Oxidative Stress and Alcoholic Liver Disease

Defeng Wu, Ph.D.,1 and Arthur I. Cederbaum, Ph.D.1

ABSTRACT

Reactive oxygen species (ROS) are highly reactive molecules that are naturally
generated in small amounts during the bodys metabolic reactions and can react with and
damage complex cellular molecules such as lipids, proteins, or DNA. This review describes
pathways involved in ROS formation, why ROS are toxic to cells, and how the liver
protects itself against ROS. Acute and chronic ethanol treatment increases the production
of ROS, lowers cellular antioxidant levels, and enhances oxidative stress in many tissues,

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especially the liver. Ethanol-induced oxidative stress plays a major role in the mechanisms
by which ethanol produces liver injury. Many pathways play a key role in how ethanol
induces oxidative stress. This review summarizes some of the leading pathways and
discusses the evidence for their contribution to alcohol-induced liver injury.

KEYWORDS: Oxidative stress, alcoholic liver injury, reactive oxygen species, free
radicals, antioxidants

A lcohol acts through numerous pathways to
affect the liver and other organs and to lead to the
development of alcoholic liver disease (ALD).13 Many
mechanisms act in concert, reflecting the spectrum of the
organisms response to a myriad of direct and indirect
actions of alcohol. One factor playing a central role in
many pathways of alcohol-induced damage is the exces-
sive generation of molecules called free radicals, which
can result in a state called oxidative stress.46 Reactive
oxygen species (ROS) can damage or cause complete
degradation (i.e., peroxidation) of essential complex mol-
ecules in the cells, including lipids, proteins, and DNA.
Both acute and chronic alcohol exposure can increase
production of ROS and enhance peroxidation of lipids,
protein, and DNA, as has been demonstrated in a variety
of systems, cells, and species, including humans.16
WHAT ARE FREE RADICALS AND
REACTIVE OXYGEN SPECIES?
A free radical is an atom, molecule, or compound that
is highly unstable because of its atomic or molecular
structure (i.e., the distribution of electrons within
the molecule). As a result, free radicals are very
reactive as they attempt to pair up with other mole-
cules or atoms to create a stable compound. The four
primary types of chemical reactions that free radicals
undergo are:

Hydrogen abstraction, in which a radical interacts
with another molecule that has a free hydrogen
atom (i.e., a hydrogen donor). As a result, the
radical binds to the hydrogen atom and becomes
stable, whereas the hydrogen donor is converted to
a free radical.
A
RH ! AH R


Addition, in which the radical binds to another,
originally stable molecule, converting the combined
molecule into a radical
A
A
RCH CHR ! CH-C
HR
R

1
Department of Pharmacology and Systems Therapeutics, Mount
Sinai School of Medicine, New York, New York.
Address for correspondence and reprint requests: Arthur I.
Cederbaum, Ph.D., Department of Pharmacology and Systems
Therapeutics, Mount Sinai School of Medicine, One Gustave L. Levy
Place, Box 1603, New York, NY 10029 (e-mail: arthur. cederbaum
@mssm.edu).
Alcohol and Liver Disease; Guest Editor, Natalia Osna, M.D., Ph.D.
Semin Liver Dis 2009;29:141154. Copyright # 2009 by
Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York,
NY 10001, USA. Tel: +1(212) 584-4662.
DOI 10.1055/s-0029-1214370. ISSN 0272-8087.
141
142 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009

Termination, in which two radicals react with each
other to form a stable compound
A
A
! A2

Disproportionation, in which two identical radicals
react with each other, with one of the radicals donat-
ing an electron to the other so that two different
molecules are formed, each of which is stable
2H



O2 O2 ! O2 H2 O2
Molecular oxygen can accept four electrons, one at
a time, and the corresponding number of protons to
generate two molecules of water. During this process,
different oxygen radicals are successively formed as
intermediate products, including superoxide (O2
); per-
oxide (O2 ), which normally exists in cells as hydrogen
peroxide (H2O2); and the hydroxyl radical (
OH).
Superoxide, peroxide, and the hydroxyl radical are con-
sidered the primary ROS. Because they are unstable and
rapidly react with additional electrons and protons, most
of these ROS are converted to water before they can
damage cells. It has been estimated that only 2 to 3% of
the O2 consumed by the respiratory chain is converted to
ROS.7 Nevertheless, the toxic effects of O2 in biological
systemssuch as the breakdown of lipids, inactivation of
enzymes, introduction of changes (i.e., mutations) in
the DNA, and destruction of cell membranes and,
ultimately, cellsare attributable to the reduction of
O2 to ROS.710
e: e: 2H e: e:
O2! O:
2 ! O2 ! H2 O2 !
H2 O : OH
!
H2 O
H H
WHAT IS OXIDATIVE STRESS?
Under certain conditions, such as acute or chronic
alcohol exposure, ROS production is enhanced and/
or the level or activity of antioxidants is reduced.
The resulting statewhich is characterized by a
disturbance in the balance between ROS production
on one hand and ROS removal and repair of dam-
aged complex molecules on the otheris called
oxidative stress.11 Many processes and factors are
involved in causing alcohol-induced oxidative stress,
including:

Changes in the NAD/NADH ratio in the cell as a
result of alcohol metabolism. Alcohol dehydrogenase
converts alcohol to acetaldehyde, a toxic and reactive
molecule. Aldehyde dehydrogenase converts the ace-
taldehyde to acetate. Each of these reactions leads to
formation of one molecule of NADH, thereby pro-
viding reducing equivalents and thus enhanced activ-
ity of the respiratory chain, including heightened O2
use and ROS formation.

Production of acetaldehyde during alcohol metabo-
lism, which through its interactions with proteins and
lipids also can lead to radical formation and cell
damage

Damage to the mitochondria resulting in decreased
adenosine triphosphate (ATP) production

Effects on cell structure and function caused by
alcohols interactions with either membrane compo-
nents (phospholipids) or enzymes and other proteins

Alcohol-induced oxygen deficiency (i.e., hypoxia),
especially in the pericentral region of the liver
where extra oxygen is required to metabolize
alcohol

Alcohols effects on the immune system, which lead to
altered production of certain signaling molecules,
cytokines, which activate biochemical processes
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Alcohol-induced increase in the ability of the bacte-
rial product endotoxin to enter the bloodstream
and liver, where it can activate certain immune
cells

Alcohol-induced increases in cytochrome P450 2E1
(CYP2E1), which metabolizes alcohol and other
molecules and generates ROS in the process

Alcohol-induced increases in the levels of free iron,
which can promote ROS generation

Effects on antioxidant enzymes and chemicals, partic-
ularly glutathione (GSH)

Biochemical reactions generating an alcohol-derived
radical (i.e., the 1-hydroxyethyl radical [HER])

Conversion of the enzyme xanthine dehydrogenase
into a form called xanthine oxidase, which can gen-
erate ROS
Many of these processes operate concurrently, and
it is likely that several, indeed many, systems contribute
to the ability of alcohol to induce a state of oxidative
stress. More details on some of these pathways will be
discussed below.
SYSTEMS PRODUCING REACTIVE
OXYGEN SPECIES
The major source of ROS production in the cell is the
mitochondrial respiratory chain, which, as described
earlier, utilizes 80 to 90% of the O2 a person consumes.
Thus, even though only a small percentage of that O2 is
converted to ROS, the mitochondrial respiratory chain
in all cells generates most of the ROS produced in the
body.12,13
Another major source of ROS, especially in the
liver, is the cytochrome P450 mixed-function oxidases.
P450s are responsible for removing or detoxifying a
variety of compounds present in our environment and
 OXIDATIVE STRESS AND ALCOHOLIC LIVER DISEASE/WU, CEDERBAUM
ingested (e.g., foods or drugs), including alcohol. Some
P450 enzymes are important for metabolizing fatty
acids, cholesterol, steroids, or bile acids. The biochem-
ical reactions catalyzed by cytochrome P450 use mo-
lecular oxygen and during these reactions small
amounts of ROS are generated. The extent of ROS
generation may vary considerably depending on the
compound to be degraded and on the cytochrome
P450 molecule involved. CYP2E1 is of particular
interest when investigating alcohol-induced oxidative
stress because its activity increases after heavy alcohol
exposure and because CYP2E1 itself also metabolizes
alcohol.14
Under normal physiological conditions, xanthine
oxidase acts as a dehydrogenasethat is, it removes
hydrogen from xanthine or hypoxanthine and transfers
it to NAD, thereby generating NADH. However, under
certain conditions, such as the disruption of blood flow to
a tissue, xanthine dehydrogenase is converted to a ROS-
producing oxidase form. Alcohol consumption also may
promote the conversion of xanthine dehydrogenase to
xanthine oxidase, which can generate ROS, thereby
enhancing oxidative stress.15
Other sources of ROS in the body are macro-
phages and neutrophils, which help defend the body
against invading microorganisms. In this case, however,
ROS production is beneficial and even essential to the
organism because it plays a central role in destroying
foreign pathogens.16 Macrophages and neutrophils con-
tain the NADPH oxidase complex, which, when acti-
vated, generates superoxide radicals and hydrogen
peroxide. Many other cellular oxidative enzymes can
produce ROS including monoamine oxidase, prosta-
glandin synthase, lipoxygenases, and peroxisomal oxi-
dases including the fatty acyl COA oxidase.
Autooxidation of hemoglobin, riboflavin, quinones cat-
echolamines, and transition metals such iron and copper
produce ROS.
Besides the ROS generation that occurs naturally
in the body, we are constantly exposed to environmental
free radicals, in the form of radiation, ultraviolet (UV)
light, smog, and tobacco smoke.
Role of Metals
Most of the systems for the production of ROS des-
cribed above produce superoxide radicals or hydrogen
peroxide. In the presence of certain metals, particularly
free iron or copper ions, a hydroxyl radical, the most
powerful ROS, can be produced via the Fenton or the
metal-catalyzed HaberWeiss reaction.17 These two
chemical reactions appears to account for most of the
hydroxyl radical production in biological systems and
explain, at least in part, why metals such as iron and
copper produce oxidative stress and ROS-induced injury
in cells.
143
PROTECTION AGAINST REACTIVE
OXYGEN SPECIES TOXICITY
Because ROS production is a naturally occurring proc-
ess, a variety of enzymatic and nonenzymatic mecha-
nisms have evolved to protect cells against ROS.11,18 At
least some of these mechanisms are impaired after long-
term alcohol consumption.
Antioxidant enzymes involved in the elimination
of ROS include superoxide dismutases (SODs), catalase,
and glutathione peroxidase. SODs catalyze removal of
superoxide radicals. A copperzinc SOD is present in the
cytosol and in the space between the two membranes
surrounding the mitochondria; a manganese-containing
SOD is present in the mitochondrial matrix. Both of
these enzymes are critical for prevention of ROS-in-
duced toxicity.19 The effects of chronic alcohol exposure
on the cellular content or activity of SODs are contro-
versial, with reports of increases, no changes, or de-
creases, depending on the model, diet, amount, and time
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of alcohol feeding. Studies employing the intragastric
infusion model found decreases in SOD activity in the
liver.20
Catalase and the glutathione peroxidase system
both help to remove hydrogen peroxide. Catalase is
found primarily in peroxisomes; it catalyzes a reaction
between two hydrogen peroxide molecules, resulting in
the formation of water and O2. In addition, catalase can
promote the interaction of hydrogen peroxide with
hydrogen donors so that the hydrogen peroxide can be
converted to one molecule of water, and the reduced
donor becomes oxidized (peroxidatic activity of catalase).
Compounds that can provide these hydrogen atoms
include ethanol and methanol, which are oxidized to
acetaldehyde and formaldehyde, respectively. The glu-
tathione peroxidase system consists of several compo-
nents, including the enzymes glutathione peroxidase and
glutathione reductase and the cofactors GSH and
NADPH. Together, these molecules effectively remove
hydrogen peroxide. GSH is an essential component of
this system and serves as a cofactor for glutathione
transferase, which helps remove certain drugs and chem-
icals as well as other reactive molecules from the cells.
Because of all its functions, GSH is probably the
most important nonenzymatic antioxidant present in
cells. Therefore, enzymes that help generate GSH are
critical to the bodys ability to protect itself against
oxidative stress. Alcohol has been shown to deplete
GSH levels, particularly in the mitochondria, which
normally are characterized by high levels of GSH needed
to eliminate the ROS generated during activity of the
respiratory chain.21 Numerous other nonenzymatic anti-
oxidants are present in the cells, most prominently
vitamin E (a-tocopherol) and vitamin C (ascorbate).
Vitamin E is a major antioxidant found in the lipid phase
of membranes and acts as a powerful terminator of lipid
peroxidation. During the reaction between vitamin E
144 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009
and a lipid radical, the vitamin E radical is formed, from
which vitamin E can be regenerated in a reaction
involving GSH and ascorbate. Alcohol also appears to
interfere with the bodys normal vitamin E content
because patients with ALD commonly exhibit reduced
vitamin E levels.22
The sections below explore in more detail some of
the major mechanisms that are believed to play an
important role in pathways contributing to alcohol-
induced oxidative stress.
KUPFFER CELLS AND ALCOHOLIC LIVER
DISEASE
Kupffer cells are stimulated by chronic ethanol treatment
to produce free radicals and cytokines, including tumor
necrosis factor a (TNFa), which plays a role in ALD.23,24
This stimulation is mediated by bacterial-derived endo-
toxin, and ALD is decreased when gram-negative bacteria
are depleted from the gut by treatment with lactobacillus
or antibiotics.25 Destruction of Kupffer cells with gado-
linium chloride attenuated ALD.23 A major advance was
the finding that anti- TNFa antibodies protect against
ALD.24 NADPH oxidase was identified as a key enzyme
for generating ROS in Kupffer cells after ethanol treat-
ment.26 Moreover, in mice deficient in a subunit of
NADPH oxidase, p47phox, the ethanol-induced increase
in ROS and TNFa and liver injury was decreased.27 The
role of TNFa in ALD was further validated by the
findings that the ethanol-induced pathology was nearly
blocked in TNFa receptor 1 knockout mice.28
The transcription factor nuclear factor-kappaB
(NF-kB) regulates activation of many inflammatory
genes, including TNFa. Endotoxin activates NF-kB,
leading to the hypothesis that inhibition of NF-kB
would prevent ALD.29 Administration of an adenovirus
encoding for the IkB superrepressor to rats chronically
infused with ethanol blunted the ethanol-induced acti-
vation of NF-kB, TNFa production, and pathologic
changes. A general scheme to explain these results is that
chronic ethanol treatment elevates endotoxin levels;
endotoxin activates Kupffer cells to produce free radicals
via NADPH oxidase; the free radicals activate NF-kB,
leading to an increase in production of TNFa, followed
eventually by tissue damage.3032
IRON- AND ALCOHOL-INDUCED
OXIDATIVE STRESS
As discussed above, iron promotes oxidative stress by
catalyzing the conversion of less-reactive oxidants such
as superoxide or H2O2 to more powerful oxidants such as
hydroxyl radical or perferryl-type oxidants. An increase
in hepatic iron concentrations occurs in alcohol-depend-
ent individuals and elevated hepatic iron uptake is seen
in patients with alcohol-induced cirrhosis.33 An increase
in the cellular pool of low-molecular-weight iron occurs
during ethanol metabolism in rat hepatocyte cultures.34
In rats, chronic ethanol feeding for 8 weeks elevated iron
content in the hepatocytes and Kupffer cells.35 Treat-
ment of rats with ethanol plus carbonyl iron strikingly
elevated liver iron levels and produced significant liver
injury.35,36 In the intragastric infusion model, addition
of a small amount of iron, which only elevated hepatic
iron levels 2- to 3-fold, enhanced lipid peroxidation,
serum transaminase levels, and induced fibrosis.37
Ethanol administration elevated the iron content of
Kupffer cells, and this was suggested to prime Kupffer
cells for NF-kB activation and ultimately for TNFa
production and ALD.38 Addition of ferrous but not
ferric increased TNFa release by rat Kupffer cells in
an NF-kB-dependent manner.39 Oral iron chelators
attenuated these effects, reducing the elevations in non-
heme iron, lipid peroxidation, and liver fat accumulation
and injury.38,40
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ROS production, lipid peroxidation, and interac-
tion with iron chelates were enhanced with microsomes
from ethanol-treated rats.41 This was associated with
elevated levels of CYP2E1 and blocked by inhibitors of
CYP2E1 or by anti-CYP2E1 immunoglobulin. In
HepG2 cells expressing CYP2E1, an iron chelator, ferric
nitrilotriacetate, produced greater toxicity than that
found with control HepG2 cells.41 Damage to the
mitochondria played a critical role in the CYP2E1 plus
iron-dependent toxicity. In the CYP2E1-expressing
HepG2 cells, synergistic interactions between iron and
polyunsaturated fatty acids were observed.41 The pro-
ceedings of a symposium on the role of iron in alcoholic
liver disease have been published in the journal Alcohol
(Vol. 30, No. 2, 2003).
1-HYDROXYETHYL RADICAL
Ethanol is a hydroxyl radical scavenger; the product of
the interaction of ethanol with hydroxyl radical is 1-
hydroxyethyl radical (HER). Liver microsomes can
oxidize ethanol to HER in an NADPH-dependent
manner.42 The mechanism involves production of super-
oxide and H2O2 by cytochrome P450, followed by an
iron-catalyzed generation of hydroxyl radical-like oxi-
dants, which interact with ethanol to yield HER.43,44
Microsomes isolated from rats treated chronically with
ethanol were more reactive in producing HER from
ethanol than control microsomes.45 This was due to
induction of CYP2E1. HER production from ethanol
has been demonstrated in vivo, as a spin-trapped HER
adduct was detected in bile from mice or rats treated
with ethanol.46 The role of HER adducts in ALD is not
known. HER binds readily to proteins to produce
ethanol-derived protein adducts, which are immuno-
genic, and production of antibodies that specifically
recognize HER protein adducts was found after chronic
 OXIDATIVE STRESS AND ALCOHOLIC LIVER DISEASE/WU, CEDERBAUM
ethanol consumption,47 as well as in patients with
alcohol-induced cirrhosis.48 Interaction of HER with
cellular antioxidants could contribute to mechanisms by
which ethanol produces a state of oxidative stress.49
GLUTATHIONE
The effects of ethanol on total hepatic GSH levels are
variable, with reports of decreases, no effects, or even an
increase.5052 Lowering of mitochondrial GSH by
chronic ethanol treatment has been a more consistent
observation and appears to be a key factor contributing to
ALD.21 Because liver mitochondria lack catalase, mito-
chondrial GSH in association with glutathione perox-
idase is the major mechanism by which H2O2 is
detoxified by mitochondria. Chronic ethanol intake
either in the LieberDeCarli model or the intragastric
infusion model selectively lowers levels of mitochondrial
GSH in hepatocytes.21,50 Depletion of mitochondrial
GSH by chronic ethanol feeding occurs preferentially in
pericentral hepatocytes, where most of the liver injury
originates.53 This depletion by ethanol is attributable to
defective transport of GSH from the cytosol into the
mitochondria and can be prevented by fluidization of
the mitochondrial membrane by S-adenosylmethionine
(SAM).53,54 Lowering of mitochondrial GSH by
ethanol has been suggested to sensitize hepatocytes to
TNFa-induced cell death, and replenishment of mito-
chondrial GSH with SAM protects hepatocytes from
alcohol-treated rats to TNF toxicity.54 Bailey et al,55
however, found that mitochondrial GSH levels were
increased after chronic ethanol feeding in the Lieber
DeCarli model by 25%. This finding was suggested to
reflect an adaptive response to counteract ethanol-
related increases in mitochondrial production of ROS.
Deaciuc et al56 reported no change in mitochondrial
GSH levels after 7 weeks of ethanol intake. Thus, the
effects of ethanol on mitochondrial GSH, as with total
GSH, remain controversial.
MITOCHONDRIA, OXIDATIVE STRESS,
AND ALD
Chronic ethanol treatment has long been known to
depress mitochondrial function.57 Ethanol impairment
of mitochondrial structure and function may produce an
increase in production of ROS and cause cell toxicity. An
increase occurs in lipid peroxidation-derived products in
mitochondria isolated from chronic ethanol-fed rats.58
An increase also occurs in the content of protein car-
bonyl groups in mitochondrial proteins as compared
with cytosolic proteins after ethanol treatment.55 In-
creased superoxide, H2O2, and hydroxyl radical produc-
tion were observed in mitochondria from ethanol-fed
rats.59,60 The excess of reducing equivalents generated
when ethanol is oxidized by liver alcohol dehydrogenase
145
produces a more-reduced electron transfer chain, which
will facilitate transfer of an electron to molecular oxygen
to produce superoxide.12,61 ROS generation will be
further elevated after chronic ethanol consumption be-
cause of the decreased activity of the respiratory chain,
resulting in accumulation of reduced respiratory carriers
in complexes I and III. Mitochondria contribute to the
increase in oxidant levels in hepatocytes exposed to
ethanol acutely or chronically. An exciting advance in
this area is the use of mitochondrial proteomics to
identify alterations to the mitochondrial proteome in
the development of ALD.62
A single oral dose of ethanol had no effect on
nuclear DNA integrity of mouse liver, whereas hepatic
mitochondrial (mt) DNA was extensively damaged and
depleted/degraded.63 This mtDNA depletion was pre-
vented by 4-methylpyrazole (4-MP), indicating a role
for ethanol metabolism. Daily ethanol administration for
4 days caused a longer-lasting depletion of mtDNA in
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mouse liver, perhaps because of an impaired ability to
synthesize mtDNA.64 This was associated with an
increase in lipid peroxidation and CYP2E1 protein
levels. A so-called common 4,977-base pair mtDNA
deletion was also found to be more prevalent in patients
with alcohol-induced liver disease than in controls.65
Feeding rats the LieberDecarli diet caused a significant
decrease in mtDNA levels after 4 to 6 months, in
association with an increase in 8-hydroxydeoxyguano-
sine (8-OHdG) levels, indicative of an increase in
oxidative modification of mtDNA.66 Despite dramatic
changes in mtDNA elicited by ethanol, significant ALD
is not observed in these models; therefore, the role of
mtDNA deletion and oxidation in the pathophysiology
of ALD remains to be determined.
Incubation of rat liver hepatocytes with
50 mmol/L ethanol increased ROS, largely H2O2
production.67 Depletion of GSH led to enhanced
ROS and loss of hepatocyte viability induced by etha-
nol. Ethanol caused a decline in the mitochondrial
membrane potential and triggered opening of the
mitochondrial permeability pore. These effects were
blocked by an inhibitor of ethanol metabolism
(4-MP) and an antioxidant.68 Interestingly, mitochon-
dria from chronic ethanol-fed rats are more sensitive
to the mitochondrial permeability pore induced by
calcium and other apoptotic stimuli than control mi-
tochondria.69 Reviews on ethanol, mitochondria, and
ROS production have been published.7072
NITROSATIVE STRESS
Depending on conditions, nitric oxide (NO) can be either
hepatoprotective or potentiate liver injury.73 Chronic
ethanol administration increases NO production in rat
liver.74 Peroxynitrite, derived from the interaction of
NO with superoxide, has been suggested to play a role
146 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009
in ethanol-induced hypoxic liver injury.75 The role of NO
in ALD is not clear. An inhibitor of nitric oxide synthase
increased the severity of ALD, whereas L-arginine sup-
plementation completely prevented the liver injury.76,77
The authors concluded that NO was protective against
ALD, although which isoform of nitric oxide synthase
was responsible for the protection was not identified. In
contrast, inducible nitric oxide synthase was shown to be
required for ALD because ethanol toxicity was signifi-
cantly blunted in inducible nitric oxide synthase (iNOS)
knockout mice.78 A similar protection was found if wild-
type mice were treated with a relatively specific iNOS
inhibitor, 1400W.78 However, N(G)-nitro-L-arginine-
methyl ester, a more effective inhibitor of endothelial
nitric oxide synthase than iNOS, enhanced liver dam-
age.78 The concept that NO produced from endothelial
nitric oxide synthase may be protective, whereas NO
derived from Kupffer cell iNOS is critical for ALD,
was advanced to explain the divergent inhibitor results.78
What regulates this balance or distribution of NO
production in ALD is not known.
Chronic ethanol consumption increased the sen-
sitivity of rat liver mitochondrial oxygen consumption to
inhibition by NO.79 Generation of NO from an NO
donor progressively inhibited mitochondrial respiration,
and there was a greater inhibition of respiration in
mitochondria from the ethanol-fed rats. This greater
sensitivity of mitochondria from ethanol-treated rats to
NO was suggested to contribute to ethanol-induced
hypoxia and to depression of the energy state of the
liver.79 Mitochondria isolated from iNOS knockout
mice fed ethanol chronically did not exhibit this in-
creased sensitivity to NO as did mitochondria from
wild-type controls.80 Somewhat surprisingly, ethanol
feeding did not decrease state 3 mitochondrial respira-
tion in mice as it does in rats; this needs to be further
studied because impairment of mitochondrial function is
believed to be an important component in alcohol-
induced liver injury.
CELLULAR REPAIR
The 26S proteosome is important for the catabolism of
damaged proteins produced by oxidative stress for
which improper removal can result in cell toxicity.
Chronic ethanol consumption causes protein accumu-
lation in the liver because of a decreased rate of protein
catabolism.81 Chronic ethanol consumption in the
intragastric infusion model, but not in the oral
LieberDeCarli model, caused a decrease in chymo-
trypsin and trypsin-like activities of the proteosome.82
An inverse correlation was found between proteosomal
chymotrypsin activity and hepatic lipid peroxidation,
suggesting that ethanol-induced oxidative stress can
inactivate the proteosome.83 This may contribute to
the accumulation of proteins, especially oxidized pro-
teins, in the liver. Intragastric ethanol administration
had no effect on proteosome activity in CYP2E1
knockout mice,84 and chlormethiazole (CMZ), a
CYP2E1 inhibitor, prevented the ethanol-mediated
decrease in proteosome activity.85 These results suggest
that CYP2E1-derived oxidant stress plays a role in the
inactivation of the proteosome by ethanol. Another
consequence of the lowering of proteosome activity by
ethanol would be an elevation of CYP2E1 levels,
because CYP2E1 is degraded by the proteosome.86,87
Oxidative DNA adducts, mutagenic apurinic/
apyrimidinic sites, and expression of DNA repair en-
zymes such as 8-oxoguanine DNA glycosylase/lyase 1,
endonuclease 1, polymerase b, and poly (ADP-ribose)
polymerase were elevated by ethanol.88 The latter repair
enzymes are a sensitive marker for oxidative stress-
induced DNA damage. No increase in any of these
endpoints was observed in ethanol-treated CYP2E1
knockout mice, but was observed in NADPH oxidase
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knockout mice.88 The increase in expression of DNA
repair enzymes was abolished by treatment with a broad
spectrum P450 inhibitor. The authors indicated that
CYP2E1 is required for the induction of oxidative stress
to DNA and may play a role in ethanol-associated
hepatocarcinogenesis. These studies emphasize the im-
portance of cellular repair enzymes such as the proteo-
some and DNA repair enzymes in preventing ethanol
toxicity.
ETHANOL-INDUCED PROTEIN ADDUCTS
Reactive aldehydes produced from ethanol metabolism
and ethanol-induced oxidant stress such as acetaldehyde,
malondialdehyde (MDA), and 4-hydroxy-2-nonenal
(HNE) can bind to proteins to produce protein adducts.
The various types of protein-adducts that can be gen-
erated as a result of ethanol consumption have been
summarized and characterized by Niemela.89 Acetalde-
hyde, MDA, and HNE adducts have been found in rats
chronically consuming ethanol, in human alcoholics, and
in a micropig model of ALD.89,90 Such adducts may
produce toxicity because the adducted protein may lose
function. Alternatively, the protein adducts may be
immunogenic and provoke an immune response.91,92
Tuma93 and others94 have characterized the malondial-
dehyde-acetaldehyde (MAA) adduct, which results from
the ability of acetaldehyde and MDA to increase each
others binding to proteins to produce hybrid adducts.
Targeting of MAA-adducted proteins to scavenger re-
ceptors on professional antigen-presenting cells appears
to be a mechanism by which antibody and T cells invoke
an immune response.95 In vitro experiments showed that
the viability of antigen-presenting cells, lymphocytes,
and hepatocytes was decreased on incubation with a
MAA hen egg lysosome adduct by necrotic and apop-
totic modes of cell death. Circulating antibodies against
 OXIDATIVE STRESS AND ALCOHOLIC LIVER DISEASE/WU, CEDERBAUM
MAA protein adducts were increased in patients with
alcohol-induced cirrhosis and hepatitis and correlated
with the severity of liver injury.96
CYTOCHROME P450 2E1
CYP2E1 metabolizes and activates many toxicologically
important compounds such as ethanol, carbon tetrachlor-
ide, acetaminophen, benzene, halothane, and many other
halogenated substrates.14,97 Procarcinogens including ni-
trosamines and azo compounds are effective substrates for
CYP2E1.98 CYP2E1 displays high NADPH oxidase
activity because it appears to be poorly coupled with
NADPH-cytochrome P450 reductase.99 The increase
in ROS production and lipid peroxidation found with
microsomes from chronic ethanol-treated rats was
blocked by chemical inhibitors of CYP2E1 and anti-
CYP2E1 immunoglobulin G.41,99 CYP2E1 is induced
by ethanol, several low-molecular-weight compounds
and under a variety of metabolic, nutritional, and path-
ophysiologic conditions.14,97,100 In the intragastric model
of ethanol feeding, large increases in microsomal lipid
peroxidation have been observed, and the ethanol-
induced liver pathology has been shown to correlate with
CYP2E1 levels and elevated lipid peroxidation.84,101103
Experimentally, a decrease in CYP2E1 induction was
found to be associated with a reduction in alcohol-induced
liver injury.84,104 A CYP2E1 transgenic mouse model was
developed that overexpressed CYP2E1. When treated
with ethanol, the CYP2E1-overexpressing mice displayed
higher transaminase levels and histologic features of
liver injury compared with control mice.105 Conversely,
studies by Thurman and colleagues106,107 suggest that
CYP2E1 may not play a role in alcohol-induced liver
injury. These issues have been discussed elsewhere.108
Clearly, further studies are necessary to resolve the
above discrepancies. As summarized in this review,
several mechanisms contribute to alcohol-induced liver
injury, and ethanol-induced oxidant stress is likely to arise
from several sources, including CYP2E1, mitochondria,
and activated Kupffer cells. Bradford et al88 recently
reported that although NADPH oxidase and not
CYP2E1 was important for ethanol-induced liver injury
in their model, CYP2E1 and not NADPH oxidase was
critical for ethanol-induced oxidative DNA damage and
ethanol-associated hepatocarcinogenesis.
An approach that our laboratory has used to
understand basic effects and actions of CYP2E1 is to
establish HepG2 cell lines that constitutively express
human CYP2E1. We have characterized the toxicity of
ethanol, polyunsaturated fatty acids, iron, the effect of
GSH depletion, and the production of ROS and
development of a state of oxidative stress in these cell
lines. Results are summarized in recent reviews.108,109
Damage to mitochondria by CYP2E1-derived oxidants
is an early event in the overall pathway of cellular
147
toxicity. Similar findings were obtained with primary
hepatocyte cultures after induction of CYP2E1 by
treatment with pyrazole.
LIPOPOLYSACCHARIDE/TNFa-CYP2E1
INTERACTIONS
As discussed above, abnormal cytokine metabolism is a
major feature of ALD. Rats chronically fed ethanol were
more sensitive to the hepatotoxic effects of administra-
tion of lipopolysaccharide (LPS) and had higher plasma
levels of TNFa than control rats.110,111 In the intra-
gastric model of chronic ethanol administration, the
development of liver injury coincided with an increase
in TNFa, associated with an increase in serum LPS.3032
Anti-TNFa antibody prevented alcohol liver injury in
rats24 and mice lacking the TNFR1 receptor did not
develop alcohol liver injury.28 Taken as a whole, these
and other studies clearly implicate TNFa as a major risk
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factor for the development of alcoholic liver injury. One
complication in this central role for TNFa is that
hepatocytes are normally resistant to TNFa induced
toxicity. This led to the hypothesis that besides elevating
TNFa, alcohol somehow sensitizes or primes the liver to
become susceptible to TNFa.112,113 Known factors that
sensitize the liver to TNFa are inhibitors of mRNA or
protein synthesis, which likely prevent the synthesis of
protective factors, inhibition of NF-kB activation to
lower synthesis of such protective factors, depletion of
GSH, especially mitochondrial GSH, lowering of SAM
coupled to elevation of S-adenosylhomocysteine (SAH)
i.e., a decline in the SAM/SAH ratio, or inhibition of
the proteasome. Combined treatment with ethanol plus
TNFa is more toxic to hepatocytes and HepG2 E47
cells, which express high levels of CYP2E1 than are
control hepatocytes with lower levels of CYP2E1 or
HepG2 C34 cells, which do not express CYP2E1.114
RALA hepatocytes with increased expression of
CYP2E1 were sensitized to TNFa  mediated cell
death.115 These results suggest that increased oxidant
stress from CYP2E1 may sensitize isolated hepatocytes
to TNFa-induced toxicity.
Because CYP2E1 and LPS/TNFa are believed to
be key risk factors in the development of alcoholic liver
injury, we evaluated possible interactions in promoting
liver injury between them in vivo.116,117 Sprague-Dawley
rats or C57BL/6 mice were treated with pyrazole (to
induce CYP2E1) in the absence or presence of LPS. The
combination of LPS plus pyrazole treatment resulted in
elevated alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) levels in rats and mice. Liver
injury was confirmed by hematoxylin and eosin (H&E)
staining. LPS alone or pyrazole alone did not elevate
transaminase levels and did not produce liver injury
under these conditions.116,117 Increased 3-nitrotyrosine
protein and 4-HNE adducts were observed after LPS
148 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009
plus pyrazole treatment. The CYP2E1 inhibitor CMZ
protected against the elevation in ALT and AST and
the histopathology changes.117 CYP2E1 knockout
mice obtained from Dr. Frank Gonzalez (National
Cancer Institute, Bethesda, MD) were treated with
pyrazole plus LPS. Compared with SV/129 wild-type
mice, ALT and AST levels were lower in the CYP2E1
null mice, histopathology was normal, and terminal
deoxynucleotidyl transferase-mediated dUTP-biotin
nick end labeling (TUNEL) staining was much
less.117 Western blot analysis confirmed the absence
of CYP2E1 in the CYP2E1 knockout mice. Similar
results were found when pyrazole-treated mice were
challenged with TNFa instead of LPS.118 We hypo-
thesize that increased production of ROS by CYP2E1
may prime or sensitize the liver to LPS/TNFa, and
such interactions may be important in alcohol-induced
liver injury.
ALCOHOL, OXIDATIVE STRESS, AND CELL
INJURY
What is the evidence that alcohol-induced oxidative
stress plays a role in cell injury, particularly damage to
the liver cells? The involvement of oxidative injury in
ethanol toxicity was first proposed by DiLuzio 45 years
ago in studies showing antioxidants could prevent acute
ethanol-induced fatty liver.119 Many studies have dem-
onstrated that alcohol increases lipid peroxidation as well
as the modification of proteins; however, it is not always
clear if these changes are the causes rather than con-
sequences of alcohol-induced tissue injury. Nevertheless,
numerous investigations have found that administering
antioxidants, agents that reduce the levels of free iron, or
agents that replenish GSH levels can prevent or ameli-
orate the toxic actions of alcohol. For example, in the
intragastric infusion model, the antioxidant vitamin E;
the chemical ebselen, which mimics the actions of
glutathione peroxidase; the copperzinc or manganese
SODs; or a GSH precursorall prevented ALD.120123
In the intragastric infusion model, ALD was
associated with enhanced lipid peroxidation, protein
modification, formation of the 1-hydroxyethyl radical
and lipid radicals, and decreases in the hepatic antiox-
idant defense, particularly GSH levels.120123 Moreover,
changes in the animals diets that helped promote or
reduce oxidative stress led to corresponding changes in
the extent of liver injury. For example, when polyunsa-
turated fats were replaced with saturated fats or medium-
chain triglycerides, lipid peroxidation as well as ALD
were reduced or prevented completely, indicating that
both alcohol and polyunsaturated fats must be present for
ALD to occur.124 The extent of the ALD was further
exacerbated when ironwhich, as mentioned earlier, is
required for the generation of the hydroxyl radical and
therefore promotes oxidative stresswas added to these
diets.37,38 Conversely, the addition of antioxidants such
as vitamin E, SOD, or GSH precursors prevented the
development of ALD, as mentioned above.
We investigated the effect of a compromised
antioxidant defense system, copper-zinc superoxide dis-
mutase (Sod1) deficiency on alcohol-induced liver in-
jury.125 C57BL/SV129 wild-type and Sod1 knockout
mice were fed dextrose or ethanol (10% total calories)
liquid diets for 3 weeks. Histologic evaluation of the liver
showed the development of liver injury ranging from
mild to extensive centrilobular necrosis and inflamma-
tion. Alanine aminotransferase levels were elevated only
in the Sod1 knockouts fed ethanol and not in the other
three groups. Hepatic ATP levels were lowered only in
the Sod1 knockout mice fed ethanol and oxidative and
nitrosative stress was found in their livers. Thus, a rather
moderate ethanol consumption promoted oxidative
stress and mitochondrial and liver injury in Sod1 knock-
out mice, an indication that compromised antioxidant
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defense promotes alcohol liver injury.
Studies with liver cells grown in culture also
showed that alcohol can produce oxidative stress and
hepatocyte toxicity.13,54,61,67,68 Studies with hepatocytes
isolated from control rats or from rats fed alcohol
indicated that alcohol metabolism via the enzyme alco-
hol dehydrogenase results in increased ROS production,
hepatocyte injury, and apoptosis. These reactions could
be blocked by antioxidants. Studies using an established
hepatocyte cell line that expresses CYP2E1 demon-
strated that adding alcohol, polyunsaturated fatty acids,
or iron, as well as reducing GSH, resulted in cell toxicity,
increased oxidative stress, and mitochondrial dam-
age.108,109 These reactions could be prevented by admin-
istering antioxidants. HepG2 cells expressing both
CYP2E1 and ADH have been very valuable in studies
on ethanol-induced oxidative stress, cell injury, and
alteration of proteasome activity.126 Taken together,
these findings indicate that alcohol-induced oxidative
stress is a pivotal factor in the development of ALD.
THE ROLE OF OXIDATIVE STRESS IN
HUMAN ALCOHOLISM
There are many reports that various parameters of
oxidative stress are elevated by alcohol in humans.
An early study assaying for the 9,11 linoleic acid
isomer in blood reported that levels were higher in
patients with chronic alcoholism immediately after
withdrawal and was evidence for increased free radical
activity in those suffering from alcoholism.127 Ethane
is a fragmentation product of the lipid peroxidation
cascade and was elevated 5-fold in alcohol abusers.128
The ethane exhalation was correlated to the daily
ethanol intake prior to hospitalization and with stea-
tosis, and was decreased during abstinence. Similarly,
plasma phosphatidylcholine hydroperoxide levels, a
 OXIDATIVE STRESS AND ALCOHOLIC LIVER DISEASE/WU, CEDERBAUM
direct peroxidation product of oxidized polyunsaturated
fatty acids, was 3-fold higher in alcoholic patients than in
controls.129 These levels decreased during abstinence.
HNE, another lipid peroxidation fragmentation product
reacts with proteins to generate HNE-protein adducts.
Strong HNE-protein adduct immunohistochemical
staining was observed in patients with ALD compared
with those with viral liver disease.130 There was a
correlation in the alcoholic patients between HNE
adducts and iron suggesting that the latter catalyze
lipid peroxidation and HNE production. Meagher et
al131 measured urinary excretion of isoprostanes, which
are free radical-catalyzed products of arachidonic acid.
Urinary isoprostanes increased in a time- and dose-
dependent manner in volunteers receiving acute
ethanol. Urinary isoprostanes markedly increased in
patients with acute alcoholic hepatitis and in patients
with cirrhosis. The antioxidant vitamin C reduced
urinary isoprostane generation by alcoholic patients
with liver disease.
Besides lipid peroxidation, there are other indices
of alcohol-induced oxidant stress in humans. Formation
of 2,3-dihydroxybenzoic acid (DHBA) from aspirin is a
hydroxyl radical catalyzed reaction. After intravenous
(IV) administration of aspirin, there was an increase in
DHBA levels in peripheral blood from patients with
alcohol dependence compared with control, suggesting
elevated hydroxyl radical generation.132 Mt DNA dele-
tions reflect oxidative damage to mt DNA. Diverse mt
DNA deletions were observed in alcoholic patients
especially in alcoholic patients with microvesicular stea-
tosis.65 Similar to HNE, HER can react with proteins to
generate HER protein adducts. The sera of alcoholic
patients with cirrhosis contained antibodies that recog-
nized HER-protein adducts; no such antibodies were
found in controls or nonalcoholic patients with cirrho-
sis.48 This suggests formation of HER from ethanol by
alcoholics, and such HER protein adducts can lead to
the development of immunologic reactions. Protein
carbonyls, were elevated in the serum of patients with
chronic alcoholism compared with controls.133
Besides reports of increased oxidative damage to
lipids, mtDNA, and proteins by alcohol, antioxidant
defense appears to be lowered. Depressed serum sele-
nium and vitamin E levels in alcoholic populations
have been reported,134 as well as reduced hepatic a-
tocopherol levels.135 Although many of the above and
other studies show increased oxidative stress in individ-
uals suffering from alcoholism, the functional signifi-
cance of these changes is not known. HNE adducts and
8-OHdG levels were higher in livers from patients with
ALD; the location of the HNE adducts and the nuclear
expression of 8-hydroxydeoxyguanosine was mainly in
hepatocytes with active inflammation.136 The grade of
necroinflammation activity and presence of Mallory
bodies and severity of steatosis were associated with
149
the expression of HNE adducts and 8OHdG. It appears
that lipid peroxidation and oxidative DNA damage
correlate with the severity of steatosis and liver injury,
leading to the conclusion that oxidative stress plays a
major role in the pathogenesis of human alcoholic liver
disease. Markers of lipid peroxidation, antioxidant
status, hepatic fibrogenesis, and liver function were
measured in blood and urine from 24 patients with
established alcoholic liver cirrhosis and 49 matched
controls. In the alcohol group, markers of lipid perox-
idation, 8-isoprostane, and MDA were significantly
increased as was the glutathione disulfide (GSSG)/
GSH ratio, whereas antioxidants selenium, GSH, and
vitamins A, C, and E were decreased. Markers of hepatic
fibrogenesis correlated with the elevated lipid peroxida-
tion and lowered antioxidants.137 Several studies have
shown the presence of antibodies against MDA,
4-HNE, and oxidized phospholipids in serum of patients
with ALD, but not in patients with fatty liver only.138
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Elevations in circulating immunoglobulin (IgG) against
MDA-albumin or oxidized cardiolipin were higher in
heavy drinkers with ALD than in heavy drinkers without
ALD or healthy controls.139 The elevation of these
antibodies was associated with higher circulating levels
of interleukin 2 (IL-2) and TNFa, but not IL-6 or IL-8.
The authors concluded that immune responses toward
oxidative stress-derived antigens promoted TNFa pro-
duction and contributed to liver damage in alcohol
abusers. Albano has recently reviewed free radical mech-
anisms in immune reactions associated with ALD.140
CONCLUSION
Alcohol-induced liver injury is probably a multifactorial
process involving several mechanisms. Future studies are
required to clarify further how alcohol produces oxida-
tive stress in various tissues. What is the role of ethanol
metabolism or ethanol metabolites like acetaldehyde in
the production of ROS, and how is oxidative stress
produced by ethanol in tissues with limited ethanol
metabolism? What are the priming or sensitizing factors
for ethanol-induced oxidant stress and cell injury? Can
markers predictive of individuals particularly sensitive to
ethanol-induced oxidant stress and liver injury be devel-
oped? Alcoholnutritional interactions require further
study, especially interactions with prooxidants, such as
iron; polyunsaturated fatty acids; or reagents that lower
oxidant defense, e.g., lower GSH levels. There is much
current interest in synergistic interactions between alco-
hol and hepatitis B or hepatitis C virus, especially with
respect to generating oxidative stress. The ability of
alcohol to promote oxidative stress and the role of free
radicals in alcohol-induced tissue injury clearly are im-
portant areas of research, particularly because such in-
formation may be of major therapeutic significance in
attempts to prevent or ameliorate alcohols toxic effects,
150 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009

e.g., by antioxidants, iron chelators, inhibitors of 7. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in
CYP2E1 or of cytokine production/actions, and GSH mammalian organisms. Physiol Rev 1979;59:527605
replenishment. As basic information continues to 8. Toyokuni S. Reactive oxygen speciesinduced molecular
damage and its application in pathology. Pathol Int 1999;
emerge regarding the role of oxidative stress in disease
49:91102
development and the mechanisms underlying ROS- 9. de Groot H. Reactive oxygen species in tissue injury.
related cellular toxicity, these findings will lead to Hepatogastroenterology 1994;41:328332
more rational antioxidant therapeutic approaches. 10. Nakazawa H, Genka C, Fujishima M. Pathological
aspects of active oxygens/free radicals. Jpn J Physiol
1996;46:1532
ABBREVIATIONS 11. Halliwell B. Antioxidant defense mechanisms: From the
1-HER 1-hydroxyethyl radical beginning to the end. Free Radic Res 1999;31:261272
4-MP 4-methylpyrazole 12. Bailey SM, Cunningham CC. Contribution of mitochon-
dria to oxidative stress associated with alcoholic liver disease.
8-OHdG 8-hydroxydeoxyguanosine
Free Radic Biol Med 2002;32:1116
ADH alcohol dehydrogenase 13. Adachi M, Ishii H. Role of mitochondria in alcoholic liver
ALD alcoholic liver disease injury. Free Radic Biol Med 2002;32:487491
ALT alanine aminotransferase 14. Lieber CS. Cytochrome P450 2E1: Its physiological and
AST aspartate aminotransferase pathological role. Physiol Rev 1997;77:517544
ATP adenosine triphosphate 15. Sultatos LG. Effects of acute ethanol administration on the
CMZ chlormethiazole hepatic xanthine dehydrogenase/xanthine oxidase system in
the rat. J Pharmacol Exp Ther 1988;246:946949

Downloaded by: East Carolina University. Copyrighted material.

CYP2E1 cytochrome P450 2E1 16. Rosen GM, Pou S, Ramos CL, et al. Free radicals and
DHBA 2,3-dihydroxy-benzoic acid phagocytic cells. FASEB J 1995;9:200209
GSSG glutathione disulfide 17. McCord JM. Iron, free radicals, and oxidative injury. Semin
GSH glutathione Hematol 1998;35:512
HNE 4-hydroxynonenal 18. Yu BP. Cellular defenses against damage from reactive
IgG immunoglobulin oxygen species. Physiol Rev 1994;74:139162
IL interleukin 19. Fridovich I. Superoxide anion radical, superoxide dismu-
tases, and related matters. J Biol Chem 1997;272:18515
iNOS inducible nitric oxide synthase 18517
LPS lipopolysaccharide 20. Polavarapu R, Spitz DR, Sim JE, et al. Increased lipid
MAA malondialdehyde-acetaldehyde adduct peroxidation and impaired antioxidant enzyme function is
MDA malondialdehyde associated with pathological liver injury in experimental
MT mitochondrial alcoholic liver disease in rats fed diets high in corn oil and
NF-kB nuclear factor kappa B fish oil. Hepatology 1998;27:13171323
NO nitric oxide 21. FernandezCheca JC, Kaplowitz N, Colell A, GarcaRuiz
C. Oxidative stress and alcoholic liver disease. Alcohol
OH hydroxyl radical Health Res World 1997;21:321324
ROS reactive oxygen species 22. Nanji AA, HillerSturmhofel S. Apoptosis and necrosis.
SAH S-adenosylhomocysteine Alcohol Health Res World 1997;21:325330
SAM S-adenosylmethionine 23. Adachi Y, Bradford BU, Gao W, Bojes HK, Thurman RG.
SOD superoxide dismutase Inactivation of Kupffer cells prevents early alcohol-induced
TNF tumor necrosis factor liver injury. Hepatology 1994;20:453460
24. Iimuro Y, Gallucci RM, Luster MI, Kono H, Thurman
RG. Antibodies to tumor necrosis factor alpha attenuate
REFERENCES hepatic necrosis and inflammation caused by chronic
exposure to ethanol in the rat. Hepatology 1997;26:1530
1. Nordmann R, Ribiere C, Rouach H. Implication of free 1537
radical mechanisms in ethanol-induced cellular injury. Free 25. Nanji AA, Khettry U, Sadrzadeh SM. Lactobacillus
Radic Biol Med 1992;12:219240 feeding reduces endotoxemia and severity of experimental
2. Bondy SC. Ethanol toxicity and oxidative stress. Toxicol alcoholic liver disease. Proc Soc Exp Biol Med 1994;205:
Lett 1992;63:231241 243247
3. Cederbaum AI. Introduction-serial review: alcohol, oxidative 26. Kono H, Rusyn I, Uesugi T, et al. Diphenyleneiodonium
stress and cell injury. Free Radic Biol Med 2001;31:1524 sulfate, an NADPH oxidase inhibitor, prevents early
1526 alcohol-induced liver injury in the rat. Am J Physiol
4. Tsukamoto H, Lu SC. Current concepts in the pathogenesis Gastrointest Liver Physiol 2001;280:G1005G1012
of alcoholic liver injury. FASEB J 2001;15:13351349 27. Kono H, Rusyn I, Yin M, et al. NADPH oxidase-derived
5. Ishii H, Kurose I, Kato S. Pathogenesis of alcoholic liver free radicals are key oxidants in alcohol-induced liver
disease with particular emphasis on oxidative stress. disease. J Clin Invest 2000;106:867872
J Gastroenterol Hepatol 1997;12:S272S282 28. Yin M, Wheeler MD, Kono H, et al. Essential role of tumor
6. Arteel GE. Oxidants and antioxidants in alcohol-induced necrosis factor alpha in alcohol-induced liver injury in mice.
liver disease. Gastroenterology 2003;124:778790 Gastroenterology 1999;117:942952
 OXIDATIVE STRESS AND ALCOHOLIC LIVER DISEASE/WU, CEDERBAUM
29. Uesugi T, Froh M, Arteel GE, et al. Delivery of IkappaB
superrepressor gene with adenovirus reduces early alcohol
induced liver injury in rats. Hepatology 2001;34:11491157
30. Thurman RG. Mechanisms of hepatic toxicity. II. Alcoholic
liver injury involves activation of Kupffer cells by endotoxin.
Am J Physiol 1998;275:G605G611
31. Wheeler MD, Kono H, Yin M, et al. The role of Kupffer
cell oxidant production in early ethanol-induced liver
disease. Free Radic Biol Med 2001;31:15441549
32. Takei Y, Arteel GE, Bergheim I, et al. Roles of Kupffer cells
in alcoholic liver disease. Alcohol Clin Exp Res 2006;29:
11161120
33. Chapman RW, Morgan MY, Bell R, Sherlock S. Hepatic
iron uptake in alcoholic liver disease. Gastroenterology
1983;84:143147
34. Sergent O, Morel I, Cogrel P, et al. Increase in cellular pool
of low-molecular-weight iron during ethanol metabolism in
rat hepatocyte cultures: Relationship with lipid peroxida-
tion. Biol Trace Elem Res 1995;47:185192
35. Valerio LG Jr, Parks T, Petersen DR. Alcohol mediates
increases in hepatic and serum nonheme iron stores in a rat
model for alcohol-induced liver injury. Alcohol Clin Exp
Res 1996;20:13521361
36. Stal P, Johansson I, Ingelman-Sundberg M, Hagen K,
Hultcrantz R. Hepatotoxicity induced by iron overload and
alcohol: studies on the role of chelatable iron, cytochrome
P450 2E1 and lipid peroxidation. J Hepatol 1996;25:538
546
37. Tsukamoto H, Horne W, Kamimura S, et al. Experimental
liver cirrhosis induced by alcohol and iron. J Clin Invest
1995;96:620630
38. Tsukamoto H, Lin M, Ohata M, et al. Iron primes hepatic
macrophages for NF-kappaB activation in alcoholic liver
injury. Am J Physiol 1999;277:G1240G1250
39. She H, Xiong S, Lin M, et al. Iron activates NF-kappaB in
Kupffer cells. Am J Physiol Gastrointest Liver Physiol
2002;283:G719G726
40. Sadrzadeh SM, Nanji AA, Price PL. The oral iron chelator,
1,2-dimethyl-3-hydroxypyrid-4-one reduces hepatic-free
iron, lipid peroxidation and fat accumulation in chronically
ethanol-fed rats. J Pharmacol Exp Ther 1994;269:632636
41. Cederbaum AI. Iron and CYP2E1-dependent oxidative
stress and toxicity. Alcohol 2003;30:115120
42. Albano E, Tomasi A, Goria-Gatti L, Dianzani MU. Spin
trapping of free radical species produced during the
microsomal metabolism of ethanol. Chem Biol Interact
1988;65:223234
43. Knecht KT, Thurman RG, Mason RP. Role of superoxide
and trace transition metals in the production of alpha-
hydroxyethyl radical from ethanol by microsomes from
alcohol dehydrogenase-deficient deer mice. Arch Biochem
Biophys 1993;303:339348
44. Rashba-Step J, Cederbaum AI. Generation of reactive
oxygen intermediates by human liver microsomes in the
presence of NADPH or NADH. Mol Pharmacol 1994;45:
150157
45. Albano E, Tomasi A, Persson JO, et al. Role of ethanol-
inducible cytochrome P450 (P450IIE1) in catalysing the
free radical activation of aliphatic alcohols. Biochem
Pharmacol 1991;41:18951902
46. Knecht KT, Bradford BU, Mason RP, Thurman RG. In
vivo formation of a free radical metabolite of ethanol. Mol
Pharmacol 1990;38:2630
151
47. Moncada C, Torres V, Varghese G, Albano E, Israel Y.
Ethanol-derived immunoreactive species formed by free
radical mechanisms. Mol Pharmacol 1994;46:786791
48. Clot P, Bellomo G, Tabone M, Arico S, Albano E.
Detection of antibodies against proteins modified by
hydroxyethyl free radicals in patients with alcoholic
cirrhosis. Gastroenterology 1995;108:201207
49. Reinke LA. Spin trapping evidence for alcohol-associated
oxidative stress. Free Radic Biol Med 2002;32:953957
50. Fernandez-Checa JC, Ookhtens M, Kaplowitz N. Effects of
chronic ethanol feeding on rat hepatocystic glutathione:
relationship of cytosolic glutathione to efflux and mitochon-
drial sequestration. J Clin Invest 1989;83:12471252
51. Iimuro Y, Bradford BU, Yamashina S, et al. The
glutathione precursor L-2-oxothiazolidine-4-carboxylic acid
protects against liver injury due to chronic enteral ethanol
exposure in the rat. Hepatology 2000;31:391398
52. Oh SI, Kim CI, Chun HJ, Park SC. Chronic ethanol
consumption affects glutathione status in rat liver. J Nutr
1998;128:758763
53. Garcia-Ruiz C, Morales A, Colell A, et al. Feeding S-
adenosyl-L-methionine attenuates both ethanol-induced
Downloaded by: East Carolina University. Copyrighted material.
depletion of mitochondrial glutathione and mitochondrial
dysfunction in periportal and perivenous rat hepatocytes.
Hepatology 1995;21:207214
54. Colell A, Garcia-Ruiz C, Miranda M, et al. Selective
glutathione depletion of mitochondria by ethanol sensitizes
hepatocytes to tumor necrosis factor. Gastroenterology 1998;
115:15411551
55. Bailey SM, Patel VB, Young TA, Asayama K, Cunningham
CC. Chronic ethanol consumption alters the glutathione/
glutathione peroxidase-1 system and protein oxidation
status in rat liver. Alcohol Clin Exp Res 2001;25:726733
56. Deaciuc IV, Fortunato F, DSouza NB, et al. Modulation of
caspase-3 activity and Fas ligand mRNA expression in rat
liver cells in vivo by alcohol and lipopolysaccharide. Alcohol
Clin Exp Res 1999;23:349356
57. Cunningham CC, Coleman WB, Spach PI. The effects of
chronic ethanol consumption on hepatic mitochondrial
energy metabolism. Alcohol Alcohol 1990;25:127136
58. Sastre J, Manana JB, Alguacil P, et al. Chronic ethanol
feeding causes oxidative stress in rat liver mitochondria:
prevention by S-adenosyl methionine. Free Radic Res 1999;
30:325327
59. Boveris A, Fraga CG, Varsavsky AI, Koch OR. Increased
chemiluminescence and superoxide production in the liver of
chronically ethanol-treated rats. Arch Biochem Biophys
1983;227:534541
60. Kukielka E, Dicker E, Cederbaum AI. Increased production
of reactive oxygen species by rat liver mitochondria after
chronic ethanol treatment. Arch Biochem Biophys 1994;309:
377386
61. Bailey SM, Cunningham CC. Acute and chronic ethanol
increases reactive oxygen species generation and decreases
viability in fresh, isolated rat hepatocytes. Hepatology 1998;
28:13181326
62. Venkatraman A, Landar A, Davis AJ, et al. Modification of
the mitochondrial proteome in response to the stress of
ethanol-dependent hepatotoxicity. J Biol Chem 2004;279:
2209222101
63. Mansouri A, Gaou I, De Kerguenec C, et al. An alcoholic
binge causes massive degradation of hepatic mitochondrial
DNA in mice. Gastroenterology 1999;117:181190
152 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009
64. Demeilliers C, Maisonneuve C, Grodet A, et al. Impaired
adaptive resynthesis and prolonged depletion of hepatic
mitochondrial DNA after repeated alcohol binges in mice.
Gastroenterology 2002;123:12781290
65. Mansouri A, Fromenty B, Berson A, et al. Multiple hepatic
mitochondrial DNA deletions suggest premature oxidative
aging in alcoholic patients. J Hepatol 1997;27:96102
66. Cahill A, Stabley GJ, Wang X, Hoek JB. Chronic ethanol
consumption causes alterations in the structural integrity of
mitochondrial DNA in aged rats. Hepatology 1999;30:881
888
67. Kurose I, Higuchi H, Kato S, et al. Oxidative stress on
mitochondria and cell membrane of cultured rat hepatocytes
and perfused liver exposed to ethanol. Gastroenterology
1997;112:13311343
68. Higuchi H, Adachi M, Miura S, Gores GJ, Ishii H. The
mitochondrial permeability transition contributes to acute
ethanol-induced apoptosis in rat hepatocytes. Hepatology
2001;34:320328
69. Pastorino JG, Marcineviciute A, Cahill A, Hoek JB.
Potentiation by chronic ethanol treatment of the mitochon-
drial permeability transition. Biochem Biophys Res Com-
mun 1999;265:405409
70. Cahill A, Cunningham CC, Adachi M, et al. Effects of
alcohol and oxidative stress on liver pathology: the role
of the mitochondrion. Alcohol Clin Exp Res 2002;26:
907915
71. Hoek JB, Cahill A, Pastorino JG. Alcohol and mitochondria:
a dysfunctional relationship. Gastroenterology 2002;122:
20492063
72. Bailey SM. A review of the role of reactive oxygen and
nitrogen species in alcohol-induced mitochondrial dysfunc-
tion. Free Radic Res 2003;37:585596
73. Hon WM, Lee KH, Khoo HE. Nitric oxide in liver
diseases: friend, foe, or just passerby? Ann N Y Acad Sci
2002;962:275295
74. Wang JF, Greenberg SS, Spitzer JJ. Chronic alcohol
administration stimulates nitric oxide formation in the rat
liver with or without pretreatment by lipopolysaccharide.
Alcohol Clin Exp Res 1995;19:387393
75. Arteel GE, Kadiiska MB, Rusyn I, et al. Oxidative stress
occurs in perfused rat liver at low oxygen tension by
mechanisms involving peroxynitrite. Mol Pharmacol
1999;55:708715
76. Nanji AA, Greenberg SS, Tahan SR, et al. Nitric oxide
production in experimental alcoholic liver disease in the rat:
role in protection from injury. Gastroenterology 1995;109:
899907
77. Nanji AA, Jokelainen K, Lau GK, et al. Arginine reverses
ethanol-induced inflammatory and fibrotic changes in liver
despite continued ethanol administration. J Pharmacol Exp
Ther 2001;299:832839
78. McKim SE, Gabele E, Isayama F, et al. Inducible nitric
oxide synthase is required in alcohol-induced liver injury:
studies with knockout mice. Gastroenterology 2003;125:
18341844
79. Venkatraman A, Shiva S, Davis AJ, et al. Chronic alcohol
consumption increases the sensitivity of rat liver mitochon-
drial respiration to inhibition by nitric oxide. Hepatology
2003;38:141147
80. Venkatraman A, Shiva S, Wigley A, et al. The role of iNOS
in alcohol-dependent hepatotoxicity and mitochondrial
dysfunction in mice. Hepatology 2004;40:565573
81. Donohue TM Jr, Zetterman RK, Tuma DJ. Effect of
chronic ethanol administration on protein catabolism in rat
liver. Alcohol Clin Exp Res 1989;13:4957
82. Donohue TM Jr, Zetterman RK, Zhang-Gouillon ZQ,
French SW. Peptidase activities of the multicatalytic
protease in rat liver after voluntary and intragastric ethanol
administration. Hepatology 1998;28:486491
83. Fataccioli V, Andraud E, Gentil M, French SW, Rouach H.
Effects of chronic ethanol administration on rat liver
proteasome activities: relationship with oxidative stress.
Hepatology 1999;29:1420
84. Bardag-Gorce F, Yuan QX, Li J, et al. The effect of
ethanol-induced cytochrome p4502E1 on the inhibition of
proteasome activity by alcohol. Biochem Biophys Res
Commun 2000;279:2329
85. Gouillon Z, Lucas D, Li J, et al. Inhibition of ethanol-
induced liver disease in the intragastric feeding rat model
by chlormethiazole. Proc Soc Exp Biol Med 2000;224:
302308
86. Goasduff T, Cederbaum AI. NADPH-dependent micro-
somal electron transfer increases degradation of CYP2E1 by
the proteasome complex: role of reactive oxygen species.
Downloaded by: East Carolina University. Copyrighted material.
Arch Biochem Biophys 1999;370:258270
87. Roberts BJ. Evidence of proteasome-mediated cytochrome
P-450 degradation. J Biol Chem 1997;272:97719778
88. Bradford BU, Kono H, Isayama F, et al. Cytochrome P450
CYP2E1, but not nicotinamide adenine dinucleotide
phosphate oxidase, is required for ethanol-induced oxidative
DNA damage in rodent liver. Hepatology 2005;41:336344
89. Niemela O. Distribution of ethanol-induced protein adducts
in vivo: relationship to tissue injury. Free Radic Biol Med
2001;31:15331538
90. Niemela O, Parkkila S, Yla-Herttuala S, et al. Sequential
acetaldehyde production, lipid peroxidation, and fibro-
genesis in micropig model of alcohol-induced liver disease.
Hepatology 1995;22:12081214
91. Albano E. Free radical mechanisms in immune reactions
associated with alcoholic liver disease. Free Radic Biol Med
2002;32:110114
92. Duryee MJ, Willis MS, Freeman TL, et al. Mechanisms of
alcohol liver damage: aldehydes, scavenger receptors, and
autoimmunity. Front Biosci 2004;9:31453155
93. Tuma DJ. Role of malondialdehyde-acetaldehyde adducts in
liver injury. Free Radic Biol Med 2002;32:303308
94. Worrall S, de Jersey J, Wilce PA. Comparison of the
formation of proteins modified by direct and indirect
ethanol metabolites in the liver and blood of rats fed
the Lieber-DeCarli liquid diet. Alcohol Alcohol 2000;35:
164170
95. Willis MS, Klassen LW, Tuma DJ, Sorrell MF, Thiele
GM. Adduction of soluble proteins with malondialdehyde-
acetaldehyde (MAA) induces antibody production and
enhances T-cell proliferation. Alcohol Clin Exp Res 2002;
26:94106
96. Rolla R, Vay D, Mottaran E, et al. Detection of circulating
antibodies against malondialdehyde-acetaldehyde adducts in
patients with alcohol-induced liver disease. Hepatology
2000;31:878884
97. Koop DR. Oxidative and reductive metabolism by cyto-
chrome P450 2E1. FASEB J 1992;6:724730
98. Yang CS, Yoo JS, Ishizaki H, Hong JY. Cytochrome
P450IIE1: roles in nitrosamine metabolism and mechanisms
of regulation. Drug Metab Rev 1990;22:147159
 OXIDATIVE STRESS AND ALCOHOLIC LIVER DISEASE/WU, CEDERBAUM
99. Ekstrom G, Ingelman-Sundberg M. Rat liver microsomal
NADPH-supported oxidase activity and lipid peroxidation
dependent on ethanol inducible cytochrome P-450 (P-
450IIE1). Biochem Pharmacol 1989;38:13131319
100. Song BJ, Cederbaum AI, Koop DR, Ingelman-Sundberg M,
Nanji A. Ethanol-inducible cytochrome P450 (CYP2E1):
biochemistry, molecular biology and clinical relevance.
Alcohol Clin Exp Res 1996;20:138A146A
101. Castillo T, Koop DR, Kamimura S, Triadafilopoulos G,
Tsukamoto H. Role of cytochrome P-450 2E1 in ethanol-,
carbon tetrachloride- and iron-dependent microsomal lipid
peroxidation. Hepatology 1992;16:992996
102. Nanji AA, Zhao S, Sadrzadeh SM, et al. Markedly
enhanced cytochrome P450 2E1 induction and lipid
peroxidation is associated with severe liver injury in fish
oil-ethanol-fed rats. Alcohol Clin Exp Res 1994;18:1280
1285
103. French SW, Wong K, Jui L, et al. Effect of ethanol on
cytochrome P450 2E1 (CYP2E1), lipid peroxidation, and
serum protein adduct formation in relation to liver
pathology pathogenesis. Exp Mol Pathol 1993;58:6175
104. Morimoto M, Hagbjork AL, Wan YJ, et al. Modulation of
experimental alcohol-induced liver disease by cytochrome
P450 2E1 inhibitors. Hepatology 1995;21:16101617
105. Morgan K, French SW, Morgan TR. Production of a
cytochrome P450 2E1 transgenic mouse and initial evalua-
tion of alcoholic liver damage. Hepatology 2002;36:122134
106. Koop DR, Klopfenstein B, Iimuro Y, Thurman RG.
Gadolinium chloride blocks alcohol-dependent liver tox-
icity in rats treated chronically with intragastric alcohol
despite the induction of CYP2E1. Mol Pharmacol 1997;51:
944950
107. Kono H, Bradford BU, Yin M, et al. CYP2E1 is not
involved in early alcohol-induced liver injury. Am J Physiol
1999;277:G1259G1267
108. Caro AA, Cederbaum AI. Oxidative stress, toxicology, and
pharmacology of CYP2E1. Annu Rev Pharmacol Toxicol
2004;44:2742
109. Kessova I, Cederbaum AI. CYP2E1: biochemistry, toxicol-
ogy, regulation and function in ethanol-induced liver injury.
Curr Mol Med 2003;3:509518
110. Honchel R, Ray MB, Marsano L, et al. Tumor necrosis
factor in alcohol enhanced endotoxin liver injury. Alcohol
Clin Exp Res 1992;16:665669
111. Kamimura S, Tsukamoto H. Cytokine gene expression by
Kupffer cells in experimental alcoholic liver disease.
Hepatology 1995;22:13041309
112. Tsukamoto H. How is the liver primed or sensitized for
alcoholic liver disease? Alcohol Clin Exp Res 2001;25:
171S181S
113. Purohit V, Brenner DA. Mechanisms of alcohol-induced
hepatic fibrosis: a summary of the Ron Thurman Sympo-
sium. Hepatology 2006;43:872878
114. Pastorino JG, Hoek JB. Ethanol potentiates tumor necrosis
factor-alpha cytotoxicity in hepatoma cells and primary rat
hepatocytes by promoting induction of the mitochondrial
permeability transition. Hepatology 2000;31:11411152
115. Liu H, Jones BE, Bradham C, Czaja MJ. Increased
cytochrome P-450 2E1 expression sensitizes hepatocytes
to c-Jun-mediated cell death from TNF-alpha. Am J
Physiol Gastrointest Liver Physiol 2002;282:G257G266
116. Lu Y, Wang X, Cederbaum AI. Lipopolysaccharide-
induced liver injury in rats treated with the CYP2E1
153
inducer pyrazole. Am J Physiol Gastrointest Liver Physiol
2005;289:G308G319
117. Lu Y, Cederbaum AI. Enhancement by pyrazole of
lipopolysaccharide-induced liver injury in mice: role of
cytochrome P450 2E1 and 2A5. Hepatology 2006;44:263
274
118. Wu D, Cederbaum AI. Cytochrome P4502E1 sensitizes to
tumor necrosis factor alpha-induced liver injury through
activation of mitogen-activeted protein kineses in mice.
Hepatology 2008;47:10051017
119. Di Luzio NR, Hartman AD. Role of lipid peroxidation in
the pathogenesis of the ethanol-induced fatty liver. Fed Proc
1967;26:14361442
120. Nanji AA, Yang EK, Fogt F, Sadrzadeh SM, Dannenberg
AJ. Medium chain triglycerides and vitamin E reduce the
severity of established experimental alcoholic liver disease. J
Pharmacol Exp Ther 1996;277:16941700
121. Kono H, Arteel GE, Rusyn I, Sies H, Thurman RG.
Ebselen prevents early alcohol-induced liver injury in rats.
Free Radic Biol Med 2001;30:403411
122. Iimuro Y, Bradford BU, Yamashina S, et al. The
glutathione precursor L-2-oxothiazolidine-4-carboxylic
Downloaded by: East Carolina University. Copyrighted material.
acid protects against liver injury due to chronic enteral
ethanol exposure in the rat. Hepatology 2000;31:391
398
123. Wheeler MD, Kono H, Yin M, et al. Delivery of the Cu/Zn-
superoxide dismutase gene with adenovirus reduces early
alcohol-induced liver injury in rats. Gastroenterology 2001;
120:12411250
124. Nanji AA, Jokelainen K, Tipoe GL, Rahemtulla A,
Dannenberg AJ. Dietary saturated fatty acids reverse
inflammatory and fibrotic changes in rat liver despite
continued ethanol administration. J Pharmacol Exp Ther
2001;299:638644
125. Kessova IG, Ho Y, Thung S, Cederbaum AI. Alcohol-
induced liver injury in mice lacking Cu, Zn- superoxide
dismutase. Hepatology 2003;38:11361145
126. Clemens DL. Use of cultured cells to study alcohol
metabolism. Alcohol Res Health 2006;29:291295
127. Fink R, Clemens MR, Marjot DH, et al. Increased free-
radical activity in alcoholics. Lancet 1985;2:291294
128. Letteron P, Duchatelle V, Berson A, et al. Increased ethane
exhalation, an in vivo index of lipid peroxidation, in alcohol-
abusers. Gut 1993;34:409414
129. Adachi J, Matsushita S, Yoshioka N, et al. Plasma
phosphatidylcholine hydroperoxide as a new marker of
oxidative stress in alcoholic patients. J Lipid Res 2004;
45:967971
130. Ohhira M, Ohtake T, Matsumoto A, et al. Immunohis-
tochemical detection of 4-hydroxy-2-nonenal-modified-
protein adducts in human alcoholic liver diseases. Alcohol
Clin Exp Res 1998;22:145S149S
131. Meagher EA, Barry OP, Burke A, et al. Alcohol-induced
generation of lipid peroxidation products in humans. J Clin
Invest 1999;104:805813
132. Thome J, Zhang J, Davids E, et al. Evidence for increased
oxidative stress in alcohol-dependent patients provided by
quantification of in vivo salicylate hydroxylation products.
Alcohol Clin Exp Res 1997;21:8285
133. Mutlu-Turkoglu U, Dogru-Abbasoglu S, Aykac-Toker G,
et al. Increased lipid and protein oxidation and DNA
damage in patients with chronic alcoholism. J Lab Clin Med
2000;135:287291
154 SEMINARS IN LIVER DISEASE/VOLUME 29, NUMBER 2 2009
134. Tanner AR, Bantock I, Hinks L, et al. Depressed
selenium and vitamin E levels in an alcoholic population:
possible relationship to hepatic injury through in-
creased lipid peroxidation. Dig Dis Sci 1986;31:1307
1312
135. Bell H, Bjorneboe A, Eidsvoll B, et al. Reduced concen-
tration of hepatic a-tocopherol in patients with alcoholic
cirrhosis. Alcohol Alcohol 1992;27:3946
136. Seki S, Kitada T, Sakaguchi H, Nakatoni K, Wakasa K.
Pathological significance of oxidative cellular damage in
human alcoholic liver disease. Histopathology 2003;42:
365371
137. Pemberton PW, Smith A, Warnes TW. Non-invasive
monitoring of oxidant stress in alcoholic liver disease. Scand
J Gastroenterol 2005;40:11021108
138. Rolla R, Vay D, Mottaran E, et al. Anti-phospholipid
antibodies associated with alcoholic liver disease specifically
recognize oxidized phospholipids. Gut 2001;49:852859
139. Vidali M, Hietale J, Occhino G, et al. Immune responses
against oxidative stress-derived antigens are associated with
increased circulating tumor necrosis factor-a in heavy
drinkers. Free Radic Biol Med 2008;45:306311
140. Albano E. Alcohol, oxidative stress and free radical damage.
Proc Nutr Soc 2006;65:278290
Downloaded by: East Carolina University. Copyrighted material.