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World J Gastroenterol 2003;9(1):125-128
World Journal of Gastroenterology
Copyright ©2003 by The WJG Press ISSN 1007-9327
• BASI C RESEARCH •
N-acetylcysteine attenuates alcohol-induced oxidative stress in
the rat
Resat Ozaras, Veysel Tahan, Seval Aydin, Hafize Uzun, Safiye Kaya, Hakan Senturk
Resat Ozaras, Department of Infectious Diseases and Clinical
Microbiology, University of Istanbul, Istanbul, Turkey
Veysel Tahan, Hakan Senturk, Department of Internal Medicine,
University of Istanbul, Istanbul, Turkey
Seval Aydin, Hafize Uzun, Safiye Kaya, Department of Biochemistry,
University of Istanbul, Istanbul, Turkey
This study was presented in Digestive Disease Week, 20-23rd May,
2001, Atlanta, Georgia as an oral presentation
Supported by the research Fund of the University of Istanbul. No:
T-589/240698
Correspondence to: Resat Ozaras, M.D., Altimermer Cad. 27/4,
Kucukhamam TR-34303 Fatih, Istanbul, Turkey. rozaras@yahoo.com
Telephone: +90-212-5882840 Fax: +90-212-5882840
Received: 2002-07-26 Accepted: 2002-11-22
Abstract
AIM: There is increasing evidence that alcohol-induced liver
damage may be associated with increased oxidative stress.
We aimed to investigate free-radical scavenger effect of nacetylcysteine in rats intragastrically fed with ethanol.
METHODS: Twenty-four rats divided into three groups were
fed with ethanol (6 g/ kg/ day, Group 1), ethanol and nacetylcysteine (1 g/ kg, Group 2), or isocaloric dextrose
(control group, Group 3) for 4 weeks. Then animals were
sacrificed under ether anesthesia, intracardiac blood and
liver tissues were obtained. Measurements were performed
b o t h i n ser u m an d i n h o m o g en i ze d l i v er t i ssu e s.
Malondialdehyde (MDA) level was measured by TBARS
method. Glutathione peroxidase (GSH-Px) and superoxide
dismutase (SOD) levels were studied by commercial kits.
Kruskal-Wallis test was used for statistical analysis.
RESULTS: ALT and AST in Group 1 (154 U/ L and 302 U/ L,
respectively) were higher than those in Group 2 (94 U/ L and
155 U/ L) and Group 3 (99 U/ L and 168 U/ L) (P= 0.001 for
both). Serum and tissue levels of MDA in Group 1 (1.84 nmol/
mL and 96 nmol/ 100 mg-protein) were higher than Group 2
(0.91 nmol/ mL and 64 nmol/ 100 mg-protein) and Group 3
(0.94 nmol/ mL and 49 nmol/ 100 mg-protein) (P< 0.001 for
both). On the other hand, serum GSH-Px level in Group 1
(8.21 U/ g-Hb) was lower than Group 2 (16 U/ g-Hb) and
Group 3 (16 U/ g-Hb) (P< 0.001). Serum and liver tissue levels
of SOD in Group 1 (11 U/ mL and 26 U/ 100 mg-protein)
were lower than Group 2 (18 U/mL and 60 U/100 mg-protein)
and Group 3 (20 U/ mL and 60 U/ 100 mg-protein) (P< 0.001
for both).
CONCLUSION: This study demonstrat ed that ethanolinduced liver damage is associated with oxidative stress,
and co-administration of n-acetylcysteine attenuates this
damage effectively in rat model.
Ozaras R, Tahan V, Aydin S, Uzun H, Kaya S, Senturk H. Nacetylcysteine attenuates alcohol-induced oxidative stress in
the rat. World J Gastroenterol 2003; 9(1): 125-128
http:/ / www.wjgnet.com/ 1007-9327/ 9/ 125.htm
INTRODUCTION
Reactive oxygen intermediates contributes to the pathogenesis
of various hepatic disorders such as paracetamol intoxication,
hemochromatosis, toxic hepatitis, and alcoholic liver injury[1-4].
Increased oxygen radical production leads to lipid peroxidation
by induced cytochrome P4502E1[5,6]. Enhanced generation of
acetaldehyde was shown to cause lipid peroxidation in isolated
perfused livers[7]. Oxidative damage correlates with the amount
of ethanol consumed[8].
Antioxidants such as vitamin E have been suggested as
therapeutic options in acute and chronic liver diseases[9,10]. Nacetylcysteine (NAC) exerts a strong antioxidant activity, and
is the treatment of choice in acetaminophen intoxication. NAC
provides protection from toxic liver damage by elevating
intacellular glutathione concentrations[1,11]. It has also been used
in the treatment of CCl4 poisoning[12].
In this study, we tested whether NAC attenuates alcoholinduced free radical damage in the liver in a rat model.
MATERIALS AND METHODS
Male Wistar-Albino rats weighing 220-250 g obtained from
University of Istanbul Animal Research Laboratory were kept
in the same unit and fed chow (Eris Chow Industry, Istanbul,
Turkey) ad libitum. All rats had free access to tap water. All
animals received humane care in compliance with the National
Institutes of Health criteria for care of laboratory animals.
Twenty-four rats divided into three groups and were given
ethanol (Group 1) or both ethanol and NAC (Group 2) or
isocaloric dextrose (Group 3). NAC and alcohol were
administered respectively 4 hours apart. Ethanol and NAC were
given intragastrically at doses of 6 g/kg/day and 1 mg/kg/day
respectively.
All rats were sacrificed at 1 month under ether anesthesia.
After exploration of the thorax, intracardiac blood and liver
samples were quickly obtained. Serum alcohol levels were
measured on the day the rats were sacrificed.
Glutathione peroxidase levels in erythrocytes, serum alcohol
level and biochemistry were studied immediately. For the
remaining studies, serum and liver tissue samples were stored
at -70 .
Tissue Homogenization: Liver samples were weighed and
homogenized in 0.15 M NaCl for lipid peroxidation parameters
and for the other studies, and homogenates of 20 % were
obtained. Tissue homogenates were sonicated two times at 30
sec. intervals. Homogenization and sonication were performed
at 4 . After sonication, homogenates for lipid peroxidation
and biochemical studies were centrifuged at 3 000 rpm for 10
minutes and at 15 000 rpm for 15 minutes respectively. Aliquots
of the supernatants were used for both studies[13]. The assayed
parameters were expressed per mg protein and protein content
of the aliquots was determined by the method of Lowry et al [14].
Lipid peroxidation
Lipid peroxidation was measured by thiobarbituric acid
method, a modified form of the procedure described by Beuge
and Aust[15]. This method measures several aldehydes derived
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World J Gastroenterol
from lipid hydroperoxides and also known as TBARS
(thiobarbituric acid reactive substances) method.
30
Superoxide dismutase (SOD) level
Serum and homogenized liver tissue SOD levels were measured
by using commercial kits. (Randox-Ransod, Cat No:SD 125).
20
January 15, 2003 Volume 9 Number 1
Serum
Serum GGT
GGT
T
issue GGT
Tissue
GGT
b
10
Glutathione level
Glutathione concentration was determined according to the
method of Beutler et al[16]. using metaphosphoric acid for
protein precipitation and 5’ 5-dithiobis-2-nitrobenzoic acid for
colour development. Erythrocyte and tissue glutathione
concentrations were expressed as mg/grHb, mg/gr wet weight
respectively.
Figure 2 Serum (U/ L) and tissue (U/ g-protein) GGT levels.
Alc: alcohol, NAC: N-acetylcysteine, bP<0.001 vs control and
Alc+NAC group.
Glutathione peroxidase level
Serum and homogenized liver tissue glutathione peroxidase
levels were also measured by using commercial kits. (RandoxRansel, Cat No: RS 505).
Biochemical studies were performed by using autoanalyser
(Hitachi 717). Hemoglobin level was measured manually by
cyanmethemoglobin method.
Although serum ALP level in Group 1 (131.38±33.84 U/L)
tend to be higher than those in Group 2 (109.50±49.75 U/L)
and Group 3 (93.13±32.42 U/L), the difference was not
significant either. But tissue ALP level in Group 1 (26.88±3.31
U/g-protein) was significantly higher than those in Group 2
(12.63±2.67 U/g-protein) and Group 3 (11.25±2.49 U/gprotein) (P<0.001) (Figure 3).
Alcohol level
Serum alcohol level was measured by fluorescent polarizing
immunoassay using commercial kits (Abbot TDx, Cat No:
378190100).
Statistical analysis
All results are expressed as mean ±standard deviation.
Comparisons between the groups were performed by KruskalWallis variance analysis and a P value <0.05 was accepted as
statistically significant.
RESULTS
Blood alcohol levels of Group 1 and 2 were comparable
(207±33 mg/dL vs 182±27 mg/dL).
AST level in Group 1 (302.00±70.68 U/L), was higher
than those in Group 2 (155.25±24.07 U/L), and in Group 3
(168.25±32.08 U/L) (P=0.001). ALT level also in Group 1
(154.13±33.59 U/L), was higher than those in Group 2
(94.25±16.02 U/L), and in Group 3 (99.00±19.86 U/L)
(P=0.001) (Figure 1).
400
400
b
0
Control
Control
300
300
b
200
200
100
100
Tissue
ALP
Tissue
ALP
160
160
140
140
120
120
100
100
80
80
60
60
b
40
40
20
20
00
Control
Control
Alc+NAC
Alc+NAC
Control
Control
Alc+NAC
Alc+NAC
Alc
Alc
Figure 1 Serum AST and ALT levels (U/ L). Alc: alcohol, NAC:
N-acetylcysteine, bP=0.001 vs control and Alc+NAC group.
Although serum GGT level in Group 1 (8.50±3.16 U/L)
tend to be higher than those in Group 2 (8.25±3.92 U/L) and
Group 3 (6.88±3.76 U/L), the difference was not significant.
However tissue GGT level in Group 1 (18.75±5.90 U/g-protein)
was significantly higher than those in Group 2 (7.23±.6.09 U/
g-protein) and Group 3 (6.25±3.33 U/g-protein) (P<0.001)
(Figure 2).
Alc
Alc
Figure 3 Serum (U/ L) and Tissue (U/ g-protein) ALP levels
Alc: alcohol, NAC: N-acetylcysteine, bP<0.001 vs control and
Alc+NAC group.
Erythrocyte glutathione level was lower in Group 1 (356.2
±18.3 mg/g-Hb) when compared to Group 2 (387.8±13.1 mg/
g-Hb) and Group 3 (398.0±18.0 mg/g-Hb) (P<0.01) (Figure 4).
R
BC-GSH
RBC-GSH
440
440
420
420
340
340
320
320
b
Control
Control
0
Alc
Alc
Serum
ALPALP
Serum
180
180
400
400
380
380
360
360
AST
ALT
ALT
AST
0
Alc+NAC
Alc+NAC
Alc+NAC
Alc+NAC
Alc
Alc
Figure 4 Erythrocyte glutathione levels (U/ g-Hb). Alc: alcohol,
NAC: N-acetylcysteine, bP<0.01 vs control and Alc+NAC group.
Blood glutathione peroxidase level was lower in Group 1
(8.21±1.15 U/g-Hb) when compared to Group 2 (16.04±2.38
U/g-Hb) and Group 3 (16.84±2.68 U/g-Hb) (P<0.001) (Figure
5). Also serum SOD level was lower in Group 1 (11.08±1.13
U/mL) when compared to Group 2 (17.92±0.81 U/mL) and
Group 3 (19.68±1.76 U/mL) (P<0.001). The same was true
for the tissue SOD levels: it was lower in Group 1 (26.04±8.49
U/ 100 mg-protein) when compared to Group 2 (59.96±10.23
U/ 100 mg-protein) and Group 3 (60.34±8.24 U/ 100 mgprotein.) (P<0.001) (Figure 6).
Ozaras R et al. N-acetylcysteine and oxidative stress
Serum MDA level was significantly higher in Group 1
(1.84±0.14 nmol/mL) than those in Group 2 (0.91±0.14 nmol/
mL) and Group 3 (0.94±0.11 nmol/mL) (P<0.001) (Figure 7).
For the tissue levels, it was higher also in Group 1 (96.00±18.20
nmol/100 mg-protein) than those in Group 2 (64.00±11.63
nmol/100 mg-protein) and Group 3 (49.63±12.11 nmol/100
mg-protein) (P<0.001) (Figure 8).
GSH-Px
GSH-Px
25
20
15
b
10
5
0
Control
Control
Alc+NAC
Alc+NAC
Alc
Alc
Figure 5 Blood glutathione peroxidase levels (U/ g-Hb). Alc: alcohol,
NAC: N-acetylcysteine, aP<0.001 vs control and Alc+NAC group.
Serum
SerumSOD
SOD
80
80
TTissue
issue SO
D
SOD
60
60
b
40
40
20
20
00
b
Control
C o n trol
AAlc+NAC
lc+NAC
A lAlc
c
Figure 6 Serum (U/ mL) and tissue (U/ 100 mg-protein) su-
pero xid e d ismutase (SOD) levels. A lc: alco ho l, NA C: Nacetylcysteine, bP<0.001 vs control and Alc+NAC group.
2.5
2.5
Serum
DA
SerumMMDA
2.02
b
1.5
1.5
1.01
0.5
0.5
00
Control
C o n trol
Alc+NAC
Alc+NAC
Alc
Alc
Figure 7 Serum MDA level (nmol/ mL). Alc: alcohol, NAC: Nacetylcysteine, bP<0.001 vs control and Alc+NAC group.
120
120
b
TTissue
issue MDA
MDA
100
100
80
80
60
60
40
40
20
20
00
Control
Control
Alc+NAC
A lc+NAC
Alc
A lc
Figure 8 Tissue MDA level (nmol/ 100 mg-pro tein). A lc:
alcoho l, NA C: N-acetylcysteine, b P <0.001 vs co ntro l and
Alc+NAC group.
127
DISCUSSION
Ethanol is capable of generating oxygen radicals, inhibiting
glutathione synthesis, producing glutathione loss from the
tissue, increasing malonyldialdehyde levels and impairing
antioxidant defense systems in humans and experimental
animals. Lipid peroxidation results from the increased oxygen
radical production by the induced 2E1[6]. Enhanced generation
of acetaldehyde was also shown to be capable of causing lipid
peroxidation in isolated perfused livers[7]. Lipid peroxidation
is not only a reflection of tissue damage, it may also play a
pathogenic role, for instance by promoting collagen
production[17]. The removal of the toxic metabolites is believed
to be the vital initial step in providing cell survival during
ethanol intoxication[18].
Genc et al[19]. used melatonin in preventing lipid peroxidation
due to acute alcohol intoxication. They found that melatonin
administration prior to alcohol did not alter MDA and GSH
levels of the tissue but an antioxidant enzyme (CuZn-SOD)
was higher in animals receiving alcohol+melatonin. However
since absorption and kinetics of this hormone are not widely
known, and its antioxidant effect depend on both the tissue
studied and the dose applied, the results of a melatonin study
may not reflect the effects of other antioxidants.
Nanji et al[20]. used thromboxane inhibitors in alcoholic liver
disease in rats. They found that treatment with thromboxane
inhibitors prevented necrosis and inflammation, and suggested
a role for the use of thromboxane inhibitors in the treatment of
alcoholic liver disease. Flora et al[21] tested NAC and a chelator
agent meso 2,3-dimercaptosuccinic acid combination in the
treatment of arsenic-induced oxidative stress, and found that
these agents had a capability of reversing the toxic effects.
Bruck et al [1] . have used NAC in the treatment of
thioacetamide-induced fulminant hepatic failure in the rat
model. They found no protective effect of NAC. In this model,
total hepatic glutathione content is not affected. Instead, other
free radical scavengers, dimethylsulfoxide and dimethylthiourea
having additional modes of action such as inhibition of nitric
oxide formation prevented liver injury.
The results of our study show that co-administration of NAC
diminishes oxidative stress, by increasing antioxidant enzymes.
This restoration of oxidant/antioxidant balance is reflected by
lower levels of transaminases, ALP, and GGT. Although the
decrease in serum level of the latter two enzymes was not
significant, tissue levels were lower. NAC has been used in
acetaminophen intoxication. It reduces the incidence of organ
failure and enhances survival[22]. It acts by reducing tissue
hypoxia, mediated by the activity of the nitric oxide/soluble
guanylate cyclase system[1]. Cysteine derived from NAC also
serves as a precursor of glutathione which forms conjugates
with carbon tetrachloride metabolites [23] and increases
intracellular glutathione concentrations[1]. In a recent study,
NAC - in a lower dose than used in the current study- has been
shown to prevent the fatty acid changes produced by ethanol
and also reduce inflammatory response by reducing the level
of prostaglandin[24].
Antioxidant protective mechanisms are both enzymatic and
nonenzymatic. Impairments in these defense systems have been
shown in alcoholics: alterations in ascorbic acid levels,
glutathione, selenium, and vitamin E have been observed[25,26].
Reduced hepatic alpha-tocopherol content after long-term
ethanol feeding in rats under adequate intake of vitamin E[27]
and also in alcoholics[28] has been reported. Alpha-tocopherol
level was found to be reduced in the blood of the alcoholics[29].
In addition to acetaldehyde and free radical generation by the
ethanol-induced microsomes, these deficient defense systems
were suggested to contribute to liver damage via lipid
peroxidation[17]. Lipid peroxidation is a reflection of tissue
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damage and plays a pathogenic role by promoting collagen
production[30].
A growing body of experimental and clinical experience
shows the importance of free radicals in ethanol-induced liver
damage. Free radical scavenging property may be beneficial
as ascertained by previous studies of NAC in both
acetaminophen and carbon tetrachloride intoxication. In this
rat model, we have used NAC to attenuate ethanol-induced
free radical damage. In both serum and tissue levels, we
observed a favorable result. The results of this study suggest a
role for NAC in the management of ethanol-induced liver
damage as a safe, cheap, and effective option.
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Edited by Xu JY and Xu XQ