Physiol Rev 94: 1–34, 2014
doi:10.1152/physrev.00017.2013
TARGETING ALDEHYDE DEHYDROGENASE 2:
NEW THERAPEUTIC OPPORTUNITIES
Che-Hong Chen, Julio Cesar Batista Ferreira, Eric R. Gross, and Daria Mochly-Rosen
Department of Chemical and Systems Biology, Stanford University, School of Medicine, Stanford, California;
Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil;
and Department of Anesthesiology, Stanford University, School of Medicine, Stanford, California
Chen C-H, Ferreira JCB, Gross ER, Mochly-Rosen D. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities. Physiol Rev 94: 1–34, 2014;
doi:10.1152/physrev.00017.2013.—A family of detoxifying enzymes called aldehyde
dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying
aldehydes that accumulate through metabolism and to which we are exposed from the
environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH
emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2,
is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less
known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction
may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also
associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an
ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in
humans, and epidemiological studies suggest a correlation between this inactivating mutation and
increased propensity for common human pathologies. These data together with studies in animal
models and the use of new pharmacological tools that activate ALDH2 depict a new picture related
to ALDH2 as a critical health-promoting enzyme.
L
I.
II.
III.
IV.
V.
INTRODUCTION
MOUSE MODELS WITH MODIFIED...
PHARMACOLOGICAL MODULATORS...
DISEASES ASSOCIATED WITH ALDH2
CONCLUDING REMARKS
1
4
6
7
24
I. INTRODUCTION
Throughout their lifespan, every organism is exposed to
numerous damaging agents. Some are formed endogenously, while others accumulate following ingestion of food
or exposure to environmental pollutants. There are three
sets of active responses to these damaging events. The first
set provides a shield from these damaging agents by quickly
detoxifying them through enzymatic reactions or through
sequestration of these agents (e.g., phase I and II detoxifying
enzymes). The second set of responses involves a damage
repair system (e.g., proteolysis, DNA repair), and the third
set activates programmed cell death (e.g., apoptosis), thus
preventing propagation of the injury. It is not surprising
that many diseases are associated with a failure of one or
more of these three sets of protective mechanisms.
Many recent reviews cover literature on the repair mechanisms and on programmed cell death activation (52, 92,
263, 328). In this review, we focus on one detoxifying enzyme that provides a critical shield from damaging agents
that arise both endogenously and exogenously from exposures to the environment: aldehyde dehydrogenase-2
(ALDH2). However, many other detoxifying enzymes have
also evolved. The best characterized detoxification enzymatic system (also termed phase I system) is encoded by the
cytochrome P-450 gene superfamily, consisting of 57 different genes, that encode the enzymes involved in ⬃75% of
all drug metabolism in humans (103, 104, 340). Aldehyde
dehydrogenase (ALDH) is another gene superfamily of
phase I oxidizing enzymes that is responsible for the detoxification of biogenic and xenogenic aldehydes. Over the
years, the field of ALDH research has been frequently reviewed and updated. Notably, Dr. Vasilis Vasiliou’s laboratory at the University of Colorado’s Health Sciences Center has maintained a database of the ALDH gene superfamily for public viewing at www.aldh.org. TABLE 1 lists these
ALDHs and some of their characteristics.
ALDH has an ancient origin and is found in all living organisms from Archaea and Eubacteria to eukaryotes (131,
291). Complete human genome and expression studies revealed that there are 19 functional ALDH genes with a wide
range of tissue expression and substrate specificity. The
ALDH gene superfamily has been reviewed recently (for
comprehensive reviews on ALDH, see Refs. 59, 194, 320).
Here, we focus solely on the physiology and pathology associated with one member of this family, the mitochondrial
0031-9333/14 Copyright © 2014 the American Physiological Society
1
CHEN ET AL.
Table 1. 19 Human ALDH isozymes
ALDH
Subcellular
Location
Preferred Substrate
ALDH1A1
Cytosol
Retinal
ALDH1A2
Cytosol
Retinal
ALDH1A3
Cytosol
Retinal
ALDH1B1
ALDH1L1
Mitochondria
Cytosol
Aliphatic aldehydes
10-Formyl tetrahydrofolate
ALDH1L2
ALDH2
Unknown
Mitochondria
ALDH3A1
Cytosol, nucleus
ALDH3A2
ALDH3B1
Microsomes,
peroxisomes
Cytosol
Unknown
Acetaldehyde, 4-HNE and
MDA
Aromatic, aliphatic
aldehydes
Fatty aldehydes
ALDH3B2
ALDH4A1
ALDH5A1
ALDH6A1
Unknown
Mitochondria
Mitochondria
Mitochondria
Unknown
Glutamate ␥-semi-aldehyde
Succinate semi-aldehyde
Malonate semi-aldehyde
ALDH7A1
Cytosol, nucleus,
mitochondria
␣-Aminoadipic semi-aldehyde
ALDH8A1
ALDH9A1
ALDH16A1
ALDH18A1
Cytosol
Cytosol
Unknown
Mitochondria
Retinal
␥-Aminobutyr-aldehyde
Unknown
Glutamic ␥-semi-aldehyde
Unknown
Disease, Mutational
Phenotypes, Functions
Alcohol sensitivity, alcoholism,
Parkinsonism with ALDH2
double knockout animal
(334)
Increased risk for neural tube
defect
Embryonic lethal in knockout
animal
Unknown
Low fertility and decreased
hepatic folate in knockout
animal
Unknown
See Figure 4 in this review
Chromosome
GeneBank
Accession No.
9q21.13
NM_000689
15q22.1
NM_003888
15q26.2
NM_000693
9q11.1
3q21.2
NM_000692
NM_012190
12q23.3
12q24.2
NM_001034173
NM_000690
Cataracts in knockout animal
17p11.2
NM_000691
Sjögren-Larsson syndrome
17p11.2
NM_000382
Unknown, locus linked to
paranoid schizophrenia
Unknown
Type II hyperprolinemia
␥-Hydroxybutric aciduria
Development delay, metabolic
abnormality, methylmalonic
aciduria
Pyridoxine-dependent
seizures, locus linked to
osteoporosis (107)
Unknown
Unknown
Unknown
Hyperammonemia,
hypoprolinemia,
neurodegeneration,
cataract
11q13.2
NM_000694
11q13.2
1p36.13
6p22.2
14q24.3
NM_000695
NM_003748
NM_001080
NM_005589
5q31
NM_001182
6q23.2
1q23.2
19q13.33
10q24.3
NM_022568
NM_000696
NM_153329
NM_002860
ALDH, aldehyde dehydrogenases. Data from the following sources: Marchitti et al. (194), Marchitti et al.
(195), http://www.aldh.org/.
ALDH2. Significant discussion is dedicated to the potential
health impact of an inactivating single point mutation in
ALDH2 that is found in ⬃560 million East Asians
(ALDH2*2 mutation), or nearly 8% of the world’s population (24).
Human ALDH2 is a 517-amino acid polypeptide encoded by a nuclear gene located at chromosome 12q24
(256). The protein is transported to the mitochondrial
matrix in a process that is dependent on its NH2 terminus
17-amino acid mitochondrial targeting sequence, which
is cleaved as part of the complete folding and maturation
of the enzyme inside the mitochondria (20). Like most
members of the ALDH family, ALDH2 is a tetrameric
enzyme with ⬃56 kDa identical subunits. The tetramer is
regarded as a dimer of dimers with only 2 of the catalytic
2
sites on each enzyme complex maintaining the activity.
Each subunit consists of three main domains: the catalytic domain, the coenzyme- or NAD⫹-binding domain,
and the oligomerization domain. In addition to its dehydrogenase activity, ALDH2 has also reductase and esterase activities (49, 163, 211). However, no physiologically relevant substrates for these two additional enzymatic activities have been reported, except for the
reductase activity-dependent bioconversion of nitroglycerin to 1,2-glyceryl dinitrate (GDN) resulting in the release of nitric oxide (NO) (48 –50, 312). ALDH2 is expressed ubiquitously in all tissues but is most abundant in
the liver and also found in high amounts in organs that
require high mitochondrial oxidative phosphorylation,
such as the heart and brain (236, 295). Over the past few
decades, the biochemical and molecular characteriza-
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TARGETING ALDEHYDE DEHYDROGENASE 2
tions of ALDH2 have been studied extensively (see reviews in Refs. 68, 330, 331).
ALDH2 is better known for its critical role in ethanol metabolism. The ethanol detoxifying pathway in humans occurs mainly in the liver and is carried out by two enzymatic
steps. The first step is catalyzed by alcohol dehydrogenase
(ADH), and the second step is mainly catalyzed by ALDH2
(FIGURE 1). The ALDH2 gene was first identified and fully
characterized in 1987 (20, 21), and the X-ray crystal structure of ALDH2 was fully elucidated in 1999 (222). Among
the 19 human ALDH isozymes, ALDH2 is the most efficient
one for the metabolism of ethanol-derived acetaldehyde; it
has the lowest Km (⬃0.2 ␮M) towards this substrate (72).
This Km is 900-fold lower than that of the abundant cytosolic ALDH, ALDH1, and therefore, in humans, ALDH2 is
probably the only ALDH enzyme that contributes significantly to acetaldehyde metabolism (149). Less known is
that ALDH2 is also capable of metabolizing numerous
other short-chain aliphatic aldehydes, as well as some aromatic and polycyclic aldehydes (148), thus providing an
important protective enzymatic function against these toxic
agents. In particular, ALDH2 plays a key role in oxidizing
endogenous aldehydic products that arise from lipid peroxidation under oxidative stress, such as 4-hydroxy-2-nonenal
(4-HNE) and malondialdehyde (MDA) as well as environmental aldehydes, such as acrolein (present, for example, in
tobacco smoke and in car exhaust; Refs. 41, 368).
Ethanol
Acetaldehyde
[ADH]
Alcohol dehydrogenase
Acetic acid
Ethanol
Acetaldehyde
ALDH2
Genetic polymorphisms of human ALDH2 have been well
surveyed among a wide range of ethnic groups (44, 99, 172,
227, 319, 366). The most relevant ALDH2 variant is the
ALDH2*2 allele, which is found in as many as ⬃35– 45%
of East Asians (i.e., Chinese, Japanese, Korean, and Taiwanese) (59, 99). The ALDH2*2 carriers have a lower
ALDH2 enzymatic activity, and this deficiency is manifested by the characteristic facial flushing, headaches, nausea, dizziness, and cardiac palpitations after consumption
of alcoholic beverages. This ethanol-induced flushing syndrome in ALDH2*2 individuals is caused by a single G to A
nucleotide change, which leads to a substitution of glutamate to lysine at position 487 (E487K, or E504K in some
literature, when the first 17 amino acid mitochondrial targeting sequence, which are removed in the mature enzyme,
are included) in the ALDH2 monomer (366). The E487K
mutation exerts a dominant effect over the wild-type monomer encoding the ALDH2*1 allele. Therefore, heterozygotic individuals (ALDH2*1/*2) are expected to have dramatically lower than 50% of the wild-type’s enzymatic activity, and ALDH2*2/*2 homozygotes have ⬍1– 4% of the
wild-type activity (77, 78, 378). With ⬃560 million (or 8%)
of the world population having this mutation, ALDH2*2 is
probably the most common human enzyme deficiency,
exceeding other well-known human enzymopathies, such
as glucose-6-phosphate dehydrogenase (G6PD; affecting
⬃400 million; Ref. 249), or sickle cell anemia and thalassemia (hemoglobin disorders; affecting ⬃350 million; Ref.
3). Remarkably, it appears that all of the ⬃560 million
ADH
Normal Alcohol Metabolsm
Acetate
[ALDH2]
Aldehyde dehydrogenase
Acetic acid
Acetaldehyde
ALDH2*2
Ethanol
ADH
Accumulation of Acetaldehyde in ALDH2*2
FIGURE 1. Ethanol metabolism. A: ethanol metabolism occurs in two steps: ethanol is metabolized quickly by
alcohol dehydrogenase (ADH) to generate acetaldehyde. Acetaldehyde is then metabolized by the mitochondrial
aldehyde dehydrogenase 2 (ALDH2) to acetate. The first step of metabolism, catalyzed by ADH, is a reversible
reaction. The second step, catalyzed by ALDH2, is the rate-limiting step in ethanol metabolism, and it takes ⬃1
h to metabolize the amount of ethanol that is found in a single alcoholic beverage. B: ethanol metabolism occurs
mainly in the liver. In normal ALDH2 individuals, acetaldehyde is quickly oxidized to nontoxic acetate. In ALDH2
enzyme-deficient (ALDH2*2) individuals, a significant amount of acetaldehyde is rapidly accumulated even after
a moderate amount of alcohol ingestion. Acetaldehyde, which is very diffusible and crosses biological membranes, can be circulated in the blood and metabolized in all tissues, as ALDH2 is present in all mitochondria.
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CHEN ET AL.
ALDH2*2 East Asians carry the identical single nucleotide
mutation, which can be traced back to the Han Chinese in
Southeast China ⬃2,000 –3,000 years ago (184).
As discussed above, although human mitochondrial aldehyde dehydrogenase was first described in the early 1980s,
much of the work on the enzyme relates to its role in ethanol
metabolism. In particular, these studies investigated the
negative health impacts of the ALDH2*2 mutation associated with alcohol consumption and reduced prevalence
of alcoholism among ALDH2*2 carriers, due to alcohol
avoidance because of the unpleasant effect of acetaldehyde
accumulation following alcohol consumption.
However, a significant amount of evidence has recently
emerged to indicate a greater role of ALDH2: its role in
preventing numerous pathologies. Critical participation of
ALDH2 in cytoprotection and its implication in many human diseases have only been recently elucidated. The following review summarizes these new data. As will be discussed, some of the observations on the protective role of
ALDH2 in a variety of pathologies were based on epidemiological studies, whereas other data include studies in
model systems of human diseases. A number of novel small
molecule modulators of ALDH2 have been discovered and
developed recently. In addition to the well-characterized
inhibitors of ALDH2, new research tools, including more
selective inhibitors, transgenic mice with altered ALDH2
activity, and selective ALDH2 activators have shed new
light on the role of ALDH2 in human diseases. The description of these tools and how they helped to determine the
role of ALDH2 in human pathologies is provided in this
review. Importantly, the ability to specifically enhance or
repair the enzymatic activity by a newly discovered class of
ALDH2 activators has stimulated new interest in this enzyme and may provide new opportunities for the development of pharmaceuticals for the treatment of important
human diseases.
II. MOUSE MODELS WITH MODIFIED
ALDH2 ACTIVITY
Since a deficiency in ALDH2 activity is common in East
Asians, transgenic mice with ALDH2 deficiency were generated to serve as models to recapitulate the phenotypes of
the diseases or physiological responses associated with this
mitochondrial enzyme deficiency. These animal models can
also be used in proof-of-concept studies of potential interventions and therapeutics targeting ALDH2 in humans.
Four types of transgenic ALDH2 mice have been developed.
TABLE 2 summarizes the description of these transgenic
mouse models.
A. ALDH2 Knockout Mice
Many manifestations of defects in alcohol metabolism, alcohol-induced pathology, and hypersensitivity to acetalde-
4
hyde observed in the ALDH2*2 East Asians can be reproduced in the ALDH2 knockout (denoted ALDH2⫺/⫺)
mouse model. Pronounced susceptibility to ischemia-reperfusion injuries and accumulation of aldehydic adducts are
also demonstrated in the ALDH2⫺/⫺ mice. Two independent ALDH2 knockout alleles, tagged with a neomycinresistant marker within the ALDH2 gene, were introduced
to the C57BL/6 mouse genome to disrupt the gene function
(80, 147). Western blot analyses confirmed that no immunoreactive ALDH2 protein was produced in the homozygous ALDH2⫺/⫺ mice. ALDH2 knockout mice provided
useful research tools to explore the physiology, phenotypes,
and pathologies derived from a complete lack of ALDH2
function. However, these mice may not completely reflect
the phenotype of the human ALDH2*2 population, since
the ALDH2*2 allele in human carries residual enzyme activity. It is conceivable that subtle biological differences may exist between the genotypes of ALDH2⫺/⫺,
ALDH2⫹/⫺, and ALDH2*1/*2, which represents the largest affected human population. A comprehensive review on
the ALDH2 knockout mouse has been published recently
by Yu et al. (371).
B. ALDH2*2 Overexpressing Mice
The E487K ALDH2 mutation exerts a dominant-negative
effect on the wild-type gene in humans; heterotetramerization of the mutated and wild-type subunits of ALDH2 results in a dramatically reduced catalytic activity. Therefore,
the first mice to carry the ALDH2*2 mutation were transgenic mice that overexpressed mouse E487K ALDH2 subunits (69, 231). These mice showed 2- to 20-fold increased
expression of ALDH2*2 gene over the endogenous wildtype ALDH2 transcripts and suppressed catalytic activity
relative to the endogenous wild-type ALDH2 enzyme (69,
120, 231). These ALDH2*2 transgenic mice better represent the human phenotype of ALDH2*2 homozygotes,
since they have some residual ALDH2 enzyme activity
(120), and unlike the ALDH2 knockout mice, they can be
used to test the ability of ALDH2 modulators that correct
the structural defect of the enzyme to reverse the phenotype.
Two strains of transgenic ALDH2*2 have been constructed
to express full-length human ALDH2 cDNA containing the
E487K point-mutation under the control of elongation factor, EF1␣ and chicken ␤-actin promoters, respectively (69,
231). In the EF1␣ ALDH2*2 transgenics, expression of
exogenous ALDH2*2 transcripts was observed in all tissues of these mice. In particular, high levels of the
ALDH2*2 transcripts were detected in olfactory bulb, cortex, hippocampus, and midbrain, where their levels were
more than twice as high as the endogenous wild-type
ALDH2 transcripts. In the second transgenic strain, overexpressing the mutated mouse E487K cDNA under the control of the chicken-␤-actin promoter, tissue specific overexpression of the ALDH2*2 mutant subunits was particularly
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TARGETING ALDEHYDE DEHYDROGENASE 2
Table 2. Transgenic ALDH2 animal models
Mouse Strain
Nature of the Change
in ALDH2
ALDH2 knockout
Endogenous wild-type
ALDH2 gene
replaced by an
inactivated ALDH2
gene
ALDH2*2 transgenic
Multiple insertions of
overexpressed
ALDH2*2 gene,
endogenous wildtype ALDH2 gene
is intact
ALDH2 overexpression
Multiple insertions of
overexpressed
ALDH2 wild-type
gene, endogenous
wild-type ALDH2
gene is intact
Endogenous wild-type
ALDH2 gene
replaced by an
ALDH2*2 gene
Endogenous wild-type
ALDH1A1 and
ALDH2 genes
replaced by
inactivated
ALDH1A1 and
ALDH2 genes
ALDH2 E487K knock-in
ALDH2, ALDH1A1 double
knockout
Phenotype
Hypersensitivity to ethanol and acetaldehyde;
elevated blood ethanol and acetaldehyde
levels; increased ethanol-induced tissue
oxidative markers, osteopenia, tissue
damage and DNA modification; increased
ischemia-reperfusion damage,
cardiomyocyte dysfunction, 4-HNE adducts
Accelerated aging, shortened lifespan,
neuronal loss and impaired cognitive
function, smaller body size, increased 4HNE adducts; mitochondria stress,
metabolic remodeling, tolerance to
ischemia-reperfusion stress;
hypersensitivity of osteoblasts to
acetaldehyde, kyphosis, osteoporosis
Reduced blood ethanol and acetaldehyde
levels, reduced 4-HNE adducts; protection
against acute and chronic ethanol
administration and oxidative stress in liver,
heart, and brain
Reference Nos.
79, 126–129, 184, 200–
202, 215, 227, 228,
235, 283
68, 230, 119
65, 95, 96, 106, 172, 185,
258, 259, 372, 374,
Hypersensitivity to ethanol and acetaldehyde
Increased neurodegeneration, agedependent motor dysfunction, increased
DOPAL, 4-HNE adduct formation
pronounced in cardiac and skeletal muscles (69). Reduced
ALDH enzyme activity correlated well with increased aldehydic load and severity of the phenotypes. It is important to
note that many developmental abnormalities and exacerbated pathophysiological conditions described in both
strains of the ALDH2*2 overexpressing mice were not observed in the ALDH2⫺/⫺ mice. Considerations should be
therefore made to determine how applicable and relevant
the phenotypes and the results derived from ALDH2*2
overexpressing transgenic mice are to humans carrying the
ALDH2*2 mutation.
C. ALDH2 Overexpressing Mice
Transgenic mice overexpressing the wild-type ALDH2 gene
driven by the chicken-␤-actin promoter have also been generated (66). These ALDH2 overexpressing mice were used
to evaluate the effect of augmented ALDH2 function on
organ protection against acetaldehyde, oxidative stress, and
lipid peroxidation-derived 4-HNE. The ALDH2 overexpressing mice have been challenged with both acute and
chronic ethanol administration and were subjected to diabetes-induced cardiomyopathy to explore different signal
transduction pathways mediated by ALDH2 (66, 96, 97,
Unpublished data
335
107, 138, 173, 185, 186, 260, 372, 375, 376). Overall, this
different genetic model demonstrated the critical role of
ALDH2 in the protection against toxic aldehydes and oxidative stress in various organs and physiological conditions.
As expected, the outcomes were, in general, the opposite of
what were observed in the ALDH2 knockout or the
ALDH2*2 overexpressing mice, which showed increased
susceptibility to aldehydes and pathological conditions.
The studies in ALDH2 overexpressing mice demonstrated,
in principle, that enhancing the ALDH2 activity can ameliorate many of the deleterious effects of aldehydes and may
provide a better protection against both acute and chronic
injuries induced by ethanol toxicity or oxidative stress.
D. ALDH2*2 Knock-in Mice
An ALDH2*2 knock-in mouse model was recently developed in our laboratory. In these mice, we replaced the
mouse wild-type ALDH2 allele with a mouse E487K substituted ALDH2 allele by homologous recombination. The
ALDH2*2 knock-in mice therefore differ only by one
single amino acid within the ALDH2 gene compared with
the wild-type mice. Western blot analysis indicated that the
ALDH2*2 heterozygous animals expressed similar levels of
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5
CHEN ET AL.
ALDH2 protein to the wild-type mice. However, the enzymatic activity of the knock-in animal is dramatically reduced as expected from the dominant-negative effect of the
E487K mutation. The ALDH2 knock-in mice carry a single
amino acid substitution that is equivalent to the E487K
substitution that defines the human ALDH2*2 mutation.
Unlike ALDH2 knockout or ALDH2 overexpression, the
overall level of ALDH2 gene expression in the ALDH2*2
knock-in mice is not affected and should mimic the expression level of human ALDH2*2. The enzymology, structural
defect, and phenotype of the ALDH2*2 mice are, therefore,
a true representation of the human ALDH2*2 carriers. Because of the identical nature of the genetic defect, the
ALDH2*2 knock-in mice can serve as an ideal experimental
model for the research of human diseases associated with
ALDH2 deficiency, and also for efficacy studies using small
molecule activators (Aldas, described below in the section
of pharmacological modulators of ALDH2 activity) in animal. Indeed, in our in vitro enzyme assays, Alda-1 was
capable of activating both human and mouse ALDH2*2
recombinant enzymes and with similar potency profile and
activation kinetics. In our view, the ALDH2*2 knock-in
mice should be the best model for the studies of human
ALDH2*2 mutation phenotype.
III. PHARMACOLOGICAL MODULATORS OF
ALDH2 ACTIVITY
6
Based on the structure of daidzin and its co-crystal structure
with ALDH2, other potent ALDH2 inhibitors have been
designed (7, 94). Most recently, CVT-10216, which was
developed based on structure-activity relationship studies
of daidzin, was found to be a potent ALDH2 inhibitor (IC50
of ⬃30 nM; ⬃2- to 3-fold better than the parent molecule,
daidzin) (7, 235). Another new class of irreversible inhibitors has been identified using an unbiased high-throughput
screening using purified ALDH2 and measuring the decline
in catalytic activity in vitro (40, 145). This class of aldehyde
dehydrogenase inhibitors (Aldis) is characterized by a core
structure of 3-amino-1-phenylpropan-1-one (145). Due to
an enzyme-mediated ␤-elimination reaction, which generates a vinyl ketone intermediate, these inhibitors covalently
modify the active site cysteine residue (Cys 302), thus irreversibly abolishing ALDH enzymatic activity. These first
generation Aldis, which are not ALDH isozyme-selective,
represent a novel class of broad and generally applicable
suicide inhibitors for ALDH. Additional ALDH2 inhibitors
have also been discovered by other efforts of high-throughput screening and virtual computational screens, based on
crystal structures of ALDH2 and the identification of critical interacting amino acids in the catalytic site of the enzyme (239). A comprehensive review of the pharmacology,
mechanism of action, isozyme selectivity, substrate specificity, and clinical applications of an additional 13 known
ALDH inhibitors is available (151).
A. Inhibitors
B. Activators
Inhibition of ALDH2 as a treatment for alcohol abuse has a
long history that predates modern pharmacological interventions using small molecule-based drugs. Kudzu (Pueraria lobata), a traditional Chinese medicine, has been used
for more than 1,000 years for its antidipsotropic effect
(144). Daidzin and daidzein are two active isoflavones identified in the root and flowers of Kudzu. Daidzin binds to the
catalytic site of ALDH2 and is a potent reversible competitive inhibitor of ALDH2 with IC50 of ⬃80 nM (181). Daidzin inhibits mitochondrial ALDH2 activity, which results in
higher blood acetaldehyde levels, thus mimicking the
ALDH2*2 phenotype (143). Disulfiram (Antabuse) is, so
far, the only United States Food and Drug Administration
(FDA)-approved drug that putatively targets ALDH2. Disulfiram and its metabolites irreversibly inactivate catalytic
Cys302 in ALDH2 by carbamylation in the substrate site of
the enzyme (178, 283). Similar to daidzin, inhibition of
ALDH2 by disulfiram leads to acetaldehyde accumulation,
facial flushing, nausea, and vertigo after alcohol drinking,
thus mimicking the ALDH2*2 phenotype. As a result of
these discomforts, disulfiram has been approved and used
as an alcohol aversion drug in the treatment of dependency
since 1951 (12). However, poor compliance of drug usage
has been an issue that compromises the effectiveness of
disulfiram as a treatment for alcoholism (297) (See further
discussion below).
Only a couple of small molecule activators for metabolic
enzymes have been identified. One example is a class of
novel molecules called Aldas, for aldehyde dehydrogenase
activators. We discovered Aldas using a fluorescence-based
high-throughput screening. A family of ALDH2 isozymespecific agonists, represented by Alda-1 [N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide, MW 324], were
found to enhance the catalytic activity of ALDH2 in vitro
and in vivo (40). Alda-1 has several useful features. 1) It
enhances, specifically, the catalytic activity of ALDH2 by
nearly twofold, with EC50 of ⬃20 ␮M. 2) It protects
ALDH2 enzymatic activity from 4-HNE-induced inactivation and is able to maintain the catalytic activity of ALDH2
in the presence of a high concentration of 4-HNE. 4-HNE,
a substrate of ALDH2, also inhibits the enzyme by forming
a Michael adduct or Schiff base with cysteine 302 at the
catalytic site (65, 182). 3) Importantly, and of potential
clinical relevance, Alda-1 effectively restores the enzymatic
activity of the E487K mutant ALDH2. Alda-1 increases the
catalytic activity of ALDH2*2 homozygotic enzyme by 11fold and brings the activity of the heterozygotic enzyme to
wild-type levels (40). Cocrystal structures of Alda-1 with
wild-type ALDH2 and Alda-1 with the E487K mutant
ALDH2 demonstrated that Alda-1 binds at the entrance of
the catalytic tunnel in close proximity to Cys302 and
Glu286, which are critical to its substrate catalysis (248)
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TARGETING ALDEHYDE DEHYDROGENASE 2
(FIGURE 2).
Kinetically, Alda-1 dramatically decreases the
Km of the ALDH2 for the cofactor NAD and increases the
Vmax of substrate catalysis of aldehydes up to four carbons
in length (14, 248). The binding of Alda-1 near the substrate entrance may facilitate productive encounters between the activated water molecule and the thio-acyl intermediate of the substrate, thus resulting in an accelerated
hydrolysis and release of the catalytic product. The close
proximity between Alda-1 and Cys302 likely also shields
this crucial catalytic residue from the nucleophilic attack of
reactive aldehydes, such as 4-HNE.
which exerts its allosteric effect to restore the structural
defect of a catalytically impaired enzyme.
Another example of generating novel ALDH agonists was
guided by the information generated from X-ray cocrystal
structure of Alda-1 with ALDH2. With the use of a computer-assisted virtual screening of chemical libraries, threedimensional molecular docking of 19,943 compounds on
ALDH2 generated 21 hits (152).
In summary, the novel small molecule modulators of ALDH
represented by Aldas and Aldis are useful tools to determine
the role of ALDH2 in a variety of human pathologies. They
may also serve as a new class of therapeutics.
Another important feature of Alda-1 is its ability to restore
the activity of a defective ALDH2*2 enzyme. E487K substitution in ALDH2 causes a loss of electron density near
residues 245–262, which form helix ␣G, and residues 466 –
478, which form the active-site loop (162, 163). As revealed
by the crystal structure of Alda-1 in complex with ALDH2
E487K mutant, binding of Alda-1 restores the ␣ helix structure and the loop at these regions nearly to the native wildtype state even though Alda-1 has no direct contact with
these residues (248) (FIGURE 2). Alda-1 is therefore an agonist and simultaneously functions as a chemical chaperone,
IV. DISEASES ASSOCIATED WITH ALDH2
Aldehydes are formed as a part of normal catabolism (e.g.,
from amino acids, lipids, and nucleotides). Aldehydes and
their precursors are present in food: ethanol is a precursor
to acetaldehyde, fermented or pickled fruits and vegetables
contain aldehydes, many spices and flavorings like vanilla
Ni
co
tin
Cl am
eft ide
Trp177
Wild-type
Glu268
Alda-1
Cys302
ALDH2*2
ALDH2*2 with Alda-1
FIGURE 2. Alda-1, an allosteric agonist of ALDH2, corrects the structural defect in the ALDH2*2 mutant
present in 8% of the human population. Top: crystal structure of Alda-1 (stick presentation) in the catalytic
tunnel (shown as filled structure) of ALDH2 (Protein Data Bank Accession Code PDB: 3INJ). Highlighted are the
critical amino acids in this interaction. Bottom: crystal structure of wild-type ALDH2 (left panel, Protein Data
Bank Accession Code PDB: 1O05) and mutant ALDH2*2 (middle panel, Protein Data Bank Accession Code,
PDB: 1ZUM). Co-crystal structure of ALDH2*2 with Alda-1 (shown as a ball-filled structure and highlighted in
yellow; see arrows) is provided in the right panel (PDB: 3INL). Circles focus on the alpha helix structure (␣G)
that is missing in the mutant ALDH2*2 enzyme (middle panel vs. left panel) and is restored (right panel) when
Alda-1 is bound to ALDH2*2. Images produced using UCSF Chimera package from the Computer Graphics
Laboratory, University of California, San Francisco.
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7
CHEN ET AL.
and cinnamon are aldehydes, and aldehydes are also products of frying or smoking meat and fish. Finally, aldehydes
are also present in our environment in a variety of aromas
and perfumes, in cigarette smoke and car exhaust, and in a
variety of chemicals and pollutants.
The potential role of aldehydes in general and aldehydes
that are the substrates of ALDH2 in particular, in human
pathologies has not been the focus of much research until
recently. ALDH2 is well conserved throughout evolution,
from bacteria to humans (131, 291, 319). Because much of
its biochemical characterization occurred about 30 years
ago (132, 177, 281), and because an inactivating point mutation present in 8% of the human population was thought
to be benign (24, 183, 285, 358), there was little active
research on the role of ALDH2 in human diseases apart
from its role in ethanol metabolism and complications associated with excessive ethanol drinking.
Yet, the toxic nature of aldehydes has been known for a
long time. Aldehydes can easily diffuse through cell membranes, form adducts with macromolecules including proteins, DNA, and lipids; such aldehydic adduction usually
modulates or disrupts the function of these macromolecules
(FIGURE 3; Refs. 73, 223, 250, 266, 307). Reactive aldehydes are readily formed during oxidative stress as products
of lipid peroxidation (FIGURE 3). Therefore, a mechanism
for rapid clearance of these highly diffusible and harmful
aldehydes is crucial to protect cells and tissues from damage. In diseases where there is excessive aldehydic load, at
least some of the pathologies may be alleviated by faster
metabolism and inactivation of these toxic aldehydes.
In the following, we argue that ALDH2 is critical to the
health of cells and that it serves as an important shield from
the damage occurring under oxidative stress. Since oxidative stress is associated with numerous human pathologies,
increasing the catalytic activity of ALDH2 may provide a
novel and effective means to reduce oxidative stress-induced cell and organ dysfunction and therefore support
human health. FIGURE 4 depicts some of the diseases in
which reports from human specimens demonstrated an increase in the aldehydic load and/or in which ALDH2 was
associated with or played a positive role in reducing the
pathology. These diseases are discussed in more detail in the
following.
A. Cardiovascular Diseases
1. Myocardial infarction, heart failure, and stroke
Cardiovascular diseases, including myocardial infarction,
cardiac hypertrophy, and heart failure, are a major cause of
morbidity and mortality worldwide. Although early research efforts focused on the damaging effects of free radicals in the heart (19, 280), recent findings revealed that
8
accumulation of cardiotoxic reactive aldehydes, derived
from reactive oxygen species-induced stress, can also impair
myocardial function in rodents and humans (40, 41, 255).
Of the 19 different ALDH isozymes expressed in the human
body, the mitochondrial ALDH2 has rapidly emerged as a
crucial enzyme involved in protecting the heart from oxidative stress. Although the contribution of ALDH2 in cardioprotection was initially discovered by our group downstream of protein kinase C type ⑀ (⑀PKC) activation (40),
⑀PKC has multiple protein partners intracellularly including ion channels (45). Additionally proteomic analysis suggests ⑀PKC in models of cardioprotection have many subcellular protein partners (323). In this regard, targeting
ALDH2, instead of ⑀PKC, will provide a more specific therapeutic target. Experimental approaches using either pharmacological activation or genetic overexpression of ALDH2
have shown that improved detoxification of reactive aldehydes, such as 4-HNE, is protective against acute (i.e., ischemia) and chronic (i.e., heart failure) cardiovascular diseases
(25, 26, 40, 41, 97, 185, 186, 259, 298, 375–377). The cardioprotective effects of ALDH2 have also been highlighted by
epidemiological studies, demonstrating that individuals carrying a point mutation in the ALDH2 gene (ALDH2*2), which
results in a dramatic reduction in enzymatic activity, are more
susceptible to cardiac diseases and to the morbidity associated
with them (16, 38).
Most of the cardiac damage occurring during ischemia and
reperfusion injury and in heart failure is due to excessive
generation of reactive oxygen species (19), which leads to
exacerbated peroxidation of polyunsaturated fatty acids
(i.e., linoleic acid, arachidonic acid, and cardiolipin) present
in biological membranes (266). These lipid hydroperoxides
can subsequently form toxic secondary end products, such
as 4-HNE and malondialdehyde (266). As mentioned
above, 4-HNE is a highly reactive carbonyl compound that
readily interacts with cysteine, histidine, and lysine residues
and forms protein adducts via Michael addition or Schiff
base reaction (314). Excessive 4-HNE adduct formation in
the myocardium during ischemia-reperfusion injury impairs cardiac contractility (1), mitochondrial bioenergetics
(116), and redox balance (154) while impairing also protein
quality control (31, 55) by targeting key metabolic enzymes
(313) and the 26S proteasome (FIGURE 3; Ref. 75).
Accumulation of 4-HNE is also associated with the establishment of cardiomyopathies from different etiologies and
with progression to heart failure (192, 218, 219). 4-HNEmodified proteins are significantly increased in myocardial
biopsies from patients with hypertrophic and dilated cardiomyopathy compared with the levels in control subjects
who do not have cardiac disease (218, 219). Of interest,
sustained treatment with a beta-blocker (carvedilol) for 9
months, which improves cardiac functions, is associated with a
40% reduction in the levels of cardiac 4-HNE-modified
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TARGETING ALDEHYDE DEHYDROGENASE 2
1
ROS
4
4-HNE
Proteasome
2
ROS
1
3
4-HNE
ETC
5
6
KGDH
9
7
ALDH2
8
Nucelus
FIGURE 3. 4-HNE, an aldehydic product of lipid peroxidation, causes cytotoxicity. Buildup of aldehydes, such
as 4-hydroxynonenal (4-HNE), due to oxidative stress promotes cell death. Listed are steps in aldehydic injury
to cells, using 4-HNE (depicted as a zigzag line), as an example. 1) 4-HNE is produced through ROS-mediated
lipid peroxidation of mitochondrial and plasma membranes. 4-HNE can in turn 2) reduce membrane integrity,
3) inhibit proteasomal function, 4) trigger unfolded protein accumulation, 5) inhibit electron transport chain
activity, 6) reduce Kreb’s cycle activity, 7) inhibit ALDH2 activity, 8) increase mitochondrial permeability and
dysfunction, or 9) cause DNA damage.
proteins in patients with dilated cardiomyopathy (218).
Mak et al. (192) reported that 4-HNE levels are consistently
elevated in the plasma of congestive heart failure patients
and are inversely correlated with left ventricular contractility. Moreover, animal models that present increased circulating levels of 4-HNE due to alcohol dehydrogenase overexpression or ALDH2 silencing are more sensitive to ischemia and reperfusion injury and are more prone to develop
cardiomyopathy and heart failure (40, 101, 138, 185, 186,
259, 298, 377). Exposure of isolated adult rat cardiomyocytes to 4-HNE (0.5– 400 ␮M) disrupts the cellular redox
balance, induces calcium overload, causes contractile dys-
function, and elicits pro-arrhythmic effects (15, 220). Thus
increased aldehydic load is detrimental to the heart.
Elevated 4-HNE-modified proteins have also been reported
in patients with hypertension or with peripheral artery disease (189, 251, 252, 299). Gastrocnemius muscle biopsy
obtained from peripheral artery disease patients presents
higher levels of protein carbonyls, lipid peroxides, and
4-HNE compared with control muscles (251). These
changes are accompanied by reduced mitochondrial respiration and reduced antioxidant activity (251). Although
4-HNE accumulation negatively associates with peripheral
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9
CHEN ET AL.
Oxidative Stress
Reactive Aldehydes
Myocardial
infarction
Ethanol toxicity/
intoxication
Nitroglycerin
intolerance
Radiation dermatitis
ALDH2
ALDH2
Osteoporosis
Cardiac arrhythmia
Squamous cell carcinoma
Fanconi Anemia
Hypertension
Stroke
Drug toxicity
Pain
Diabetic
complications
Parkinson’s
disease
Alzheimer’s
disease
FIGURE 4. Diseases in which activators of ALDH2 may be beneficial. See text for full discussion. Red
highlights diseases where the evidence for ALDH2 role came from preclinical proof-of-concept studies. Blue
highlights diseases where ALDH2 role was supported by clinical observations. Black highlights diseases where
evidence from both preclinical studies and from human epidemiological or pathohistological studies supports
a role for ALDH2.
artery diseases, the mechanisms involved in this process,
including the contribution of ALDH2 to the pathophysiology of peripheral artery disease, remain to be elucidated.
Acetaldehyde, the first oxidized metabolic product of ethanol, has been considered another important candidate for
the pathogenesis of cardiovascular diseases. Accumulation
of acetaldehyde is very harmful to the heart, causing cardiac
hypertrophy and contractile dysfunction in mouse models
of alcohol dehydrogenase overexpression (67, 117). Acetaldehyde directly impairs cardiac excitation-contraction
coupling and inhibits sarco(endo)plasmic reticulum calcium-ATPase (SERCA) function in rodents (117, 261). However, the molecular basis of acetaldehyde cardiotoxicity has
remained elusive since acetaldehyde has very high chemical reactivity, it is volatile and it is metabolized quite
quickly (67).
The cardioprotective effects of mitochondrial ALDH2 during cardiac ischemia and reperfusion injury, in models of
ethanol-mediated cardiomyopathy and in heart failure,
have been demonstrated by the use of a variety of ALDH2
transgenic mice (96, 97, 173, 185, 186, 259, 377). Overexpression of ALDH2 wild-type enzyme attenuates infarct
size and improves fractional shortening in an in vivo mouse
model of acute myocardial infarction (185). Conversely,
ALDH2 null mice display exacerbated cardiac damage fol-
10
lowing ischemia-reperfusion along with increased formation of mitochondrial reactive oxygen species and endothelial dysfunction (185, 334). In agreement with the cardioprotective role ALDH2, Endo et al. (69) reported that mice
overexpressing the inactive ALDH2*2 exhibited impairment in mitochondrial bioenergetics along with increased
oxidative stress, and elevated levels of 4-HNE-protein adducts.
Although not tested for resistance to acute myocardial infarction injury, adult transgenic mice overexpressing
ALDH2, which results in increased ALDH2 enzymatic activity by fourfold, are more resistant to acute ethanol cardiotoxicity (186). Acute intraperitoneal ethanol injection (3
g/kg) in mice with wild-type ALDH2 expression drastically
increases cardiac acetaldehyde levels along with reduced ex
vivo left ventricular developed pressure and mitochondrial
uncoupling (186, 187). This cardiac toxicity was attenuated
and accentuated in ALDH2 overexpressing and ALDH2
knockout mice, respectively (186, 187). Mice overexpressing ALDH2 exhibited a significantly reduced ethanol-mediated cardiac damage by decreasing acetaldehyde levels, thus
improving myocardial contractility and restoring mitochondrial membrane potential (186).
The sensitivity of the myocardium to aldehydic load has
also been examined in another animal model, in which
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TARGETING ALDEHYDE DEHYDROGENASE 2
chronic ingestion of a 4% ethanol diet for 12 wk was found
to induce cardiac hypertrophy, myocardial fibrosis, reduced
fractional shortening, cell shortening, impaired intracellular Ca2⫹, and homeostasis in wild-type mice. These hallmarks of alcohol cardiomyopathy, described first in humans (79, 317), were associated with elevated blood
acetaldehyde levels, increased oxidative stress, lipid
peroxidation, and protein carbonyl formation in the wildtype, nontransgenic mice (66, 259). Hyperphosphorylation
of oxidative stress related kinases, glycogen synthase kinase-3␤ (GSK-3␤) and apoptosis signaling regulated kinase
(ASK-1), and increased phosphorylation of the transcription factors, GATA4 and CREB cAMP-response element
binding protein (CREB), are reported as the mediators of
ethanol-induced cardiac cardiomyopathy (66, 376).
Chronic administration of ethanol also induced impaired
glucose tolerance, cardiac insulin insensitivity, cardiomyopathy, and contractile defects (173), similar to those observed in diabetic cardiomyopathy from streptozotocininduced diabetic mice (375). In both animal models of ethanol-induced cardiomyopathy and streptozotocin-induced
cardiomyopathy, reduced phosphorylation of Akt, GSK3␤, and forkhead transcription factor of the O subtype
(Foxo3a) was the key signaling cascade that led to apoptosis and mitochondrial dysfunction. Importantly, both mo-
lecular and pathological abnormalities were prevented in
the ALDH2 overexpressing mice. In addition, in an acute
model of ethanol challenge (3 g/kg), ALDH2 overexpression reduced blood acetaldehyde levels and prevented myocardial and cardiomyocyte contractile dysfunction (186).
Therefore, in both acute and chronic ethanol-induced myocardial injuries, a common protective mechanism that is mediated
by ALDH2 appears to exist. As illustrated in FIGURE 5,
ALDH2 conferred its protection against ethanol toxicity by
the regulation of two key kinases, Akt and AMPK. Akt and
AMPK, in turn, regulate transcription factor Foxo3a phosphorylation by ALDH2-mediated decreased phosphatases,
PP2A and PP2C (138, 186, 376). Simultaneously, it was
demonstrated that ALDH2 overexpression also conferred
its protection by the regulation of autophagy and apoptosis
through the balance between Akt and AMPK and their
downstream substrates mTOR, STAT3, and Notch1 (95,
96). Detoxification of acetaldehyde in ALDH2 overexpressing mice also led to attenuation of chronic ethanol exposure-induced damages in brain and liver by a similar mechanism involving Akt and apoptotic regulation (FIGURE 5;
Refs. 107, 260). More recently, Zhang et al. (372) described
that genetic or pharmacological ALDH2 activation protects
against endoplasmic reticulum stress-induced cardiomyo-
Ethanol-induced cardiotoxicity
ALDH2*2 knock-in
or
Alda-1
Ethanol
ADH
ALDH2 knock-out
or
ALDH2 over-expression
Toxic aldehydes
ALDH2
↑ Toxic aldehydes
↓ Toxic aldehydes
Non-reactive acids
pAkt
pAMPK
mTOR
Foxo3
Hypertrophic
PP2A
PP2C
GSK3β
pNotch1
pSTAT3
Contractile dysfunction
Cardiac hypertrophy
Excessive autophagy
Normal
FIGURE 5. Ethanol-induced cardiotoxicity. ALDH2 confers protection against ethanol toxicity by affecting the
activity of key proteins in cardiac myocytes. The scheme summarizes data from transgenic mice that elucidated
the potential pathways that are regulated by ALDH2 and acetaldehydes. Activation of ALDH2 or ALDH2
overexpression were found to confer cardiac protection by regulating autophagy and apoptosis through the
balance between Akt and AMPK and their downstream substrates, such as mTOR, STAT3, Notch1, PP2A,
and PP2C (93, 98, 112).
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11
CHEN ET AL.
cyte contractile dysfunction possibly through reduction of
autophagy (317).
Consistent with the role of ALDH2-mediated cardioprotection against ischemia-reperfusion injury, ALDH2 overexpression prevents 4-HNE-protein adduct formation and attenuates myocardial damage from ischemia-reperfusion
also via the Akt- and AMPK-mTOR signaling cascades
(185). Hence, ethanol toxicity and ischemia-reperfusion appear to trigger a common molecular signaling event that
leads to mitochondrial dysfunction, apoptosis, and autophagy. These studies illustrate the intricacy of signaling
networks that are elicited by acetaldehyde and/or oxidative
stress-derived 4-HNE. These rodent models of cardiac damage associated with increased aldehydic load recapitulate
well the pathological findings in humans (see in the following segment), and the data therefore suggest that ALDH2
activation may benefit patients with any of these cardiac
diseases.
The ALDH2 isozyme-selective activator Alda-1 enabled a
direct examination of whether ALDH2 activation is sufficient to produce cardiac protection. It was shown that pharmacological ALDH2 activation by Alda-1 treatment is sufficient to protect hearts against ischemia-reperfusion injury
in a rat model of myocardial infarction (40). Administration of 20 ␮M Alda-1 for 10 min prior to 35 min of ischemia followed by 60 min of reperfusion increases ALDH2
activity by twofold and significantly reduced cardiac damage by 60% in an ex vivo model of ischemia-reperfusion in
rats (40). Alda-1-induced cardioprotection is also effective
in an open-chest model of acute myocardial infarction in
rats (40, 41). Injection of Alda-1 (8 mg/kg) into the left
ventricle 5 min before ligating the left anterior descending
coronary artery decreases the extent of myocardial infarction and creatine kinase release into the blood by 50% (40).
Conversely, ALDH2 inactivation increases infarct size with
a tight inverse correlation between infarct size and ALDH2
activity (40). In another study, it was demonstrated that
selective ALDH2 activation using an Alda-1 analog, Alda44, circumvents the requirement of ⑀PKC to induce cardioprotection (25). In an independent study, Lagranha et al.
(158) reported that Alda-1 reduces infarct size and postischemic contractile dysfunction in male but not in female
rats (because females already have phosphorylated/activated ALDH2) after ischemia-reperfusion injury (158).
More recently, in another study, Gong et al. (102) demonstrated that addition of Alda-1 to cardioplegic solution (histidine-tryptophan-ketoglutarate) significantly reduces cardiac ischemia-reperfusion injury in an ex vivo rat model.
Alda-1 supplementation improves the cardioprotective effects of the cardioplegic solution by increasing ALDH2 activity, which results in reduction of cardiac 4-HNE levels
and better myocardial contractility depicted by improved
left ventricular developed pressure (102). Such applications
of Alda-1 as an adjuvant in cardioplegic solutions for sched-
12
uled open-heart surgery may have a wide utility for organ
preservation in open-heart surgery or other solid organ
transplantations.
In addition to its direct effect on cardiomyocytes, Koda et
al. (150) have shown that Alda-1 plays a key role in cardiac
mast cell biology during ischemia-reperfusion injury. In this
ex vivo study using a guinea pig model, it was found that
ALDH2 activation prevents degranulation of cardiac mast
cells and subsequent local renin-angiotensin activation during ischemia-reperfusion injury (150). Alda-1 treatment
also drastically reduces mast cell degranulation and release
of renin (the rate-limiting step in renin/angiotensin system
activation) elicited by acetaldehyde, 4-HNE, or during cardiac ischemia-reperfusion. Preincubation with ALDH2 inhibitors, such as cyanamide or high levels of nitroglycerin,
prevents the benefits of Alda-1 (150). Arrhythmias in patients with myocardial infarction are common and a major
cause for sudden death in this patient population (267).
Thus, in addition to protecting the heart muscle functions,
activation of ALDH2 by Alda-1-like compounds may reduce the damage resulting from reperfusion-induced arrhythmias via enhanced aldehyde detoxification (265). Together, these results uncover novel basic mechanisms of
pharmacological cardioprotection induced by ALDH2 activation.
Finally, Ge et al. (96) demonstrated the use of the ALDH2
activator, Alda-1, in protecting cardiac cells from ethanolinduced contractile dysfunction and exacerbated autophagy. Administration of Alda-1 to ethanol-treated adult
mouse cardiomyocytes significantly improves intracellular
calcium handling, abolishes the ethanol-mediated cardiomyocyte mechanical abnormalities characterized by depressed peak shortening and relaxation, and reduces
caspase-3 activity, a key player in programmed cell death
(96). Together, these findings highlight the potential use of
ALDH2 activators and the critical role of activating
ALDH2 to protect against cardiac damage caused by toxic
aldehydes that accumulate following myocardial infarction,
or under conditions leading to alcoholic cardiomyopathy
and to heart failure.
The ALDH2-mediated protection also extends to ischemic
brain injury, or ischemic stroke (155). Okun et al. (234)
found elevated levels of 4-HNE-protein adducts in the ischemic cerebral cortex when measured within 2 h of stroke
induction in mice. More recently, Lee et al. (168) showed
that plasma 4-HNE levels are elevated in patients with
stroke and in two different animal models of cerebral ischemia. Indeed, intravenous administration of 4-HNE before
stroke not only enlarged the cerebral ischemia-induced infarct area, but also increased oxidative stress in rats (168).
Furthermore, ALDH2 overexpression increases neuronal
survival in the presence of 4-HNE toxicity in PC12 cells.
Cerebral damage and neuronal cell death are also reduced
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TARGETING ALDEHYDE DEHYDROGENASE 2
in ALDH2 transgenic mice after 12 wk of ethanol consumption (260). Two studies have evaluated the association between ALDH2*2 allele and stroke, a common cause of
death in East Asia, where no correlation between the incidence of stroke and the genotype was found (215, 353).
However, considering that heavy alcohol consumption increases the risk of stroke (125, 247), further large cohort
studies are needed to better understand the contribution of
ALDH2 mutation to stroke.
2. Epidemiological human studies suggesting a role
for ALDH2 in the health of the myocardium
Over the past decade, the reduced ALDH2 activity due to
the ALDH2*2 mutation has been shown to be an independent risk factor for acute coronary syndrome in humans.
Takagi et al. (306) demonstrated that the ALDH2*2 allele
is a risk factor for myocardial infarction in Japanese men.
More recently, Xu et al. (348) showed that Chinese male
carriers of the ALDH2*2 inactivating mutation have a significantly higher incidence of acute coronary syndrome
compared with noncarriers. Similar results were found in a
subgroup analysis of patients with primary ST-segment elevation myocardial infarction (STEMI) (348). However,
this ALDH2 mutation is not associated with the number of
coronary arteries with significant stenosis (348). [In this
latter study ALDH2*1/*2 and ALDH2*2/*2 were pooled
into a single group for the analysis.] A case control study of
an elderly Korean male population found that the
ALDH2*2 allele was significantly linked to increased risk
of developing acute coronary syndrome (134). Furthermore, the ALDH2 mutation along with the abnormal highdensity lipoprotein, cholesterol, and elevated body index
mass is an independent risk marker for myocardial infarction in these elderly Korean males. A multivariant regression analysis showed that subjects carrying the ALDH2*2
allele displayed elevated plasma levels of inflammatory
markers (i.e., high C-sensitive protein, hs-CRP) after the
onset of acute myocardial infarction, suggesting higher
myocardial injuries following ischemic insult (16).
The ALDH2*2 allele has also been linked to chronic cardiovascular pathophysiologies. In a large scale meta-analysis of genome-wide association studies from 19,608 subjects, ALDH2*2 (G to A exchange, which is also designated
as SNP rs671) was identified as the most prominent SNP
that is associated with blood pressure variation in East
Asians (139). Other studies confirmed that ALDH2*2 is a
strong risk factor for elevated blood pressure and hypertension among males who consume ethanol excessively (118,
217, 305). Chang et al. (38) recently reported that homozygous carriers of the ALDH2*2 allele were more likely to
progress to hypertension compared with noncarriers over a
follow-up period of 5.7 yr. The risk associated with the
ALDH2*2 mutation is the strongest in alcohol drinkers
relative to nondrinkers, providing further evidence for an
association between ALDH2 genetic variants and alcohol
intake on the risk of hypertension (38). Chen et al. (43)
carried out a meta-analysis to assess the association between the ALDH2 genotype and blood pressure (5 studies,
n ⫽ 7,658) and hypertension (3 studies, n ⫽ 4,219). Unlike
the previous publication, they found that individuals carrying the ALDH2*1/*1 genotype, with average alcohol intake of 25 g/day (or 2 alcoholic beverages), had strikingly
higher systolic and diastolic blood pressure than those with
the ALDH2*2/*2 genotype. Systolic blood pressure was
7.44 mmHg greater for ALDH2*1/*1 than for ALDH2*2/*2
and 4.24 mmHg greater among ALDH2*1/*2 than among
ALDH2*2/*2. Moreover, the risk for hypertension among
ALDH2*1/*1 was found to be ⬃2.5-fold higher than that
among ALDH2*2/*2. Since, unlike the study of Chang et al.
(38), this latter study did not examine heavy drinkers (e.g., ⬎4
alcoholic beverages/day), the data in these two studies may not
be inconsistent. Together, these epidemiological findings highlight an association between the ALDH2 inactivating mutation and alcohol intake on blood pressure and on the risk of
developing hypertension. To date, there are no epidemiological studies that examine the association between ALDH2 mutation and heart failure.
3. Nitroglycerin tolerance
Another aspect relevant to cardiac disease and ALDH2 relates to nitroglycerin metabolism. Nitroglycerin has been
widely used as a vasodilator to treat ischemic and congestive cardiac diseases, including angina pectoris, myocardial
infarction, and heart failure (82, 242). Chen et al. (48)
demonstrated that ALDH2 plays an essential role for the
bioconversion of nitroglycerin to NO to achieve its vasodilating effects. However, nitroglycerin is a potent ALDH2
inhibitor, and continued treatment of nitroglycerin leads to
insensitivity to its vasodilating effect in patients with angina
pectoris (82). As expected, in East Asian patients carrying
the inactivating ALDH2*2 mutation, nitroglycerin is less
effective in eliciting a cardiovascular response compared
with ALDH2 wild-type subjects receiving the same treatment (106, 174, 374). Yet nitroglycerin use in East Asia for
patients with acute myocardial infarction and in congestive
heart failure is very common.
Because nitroglycerin is widely used in patients with acute
myocardial infarction, its inactivating effect on the cytoprotective enzyme ALDH2 may be of concern. Chen et al. (40)
examined this question using a rat model of myocardial
infarction. It was found that 16 h use of nitroglycerin (7.2
mg·kg⫺1·day⫺1) just prior to acute myocardial infarction
resulted in an infarct size that was 31% bigger than in
nontreated rats (40). Furthermore, a worse cardiac function
was observed in rats treated with nitroglycerin overnight, at
clinically relevant concentrations (298). Importantly, we
found that a sustained treatment with Alda-1 (16
mg·kg⫺1·day⫺1) during nitroglycerin use blocked nitroglycerin-induced inhibition of ALDH2 activity and prevented
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13
CHEN ET AL.
the nitroglycerin-induced decrease in cardiac function and
nitroglycerin-induced increase in infarct size (298). Sustained treatment with nitroglycerin also increased protein
carbonylation in the infarct region of the myocardium. In
agreement with the protective role of Alda-1 on ALDH2,
cotreatment of Alda-1 and nitroglycerin greatly reduced the
level of protein carbonylation after myocardial infarction,
in vivo (298).
consumption was required to develop insulin resistance
(135). Hemoglobin A1C, a long-term marker of glycemic
control, was also higher for those ALDH2*2 diabetics
who drank a light to moderate amount of ethanol compared with carriers of the wild-type enzyme (212). These
studies further lead to determining whether ALDH2*2,
independent of alcohol consumption, is a risk factor for
diabetes.
Other studies have shown that NO-induced nitrosylation
on critical cysteines can lead to ALDH2 inactivation (175).
It is likely that the protection offered by Alda-1 against
increased myocardial infarction by nitroglycerin is mediated by a mechanism which provides a protective shield for
Cys302 from nitrosylation, similar to the protection mediated by Alda-1 against carbonylation by 4-HNE. Regardless of the exact mechanism, these data suggest that protecting ALDH2 from nitroglycerin-induced inactivation
will benefit patients treated with nitroglycerin in a sustained fashion. The therapeutic value of an Alda-like
drug which can enhance nitroglycerin bioconversion and
prevent the inhibitory effect of nitroglycerin has been
reviewed recently (82).
Several studies have suggested that the maternal ALDH2
genotype may contribute to NIDDM (213, 301). An association between ALDH2*2 genotype and diabetes prevalence is greater in people with diabetic mothers (32.6%)
compared with those whose mothers have the wild-type
ALDH2 (19.2%) (301). A follow-up study found that those
with an ALDH2*2 genotype were five times more likely to
have a diabetic mother compared with those with wild-type
ALDH2 (300). The authors of these studies suggest the
ALDH2*2 genotype may lead to mitochondrial DNA damage, which in turn may contribute to diabetic complications
in the offspring of diabetic mothers (302). In 542 coronary
artery disease patients, all Han Chinese, a genetic link was
investigated between the ALDH2*2 genotype and NIDDM. Interestingly, the ALDH2*2 genotype was suggested to be a risk factor for NIDDM only in females, and
not males (347).
B. Diabetes Mellitus
Generally considered a disease of the Western world, diabetes is now a global epidemic, predicted to affect 380 million people by 2025, and Asian countries are predicted to
make up 60% of the diabetic world’s population (36). Unlike the Western countries, the diabetic population in Asian
countries is proportionally younger (36). A number of potential genes identified in the Asian population may link to
developing diabetes. These include ALDH2*2, which may
be a risk factor for non-insulin-dependent diabetes mellitus
(NIDDM).
Initial observations by Pyke and Leslie (171, 254) in 1978
for a Caucasian population suggested that chlorpropamide,
a sulfonylurea anti-diabetic agent, taken prior to consumption of alcohol, caused a high percentage of NIDDM to
have facial flushing. The initial studies identified this effect
to be specific for chlorpropamide, and in a study where
patients first received a placebo instead of chlorpropamide
prior to alcohol consumption, facial flushing was not observed. The researchers also found that this flushing effect
of chlorpropamide did not occur in insulin-dependent diabetics. Although chlorpropamide is no longer used clinically, in 2001, chlorpropamide was found to inhibit ALDH,
suggesting a possible link between ALDH and diabetes.
Another study investigating the correlation between ethanol and diabetes concluded that, among those with a
normal ALDH2 genotype, ethanol consumption greater
than 20 drinks per week may increase insulin resistance.
Among those with the ALDH2*2 genotype, less ethanol
14
Preclinical studies in rats also suggest that ALDH2 activity
may affect diabetic pathology (326, 375). Furthermore, in
transgenic mice, overexpression of ALDH2 has been shown
to be protective against streptozotocin-induced diabetic
cardiomyopathy. In this setting of diabetic complications,
Alda-1 was able to recapture the protective effect against
high glucose-induced mitochondrial damage and contractile function in cardiomyocytes (375). Together, this work
suggests an association between diabetes and ALDH2 activity. However, more studies are needed to further examine
the underlying pathology associated with ALDH2 and diabetes.
C. Neurodegenerative Diseases
Brain is an organ that is rich in polyunsaturated fatty acid
and, at the same time, harbors a relatively high concentration of oxygen in the lipid bilayer (257). Oxidative stressinduced lipid peroxidation, mitochondria dysfunction, and
accumulation of aldehydic products are key features in aging, memory loss, cognitive decline, and neurodegeneration
(133, 258, 379). The role of ALDH2 and aldehydic load in
a variety of neurodegenerative diseases has been suggested
based on animal studies, human pathological findings, and
data from epidemiological studies. Here we focus on two
neurodegenerative diseases, Parkinson’s disease and Alzheimer’s disease.
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1. Parkinson’s disease
Parkinson’s disease is a progressive neurodegenerative disease manifested as a loss of movement control and varying
degree of dementia. Parkinson’s disease afflicts ⬃1% of the
population at age 65, and the incidence increases to 4 –5%
of the population at age 85 (74, 76). The disease is associated with a progressive loss of neurons in deep nuclei in the
brain, in particular dopaminergic neurons in the substantia
nigra. Both genetic and environmental toxins have been
associated with the disease. Relevant to this review, a number of years ago, Burke and collaborators proposed and
have since accumulated substantial evidence that toxic aldehydes play a causal role in the disease (27–29, 153, 238).
In particular, a very reactive aldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), has been implicated in the disease. DOPAL is a dopamine metabolite and a product of
monoamine oxidase in dopaminergic neurons of the brain
(29). Studies in animals demonstrated that DOPAL is a
neutoxin, and its injection induces Parkinsonism (238,
336). As DOPAL is converted to its nontoxic metabolite
3,4-dihydroxyphenylacetic acid (DOPAC) by ALDH2, increasing the activity of ALDH2 may provide a means to
reduce dopaminergic neuronal cell death. Indeed, accumulation of DOPAL in the substantia nigra is documented in
animal models of and in humans with Parkinson’s disease
(28, 29, 93, 193, 335, 336).
Other aldehydes contribute to neurotoxicity in general and
to the loss of dopaminergic neurons in particular. Culture
studies demonstrate that 4-HNE induces neuronal cell apoptosis (155). Post mortem analysis of brain samples from
patients with Parkinson’s disease and control patients demonstrated that ⬃60% of substantia nigra neurons are positively stained for 4-HNE-modified proteins in brains of the
patients with Parkinson’s disease; in contrast, only 9% of
the control subjects showed such staining (365). Furthermore, physiologically relevant concentrations of 4-HNE
(low micromolar) inhibit biotransformation of the toxic
DOPAL to the inactive DOPAC (86). As discussed earlier,
even though 4-HNE is a substrate of ALDH2, it can also
inactivate ALDH2 by covalently adducting to the Cys in the
catalytic site of the enzyme (65). Thus increased accumulation of 4-HNE in neurons may, in turn, increase the levels of
DOPAL, thus linking oxidative stress to the uncontrolled
production of an endogenous neurotoxin that has been
linked to Parkinsonism (86).
Epidemiological studies show a correlation between Parkinson’s disease with living in a rural area and/or exposure
to agricultural pesticides (9, 62, 84, 308). Many of these
pesticides, such as maneb, mancozeb, and benomyl, were
found to directly inhibit the activity of purified rat ALDH2
(and ALDH5A), thus decreasing the detoxification of lipid
peroxidation-derived aldehydes, such as 4-HNE and
DOPAL (170). For example, Benomyl is a benzimidazole
fungicide and a potent inhibitor of ALDH2 in vitro and in
vivo. Benomyl was found to have an IC50 of 24 ␮M/kg
when injected intraperitoneally to animals (151, 293, 294).
Recently, Fitzmaurice et al. (85) provided substantial supportive evidence for the inhibition of mitochondria ALDH
by benomyl as a plausible pathogenic mechanism of Parkinson’s disease. In that study, an in vivo metabolite of benomyl which inhibits mitochondria ALDH activity at nanomolar concentrations was identified. This inhibitor led to
accumulation of DOPAL and to selective damage of dopaminergic neurons in primary cell cultures and in a zebrafish
model system (85). Epidemiological studies also confirmed
the association between exposure to benomyl and Parkinsonism; there is a twofold increase of Parkinson’s disease
risk following chronic exposure to ambient benomyl at the
workplace (85). Genetic studies on the association of
ALDH2*2 mutation and Parkinson’s disease has been
sparse. Only one case-control study has been reported,
comparing 93 genotyped Parkinson’s disease patients with
297 healthy controls. In this study, no association between
ALDH2 genotype and Parkinson’s disease was found (90).
Finally, in animals, diminished capacity for toxic biogenic aldehyde removal in double knock-out mice of
ALDH1A1⫺/⫺ and ALDH2⫺/⫺ was found to lead to
4-HNE and DOPAL accumulation in the substantia nigra.
Importantly, increased neurodegeneration and age-dependent motor dysfunction were also found in this transgenic
model compared with wild-type mice (336).
2. Alzheimer’s disease
Alzheimer’s disease is a degenerative disease for which there
is currently no cure. It is the most common cause of dementia, and although a less prevalent early-onset form exists, it
is the most common form of dementia afflicting people over
the age of 65. More than 4.5 million Americans are believed
to have Alzheimer’s disease. By 2050, because of the increase in life span, the number is expected to increase to
13.2 million (22). Currently, the only therapeutic treatment
of Alzheimer’s disease involves managing the symptoms,
with limited success.
Progressive loss of mitochondrial function or defects in mitochondrial metabolism in the later stages of Alzheimer’s
disease can lead to the generation of reactive oxygen species
resulting in oxidation of membrane lipids and accumulation of 4-HNE in the brain (232) and blood (205). Additionally, 4-HNE accumulates in the hippocampal regions of
patients with mild cognitive impairment and patients with
early Alzheimer’s disease (342). 4-HNE adduction was
found on amyloid proteins (277), and this 4-HNE adduction is thought to contribute to amyloid plaque formation in
a later stage of the disease (5, 205). The accumulation of
amyloid plaques over time correlates with neurodegeneration and subsequent atrophy of the affected regions of the
brain. It is therefore possible that diminishing 4-HNE accumulation during the early stages of Alzheimer’s disease may
reduce or prevent the disease progression.
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CHEN ET AL.
As we discussed earlier (see also FIGURE 3), ALDH2 protects mitochondrial functions through the detoxification of
4-HNE that accumulate in this organelle. Recent human
epidemiological studies correlate a higher incidence of Alzheimer’s disease in Asian patients with the inactivating
ALDH2*2 mutation (110, 136, 324). In a Japanese study
involving more than 2,200 subjects between the ages of 40
and 70 yr, levels of serum lipid peroxides were clearly
higher in female ALDH2*2 carriers, after exclusion of alcohol drinking behavior (230). There is also a synergistic
risk increase of Alzheimer disease between ALDH2*2 and
APOE-⑀4 allele (136, 324). Apolipoprotein E protein,
APOE-⑀4, is the least effective 4-HNE-eliminating apolipoprotein, compared with APOE-⑀2 and APOE-⑀3 proteins
(245). This may explain the increased risk of Alzheimer’s
disease associated with the APOE-⑀4 allele and its combined vulnerability with ALDH2*2 to this disease.
Relative to wild-type mice, transgenic mice overexpressing
ALDH2*2 exhibit accelerated neurodegeneration in the
hippocampus as well as diminished 4-HNE clearance as
the mice age (231, 233). Therefore, agents that accelerate
4-HNE removal may provide a possible therapeutic mode
of intervention to prevent or reduce disease progression.
The same study showed that primary cortical neuronal cells
derived from the ALDH2*2 transgenic mice are more susceptible to 4-HNE-induced cell death in culture (231). Concomitantly, elevated 4-HNE accumulation and premature
brain degeneration were observed in these animals. Furthermore, accumulation of hyperphosphorylated tau protein,
which is found also in human patients, was increased in the
transgenic mice compared with wild-type mice (231). Importantly, these ALDH2*2 transgenic mice developed
marked age-related neuronal loss in the brain with cognitive
impairment of memory loss, premature aging, and shortened life span (221, 231, 232). These phenotypes of neurodegeneration mimic those seen in patients with Alzheimer’s
disease and were enhanced by knocking-out the ApoE gene,
a well-known risk factor in Alzheimer’s disease in humans
(179). Therefore, agents that increase the activity of ALDH
and thereby increase 4-HNE detoxification hold a great
potential for slowing down or preventing Alzheimer’s disease progression. This was demonstrated in a recent pilot
study, using the ALDH2 activator Alda-1, to enhance
4-HNE detoxification; Alda-1 treatment prevented amyloid
␤ peptide-induced impairment of angiogenesis in cultured
endothelial cells (290).
D. Alcohol-Induced Pathophysiology
An estimated 1.9 billion adults (⫽15 yr old) worldwide
consume at least a daily average of one alcoholic beverage
(13 g of ethanol) regularly (11, 338). About 140 million
people consume ethanol in excess, which results in alcoholism or alcohol dependence (337, 339). The mechanism by
which ethanol addiction (alcoholism) relates to activation
16
of the reward system in the brain, the genetic predisposition
to alcoholism, its direct effect on the functions of the central
nervous system, and the mechanism of end organ injuries
due to alcohol consumption have all been topics of active
research (23, 225, 367). Here we focus on the roles of
acetaldehydes in these pathologies.
1. Alcohol intoxication and Asian acetaldehyde
toxicity syndrome
Much of the damage by ethanol can be attributed to acetaldehyde accumulation. However, acetaldehyde toxicity
has attracted much less public awareness than the emphasis
on the dangers of alcohol. For example, in 2007, The International Agency for Research on Cancer (IARC) classified
ethanol in alcoholic beverages as a group 1 carcinogen in
humans based on epidemiological studies (11). In fact, it is
well known that ethanol is not a carcinogen (142); rather, it
is acetaldehyde that causes malignancy. This highlights the
under-recognized cytotoxicity and genotoxicity risks of acetaldehyde.
Ingested ethanol is quickly metabolized to acetaldehyde (by
alcohol dehydrogenase) and then to acetate, by the mitochondrial enzyme ALDH2 (FIGURE 1). In ALDH2-deficient
subjects, blood acetaldehyde concentrations are significantly higher even after a moderate amount of ethanol (46,
47, 246, 318, 364). One to two alcoholic beverages of any
kind (or an equivalent of 0.2– 0.6 g/kg ethanol) results in
peak blood acetaldehyde levels of 73.7 ␮M ⬃30 min after
consumption in ALDH2*2 heterozygotes compared with
4.4 ␮M blood acetaldehyde levels in subjects with wild-type
enzyme. Roh et al. (268) used the technique of “alcohol
clamp” and demonstrated the contribution of acetaldehyde
to intoxicated behaviors in social drinkers. In this study, volunteers with either wild-type ALDH2*1/*1 or ALDH2*1/*2
genotypes were infused with ethanol intravenously to maintain an identical stable breath ethanol concentration of 50
mg/dl for 165 min. Significant differences in subjective physiological responses and stimulant and sedative effects of ethanol were clearly distinguishable between the two genotypes,
including increased central stimulation sensation, anesthetic
sensation, and impaired function of skills and abilities in the
ALDH2*1/*2 relative to wild-type subjects (268). Therefore,
the physiological and behavioral consequences of ethanol consumption are due to effects of both ethanol and acetaldehyde (60).
In 6- to 8-wk free-choice ethanol and water experiments,
ALDH2 knockout mice drank much less ethanol compared
with the wild-type control mice (80, 128), thus mimicking
the phenotype of ALDH2*2 East Asians (24). Studies in
mice demonstrated that oral administration of increasing
doses of ethanol from 0.5 to 5.0 g/kg in wild-type and
ALDH2 knockout mice leads to significantly higher blood
acetaldehyde levels in the knockout mice (127). At high
ethanol doses, a significant delay in ethanol clearance was
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also observed in the ALDH2 knockout mice relative to
wild-type mice. Furthermore, increased mortality rate was
reported following an 8-day subacute administration of
ethanol at 2 g·kg⫺1·day⫺1 in the mutant mice compared
with the wild-type ALDH2 mice (237), suggesting that
ALDH2 is a protective enzyme from ethanol-induced injury.
A single dose of 5 g/kg ethanol did not seem to induce severe
oxidative stress to liver tissue in these mice, as measured by
the levels of malondialdehyde or glutathione. On the contrary, the deficiency of ALDH2 was suggested to attenuate
ethanol-induced oxidative stress in rodent liver tissue (201).
The mechanism of this attenuation in ALDH2 knockout
mice is likely to be mediated by downregulation of
CYP2E1, which is a known oxidative stress generator by
ethanol (202). In a separate chronic ethanol study, which
subjected the mice to 5 wk of free-drinking ethanol solution, ALDH2 knockout mice showed lower levels of injury
markers of malondialdehyde, tumor necrosis factor
(TNF)-␣, and alanine aminotransferase (ALT) in their serum, compared with wild-type mice (203). However, there
was increased ballooning of hepatocytes and DNA adducts
of N2-ethylidene-2=-deoxyguanosine in hepatocytes and
gastric tissues, indicating a significant increase in the hepatic and gastric damage in the ALDH2 knockout mice
(198, 203, 216). Increased levels of DNA adducts were
similarly detected in liver, stomach, and kidney of ALDH2
knockout mice compared with wild-type mice when using
radioactive-labeled ethanol in the study (229). Therefore,
both protective and injurious effects on the liver and the gut
appeared to be observed in ALDH2 knockout mice following ethanol administration. It is not known whether different genetic backgrounds of these transgenic mice and/or
activation of other enzymes contribute to the differences in
these studies.
Hypersensitivity to acetaldehyde vapor was also demonstrated in ALDH2 knockout mice when exposed to vapor
inhalation (129). Blood acetaldehyde levels were 2- to 3.2fold higher in ALDH2 knockout mice compared with wildtype mice, when measured after 4 h of inhaling acetaldehyde at a concentration of 5,000 ppm. Concomitantly, behavioral and physiological symptoms of augmented acute
acetaldehyde toxicity, such as staggering gaits, prone position, hypoactivity, abnormal deep breathing, and dyspnea,
were observed in the ALDH2 knockout mice relative to
wild-type mice (129). These symptoms of acetaldehyde toxicity were reproduced by a single injection of a sublethal
dose of acetaldehyde. In a study of intraperitoneal injection
of 400 mg/kg acetaldehyde, ALDH2 deficiency delayed the
recovery of physiological and behavioral symptoms and the
elimination of acetaldehyde compared with wild-type mice
(130). Interestingly, mitochondrial liver homogenates derived from these ALDH2 knockout mice also showed a
dramatic deficiency in catalytic activity toward 2-methoxy-
acetaldehyde, a major metabolite of toxic 2-methoxyethanol (ethylene glycol monoethyl ether), which is commonly
used as a water-soluble solvent in the chemical industry
with known adverse reproductive and central nervous system effects in factory workers (147). These data confirm
that ALDH2 is the major acetaldehyde-metabolizing enzyme in mice and that deficiency in ALDH2 leads to elevated acetaldehyde levels after ethanol intake, and to increased susceptibility to prolonged exposure to acetaldehyde and other toxic aldehydes. They also highlight the
potential health risks of toxic aldehydes for human carriers
of ALDH2*2 relative to the rest of the population.
Acute exposure to acetaldehyde causes much of the unpleasant effects associated with ethanol intoxication, including nausea, palpitations, vomiting, dizziness, and headaches (24). These negative effects of acetaldehyde are also
deterrents, preventing reinstatement of alcoholism. After
excessive drinking was interrupted, the drug disulfiram, an
inhibitor of ALDH2, is used to trigger aversion to ethanol
consumption (297), as it causes acute acetaldehyde accumulation and the corresponding unpleasant effects. Disulfiram’s effects in the presence of ethanol are not different
from the Asian Acetaldehyde (Flushing)-Toxicity Syndrome, which results from the inactivating point mutation
in ALDH2 (ALDH2*2) (24). The finding that alcoholism
was less common in ALDH2*1/*2 and is nonexistent in
ALDH2*2/*2 individuals led to the notion that acetaldehyde is a strong deterrent from ethanol drinking (39, 114,
115). However, social pressure and changes in the work
and life-style of urban East Asians led to a greatly increased
incidence of alcoholism in this population. Higuchi reported a significant increase of alcoholics in Japan from 2.5
to 13% in the ALDH2*1/*2 heterozygous group from
1979 to 1992 (114). In a more recent study conducted in
Japan in 2002, 26% of the heavy drinkers (⬎400 g ethanol
consumption per week) in the Tokyo area were ALDH2*1/*2
heterozygotes (356); ALDH2*1/*2 in the general population
is ⬃30%. A similar trend was observed in Taiwan, where
17% of alcoholics were found to carry the ALDH2*1/*2
heterozygous genotype in 1999 (39). As will be discussed later,
ALDH2*1/*2 subjects that drink excessively are at a much
great risk of developing esophageal and other upper aerodigestive track cancers, a likely result of DNA damaging effects
of acetaldehyde.
2. Alcoholism
Another mechanism by which ALDH2 inhibitors can be
beneficial for the treatment of alcoholism has been recently
reported. A study by Yao et al. (354) suggests that alcoholism may be induced, in part, by chemical adduction of dopamine, and its monoamine oxidase aldehyde metabolite
DOPAL, to form tetrahydro-papaveroline (THP) in the
neurons of the ventral tagmental area. Inhibition of ALDH2
leads to an increase of DOPAL and hence accumulation of
THP, which selectively decreased dopamine release and
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CHEN ET AL.
hence led to inhibition of pro-addiction reward signaling
through a negative-feedback loop (7, 354). As previously
mentioned, CVT-10216 is a potent ALDH2 inhibitor with
IC50 of ⬃30 nM. In these studies, CVT-10216 prevented
the increase of alcohol-induced rewarding dopamine release in brain in the absence of acetaldehyde (7, 235). Because of this unique property, CVT-10216 may therefore be
developed as a drug lead for alcoholism. In an animal model
of drug addiction, CVT-10216 also suppressed cocaine selfadministration and prevented relapse-like behavior, possibly by a similar ALDH2 inhibition-mediated increase of
THP via DOPAL and dopamine condensation (354).
These data demonstrate that acetaldehyde has a clear role in
the intoxication associated with ethanol ingestion and that
a neuronal-derived ALDH2 substrate, DOPAL, may play a
direct role in addiction (332). Inhibiting ALDH2 (e.g., by
disulfiram) provides ethanol aversion therapy, whereas selective inhibition of ALDH2 by compounds, such as CVT10216, in the brain may prevent activation of the addictive
behavior for ethanol and other drugs of abuse. However,
care should be given not to affect ALDH2 in the liver, as
acetaldehyde accumulation causes a number of other pathologies, as discussed in this review. Further consideration
should be given to the role of ALDH2 in metabolizing aldehydes such as DOPAL and 4-HNE. As we discussed earlier, these aldehydes have been connected with progression
of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases.
3. Fetal alcohol syndrome
Finally, a discussion about alcohol and acetaldehyde must
include a very important consequence of acetaldehyde accumulation on early embryonic development. It is well-recognized that exposure to ethanol during the first trimester
of embryo development can lead to severe developmental
disorders, including craniofacial abnormalities, behavioral
and intellectual deficits (collectively referred to as fetal alcohol syndrome), as well as to fetal death (105, 253). Although most of these features were attributed to exposure
to ethanol’s effects at a critical stage of embryonic development, more recent work suggests that these abnormalities
relate directly to acetaldehyde toxicity and DNA damage
during this critical developmental period. Exciting new observations highlight a similarity between fetal alcohol syndrome, acute childhood leukemia (164, 190), and Fanconi
anemia (161) and demonstrate a role of acetaldehyde in
these pathologies. The role of acetaldehyde toxicity in early
embryo development will be discussed more in the section
related to Fanconi anemia.
E. Upper Aerodigestive Track Cancer
Chronic alcohol (ethanol) consumption is a prominent
cause of morbidity and mortality worldwide. The amount
18
of ethanol consumption has remained relatively stable in
most parts of the world over the past 40 years; e.g., 6.8 and
6.5 liters of pure ethanol are consumed per capita in the
United States and Japan, respectively (344). The only region
in which there is a significant increase in ethanol consumption is the Western Pacific; there is about a fivefold increase
in ethanol consumption in China alone (11, 338). This rise
in numbers in the Western Pacific is alarming, since ⬃3.6%
of all cancers and ⬃3.5% of all cancer death are attributable to ethanol drinking based on data from 2002 (18). As
discussed above, alcoholic beverages have been classified as
group 1 human carcinogens by IARC, and chronic consumption of ethanol has been causally linked to the development of upper aerodigestive track (UADT) cancers, liver
cancer, colorectal cancer, and breast cancer in women
(11, 32).
UADT cancers include the cancers of the head and neck
region (oral cavity, oropharynx, hypopharynx, larynx) and
esophageal cancers. About 600,000 new cases of neck cancer occur each year worldwide. Only 40 –50% of these patients survive 5 years after diagnosis, thus leading to annual
deaths of 270,000 from these cancers (119, 169). Esophageal cancer alone is the eighth most common and the sixth
most deadly cancer in the world (54, 243, 304, 315). Esophageal neoplasms are often undetected. Therefore, more than
50% of the patients have unresectable or metastatic tumors
at the time of diagnosis (71). Acetaldehyde, the toxic metabolite of alcohol, forms DNA-DNA and DNA-protein
crosslinks. In many experimental animal models, acetaldehyde has been proven unequivocally to be a carcinogen
(209, 345, 346) and is the most likely culprit of DNA damage in the development of UADT tumors (17, 278).
Yokoyama et al. (357) first reported a dramatic 7- to 12fold increase of esophageal cancer risk in both alcoholic and
nonalcoholic ALDH2*2 carrier drinkers than their respective wild-type ALDH2 controls. Many other studies from
Japan, Taiwan, and China have since overwhelmingly confirmed the significant association between ALDH2 enzyme
deficiency and UADT cancer risk (8, 64, 167, 172, 214,
352, 361, 362). Reviews on many primary articles and
meta-analyses consistently found ALDH2*2 as one of the
most common genetic polymorphisms involved in UADT
cancers (30, 119). Considering that cigarette smoke is also a
source of acetaldehyde, it is not surprising that tobacco use
and heavy drinking combined with the ALDH2*2 genotype
carry the greatest cancer risk (167, 207); indeed, ALDH2*2
carriers are the youngest patients with esophageal cancer.
Both blood and saliva acetaldehyde concentrations are significantly higher in ALDH2*2 subjects after even a moderate amount (0.2– 0.6 g/kg or 1–2 drinks) of ethanol consumption (46, 47, 246, 318, 364). Salaspuro et al. (276)
demonstrated the interaction between alcohol drinking and
smoking and their effect on the concentration of acetaldehyde in saliva, in vivo. When given the same amount of
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ethanol, acetaldehyde concentration was found to be two
times higher in smokers than in nonsmokers. Furthermore,
with concomitant cigarette smoking during ethanol challenge, smokers had seven times higher saliva acetaldehyde
concentration compared with nonsmokers (276). A synergistic effect of increased cancer risks associated with ethanol consumption and smoking has been reported for oral
cavity, pharynx, larynx, and esophageal cancers (33, 34,
87, 166, 207, 208, 275). Heavy ethanol consumption and
smoking in subjects carrying the ALDH2*2 genotype contribute to probably one of the highest known cancer risks
(odds ratio of 50.1) (207) and up to a 25-yr earlier onset of
carcinoma (45 vs. 70 yr old) compared with nonsmoker,
nondrinker wild-type ALDH2 subjects (24, 167, 207). The
recognition of the clear association between ALDH2 enzyme deficiency, acetaldehyde toxicity, and the risk of
UADT cancer and other cancers cannot therefore be overemphasized (24, 279).
The molecular mechanism underlying acetaldehyde-induced UADT cancers in ALDH2*2 subjects has been actively studied (310, 370). There is direct evidence showing
increased DNA modification and chromosomal damage in
ALDH2*2 individuals relative to subjects with active
ALDH2 after they consume the same amount of alcohol
(200). Matusta et al. (200) identified three acetaldehydederived DNA adducts that are significantly higher in blood
lymphocyte samples of 44 alcoholic patients. One of the
acetaldehyde-modified DNA adducts, N(2)-ethyl-2=-deoxyguanosine (200), was identical to its more stable precursor,
N(2)-ethylidene-2=-deoxyguanosine, detected in gastric and
hepatic samples of ALDH2 knockout mice fed with
20% ethanol for 5 wk (198, 216). N(2)-ethyl-2=-deoxyguanosine is a known blocker of translesion DNA synthesis
and may contribute to the hindrance of DNA repair (199,
288, 316). Chromosomal damage or genome instability in
ALDH2*2 subjects who consume ethanol were also measured by biomarkers such as cytokinesis-block micronucleus assay (126) or by alkaline comet assay using peripheral blood lymphocytes from the ALDH2*2 subjects (287,
333). Blocked micronuclei frequency was significantly
higher in the heavy or moderate alcohol drinkers with the
ALDH2*2 allele compared with their ALDH2*1 wild-type
counterparts (126). Similarly, using the alkaline comet assay to measure the mobility of DNA as an index of DNA
damage in peripheral mononuclear cells in a study of 122
healthy volunteers, increased DNA damage was noted in
ALDH2*2 subjects who were frequent alcohol drinkers
(333). Although these data paint a clear connection between ethanol consumption, ALDH2 activity, and cancer,
much research is still required to identify key genetic and
epigenetic changes, which contribute to the high incidence
of UADT cancers among the ALDH2*2 populations.
In recent years, UADT cancer prevention has become a
public health priority in East Asian countries such as Japan
and Taiwan. New measures were taken in these countries.
These include identifying high risk groups at the early stage
of disease, screening by image-enhanced endoscopy, oral
examinations, the use of alcohol flushing questionnaires to
identify subjects that carry the ALDH2*2 gene, and intensive surveillance for secondary recurrence in those who
were diagnosed and treated for the disease (54, 207, 363).
Intensive efforts are also underway to develop better biomarkers and diagnostics for UADT cancers (54). Considering that ALDH2*2 is a very common mutation in all East
Asian countries and that ALDH2 genotype is a very strong
predictor of multiple squamous cell carcinoma in UADT, it
is warranted that the determination of ALDH2 genotype
should be included in the overall screening and preventive
strategies for UADT cancers. Alternatively, since elimination of primary risk factors (e.g., smoking and drinking)
remains a difficult obstacle to overcome, enhancing the
ALDH2 activity by its activators in ALDH2*2 enzymedeficient individuals may help to reduce the exposure of
acetaldehyde genotoxicity from saliva and in the UADT
region in this very high risk East Asian group.
F. Radiation Dermatitis
Ionized radiation used as a treatment for solid tumors often
causes burnlike injury to the skin due to cellular oxidative
stress. This painful and debilitating side effect occurs in
⬃95% of patients receiving radiation therapy for cancer,
and ranges in severity from mild erythema to severe ulceration (274). Although intensity-modulated radiotherapy
enables better sparing of normal tissues, this burnlike skin
reaction is unavoidable (13). Meta-analysis of 20 clinical
trials involving ⬃3,000 patients demonstrated that both
prophylactic and treatment trials to prevent radiation dermatitis failed when using a variety of topical agents (188).
The trials analyzed in that meta-analysis study included use
of topical agents such as mometasone furoate, beta-methasone, and beclomethasone (all anti-inflammatory corticosteroids); Gentian violet (a topical antiseptic agent); urea,
Lianbai liquid, and aloe vera (traditional medicine remedies
with possible antioxidative stress properties); hyaluronic
acid, Biafine, dexpanthenol, and Sucralfate (agents that induce wound barrier and/or are moisturizers); and Trolamine (a barrier and analgesic agent) (188). The failure of all
these clinical trials emphasizes that radiation dermatitis remains an unmet clinical problem in cancer patients undergoing radiation therapy.
Ionizing radiation causes a robust production of reactive
oxygen species (341), which then triggers an inflammatory
response as well as a local cell proliferative response, especially keratinocytes. Since anti-inflammatory agents appear
to provide little benefit (see above), the source of injury lies
elsewhere, perhaps an increased aldehydic load. Early studies attributed the sustained radiation-induced skin injuries
to increased levels of aldehydes, such as 4-HNE and MDA,
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CHEN ET AL.
the lipid peroxidation products (2, 58). Therefore, prophylaxis and treatment with agents that prevent aldehyde accumulation, such as activators of ALDH2, may be beneficial for these patients.
In a proof-of-concept mouse model, we found that direct
topical application of Alda-1 on the skin of irradiated animals effectively delayed the onset and reduced the severity
of radiation dermatitis. This Alda-1-induced protection
correlated with increase in ALDH2 activity, a concomitant
reduction in epidermal thickness, and a decrease in 4-HNEadduct formation in irradiated skin biopsy samples from
these mice (224). Importantly, we found that Alda-1 did not
protect solid tumors in mice against radiation therapy. It is
conceivable that the skin protection offered by ALDH2 activation may be extended to other damage caused by reactive oxygen species-generating radiations such as ultraviolet
and radioactive nuclear exposure. Furthermore, whether
the severity of the response of East Asian carriers of
ALDH2*2 to the damaging ionizing radiation is higher
than those with wild-type ALDH2 remains to be determined.
G. Fanconi Anemia
Fanconi anemia (FA) is a rare genetic disease, first discovered by Guido Fanconi, a Swiss Physician, in 1927. FA is
characterized by a progressive bone marrow failure (BMF)
accompanied by increased risk of cancer as well as developmental abnormalities, such as short stature and malformations of the thumbs, forearms, skeletal system, eyes, kidneys, and urinary tract. In addition to hematological abnormalities, FA is clinically diagnosed by the presence of
chromosomal breakage, and by hypersensitivity to DNA
cross-linking agents such as mitomycin C, diepoxybutane,
and cisplatin (140, 141). No effective treatment is currently
available for this rare genetic disorder, which occurs in
⬃1/350,000 individuals in all ethnic groups (10, 321).
In the United States, ⬃31 newborns are diagnosed with
Fanconi anemia (FA) each year (270). Based on International Fanconi Anemia Registry, the average life expectancy
of FA patients is 29 years. With blood transfusions, many
patients can live at times twice as long as life expectancy. FA
is caused by homozygous recessive mutations in any of the
15 identified FA genes (FNAC genes), which encode proteins that serve an essential function in DNA repair of damages caused by DNA cross-linking agents (206). The FA
gene products play a crucial role in guarding the integrity of
the human genome; mutations in any of the FA genes invariably lead to catastrophic consequences of genomic instability due to failure to repair DNA crosslinking damage.
Until recently, the source and chemical agents that cause
excessive genomic instability leading to the phenotype of
developmental abnormality, BMF, and predisposition to
20
malignancy in FA patients have been elusive. Environmental
pollutants, carcinogens, and biogenic reactive chemical species that are capable of attacking DNA and causing genome
instability under physiological conditions were the prime suspects of the molecular triggers of FA. Reactive aldehydes are
known toxic molecules that can damage DNA by forming
DNA-protein or DNA-DNA interstrand crosslinking (53, 61,
156, 296, 309). Ridpath et al. (262) first reported the genotoxic effect of formaldehyde in cells that are deficient in the
FANC/BRCA pathway. In a chicken B lymphocyte-derived
cell line, DT40, hypersensitivity to low micromolar concentrations of formaldehyde was demonstrated in isogenic DT40 cell
lines carrying mutations in several DNA repair genes including
FANCD2, FANCD1 (better known as BRCA2, which plays a
critical role in genetic forms of breast cancer). In these DT40
mutant cell lines, cell survival was dramatically reduced by
exposure to low concentrations of formaldehyde compared
with survival of the parental wild-type DT40 cells under the
same conditions (262). Hypersensitivity to formaldehyde was
similarly shown in human colorectal RKO cancer cells in
which the FANCC and FANCG genes were disrupted. These
findings suggest that exposure to formaldehyde at levels observed in human plasma (13–97 ␮M) (113) could cause severe
cellular damage due to failure of DNA repair. Interestingly,
acetaldehyde, but not other aldehydes such as acrolein, crotonaldehyde, glyoxal, or methylglyoxal, also caused hypersensitivity in a FANCD2-deficient DT40 cell line (262). Endogenous formaldehyde is known to be generated within the nuclei
as a by-product of histone demethylation or dealkylation of
methylated DNA (210, 272). In vertebrate cells, alcohol dehydrogenase 5 (ADH5) is the major dehydrogenase that metabolizes formaldehyde and protects the toxic effect of formaldehyde (124). With the use of a cell line with ADH5 knockout, it
was demonstrated that deficiency in FA DNA-repair pathway
became lethal, presumably due the accumulation of endogenous formaldehyde (269).
Chronic exposure to low concentration of acetaldehyde vapor at 125 and 500 ppm for 6 –12 days led to a significant
elevation of the DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) in urine samples of the ALDH2
knockout mice (228). As discussed earlier, acetaldehyde,
commonly found in many food sources (81), environmental
pollutants (121, 311), cigarette smoke (264), or as a metabolite of drinking alcohol, can also react in cells to generate
interstrand DNA crosslinks (309). Acetaldehyde can also be
generated as a by-product of endogenous metabolism from
alanine by myeloperoxidase (112) or from deoxyribose
phosphate by aldolase (226). Marietta et al. (196) showed
that exposure to acetaldehyde at biologically relevant concentrations (0.1–1 mM) resulted in concentration-dependent FANCD2 mono-ubiquitination, in phosphorylation of
the FANC gene product BRCA1, and in appearance of a
hallmark of DNA damage, histone ␥H2AX phosphorylation, in human lymphoblastoid cells.
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TARGETING ALDEHYDE DEHYDROGENASE 2
Using a genetic approach, Langevin et al. (161) showed that
maternal ALDH2 is essential to counteract the acetaldehyde toxicity for the normal development of FANCD2
knock-out embryos. Mouse embryos that carried double
knockout deficiencies, ALDH2⫺/⫺ and FANCD2⫺/⫺, were
not viable, unless they were protected by at least one copy of
a functional maternal ALDH2 allele of a ALDH2⫹/⫺ females. Moreover, these rescued double mutant ALDH2⫺/⫺
and FANCD2⫺/⫺ embryos were hypersensitive to developmental deformities in the uterus if the mothers were exposed to ethanol. In the postnatal stage, the ALDH2⫺/⫺ and
FANCD2⫺/⫺ mice rapidly developed bone marrow failure
when ethanol was administered in the drinking water. Incidences of spontaneously acute lymphoblastic leukemia
were also observed in mice 3–5 months after weaning in the
absence of ethanol exposure. Importantly, in adult
ALDH2⫺/⫺ and FANCD2⫺/⫺ mice that were spared of leukemia, the hematopoietic stem and progenitor cells, and not
the more mature blood precursors, appeared to be the most
sensitive to acetaldehyde-induced DNA damage (95). There
was a more than 600-fold reduction in the hematopoietic stem
cell pool in these mice that are defective in both acetaldehyde
detoxification and the FA DNA repair pathway. These
ground-breaking studies established a molecular link between
toxic acetaldehyde and DNA damage in FA (FIGURE 6). These
findings also underlie the crucial role of ALDH2 activity
during embryogenesis and in protecting bone marrow hematopoietic stem cells. Finally, as briefly discussed earlier,
because many of the developmental abnormalities observed
in FA patients are similar to those with fetal alcohol syndrome, aldehyde-induced DNA damage may underlie both
pathologies (FIGURE 6).
It is of interest to note that in adult FA patients, upper
aerodigestive track cancers or head and neck cancers are the
most common cancer types (4, 157). These are the very
same cancer types that are most prevalent in the ALDH2*2
East Asians who are exposed to the high level of acetaldehyde genotoxicity due to heavy alcohol drinking (359 –
361). Significant numbers of FA patients have been reported
in ALDH2*2 in Japan and Korea (240, 350). However,
very few cases of FA have been reported in China or Taiwan, where ALDH2*2 mutation accounts for a very large
percentage of the population (i.e., ⬎40% in Taiwan). It is
possible that in these ALDH2*2 prevalent countries, germline FA, or non-germline FA mutations are under-diagnosed. Alternatively, human embryos that have both a mutated FA gene and mutated ALDH2 gene may die at early
embryonic stage, thus reducing the FA incidence seen in
children and adults in these countries.
Exactly what types of xenogenic or biogenic reactive aldehydes that may serve as potent physiological triggers for
different pathological features of FA remain to be identified
(244). Regardless, the relationship between acetaldehyde
toxicity and DNA damage is likely to be especially relevant
to the health of East Asian FA patients who carry the
ALDH2*2 mutation (FIGURE 6).
Therapeutic agents that target ALDH2 may also provide a
viable strategy to address the national health problem of pain,
resulting in approximately $50 billion annually in health care
cost and productivity loss (109). Recent studies suggest that
the ability to reduce aldehydic load may address a broad range
of pain pathologies, including peripheral neuropathy and perhaps even acute and chronic inflammatory pain.
For both clinical practice and research, it will be important to
explore the connection (if any) between ALDH2 genotype
status and FA patients who are from the East Asian group. It
will be equally important to determine if there is a correlation
between ALDH2*2 mutation and FA disease severity and
prognosis. Such basic information may help guide future treatment options. Enhancing the function of ALDH2 by either
gene regulation or direct enzyme activation may conceivably
provide the needed clinical intervention for FA patients who
are hypersensitive to reactive aldehydes. This may be especially critical for FA patients who are also ALDH2*2 carriers,
but should be relevant to the general population, too.
H. Pain
Solid tumors
O
DNA
damage
ALDH2
Acetate
H3C
Fanconi
mutations
Leukemia
H
Acetaldehyde
Bone marrow failure
Damaged DNA
Fetal alcohol syndrome
ALDH2*2
Developmental abnormalities
FIGURE 6. ALDH2 and Fanconi anemia. In a mouse model of Fanconi anemia (FANCD), ALDH2 is essential
in counteracting acetaldehyde toxicity. In the absence of a functional ALDH2, DNA damage triggered by
elevated acetaldehyde toxicity cannot be repaired efficiently due to Fanconi mutations. Accumulation of such
unrepaired damaged DNA leads to the manifestation of multiple pathological conditions and health complications seen also in patients with Fanconi anemia (95, 161, 244).
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21
CHEN ET AL.
1. Peripheral neuropathy
Peripheral neuropathy, characterized by allodynia (pain in
response to a nonnociceptive stimulus) and hyperalgesia
(increased response to painful stimuli), is present in ⬃24
million people. More than 100 causes of peripheral neuropathy have been identified, and peripheral neuropathy is idiopathic in ⬃33% of the patients (286). Diabetic neuropathy is likely the most common cause of this pathology.
Relevant to this review, ethanol, chemotherapeutic agents,
and the ALDH2 inhibitor disulfiram are also documented
causes of peripheral neuropathy. As discussed in the following, pharmacological and genetic evidence suggests that reducing ALDH2 enzymatic activity may contribute to peripheral neuropathy, whereas ALDH2 activators may perhaps help ameliorate pain.
One side effect of disulfiram in humans is peripheral neuropathy, which can be reversed by stopping or reducing the
disulfiram dose (83, 89). The largest case report of 37 patients with disulfiram-induced neuropathy showed that the
effect is dose-dependent, with doses greater than 250 mg/
day being the greatest factor in causing neuropathic symptoms (89). Since this form of neuropathy is drug induced,
given that the disulfiram metabolite S-methyl-N,N-diethylthiocarbamate inhibits ALDH2 (204), these data suggest
that decreased ALDH2 activity is sufficient to induce peripheral neuropathy in humans.
Indeed, some reports suggest that individual carriers of the
ALDH2*2 mutation are more susceptible to developing
alcohol- or diabetic-induced peripheral neuropathy (197,
303). Chronic alcoholism in ALDH2*1/*2 subjects is associated with reduced sensory nerve action potential amplitude, a characteristic of peripheral neuropathy (273), compared with chronic alcoholics with wild-type ALDH2
(197). Although this study included a small number of subjects (21 per group), the findings suggest that further studies
are needed to investigate ALDH2 as a genetic cause of alcohol-induced neuropathy. A study of 158 type 2 diabetics
investigated how ADH and ALDH2 activity altered diabetic clinical symptoms, including neuropathy (303).
Among the patients studied, although hemoglobin A1C,
age, and weight were similar between groups, patients with
an ALDH2*2 mutation with normal ADH activity had the
highest incidence of peripheral neuropathy (16 of 32 people
or 50%). The lowest incidence of neuropathy occurred in
those patients who had normal ADH and ALDH2 activity
(8 of 44 or 18.2%). Although an increase in ADH activity
was also considered in this study to contribute to diabetic
pathology, reanalyzing the groups in this study without
considering ADH activity suggests that an inactive ALDH2
enzyme is correlated with a higher incidence of diabeticinduced neuropathy (36 vs. 26%).
Studies regarding ethnicity and neuropathy prevalence are
at best inconclusive (292). Although in both alcohol- and
22
diabetes-induced neuropathy studies the number of patients
was small, these findings, in addition to the evidence that
disulfiram can induce peripheral neuropathy, suggest that
further studies are needed to investigate ALDH2 as a genetic contributor to neuropathy.
2. Inflammatory pain
As compared with other ethnic populations, Asians are
more sensitive to painful stimuli such as skin irritation
(271), capsaicin treatment (325), exposure to heat (329),
cold (122), or mechanical pressure (343). These differences
are attributed primarily to social or cultural factors (37,
56). However, the high prevalence of the ALDH2*2 inactivating mutation among East Asians [⬃36% on average
(24) and in some regions as high as 45% (70)] suggests that
increased pain sensitivity in Asians may be attributed to the
ALDH2*2 genotype. We recently tested this hypothesis in
our laboratory using a C57/BL6 mouse with a knock-in
mutation for the ALDH2*2 gene and found that male
ALDH2*1/*2 heterozygotic mice subjected to carrageenaninduced inflammation were more hyperalgesic compared with matched male littermates with ALDH2*1/*1
(wild-type) genotype (unpublished data). Although more
studies are needed, our findings suggest that ALDH2 activity may regulate inflammatory-induced pain sensation. If
correct, restoring the ALDH2 activity in East Asians in
particular and increasing ALDH2 activity for subjects with
a wild-type ALDH2 could provide a novel means to treat
pain.
I. Osteoporosis
Osteoporosis is a disease characterized by decreased bone
mineral density, bone mass, and bone strength, which leads
to increased risk of bone fracture. Osteoporotic fractures
are associated with high morbidity and mortality, as well as
a high burden of health care costs, especially with the aging
population worldwide (57). There are multiple causes for
osteoporosis, including environmental, dietary, and genetic
factors. Relevant to this review, excess alcohol consumption and oxidative stress are associated with osteoporosis,
decreased bone density, and increased fracture incidence
(63, 91, 137, 165, 191); heavy ethanol drinking was found
to be a significant risk factor for increased hip fracture and
vertebrate deformation in large cohorts of Japanese and
Chinese women (91, 165).
In humans, both bone mineral density and osteoporotic fractures have high genetic determinants (180, 349). In a genetic
screening of osteoporosis and DNA polymorphism among
403 elderly Japanese, ALDH2*2 was the only mutation that
was associated with osteoporosis among 11 other known osteoporosis-related genes (351). This association is particularly
pronounced in individuals with the homozygous ALDH2*2/*2
genotype and in women.
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TARGETING ALDEHYDE DEHYDROGENASE 2
In bone marrow cultures derived from rodents, treatment
with ethanol and acetaldehyde greatly inhibits early osteoblast progenitor formation (98). The inhibitory effect of
ethanol was observed at the physiological concentrations of
0.04 – 0.6%, similar to those reached in humans. Acetaldehyde exerted an even stronger toxic effect toward osteoblastic progenitor cells, at concentrations as low as 0.004 to
0.02%. At 0.06% acetaldehyde, pluripotent colony-forming units for osteoblast progenitors were completely abolished in cultured murine cells. Acetaldehyde also inhibited
cell proliferation in two human osteoblastic cell lines. These
observations were also confirmed in human bone marrow
cell cultures derived from young, chronic alcoholics (98);
the number of colony-forming units for osteoblast progenitors obtained from nine alcoholics was reduced to only
⬃30% compared with cells derived from seven agematched normal control subjects. These data demonstrate a
direct effect of both ethanol and acetaldehyde on the formation of osteoblastic progenitor cells and on bone marrow
cell proliferation. Taken together, these studies provide
strong evidence that the ALDH2*2 mutation in East Asians
may be a contributing risk factor to osteoporosis, due to the
lack of enzyme protection against aldehyde toxicity for osteoblasts and their progenitor cells. Furthermore, activators
of ALDH2 in these patients, and in the general population
as well, may prevent or slow down the progression of osteoporosis.
The notion of ALDH2 as a therapeutic target for osteoporosis
is supported by two separate animal models of ALDH2
knockout and ALDH2*2 overexpressing transgenic mice. In
ALDH2 knockout mice, administration of 5% ethanol for 4
wk resulted in a reduction of trabecular bone and bone volume
formation, with a concomitant decrease in gene expression of
osteoblast markers, type I collagen, osterix, osteopontin, and
osteocalcin (284). Mineralized nodule formation was also decreased in bone marrow cell cultures derived from the ethanolfed ALDH2 knockout mice. No such changes were observed
in ethanol-fed wild-type ALDH2 mice. Independently, in a
model of ALDH2*2 overexpressing transgenic mice that exhibited a dominant-negative effect on the endogenous
ALDH2, Endo et al. (80) observed phenotypes of small body
size, reduced muscle mass and fat, osteopenia, and kyphosis,
that are related to osteoporosis. Reduced bone mineral density
was confirmed in these ALDH2 transgenic mice by dual-energy X-ray absorptiometry (120). In these ALDH2*2 osteoporotic mice, elevated blood acetaldehyde levels were also detected even in the absence of ethanol ingestion. The severity of
osteoporosis also correlated with the level of ALDH2*2 expression, depending on different copy numbers of the inserted
gene. Furthermore, acetaldehyde treatment at a concentration
as low as 0.04% effectively inhibited proliferation and induced apoptosis in osteoblast primary cultures of wild-type
osteoblastic cells or the osteoblast cell line MC3T3-E1. Expression of either ALDH2*2 or treatment with acetaldehyde
resulted in excessive accumulation of 4-HNE and increased
PPAR␥ expression, a transcription factor that inhibits osteoblastogenesis. These studies indicated that aldehydic stress resulting from ALDH2*2 mutation leads to impaired osteoblastogenesis and osteoporosis. Independently, severe ethanol-induced suppression of osteoblast differentiation, mineralized
nodule formation, and osteogenic gene expressions that led to
osteopenia was also observed in ALDH2 knockout mice
(284). This is in agreement with the hypothesis that ALDH2
protects normal development and differentiation of osteoblastic stem cells in the bone marrow and maintenance of bone
homeostasis.
Interestingly, in a genome-wide association study involving
11,500 individuals from well-defined and homogeneous
populations, Guo et al. (108) recently identified a SNP in
ALDH7A1 as a highly significant susceptible gene for osteoporosis. These studies provided insights into the crucial
function of ALDH in protecting osteoblasts against toxic
aldehydes, oxidative stress, and perhaps maintaining
proper osmotic pressure and homeostasis for osteoblastic
bone marrow cells.
J. Aging
Denham Harman in 1956 proposed the free radical theory of
aging, suggesting that oxygen free radicals cause the aging
process (111). This theory is also supported by the premature
aging disease Hutchinson Gilford progeria, where the average
human life span is only ⬃13 yr. Fibroblasts of progeria patients have a 1.6-fold higher fluorescence emission measured
by intracellular oxidation of 2,7-di-chlorofluorescein (DCF)
and a 1.7- to 4-fold increase in carbonylated proteins compared with age-matched control fibroblasts (322). Moreover,
in 2007, an inverse correlation was found in 12 species between the rate of hydrogen peroxide (H2O2) generation by the
myocardium mitochondria and maximum life span (160).
This inverse correlation (R2 ⫽ 0.54) existed when measuring
H2O2 formation by reverse electron transfer from succinate to
complex I in diverse species. In turn, therapeutics that reduce
mitochondrial reactive oxygen species generation are considered potential means to reduce aging-induced damage, and
mitochondrial targeted anti-oxidants, such as SkQ1, have
been under development in an attempt to slow down the aging
process (289). As discussed earlier, reactive oxygen generation
triggers lipid peroxidation and accumulation of reactive aldehydes (73, 194).
Protecting the mitochondria from reactive oxygen species
may increase life span. Decrease in mitochondrial detoxification of lipid peroxidative aldehydes has been observed
and proposed as an underlying mechanism of aging in animals (231, 369). It was shown in rats that the capacity of
4-HNE removal by mitochondrial ALDH declines with aging in rats (42). Therefore, ALDH2 activation may slow
down, at least to some extent, the aging process. To that
end, specifically targeting mitochondrial ALDH2 may re-
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23
CHEN ET AL.
duce or prevent many biological effects associated with free
radical-induced aging. Indeed, deleting the ALDH2 homolog in Drosophila reduced survival and longevity when
flies were challenged with hyperoxia or with starvation
(35). There was also a reduced reproductive rate and increased protein carbonyl level in flies in which ALDH2 was
deleted compared with wild-type ALDH2 flies. Consistent
with these findings, transgenic mice that overexpress
ALDH2*2 (called DAL101) showed a significant reduction
in median life span compared with control mice; their life
span was 96 wk compared with 126 wk for the wild-type
mice. In addition, the ALDH2*2 mice showed a greater
muscle weakness and hair discoloration in males (231).
In humans, a high throughput candidate gene study in 137
Koreans over 90 yr old also found that the ALDH2 gene
may predict longevity (241). This study suggested, however, that the ALDH2*2 genotype, not the wild-type, was
associated with living longer for male Koreans. Contrary to
this suggestion, a study conducted in Japan involving more
than 2,200 human subjects showed that the frequency of
ALDH2*2 homozygous genotype was significantly reduced
in females in the 60 –70s age group versus 40 –50s group
(230). No age-related difference in stomach and colon
ALDH activity was found in human biopsy samples (159,
355). However, Wang et al. (327) did observe a slight decline in liver ALDH activity in older human subjects. The
rate of loss in telomeric repeat sequences in human peripheral leukocytes, as a cellular marker of senescence, has been
well correlated with aging (88). It may be warranted for
future research to measure the telomere length among the
ALDH2*2 individuals and compare that to the wild-type
individuals. Whether therapeutic agents that activate
ALDH2 are beneficial in diseases of accelerated aging, such
as progeria, will help determine the role of aldehydic load in
aging. Even if association between ALDH2*2 and life span
is not found in humans, we should examine the association
between ALDH2 activity and aging-associated damage;
in light of the number of studies linking the ADLH2*2
genotype and aldehydic load to many diseases that shorten
life expectancy (TABLE 3), an association between agingassociated damage and ALDH2 function is expected.
V. CONCLUDING REMARKS
As discussed here, ALDH2 is a well-known member of 19
different aldehyde dehydrogenases that are present in humans. Much of what we knew about ALDH2 stemmed
from an interest in ethanol metabolism. However, an exciting body of more recent research indicates that this enzyme
provides an important shield from oxidative stress and increased aldehydic load, thus contributing to better health.
Many human diseases are associated with increased aldehydic load. In fact, positive thiobarbituric acid reactive substance (TBARS) assay, measuring aldehydes in the blood,
which was a standard clinical test to determine inflammation and other general stresses, correlates with severity of
many human diseases (51, 123, 282). Accordingly, are aldehydes a marker of cellular stress in a variety of diseases or
do they actually contribute to these human pathologies?
In this review, we examined the evidence from animal studies and from human epidemiological studies; we believe that
an important new picture emerges. It appears that aldehydes are major contributors to the pathology of a variety
of human diseases. From developmental abnormalities,
such as Fanconi anemia, to diseases often associated with
life-style, including UADT cancer, diabetes, and heart disease; as well as diseases associated with aging, including
Alzheimer’s and Parkinson’s disease, reducing aldehydic
load appears to correspond to a better outcome. The role of
other aldehyde dehydrogenases in a number of human diseases has also been documented recently (194, 195). However, much of the data strongly support a specific role for
one aldehyde dehydrogenase, the ubiquitous mitochondrial
ALDH2.
Why is there such a central role for the mitochondrial
ALDH? Mitochondria are not only critical for energy generation. They are also major determinants in triggering both
apoptotic and necrotic cell death (6, 146). Therefore, protecting mitochondrial integrity by removing toxic aldehydes that accumulate in this organelle may be critical for
the cell. In particular, emerging data connecting the common ALDH2 inactivating mutation, ALDH2*2, in East
Table 3. Role of ALDH2 in aging
Findings Supporting An Active and Healthy ALDH2 May Improve Longevity
Reduces myocardial injury
Reduces cardiac arrhythmias
ALDH2*2 increases cancer risk
ALDH2*2 increases osteoporosis risk
Improves radiation-induced dermatitis
ALDH2*2 increases peripheral neuropathy risk
Increases neuronal survival from reactive aldehyde-induced toxicity
ALDH2*2 increases DNA damage, including mitochondrial DNA
ALDH2 knockout mice have a shortened lifespan
24
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Reference Nos.
25, 40
149
355
349
223
302
167
227, 301
230
TARGETING ALDEHYDE DEHYDROGENASE 2
Asians with certain diseases suggest that together with other
factors, ALDH2 provides an important protective enzyme.
acknowledged for their critical reading of and useful comments for the manuscript.
It should be noted that the inactivating mutation of
ALDH2*2 is still regarded as a relatively benign mutation.
ALDH2*2 was thought to cause a deficiency only in ethanol metabolism, and the resulting acetaldehyde toxicity or
“Asian flushing” was thought to be of no medical consequence. Together, this unpleasant flushing and associated
phenotypes after ethanol consumption were thought to be
positive, as they acted as a deterrent from alcoholism. However, much recent work elucidates the danger of acetaldehyde toxicity. Acetaldehyde is a cytotoxin and carcinogen.
Furthermore, ALDH2 inactivating mutation increases the
risk of subjects to the damaging effects of many other biogenic and environmental aldehydes. As ALDH2*2 affects
560 million East Asians, public education and awareness
are clearly needed to achieve better healthcare of the general
public. In one estimate, using population case-control data
from Japan for risk factor assessment, it was estimated that
if moderate-to-heavy drinkers of the ALDH2*1/*2
heterozygotes were instead only light drinkers, it could reduce 53% of the esophageal cancers in Japanese man (24).
With the increased emphasis on personalized medical care,
it may also be an interesting question for the medical community to consider whether individual carriers of ALDH2*2
should be treated as a unique subpopulation for disease prevention and treatment.
Address for reprint requests and other correspondence: D.
Mochly-Rosen, Dept. of Chemical and Systems Biology,
269 Campus Dr., CCSR Building, Room 3145, Stanford
University, School of Medicine, Stanford, CA 94305-5174
(e-mail: mochly@stanford.edu).
As a last note, an intriguing biological mystery remains:
What is the explanation for the existence of the widespread
ALDH2*2 mutation in East Asians? How come a single
monogenetic mutation that occurred ⬃2,000 –3,000 years
ago became the most common and uniform genetic enzymopathy, affecting an estimated 8% of the human race?
Could there be a selective advantage or added fitness for
having this mutation? A few hypotheses have been postulated, but none has been confirmed thus far (100, 176).
2. Aldini G, Granata P, Orioli M, Santaniello E, Carini M. Detoxification of 4-hydroxynonenal (HNE) in keratinocytes: characterization of conjugated metabolites by liquid
chromatography/electrospray ionization tandem mass spectrometry. J Mass Spectrom
38: 1160 –1168, 2003.
Finally, based on the discussion above on the critical role of
ALDH2 in cytoprotection and in how aldehydes contribute
to the pathology of a variety of human diseases, there may
be a role for new therapeutic interventions. One such intervention could be a class of activators of ALDH2, which
increase ALDH2 catalytic activity, protect it from inactivation by its toxic substrates, and correct the structural defect
in the common human ALDH2*2 mutation. Whether such
ALDH2 activators will slow down the progression of some
human diseases or actually prevent them and whether this
class of drugs will affect life span or only the quality of life
of humans will be interesting areas for both basic research
and potential pharmaceutical development.
GRANTS
The work in the Mochly-Rosen laboratory was supported
by National Institutes of Health (NIH) Grant R37
AA11147. E. R. Gross was supported by NIH K Award
HL-109212. J. C. B. Ferreira was supported by FAPESP
(2012/05765–2) and CNPq.
DISCLOSURES
D. Mochly-Rosen and C.-H. Chen are founders of ALDEA
Pharmaceuticals. C.-H. Chen is also a consultant to the
company.
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