International Journal of

Molecular Sciences

Review
Pathophysiological Aspects of Alcohol Metabolism in the Liver
Jeongeun Hyun 1,2,3 , Jinsol Han 4 , Chanbin Lee 4 , Myunghee Yoon 5 and Youngmi Jung 4,6, *

1 Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 31116, Korea;
[email protected]
2 Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative
Medicine, Dankook University, Cheonan 31116, Korea
3 Department of Regenerative Dental Medicine, College of Dentistry, Dankook University,
Cheonan 31116, Korea
4 Department of Integrated Biological Science, Pusan National University, Pusan 46241, Korea;
[email protected] (J.H.); [email protected] (C.L.)
5 Department of Surgery, Division of Hepatobiliary and Pancreas Surgery, Biomedical Research Institute,
Pusan National University, Pusan 46241, Korea; [email protected]
6 Department of Biological Sciences, Pusan National University, Pusan 46241, Korea
* Correspondence: [email protected]; Tel.: +82-51-510-2262

Abstract: Alcoholic liver disease (ALD) is a globally prevalent chronic liver disease caused by chronic
or binge consumption of alcohol. The liver is the major organ that metabolizes alcohol; therefore,
it is particularly sensitive to alcohol intake. Metabolites and byproducts generated during alcohol
metabolism cause liver damage, leading to ALD via several mechanisms, such as impairing lipid
metabolism, intensifying inflammatory reactions, and inducing fibrosis. Despite the severity of ALD,
the development of novel treatments has been hampered by the lack of animal models that fully
mimic human ALD. To overcome the current limitations of ALD studies and therapy development, it



is necessary to understand the molecular mechanisms underlying alcohol-induced liver injury. Hence,


to provide insights into the progression of ALD, this review examines previous studies conducted
Citation: Hyun, J.; Han, J.; Lee, C.;
on alcohol metabolism in the liver. There is a particular focus on the occurrence of ALD caused by
Yoon, M.; Jung, Y. Pathophysiological
hepatotoxicity originating from alcohol metabolism.
Aspects of Alcohol Metabolism in the
Liver. Int. J. Mol. Sci. 2021, 22, 5717.
https://doi.org/10.3390/ijms22115717
Keywords: alcoholic liver disease; alcohol metabolism; lipotoxicity; inflammation; fibrosis

Academic Editor:
Akiko Kojima-Yuasa
1. Introduction
Received: 10 May 2021 Alcohol has long been closely related to human culture, used as a drug, and considered
Accepted: 24 May 2021 an indulgence [1,2]. As the consumption of alcoholic beverages increases, it contributes to
Published: 27 May 2021 the significant elevation of morbidity and mortality worldwide [3,4]. In 2018, the World
Health Organization (WHO) estimated that alcohol consumption is responsible for three
Publisher’s Note: MDPI stays neutral million deaths worldwide annually, accounting for nearly 14% of the total mortality in
with regard to jurisdictional claims in people aged 20 to 40 years old [5]. In addition to social and psychiatric problems, more
published maps and institutional affil-
than 200 diseases that affect several organs, such as the brain, heart, gastrointestinal
iations.
tract, and liver, are related to habitual alcohol consumption [6–8]. Among these alcohol-
damaged organs, the liver is specifically susceptible to damage because the liver is the
primary site of alcohol metabolism in the body [9,10]. Alcohol metabolism generates
products that damage the liver, resulting in alcoholic liver disease (ALD), a main cause of
Copyright: © 2021 by the authors. chronic liver disease [4,9–11]. ALD encompasses a broad spectrum of conditions, including
Licensee MDPI, Basel, Switzerland. alcoholic fatty liver (simple steatosis), alcoholic hepatitis, alcoholic cirrhosis, and liver
This article is an open access article cancer [4,11–13]. Alcoholic fatty liver is defined by fat accumulation in hepatocytes without
distributed under the terms and
substantial inflammation, or hepatic fibrosis, and is observed in up to 90% of heavy
conditions of the Creative Commons
drinkers [9,11–14]. Heavy long-term alcohol consumption accelerates the transition of
Attribution (CC BY) license (https://
alcoholic fatty liver into alcoholic hepatitis, which is characterized by steatosis, ballooning
creativecommons.org/licenses/by/
hepatocytes, and infiltration of neutrophils with or without fibrosis [11–16]. Alcoholic
4.0/).

Int. J. Mol. Sci. 2021, 22, 5717. https://doi.org/10.3390/ijms22115717 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 5717 2 of 16

cirrhosis is a more severe form of ALD and is defined by disorganized liver architecture
with fibrosis [11–16].
Although public awareness of the hepatotoxicity caused by alcohol is rising, the
trend of alcohol consumption is steadily increasing [4,17]. Alcoholic cirrhosis accounts
for approximately half of the deaths related to liver cirrhosis, and alcohol is known to
accelerate liver injury in people infected with hepatitis virus [18,19]. To prevent and/or
reverse ALD progression, abstinence from alcohol is the most fundamental solution, but
it is difficult to suppress the recurrence of drinking because most patients with ALD are
alcoholics [20]. Liver transplantation is considered the definitive treatment for ALD, like
other end-stage liver diseases [21,22]. However, liver transplantation is a complicated
treatment option because it depends on donor availability and demands abstinence from
alcohol for at least six months before and after the transplant [23]. There is also an ethical
concern that the transplanted liver could be “wasted” on a patient who eventually relapses
to drinking and damages the transplanted liver [21]. In addition, there is no effective
Food and Drug Administration (FDA)-approved drug for treating patients with ALD [24].
Therefore, it is important to develop effective therapeutics for ALD. Understanding the
process of alcohol metabolism in the liver and the actions of hepatotoxic intermediates
involved in ALD progression could provide clues for developing ALD treatment strategies.
Herein, the metabolic processes of alcohol are summarized, and ALD pathogenesis is
reviewed, providing knowledge into the underlying mechanism of ALD.

2. Alcohol Metabolism
Ingested alcohol is absorbed through the stomach and intestines. Less than 10% of
absorbed alcohol is excreted in breath, sweat, and urine. This means that more than 90% of
the absorbed alcohol circulates through the body and is eventually transported to the liver
via the portal vein [10,25]. Due to the high levels of alcohol metabolizing enzymes in the
liver, the liver plays a major role in alcohol metabolism [10]. In the liver, alcohol is metabo-
lized by oxidative and non-oxidative pathways (Figure 1) [26,27]. The oxidative pathway
is the major pathway for alcohol metabolism and is composed of two steps. First, alcohol
is oxidized to acetaldehyde by alcohol dehydrogenase (ADH), a primary enzyme that
converts alcohol to acetaldehyde [28]. Excess alcohol consumption increases the expression
and activity of cytochrome P450 2E1 (CYP2E1), not ADH. Activated CYP2E1 promotes the
production of acetaldehyde through the formation of reactive oxygen species (ROS) [29,30].
In addition, peroxisomal catalase breaks down alcohol to acetaldehyde, but its action is
considered a minor pathway because of its small contribution to alcohol digestion [10,27].
The second step in the oxidative pathway involves the rapid conversion of acetaldehyde to
acetate by aldehyde dehydrogenase (ALDH). Acetate is metabolized into carbon dioxide
(CO2 ), fatty acids (FAs), and water (H2 O) in peripheral tissues, not the liver [10,27]. The
nonoxidative pathway accounts for a minor portion of alcohol metabolism in quantita-
tive terms [31–33]. A small amount of alcohol is nonoxidatively conjugated to various
endogenous metabolites by different enzymes. For example, enzymatic esterification of
alcohol with FAs forms fatty acid ethyl ester (FAEE) and phospholipase D (PLD) catalyzes
transphosphatidylation of phosphatidylcholine with ethanol to form phosphatidylethanol
(PEth). In addition, alcohol conjugated to glucuronic acid and sulfate generates ethyl
glucuronide (EtG) and ethyl sulfate (EtS), respectively [31–33].
Products generated during alcohol metabolism damage the liver and act as a driv-
ing force of ALD progression from alcoholic steatosis to alcoholic cirrhosis [34,35]. The
most well-known toxic compound produced by alcohol metabolism is acetaldehyde [36].
Acetaldehyde interacts directly with DNA and causes point mutations and chromosomal
damage. It also binds to a variety of proteins to form acetaldehyde adducts, which distorts
liver function and structure [37,38]. These protein adducts upregulate CYP2E1 expression
and enhance oxidative stress [39,40]. In addition, Holstege et al. [41] demonstrated that
protein adducts contribute to lipid accumulation, inflammation, and fibrosis, playing a key
role in the pathogenesis of various stages of ALD. Acetate has been reported to increase
Int. J. Mol. Sci. 2021, 22, 5717 3 of 16

portal blood flow by circulating in the bloodstream, although it is less toxic than acetalde-
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 16
hyde [42]. Nonoxidative pathway-derived metabolites, including PEth and FAEE, are also
known to cause alcohol toxicity, but the mechanisms have not yet been determined [31,43].

Figure 1. Scheme
Scheme for
for alcohol
alcohol metabolism
metabolism in in the
the liver.
liver. In
In the
theliver,
liver, alcohol
alcohol isis metabolized
metabolized by by the
the oxidative
oxidative and
and non-oxidative
non-oxidative
pathway.
pathway. In In the
the oxidative
oxidative pathway, the major
pathway, the major pathway
pathway of of alcohol
alcohol digestion,
digestion, alcohol
alcohol is
is oxidized
oxidized to to acetaldehyde
acetaldehyde byby various
various
enzymes
enzymes including
including alcohol
alcohol dehydrogenase
dehydrogenase (ADH), (ADH), cytochrome
cytochrome P450 P450 2E1
2E1 (CYP2E1),
(CYP2E1), and catalase. Then,
and catalase. Then, acetaldehyde
acetaldehyde isis
broken down into acetate, which is excreted out of the liver. Especially when excessive alcohol is consumed, CYP2E1 is
broken down into acetate, which is excreted out of the liver. Especially when excessive alcohol is consumed, CYP2E1
activated and promotes formation of reactive oxygen species (ROS). The non-oxidative pathway accounts for a small
is activated and promotes formation of reactive oxygen species (ROS). The non-oxidative pathway accounts for a small
amount of alcohol metabolism. Various enzymes nonoxidatively conjugates alcohol with different endogenous metabo-
amount
lites, of alcohol
producing metabolism.
fatty acid ethyl Various enzymes
ester (FAEE), nonoxidatively conjugates
phosphatidylethanol alcohol
(PEth), ethyl with different
glucuronide (EtG), endogenous metabolites,
and ethyl sulfate (EtS).
producing fatty acid ethyl ester (FAEE), phosphatidylethanol (PEth), ethyl glucuronide (EtG),
The byproducts generated during alcohol metabolism injure the liver by increasing lipid accumulation, inflammation, and ethyl sulfate (EtS). and
The
byproducts generated during alcohol metabolism injure the liver by increasing lipid accumulation,
fibrosis. Especially, acetaldehyde, the first metabolite of alcohol metabolism, is well known for toxic compounds. ROS, inflammation, and
fibrosis.
which areEspecially,
generatedacetaldehyde,
by activation the first metabolite
of CYP2E1, are alsoofconsidered
alcohol metabolism,
as one of theis well
majorknown for toxicofcompounds.
contributors liver damage.ROS,
In
addition,
which areacetate and non-oxidative
generated by activation ofmetabolites
CYP2E1, are are also
known to damage
considered as the
oneliver.
of the major contributors of liver damage. In
addition, acetate and non-oxidative metabolites are known to damage the liver.
Products generated during alcohol metabolism damage the liver and act as a driving
3. Oxidative
force of ALD Ethanol
progressionMetabolites Are Involved
from alcoholic steatosisintoALD Pathogenesis
alcoholic cirrhosis [34,35]. The most
Oxidative
well-known ethanol-derived
toxic metabolites
compound produced exert a metabolism
by alcohol broad spectrum of damage in
is acetaldehyde theAcet-
[36]. liver,
ranging from
aldehyde lipid directly
interacts accumulation in hepatocytes
with DNA and causestopoint
inflammation,
mutationsfibrosis, and carcinogene-
and chromosomal dam-
sis (Figure
age. It also 2). Excessive
binds lipid accumulation
to a variety of proteins toinform
hepatocytes resultsadducts,
acetaldehyde in massive hepatocyte
which distorts
death, which triggers pro-inflammatory and pro-fibrogenic responses, increasing
liver function and structure [37,38]. These protein adducts upregulate CYP2E1 expression the risk
of liver
and cancer
enhance [44]. Because
oxidative there is In
stress [39,40]. a dearth of Holstege
addition, information about
et al. [41] ALD pathogenesis
demonstrated that
exerted adducts
protein by non-oxidative
contributeethanol
to lipidmetabolism,
accumulation,thisinflammation,
section reviews andthe mechanisms
fibrosis, playingun-a
derlying
key role inoxidative ethanol metabolite-mediated
the pathogenesis of various stages ofhepatotoxicity
ALD. Acetate caused
has been byreported
excess alcohol
to in-
exposure
crease [45].blood flow by circulating in the bloodstream, although it is less toxic than
portal
acetaldehyde [42]. Nonoxidative pathway-derived metabolites, including PEth and FAEE,
are also known to cause alcohol toxicity, but the mechanisms have not yet been deter-
mined [31,43].

3. Oxidative Ethanol Metabolites Are Involved in ALD Pathogenesis

Oxidative ethanol-derived metabolites exert a broad spectrum of damage in the liver,
ranging from lipid accumulation in hepatocytes to inflammation, fibrosis, and carcino-
genesis (Figure 2). Excessive lipid accumulation in hepatocytes results in massive hepato-
cyte death, which triggers pro-inflammatory and pro-fibrogenic responses, increasing the
risk of liver cancer [44]. Because there is a dearth of information about ALD pathogenesis
exerted by non-oxidative ethanol metabolism, this section reviews the mechanisms un-
derlying oxidative ethanol metabolite-mediated hepatotoxicity caused by excess alcohol
exposure [45].
Int. J. Mol. Sci. 2021, 22, 5717 4 of 16
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 16

Figure 2.
Figure 2. Harmful
Harmful effects
effects of
of acetaldehyde
acetaldehyde on on ALD.
ALD. Acetaldehyde,
Acetaldehyde, one one of of the
the oxidative
oxidative ethanol-derived
ethanol-derived metabolites,
metabolites, exerts
exerts
a broad spectrum of damage to the liver, ranging from lipid accumulation in hepatocytes to
a broad spectrum of damage to the liver, ranging from lipid accumulation in hepatocytes to inflammation, fibrosis, and inflammation, fibrosis, and
carcinogenesis. The hepatocytes accumulate lipids by the lipid oversupply (increase of fatty acid
carcinogenesis. The hepatocytes accumulate lipids by the lipid oversupply (increase of fatty acid (FA) uptake to the liver (FA) uptake to the liver
andde
and denovo
novolipogenesis
lipogenesisininthe
theliver)
liver) and/orthe
and/or theimpaired
impaired pathway
pathway of of lipid
lipid clearance
clearance (decrease
(decrease of mitochondrial
of mitochondrial β-oxida-
β-oxidation
tion and secretion of excessive lipids in very-low density lipoprotein (VLDL)). In addition, acetaldehyde activates Kupffer
and secretion of excessive lipids in very-low density lipoprotein (VLDL)). In addition, acetaldehyde activates Kupffer
cells, the liver-resident macrophage, to release reactive oxygen species (ROS) and cytokines that recruit other immune
cells, the liver-resident macrophage, to release reactive oxygen species (ROS) and cytokines that recruit other immune
cells. When exposed to acetaldehyde, Kupffer cells activate the toll-like receptor 4 (TLR4)-mediated nuclear factor (NF)-
cells. When exposed
κB signaling pathway,to acetaldehyde, Kupffer cells
triggering inflammatory activate the
responses. Thetoll-like receptor 4 (TLR4)-mediated
acetaldehyde-protein adducts promotenuclear
thefactor (NF)-κB
collagen pro-
signaling
duction by pathway, triggering
activated inflammatory
hepatic stellate responses.
cells (HSCs) Theprotein
via the acetaldehyde-protein
kinase C (PKC)adductsand thepromote the collagen
TGF-β signaling production
pathway. ROS-
by activated
mediated hepatic stellate
oxidative cellsaccelerates
stress also (HSCs) vialiverthe protein
fibrosis.kinase C (PKC)
Moreover, and the
protein andTGF-β
DNAsignaling
adducts withpathway. ROS-mediated
acetaldehyde cause
hepatic carcinogenesis.
oxidative They weaken
stress also accelerates liverDNA integrity
fibrosis. and interfere
Moreover, proteinwith
and DNA DNArepair system,
adducts withincreasing
acetaldehydecarcinogenic DNA
cause hepatic
mutation. Genetic
carcinogenesis. Theyvariations
weaken DNA in theintegrity
alcohol-metabolizing
and interfere with enzymes,
DNA suchrepairassystem,
acetaldehyde dehydrogenase
increasing carcinogenic (ALDH)2*2
DNA mutation. and
alcohol dehydrogenase (ADH)1C*1 alleles, suppress the activity of the enzymes, enhancing
Genetic variations in the alcohol-metabolizing enzymes, such as acetaldehyde dehydrogenase (ALDH)2*2 and alcohol the amount of acetaldehyde.
dehydrogenase (ADH)1C*1 alleles, suppress the activity of the enzymes, enhancing the amount of acetaldehyde.
3.1. Excessive Lipid Accumulation in Hepatocytes
3.1. Excessive
HepatocytesLipid accumulate
Accumulation in Hepatocytes
lipids when there is an oversupply of lipids and the lipid
removal pathway accumulate
Hepatocytes is impaired lipids [46]. Thewhenformer
thereoccurs when the liver
is an oversupply increases
of lipids and uptake
the lipid of
exogenous
removal FAs or is
pathway lipid production
impaired in the
[46]. The liver, occurs
former called de novo
when thelipogenesis. Acetaldehyde,
liver increases uptake of
in particular,
exogenous FAspromotes lipolysis ininadipose
or lipid production the liver, tissue
calledand
de elevates the amount
novo lipogenesis. of free FAs
Acetaldehyde,
in particular,
(FFAs) that arepromotes
absorbed lipolysis
by the in adipose
liver [15,47].tissue
The and elevates
hepatic the amount of
FA transporters, suchfreeasFAs
FA
(FFAs) that proteins
transporter are absorbed (FATPs) by and
the liver [15,47]. The
FA translocase hepatic FA
(FAT/CD36), aretransporters,
involved in this such as FA
process,
transporter proteins
and upregulation of(FATPs)
FATP2, andFATP5, FA translocase
and FAT/CD36 (FAT/CD36),
has beenare involved
observed in in thistissues
liver process,in
and upregulation
rodent models of of ALD FATP2, FATP5,
[48,49]. and FAT/CD36
In addition, has been
acetaldehyde observed inthe
dysregulates liver tissues in
5′ adenosine
rodent models of ALD [48,49].
monophosphate-activated proteinIn addition, acetaldehyde
kinase (AMPK) pathway,dysregulates
which regulatesthe 50 the
adenosine
expres-
monophosphate-activated protein kinase (AMPK) pathway, which
sion of lipogenic transcription factors, including sterol regulatory element-binding pro-regulates the expression
of lipogenic
tein transcription
1c (SREBP-1c) factors, including sterolelement-binding
and carbohydrate-responsive regulatory element-binding
protein (ChREBP) protein 1c
[50].
(SREBP-1c)
Thus, there andis ancarbohydrate-responsive
increase in the expressionelement-binding
of lipogenic enzyme protein (ChREBP)
genes, [50]. Thus,
such as acetyl-CoA
there is an increase
carboxylase 1 (ACC1), in the expression
fatty of lipogenic
acid synthase (FASN), enzyme genes, such
and sterol-CoA as acetyl-CoA
desaturase 1 (SCD1)car-
boxylase 1 (ACC1), fatty acid synthase (FASN), and sterol-CoA desaturase
[15,51]. LIPIN1 plays a role in lipid homeostasis. Hepatic LIPIN1 either acts as a transcrip- 1 (SCD1) [15,51].
LIPIN1 plays a role
tional coactivator in lipid homeostasis.
(LIPIN1α) in the nucleus Hepatic LIPIN1aseither
or functions a Mg2+ acts as a transcriptional
-dependent phosphati-
coactivator (LIPIN1α) in the nucleus or functions as a Mg 2+ -dependent phosphatidate
date phosphatase (LIPIN1β) promoting biosynthesis of triglyceride and phospholipid in
phosphatase
the cytoplasm(LIPIN1β)
[52]. It haspromoting
been reported biosynthesis
that ethanol of triglyceride
upregulates and phospholipid
LIPIN1 in the
via the activation
cytoplasm [52]. It has been reported that ethanol upregulates LIPIN1 via the activation
Int. J. Mol. Sci. 2021, 22, 5717 5 of 16

of SREBP-1c in an AMPK-dependent manner, leading to increased lipid accumulation in

hepatocytes [52].
Lipid clearance in the liver is mediated by mitochondrial β-oxidation and secretion of
excessive stored triglycerides that are packed into very-low density lipoprotein (VLDL) [53].
It should be noted that alcohol-induced dysregulation of mitochondrial β-oxidation is
considered the most significant contribution to lipid accumulation in the liver [46]. Splicing
factor arginine/serine-rich 10 (SFRS10), an RNA splicing factor, promotes the skipping
of the LIPIN1 exon and generates the LIPIN1α variant [52]. Yin et al. [54,55] demon-
strated that sirtuin 1 (SIRT1) activating SFRS 10 is inhibited by ethanol via upregulation of
microRNA-217, and that suppression of SIRT1 increases the LIPIN1β/α ratio, enhancing
the lipid biosynthetic activity of LIPIN1β and reducing its capacity for mitochondrial FA β-
oxidation. In addition, overexpression of LIPIN1 in mice has been shown to alleviate VLDL
secretion without altering two essential proteins for VLDL formation: apolipoprotein B
(ApoB) and microsomal triglyceride transfer protein (MTTP) [52]. Peroxisome proliferator-
activated receptor alpha (PPARα), a transcriptional factor, regulates the expression of genes
participating in FA oxidation in the mitochondria [56]. Acetaldehyde inactivates PPARα
by suppressing the DNA binding affinity of PPARα [57]. Additionally, inactivated PPARα
inhibits transcription of FA oxidation-related genes, including the gene encoding carnitine
palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of mitochondrial β-oxidation, by
translocating FA into mitochondria [58]. Autophagy is also an important mechanism for
removing lipid droplets accumulated by ethanol. Acute exposure to ethanol activates
autophagy by inhibiting mammalian targets of rapamycin (mTOR) [59]. However, chronic
exposure to ethanol inhibits autophagy, which seems to be due to a decrease in AMPK
activity caused by acetaldehyde [60].
The ethanol-derived toxic metabolites dysregulate multiple aspects of hepatic lipid
metabolism—they increase hepatic FA uptake and de novo lipid synthesis and decrease FA
oxidation and lipid export. The effects converge to cause hepatocellular lipid accumulation.
Since hepatocellular lipid accumulation is the earliest sign of ALD, further study on lipid
metabolism in hepatocytes could create opportunities for early therapeutic intervention for
people at risk of advanced ALD.

3.2. Dysregulated Immune System

Accumulating evidence indicates that chronic alcohol consumption disrupts the in-
testinal barrier and the tight and adherent junctions in the colonic mucosa, which promotes
the translocation of lipopolysaccharide (LPS) to the circulation [61–63]. The detrimental
effects of acetaldehyde contribute to this via upregulation of microRNA-212 in enterocytes
and downregulation of zonula occludens 1 (ZO-1), a tight junction component [64]. Both
acetaldehyde and LPS activate Kupffer cells, the liver-resident macrophages, to release ROS
and chemokines that recruit bone marrow-derived neutrophils and blood-derived mono-
cytes into the liver [15,65]. This stage of ALD is called alcoholic hepatitis. ROS released from
Kupffer cells mediates the activation of the toll-like receptor 4 (TLR4)/mitogen-activated
protein kinase (MAPK)/nuclear factor (NF)-κB axis [66]. It has also been reported that FFA
promotes the activation of the TLR4-mediated NF-κB signaling pathway and inflammatory
cyclooxygenase 2 (COX2) expression in macrophages in vitro [67]. Furthermore, it has been
demonstrated in in vivo experiments; TLR4 knockout (KO) mice were found to be resistant
to alcohol-induced hepatic steatosis [68,69].
The levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα),
interleukin (IL)-1β, and IL-8, are upregulated in patients with ALD [70]. When exposed
to oxidative ethanol metabolites, such as acetaldehyde and acetate, the NF-κB signaling
pathway is activated and the level of TNFα is elevated in rodent macrophages [71]. In
a mouse model of ALD generated by chronic alcohol consumption, the NF-κB pathway
was activated in Kupffer cells, and systemic release of pro-inflammatory factors, including
TNFα, IL-6, and macrophage chemoattractant protein 1 (MCP1), was observed [71]. Inhibi-
tion of SIRT1, an NF-κB antagonist, also partly induces the activation of NF-κB signaling.
Int. J. Mol. Sci. 2021, 22, 5717
Int. x FOR PEER REVIEW 66 of
of 16
16

signaling.
The NF-κBThe NF-κBmediates
signaling signaling mediates ROS-triggered
ROS-triggered inflammatoryinflammatory
responses viaresponses via
downstream
downstream effectors [72], such as intercellular adhesion molecule 1 (ICAM1). ICAM1
effectors [72], such as intercellular adhesion molecule 1 (ICAM1). ICAM1 is involved in is
involved in the contact of hepatocytes with neutrophils, promoting neutrophil-mediated
the contact of hepatocytes with neutrophils, promoting neutrophil-mediated hepatocyte
hepatocyte
killing killing
(Figure (Figure 3) [73].
3) [73].

Figure 3. Cellular
Figure 3. Cellularinteractions
interactionsduring
duringthe thepathogenesis
pathogenesisofof ALD.
ALD. Acetaldehyde
Acetaldehyde (Ac) produced
(Ac) produced by by
hepatocytes during
hepatocytes ethanol
during eth-
oxidation activates
anol oxidation Kupffer
activates cells cells
Kupffer to release reactive
to release oxygen
reactive species
oxygen (ROS)
species which
(ROS) triggers
which triggersinflammatory
inflammatory responses
responses in in
a
a TLR4/NF-κB-dependent
TLR4/NF-κB-dependent manner
manner and and leads
leads to massive
to massive hepatocyte
hepatocyte death.
death. In vitro,
In vitro, Ac secreted
Ac secreted fromfrom hepatocytes
hepatocytes entersenters
into
intohepatic
the the hepatic stellate
stellate cells (HSCs),
cells (HSCs), promotespromotes the binding
the binding of Ac-responsive
of Ac-responsive transcription
transcription factors factors (AcR-TFs)
(AcR-TFs) to the acetal-
to the acetaldehyde-
dehyde-responsive
responsive elementin(AcRE)
element (AcRE) in the promoter
the promoter region of region of type I (Col)
type I collagen collagen
genes(Col) genesoftotranscription.
to onset onset of transcription. Ac also
Ac also activates
activates
HSCs HSCspro-fibrogenic
via other via other pro-fibrogenic
mechanisms, mechanisms, increasing of
increasing deposition deposition of extracellular
extracellular matrix (ECM) matrix (ECM)
proteins. proteins. effect
Anti-fibrotic Anti-
fibrotic effect of natural killer (NK) cells that are cytotoxic to activated HSCs is inhibited by chronic alcohol consumption.
of natural killer (NK) cells that are cytotoxic to activated HSCs is inhibited by chronic alcohol consumption.

Oxidative ethanol-derived
Oxidative ethanol-derived metabolites
metabolites may may influence
influence adaptive
adaptive immunity
immunity and and impair
impair
antigen presentation,
antigen presentation, which
which isisrequired
requiredfor forT- T-and
andB-cell
B-cellactivation
activation[74].
[74].The amounts
The amounts of
circulating antibodies against hydroxyethyl radicals (HER)-protein adducts
of circulating antibodies against hydroxyethyl radicals (HER)-protein adducts and lipid and lipid pe-
roxidation-derived aldehydes,
peroxidation-derived aldehydes,such
suchasasmalondialdehyde
malondialdehyde(MDA), (MDA), are
are elevated
elevated in patients
in patients
with alcoholic hepatitis [74]. The increased levels of anti-HER and anti-MDA
with alcoholic hepatitis [74]. The increased levels of anti-HER and anti-MDA antibod- antibodies
are are
ies associated withwith
associated the presence
the presenceof activated
of activatedCD4+CD4+
T cellsT in peripheral
cells blood.blood.The
in peripheral The mac-
rophage’s proteasome
macrophage’s function
proteasome functionis also
is alsoaltered
alteredbybyoxidative
oxidativeethanol
ethanol metabolism, which
metabolism, which
consequently reduces
consequently reducesantigen
antigenpresentation
presentation[60,75].
[60,75].Ethanol
Ethanol inhibits
inhibits antigen
antigen presentation
presentation in
in macrophages
macrophages and and dendritic
dendritic cells
cells [76].
[76]. Chronic
Chronic alcohol
alcohol ingestion
ingestion decreases
decreases thethe number
number of
of F4/80+
F4/80+ cells
cells expressing
expressing major
major histocompatibility
histocompatibility complex
complex (MHC)-I
(MHC)-I andand MHC-II
MHC-II [77].
[77].
FAs sensitize
sensitize non-immune
non-immune cells,cells, such
such as as hepatocytes,
hepatocytes, to to pro-inflammatory
pro-inflammatory signalssignals
respond to
and impair their ability to respond to hepatoprotective
hepatoprotective signalsignal proteins,
proteins, such
such asas signal
signal
transducer and activator of transcription 3 (STAT3) (STAT3) [78,79].
[78,79]. Furthermore,
Furthermore, the the production
production
of ROS during ethanol metabolism rapidly increases the fluidity of the the hepatocyte
hepatocyte cellcell
membrane
membrane and andpromotes
promotescytoplasmic
cytoplasmiciron ironoverload
overload [80] and
[80] andaccelerated lipid
accelerated peroxidation,
lipid peroxida-
eventually leading
tion, eventually to massive
leading hepatocyte
to massive death.death.
hepatocyte

3.3. Induction of
3.3. Induction of Fibrosis
Fibrosis
Massive
Massive hepatocyte
hepatocyte death
death provokes
provokes thethe fibrotic
fibrotic repair
repair process
processinin the
the liver
liver [81].
[81]. When
When
the hepatic parenchyma is progressively replaced by scar tissue, the metabolic
the hepatic parenchyma is progressively replaced by scar tissue, the metabolic function function
of
of
thethe liver
liver is compromised,
is compromised, leading
leading to liver
to liver failure
failure [15].
[15]. Hepatic
Hepatic stellate
stellate cellscells
(HSCs)(HSCs)
are
are activated
activated in livers
in the the livers of patients
of patients withwith alcoholic
alcoholic steatohepatitis
steatohepatitis to become
to become the main
the main pro-
producers of extracellular matrix proteins, such as collagens and
ducers of extracellular matrix proteins, such as collagens and fibronectin, causing fibronectin, causing
liver
liver fibrosis
fibrosis [15].fibrogenic
[15]. The The fibrogenic mechanisms
mechanisms are initiated
are initiated and perpetuated
and perpetuated by alcohol
by alcohol metab-
metabolism (Figure 3) [11]. The protein adducts generated by ethanol-derived acetaldehyde
olism (Figure 3) [11]. The protein adducts generated by ethanol-derived acetaldehyde and
and the aldehydes produced from lipid peroxidation, such as MDA, turn on pro-fibrogenic
the aldehydes produced from lipid peroxidation, such as MDA, turn on pro-fibrogenic
pathways in activated HSCs [11,15]. It has been shown that the ALDH2-KO mouse hav-
pathways in activated HSCs [11,15]. It has been shown that the ALDH2-KO mouse having
ing highly accumulated acetaldehyde in the liver is more susceptible to alcohol-induced
highly accumulated acetaldehyde in the liver is more susceptible to alcohol-induced liver
Int. J. Mol. Sci. 2021, 22, 5717 7 of 16

liver inflammation and fibrosis compared to wide-type mice because of the higher level
of MDA-acetaldehyde adducts [82]. The results of an in vitro experiment showed that
acetaldehyde released from hepatocytes entered HSCs and directly led HSCs to express
type I collagen genes [83]. Acetaldehyde regulates the expression of collagen genes in a
protein kinase C (PKC)-dependent manner [84]. In human HSCs, PKC phosphorylates
p70 S6 kinase (p70S6K) by activating extracellular signaling-regulated kinase (ERK) and
phosphoinositide 3 kinase (PI3K), leading to collagen gene expression [84]. Another pro-
fibrogenic mechanism stimulated by acetaldehyde is the transforming growth factor beta
(TGFβ) pathway, which is essential in liver fibrogenesis [85]. In human HSCs treated with
acetaldehyde, the transcription levels of type I collagen and fibronectin genes were found
to be elevated in a TGFβ-independent manner in the early phase of treatment [86,87], while
TGFβ-dependent responses, including the secretion of latent TGFβ1 and the expression of
type II TGFβ receptor, occurred in the late phase [85]. LPS from the gastrointestinal tract
also contributes to HSC activation by increasing the susceptibility of HSCs to acetaldehyde
and TGFβ [88–90]. Moreover, acetaldehyde has been shown to inhibit β-catenin phos-
phorylation and degradation by blocking glycogen synthase kinase (GSK)3B, thereby the
active β-catenin translocalizes into the nucleus and upregulates the expression of fibrogenic
genes [91].
An accumulating body of evidence indicates that acetaldehyde activates HSCs in
ALD by inducing oxidative stress [60]. ROS generated during CYP2E1-dependent oxida-
tive ethanol metabolism enhance collagen production in HSCs co-cultured with hepato-
cytes [92]. Hydrogen peroxide (H2 O2 ), an ROS, also aids acetaldehyde-mediated hepatic
fibrogenesis. DNA binding affinity and activity of CCAT/enhanced binding protein beta
(C/EBPβ) that promotes the transcription of collagen I α1 (COLI α1) are increased by
acetaldehyde, and the actions of C/EBPβ are mediated by H2 O2 [93]. Similarly, transcrip-
tional activity of peroxisome proliferator-activated receptor gamma (PPARγ) is inhibited
by acetaldehyde in the H2 O2 -dependent pathway in activated HSCs [94]. In addition,
acetaldehyde binds to glutathione (GSH) and weakens its antioxidant activity [95]. Nuclear
erythroid 2-related factor 2 (NRF2), a transcription factor activated by oxidative stress, is
presented to upregulate antioxidant gene expression [96]. Thus, Nrf2 overexpression in
mice improves ALD by preventing oxidative stress [97], whereas Nrf2 deficiency promotes
ALD progression [98].
Moreover, chronic alcohol consumption suppresses the anti-fibrotic function of natural
killer cells that are cytotoxic to activated HSCs, accelerating hepatic fibrogenesis. This
suppression is mediated by TNF-related apoptosis-inducing ligand (TRAIL)-TRAIL re-
ceptor interaction and interferon gamma (IFNγ) [78,99]. In addition, IL-22 released by
natural killer cells and T helper cells is shown to inhibit activation and proliferation of
HSCs stimulated by acetaldehyde [100]. It was demonstrated that IL-22 promoted the
nuclear translocation of Nrf2 in HSCs and led to HSC inactivation by arresting them at
G1/S phase. Overexpression of IL-22 was shown to induce HSC senescence in carbon
tetrachloride (CCl4 )-induced fibrotic livers of mice by upregulating p53, a mediator of
cellular senescence [101]. IL-22 also has anti-apoptotic, anti-oxidative, and pro-regenerative
effects against alcohol-induced liver damage, and drives the onset of clinical trials of IL-22
for the treatment of ALD [102].
The ROS also stimulate intracellular pro-fibrogenic pathways in HSCs, including the
ERK, protein kinase B (PKB/Akt), c-Jun N-terminal kinase (JNK), and tissue inhibitor of
metalloproteinase 1 (TIMP1) pathways. Whereas, the activity of matrix metalloproteinases
(MMPs) is inhibited by ROS [60]. The pro-fibrotic effect of ROS was confirmed by the find-
ing that ROS-scavenging enzymes prevent hepatic fibrosis in animal models of ALD [103].
In addition, ethanol-mediated lipid peroxidation is blocked and liver injury is reduced
in Cyp2e1-KO mice [104]. These findings suggest that the suppression of oxidative stress,
potentially by antioxidants, could attenuate ALD-related fibrosis. For example, helenalin
isolated from Centipeda minima prevented liver injury and fibrosis in rats with intragastric al-
cohol infusions up to 24 weeks by enhancing the activities of ethanol-detoxifying enzymes,
Int. J. Mol. Sci. 2021, 22, 5717 8 of 16

ADH and ALDH [105]. Helenalin also reduced CYP2E1 activity, inhibited HSC activation
by suppressing the TGFβ signaling pathway, and decreased fibrosis by upregulating the
levels of MMP1 and MMP9. Similarly, other antioxidants, such as hesperidin and fraxetin,
were shown to attenuate alcohol-induced HSC activation and fibrosis [106,107]. However,
any pharmacological treatment has not been successful in clinical trials of patients with
alcoholic cirrhosis [108].

3.4. Development of Cancer

Alcohol is recognized as a carcinogen by the International Agency for Research on
Cancer. This is because alcohol causes many types of cancers, including hepatocellular
carcinoma (HCC) in humans [109]. It is known that excessive alcohol consumption leads
to a 3- to 10-fold increase in the risk of HCC and accounts for 30% of HCC cases world-
wide [109,110]. Several mechanisms by which alcohol induces carcinogenesis in the liver
have been suggested. Among them, the carcinogenic effect of ethanol-derived acetalde-
hyde forming protein and DNA adducts may be specific to ALD-associated HCC [111,112].
For example, N2-ethyl-deoxyguanosine (N2-Et-dG), an acetaldehyde-DNA adduct, has
been detected in patients with ALD [113]. It has also been reported that N2-propano-20 -
deoxyguanosine (N2-Et-dGTP), another DNA adduct, changes DNA integrity [114]. More-
over, acetaldehyde directly binds to amino acids, and the resulting protein adduct (with
O6-methylguanine methyltransferase) interferes with the DNA repair system, contributing
to liver carcinogenesis [115]. It has been shown in animal studies that acetaldehyde in-
creases point mutation frequency in the hypoxanthine phosphoribosyl transferase (HPRT)
gene in lymphocytes, a biomarker of lymphocyte functional deficiency [116].
Genetic variations in alcohol-metabolizing enzymes are the main causes of carcino-
genic acetaldehyde production, and thus have potential as biomarkers of alcohol-induced
liver cancer [117]. In particular, a deficiency of mitochondrial ALDH2 activity leads to
extreme accumulation of acetaldehyde because ALDH2 is a key acetaldehyde-detoxifying
enzyme. The ALDH2 mutant genotype, ALDH2*2 allele (also known as ALDH2(E487K)
or ALDH2 rs671 polymorphism), has been found to be associated with a high incidence
of liver cancer in heavy drinkers [118–120]. The relationship between ALDH2 deficiency
and the development of liver cancer was recently proven. Seo et al. [121] showed that after
exposure to carbon tetrachloride (a hepatotoxin) and ethanol, Aldh2-deficient hepatocytes
produced abundant oxidized mitochondrial DNA that was favorable for HCC progression
by activation of oncogenic pathways together with acetaldehyde. They also demonstrated
that ALDH2 deficiency increases the risk of HCC development in patients who have hep-
atitis B virus-cirrhosis and abuse alcohol. These findings might explain why the prevalence
of ALD-associated liver cancer is high in the Asian population, among whom 30–40% have
an ALDH2 deficiency. In addition, the homozygous ADH1C*1 allele has been discovered
to be a predictor of an increased risk of HCC in ALD patients, since the ADH1C*1 allele
encodes a 2.5-fold higher ethanol oxidizing capacity to generate more acetaldehyde than
the ADH1C*2 allele [122].
Besides the accumulation of harmful acetaldehyde, alcohol can induce hepatocar-
cinogenesis by promoting oxidative stress contingent on ROS production from alcohol
metabolism [109,110]. Accumulated ROS generate lipid peroxides, such as 4-hydroxy-
nonenal (4-HNE), and alter gene expression, leading to the upregulation of pro-inflammatory
cytokines and the activation of immune cells [109]. ROS also upregulate angiogenesis and
the metastatic process [109,123].

4. Models Used in the Study of Pathophysiology Related to Alcohol Metabolism

Progress in research and treatment of ALD is slowed, at least in part, due to the
lack of animal models that fully reflect the spectrum of human ALD. Although various
animal models such as primates and micropigs have been proposed for ALD study, rodent
models are commonly used because of better handiness in experiments and efficiency in
terms of cost and time [124]. However, there are fundamental differences in physiological
Int. J. Mol. Sci. 2021, 22, 5717 9 of 16

and biological processes for alcohol between rodents and human [125]. Unlike humans,
rodents rarely increase the amount of alcohol consumption over time and stop drinking
when blood acetaldehyde level is upregulated [126]. Additionally, rodents have a natural
aversion to alcohol. Notably, it is known that the alcohol-catabolizing rate is much faster
in rodents than humans. In addition, neutrophil infiltration is hardly detected in rodents
during ALD pathogenesis, whereas it is one of the key features of alcoholic steatohepatitis
in humans [73]. Furthermore, the sex, age, and genetic background of the animal, and even
the animal facility environment, impact susceptibility to alcohol-induced liver damage, and
bring to the high inter-variability in each experimental animal, hindering the generation
of rodent models of ALD [127]. Therefore, scientists have been developing rodent models
which could minimize the differences between rodent models of ALD and human ALD
(Table 1). The simplest model of ALD is to give the rodent alcohol-mixed drinking water
with normal chow-diet and let them drink it ad libitum. However, this model brings a
slight increase of blood alcohol levels (BALs) and mild liver injury [128,129]. DeCarli and
Lieber [130,131] developed the alcohol-containing liquid diet formula, called the Lieber-
DeCarli liquid diet. Rats and mice are fed ad libitum the Lieber–DeCarli liquid diet without
any other foods or drinks. The Lieber–DeCarli liquid diet partially overcomes the rodent’s
aversion to alcohol and induces elevated aminotransferases and hepatic steatosis. However,
inflammation is mild and fibrosis occurs rarely in the model [132]. The fundamental
limitation of the ad libitum alcohol feeding models is that animals do not drink alcoholic
water as well as humans do. Hence, the intragastric infusion model, called the Tsukamoto–
French (TF) model, was developed by Tsukamoto et al. [133,134]. In this model, alcohol
is directly injected into rodents through a surgically implanted intragastric cannula, and
higher BALs and more severe liver injuries than ad libitum alcohol-feeding models are
observed. However, the levels of hepatic inflammation and fibrosis in TF model are still less
than them in human alcoholic hepatitis. Moreover, requirements of technical competence,
intensive medical care, and expensive equipment are major obstacles to reproducing the
TF model [135]. In 2013, the Dr. Gao group at the National Institute on Alcohol Abuse and
Alcoholism of National Institute of Health [136] reported a mouse model of chronic and
binge alcohol feeding, termed the NIAAA model (commonly referred to as the Gao-binge
model). The Gao-binge model is produced by the Lieber–DeCarli liquid diet for many
consecutive days (>10 days) followed by single or multiple binge(s). According to the
periods of ethanol feeding, high BALs, elevated level of serum alanine aminotransferase
hepatic neutrophil infiltration, and even severe liver damage can be generated. To evaluate
the effects of alcohol on late phase of ALD such as cirrhosis or liver cancer, a combination
of the Lieber–DeCarli liquid diet with other hepatotoxins, such as CCl4 , diethylnitrosamine
(DEN) or LPS, as a “second hit”, is given to rodents [137–139].
In human ALD, blood acetaldehyde level is significantly upregulated and its increase
parallels the elevation of ethanol concentration [140–143]. The high level of blood acetalde-
hyde in human alcoholics after ethanol ingestion is caused by the decreased oxidation of
acetaldehyde rather than its increased formation from ethanol. It is associated with low
hepatic ALDH activity in alcoholic subjects [144]. Although the acetaldehyde level also
increases in rodents after ethanol treatment, its amount varies depending on the dose of
alcohol and duration of alcohol feeding. ALDH2-KO mice may recapitulate the ALDH2*2
allele that encodes ALDH2 with low acetaldehyde-oxidizing activity. As mentioned above,
ALDH2-KO mice have higher levels of acetaldehyde and acetaldehyde adducts in blood
and/or liver than wild-type mice when exposed to ethanol [145]. It is reported that gut
permeability, the concentration of plasma transaminases, and the amount of hepatic triglyc-
eride are higher in heterozygous ALDH2-KO (ALDH2+/−) mice fed the Lieber–DeCarli
liquid diet than in alcohol-fed wild-type mice [146]. In addition, ALDH2-KO mice have
been shown to be more sensitive to ethanol-induced liver inflammation, fibrosis, and
HCC, except hepatic steatosis, than wild-type mice [82,121]. Genes responsible for alcohol
oxidization, such as ADH1 and CYP2E1, are also targeted for modification in mice in
order to investigate the role of these enzymes in ALD pathogenesis. The ADH1 global
Int. J. Mol. Sci. 2021, 22, 5717 10 of 16

KO mice was shown to have a problem in blood ethanol clearance [147], but the effect of
knock-out-ADH1 on ALD has been poorly studied. The genetic deficiency of CYP2E1 rarely
induced hepatic steatosis and oxidative stress in mice fed a high-fat Lieber–DeCarli liquid
diet, whereas overexpression of CYP2E1 increased serum transaminases and accelerated
other liver damages, such as upregulation of fibrosis markers including α-smooth muscle
actin (α-SMA) and type I collagen, and elevated necroinflammation [104,148].

Table 1. Models of alcohol administration to rodent.

Models Administration Characteristics Feasibility

Ad libitum Oral alcohol consumption by Low BAL; minimal elevation
Easy to perform
alcohol-drinking water drinking water of ALT; mild steatosis
Oral alcohol consumption
Ad libitum Variable elevation range of
with alcohol-containing liquid
liquid diet ALT; marked steatosis; mild Easy to perform
diet formula but with no other
(Lieber–DeCarli diet) inflammation
food or drink
Direct enteral feeding through High BAL; marked elevation
Intragastric infusion
a surgically implanted of ALT; severe steatosis; mild Difficult to perform
(The Tsukamoto-French model)
intragastric cannula inflammation; fibrosis
A single or repeated
High BAL; marked elevation
Chronic and binge intragastric gavage of alcohol
of ALT; steatosis; neutrophil
alcohol feeding following chronic feeding Easy to perform
infiltration; necrosis;
(Gao-binge model) with the Lieber–DeCarli liquid
no fibrosis
diet
Addition of hepatotoxins such
Lieber–DeCarli diet Marked elevation of ALT;
as DEN, LPS or CCl4 during
+ other hepatotoxins high mortality rate; significant Easy to perform
the chronic feeding phase of
(Second hit model) liver fibrosis
the Lieber–DeCarli liquid diet
ALT, alanine aminotransferase; BAL, blood alcohol level; CCl4 , carbon tetrachloride; DEN, diethylnitrosamine; LPS, lipopolysaccharide.

Advances in bioengineering techniques lead to the development of in vitro and ex

vivo models which possibly compromise the limitations that animal models have. To
study ALD pathogenesis, Lee et al. [149] developed a three-dimensional in vitro model
of ALD by co-culturing rat primary hepatocytes and HSCs in a microfluidic chip where
the cells were exposed to ethanol via the flow. Recently, Wang et al. [150] generated
the expandable hepatic organoids derived from human embryonic stem cells. These
organoids phenocopied the bipotent liver progenitor cells that could differentiate into
both hepatocytes and cholangiocytes. The hepatic organoids co-cultured with human
fetal liver mesenchymal cells reflected the pathophysiology of human ALD by showing
the increased CYP2E1 activity, expression of fibrosis markers, fibrous accumulation, pro-
collagen secretion, oxidative stress, steatosis, and expression of lipogenic genes after
ethanol exposure. In addition, secretome analysis for inflammatory mediators showed
that a number of pro-inflammatory cytokines were upregulated in this model after alcohol
treatment. Therefore, these findings suggest that in vitro or ex vivo pathophysiological
models are promising to study ALD. However, further research is needed to verify these
models and to standardize the culture conditions.

5. Conclusions
Consumption of alcohol is deeply ingrained in the daily lives of many people; there-
fore, it is difficult to suppress consumption and intervene in the progression of ALD.
Nevertheless, efforts to prevent and treat ALD by intervening in alcohol-related diseases
are considered urgent tasks because of the increasing incidence of ALD and the absence
of FDA-approved therapy. To reverse or inhibit the progression of ALD, identification of
ALD risk factors and of mechanisms explaining their actions in ALD progression provides
vital information for developing therapeutic targets for ALD. Therefore, in this review, we
Int. J. Mol. Sci. 2021, 22, 5717 11 of 16

have briefly explained alcohol metabolism and its products, and reviewed the effects of
these metabolites in ALD progression. We focused on the hepatotoxicity of ethanol-derived
metabolites produced by oxidative alcohol metabolism (acetaldehyde and ROS) during
excessive exposure to alcohol. As discussed above, the products of alcohol metabolism
directly or indirectly damage the liver, leading to liver cancer. However, the mechanisms of
ALD pathogenesis remain unclear because of the lack of ALD patient samples and reliable
animal models for ALD that reflect human ALD. Limited understanding of ALD hampers
the development of novel therapies for ALD. Even though possible therapeutic candidates
have been proposed in many studies based on animal models, their effects are controversial
or not proven in the clinical stage. Therefore, further studies are necessary to elucidate the
detailed mechanisms of ALD and to discover potential therapeutic targets for treating ALD.

Author Contributions: J.H. (Jeongeun Hyun) contributed to this paper with literature review and
analysis, drafting the manuscript; J.H. (Jinsol Han) contributed to this paper with conception, litera-
ture review and analysis; M.Y. and C.L. contributed to this paper with literature review and analysis;
Y.J. contributed to this paper with conception, review, analysis, drafting and editing the manuscript,
and supervision. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by Y. Jung, National Research Foundation of Korea to J.H.
(Jeongeun Hyun) (2021R1C1C1003904), and Biomedical Research Institute Grant of Pusan National
University Hospital to M.Y. (20200216).
Conflicts of Interest: The authors declare no conflict of interest.

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