Blue Biotechnology Journal
Volume 2, Number 2
ISSN: 2163-3886
© 2014 Nova Science Publishers, Inc.
ETHANOL-INDUCED HEPATOPATHY IN RATS
Aziza A. Saad1, Elhassan Mokhamer2, Mohamed Abdel Mohsen1,
and Gaylan Fadaly3
1
Applied Medical Chemistry Department, Medical Research Institute,
Alexandria University, Egypt
2
Pharmacology and Toxicology Department,
Faculty of Pharmacy and Drug Manufacturing,
Pharos University, Alexandria, Egypt
3
Pathology Department, Medical Research Institute,
Alexandria University, Egypt
ABSTRACT
A chronic consumption of alcohol can lead to the generation of oxygen free radicals
and reactive aldehydic species which alter the activities of lysosomes. Furthermore, the
reduction in protein degradation contributes to excessive protein accumulation.This was
associated with reduced volume densities of autophagosomes and autolysosomes, as
determined morphometrically. In this respect the current study was endevoured to the
detection of the oxidative stress (serum MDA) as well as the liver tissue homogenate
enzymes as arylsulphatase A, Aryl sulphatase B, B-glucoronidase, in addition to the liver
histopathological examination of alcohol fed rat livers showed histopathological
changes. These findings confirmed the biochemical results which demonstrated that
the ingestion of alcohol to rats induced oxidative stressor which was manifested by the
strikingly significantly increase in the level of serum MDA associated with significant
decrease in the hepatic specific enzymatic activities of ASA, ASB and β-Glase in rats
ingested alcohol.
Keywords: Ethanol, free radicals, oxidative stress, hepatopathy, autophagy
1. INTRODUCTION
Most alcoholic drinks consist of ethanol (ethyl alcohol). Ethanol is soluble in water and
many organic solvents. The solubility of ethanol enables it to cross the cell membrane and
more importantly the blood-brain barrier [1] . A chronic consumption of alcohol can lead to

Corresponding author: El Hassan Mokhamer, E.mail: Hassan.mokhamer@pua.edu.eg, Tel: +203/01222510555,
+203/01122244233
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Aziza A. Saad, Elhassan Mokhamer, Mohamed Abdel Mohsen et al.
the generation of oxygen free radicals and reactive aldehydic species. The metabolism of
ethanol also leads to the generation of hydroxyethyl radicals that can be associated with
stimulation of lipid peroxidation. The lipid peroxidation is associated with an increase in liver
disease, development of hepatic fibrosis, and cirrhosis [2].
Strong evidence suggests that alcohol consumption can alter the activities of
lysosomes. One effect of alcohol consumption is an elevation in lysosomal pH. This shift to
alkalinity reduces the activities of lysosomal proteinases and results in less-than-optimal
protein hydrolysis in the lysosome. Furthermore, the reduction in protein degradation
contributes to excessive protein accumulation, which has been observed in livers of human
alcoholics and alcohol-fed laboratory animals [3].
In addition, both acute and chronic ethanol administration cause enhanced formation of
cytokines, especially TNF-alpha by hepatic Kupffer cells, which have a significant role in
liver injury [4-6]. Besides the development of fatty liver (steatosis), another early sign of
excessive ethanol consumption is liver enlargement and protein accumulation, both of which
are common findings in alcoholics and heavy drinkers. [7-12]
One of that oxidants (e.g. peroxynitrite) and reactive species (e.g. acetaldehyde and
malondialdehyde-acetaldehyde) [13] derived from ethanol metabolism may impair
autophagy, similar to that which occurs with the proteasome [14-16]. Some of the current
evidence for this is circumstantial, but nevertheless compelling as there is evidence of
lysosomal damage, as judged by enhanced lysosomal fragility, which could result from either
altered lipid metabolism, oxidative stress or both [17].
In this respect, the objective of the present study was launched to the detection of lipid
peroxidation markers in the serum of alcohol fed rats that showed relationship between
alcohol induced oxidative stress and the development of liver pathology.
2. MATERIALS AND METHODS
2.1. Chemicals
The fine chemicals used in this study. p- nitrophenyl-β-D-glucuronide, p-nitrophenol,
thiobarbituric acid (TBA), malonaldehyde-bis-diethyl-acetate (1, 1, 3, 3-tetraethoxy-propone),
p-nitrocatechal sulfate bovine serum albumin were purchased from sigma chemical Co, st.
Louis MO, USA. All other chemicals used for the preparation of the buffers used were of the
analytical grade.
2.2. Animals
Male wistar rats weighing 200 – 230 g were obtained from Animal House, Medical
Technology Center, Medical Research Institute, University of Alexandria. Animals received
standard laboratory chow (Purina) and water ad libitum, and were kept in identical housing
units on a twelve hours light/ dark cycle, they were treated humanly according to the national
guideline for the care of animal. The animals were divided into two groups.
Group 1: comprised of ten rats which fed on normal balanced diet serve as controls.
Ethanol-Induced Hepatopathy in Rats
367
Group 2: comprised of ten rats orally ingested with 2 gm/kg body weight ethanol, 20%
(wt/v) once daily for 4 weeks [18].
Four weeks post ethanol treatment; rats were sacrificed by cervical dislocation. Blood
samples were collected by cardiac puncture and the separated sera were kept at -20 C till
analysis for malondialdehyde (MDA), as a marker for lipid peroxidation, [19] were carried
out. Supernatant of liver homogenates were used for the determination of the specific
activities of arylsulfatases A (ASA) and arylsulfatases B (ASB) [20] as well as the specific
enzymatic activity of β-glucurondiase (β-Glase) . [21] Total hepatic protein level was measured
by Biuret method. [22]
2.3. Histopathology [23]
Liver samples were obtained from the same lobe in all animals and fixed in 10% buffered
formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H & E).
2.4. Statistical Analysis
The comparison between the studied groups was carried out by Student "t" test. The
mean differences were considered significant at level p < 0.05.
3. RESULTS
3.1. Biochemical Results
The results of the present study revealed that there was a highly significant increase in the
level of malondialdehyde (MDA) in the sera of ethanol treated rats than that of control group
(figure 1) (table 1) ( p<0.05).
Table 1. Malondialdehyde (MDA) level (nmol/ml) in the sera of the studied groups
MDA( nmol/ml)
Control (n=10)
32.4±4.0
Ethanol (n=10)
7.8±2.9*
Results are expressed as mean ±S.D.; p values shown as p* < 0.05 compared with control group.
*Statistically Significant.
Table 2. Arylsulfatase A, Arylsulfatase B and β-glucuronidase enzymatic specific
activities level (U/g protein) in the liver homogenates of rates in the studied groups
ASA (U/g Protein)
ASB (U/g Protein)
β-Glase (U/g protein)
Control (n=10)
32.4±4.0
36.7±5.78
2.02±0.22
Ethanol (n=10)
7.8±2.9*
26.8±7.44*
1.42±0.29*
Results are expressed as mean ±S.D.; p values shown as p* < 0.05 compared with control group.
*: Statistically Significant.
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Aziza A. Saad, Elhassan Mokhamer, Mohamed Abdel Mohsen et al.
On the other hand there were high significant decrease of the specific enzymatic activities
levels of β-glase , ASA, ASB in the liver homogenates of ethanol treated rats than that of
control group (figure.1)(table.2) ( p<0.05).
*Statistically Significant; p < 0.05.
Figure 1. Mean Level of Serum MDA (nmol/ml) and the Mean Specific Enzymatic Activities of Glase, ASA and ASB (U/g protein) in the Control and Ethanol Ingested Group.
3.2. Histopathological Results
Paraffin sections from various parts of control rat livers revealed normal configuration of
hepatic parenchyma, made up of compact lobules with centrally located veins, surrounded by
radiating single row cords of hepatocytes separated by thin sinusoids.
The hepatocytes have small central round unclei with tiny prominent nucleoli and
abundant eosinophilic granular cytoplasm. The sinusoids are lined by flat endothelium. Portal
tracts are loose fibrous including hepatic ducts, portal vein and artery with no inflammatory
cells and no fibrous tissues (Figure 2).
Ethanol-Induced Hepatopathy in Rats
369
Figure 2. Paraffin hepatic section of control hepatic parenchyma showing centrally located vein (V),
surrounded by radiating single row cords of hepatocytes (H) separated by thin sinusoids (S) (H&E 
200).
Paraffin sections from various parts of alcohol treated rat livers revealed scattered areas
of mild to moderate macrovesicular steatosis evidenced by clear hepatocytes showing
vaculated lipid laden cytoplasm, mild intraparnechymal lobular infiltration by focal chronic
mononuclear inflammatory cells is evident forming mild form of steatohepatitis. Liver cells
showed scattered apoptotic changes manifested by small pyknotic densely stained nuclei and
abundant eosinophilic cytoplasm. Portal tracts are mildly fibrotic with moderate infiltration
by chronic inflammatory cells. Occasional thin interrupted fibrous septa are traversing the
hepatic parenchyma. No evident cirrhosis is identified in any of the rat liver (Figure 3 – 6).
Figure 3. Paraffin hepatic section of alcohol ingested group showing scattered apoptotic changes () in
the liver cells and moderate infiltration of portal tract (PT) by lymphocytic infiltration (L) (H&E 
200).
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Aziza A. Saad, Elhassan Mokhamer, Mohamed Abdel Mohsen et al.
Figure 4. Paraffin hepatic section of alcohol ingested group showing cords of hepatocytes expressing
diffuse macrosteatosis () (H&E  200).
Figure 5. Paraffin hepatic section of alcohol ingested group showing hepatic parenchyma traversed by
fibrous tissue septa moderately infiltrated by chronic lymphocytic infiltrate (L) (H&E  200).
Figure 6. Paraffin hepatic section of alcohol ingested group showing portal tract (PT) with moderate
fibrosis (F) and moderate lymphocytic infiltration (L) (H&E  200).
Ethanol-Induced Hepatopathy in Rats
371
DISCUSSION AND CONCLUSION
The pathogenesis of liver disease from alcohol abuse comes from the interaction of
several factors, including the generation of oxidants and reactive metabolites from ethanol
oxidation, which in turn, causes other metabolic derangements.[4-6] Liver injury may be
caused by direct toxicity of metabolic by-products of alcohol as well as by inflammation
induced by these by-products [24].
It is now well accepted that the progression of liver injury consequent to chronic alcohol
abuse is a multifactorial event that involves a number of genetic and environmental factors.
Among these factors there is growing interest in the role of free radical mediated oxidative
stress [25]. In this respect, the object of the present study was launched to detect the lipid
peroxidation marker in the serum of alcohol fed rats that showed relationship between alcohol
induced oxidative stress and the development of liver pathology.
There is a considerable interest in the role of oxidative stress and ethanol generation of
ROS in the mechanism by which ethanol is hepatotoxic [26-30]. It is probable that
acetaldehyde and free radicals (oxidative stress) destroy the lysosomal membranes [31,32]. It
was showed that ethanol administration increased the in situ pH of hepatic lysosomes by 0.15
to 0.2 pH units. This pH increase was sufficient to cause a significant reduction in lysosomal
protein degradation [3].
Ethanol administration causes a decrease in intralysosomal proteolysis, which is
responsible in part for a net hepatic protein gain in ethanol-fed animals [9,17]. It was showed
that decreased degradation is associated with a deficiency in the content of cathepsins B and
L in lysosomes of ethanol-fed rats [33]. This deficiency may arise from ethanol-induced
alterations in the trafficking and processing of lysosomal hydrolases, as indicated by
experiments which showed that ethanol consumption by rats delays the processing of
procathepsin L to its mature form in hepatocytes from these animals [34]. A similar influence
on the maturation of lysosomal  glucuronidase was reported after acute ethanol
administration [35].
Hepatic ASA activity was reduced due to ethanol administration. One intriguing
possibility to explain the observed decrease in the activity of ASA may be related to the coor posttranslational modification of ASA by formylglycine-generating enzyme that is required
for its catalytic activity. This modification involves the oxidation of cysteine to formylglycine
and is required for activation of all members of the sulfatase family. Furthermore, this
activation is inhibited by reactive oxygen molecules [36]. It was hypothesized that chronic,
high levels of ethanol in humans inhibit the formylglycine-generating enzyme activity by
production of reactive oxygen species and consequently will cause intermittent hepatic
multiple sulfatase deficiency, i.e., a deficiency of many sulfatase activities specifically in the
liver [37].
The aforementioned findings confirmed and represented a very excellent interpretation of
the present results which demonstrated the high significant decrease in the enzymatic specific
activities of -glucouronidase and arylsulfatases A and B.
The liver is the predominant organ that metabolizes ethanol and liver injury is the
principal clinical complication of alcohol abuse [38]. This will focus on the process of
autophagy, its role in normal hepatic function and its alteration due to ethanol consumption. It
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Aziza A. Saad, Elhassan Mokhamer, Mohamed Abdel Mohsen et al.
will consider how ethanol disrupts protein catabolism, which is believed to have a significant
role in liver injury [39].
The oxidants (e.g. peroxynitrite) and reactive species (e.g. acetaldehyde and
malondialdehyde-acetaldehyde) [13] derived from ethanol metabolism may impair autophagy,
similar to that which occurs with the proteasome [14-16].
Further, the finding that acetaldehyde can undergo secondary reactions with
malondialdehyde (MDA) to form more bulky substituents on proteins, known as
malondialdehyde-acetaldehyde adducts (MAA) with proteins, makes this mechanism of
autophagic suppression an attractive hypothesis [39].
Because autophagy is an energy-dependent process and requires ATP,[40] it follows that
ATP depletion naturally suppresses autophagy. Ethanol consumption has been reported to
inhibit ATP production by mitochondria [41], in part by enhancing oxidative modification
and inactivation of proteins within these organelles [42].
Adenosine monophosphate-activated protein kinase (AMPK) is a heterotrimeric protein
that is, itself, activated by elevated ratios of AMP/ATP and is a significant regulator of a
variety of metabolic and signal transduction pathways, including autophagy. Elevated
AMP/ATP ratios are indicators of low intracellular energy charge. Therefore, when AMP
activates the AMPK it generally down-regulates energy requiring pathways and stimulates
catabolic pathways. Interestingly, however, inhibition of AMPK also suppresses
autophagy.[43] These findings are consistent with the suppression of autophagy by ethanol
consumption, which significantly reduces AMPK activity in liver [44].
Thus, while ethanol metabolism similarly generates oxidants and reactive species,
including acetaldehyde, these molecules down-regulate upstream kinases and up-regulate the
downstream phosphatase, PP2A, resulting in AMPK inactivation, which, in turn, can cause
autophagic suppression. AMPK also has an important role in regulating lipid metabolism and
AMPK suppression by ethanol allows activation of rate-limiting enzymes involved in lipid
biosynthesis, which contribute to ethanol-induced fatty liver depicts the putative mechanisms
of autophagic suppression [45,46].
Fatty liver (steatosis) is the earliest, most common response of the liver to moderate or
large doses (i.e. binge drinking) of alcohol as well as to chronic ethanol consumption.[4]
Alcohol-induced fatty liver enhances susceptibility of the liver to more advanced pathology if
drinking continues. Individuals with alcohol-induced steatosis are vulnerable to developing
alcoholic steatohepatitis (ASH), hepatic fibrosis, cirrhosis and even hepatocellular carcinoma
[47].
It is worth to note that the results of the present histopathological examination elucidated
that in the liver of ethanol group of rats (Group II) there were scattered areas of mild to
moderate macrovesicular steatosis evidenced by clear hepatocytes showing vacuolated lipid
laden cytoplasm. Mild intraparenchymal lobular infiltration by focal chronic monovacuolar
inflammatory cells is evident forming mild form of steatohepatitis, liver cells showed
scattered apoptotic changes manifested by small pyknotic densely stained nuclei and
abundant eosinophilic cytoplasm. Thus the present findings were consistent with a lot of
studies which reported that alcohol induce steatosis as well as apoptosis (Figure 3-6).
Alcoholic liver disease (ALD) involves several stages of liver injury: steatosis, alcoholic
steatohepatitis, alcoholic hepatitis, and cirrhosis [48]. Kuppfer cells play an important role in
alcohol-induced liver damage. Alcohol increases the gut permeability to endotoxin or
lipopolysaccharide (LPS), a major constituent of the outer membrane of gram-negative
Ethanol-Induced Hepatopathy in Rats
373
bacteria, which triggers a variety of inflammatory reactions, including the release of
proinflammatory cytokines and other soluble factors [49].
Activated Kupffer cells release a number of soluble agents, including cytokines, such as
transforming growth factor-beta (TGF-), platelet-derived growth factor (PDGF), TNF-, ROS,
and other factors.[50] These factors act on hepatic stellate cells (HSC), which are localized in
the parasinusoidal space, and store most of the vitamin A in the body [51].
Activated HSC produce then large amounts of extracellular matrix components e.g.
collagen I in an accelerated fashion, triggering a fibrogenic response. During the cross-talk of
both liver cell types, mediated by different cytokines, reactive oxygen species, and other
soluble factors, hepatocellular damage is initiated, and subsequent establishment of liver
fibrosis occurs [52-55]. The histopathological examination of the livers of alcoholic group (gp
II) in the current study emphasized that portal tracts are mildly fibrotic with moderate
infiltration by chronic inflammatory cells. Occasionally their interrupted fibrous septa are
traversing the hepatic parenchyma.
Conclusively, the present findings speculated the injury and damage induced by ethanol
intoxication in rats that was demonstrated obviously by the significant increase in the level of
MDA in the sera of ethanol treated group as a marker of lipid peroxidation (acetyaldehyde
free radical mediated oxidative stress) and the significant decrease in the enzymatic specific
activities of -glucuronidase and arylsulfatases A and B. The biochemical results were
confirmed by the histopathological changes induced by ethanol ingestion.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
S. Zakhari, “How is alcohol metabolized by the body?”, Alc. Res. Health. 29 (2006)
245-254.
S. K. Das, D. M. Vas Udevan, “Alcohol-induced oxidative stress”, Life Sc. 81 (2007)
177-187.
A. Dolganiuc A., P. G. Thomes, W. X. Ding, J. J. Lemasters, T. M. Donohue T. M.,“
Autophagy in alcohol-induced liver diseases ”, Alcohol Clin Exp Res. 36 (2012 ) 13011308.
Z. Zhou, L. Wang, Z. Song, J.C. Lambert, C.J. McClain, Y.J. Kang, “A critical
involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha
production”, Am. J. Pathol. 163 (2003) 1137-1146.
P. Mandrekar, G. Szabo. “Signalling pathways in alcohol-induced liver inflammation”,
J. Hepatol. 50 (2009) 1258-1266.
X. Wang, Y. Lu, B. Xie, A. I. Cederbaum, “Chronic ethanol feeding potentiates Fas
Jo2-induced hepatotoxicity: role of CYP2E1 and TNF-alpha and activation of JNK and
P38 MAP kinase”, Free Radic. Biol. Med. 47 (2009) 518-528.
S. Patouraux, S. Bonnafous, C.S. Voican, R. Anty, M.C. Saint-Paul, M.A. RosenthalAllieri, H. Agostini, M. Njike, N. Barri-Ova, S. Naveau, Y. Le Marchand-Brustel, P.
Veillon, P. Calès, G. Perlemuter, A. Tran, P. Gual, “The osteopontin level in liver,
adipose tissue and serum is correlated with fibrosis in patients with alcoholic liver
disease”, PLoS One. 7 (2012) e35612.
374
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Aziza A. Saad, Elhassan Mokhamer, Mohamed Abdel Mohsen et al.
H. Koivisto, J. Hietala, O. Niemelä, “An inverse relationship between markers of
fibrogenesis and collagen degradation in patients with or without alcoholic liver
disease”, Am. J. Gastroenterol. 102 (2007): 773-779.
Jr. Donohue, “Autophagy and ethanol-induced liver injury”, World J. Gastroenterol. 15
(2009) 1178-1185.
A. M. Karinch, J. H. Martin, T. C. Vary, “Acute and chronic ethanol consumption
differentially impact pathways limiting hepatic protein synthesis”, Am. J. Physiol.
Endocrinol. Metab. 295 (2008) E3-9.
F. Fortunato, H. Bürgers, F. Bergmann, P. Rieger, MW. Büchler, G. Kroemer, J.
Werner, “Impaired autolysosome formation correlates with Lamp-2 depletion: role of
apoptosis, autophagy, and necrosis in pancreatitis”, Gastroenterology. 1 (2009): 73507360.
F. Bardag-Gorce, “Effects of ethanol on the proteasome interacting proteins”, World J.
Gastroenterol. 16 (2010) 1349-1357.
D. J. Tuma, “Role of malondialdehyde- acetaldehyde adducts in liver injury”, Free
Radic. Bio. Med. 32 (2002) 303-308.
F. Barda-Gorce , B.A. French, I. Nan, H. Song, S. K. Nguyen, H. Yong, J. Dede, S. W.
Ferench, “ CYP2E1 induced by ethanol cases oxidative stress, proteasome inhibition
and cytokeratin aggresome (Mallory body-like) formation”, Exp. Mol. Pathol. 81
(2006) 191-201.
B. Levine, G. Kroemer, “Autophagy in the pathogenesis of disease”, Cell. 132 (2008)
27-42.
T. M. Donohue, A. L Cederbaum, S.W. French, S. Brave, B. Gao, N. A. Osna, “Role of
the proteasome in ethanol- induced liver pathology”, Alcohol Clin. Exp. Res. 31 (2007)
1446-1459.
W. X. Ding, M. Li, X. Chen , H.M. Ni , C.W. Lin, W. Gao, B. Lu, D.B. Stolz, D.L.
Clemens, X.M. Yin. Autophagy reduces acute ethanol-induced hepatotoxicity and
steatosis in mice. Gastroenterology 139 (2010) 1740-1752.
K. Husain, B. R. Scott, S. K. Redy, S. M. S. Somani, “Chronic ethanol and nicotine
interaction on rat tissue antioxidant defense system”, Alcohol. 25 (2001) 89-97.
K. Stoh, “Serum lipid peroxides in cerebrovascular disorders determined by a new
colorimetric method”, Clin. Chim. Acta. 90 (1978) 37-43.
H. Baum, K.S. Dodgson, B. Spencer, “The anomalous kinetics of arylsulfatase of
human tissue”, The anomalies Biochem. J. 69 (1958) 567-572.
Y. Katok, H. Tsukamto, M. Nobunaga, T. Masuya, T. Sawada, “ Synthesis of Pnitrophenyl β-D- glucopyranosiduronic acid and its utilization as substrate for assay of
β-glucuronidase activity”, Chem. Pharm. Bull. Tokio. 8 (1960) 239.
L. M. Silverman, R. H. Christenson, Amino acids and proteins, In Tietz Fundamentals
of clinical chemistry, Edited by C. A. Burtis, E. R. Ashwood, W. B. Saunders
Company, 1996, 240-282.
T. M. Worner, C.S. Leiber, “Perivenualr fibrosis as precursors lesion of cirrhosis”,
JAMA 254 (1985) 627-30.
H. Jaeschke, J. Gregory. G. J. Gores, A. I. Cederbaum, J. A. Hinson, D. Pessayre, J. J.
Lemasters, “Mechanisms of Hepatotoxicity”, Toxicol. Sci. 65 (2002) 166-176.
E. Albano, “Alcohol, oxidative stress and free radical damage”, Nutrition and
Alcoholism. 65 (2006) 278–90.
Ethanol-Induced Hepatopathy in Rats
375
[26] S. V. Siegmund, S. Dooley, D. A. Brenner, “Molecular mechanisms of alcohol- induced
hepatic fibrosis”, Dig. Dis. 23 (2005) 264-274.
[27] C. S. Lieber, “Metabolism of alcohol”, Clin. Liver. Dis. 9 (2005) 1-35.
[28] D. E. Wu, A.I. Cederbaum, “Alcohol, oxidative stress and free radical damage”,
Alcohol Res. Health. 27 (2003) 277-284.
[29] A. Cahill, C. C. Cunningham, M. Adachi, H. Ishii, S. M. Bailey, B. Fromenty, A.
Davies, “Effects of alcohol and oxidative stress on liver pathology: the role of the
mitochondrion”, Alcohol Clin. Exp. Res. 26 (2002) 907-915.
[30] A. Dey, A. I. Cederbaum, “Alcohol and oxidative liver injury”, Hepatology. 43 (2006)
563-574.
[31] A. Ambade, P. Mandrekar, “Oxidative stress and inflammation: essential partners in
alcoholic liver disease”, Int. J. Hepatol. 2012 (2012) 853175-853184.
[32] Y. Guo, X. Q. Wu, C. Zhang, Z. X. Liao, Y. Wu, Z. Y. Xia, H. Wang, “Effect of
indole-3-carbinol on ethanol-induced liver injury and acetaldehyde-stimulated hepatic
stellate cells activation using precision-cut rat liver slices”, Clin. Exp. Pharmacol.
Physiol. 37 (2010) 1107-1113.
[33] T. M. Donohue, T. V. Curry-McCoy, A. A. Nanji , K. K. Kharbanda, N. A. Osna, S. J.
Radio, S.L. Todero , R.L. White, C.A. Casey, “Lysosomal leakage and lack of
adaptation of hepatoprotective enzyme contribute to enhanced susceptibility to ethanolinduced liver injury in female rats”, Alcohol Clin. Exp. Res. 31 (2007) 1944-1952.
[34] D. Wu, X. Wang, R. Zhou, A. Cederbaum, “CYP2E1 enhances ethanol-induced lipid
accumulation but impairs autophagy in HepG2 E47 cells”, Biochem. Biophys. Res.
Commun. 402 (2010) 116-122.
[35] P. G. Thomes, C.S.Trambly, G.M. Thiele, M.J. Duryee, H.S. Fox, J. Haorah, T.M.
Donohue, “ Proteasome activity and autophagosome content in liver are reciprocally
regulated by ethanol treatment”, Biochem. Biophys. Res. Commun. 6; 417(1) (2012)
262-267.
[36] T. Dierks, A. Dickmanns, A. Preusser- Kunze, B. Schmidt, M. Mariappan, K. Von
Figura, R. Ficner, M.G. Rudolph, “Molecular basis for multiple sulfatese deficiency and
mechanism for formylglycine generation of human formylglycine- generating enzyme”,
Cell. 121 (2005) 541-552.
[37] S. Jean, C. Kasinathan, S. Buyske, P. Manowitz, “Ethanol decreases rat hepatic
arylsulfatase A activity levels”, Alcohol Clin Exp Res. 30 (2006) 1950-1955.
[38] S. Zakhari, T.K. Li, “Determinants of alcohol use and abuse: impact of quantity and
frequency patteren on liver disease”, Hepatology. 46 (2007) 2032-2039.
[39] D. Wu, X. Wang, R. Zhou, L.Yang, A.I. Cederbaum, “Alcohol steatosis and
cytotoxicity: the role of cytochrome P4502E1 and autophagy”, Free Radic. Biol. Med.
53 (2012) 1346-1357.
[40] A. J. Meijer, P. Codogno, “Regulation and role of autophagy in mammalian cells”, Int.
J. Biochem. Cell. Biol. 36 (2004) 2445-2462.
[41] T. A. Young, S.M. Bailey, C.G. Van Horn, “Chronic thanol consumption decrease
mitochondrial and glycoltic production of ATP in liver”, Alcohol. 41(2006) 254-260.
[42] K. H. Moon, B.L. Hood, B.J. Kim, J.P. Hardwick, T.P. Conrads, T.D. Veenstra, B.K.
Song, “Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic
fatty liver of rats”, Hepatology. 44 (2006) 1218-1230.
376
Aziza A. Saad, Elhassan Mokhamer, Mohamed Abdel Mohsen et al.
[43] D. Meley, C. Bauvy, J.H. Houben-weerts, P.F. Dubbelhuis, M.T. Helmond, P.
Codogno, A.J. Meijer A.J, “AMP-activated protein kinase and the regulation of
autophagic proteolysis”, J. Biol. Chem. 281 (2006) 34870-34879.
[44] M. You, M. Matsumoto, C.M. Pacoid, W.K. Cho, D.W. Crabb, “The role of AMPactivated protein kinase in the action of ethanol in the liver”, Gastroenterology. 127
(2004) 1798-1808.
[45] H. C. Choi, P. Song, Z. Xie, Y. Wu, J. Xu, M. Zhang, Y. Dong, S. Wang, K. Lau, M.H.
Zau, “Reactive nitrogen species is required for the activation of the AMP-activated
protein kinase by statin in vivo”, J. Biol. Chem. 283 (2008) 20186-20197.
[46] D. W. Crabb, S. Liangpunsakul, “Alcohol and lipid metabolism”, J. Gastroenterol.
Hepatol. 21 (2006) 558-560.
[47] T. M. Donohue, “Alcohol-induced steatosis in liver cells”, World J. Gastroenterol. 13
(2007) 4974-4978.
[48] I.G. Kesova, S. Hoys, S. Thung, A.I. Cederbaum, “Alcohol- induced liver injury in
mice lacking Cu, Zn-superoxide dismutase”, Hepatology. 38 ( 2003) 1136-1145.
[49] G. Szabo, S. Bala, “Alcoholic liver disease and the gut-liver axis”, World J.
Gastroenterol. 16 (11) (2010) 1321-1329.
[50] S.L. Friedman, “Mechanisms of hepatic fibrogenesis”, Gastroenterology 134(6) (2008)
1655-1669.
[51] F. Tacke, R. Weiskirchen, “Update on hepatic stellate cells: pathogenic role in liver
fibrosis and novel isolation techniques”, Expert. Rev. Gastroenterol. Hepatol. 6(1)
(2012) 67-80.
[52] J. Mann, D.A. Mann, “Transcriptional regulation of hepatic stellate cells”, Adv. Drug.
Deliv. Rev. 61(7-8) (2009) 497-512.
[53] S. T. Rashid, J. D. Humphries, A. Byron, A. Dhar, J. A. Askari, J. N. Selley, D. Knight,
R. D. Goldin, M. Thursz, M. J. Humphries, “ Proteomic analysis of extracellular matrix
from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel
constituents of fibrotic liver”, J. Proteome. Res. 3;11(8) (2012) 4052-4064.
[54] J. Ji, F. Yu, Q. Ji, Z. Li, K. Wang, J. Zhang, J. Lu, L. Chen, Q.E.Y. Zeng, Y. Ji,
“Comparative proteomic analysis of rat hepatic stellate cell activation: a comprehensive
view and suppressed immune response”, Hepatology. 56(1) (2012) 332-349.
[55] K. Iwaisako, DA. Brenner, T. Kisseleva, “What's new in liver fibrosis? The origin of
myofibroblasts in liver fibrosis”, J. Gastroenterol. Hepatol. 27 (2012) 65-68.
Received 1 September 2013; accepted in final form 26 September 2013.