JOURNAL OF NEUROCHEMISTRY | 2014 | 129 | 770–780
12677
Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center,
Lubbock, Texas, USA
Abstract
Excessive alcohol consumption is a prominent problem and one
of the major causes of mortality and morbidity around the world.
Long-term, heavy alcohol consumption is associated with a
number of deleterious health consequences, such as cancer,
heart and liver disease, a variety of neurological, cognitive, and
behavioral deficits. Alcohol consumption is also associated with
developmental defects. The causes of alcohol-induced toxicity
are presently unclear. One of the mechanisms underlying alcohol
toxicity has to do with its interaction with folic acid/homocysteine
or one-carbon metabolism (OCM). OCM is a major donor of
Millions of people worldwide have alcohol-use disorders
(Harper 2009). Numerous mechanisms may account for all
the effects of alcohol on both adult and developing
organisms. Yet, many gaps remain in our knowledge of
their molecular basis. One of mechanisms underlying alcohol
toxicity has to do with one-carbon metabolism (OCM)
impairment. OCM is a complex metabolic network of
pathways involved in the transfer of one-carbon groups and
the main source of these groups for methylation reactions in
the cell (Stover 2009). Hyperhomocysteinemia or elevated
homocysteine (Hcy) - an indicator of OCM dysfunction - is
often observed in chronic alcoholism (Seitz and Stickel
2007).
The physiological importance of OCM is based on its two
main functions: (i) de novo nucleotide biosynthesis, essential
for DNA replication and repair and (ii) provision of methyl
groups for methylation, including that of DNA methylation.
The latter is an important epigenetic determinant in gene
expression (Mason 2003; Villanueva et al. 2007). Other
epigenetic phenomena include histone modifications, which
are in turn associated with chromatin remodeling. Chromatin
is a fairly dynamic system for packaging and condensing
genomic DNA, critical for controlling the accessibility of
DNA for transcription. Since epigenetic mechanisms are
critical for gene expression and stably alter DNA function
without changing the sequence, structural chromatin
770 © 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
doi: 10.1111/jnc.
methyl groups for methylation, particularly DNA methylation
critical for epigenetic regulation of gene expression, and its
disturbance may compromise DNA methylation, thereby affect-
ing gene expression. OCM disturbance mediated by nutrient
deficits is a well-known risk factor for various disorders and
developmental defects (e.g., neural tube defects). In this review,
we summarize the role of OCM disturbance and associated
epigenetic aberrations in chronic alcohol-induced toxicity.
Keywords: alcohol toxicity, DNA methylation, one-carbon
metabolism.
J. Neurochem. (2014) 129, 770–780.
modifications can significantly impact gene expression and
thereby cellular function (Szyf 2011). Ethanol-related OCM
dysfunction may therefore induce epigenetic modifications.
This can explain why long-term excessive ethanol consump-
tion adversely impacts nearly every organ of the body. Since
epigenetic alterations leading to modified physiological
processes may be reversible, they can serve as targets for
potential intervention (Choi and Friso 2010).
Although alcohol is a known antagonist to OCM
(Giovannucci 2004; Kharbanda 2009), the etiology of
OCM disturbance in alcoholism is not entirely understood.
Received August 19, 2013; revised manuscript received January 30,
2014; accepted February 3, 2014.
Address correspondence and reprint requests to Inna I. Kruman,
Department of Pharmacology and Neuroscience, Texas Tech University
Health Sciences Center, Lubbock, TX 79430, USA.
E-mail: inna.kruman@ttuhsc.edu
Abbreviations used: Dnmt, DNA methyltransferases; dNTP, deoxyri-
bonucleotide triphosphates; FASD, fetal alcohol spectrum disorders;
HAT, histone acetyltransferase; Hcy, homocysteine; HDAC, histone
deacetylase; HMTase, histone methyltransferase; MBD, methylated
DNA-binding domain proteins; MS, methionine synthase; MTHFR,
methylene tetrahydrofolate reductase; MTRR, methionine synthase
reductase; NR2B, N-methyl D-aspartate receptor subtype 2B; OCM,
one-carbon metabolism; ROS, reactive oxygen species; SAH, S-adenosyl
homocysteine; SAM, S-adenosyl methionine; THF, 5-methyl tetrahy-
drofolate; TSA, trichostatin A.

It may result from the deficiency of folate, an essential
vitamin involved in OCM. Well documented in alcoholics,
folate deficiency may be caused by poor diet, malabsorption,
increased urinary excretion, or a combination of these factors
(Halsted and Medici 2011). OCM dysfunction, however,
may result from a direct interaction of ethanol or its
metabolites with OCM enzymes, such as methionine
synthase (MS), and this is not associated with reduced
availability of folate (Barak et al. 1993; Villanueva and
Halsted 2004). Given that MS is critical for OCM function,
its inactivation by ethanol may play a significant role in
alcohol-induced OCM impairment.
Toxins that may directly impact OCM are generated from
ethanol metabolism by alcohol dehydrogenase (ADH),
cytochrome P450 2E1, or catalase. These enzymes convert
ethanol to acetaldehyde (Wu et al. 2010; Cederbaum 2012).
Cytochrome P450 2E1 (Cyp2E1) is one of P450 isozymes
inducible by ethanol. Cyp2E1 is a major component of the
microsomal ethanol oxidizing system in the liver (Carpenter
et al. 1996; Lieber 2004), it is also inducible by ethanol in
other tissues including brain (Zerilli et al. 1995; Warner and
Gustafsson 1994; Zimatkin et al. 2006; Deitrich 2011). As
demonstrated in vitro studies, in the brain, where ADH
activity is low, catalase and Cyp2E1 generate 60 to 70% and

5 to 20% of acetaldehyde, respectively (Zimatkin et al.
2006). Acetaldehyde production is the first step of ethanol
metabolism, which is accompanied by formation of reactive
oxygen species (ROS) which has been shown to occur in
brain as well (Montoliu et al. 1994; Haorah et al. 2008).
Given that 8-hydroxy-20 -deoxyguanosine is a major product
of oxidative DNA damage, its formation in mouse brains
following alcohol exposure also confirms that alcohol gen-
erates ROS in the brain (Hirano 2011; Fowler et al. 2012).
Fig. 1 Ethanol interferes with one-carbon metab-
olism. CBS, cystathionine b-synthase; DHF,
dihydrofolate; dNTP, deoxyribonucleotide triphos-
phate; DMG, dimethylglycine; dTMP, deoxyt-
hymidine monophosphate; dUMP, deoxyuridine
monophosphate; Hcy, homocysteine; MS, methio-
nine synthase; MTHFR, methylene THF reductase;
5-methylTHF, SAH, S-adenosyl homocysteine;
SHMT, serine hydroxymethyltransferase.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
Alcohol toxicity and DNA methylation 771
Acetaldehyde is further metabolized to acetate by aldehyde
dehydrogenase 2. Acetaldehyde and ROS damage cellular
macromolecules such as lipids, proteins, and DNA and
contribute to alcohol-produced cell and tissue destruction
(Deng and Deitrich 2008) when acting in concert as factors
involved in alcohol toxicity.
One-carbon metabolism and risk factors for its
disturbance
OCM (Fig. 1) is a complex biochemical pathway responsible
for generating DNA nucleotides, precursors for DNA
biosynthesis and repair, as well as production and utilization
of S-adenosyl methionine (SAM), the universal methyl donor
in cellular methylation reactions (Stover 2004). B vitamins
including folic acid and its derivatives, folates together with
OCM enzymes such as MS, methylene tetrahydrofolate
reductase (MTHFR) and cystathinine beta-synthase (Singh
and Jaiswal 2013) represent essential OCM components.
MTHFR converts 5-methyltetrahydrofolate (5-methylTHF)
to 5-methylTHF. Once absorbed into the bloodstream, folate
molecules are taken up by the liver and retained or released
into blood or bile as 5-methylTHF, the main circulating form
of folate, which is then transported into various organs by
active transporters. MS converts Hcy to methionine utilizing
5-methylTHF as a donor of one-carbon moiety to methylate
Hcy. This yields methionine and tetrahydrofolate (THF).
Methionine is converted to the direct methyl donor, SAM
(Stover 2009). The methyl groups methylate biomolecules
(histone proteins and DNA), whereas catalyzed by DNA
methyltransferases (Dnmts) (Fuso 2013). SAM donates
methyl groups and converts to S-adenosyl-homocysteine
(SAH) which is hydrolyzed to Hcy. Then Hcy is catabolyzed
772 I. I. Kruman and A.-K. Fowler
via the trans-sulfuration pathway initiated by cystathionine b
synthase. Hcy accumulation increases SAH because of the
reversibility of the reaction converting SAH to Hcy; the
equilibrium favors SAH synthesis, whereas the hydrolytic
direction is favored only if Hcy is efficiently lowered (Stover
2009). SAH is a strong DNA methyltransferase inhibitor,
reinforcing DNA hypomethylation (Chiang et al. 1996; Lu
2000). Alteration in OCM can lead to a decrease in the SAM/
SAH ratio, eventually causing aberrant methylation resulting
in modified gene expression (Fuso and Scarpa 2011).
Biosynthesis of the deoxynucleotides in OCM involves
serine and is catalyzed by serine hydroxymethyltransferase 1
(SHMT) (Fig. 1). For this synthesis, one-carbon moiety is
obtained from THF, yielding 5,10-methyleneTHF, a
common substrate to methylation and synthesis of DNA
precursors - deoxyribonucleotide triphosphates (dNTPs).
Insufficiency of folate and other vitamins such as B6, and
B12 compromises the steady-state level of dNTPs which
may lead to misincorporation of uracil into the DNA
backbone in place of thymine (Singh and Jaiswal 2013).
Persistent uracil misincorporation results in futile cycles of
DNA repair, chromosome breakage and genomic instability
(Stover 2009). This scenario is also possible in conditions of
‘methyl trap’. Once 5,10-methyleneTHF is converted by
MTHFR to 5-methylTHF, the reverse reaction cannot occur,
and the only way for the recycling of 5-methylTHF to THF -
and thus participation in dNTP biosynthesis - is through MS.
However, MS inactivation which can be caused, for example,
by the oxidation of its coenzyme, vitamin B12 (Lim and
Song 2012) or by ethanol-produced damage to MS (Barak
et al. 1993; Villanueva and Halsted 2004), prevents the
conversion of 5-methylTHF to THF, resulting in trapping of
the cellular folate as 5-methylTHF. This leads to a cellular
pseudo-folate deficiency, where despite adequate amounts of
folate, OCM is impaired in a manner that is identical to that
seen in true folate deficiency (Stover 2009). The loss of MS
activity will also compromise conversion of Hcy to methi-
onine, since MS catalyzes this conversion. Hcy, however,
can be remethylated via an alternative mechanism using
betaine, supplied from dietary choline, in the kidney and
liver. Thus, OCM regulates two vital processes - DNA
synthesis and DNA methylation. Consequently, disturbance
of OCM enzyme activity or deficiency in its vitamins affects
both methylation and DNA synthesis, which may lead to
disease (Singh and Jaiswal 2013). Particularly, elevated
blood- and CSF levels of Hcy, and reduced SAM in CSF and
brain tissue are associated with neurological diseases and
mood disorders (D’Anci and Rosenberg 2004). OCM which
is essential for CNS function is different from OCM in the
liver or kidney, where remethylation can be performed
through betaine-homocysteine-methyltransferase which is
not detected in the brain (Sunden et al. 1997). In addition,
in contrast to the liver and kidney, the brain exhibits a reduced
activity of the trans-sulfuration pathway (Miller 2003).
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
Methylating function of OCM through SAM formation
is indispensable for various CNS reactions including syn-
thesis of neurotransmitters and phospholipids and methyla-
tion of DNA and proteins (Stanger et al. 2009). The
importance of OCM for the CNS was initially demonstrated
in studies that revealed an increased risk of neural tube
defects under conditions of folate deficiency during preg-
nancy (Laurence et al. 1981; Blom et al. 2006). Epidemio-
logical studies have demonstrated that OCM disturbance is
also involved in pathogenesis of neurodegenerative and
neuropsychiatric diseases, including stroke, Parkinson’s and
Alzheimer’s diseases, dementia, and depression (Reynolds
2002; He et al. 2004; Irizarry et al. 2005; Lamberti et al.
2005; Choi and Friso 2010). Animal and in vitro studies
supported direct impact of folate deficiency and hyperhom-
ocysteinemia in neurotoxicity (Kruman et al. 2002; Fuso
et al. 2012).
DNA methylation
DNA methylation is the most extensively studied epigenetic
mechanism, important for regulation of transcription, histone
modification, and genomic stability. DNA methylation
involves a covalent modification of DNA by addition of a
methyl group to cytosine base at the CpG dinucleotide
residues which can cause a stable alteration of DNA function
without changing the sequence (reviewed in Choi and Friso
2010; Szyf 2011). DNA methylation is a heritable epigenetic
mechanism by which a methylated cytosine in CpG
accurately replicates its methylation pattern (Gruenbaum
et al. 1981; Bohacek et al., 2013) and participates in
establishing differentiated cell types during embryogenesis
(Razin et al. 1984; Szyf 2011; Lister et al. 2009). In
vertebrates, the CpG dinucleotide sequence is a principal
target of DNA methylation, as it is preferentially recognized
by Dnmts (Gruenbaum et al. 1982). Five different Dnmts
catalyze DNA methylation at the CpG sequence, responsible
for maintenance (Dnmt1) and de novo (Dnmt 3a and 3b)
methylation. The function of Dnmt2 is not yet clear.
Knockout studies in mice have shown that de novo DNA
methylation is essential for development, since Dnmt3a-
deficient animals die several weeks after birth and Dnmt3b-
deficient animals die in utero (Okano et al. 1999). Changes
in Dnmt activity or expression can affect global DNA
methylation. Dnmt1 expression is known to decrease with
age, which may alter DNA methylation status and DNA
methylation-mediated gene expression (Li et al. 2010). This
can be associated with pathogenesis of age-associated
diseases. DNA methylation, however, not always occurs in
the context of CpG. Recent data suggest that DNA methyl-
ation occurs in other dinucleotide sequences in regions
beyond the traditional promoters (Lister et al. 2009; Fuso
et al. 2010; McGowan et al. 2011). This, in turn, suggests
that differentially methylated regions are not limited to

promoters, as well as that methylation of these regions may
play a role in controlling genome function beyond promoter
regulation of gene expression. The presence of non-CpG
methylation in the genome suggests that DNA methylation is
not always mitotically heritable, and can thus be maintained
by a balance of methylating and demethylating enzymes
(McGowan et al. 2011). Indeed, active demethylation has
been confirmed in this context (Lucarelli et al. 2001;
Bruniquel and Schwartz 2003). The dynamic methylation-
demethylation equilibrium has been observed in postmitotic
cells, particularly in postmitotic neurons (Weaver et al.
2004; Feng and Fan 2009), where conditional knockout of
Dnmt1 resulted in DNA demethylation (Feng and Fan 2009).
The mechanism involved in regulation of gene expression
not associated with promoter DNA methylation includes a
recruitment of methylated DNA-binding domain proteins
(MBD) to methylated cytosines in promoters (Nan et al.
1997). MBDs recruit complexes containing histone deacet-
ylases and histone methyltransferases to promoters which
leads to shaping inactive chromatin configuration around the
genes (Dobosy and Selker 2001). Several lines of evidence
indicate that chromatin modification and DNA methylation
are correlated (Cheng and Blumenthal 2010). Methylated
DNA is enriched in regions of the genome that are packaged
in inactive chromatin (Razin and Cedar 1977), suggesting a
bilateral relationship between chromatin modification and
DNA methylation. Relationship between histone acetylation
and DNA methylation is associated with a well-known
connection between chromatin compaction and DNA meth-
ylation. The commonly used histone deacetylase inhibitors,
trichostatin A, and valproic acid induce loss of DNA
methylation, chromatin decondensation, and transcriptional
activation (Cervoni and Szyf 2001; Ou et al. 2007; Dong
et al. 2008). This may result from the facilitated access of
gene regulatory regions to demethylases (Ou et al. 2007).
Since chromatin modification is dynamic, it makes altera-
tions in DNA methylation also dynamic, with critical
implications in the brain (Gr€aff et al. 2012; Zentner and
Henikoff 2013).
Although the mechanisms of DNA demethylation are not
fully understood, it has been suggested that DNA demethy-
lation may involve (i) DNA base excision repair; (ii) DNA
nucleotide excision repair; (iii) oxidative or (iv) hydrolytic
removal of the methyl group (Zhu 2009). Most recently,
5-methylcytosine in mammalian DNA was found to be
converted to 5-hydroxymethylcytosine by methylcytosine
oxygenase TET1 (Bottiglieri and Hyland 1994; Tahiliani
et al. 2009). 5-hydroxymethylcytosine might also be pro-
duced by Dnmts via addition of formaldehyde to cytosines in
DNA (Liutkeviciute et al. 2009). Unmodified cytosine is
believed to be generated as a result of the reversible Dnmt-
catalyzed reaction from 5-hydroxymethylcytosine which may
therefore be an intermediate in direct DNA demethylation.
Significant levels of 5-hydroxymethylcytosine observed in
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
Alcohol toxicity and DNA methylation 773
mammalian DNA support this suggestion (Jin et al. 2010).
The formation of differential methylation patterns is very
important to preserve the stability of the differentiated cell
phenotype. Inappropriate modulations of DNA methylation
and associated alterations in gene expression can result in
disease (Razin and Riggs 1980; Singh and Jaiswal 2013).
There are two types of identity: the individual identity
encoded by DNA sequence and a cell-type identity encoded
in the distribution of methyl groups in the same molecule of
DNA. Cell type-specific, differentially methylated regions of
DNA produce multiple phenotypes in a multicellular organ-
ism, explaining how the same genome can encode multiple
phenotypes. DNA methylation is cell type- and tissue-
specific. There are known cell type-specific patterns of
methylation or tissue-specific differentially methylated
regions (Szyf 2011). For example, DNA methylation in
differentiated neurons is very dynamic, and Dnmt expression
in these cells is high (Feng et al. 2010). However, increase in
Hcy levels and thus high levels of SAH inhibit Dnmts (James
et al. 2002) which is especially important for brain tissue due
to the absence of betaine-homocysteine remethylation activ-
ity (Sunden et al. 1997) and significantly reduced capacity of
the trans-sulfuration pathway (Miller 2003). Thus, alterations
in DNA methylation can have tissue-specific manifestations
(Maegawa et al. 2010).
The role of epigenetics in normal physiology and
disease
Since OCM is the main source of methyl groups, its
disturbance by ethanol and nutrients may alter DNA and
histone methylation, thereby affecting gene expression at the
transcriptional level. Such epigenetic changes can be inher-
ited during cell division (Slatkin 2009), resulting in perma-
nent maintenance of the acquired phenotype. Nutrient deficits
involved in OCM can alter OCM function by affecting a
methyl group provider, SAM, and an inhibitor of Dnmts,
SAH (Choi and Friso 2010). This may lead to disturbed
DNA and histone methylation, possibly causing various
disorders. Indeed, ethanol-induced liver cirrhosis is associ-
ated with global hypomethylation of liver DNA (Purohit
et al. 2007). Global changes in DNA methylation are
common in chronic diseases such as lupus (Cornacchia
et al. 1988; Yung and Richardson 1994) and diabetes (Choi
and Friso 2010). Aberrations in DNA methylation are also
associated with cardiovascular disease (Gluckman et al.
2009), oncogene activation, chromosome instability, and
carcinogenesis (Ji et al. 1997). An increasing body of
evidence indicates that many psychiatric and neurodegener-
ative diseases such as Alzheimer’s, Parkinson’s, schizophre-
nia, and autism are also linked to epigenetics or at least have
an epigenetic component (Qureshi and Mehler 2012; Fuso
2013). In fact, only a small percentage of neurodegenerative
disorders are caused by evident genetic mutations – for
774 I. I. Kruman and A.-K. Fowler
instance, Huntington’s disease and the familial forms of
Parkinson’s and Alzheimer’s diseases (Lill and Bertram
2011). The pathogenesis of sporadic onset versus Mendelian
inheritance of most cases of neurodegenerative diseases
remains elusive. Nevertheless, it has been suggested that the
sporadic forms may be associated with environmental factors
mediating epigenetic changes, particularly evidenced in
Alzheimer’s disease (Migliore and Coppede 2009; Fuso
and Scarpa 2011; Mastroeni et al. 2011). This suggestion is
supported by observation that the treatment of a transgenic
mouse model of Alzheimer’s disease with a diet deficient in
vitamin B12, B6, and folate, fundamental for OCM homeo-
stasis and therefore methylation potential, resulted in aberrant
methylation, increased amyloid processing, and cognitive
impairment. At the same time, supplementation with SAM
attenuated these effects (Fuso and Scarpa 2011; Fuso et al.
2012). Altered levels of SAM were observed in neurological
disorders, including Alzheimer’s disease (Bottiglieri and
Hyland 1994; Tchantchou et al. 2006). Together, these
findings demonstrate the involvement of epigenetic altera-
tions in sporadic diseases and a potential role for OCM
impairment in such alterations. This, in turn, suggests that
nutritional deficiency, excessive alcohol consumption, and
other environmental factors are able to modulate methylation
metabolism. This also suggests that environmental factors
can have both therapeutic and toxic implications.
Since embryonic development is characterized by rapid
cell replication and formation of DNA methylation patterns,
the impairment of OCM or other elements of DNA
methylation machinery such as Dnmts may cause alterations
in DNA methylation (Dolinoy et al. 2007). Deficiency in
methylated DNA-binding domain proteins Mbd1 in mutant
(Mbd1
/
) mice was characterized by several deficits
associated with autism such as social interaction difficulties,
learning deficits, anxiety, and depression (Allan et al. 2008).
Folic acid deficiency during gestation resulted in multiple
teratogenic effects, which might be explained by reduced
methyl availability due to OCM dysfunction and consequent
epigenetic aberrations (Pogribny et al. 1997; Brunaud et al.
2003). If these aberrations affect the expression of genes
involved in immune programming, the offspring develops
allergic disorders (Bach 2002). Agents interfering with OCM
and therefore DNA methylation during certain time when
these patterns are generated could disrupt the DNA methyl-
ation pattern, cellular differentiation, and organogenesis,
given the importance of epigenetic programing for cell
differentiation (Huang and Fan 2010). Aberrations in DNA
methylation patterns can target different regions of the
genome and cause teratogenesis or manifest later in life,
transforming the adult health-related phenotype (Sinclair
et al. 2007). For example, immunodeficiency and facial
anomalies syndrome (ICF) - a rare chromosome breakage
disease - are characterized by global hypomethylation
(Hansen et al. 1999; Okano et al. 1999; Ehrlich 2003). Rett
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
syndrome which is also characterized by hypomethylation,
results from mutations in genes encoding proteins that
regulate DNA methylation homeostasis (Berdasco and
Esteller 2013). As epigenetic information can be inherited,
and changes in DNA methylation pattern could be main-
tained through DNA replication cycles, transient exposure to
an environmental agent such as diet, nutrients, exposure to
chemicals, and alcohol could have a lasting impact on DNA
methylation and thereby, phenotype. The honeybee model
clearly demonstrates the epigenetic effects of diet on the
phenotype. Honeybees grow into either long-lived and very
reproductive queens or short-lived functionally sterile work-
ers, depending on whether they are fed royal jelly or
beebread. The difference in phenotype has been suggested to
be caused by epigenetic modifications in DNA methylation
patterns induced by the different types of diet (Kucharski
et al. 2008).
Social factors (e.g., maternal care) may also be responsible
for these epigenetic changes. Early childhood is known to be
extremely critical in shaping these behavioral patterns in
mammals (Champagne 2010). Maternal behavior defines
life-long changes in the offspring’s responsiveness to stress
(Liu et al. 1997; Francis et al. 1999). For illustration, it is
known that exposure of infant rats to stressed caretakers with
abusive behavior produced persisting changes in methylation
pattern of the adult prefrontal cortex (Roth et al. 2009);
early-life stress in mice caused sustained DNA hypomethy-
lation of an important regulatory region of the arginine
vasopressin gene (Murgatroyd et al. 2009). It has been well
documented that maternal care differences modifies epige-
netic status of the glucocorticoid receptor gene in the
hippocampus and has a global effect on the hippocampal
transcriptome. This impacts the stress response, learning, and
memory of the offspring. As early-life experience has long-
term consequences on behavior, it is potentially reversible in
adulthood (Weaver et al. 2004, 2006) and may also be
reversed by certain interventions such as changes in meth-
ylation pattern (Cervoni and Szyf 2001; Cervoni et al. 2002;
Detich et al. 2003). Thus, both adverse chemical and social
exposures at this early period of life could have a long-lasting
impact on behavior and memory through changes in DNA
methylation (Lahiri et al. 2009). These findings support the
idea that although epigenetic programs established early in
life are sustained throughout life, they are maintained by a
dynamic balance between DNA methylation and demethy-
lation and can be potentially changed.
Alcohol-induced aberrant DNA methylation in the
adult organism
Alcohol metabolites have been shown to directly affect the
epigenome (Knezovich and Ramsay 2012). There is increas-
ing evidence that alcohol-induced epigenetic changes may
result from OCM dysfunction lowering the availability of

SAM which compromises DNA and histone methylation
(Hamid et al. 2009). Alcoholic patients often have reduced
blood folate and elevated plasma Hcy (Cravo et al. 1996),
both indicative of OCM impairment. However, toxic ethanol
metabolites such as acetaldehyde, may directly interact with
different components of OCM (e.g. MS) thereby disturbing
methylation reactions (Barak et al. 1993; Villanueva and
Halsted 2004). Indeed, ethanol exposure leads to reduced
levels of the major methyl donor, SAM, increased levels of
SAH, lower SAM⁄SAH ratio and as a consequence, global
hypomethylation (Stickel et al. 2000; Murillo-Fuentes et al.
2005). A significant decrease of mRNA expression of
Dnmt3a Dnmt3b in alcoholics (Bonsch et al. 2006) has also
been demonstrated. Ethanol has been found to cause site-
specific methylation and acetylation of histones. For exam-
ple, by activating histone acetyltransferase, ethanol exposure
induced increased histone acetylation (H3K9) in a promoter
region of the gene encoding the alcohol-metabolizing
enzyme ADH 1 in rat hepatocytes (Park et al. 2005). In
addition, exposure of hepatocytes to ethanol resulted in
distinct histone H3 methylation patterns at lysine 9 and lysine
4, which correlated with increases and decreases in gene
expression, respectively (Pal-Bhadra et al. 2007). Further-
more, epidemiological observations implicate excess ethanol
ingestion, as well as low dietary folate (the latter being one of
the most studied aspects of the toxic role of OCM
impairment; Fuso and Scarpa 2011) as risk factors for
several cancers of the oral cavity, esophagus, liver, colon,
rectum, and breast (Giovannucci 2002; Dumitrescu and
Shields 2005; Boffetta et al. 2006). Alcohol drinking was
associated with hypermethylation at DNA repair genes such
as the O6-methyl guanine methyl transferase involved in the
removal of mutagenic methyl adducts from guanine (Puri
et al. 2005) which may impact DNA repair leading to
genomic instability which plays a causative role in carcino-
genesis. These findings suggest the involvement of OCM
impairment and consequent aberrations in DNA methylation
in the mechanisms underlying cancer. The role of abnormal
hepatic OCM in alcohol liver disease is well documented
(Medici and Halsted 2013) and supported by the protective
effects of supplementation with the universal methyl donor in
eukaryotes, SAM (Song et al. 2003; Bailey et al. 2006;
Sykora et al. 2009; Andringa et al. 2010). Ethanol has been
found to alter DNA methylation in the brain (Pignataro et al.
2009). Under conditions of chronic alcohol exposure,
hyperhomocysteinemia - a hallmark of OCM dysfunction is
correlated with loss of brain volume, as well as cognitive and
memory decline and withdrawal seizures (Vogiatzoglou
et al. 2008; Bleich and Hillemacher 2009; Coppede 2010;
Tangney et al. 2011). Recently, we demonstrated that
chronic ethanol exposure results in global DNA hypomethy-
lation of brain tissue and neurotoxicity, and this alcohol
neurotoxicity was exaggerated by the additional OCM
impairment in mice deficient in MTHFR (Fowler et al. 2012),
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
Alcohol toxicity and DNA methylation 775
suggesting a causal role for OCM impairment in alcohol
neurotoxicity. Alcohol has been found to modify DNA
methylation (and expression) of specific genes. Bonsch and
associates reported significant increase of DNA methylation
at a synuclein (Bonsch et al. 2005), which is also observed
in Alzheimer’s disease (Alim et al. 2004). Alcohol epige-
netically increased expression of the glutamate N-methyl D-
aspartate receptor subtype 2B by demethylation of corre-
sponding gene (Qiang et al. 2010). Increased N-methyl D-
aspartate receptor subtype 2B expression has been implicated
in ethanol-associated phenotypes (Krystal et al. 2003). Such
epigenetic alterations induced by alcohol may play a role in
the pathophysiology of psychiatric disorders including
depression (Frieling et al. 2007; Hillemacher 2011) and
schizophrenia (Bleich et al. 2006).
Developmental aspect of alcohol-induced aberrant
methylation
Alcohol exposure during development is associated with a
multifactorial developmental deficit known as fetal alcohol
spectrum disorders (FASD) wherein its mechanism is not
clear. However, such alcohol effects may be caused by its
interference with OCM and induction of aberrant methylation
(Liu et al. 2009; Zhou et al. 2011). Recent animal study
performed on a model of gestational alcohol exposure
demonstrated changes in the expression of an epigenetically
sensitive allele, Agouti viable yellow (Avy), in the offspring
after maternal ethanol consumption (Kaminen-Ahola et al.
2010). As a result, the probability of transcriptional silencing
at this locus increased, enhancing the number of offspring
with an agouti-colored coat. This transcriptional silencing
correlated with hypermethylation at Avy. This reveals that
ethanol can affect adult phenotype epigenetically changing
the early embryo. Alcohol-induced changes in DNA meth-
ylation which are highly regulated during development, and
which disrupts key developmental events, such as differen-
tiation and the fate of early developing precursor cells
(Sachdev 2005; Liu et al. 2009; Wu and Zhang 2011; Zhou
et al. 2011), can underlie FASD, analogous to developmen-
tal defects associated with aberrant DNA methylation under
conditions of vitamin B12 or folic acid deficiency (Cravo and
Camilo 2000; Mason and Choi 2005). Indeed, chronic
ethanol consumption in pregnant mice resulted in demethy-
lation of fetal DNA (Garro et al. 1991). Such epigenetic
alterations have been found to disrupt migration, neural cell
lineage differentiation during early brain development (Feng
et al. 2007; MacDonald and Roskams 2009). In vitro
experiments confirmed that ethanol effects associated with
compromised DNA methylation patterns also inhibited
neural stem cell differentiation, similar to treatment with
the methylation inhibitor 5-aza-cytidine (Zhou et al. 2011).
Furthermore, it has been demonstrated that the alcohol-
induced changes in gene expression were linked to changes
776 I. I. Kruman and A.-K. Fowler
in DNA methylation in the hippocampus and prefrontal
cortex of animal FASD models (Otero et al. 2012; Resendiz
et al. 2013). Ethanol-induced alterations in methylation of
particular genes were also demonstrated. For example, fetal
alcohol exposure induced long-lasting hypermethylation of
Pomc gene involved in stress control, metabolic, and immune
functions (Govorko et al. 2012). These effects were found
during early gastrulation periods (Kaminen-Ahola et al.
2010). Gastrulation is generally considered the most sensitive
to teratogenic insults, because differentiating cells appear to
be particularly vulnerable to the teratogenic effects of alcohol
(Kobor 2011). For instance, ethanol exposure of pregnant
mice from the 9th through the 11th day of gestation resulted
in global hypomethylation of fetal DNA (Garro et al. 1991).
Accumulating evidence suggests that alteration in DNA
methylation may affect not only one generation but produce
effects across generations (Anway et al. 2005). This concept
of transgenerational transmission of altered DNA methyla-
tion is supported by work showing specific exposure-induced
sperm methylation patterns reproduced between generations
(Guerrero-Bosagna et al. 2010). Indeed, the offspring sired
by male mice chronically exposed to ethanol prior to
conception inherited aberrant epigenetic marks and revealed
growth retardation phenotype in the immediate post weaning
period (Knezovich and Ramsay 2012). Importantly, male
gametes in both mice and humans chronically exposed to
alcohol underwent site-specific hypomethylation (Ouko
et al. 2009; Stouder et al. 2011), consistent with a significant
decrease in postnatal growth of children of alcoholic men
(Little and Sing 1987). Consistent with these profound
epigenetic effects of alcohol, are the beneficial effects of
supplementation of animals with choline, betaine or folic
acid before mating, throughout prenatal or neonatal periods.
Indeed, the supplementation has been shown to reverse
alcohol-induced changes in methylation and partially ame-
liorated ethanol teratogenesis (Thomas et al. 2010; Downing
et al. 2011; Otero et al. 2012). Together, these findings
suggest that alcohol has a substantial impact on the
epigenome via OCM disturbance, and these epigenetic
aberrations may play an important role in alcohol-induced
developmental defects.
Conclusion
Excessive alcohol consumption has adverse effects on many
tissues and organ systems (Harper 2009). As alcohol’s harmful
health effects are well known, the underlying mechanisms are
not fully understood. One of the mechanisms underlying
alcohol toxicity is OCM impairment induced by ethanol or its
metabolites (Cravo et al. 1996; Giovannucci 2004; Fowler
et al. 2012). OCM is essential for cellular homeostasis,
providing cells with DNA precursors, necessary for cell
proliferation and DNA repair. It is likewise the main source of
methyl groups for methylation reactions, including DNA and
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 770--780
chromatin methylation. DNA methylation is an important
epigenetic determinant in gene expression (Mason 2003). As
epigenetic information can be inherited, and maintained
through DNA replication cycles, it can also be modified by
exposure to agents that interfere with OCM or other elements of
DNA methylation machinery (e.g., DNMTs) such as diet,
nutrients, or chemicals and alcohol (Dolinoy et al. 2007; Choi
and Friso 2010). Aberrant OCM therefore could result in a
lasting impact on gene expression at the transcriptional level
associated with various abnormalities (Anway et al. 2005).
Indeed, both folate deficiency and ethanol exposure - which
lead to OCM dysfunction - are associated with liver (Guerrerio
et al. 2012; Medici and Halsted 2013) and heart (Verhaar et al.
2002; Djousse and Gaziano 2008) diseases, cancer (Gio-
vannucci 2002; Dumitrescu and Shields 2005; Boffetta et al.
2006), and neurodegeneration (Harper 2009; Fuso 2013).
Since epigenetic alterations are especially important during
differentiation (Wu and Zhang 2011), agents that interfere with
OCM or other factors involved in methylation, may disturb
cellular differentiation and organogenesis during embryonic
development (Sinclair et al. 2007). Indeed, both folate defi-
ciency and alcohol are teratogenic (Pogribny et al. 1997;
Brunaud et al. 2003; Downing et al. 2011; Kobor 2011).
These observations strongly support the role of OCM impair-
ment and consequent epigenetic alterations in alcohol toxicity.
Acknowledgments and conflict of interest
disclosure
This work was supported by the Foundation for Alcohol Research,
the South Plains Alcohol and Addiction Research Center
(SPAARC).
All experiments were conducted in compliance with the ARRIVE
guidelines. The authors have no conflict of interest to declare.
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