethanol.
GSSG-R activity was the lowest in the brain of 2-month-
old rats and amounted to 2.25 U/mg protein but increased
with age to the highest value of 2.91 U/mg protein in the
brain of 24-month-old rats. Green tea caused significant in-
crease in the activity of this enzyme only in 12-month-old
rats, while ethanol intoxication caused significant increase
in young rats and a decrease in older ones. The activity
of GSSG-R in all study groups given ethanol with green
tea was decreased in comparison with the activity of the
group given green tea.
Changes in nonenzymatic antioxidative parameters in
the brain of aged rats are shown in Table 2. Ethanol caused
gradual decrease in the reduced GSH level and it was the
lowest in the brain of 24-month-old rats and amounted to
0.63 mmol/g tissue. Green tea given with ethanol restored
GSH level totally in 2- and 12-month-old rats, compared
to control and green tea groups. In 24-month-old rat groups,
green tea only partially protected GSH against ethanol
action.
The level of oxidized glutathione increased with the age
and amounted to 0.11 mmol/g tissue in 2-month-old rats and
0.15 mmol/g tissue in 24-month-old rats. The differences
between these groups were statistically significant. Ethanol
intake gradually raised GSSG level in all age groups to
reach its highest level of 0.27 mmol/g tissue in 24-month-
old rats. Green tea given with ethanol prevented changes
in oxidized glutathione level, compared to control and
green tea.
Total glutathione level in the brain of rats decreased with
age and was the least in 24-month-old rats. Ethanol admin-
istration similarly caused a decrease in this parameter.
Green tea given alone did not alter the total glutathione
level, while green tea given with ethanol to 2-month-old
rats, prevented changes observed after ethanol intoxication.
92 E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

5. Selenium level was the highest in the brain of 2-month-
old rats and amounted to 208 ng/g tissue. The total level of
this metal was gradually decreased in the brain of 12- and
24-month-old rats and it amounted to 201 and 189 ng/g tis-
sue, respectively. The values of this parameter increased
slightly after green tea ingestion, and significantly de-
creased after ingestion of ethanol. Green tea given with
ethanol only partially protected selenium brain level in
comparison with green tea given alone.
The levels of vitamins in the brain of rats decreased with
age. This was particularly evident in 24-month-old rats.
Ethanol ingestion additionally reduced the level of these
parameters, especially the level of vitamin A that was
decreased by about 37% in comparison to that found in
the brain of 24-month-old rats in the control group. Green
tea given with ethanol totally restored the contents of vita-
mins C and E only in 2-month-old rats, but partially re-
stored in 12- and 24-month-old rats.
The concentration of b-carotene in the brain decreased
gradually with age. Moreover, green tea also caused a de-
crease in this antioxidant level in 2- and 12-month-old rats’
brains. After ethanol ingestion, b-carotene concentration
decreased in all examined groups. Green tea given with
ethanol caused an increase in the content of b-carotene in
comparison with ethanol group but did not restore its level
totally in comparison to the control as well as to green tea
group.
The levels of lipid peroxidation products in the brain of
aged rats receiving ethanol and green tea are presented in
Table 3. The brain levels of the first lipid peroxidation prod-
ucts, lipid hydroperoxides (LOOH), and final lipid peroxi-
dation products, malondialdehyde and 4-HNE, increased
with age in healthy rats. The differences are statistically sig-
nificant when p ! 0.05. Green tea significantly decreased
the levels of the determined compounds, while ethanol in-
gestion caused significant increase in the level of these pa-
rameters. The level of LOOH increased higher than
twofold in comparison with control group. Green tea given
with ethanol only partly protected lipid against peroxidation
caused by ethanol, and the brain levels of lipid peroxidation
products were slightly lower than in ethanol groups but con-
tinually slightly higher than in green tea groups.
The levels of protein oxidation products in the brain of
control rats increased with age (Table 4). In the brain of
the control group, the levels of protein carbonyl groups
and bistyrosine were the highest in 24-month-old rats (in-
creased by 36% and 25%, respectively, in comparison with
2-month-old rats). The contents of carbonyl groups and bis-
tyrosine in the brain were not altered by green tea alone in
comparison with those in the brain of rats fed the control
diet. Ethanol ingestion caused a statistically significant in-
crease in the level of those markers independently of age
compared to control group. Green tea given with ethanol
significantly reduced the levels of carbonyl groups and bis-
tyrosine in all age groups in comparison with ethanol
groups.
The levels of catechins in the brain of aged rats receiving
green tea are presented in Table 5. The brain levels of epi-
catechin, epigallocatechin, and epigallocatechin gallate de-
creased with age in healthy rats. The level of epicatechin in
the brain of 24-month-old rats was the lowest (decreased by
20% in comparison to 2-month-old rats). Decrease in the
level of the examined catechins in all age groups, especially
in the brain of 24-month-old animals, where the levels of
Table 1
Activity of antioxidant enzymes [superoxide dismutase (Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase
(GSSG-R)] in the brain of rats of different ages receiving control or ethanol Lieber de Carli liquid diet with or without green tea
Enzyme Age of rats
Group of rats
Control Green tea Ethanol Ethanol 1 green tea
Cu,Zn-SOD (U/mg protein) 2 months 74.6 6 6.8 51.5 6 3.9* 26.9 6 2.7* 63.8 6 4.6* ** ***
12 months 52.6 6 5.4#
45.1 6 4.2* #
23.9 6 2.6* 57.1 6 5.3** *** #
24 months 39.8 6 4.1# ##
35.7 6 3.2# ##
15.4 6 1.6* # ##
33.6 6 3.1* *** # ##
CAT (U/mg protein) 2 months 0.36 6 0.02 0.41 6 0.03* 0.29 6 0.03* 0.37 6 0.04***
12 months 0.47 6 0.03#
0.59 6 0.05* #
0.39 6 0.04* #
0.49 6 0.04** *** #
24 months 0.41 6 0.03# ##
0.45 6 0.03* # ##
0.30 6 0.04* ##
0.35 6 0.04* ** ##
GSH-Px (U/mg protein) 2 months 17.4 6 1.1 11.2 6 0.8* 14.7 6 1.1* 13.5 6 1.1* **
12 months 16.1 6 1.2 13.2 6 1.0* #
13.6 6 1.1* 13.5 6 1.1*
24 months 13.8 6 1.0# ##
14.7 6 1.0# ##
10.9 6 0.9* # ##
12.1 6 1.1* **
GSSG-R (U/mg protein) 2 months 2.25 6 0.16 2.41 6 0.17 2.64 6 0.21* 2.30 6 0.18***
12 months 2.57 6 0.17#
2.96 6 0.20* #
2.49 6 0.20 2.63 6 0.20** #
24 months 2.91 6 0.22# ##
2.98 6 0.21#
2.45 6 0.21* 2.74 6 0.21*** #
Control group was fed a control Lieber de Carli liquid diet for 5 weeks; green tea group was fed a control Lieber de Carli liquid diet containing green tea
(7 g/l) for 5 weeks; ethanol group was fed a control Lieber de Carli liquid diet for 1 week, followed by a Lieber de Carli liquid diet containing ethanol for the
next 4 weeks; ethanol 1 green tea group was fed a control Lieber de Carli liquid diet containing green tea (7 g/l) for 1 week, followed by a Lieber de Carli
liquid diet containing ethanol as well as green tea (7 g/l) for the next 4 weeks. Data points represent mean 6 S.D., n 5 6 (*p ! 0.05 in comparison with
values for control group; **p ! 0.05 in comparison with values for green tea group; ***p ! 0.05 in comparison with values for ethanol group;
#
p ! 0.05 in comparison with values for the 2 months group; ##
p ! 0.05 in comparison with values for the 12 months group.
93E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

6. epicatechin, epigallocatechin, and epigallocatechin gallate
were decreased by 15%, 16%, and 12%, respectively, in
comparison with 2-month-old rats was due to ethanol
ingestion.
4. Discussion
Ethanol-induced oxidative stress in the rat brain affects
a variety of biochemical processes involved in the regula-
tion of the defense system, that is, antioxidant system. It
is well known that Cu,Zn-SOD is the principal enzyme
by which the nervous tissue defends itself from reactive
oxygen metabolites (Ledig et al., 1980). This high activity
has to be maintained mainly for the normal brain function,
especially when the protection of the preferentially vulner-
able brain neurons is imperiled. After ethanol ingestion
Cu,Zn-SOD activity is statistically significantly decreased
exacerbating neuronal cell damage. This endangerment
consistently correlates with disruption of energy pathways
and low energy availability (Somani et al., 1996).
Table 3
Concentrations of lipid peroxidation products [lipid hydroperoxides (LOOH), malondialdehyde (MDA), and 4-hydroxynonenal (4–HNE)] in
the brain of rats of different ages receiving control or ethanol Lieber de Carli liquid diet with or without green tea
Analyzed parameter Age of rats
Group of rats
Control Green tea Ethanol Green tea 1 ethanol
LOOH (mmol/g tissue) 2 months 103 6 6 82 6 5* 231 6 9* 115 6 7* ** ***
12 months 109 6 7 96 6 8* #
249 6 13* #
137 6 11* ** *** #
24 months 132 6 10# ##
121 6 10# ##
311 6 15* # ##
217 6 12* ** *** # ##
MDA (nmol/g tissue) 2 months 31.0 6 1.9 15.7 6 1.1* 40.6 6 1.9* 27 6 1.7* ** ***
12 months 38.2 6 2.2#
25.2 6 1.8* #
51.2 6 2.7* #
35.2 6 2.5** *** #
24 months 50.1 6 2.4# ##
46.1 6 2.3* # ##
69.1 6 3.3* # ##
54.3 6 3.5* ** *** # ##
4-HNE (nmol/g tissue) 2 months 4.03 6 0.23 3.11 6 0.21* 5.19 6 0.31* 4.23 6 0.28** ***
12 months 7.72 6 0.45#
6.50 6 0.44* #
9.34 6 0.52* #
8.19 6 0.28** *** #
24 months 11.27 6 0.63# ##
10.89 6 0.59# ##
16.29 6 0.91* # ##
12.75 6 0.71* ** *** # ##
Explanations are given in the legend to Table 1.
Table 2
Concentrations of nonenzymatic antioxidative parameters [reduced glutathione (GSH), oxidized glutathione (GSSG), total glutathione, selenium,
vitamins C, E, and A, and b-carotene] in the brain of rats of different ages receiving control or ethanol Lieber de Carli liquid diet
with or without green tea
Analyzed parameter Age of rats
Group of rats
Control Green tea Ethanol Green tea 1 ethanol
GSH (mmol/g tissue) 2 months 0.89 6 0.06 0.91 6 0.06 0.71 6 0.06* 0.89 6 0.06***
12 months 0.85 6 0.06 0.87 6 0.06 0.67 6 0.06* 0.81 6 0.07***
24 months 0.80 6 0.07#
0.81 6 0.06#
0.63 6 0.07* 0.71 6 0.07** # ##
GSSG (mmol/g tissue) 2 months 0.11 6 0.01 0.11 6 0.01 0.21 6 0.02* 0.13 6 0.01* ** ***
12 months 0.12 6 0.01 0.10 6 0.01* 0.24 6 0.02* #
0.16 6 0.01* ** *** #
24 months 0.15 6 0.01# ##
0.12 6 0.01* ##
0.27 6 0.03* #
0.20 6 0.02* ** *** # ##
Total glutathione (mmol/g tissue) 2 months 1.00 6 0.07 1.02 6 0.06 0.92 6 0.07 1.02 6 0.07***
12 months 0.97 6 0.06 0.97 6 0.07 0.91 6 0.07 0.97 6 0.07
24 months 0.95 6 0.07 0.93 6 0.07* #
0.90 6 0.08 0.91 6 0.08#
Selenium (ng/g tissue) 2 months 208 6 12 216 6 12 189 6 13* 196 6 13**
12 months 201 6 13 221 6 14* 187 6 13 199 6 15**
24 months 189 6 12#
202 6 14##
167 6 14* # ##
182 6 14**
Vitamin C (mmol/g tissue) 2 months 1.98 6 0.07 2.08 6 0.09 1.76 6 0.09* 1.99 6 0.09***
12 months 1.92 6 0.08 1.90 6 0.08#
1.70 6 0.09* 1.83 6 0.09*** #
24 months 1.73 6 0.08# ##
1.60 6 0.07* # ##
1.31 6 0.07* # ##
1.43 6 0.07* ** *** # ##
Vitamin E (nmol/g tissue) 2 months 27.3 6 1.1 26.8 6 0.9 21.1 6 1.0* 27.0 6 1.1***
12 months 25.4 6 0.9#
27.3 6 1.0* 18.4 6 0.9* #
24.9 6 1.0** *** #
24 months 21.6 6 1.0# ##
21.1 6 1.0# ##
16.0 6 0.9* # ##
18.5 6 1.0* ** *** # ##
Vitamin A (nmol/g tissue) 2 months 0.35 6 0.06 0.33 6 0.05 0.24 6 0.05* 0.29 6 0.06
12 months 0.32 6 0.07 0.34 6 0.08 0.20 6 0.08* 0.29 6 0.09
24 months 0.27 6 0.06#
0.24 6 0.05# ##
0.17 6 0.05* #
0.19 6 0.05* # ##
b-Carotene (nmol/g tissue) 2 months 1.83 6 0.05 1.75 6 0.04* 1.42 6 0.04* 1.67 6 0.05* ** ***
12 months 1.80 6 0.04 1.67 6 0.03* #
1.37 6 0.04* 1.56 6 0.05* ** *** #
24 months 1.57 6 0.08# ##
1.63 6 0.07#
1.32 6 0.06* #
1.45 6 0.07* ** *** # ##
Explanations are given in the legend to Table 1.
94 E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

7. Generated as a result of Cu,Zn-SOD action hydrogen per-
oxide is removed by GSH-Px and CAT that have common
catalytic activity. However, GSH-Px in the brain has low
basal activity, and the protection of cells is much more ef-
fective when CAT and GSH-Px act together (Michiels et al.,
1994). GSH-Px is a selenoenzyme that catalyses detoxifica-
tions of peroxides including lipid peroxides. Its expression
therefore depends on the availability of selenium com-
pounds capable of acting as precursors of selenoprotein bio-
synthesis. Ethanol intoxication leads to the statistically
significant decrease in the selenium content, and the de-
crease in GSH-Px activity has been observed in all age
groups of rats. It should be emphasized that selenium defi-
ciency in rat brain may increase susceptibility to oxidative
damage, particularly to glutamate-induced excitotoxicity.
Alterations of the brain antioxidant enzyme activities
differ between enzymes with aging. The activities of
Cu,Zn-SOD and GSH-Px are decreased during aging
indicating less probable removal of the radicals. In such
a situation, the increase in the activity of GSSG-R, which
controls the endogenous level of GSH–GSH-Px coenzyme,
does not improve antioxidant activity of this enzyme. A
small but significant increase in GSSG-R activity in the
rat brain with aging has also been reported by other authors
(Sohal et al., 1990). This increase in GSSG-R activity in
the rat brain may be explained by the adaptation response
of cells to the oxidative stress enhanced with age and
caused by influencing a small number of regulatory pro-
teinsdtranscription factors, NF-kBdcentral to stress and
immune responses. Transcription factors are regulated in
vivo by dietary factors (Pahlavani et al., 1997; Storz
et al., 1990). Moreover, it was shown that oxidants may ac-
tivate gene expression through the antioxidant responsive
elements via electrophilic thiol modification (Rushmore
et al., 1991), and thus it might be speculated that the over-
expression in the enzyme proteins took place in in vivo
conditions. Increase in GSSG-R activity due to chronic eth-
anol intoxication is likely to result from the tendency of the
cells to maintain proper GSH level or is the adaptative re-
sponse to reduced NADPH level, which accompanies etha-
nol intoxication (Somani et al., 1996). It should be
emphasized that to function properly GSSG-R enzyme
needs adequate ratio of NADP1
:NADPH and the decrease
of this ratio in NADP1
favor causes inefficient elimination
of excess peroxides due to oxidative stress despite enzyme
high activity (Somani et al., 1996). In such a situation,
GSSG-R overexpression is negatively correlated with the
lower efficiency of another antioxidative parameter, GSH.
At the same time, GSH level decreases significantly lead-
ing to enhanced lipid peroxidation in the brain. It is also
Table 4
Concentrations of protein oxidation products (carbonyl groups and bistyrosine) in the brain of rats of different ages receiving control
or ethanol Lieber de Carli liquid diet with or without green tea
Analyzed parameter Age of rats
Group of rats
Control Green tea Ethanol Green tea 1 ethanol
Carbonyl group (nmol/mg protein) 2 months 2.05 6 0.13 2.11 6 0.12 2.65 6 0.19* 2.24 6 0.19***
12 months 2.23 6 0.12#
2.07 6 0.14 3.15 6 0.25* #
2.47 6 0.22* ** ***
24 months 2.98 6 0.23# ##
2.92 6 0.17# ##
4.51 6 0.37* # ##
3.94 6 0.32* ** *** # ##
Bistyrosine (nmol/mg protein) 2 months 0.75 6 0.04 0.71 6 0.05 0.92 6 0.07* 0.84 6 0.06* **
12 months 0.73 6 0.04 0.78 6 0.04#
1.03 6 0.07* #
0.90 6 0.08* ** ***
24 months 0.98 6 0.06# ##
0.91 6 0.06# ##
2.09 6 0.17* # ##
1.68 6 0.13* ** *** # ##
Explanations are given in the legend to Table 1.
Table 5
Concentrations of epicatechin (EC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG) in the brain of rats of different
ages receiving control or ethanol Lieber de Carli liquid diet with or without green tea
Catechin Age of rats
Group of rats
Control Green tea Ethanol Green tea 1 ethanol
EC (ng/g tissue) 2 months ND 8.1 6 0.6 ND 7.7 6 0.7
12 months ND 7.6 6 0.4 ND 6.9 6 0.6**
24 months ND 6.4 6 0.5 ND 5.4 6 0.6** # ##
EGC (ng/g tissue) 2 months ND 75 6 6 ND 67 6 5**
12 months ND 73 6 6 ND 61 6 3** #
24 months ND 68 6 4 ND 57 6 4** #
EGCG (ng/g tissue) 2 months ND 82 6 6 ND 77 6 4
12 months ND 76 6 7 ND 70 6 6#
24 months ND 78 6 7 ND 68 6 6** #
ND, not detectable.
Explanations are given in the legend to Table 1.
95E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

8. possible that the decreased GSH level after ethanol admin-
istration is caused by a decrease in its synthesis (Liu
Choi, 2000).
Green tea has been implicated as a regulatory factor for
antioxidant enzymes. This is possible due to the content of
polyphenols that are characterized by their ability to scav-
enge free radicals produced during the aging process as
well as ethanol metabolism. Efficacy of their activity in
other tissues such as the liver and blood has already been
demonstrated (Dobrzynska et al., 2005; Luczaj et al.,
2004; Ostrowska et al., 2004). These antioxidants have
demonstrated neuroprotection in tea action with glutami-
nergic challenges (Dugan Choi, 1994; Komatsu Hira-
matsu, 2000) and they may affect oxidative breakdown of
tissue during neurodegenerative diseases like Alzheimer’s
(McIntosh et al., 1997).
During ethanol-induced oxidative stress, the diminution
in vitamin C level, particularly in the brain of 24-month-old
rats, has been observed. Vitamin C appears to be a particu-
larly important antioxidant because it is not synthesized in
the brain cells but must be transported from plasma, distrib-
uted, and accumulated in brain cells (Tsukaguchu et al.,
1999). It determines the first line of antioxidant defense
and effectively protects the phospholipids against detect-
able peroxidative damage, even in the presence of free,
redox-active iron (Berger et al., 1997). Its concentration
in the brain is the highest in any tissue and together with
vitamin E appears to have a protective role against brain
lipid peroxidation. The diminished plasma level of vitamin
C after oxidative stress correlates with the decreased cere-
brospinal fluid level and with rat brain damage (unpub-
lished data). The restored level of this vitamin revealed in
the present study together with vitamin E in the gray and
white matter of the brain of 2- and 12-month-old rats that
were administered green tea with ethanol could protect
against oxidative damage induced by ethanol alone. High
level of reactive oxygen metabolites formed during ethanol
metabolism also depletes other cellular nonenzymatic lipid-
soluble antioxidants such as vitamins A and E and b-caro-
tene. Such a depletion of low-molecular weight antioxi-
dants can cause neurological damage in the brain (Calvin
et al., 1986). Vitamin E is an important chain-breaking an-
tioxidant in membranes, which prevents oxidative damage
to polyunsaturated lipids in the nervous system. Green tea
normalizes the content of vitamins that convert reactive
oxygen metabolites into stable components, before they in-
flict major damage to cellular macromolecules. It helps in
antioxidative capacity of the brain tissue and as a conse-
quence partly lowers the level of lipid peroxidation prod-
ucts, very deteriorative for the brain cells. Lipid peroxides
and hydroperoxides within the membrane formed as a result
of lipid decomposition have a devastating effect on the
functional state of the brain cell membranes because they
alter their fluidity. In addition, low-molecular aldehydes
formed during lipid peroxidation cause modifications of
proteins resulting in the formation of carbonyl-modified
and nitrated neurofilament proteins, which are active fac-
tors such as cytotoxic, atherogenic, mutagenic, carcino-
genic, or enzyme inhibitory substances (Rottkamp et al.,
2000; Smith Johnson, 1989). The initial reports did not
reveal ethanol-induced increase in peroxide level of rat or-
gan homogenates (Comporti et al., 1967). Later, however, it
was shown that ethanol administration could induce lipid
peroxidation in neuron tissue in experimental animals
(Nordman et al., 1987; Rouach et al., 1997). In our studies,
we have found an increase in lipid hydroperoxides, malon-
dialdehyde, and 4-hydroxynonenal as well as protein oxida-
tion products. The most abundant polyphenols such as
epigallocatechin gallate and epicatechin gallate contained
in green tea scavenge a wide range of free radicals includ-
ing the most active hydroxyl radical, which may initiate
lipid and protein oxidative modifications, therefore chemi-
cal structure of catechins is crucial to their antioxidant ef-
fect. They may decrease the formation and concentration
of lipid free radicals and terminate the initiation and propa-
gation of lipid peroxidation. They also decrease the level of
bistyrosine and may thus affect the functioning of membrane
bound enzymes, neurotransmitter receptor systems, and ion
channels. Catechins may chelate metal ions especially iron
and copper, which, in turn, inhibit the generation of hydroxyl
radicals and degradation of lipid hydroperoxides that cause
formation of more reactive aldehydes. Furthermore, the
green tea polyphenols have been demonstrated to inhibit
iron-induced oxidation of synaptosomes by scavenging hy-
droxyl 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).
The decreasing amounts of lipid peroxidation products after
administration of green tea with ethanol were accompanied
by a concomitant increase in the activities of antioxidant
defense enzymes, for example, superoxide dismutase and
catalase, and partially in nonenzymatic low-molecular anti-
oxidants, glutathione, vitamins A, E, and C, and b-carotene.
The combination of green tea with ethanol, despite the slight
beneficial effect of green tea and the adverse effect of ethanol
alone, resulted in a reduction of oxidative potential in brain
cells especially of young and partially of aging rats. The in
vivo evidence presented here clearly suggests the necessity
of therapeutic intervention to enhance antioxidant capacity
of brain cells and modulation of the microglial function,
which may bring about some favorable changes in the aging
processes.
In the light of these conclusions, we can suggest that com-
ponents of green tea are partially efficacious in preventing
disturbances of antioxidant defense system in the brain of
rats. They may reduce neurodegeneration induced by etha-
nol ingestion and promote healthy aging. These beneficial
effects of green tea can result from the inhibition of free rad-
ical chain reactions generated during oxidative stress caused
by ethanol and from an increase in antioxidant enzyme ca-
pacity. It is also likely that polyphenol metabolites may act
favorably by exerting effects on specific signaling pathway.
96 E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

9. Acknowledgments
This investigation was supported by a grant from the
Polish Committee of Scientific Research No. 6P05F01720.
References
Adachi, Y. U., Watanabe, K., Higuchi, H., Satoh, T., Vizi, E. S. (2001).
Oxygen inhalation enhances striatal dopamine metabolism and mono-
amineoxidase enzyme inhibition prevents it: a microdialysis study. Eur
J Pharmacol 422, 61–68.
Aebi, H. (1984). Catalase in vitro. Methods Enzymol 105, 121–127.
Berger, T. M., Polidori, M. C., Morrow, J., Evans, P. J., Halliwell, B.,
Morrow, J. D., Roberts, L. J., Frei, B. (1997). Antioxidant activity
of vitamin C in iron-overloaded human plasma. J Biol Chem 272,
15656–15660.
Cakatay, U., Telci, A., Kayali, R., Tekili, F., Akcay, T., Sivas, A. (2001).
Relation of oxidative protein damage and nitrotyrosine levels in the
aging rat brain. Exp Gerontol 36, 221–229.
Calvin, H. T., Medvedovsky, C., Worgul, B. V. (1986). Near-total gluta-
thione depletion and age-specific cataracts induced by buthionine
sulfoximine in mice. Science 233, 553–555.
Cereser, C., Guichard, J., Drai, J., Bannier, E., Garcia, I., Boget, S.,
Parvaz, R., Revol, A. (2001). Quantification of reduced and total glu-
tathione at the femtomole level by high-performance liquid chromatog-
raphy with fluorescence detection: application to red blood cells and
cultured fibroblasts. J Chromatogr B 752, 123–132.
Comporti, M., Hartman, A., Di Luzio, N. R. (1967). Effect of in vivo
and in vitro ethanol administration on liver lipid peroxidation. Lab
Invest 16, 616–624.
De Leenher, A., De Bevere, V., De Rutyer, M. G., Claeys, A. C. (1979).
Simultaneous determination of retinol and a-tocopherol in human
serum by HPLC. J Chromatogr A 162, 408–413.
Dobrzynska, I., Szachowicz-Petelska, B., Ostrowska, J., Skrzydlewska, E.,
Figaszewski, Z. (2005). Protective effect of green tea on erythrocyte
membrane of different age rats intoxicated with ethanol. Chem Biol
Interact 156, 41–53.
Dugan, L., Choi, D. (1994). Excitotoxicity, free radicals and cell mem-
brane changes. Ann Neurol 35, 17–21.
Elinder, L. S., Walldius, G. (1992). Simultaneous measurement of serum
probucol and lipid soluble antioxidant. J Lipid Res 33, 131–139.
Gerlach, M., Ben-Shachar, D., Riederer, P., Youdim, M. B. H. (1994).
Altered brain metabolism of iron as a cause of neurodegenerative
diseases? J Neurochem 63, 793–807.
Graham, H. N. (1992). Green tea composition, consumption and polyphe-
nols chemistry. Prev Med 21, 334–350.
Guo, Q., Zhao, B., Li, M., Shen, S., Xin, W. (1996). Studies on protec-
tive mechanisms of four components of green tea polyphenols against
lipid peroxidation in synaptosomes. Biochim Biophys Acta 1304,
210–222.
Halliwell, B. (1992). Reactive oxygen species and the central nervous sys-
tem. J Neurochem 59, 1609–1623.
Halliwell, B. (2001). Role of free radicals in the neurodegenerative dis-
eases. Therapeutic implication for antioxidative treatment. Drug Aging
18, 685–716.
Hansson, L., Petterson, J., Eriksson, L., Olin, A. (1989). Atomic absorp-
tion spectrometric determination of selenium in human blood compo-
nents. Clin Chem 35, 537–540.
Harman, D.(1998).Extendingfunctionallifespan.ExpGerontol33, 95–112.
Ivanovic, D., Popovic, A., Radulovic, D., Medenica, M. (1999).
Reversed phase ion-pair HPLC determination of some water-soluble
vitamins in pharmaceuticals. J Pharm Biomed Anal 18, 999–1004.
Jazwinski, S. M. (1998). Genetics of aging. Exp Gerontol 53, 778–783.
Kato, S., Kawase, T., Alderman, J., Inatori, N., Lieber, C. S. (1990).
Role of xanthine oxidase in ethanol-induced lipid peroxidation.
Gastroenterology 98, 203–210.
Komatsu, M., Hiramatsu, M. (2000). The efficacy of an antioxidant
cocktail on lipid peroxide level and superoxide dismutase activity in
aged rat brain and DNA damage in iron-induced epileptogenic foci.
Toxicology 148, 143–148.
Lee, M. J., Wang, Z. Y., Li, H., Chen, L., Sun, Y., Gobbo, S.,
Balentine, D. A., Yang, C. S. (1995). Analysis of plasma and urinary
tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev
4, 393–399.
Ledig, M., M’ Paria, J. R., Louis, J. C., Freid, R., Mandel, P. (1980).
Effect of ethanol on superoxide dismutase activity in cultured neural
cells. Neurochem Res 5, 1155–1162.
Levine, R. L., Garaland, D., Iliver, C. N., Amici, A., Climent, I.,
Lenz, A. G., Ahn, B., Shaltiel, S., Stadtman, E. R. (1990). Determi-
nation of carbonyl content in oxidatively modified proteins. Methods
Enzymol 186, 464–478.
Lieber, C. S., De Carli, L. M. (1982). The feeding of alcohol in liquid
diets: two decades of applications and 1982 update. Alcohol Clin Exp
Res 6, 523–531.
Liu, R. M., Choi, J. (2000). Age-associated decline in g-glutamylcys-
teine synthetase gene expression in rats. Free Radic Biol Med 28,
566–574.
Londero, G., Greco, P. L. (1996). Automated HPLC separation with
spectrofluorometric detection of malondialdehyde–thiobarbituric acid
adduct in plasma. J Chromatogr A 729, 207–210.
Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J. (1951).
Protein measurement with Folin phenol reagent. J Biol Chem 153,
265–275.
Luczaj, W., Waszkiewicz, E., Skrzydlewska, E., Roszkowska-
Jakimiec, W. (2004). Green tea protection against age-dependent
ethanol-induced oxidative stress. J Toxicol Environ Health A 67,
595–606.
Maiani, G., Serafini, M., Salucci, M., Azzini, E., Ferro-Luzzi, A. (1997).
Application of new HPLC method for measuring selected polyphenols
in human plasma. J Chromatogr B 692, 311–317.
McIntosh, L., Trush, M., Troncoso, J. (1997). Increased susceptibility of
Alzheimer’s disease temporal cortex to oxygen free radical-mediated
processes. Free Radic Biol Med 23, 183–190.
Michiels, C., Raes, M., Toussaint, O., Remacle, J. (1994). Importance of
Se-glutathione peroxidase, catalase and Cu/Zn SOD for cell survival
against oxidative stress. Free Radic Biol Med 17, 235–248.
Misra, H. P., Fridovich, I. (1972). The role of superoxide anion in the
autooxidation of epinephrine and a simple assay for superoxide dismu-
tase. J Biol Chem 247, 3170–3175.
Mize, C. E., Langdon, R. G. (1962). Hepatic glutathione reductase. Pu-
rification and general kinetic properties. J Biol Chem 237, 1589–1595.
Morrison, J. H., Hof, P. R. (1997). Life and death of neurons in the aging
brain. Science 278, 412–419.
Neuschwander-Tetri, B. A., Roll, F. J. (1989). Glutathione measurement
by high-performance liquid chromatography separation and fluoromet-
ric detection of the glutathione–orthophtalealdehyde adduct. Anal
Biochem 179, 236–241.
Nordman, R., Ribiere, C., Rouach, H. (1987). Involvement of iron and
iron-catalyzed free radical production in ethanol metabolism and
toxicity. Enzyme 37, 57–61.
Ostrowska, J., Luczaj, W., Kasacka, I., Rozanski, A., Skrzydlewska, E.
(2004). Green tea protects against ethanol-induced lipid peroxidation
in rat organs. Alcohol 32, 25–32.
Paglia, D. E., Valentine, W. N. (1967). Studies on the quantitative and
qualitative characterization of erythrocyte glutathione peroxidase.
J Lab Clin Med 70, 58–169.
Pahlavani, M. A., Harris, M. D., Richardson, A. (1997). The increase in
the induction of IL-2 expression with caloric restriction is correlated to
changes in the transcription factor NFAT. Cell Immunol 180, 10–19.
Ponnappa, B. C., Rubin, E. (2000). Modeling alcohol’s effects on organs
in animal models. Alcohol Res Health 24, 93–104.
Porter, N. A. (1984). Chemistry of lipid peroxidation. Methods Enzymol
105, 273–282.
97E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98

10. Prutz, W. A., Butler, J., Land, E. J. (1983). Phenol coupling initiated by
one-electron oxidation of tyrosine units in peptides and histone. Int J
Radiat Biol Relat Stud Phys Chem Med 44, 183–196.
Puntarulo, L. A., Cederbaum, A. I. (1989). Chemiluminescence from ac-
etaldehyde oxidation by xanthine oxidase involves generation of and
interactions with hydroxyl radicals. Alcohol Clin Exp Res 13, 84–90.
Rice-Evans, C. A., Miller, N. J., Paganga, G. (1996). Structure–antiox-
idant activity relationship of flavonoids and phenolic acids. Free Radic
Biol Med 20, 933–956.
Rottkamp, C. A., Nunomura, A., Raina, A. K., Sayre, L. M., Perry, G.,
Smith, M. A. (2000). Oxidative stress, antioxidant, and Alzheimer
disease. Alzheimer Dis Assoc Disord 14, 62–66.
Rouach, H., Fataccioli, V., Gentil, M. (1997). Effect of chronic ethanol
feeding on lipid peroxidation and protein oxidation in relation to liver
pathology. Hepatology 25, 351–355.
Rushmore, T. H., Morton, M. R., Pickett, C. B. (1991). The antioxidant
responsive element. Activation by oxidative stress and identification of
the DNA consensus sequence required for functional activity. J Biol
Chem 266, 11632–11659.
Samson, F. E., Nelson, R. S. (2000). The aging brain, metals and oxygen
free radicals. Cell Mol Biol 46, 699–707.
Smith, L. L., Johnson, B. H. (1989). Biological activities of oxysterols.
Free Radic Biol Med 7, 285–332.
Sun, A. Y., Sun, G. Y. (2001). Ethanol and oxidative mechanisms in the
brain. J Biomed Sci 8, 37–43.
Sohal, R. S., Arnold, L. A., Sohal, B. A. (1990). Age-related changes in
antioxidant enzymes and prooxidant generation in tissues of the rat
with special reference to parameters in two insect species. Free Radic
Biol Med 10, 495–500.
Somani, S. M., Husain, K., Diaz-Phillips, L., Lanzotti, D. J., Kareti, K. R.,
Trammell, G. L. (1996). Interaction of exercise and ethanol on
antioxidant enzymes in brain regions of the rat. Alcohol 13, 603–610.
Storz, G., Tartaglia, L. A., Ames, B. N. (1990). Transcriptional regulator
of oxidative stress-inducible genes: direct activation by oxidation.
Science 248, 189–194.
Sykes, A., McCormac, F. X. Jr., O’Brien, T. J. (1978). Preliminary study
of the superoxide dismutase content of some human tumors. Cancer
Res 38, 2759–2762.
Tokumaru, S., Tsukamoto, I., Iguchi, H., Kojo, S. (1995). Specific and
sensitive determination of lipid hydroperoxides with chemical deriva-
tization into 1-naephtyldiphenylphosphine oxide and HPLC. Anal
Chim Acta 307, 97–102.
Tsukaguchu, H., Tokui, T., Mackensie, B., Berger, U., Chen, X. X.,
Wang, Y., Brubaker, R. F., Hediger, M. A. (1999). A family of
mammalian Na1
dependent L-ascorbic acid transporters. Nature 399,
70–75.
Vallet, M., Tabatabale, T., Briscoe, R. J., Baird, T. J., Beatty, W. W.,
Floyd, R. A., Gauvin, D. V. (1997). Free radical production during
ethanol intoxication, dependence, and withdrawal. Alcohol Clin Exp
Res 21, 275–285.
Yoshino, K., Matsuura, T., Sano, M., Saito, S., Tomita, I. (1986).
Fluorometric liquid chromatographic determination of aliphatic alde-
hydes arising from lipid peroxides. Chem Pharm Bull 34, 1694–
1700.
98 E. Skrzydlewska et al. / Alcohol 37 (2005) 89–98