Effects of alcohol on histone deacetylase 2 (HDAC2) and the neuroprotective role of trichostatin A (TSA).
ABSTRACT Previous studies have implicated histone deacetylases (HDACs) and HDAC inhibitors (HDIs) such as trichostatin A (TSA) in the regulation of gene expression during drug addiction. Furthermore, an increase in HDAC activity has been linked to neurodegeneration. Alcohol has also been shown to promote abundant generation of reactive oxygen species (ROS) resulting in oxidative stress. TSA inhibits HDACs and has been shown to be neuroprotective in other neurodegenerative disease models. Although HDACs and HDIs have been associated with drug addiction, there is no evidence of the neurodegenerative role of HDAC2 and neuroprotective role of TSA in alcohol addiction. Therefore, we hypothesize that alcohol modulates HDAC2 through mechanisms involving oxidative stress.
To test our hypothesis, the human neuronal cell line, SK-N-MC, was treated with different concentrations of ethanol (EtOH); HDAC2 gene and protein expression were assessed at different time points. Pharmacological inhibition of HDAC2 with TSA was evaluated at the gene level using qRT-PCR and at the protein level using Western blot and flow cytometry. ROS production was measured with a fluorescence microplate reader and fluorescence microscopy.
Our results showed a dose-dependent increase in HDAC2 expression with EtOH treatment. Additionally, alcohol significantly induced ROS, and pharmacological inhibition of HDAC2 with TSA was shown to be neuroprotective by significantly inhibiting HDAC2 and ROS.
These results suggest that EtOH can upregulate HDAC2 through mechanisms involving oxidative stress and HDACs may play an important role in alcohol use disorders (AUDs). Moreover, the use of HDIs may be of therapeutic significance for the treatment of neurodegenerative disorders including AUDs.
- SourceAvailable from: Hoa K Nguyen[Show abstract] [Hide abstract]
ABSTRACT: Prenatal ethanol exposure causes cellular stress, insulin resistance, and glucose intolerance in adult offspring, with increased gluconeogenesis and reduced muscle glucose transporter-4 (glut4) expression. Impaired insulin activation of Akt and nuclear translocation of histone deacetylases (HDACs) in the liver partly explain increased gluconeogenesis. The mechanism for the reduced glut4 is unknown. Pregnant rats were gavaged with ethanol over the last week of gestation and adult female offspring were studied. Some ethanol exposed offspring was treated with tauroursodeoxycholic acid (TUDCA) for 3 weeks. All these rats underwent intraperitoneal glucose tolerance and insulin tolerance tests. The expression of glut4, HDACs, and markers of endoplasmic reticulum (ER) unfolded protein response (XBP1, CHOP, ATF6) was examined in the gastrocnemius muscle fractions, and in C2C12 muscle cells cultured with ethanol, TUDCA, and HDAC inhibitors. Non-TUDCA-treated rats exposed to prenatal ethanol were insulin resistant and glucose intolerant with reduced muscle glut4 expression, increased ER marker expression, and increased nuclear HDACs, whereas TUDCA-treated rats had normal insulin sensitivity and glucose tolerance with normal glut4 expression, ER marker expression, and HDAC levels. In C2C12 cells, ethanol reduced glut4 expression, but increased ER makers. While TUDCA restored glut4 and ER markers to control levels and HDAC inhibition rescued glut4 expression, HDAC inhibition had no effect on ER markers. The increase in nuclear HDAC levels consequent to prenatal ethanol exposure reduces glut4 expression in adult rat offspring, and this HDAC effect is independent of ER unfolded protein response. HDAC inhibition by TUDCA restores glut4 expression, with improvement in insulin sensitivity and glucose tolerance.Physiological Reports. 12/2014; 2(12).
- [Show abstract] [Hide abstract]
ABSTRACT: We aimed to elucidate the effects of two epigenetic inhibitors, 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA), on several key secretory mediators of diabetic retinopathy (DR) in human retinal endothelial cells (HRECs) and human retinal pigment epithelial (HRPE) cells treated with high glucose or interleukin-1β (IL-1β).Molecular vision 01/2014; 20:1411-21. · 2.25 Impact Factor
Effects of Alcohol on Histone Deacetylase 2 (HDAC2)
and the Neuroprotective Role of Trichostatin A (TSA)
Marisela Agudelo, Nimisha Gandhi, Zainulabedin Saiyed, Vijaya Pichili,
Samikkannu Thangavel, Pradnya Khatavkar, Adriana Yndart-Arias, and Madhavan Nair
Background: Previous studies have implicated histone deacetylases (HDACs) and HDAC
inhibitors (HDIs) such as trichostatin A (TSA) in the regulation of gene expression during drug
addiction. Furthermore, an increase in HDAC activity has been linked to neurodegeneration.
Alcohol has also been shown to promote abundant generation of reactive oxygen species (ROS)
resulting in oxidative stress. TSA inhibits HDACs and has been shown to be neuroprotective in
other neurodegenerative disease models. Although HDACs and HDIs have been associated with
drug addiction, there is no evidence of the neurodegenerative role of HDAC2 and neuroprotective
role of TSA in alcohol addiction. Therefore, we hypothesize that alcohol modulates HDAC2
through mechanisms involving oxidative stress.
Methods: To test our hypothesis, the human neuronal cell line, SK-N-MC, was treated with
different concentrations of ethanol (EtOH); HDAC2 gene and protein expression were assessed at
different time points. Pharmacological inhibition of HDAC2 with TSA was evaluated at the gene
level using qRT-PCR and at the protein level using Western blot and flow cytometry. ROS pro-
duction was measured with a fluorescence microplate reader and fluorescence microscopy.
Results: Our results showed a dose-dependent increase in HDAC2 expression with EtOH treat-
ment. Additionally, alcohol significantly induced ROS, and pharmacological inhibition of
HDAC2 with TSA was shown to be neuroprotective by significantly inhibiting HDAC2 and
Conclusions: These results suggest that EtOH can upregulate HDAC2 through mechanisms
involving oxidative stress and HDACs may play an important role in alcohol use disorders
(AUDs). Moreover, the use of HDIs may be of therapeutic significance for the treatment of neu-
rodegenerative disorders including AUDs.
Key Words: Ethanol, Histone Deacetylases, Trichostatin A, SK-N-MC, Oxidative Stress.
more effective treatments. According to National Institute of
Alcohol Abuse and Alcoholism (NIAAA), approximately 18
million Americans suffer from AUDs and only 7% of these
individuals received any form of treatment (NIAAA, 2009).
Alcohol dependence is a complex addiction regulated by mul-
tiple mechanisms including neurotransmitters and enzymes.
Histone deacetylases (HDACs) as well as their inhibitor, tri-
chostatin A (TSA), have been found to regulate various genes
during drug addiction. Although HDACs have been associ-
ated with drug addiction or addictive behavior, the role of
HDAC dysregulation has not been examined in alcohol
addiction. Among class I HDACs that are ubiquitously
ESPITE THE EFFORTS to develop new medications
for alcohol use disorders (AUDs), there is still a lack of
expressed in various tissues and cell types, HDAC1 and
HDAC2 have been shown to be involved in neuronal fate.
Moreover, HDAC2 has also been shown to be involved in
neuronal cell differentiation (Bai et al., 2005). In the current
study, we explore the role of alcohol on HDAC2 expression
in the human neuronal cell line, SK-N-MC.
Histone acetyltransferases (HATs) and HDACs are
enzymes that remove acetyl groups to and from target lysine
residues within histones (Min-Hao and Allis, 1998; Peterson
and Laniel, 2004) resulting in regulation of gene expression.
HATs tend to be transcriptional activators, whereas HDACs
tend to be repressors (Bannister, 2010). Regulation of gene
expression is known to contribute to the long-term effects in
response to drugs of abuse, and recent studies have demon-
strated that HDACs and HDAC inhibitors (HDIs) play a
major role in drug dependence (Romieu et al., 2008). Further-
more, these enzymes have been implicated in human disease,
making them important drug targets. Recent studies have
indicated that ethanol (EtOH) exposure induces global pro-
tein hyperacetylation which likely leads to alcohol-induced
hepatotoxicity (Shepard and Tuma, 2009). Therefore, such
histone modifications may underlie the mechanisms involved
in EtOH-induced cellular injury (Shukla and Aroor, 2006).
However, most of the studies have been focused on the effects
From the Department of Immunology (MA, NG, ZS, VP, ST,
PK, AY-A, MN), Florida International University, Miami, Florida.
Received for publication November 24, 2010; accepted January 7, 2011.
Reprint requests: Madhavan Nair, PhD, Department of Immunol-
ogy, Institute of NeuroImmune Pharmacology, College of Medicine,
HLS-I 308, Florida International University, 11200 SW 8th Street,
Miami, FL 33199; Tel.: 305-348-1491; Fax: 305-348-1109; E-mail:
Copyright ? 2011 by the Research Society on Alcoholism.
Alcoholism: Clinical and Experimental Research
Vol. 35, No. 8
1550Alcohol Clin Exp Res, Vol 35, No 8, 2011: pp 1550–1556
of alcohol in hepatocytes and liver injury; very little is known
about the effects of alcohol on HDAC2 and the role of its
inhibitor TSA in alcohol-mediated effects in human central
nervous system cells.
While HDACs are expressed abundantly in the brain, little
is known about their roles in brain function. Evidence sug-
gests that these enzymes regulate neuronal survival and neu-
rodegeneration (D’Mello, 2009). Furthermore, HDIs have
recently been known to modulate genes involved in drug
addiction such as the l opioid receptor gene (Lin et al.,
2008) and decrease cocaine self-administration in rats (Ro-
mieu et al., 2008). Impressively, HDIs have been found to be
neuroprotective in cellular and animal models of acute and
chronic neurodegenerative diseases such as Alzheimer’s dis-
ease (Alan et al., 2009), and the neuroprotective role of
HDIs seems to extend to other diseases that share mecha-
nisms of oxidative stress, inflammation, and neuronal cell
apoptosis (Gray and Dangond, 2006). TSA can be used to
alter gene expression by interfering with the removal of ace-
tyl groups from histones and therefore can interfere with
transcription factors and their ability to access the DNA
molecules inside chromatin. Although HDACs have been
associated with drug addiction and TSA has been shown to
be neuroprotective in other disease models, there is no evi-
dence of the neuroprotective role of HDACs and TSA in
alcohol addiction. Therefore, it is hypothesized that alcohol
modulates HDAC2 through mechanisms involving oxidative
stress. In the current study, we report the effects of alcohol
on the expression of HDAC2 in the neuronal cell line, SK-
N-MC. Moreover, pharmacological inhibition with TSA was
also performed to test the specific alcohol regulation of
HDAC2. To delineate the mechanisms of action of alcohol,
we measured reactive oxygen species (ROS) production after
treatment with EtOH, TSA, or antioxidants; the effect of an-
tioxidants on HDAC2 gene expression was further analyzed.
Our results show that alcohol upregulates HDAC2 through
mechanisms involving oxidative stress and these effects can
be blocked with pharmacological inhibition of HDACs.
MATERIALS AND METHODS
SK-N-MC Cell Culture
The neuronal cell line, SK-N-MC, was purchased from ATCC
(catalog # HTB-10; Manassas, VA) and cultured in Eagle’s minimum
essential medium (MEM) (catalog # 30-2003) supplemented with
fetal bovine serum to a final concentration of 10% (catalog # 30-
2020) and 1% antibiotic⁄antimycotic solution (Sigma-Aldrich, St.
Louis, MO). For all the gene and protein experiments, SK-N-MC
were cultured at a concentration of 5 · 105cells⁄ml in 6-well plates
overnight to allow them to reach at least 60% confluency before any
EtOH and TSA Treatment of SK-N-MC
After incubating the cells overnight and allowing them to reach
60% confluency, SK-N-MC cells were treated with 0.05%
(?10 mM), 0.1% (?20 mM), and 0.2% (?40 mM) EtOH (catalog #
E7023). For the TSA experiments, after reaching confluency, the cells
were pretreated for 2 hours with 50 to 250 nM TSA and then treated
with 20 mM EtOH for 24 and 48 hours. Based on dose–response
results (data not shown), the lower concentration of TSA (50 nM)
was used for subsequent studies. The EtOH and TSA (catalog #
T1952) used in the present experiments were obtained from Sigma-
Aldrich. The medium was changed and reagents were replenished
every 24 hours. At these concentrations, alcohol and TSA did not
affect the viability of the cells as tested by MTT assay (data not
RNA Extraction and Quantitative Real-Time PCR
SK-N-MC cells were harvested at 24 and 48 hours, and the RNA
was extracted from the cell pellets using the RNAeasy mini kit from
Qiagen (Valencia, CA). Equal quantities of RNA (1 lg) from all the
samples were reversed transcribed using the high-capacity cDNA
reverse transcription kit from Applied Biosystems (Foster City, CA)
to perform qRT-PCR using the Taqman gene expression assays
(Applied Biosystems) for the expression of HDAC2 (Assay ID
Hs00231032_m1) gene. GAPDH (Hs99999905_m1) and 18S rRNA
(catalog # 4333760F) were used as endogenous controls. Relative
abundance of each mRNA species was assessed using the Brilliant
qRT-PCR master mix from Stratagene (Cedar Creek, TX) as previ-
ously described by us (Gandhi et al., 2010). All data were controlled
for quantity of RNA input by performing measurements on 2 endo-
genous reference genes GAPDH and 18S rRNA. In addition, results
on RNA from treated samples were normalized to results obtained
on RNA from control, untreated samples.
Intracellular HDAC2 Analysis by Flow Cytometry
To assess the levels of HDAC2 protein in SK-N-MC, the cells
were treated with ?20 mM EtOH, or 50 nM TSA, or pretreated
with TSA (50 nM) 2 hours prior to EtOH treatment. The cells were
then harvested at 48 hours after EtOH treatment and counted;
equal amounts of cells (1 · 106) were aliquoted in 12 · 75 mm poly-
styrene falcon tubes, catalog # 352058 (BD Biosciences, San Jose,
CA), blocked with human serum and normal goat serum (Chem-
icon International, Temecula, CA), and fixed and permeabilized
with Cytofix⁄Cytoperm solution (BD Bioscience). The HDAC2
protein was detected with the primary monoclonal antibody, rabbit
anti-HDAC2 (Millipore, Temecula, CA) and secondary antibody,
FITC-conjugated goat anti-rabbit IgG antibody (Millipore). Cells
were acquired on a Beckman Coulter instrument (Beckman Coulter,
Inc., Brea, CA) and analyzed with WinMDI (Windows Multiple
Document Interface for Flow Cytometry) 2.5 software (The Scripps
Research Institute, La Jolla, CA). A total of 10,000 events were col-
lected for each sample. Cells were gated based on unlabeled and sec-
ondary antibody controls. Cells positive for specific protein are
shown in the histograms with shifted mean fluorescence intensity
compared to controls.
HDAC2 Protein Levels by Western Blot Analysis
For the HDAC2 Western blot analysis, the cells were treated for
48 hours with ?20 mM EtOH, or 50 nM TSA, or pretreated with
TSA 2 hours prior to EtOH treatment; the cells were harvested and
the cell lysates were prepared in protein extraction reagent (Pierce
Biotechnology, Rockford, IL) containing protease inhibitor (Pierce
Biotechnology) following manufacture’s recommendations. The pro-
tein levels were quantified using the protein assay reagent (Bio-Rad
Laboratories, Hercules, CA). Equal quantities of protein (10 lg)
were denatured and subjected to SDS-PAGE and transferred into a
nitrocellulose membrane (Bio-Rad Laboratories), blocked with 10%
nonfat dry milk, washed with tris-buffered saline-tween 20, and incu-
bated overnight with primary antibodies, rabbit anti-HDAC2
(Millipore). After overnight incubation, the membranes were washed
and incubated for 1 hour with secondary antibody, horseradish
EFFECTS OF ALCOHOL ON HDAC2 AND THE NEUROPROTECTIVE ROLE OF TSA
peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Milli-
pore). The blot was developed using the super signal west pico chemi-
luminescent substrate (Pierce Biotechnology).
ROS levels in SK-N-MC were detected using dichlorofluorescein
diacetate assay (DCF-DA; Molecular Probes, Eugene, OR). Cells
were cultured in 96-well plates (100,000 cells⁄well) overnight to allow
60% confluency. The next day, the cells were washed and pretreated
with antioxidants, catalase (0.001 mg) or uric acid (50 lM), or TSA
for 2 hours. Next, the cells were treated with DCF-DA (100 lM) for
30 minutes at 37?C and then treated with EtOH (0.1 to 0.2%) or
H2O2(50 lM) for 2 hours, and finally read in a BioTek Synergy HT
microplate reader (excitation 485 nm and emission 528 nm; BioTek,
Winooski, VT). Proper controls with cells treated with antioxidants
or H2O2alone were included. Furthermore, fluorescence was visual-
ized in an Olympus IX51 microscope (Olympus America Inc., Center
Valley, PA) and image captured and analyzed with the Qimaging
camera and software (QImaging, British Columbia, Canada).
All experiments were performed at least 3 times in triplicates and
the values obtained were averaged. Data are represented as the
mean ± SE. Comparisons between 2 groups were performed using
the Student’s paired t-test. Differences were considered significant at
p £ 0.05.
Alcohol Induces HDAC2 Gene Expression
Data presented in Fig. 1 show the kinetic as well as the
dose–response (0.05 to 0.2%) effects of alcohol on HDAC2
in SK-N-MC cells at 4 to 48 hours. There is a significant
dose-dependent increase in HDAC2 gene at 0.1 and 0.2%
EtOH concentrations compared to the untreated controls.
Because there were no significant differences observed
between time points, the 24-hour treatment period and the
0.1% EtOH concentration were selected for subsequent
experiments. The EtOH concentrations (0.05 to 0.2%) used
are nontoxic and do not affect cell viability as demonstrated
by MTT cell viability assay and trypan blue cell count (data
TSA Inhibits HDAC2 Gene Expression
TSA is a widely used HDI known to selectively inhibit the
class I and II mammalian HDAC families of enzymes; there-
fore, in this set of experiments, we used it to inhibit the
alcohol-mediated upregulation of HDAC2. Data in Fig. 2
show a significant inhibition of HDAC2 gene expression in
SK-N-MC cells by TSA (50 nM) treatment prior to EtOH
(?20 mM) treatment (24 hours).
TSA Inhibits HDAC2 Protein Levels
Besides gene expression studies, we also determined pro-
tein levels using Western blot and flow cytometry. SK-N-
MC cells were treated with EtOH (20 mM), TSA (50 nM),
or pretreated for 2 hours with TSA and then treated with
EtOH for 48 hours. The cell lysates or the cell pellets were
analyzed using Western blot (Fig. 3A) and flow cytometry
(Fig. 3B), respectively. Results from Fig. 3A and 3B show
an increase in protein levels of HDAC2 after treatment
with EtOH, and this effect is inhibited by TSA. These
results are consistent with the gene expression results pre-
sented in Fig. 2.
TSA Inhibits Alcohol-Induced ROS Production
Previous studies have reported that EtOH induces ROS
production in human primary neurons leading to oxidative
stress and neuronal injury (Haorah et al., 2008). However,
the effects of TSA on ROS production and the ability of TSA
to prevent alcohol-induced ROS have not been reported yet.
In this set of experiments, we tested the effect of TSA on
ControlEtOH 0.05%EtOH 0.1% EtOH 0.2%
Transcript Accumulation Index (TAI)
Fig. 1. Alcohol induces HDAC2 gene expression. SK-N-MC (5 · 105cells⁄ml) were incubated in 6-well plates overnight to allow attachment and 60%
confluency. They were treated with ethanol (EtOH) (0.05 to 0.2%) for 4 to 48 hours. After incubation, the cells were harvested and RNA was extracted and
reverse transcribed followed by qRT-PCR for HDAC2 and endogenous GAPDH and 18S rRNA gene expression. Data are expressed as mean ± SEM of
TAI values of 3 independent experiments. A value of p £ 0.05 was indicative of significance.
AGUDELO ET AL.
alcohol-induced production of ROS. TSA was shown to
decrease the levels of ROS to levels similar to those shown
by antioxidants, catalase, and uric acid. The fluorescence
was measured by a fluorescence plate reader (Fig. 4) and
results were expressed as mean relative fluorescence units.
Fluorescence images were captured with an Olympus IX51
microscope (Fig. 5). According to our results, TSA is acting
as an antioxidant by inhibiting ROS production.
ControlTSAEtOH TSA + EtOH
Transcript Accumulation Index (TAI)
Fig. 2. Alcohol induces HDAC2 gene and this effect is inhibited by Trichostatin A (TSA). After reaching confluency, SK-N-MC were pretreated with TSA
(50 nM) for 2 hours and then treated with ethanol (EtOH) (0.1%) for 24 hours. After incubation, cells were harvested and RNA was extracted and reverse
transcribed followed by qRT-PCR for HDAC2 and housekeeping GAPDH and 18sRNA gene expression. EtOH significantly enhanced HDAC2, while
TSA + EtOH treatment significantly inhibited HDAC2 gene. Data are expressed as mean ± SEM of TAI values of at least 3 independent experiments. # rep-
resents significance compared to control. * represents significance compared to EtOH treatment.
Fig. 3. Alcohol induces HDAC2 protein and this effect is inhibited by Trichostatin A (TSA). After reaching confluency, SK-N-MC were preincubated with
TSA for 2 hours and then treated with ethanol (EtOH) (0.1%) for 48 hours. In (A), 10 lg of protein was analyzed using Western blot with primary anti-
HDAC2 and secondary anti-IgG-HRP antibodies. GAPDH was used as a loading control. Data presented show a representative blot indicating modulation
of HDAC2 protein expression and a bar graph representing the mean ± SE of % densitometry values of HDAC2 protein levels (% control) of 3 independent
experiments. # represents significance compared to control. * represents significance compared to EtOH treatment. For the flow cytometry experiments,
1 · 106cells were fixed and permeabilized prior to intracellular staining with primary anti-HDAC2 and secondary anti-IgG-FITC antibody. Data presented in
(B) show a representative histogram overlay of the gated cells. The bar graph represents the mean ± SE of % of gated cells expressing HDAC2, and
10,000 events were analyzed per sample. The gray and black histograms represent the unlabeled and secondary antibody controls, respectively; the green
histogram is the untreated control (?52%), blue represents EtOH (?69%), purple represents TSA (?45%), and orange represents TSA + EtOH (?49%)-
treated cells. Data are representative of 3 independent experiments.
EFFECTS OF ALCOHOL ON HDAC2 AND THE NEUROPROTECTIVE ROLE OF TSA
Catalase TSA EtOHEtOH + UAEtOH +
TSA + EtOH H2O2
ROS Production (RFU)
Fig. 4. Alcohol induces reactive oxygen species (ROS) production and this effect is inhibited by antioxidants and Trichostatin A (TSA). SK-N-MC
(1 · 105cells) were incubated in 96-well plates overnight. Then, the cells were preincubated with TSA or antioxidants: uric acid (50 lM) or catalase
(0.001 mg) for 2 hours, DCF-DA (100 lM) for 1 hour and then treated with ethanol (EtOH) or H2O2(50 lM). Levels of ROS were measured with the Biotek
Synergy HT microplate reader. The fluorescence was detected at 485 excitation and at 528 emission spectra. Data are expressed as mean ± SE of relative
fluorescence units values of 6 independent experiments. A value of p £ 0.05 was indicative of significance. # represents significance compared to control.
* represents significance compared to EtOH treatment.
Fig. 5. Alcohol-induced reactive oxygen species (ROS) production as measured by fluorescence microscopy. SK-N-MC (1 · 105cells) were incubated
in 96-well plates overnight. Then, the cells were preincubated with Trichostatin A (TSA) or antioxidants, uric acid (50 lM) or catalase (0.001 mg), for 2 hours,
DCF-DA (100 lM) for 1 hour, and then treated with ethanol (EtOH) (0.1 to 0.2%) or H2O2(50 lM), which was used as a positive control. ROS were
observed using fluorescence microcopy. Fluorescence images were captured with an Olympus IX51 microscope. Images are representative of 3 indepen-
AGUDELO ET AL.
Treatment with Antioxidants Inhibits HDAC2 Gene
Our data above showed that alcohol is upregulating
HDAC2 in a dose-dependent manner, inducing oxidative
stress by an observed increase in ROS production, and these
effects were blocked by pharmacological inhibition of
HDAC2. TSA is acting similar to the antioxidants, catalase
and uric acid, by inhibiting ROS production (Figs. 4 and 5).
Therefore, in this set of experiments, we tested the effects of
these antioxidants on HDAC2 gene expression to further con-
firm the involvement of oxidative stress on the alcohol-
induced effect on HDAC2. Results in Fig. 6 show there is a
significant inhibition of HDAC2 gene in the antioxidant pre-
treated cells (p = 0.01) compared to the alcohol-treated cells.
Alcohol acts through numerous pathways to affect the
brain and other organs leading to the development of neu-
rodegeneration. There is no single process that can account
for all the effects of alcohol on an organism; instead, many
mechanisms act together to reflect the actions of alcohol.
Recently, epigenetic mechanisms involving HDACs and their
inhibitors have been the main focus of brain research. For
instance, HDIs have been shown to have antiinflammatory
and neuroprotective effects in the brain (Kim et al., 2007).
The neuroprotective role of HDIs seems to extend to diseases
that share mechanisms of oxidative stress, inflammation, and
neuronal cell apoptosis (Gray and Dangond, 2006). There-
fore, to elucidate the mechanisms of alcohol and eventually
identify genes that will have novel therapeutic relevance, we
analyzed the effects of acute alcohol treatment on the
HDAC2 gene and the role of the HDI, TSA, on EtOH-
induced effects. It is evident from our kinetic studies that
acute alcohol treatment enhanced HDAC2 expression in a
dose-dependent manner (Fig. 1); TSA inhibited HDAC2 gene
(Fig. 2) and protein (Fig. 3) expression.
One factor that has been suggested to play a central role in
many pathways of alcohol-induced damage is the excessive
generation of free radicals, which can result in oxidative
stress. Different mechanisms have been suggested for alcohol
toxicity including an increase in oxidative stress, but the asso-
ciation between oxidative stress in the brain and EtOH-
induced effects on HDACs is poorly understood. Interest-
ingly, HDIs have been shown to protect cortical neurons from
oxidative stress-induced cell death (Ryu et al., 2003), and they
have been found to be neuroprotective in cellular and animal
models of acute and chronic neurodegenerative diseases such
as Alzheimer’s (Alan et al., 2009). Furthermore, HDIs have
also been shown to inhibit the nuclear factor kappa B
(NFjB)-mediated inflammatory responses by a variety of
mechanisms in other neurodegenerative disease models
(Segain et al., 2000). Although it has been shown that HDIs
can protect neuronal cells from oxidative stress-induced cell
death, the studies were carried out in other neurodegenerative
disease models and there is no evidence of TSA’s neuropro-
tective effects in alcohol-induced toxicity. Because quantita-
tive methods of ROS detection help elucidate the mechanisms
of alcohol-induced neurodegeneration, ROS levels were mea-
sured after EtOH and TSA treatments. Because alcohol is
inducing oxidative stress as shown by an increase in ROS pro-
duction and TSA has the ability to prevent ROS (Figs. 4 and
5), it is possible that the molecular basis of the effects
observed on HDACs after alcohol treatment may be medi-
ated via oxidative stress.
In the current study, we are the first ones to show that TSA
has neuroprotective properties and reduces EtOH-induced
oxidative stress as shown by a decrease in ROS production
(Figs. 4 and 5). The oxidative stress effects caused by alcohol
ControlTSA Uric Acid
CatalaseEtOH TSA + EtOH UA + EtOH Catalase +
Transcript Accumulation Index (TAI)
Fig. 6. Alcohol induces HDAC2 gene expression and this effect is inhibited by antioxidants. After reaching confluency, SK-N-MC were pretreated with
Trichostatin A (TSA) (50 nM) for 2 hours and then treated with ethanol (EtOH) (0.1%) for 24 hours. After incubation, cells were harvested and RNA was
extracted and reverse transcribed followed by qRT-PCR for HDAC2 and endogenous GAPDH and 18S rRNA gene expression. Data are expressed as
mean ± SEM of TAI values of 3 independent experiments. # represents significance compared to control. * represents significance compared to EtOH
EFFECTS OF ALCOHOL ON HDAC2 AND THE NEUROPROTECTIVE ROLE OF TSA
were observed at early time points as shown by an increase in
ROS after 2 hours of treatment and may subsequently result
in the late onset effects observed in reference to HDAC2 gene
and protein induced by EtOH. These results provide evidence
for a possible mechanistic role of oxidative stress in the alco-
hol-induced upregulation of HDAC2. To further confirm the
involvement of oxidative stress on the alcohol-induced effect
on HDACs, we tested the effects of antioxidants, uric acid
and catalase, on HDAC2 gene expression. Inhibition of
HDAC2 gene was evident when the cells were treated with an-
tioxidants prior to EtOH treatment. In summary, the results
presented in this study show for the first time a crucial
involvement of HDAC2 in alcohol effects and demonstrate
some of the novel neuroprotective properties of TSA for its
use in AUD.
This research was supported by grants from the National
Institutes of Health (NIH): R37DA025576, R01DA012366,
Alan PK, Yihua C, Tapadar S, Nancy EL, Peter MB, Zhenyu Z, Melissa
ADA, Weng-Long W, Yong S, Brett L (2009) Searching for disease
modifiers-PKC activation and HDAC inhibition-a dual drug approach to
Alzheimer’s disease that decreases Abeta production while blocking oxida-
tive stress. ChemMedChem 4:1095–1105.
Bai S, Ghoshal K, Datta J, Majumder S, Yoon SO, Jacob ST (2005) DNA
methyltransferase 3b regulates nerve growth factor-induced differentiation
of PC12 cells by recruiting histone deacetylase 2. Mol Cell Biol 25:751–766.
Bannister A (2010) The role of epigenetics in cancer. Key epigenetic processes
& links to cancer by Dr. Andy Bannister (Cambridge University). Available
pid=10628#affil. Accessed November 9, 2010.
D’Mello SR (2009) Histone deacetylases as targets for the treatment of human
neurodegenerative diseases. Drug News Perspect 22:513–524.
Gandhi N, Saiyed ZM, Napuri J, Samikkannu T, Reddy PVB, Agudelo M,
Khatavkar P, Saxena SK, Nair MPN (2010) Interactive role of human
immunodeficiency virus type 1 (HIV-1) clade-specific Tat protein and
cocaine in blood-brain barrier dysfunction: implications for HIV-1 associ-
ated neurocognitive disorder. J Neurovirol 16:294–305.
Gray S, Dangond F (2006) Rationale for the use of histone deacetylase inhibi-
tors as a dual therapeutic modality in multiple sclerosis. Epigenetics 1:67–
Haorah J, Ramirez SH, Floreani N, Gorantla S, Morsey B, Persidsky Y
(2008) Mechanism of alcohol-induced oxidative stress and neuronal injury.
Free Radic Biol Med 45:1542–1550.
Kim HJ, Rowe M, Ren M, Hong J-S, Chen P-S, Chuang D-M (2007) Histone
deacetylase inhibitors exhibit anti-inflammatory and neuroprotective effects
in a rat permanent ischemic model of stroke: multiple mechanisms of action.
J Pharmacol Exp Ther 321:892–901.
Lin Y-C, Flock KE, Cook RJ, Hunkele AJ, Loh HH, Ko JL (2008) Effects of
trichostatin A on neuronal mu-opioid receptor gene expression. Brain Res
Min-Hao K, Allis CD (1998) Roles of histone acetyltransferases and deacety-
lases in gene regulation. Bioessays 20:615–626.
NIAAA (2009) National institute on alcohol abuse and alcoholism: five year
strategic plan, FY07-11. Alcohol Across the Lifespan Available at: http://
htm. Accessed November 9, 2010.
Peterson CL, Laniel M (2004) Histones and histone modifications. Curr Biol
Romieu P, Host L, Gobaille S, Sandner G, Aunis D, Zwiller J (2008) Histone
deacetylase inhibitors decrease cocaine but not sucrose self-administration
in rats. J Neurosci 28:9342–9348.
Ryu H, Lee J, Olofsson BA, Mwidau A, Deodoglu A, Escudero M, Fleming-
ton E, Azizkhan-Clifford J, Ferrante RJ, Ratan RR (2003) Histone deacety-
lase inhibitors prevent oxidative neuronal death independent of expanded
polyglutamine repeats via an Sp1-dependent pathway. Proc Natl Acad Sci
Segain JP, De La Ble ´tie `re DR, Bourreille A, Leray V, Gervois N, Rosales C,
Ferrier L, Bonnet C, Blottia ˜ re HM, Galmiche JP (2000) Butyrate inhibits
inflammatory responses through NFkB inhibition: implications for Crohn’s
disease. Gut 47:397–403.
Shepard B, Tuma P (2009) Alcohol-induced protein hyperacetylation: mecha-
nisms and consequences. World J Gastroenterol 15:1219–1230.
Shukla S, Aroor A (2006) Epigenetic effects of ethanol on liver and gastroin-
testinal injury. World J Gastroenterol 12:5265–5271.
AGUDELO ET AL.