Astrocytes Protect Neurons From
Ethanol-Induced Oxidative Stress
and Apoptotic Death
Lora Talley Watts,1Mary Latha Rathinam,2Steven Schenker,2and
George I. Henderson1,2*
1Department of Pharmacology, The University of Texas Health Science Center, San Antonio, Texas
2Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas
Ethanol induces oxidative stress in cultured fetal rat
cortical neurons and this is followed by apoptotic
death, which can be prevented by normalization of cell
content of reduced glutathione (GSH). Because astro-
cytes can play a central role in maintenance of neuron
GSH homeostasis, the following experiments utilized
cocultures of neonatal rat cortical astrocytes and fetal
cortical neurons to determine if astrocytes could pro-
tect neurons from ethanol-mediated apoptotic death
via this mechanism. In cortical neurons cultured in the
absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6-,
12-, and 24-hr exposures) decreased trypan blue exclu-
sion and the MTT viability measures by up to 45% (P <
0.05), increased levels of reactive oxygen species
(ROS) by up to 81% (P < 0.05), and decreased GSH
within 1 hr of treatment by 49 and 51% for 2.5 and
4 mg/ml, respectively (P < 0.05). This was followed by
increased Annexin V binding and DNA fragmentation by
12 hr of ethanol exposure. Coculturing neurons with
astrocytes prevented GSH depletion by 2.5 mg/ml
ethanol, whereas GSH content was increased over con-
trols in neurons exposed to 4 mg/ml ethanol (by up to
341%; P < 0.05). Ethanol generated increases in neu-
ron ROS and apoptosis; decreases in viability were also
prevented by coculture. Astrocytes were largely insen-
sitive to ethanol, using the same measures. Only expo-
sure to 4.0 mg/ml ethanol decreased GSH content in
astrocytes, concomitant with a 204% increase in GSH
efflux (P < 0.05). These studies illustrate that astrocytes
can protect neurons from ethanol-mediated apoptotic
death and that this may be related to maintenance of
C 2005 Wiley-Liss, Inc.
Key words: oxidative stress; fetal alcohol syndrome;
astrocytes; neurons; ethanol; apoptosis; coculture
Alcohol abuse during pregnancy can have a devas-
tating impact on the developing nervous system. The
fetotoxic responses are characterized by abnormal brain
development, with the cerebellum, hippocampus, and
cerebral cortex being the most affected areas (Barnes and
Walker, 1981; West et al., 1984, 1990; Miller and
Potempa, 1990; Goodlett et al., 1991; Miller and Kuhn,
1995). Several in vivo and in vitro studies have shown
multiple adverse effects of ethanol on the developing
brain’s constituent cells in these areas (West et al., 1984;
Bonthius and West, 1990, 1991; Miller and Kuhn, 1995;
Miller, 1996a,b), including a loss of both neurons and
glia (Miller and Potempa, 1990). Prenatal ethanol expo-
sure decreased hippocampal pyramidal cell populations,
decreased spines on pyramidal cell dendrites, caused
abnormal projections of granule cell axons, and altered
proliferation of precursor cells in the cerebral cortex
(Barnes and Walker, 1981; Miller and al-Rabiai, 1994;
Miller and Kuhn, 1995, Miller, 1996a). Clearly, the tim-
ing and degree of ethanol exposure are critical in deter-
mining regional differences in cell loss (Maier et al.,
1999) as well as those within specific populations of
neurons (Ikonomidou et al., 2000).
Oxidative stress can elicit apoptotic death of neu-
rons (Kruman et al., 1997; Valencia and Moran, 2001)
and reactive oxygen species (ROS) may be the first-stage
initiators that commit neurons to apoptosis or they may
play additive or signaling roles within the subsequent
cascade of apoptosis. We have reported previously that
in utero, ethanol exposure of fetal rat brain activated
components of the intrinsic (mitochondrial) apoptotic
pathway along with increased mitochondrial 4-hydroxy-
Contact grant sponsor: NIH; Contract grant number: F31 AA13675-01;
Contract grant sponsor: NIAAA; Contract grant number: R21 AA013431.
*Correspondence to: George I. Henderson, PhD, 7703 Floyd Curl
Drive, San Antonio, TX 78229. E-mail: email@example.com
Received 26 August 2004; Revised 26 January 2005; Accepted 23
Published online 5 May 2005 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.20502
Journal of Neuroscience Research 80:655–666 (2005)
' 2005 Wiley-Liss, Inc.
nonenal (HNE) content, a toxic product of lipid peroxi-
dation. Additionally, in vitro treatment of mitochondria
with HNE mimicked these ethanol effects by inducing
permeability transition and releasing cytochrome c and
apoptosis inducing factor (AIF) (Ramachandran et al.,
2001). Subsequent studies with primary cultures of fetal
rat cortical neurons illustrated that ethanol can cause
rapid increases of ROS formation followed by enhanced
mitochondrial HNE, release of cytochrome c, activation
of caspase-3, and finally, apoptotic death (Ramachandran
et al., 2003). To substantiate further the role of oxidative
stress, pretreatment of cortical neurons with N-acetylcys-
teine (NAC), a maneuver that increases content of the
antioxidant reduced glutathione (GSH), prevented etha-
nol-induced apoptosis. This causative role of ethanol-
mediated oxidative stress is consistent with other reports
in liver, in mouse neural crest cells, and in postnatal rat
cerebral cortex (Chen et al., 2000; Navasumrit et al.,
2001; Heaton et al., 2003).
In most regions of the central nervous system
(CNS), astrocytes form a nearly continuous membrane
around neuron cell bodies and processes, placing them
in close to large neuronal populations. Neuron–astrocyte
interactions clearly are important factors optimizing neu-
ron function and limiting neuron death from a number
of stressors including excitotoxins and oxidants, such as
glutamate and ROS (Rosenberg and Aizenman, 1989;
Desagher et al., 1996; Wilson, 1997; Ye and Son-
theimer, 1998; Dringen et al., 2000). One intriguing
and important role of astrocytes is the generation and
export of GSH (Sagara et al., 1996). Due to the high
oxygen consumption of the brain and the prevalence of
polyunsaturated fatty acids (avid substrates for lipid oxi-
dation), this organ must be well protected from oxi-
dative damage. An important line of defense is GSH
(Sagara et al., 1993; Monks et al., 1999). Neurons, like
other cells, contain a variety of enzymatic antioxidants as
well as GSH synthesis machinery; however, there is con-
siderable evidence that astrocytes are required to main-
tain optimal thiol status of neurons, which protects these
cells from oxidative damage (Pellmar et al., 1992; Dringen
and Hamprecht, 1998; Dringen et al., 1999a; Iwata-
Ichikawa et al., 1999; Wang and Cynader, 2000). Generally,
astrocytes have more efficient GSH synthesis systems
(Dringen et al., 1999b) and higher levels of GSH than
do neurons (Wilson, 1997). They buttress GSH content
of neurons by the export of GSH into the intracellular
milieu. Although this exported GSH likely plays direct
(Drukarch et al., 1989), it also increases neuronal GSH
synthesis by increasing cellular cysteine levels (Wang and
Cynader 2000) and possibly by supporting the neuronal
uptake of the CysGly dipeptide (Dringen et al., 1999a).
The following studies illustrate that: (1) unlike neu-
rons, astrocytes are resistant to ethanol-mediated oxida-
tive stress and apoptosis; (2) the presence of astrocytes
protects neurons from oxidative stress (ethanol)-induced
apoptosis; and (3) this protective capacity likely occurs
through the efflux of GSH from astrocytes, which can
be subsequently utilized by neurons, thereby accelerating
neuronal GSH synthesis.
MATERIALS AND METHODS
Secondary cortical astrocyte cultures were prepared from
2-day-old Sprague-Dawley rats as described previously by
McCarthy and de Vellis (1980). Brains were removed after
decapitation, the meninges stripped, and forebrain cortices
were collected and dissociated with trypsin and DNase. The
dissociated cells were washed and suspended in a culture
medium consisting of Eagle minimal essential medium (MEM)
with 10% fetal bovine serum (FBS) and 2 mM glutamine and
plated in 75-cm2cell culture flasks. At confluency, astrocytes
were lightly trypsinized and replated onto cell culture inserts
(Fisher-Costar). The astrocytes were used for cocultures 7 days
after replating, at which time they formed a confluent layer
across the surface of the insert.
Primary cortical neuron cultures were prepared from
embryonic Day 16–17 rats as described by Dutton (1990).
The neurons were suspended in glial conditioned medium
(MEM containing 10% horse serum). They were plated into
6- or 12-well culture plates (1.0 ? 106or 2.5 ? 105cells/well,
respectively) coated previously with poly-d-lysine. On the next
day, neurons were treated with mitotic inhibitors to prevent
astrocyte contamination. On the third day, in vitro inserts con-
taining confluent astrocytes were placed in each well containing
neurons. Astrocytes on the inserts faced upwards from the neu-
ron monolayer and compounds extruded from the astrocytes
diffused across a 1-mm space to the underlying neurons.
Ethanol was added to media to yield a concentration of
2.5 or 4.0 mg/ml on the fifth day for neurons and on the
14th day for astrocytes. For ethanol treatment, the incubator
contained a beaker filled with ethanol to maintain the ethanol
concentration in the media (Heitman et al., 1987; Devi et al.,
1993). Ethanol concentration was determined using the alco-
hol dehydrogenase method (Sigma). An initial ethanol con-
centration of 2.5 or 4.0 mg/ml declined to no more than 2.0
or 3.5 mg/ml, respectively, after 24 hr using this method.
Controls were used for each duration of ethanol exposure to
account for variations in baseline measures with time in cul-
Trypan blue exclusion. Cells were cultured as stated
above and treated in the presence or absence of ethanol. Sub-
sequently, cells were harvested by light trypsinization and sus-
pended in defined media. A suspension of 0.2 ml was added
to 0.5 ml trypan blue (0.4%) and 0.3 ml Hanks’ balanced salt
solution (HBSS). The total number of cells and the number
of trypan blue-positive cells was counted using a hemocytom-
eter to determine percentage of viable cells.
MTT assay. Viability was determined using the MTT
kit from Sigma. Cultures were removed from the incubator,
656Watts et al.
washed, and MTT was added in an amount equal to 10% of
the culture medium volume. Cultures were then returned to
the incubator for 2–4 hr. After incubation, the resulting for-
mazan crystals were dissolved by adding an amount of MTT
solubilization solution equal to the original media volume.
This was mixed until all crystals are dissolved. Fluorescence
intensity was then measured at 570 nm, with background
measured at 690 nm.
Flow cytometry with Annexin V. Annexin V bind-
ing was used as a measure of early apoptotic cell death. After
treatment, cells (floating and adherent) were harvested and
stained with Annexin V–fluorescein isothiocyanate (Annexin
V–FITC; 1 mg/ml cell suspension; Molecular Probes) for
15 min in the dark in phosphate-buffered saline (PBS) con-
taining 1% bovine serum albumin (BSA). Cell suspensions
were adjusted to 1 ? 106cells/ml. Acquisition and analysis
were carried out on a fluorescence-activated cell sorting
(FACS) flow cytometer (argon laser; excitation 488 nm).
DNA fragmentation. A cell death detection ELISA-
plus kit from Roche Diagnostics (Mannheim, Germany) was
used to quantitate DNA fragmentation as a late marker for
apoptosis. After treatment, 20 ml of cell lysate was added to a
streptavidin-coated multiwell plate. To each well, 80 ml of
immunoreagent containing a mixture of monoclonal antibod-
ies (antihistone-biotin and anti-DNA-POD) was added. The
plate was incubated for 2 hr at room temperature in a plate
shaker at 200 rpm. After incubation, each well was washed
thrice with incubation buffer, and then 2,20axino-di-[3-ethyl-
benzthiazoline sulfonate] diammonium salt crystals (ABTS)
was added and the mixture was incubated for 10 min on the
plate shaker at 200 rpm. Absorbance was measured at 405 nm
against ABTS solution.
Measurement of ROS with 2,70-Dichlorofluorescein
Aliquots of cells were removed and analyzed for the
generation of ROS using the fluorescent probe, 2,70-dichloro-
fluorescein diacetate (DCF-DA) as described previously by
Jung et al. (2001). Cells were harvested and washed with PBS
and resuspended at 1 ? 106cells/ml. DCF-DA was added to
a final concentration of 20 mM and incubated for 30 min at
378C. After incubation, the cells were washed with PBS and
resuspended. ROS generation was measured by fluorescence
intensity (FL-1; 530 nm) of 10,000 cells with a FACS flow
Measurement of Reduced Glutathione
Cells were washed with PBS, harvested, and resus-
pended in PBS. Acivicin (0.5 mM) was added to prevent deg-
radation of GSH, and monochlorobimane (100 mM) was
added to each sample. The samples were incubated for
10 min at 378C, and then 0.02% Triton X-100 was added to
lyse the cell membrane. Cells were centrifuged for 6 min at
15,000 rpm ? g. Fluorescence was measured on a fluorescent
plate reader (Perkin-Elmer HTS 7000 Bio Assay Reader; exci-
tation/emission 400/480 nm).
Measurement of DNA Content
DNA content was measured using the TACS Hoechst
Cell Proliferation Assay from Trevigen, Inc. (Gaithersburg,
MD) as per the manufacturer’s instructions. Briefly, after treat-
ment of cells, the cells were washed once in serum-free
medium. Cells were scraped and collected by centrifugation,
resuspended in PBS, and then 150 ml of cell suspension was
removed, placed in a 96-well plate, and incubated with 50 ml
diluted H33342 dye solution for 1 hr at 378C. Fluorescence
was recorded using 355 nm excitation filter and a 460 nm
emission filter. DNA content was determined from standard
reference curve using genomic DNA.
Measurement of Glutathione Efflux
Astrocytes were treated in the presence or absence of
ethanol for 24 hr. Cells were washed with Krebs-Henseleit
buffer supplemented with HEPES and incubated in Krebs and
ethanol for 1 hr (0.5 mM acivicin). Aliquots were removed
for GSH determination using monochlorobimane (100 mM
for 10 min at 378C). Mean fluorescence of the monochlorobi-
mane–GSH complex was measured (excitation/emission 400/
480 nm) using a fluorescence plate reader.
Two-way analysis of variance (ANOVA) was used to
analyze the statistical significance of the data with time and
dose of ethanol being independent variables. Significant differ-
ences between control and ethanol treatment groups were cal-
culated using the Student-Newman-Keuls method of pairwise
multiple comparisons. P < 0.05 was considered statistically
Ethanol Alters Viability of Neurons and Astrocytes
Using Trypan Blue Exclusion
The experiments represented in Figure 1 and 2
illustrate effects of exposure of cortical neurons and cort-
ical astrocytes to ethanol on two standard cell viability
(MTT) and one that gauges plasma membrane perme-
ability (trypan blue exclusion). Controls were included
for each ethanol exposure period and ethanol treatment
values were compared statistically to their corresponding
time-dependent controls. Additionally, duration of cul-
ture effects were assessed by comparing control values
for the various time points to the ‘‘0’’-time controls. For
neurons, in the absence of ethanol (controls), there was
a downward trend in trypan blue exclusion with time in
culture, to 92, 90, and 85% at 6, 12, and 24 hr, respec-
tively (Fig. 1); however, only the 24-hr value differed
significantly from the earlier controls (P < 0.05). Expo-
sure to 2.5 mg/ml ethanol decreased trypan blue exclu-
sion to 82, 74, and 58% of their corresponding controls
(P < 0.001) after 6, 12, and 24 hr of treatment, respec-
tively. Likewise, 4.0 mg/ml ethanol decreased viability
to 77, 70, 55% of controls (P < 0.001) after 6, 12, and
24 hr of exposure, respectively. There was no statistical
difference between the 2.5 mg/ml and 4.0 mg/ml etha-
Astrocytes Protect Neurons From Apoptosis657
nol groups (P > 0.05). Unlike neurons, trypan blue
exclusion by astrocytes did not decrease with time in
culture in the absence of ethanol (data not shown) nor
did up to a 24-hr exposure to either ethanol level impact
on this measure of astrocyte viability (Fig. 1; P > 0.05).
The MTT assay is used commonly as an estimate
of cell viability based on the activity of mitochondrial
succinate dehydrogenase. As above, control values were
determined at each time interval but these did not
change throughout the 24-hr treatment period (P >
0.05) (Fig. 2). With this measure, neuron ‘‘viability’’
decreased significantly within 6 hr of ethanol exposure
(2.5 mg/ml; 42%; P < 0.001) and remained decreased
with further exposure time (54 and 55% decreases by 12
and 24 hr, respectively; P < 0.001). The 4.0 mg/ml
level of ethanol depressed viability to a similar degree,
by 48, 57, and 57% with 6, 12, and 24 hr of exposure
(P < 0.001). There was no difference in effect between
the two concentrations of ethanol (P > 0.05). Exposure
to 2.5 mg/ml ethanol did not significantly reduce viabil-
ity of astrocytes for up to 24 hr of exposure (Fig. 2);
however, the MTT viability measure was reduced by a
24 hr exposure to 4.0 mg/ml ethanol (28%, P < 0.001).
Astrocyte Protection of Neurons From Decreased
The presence of astrocytes during ethanol treat-
ment mostly prevented the decreases in neuron viability.
Neurons in coculture with astrocytes for 3 days, were
treated with ethanol (2.5 or 4.0 mg/ml) for 6, 12, or
24 hr. In this setting, ethanol did not significantly reduce
neuron trypan blue exclusion with 2.5 or 4.0 mg/ml
treatments (P > 0.05) except for a 15% reduction by
24 hr of exposure to 4.0 mg/ml ethanol (P ¼ 0.04;
Fig. 3A). With the MTT test (Fig. 3B), neither ethanol
exposure regimen had a significant effect throughout the
24-hr exposures (P > 0.05).
Astrocyte Protection Against Oxidative Stress
Prior studies in our laboratory have defined a rapid
ethanol-related increase in ROS in cultured cortical
neurons (Ramachandran et al., 2003). This response is
illustrated in Figure 4A using DCF fluorescence. The
baseline DCF fluorescence in control neurons remained
unchanged (P > 0.05) for the 1- and 2-hr treatment
regimens (data not shown). Ethanol at 2.5 and 4.0 mg/
ml increased levels of ROS by 53 and 71%, respectively,
within 1 hr of exposure (P < 0.001 compared to corre-
sponding controls). The 4.0 mg/ml ethanol treatment
value significantly exceeded that for the 2.5 mg/ml
treatment (P < 0.001). For both ethanol concentrations,
there were further increases to 68 and 81% above con-
trol values within 2 hr (P < 0.001), although the differ-
ence between the two treatments was not significant
(P > 0.05). Figure 4B illustrates the same flow cytome-
try measures of ROS in neurons that were cocultured
Fig. 1. Ethanol effects on neuron and astrocyte viability using trypan
blue exclusion. Fetal cortical neurons were exposed to ethanol (2.5
or 4.0 mg/ml) in an ethanol vapor-saturated incubator for 6, 12, and
24 hr and neonatal cortical astrocytes were exposed to the same con-
centrations of ethanol for 24 hr in an ethanol vapor-saturated incuba-
tor. Fresh ethanol containing media was added every 24 hr to main-
tain constant ethanol concentrations. Control cells from the same iso-
lation were incubated in a separate incubator without ethanol. Cells
were harvested and cell counts were carried out in triplicate using a
hemocytometer and are shown as a mean 6 standard error of the
mean (SEM) for n ¼ 10. *P < 0.05 compared to the corresponding
Fig. 2. Ethanol effects on neuron and astrocyte mitochondrial func-
tion as an estimate of cell viability (MTT assay). Fetal cortical neu-
rons were exposed to ethanol (2.5 or 4.0 mg/ml) in an ethanol
vapor-saturated incubator for 6, 12, and 24 hr neurons and astrocytes
were exposed to ethanol (2.5 or 4.0 mg/ml) for 24 hr. Fresh media
containing ethanol was added every 24 hr to maintain constant etha-
nol concentrations. Control cells were incubated in a separate incu-
bator without ethanol. MTT was added at the end of each treatment
and the cells incubated for 2 hr. Data are represented as mean 6
SEM for n ¼ 12. *P < 0.05 compared to corresponding time-
658Watts et al.
with astrocytes. As in the absence of astrocytes, ROS
content of neurons increased initially (126 and 286%; P
< 0.001) within 1 hr of ethanol exposure at 2.5 or 4.0
mg/ml, respectively. This initial increase was greater for
the 4.0 mg/ml ethanol exposure than for the 2.5 mg/ml
treatment (P < 0.001). In the presence of astrocytes,
however, neuron ROS levels returned to control values
within 2 hr of ethanol exposure (P > 0.05). As with the
absence of astrocytes, control neuron ROS estimates did
not differ over the 2-hr treatment period (P > 0.05, data
Astrocytes Protect Neurons From
The experiments represented in Figure 5A and 5B
estimated cell binding of an FITC conjugate of Annexin
V, an early marker of apoptosis that reflects increased
external expression of phosphatidylserine (PS). Annexin V
binding did not change in control cells over 24 hr of treat-
ment (P > 0.05) and the control bars in Figure 5 are the
‘‘0’’-time values. All statistical comparisons with ethanol-
Fig. 3. Astrocytes protect neurons from ethanol-related decreased
viability. These experiments were carried out as those in Figures 1
and 2 except that neurons were cocultured with astrocytes. A: Try-
pan blue exclusion was estimated after 6, 12, or 24 hr of exposure to
2.5 or 4 mg/ml ethanol. B: The MTT assay was carried out on neu-
rons exposed to ethanol (2.5 or 4 mg/ml) for 6, 12, or 24 hr. Expo-
sure to ethanol was in an ethanol vapor-saturated incubator. Control
cells were incubated in a separate incubator without ethanol. Astro-
cytes were removed after treatment with ethanol and the MTT assay
or trypan blue exclusion were carried out on the neurons alone. Data
are represented as mean 6 SEM for n ¼ 6. *P < 0.05 compared to
the corresponding time-dependent controls.
Fig. 4. Ethanol stimulates reactive oxygen species (ROS) formation
in neurons and coculturing with astrocytes can mitigate this response.
Flow cytometry determinations of 2.70-dichlorofluorescein diacetate
(DCF-DA) fluorescence were used to estimate ROS levels in neu-
rons either cultured alone (A) or cocultured with astrocytes (B). Fetal
cortical neurons were loaded with DCF-DA and treated with ethanol
(2.5 and 4.0 mg/ml) for 1 or 2 hr in the presence or absence of
astrocytes. Nonspecific background fluorescence in cell suspensions
not treated with DCF-DA was subtracted. In the cocultures, astro-
cytes were removed before DCF-DA treatment. ROS generation
from neurons was measured by fluorescence intensity (FL-1; 530nm)
with an FASC flow cytometer. Values are shown as mean 6 SEM
for n ¼ 6. *P < 0.05 compared to corresponding time-dependent
Astrocytes Protect Neurons From Apoptosis659
exposed cells, however, utilized the corresponding time-
dependent controls. Neurons cultured without astrocytes
and exposed to ethanol (2.5 and 4.0 mg/ml) exhibited an
increase in Annexin V binding within 12 hr of ethanol
treatment (Fig. 5A). For both ethanol concentrations (2.5
and 4.0 mg/ml), there was an upward trend in Annexin V
binding within 2 and 6 hr of exposure; however, these val-
ues did not differ significantly from their corresponding
controls (P > 0.05). Further exposure to ethanol increased
Annexin V binding by 146% (P < 0.001) and 269% (P <
0.001) above control with 2.5 mg/ml ethanol for 12 and
24 hr of exposure, respectively. With 4.0 mg/ml ethanol,
Annexin V binding increased 163 and 264% (both P <
0.001) above control values for 12 and 24 hr of exposure,
respectively (Fig. 5A). There was no significant difference
between the two ethanol treatment groups at each time
point (P > 0.05). Coculturing neurons with astrocytes
largely blocked the increased externalization of phosphati-
dylserine caused by ethanol (Fig. 5B). Neurons in
coculture with astrocytes were treated as in the above
experiments and the increase in Annexin V binding seen
in neurons alone was not present at all time points
and concentrations except for the 24-hr exposure to
4.0 mg/ml ethanol. In the latter setting, Annexin V bind-
ing was increased to 104% above control (P < 0.001). This
increase was half that seen without the presence of astro-
DNA fragmentation is an end-point measure of
apoptotic cell death. This was assessed by ELISA using
monoclonal antibodies, antihistone-biotin, and anti-
DNA-POD. Figure 6 illustrates the effect of ethanol (2.5
and 4.0 mg/ml) on DNA fragmentation in neurons
alone and neurons in coculture with astrocytes during
ethanol exposure. There was no time-dependent effect
on DNA fragmentation in control neurons; however,
neurons cultured in the absence of astrocytes (Fig. 6A)
presented significantly increased DNA fragmentation
within 12 hr of exposure to 2.5 mg/ml ethanol (56%;
P < 0.001 compared to the time-dependent control).
This measure was also increased by 24 hr of ethanol
treatment (73%; P < 0.001), but it did not differ signifi-
cantly from the 12-hr value (P > 0.05). The 12- and
24-hr treatments with 4.0 mg/ml ethanol increased
DNA fragmentation by 67 and 83%, respectively (P <
0.001). There was no significant difference at each time
point between the two ethanol doses used (P > 0.05).
Neurons in coculture with astrocytes were likewise
treated for 12 and 24 hr with 2.5 and 4.0 mg/ml ethanol
(Fig. 6B). The increase in DNA fragmentation seen pre-
viously in neurons alone was completely prevented by
the presence of astrocytes (P > 0.05).
Ethanol Alters GSH Homeostasis in Neurons
Prior studies in our laboratory have demonstrated
that ethanol-mediated apoptosis of neurons can be
blocked by enhancing their GSH content (Ramachan-
dran et al., 2003). Additionally, others have documented
that astrocyte-mediated control of neuron GSH homeo-
stasis may play a protective role against nitric oxide dam-
age to neurons (Gegg et al., 2003). Because this may
likewise be the mechanism underlying astrocyte protec-
tion of neurons from ethanol-related apoptosis, the fol-
lowing experiments determined the ability of these glia
to maintain neuron GSH homeostasis during ethanol
challenge and their ability to export GSH. Neurons were
exposed to ethanol (2.5 and 4.0 mg/ml) for 1, 2, 6, 12,
or 24 hr in the presence or absence of astrocytes.
Figure 7A illustrates ethanol-related changes in GSH
content of neurons cultured in the absence of astrocytes.
GSH levels in controls did not differ significantly
Fig. 5. Astrocytes protect neurons from increased Annexin V bind-
ing. Neurons were exposed to ethanol (2.5 or 4.0 mg/ml) for 2, 6,
12, or 24 hr in the absence (A) or presence (B) of astrocytes. After
treatment, neurons were harvested, Annexin V was added to cell sus-
pensions, and the resuspended cells were immediately read by flow
cytometry. The values are represented as percent of Annexin V-posi-
tive cells shown as the mean 6 SEM for six experiments. Although
not illustrated, controls were done for all time points and these did
not change over the 24-hr treatment period. *P < 0.05 compared to
the corresponding time-dependent control.
660 Watts et al.
throughout the 24-hr treatment period (P > 0.05); how-
ever, 1-hr ethanol exposure at 2.5 and 4.0 mg/ml
reduced GSH content of neurons compared to that of
controls by 49% (P ¼ 0.043) and 51% (P ¼ 0.021),
respectively. A 2-hr ethanol treatment also decreased
GSH content by 73 and 83% of control values (P <
0.001). Between 2 and 6 hr of ethanol exposure, GSH
content rebounded to normal levels (P > 0.05) and this
remained unchanged for the remainder of a 24-hr etha-
nol exposure (Fig. 7A).
The presence of astrocytes in coculture with neu-
rons not only prevented the early reduction in GSH con-
tent seen previously, but it also produced some striking
increases in neuron GSH after treatment with 4 mg/ml
ethanol (Fig. 7B). A 1-hr exposure to 2.5 mg/ml ethanol
in the presence of astrocytes had no significant effect on
neuron GSH content (P > 0.05), whereas treatment with
4 mg/ml increased neuron GSH by 341% of control
(P ¼ 0.005). A similar pattern of normalized GSH
occurred with the 2.5 mg/ml ethanol level throughout
12 hr of treatment (P > 0.05 compared to the corre-
sponding controls) and GSH was elevated in neurons
exposed to 4 mg/ml (P < 0.05). At 24 hr of exposure,
neuron GSH content was increased by 55% (P ¼ 0.008)
and 75% (P ¼ 0.001) for 2.5 mg/ml and 4 mg/ml
Fig. 7. Ethanol decreases whole-cell GSH content in neurons and the
presence of astrocytes prevents this response. Cells were exposed to
ethanol (2.5 and 4.0 mg/ml) for 1–24 hr in the absence (A) or presence
(B) of astrocytes. After treatment, neurons were harvested and cell sus-
pensions were incubated with monochlorobimane (100 mmol for 1 min
at 378C). Acivicin (0.5 mM) was added to prevent degradation of GSH
by g-glutamyl transpeptidase. Mean fluorescence of the monochlorobi-
mane-GSH complex was measured (excitation/emission 400/480 nm)
using a fluorescence plate reader. Values are shown as mean 6 SEM for
n ¼ 10. *P < 0.05 compared to the corresponding time-dependent
control; **P < 0.05 for a control value significantly different from con-
trol values at different treatment periods.
Fig. 6. Astrocytes protect neurons from increased DNA fragmenta-
tion. Neurons were exposed to ethanol (2.5 and 4.0 mg/ml) in the
absence (A) or presence (B) of astrocytes for 12 or 24 hr. The cells
tested were those that remained attached to the matrix. Control val-
ues did not change throughout the treatment period and the control
value in each figure is a composite of all controls. Values are shown
as the mean 6 SEM for n ¼ 10. *P < 0.05 compared to the corre-
sponding time-dependent control.
Astrocytes Protect Neurons From Apoptosis661
ethanol, respectively. There was no significant difference
in GSH content of control neurons in the presence or
absence of astrocytes until 24 hr of treatment, at which
time GSH content was five times that of the 1-hr control
(P ¼ 0.002). Control values for GSH in neurons cultured
in the absence of astrocytes throughout all time points
typically ranged between 2.5 and 3.7 mmol/mg DNA.
Initial control values of GSH in cocultured neurons at
time 0 through 12 hr was approximately 3 mmol/mg
DNA; 24 hr of coculture significantly increased the GSH
content of the control neuron to 8.9 mmol/mg DNA (P
< 0.05; Fig. 7B).
Astrocytes efflux GSH, which is hydrolyzed by g-
glutamyl transpeptidase to the dipeptide CysGly, the lat-
ter compound being subsequently cleaved to cysteine
and glycine at the neuron surface (Sagara et al., 1993;
Dringen et al., 1999a; 2001; see Fig. 9 for a schematic).
The increased availability of cysteine to the neuron elic-
its increased synthesis of GSH, as availability of cysteine
is a critical control point in the synthesis of GSH (Kra-
nich et al., 1998; Wang and Cynader, 2000). Figure 8
illustrates the occurrence of GSH efflux from astrocyte
and an upregulation of this by a 24-hr exposure to
4 mg/ml ethanol. In our system, baseline efflux of GSH
by astrocytes (mean 6 standard error of the mean) was
18.04 6 2.19 mmol/min/mg DNA. Exposure to
4.0 mg/ml ethanol for 24 hr increased the rate of efflux
from the astrocyte into the media by 204% (P < 0.05;
Fig. 8). Although there was an upward trend in GSH
efflux with the 2.5 mg/ml ethanol treatment, this did
not reach statistical significance.
There are a variety of potential mechanisms under-
lying the neurotoxic effects of ethanol on the developing
CNS (for reviews see Abel and Hannigan, 1995; Abel,
1998; Reynolds and Brien, 1995; West et al., 1994).
One that we have addressed frequently is apoptotic loss
of neurons secondary to ethanol-induced oxidative stress.
ROS can induce apoptosis (Beaver and Waring, 1995;
Slater et al., 1996; for review see Buttke and Sandstrom,
1994) and may mediate apoptotic cell death in neurode-
generative diseases such as Alzheimer’s and Parkinson’s
diseases (Beal, 1995; Sano et al., 1997). We have shown
that oxidative stress precedes processes of mitochondri-
ally mediated apoptotic death of cultured fetal cortical
neurons exposed to ethanol and that this can be reversed
by augmenting glutathione levels (Ramachandran et al.,
2003). Additionally, oxidative stress induced apoptotic
cell death can also occur in vivo after a 2-day binge
alcohol exposure (Ramachandran et al., 2001) and
there is compelling evidence from other laboratories that
ethanol can stimulate apoptotic systems in the intact
cortex (in vivo) and in cultured cortical neurons (Jacobs
and Miller, Mooney and Miller, 2001; 2001; Heaton
et al., 2003). In the in vivo setting, however, neurons
exist in conjunction with glia, and the present studies
demonstrate that the presence of astrocytes can alter
this toxic effect of ethanol on neurons and that one
mechanism may be maintenance of neuron GSH
Vulnerability of Neurons to Ethanol and a
Role for Oxidative Stress
The current studies illustrate that viability of cul-
tured fetal cortical neurons is reduced significantly
within 6 hr of ethanol exposure (2.5 or 4.0 mg/ml) with
increasingly fewer viable cells at 12 and 24 hr (Fig. 1
and 2). This has also been reported by other laboratories
using multiple assays (Luo et al., 1997; Jacobs and Miller,
2001; Mooney and Miller, 2001). In comparison, astro-
cytes are far more resistant to the toxic effects of ethanol
as evidenced by trypan blue exclusion and reduction of
formazan crystal formation (MTT assay; Fig. 1 and 2).
Additionally, oxidative stress occurs rapidly in cultured
fetal cortical neurons and precedes apoptotic measure-
ments, Annexin V, and DNA fragmentation (Fig. 4–6),
all of which can be reversed to control levels by aug-
menting glutathione levels with NAC (Ramachandran
et al., 2003). This early onset of increased ROS (within
minutes of ethanol exposure; Ramachandran et al.,
2003) suggests that oxidative stress may play a causal
role in ethanol-mediated apoptosis of these neurons,
which is in agreement with conclusions from several
other laboratories (Cartwright and Smith, 1995; Chen
and Sulik., 1996; Maier et al., 1996; Heaton et al.,
2003) and our past observations (Ramachandran et al.,
Fig. 8. GSH efflux from astrocytes and effects of ethanol on this
process. Astrocytes were exposed to ethanol (2.5 and 4.0 mg/ml) for
24 hr. After treatment, aliquots of media were removed for GSH
efflux determination. These aliquots were incubated with mono-
chlorobimane (100 mM for 10 min at 378C). Acivicin (0.5 mM) was
added to prevent degradation of GSH by g-glutamyl transpeptidase.
Mean fluorescence of the monochlorobimane-glutathione complex
was measured (excitation/emission 400/480 nm) using a fluorescence
plate reader. Values are shown as mean 6 SEM for n ¼ 10. *P <
0.05 compared to control.
662Watts et al.
Astrocytes Protect Neurons From Apoptotic
The presence of astrocytes in culture protected
neurons from ethanol-induced apoptosis. This could
reflect several underlying mechanisms, including neuro-
trophin support (Mattson and Rychlik, 1990; Mattson
et al., 1997, Heaton et al., 2003); however, there is
compelling evidence that antioxidant augmentation is a
key player. Neurons cocultured in the presence of astro-
cytes displayed the same ‘‘reversal’’ phenomenon seen
previously with NAC enhancement of neuron GSH
content, a setting with no alteration in neurotrophin
support (Ramachandran et al., 2003). These cocultured
neurons did not show decreased viability as with etha-
nol-exposed neurons alone, even in the presence of
increasing concentrations of ethanol (up to 4 mg/ml)
and astrocytes provided protection from increased pro-
duction of ROS within 2 hr of ethanol exposure. The
cause of this initial increase in ROS by ethanol remains
to be determined but elegant studies with hepatocytes
suggest that this may be caused by depletion of mito-
chondrial GSH (Garcia-Ruiz et al., 1994). Our experi-
ments suggest that there is a window during which
reversal of this ROS increase can prevent neuron com-
mitment to apoptosis; thus, an ethanol perturbation of
GSH homeostasis may act as both initiating and propa-
gating events. Clearly, if the increase in ROS is reversed
within 2 hr of ethanol exposure, apoptosis can be pre-
vented (Fig. 5 and 6). The increase in ROS in cocul-
tured neurons at the 1-hr point (Fig. 4), even in the
presence of elevated GSH, could reflect a temporary
delay in the available GSH blocking this ethanol-related
event that is generating the enhanced ROS levels. At
this juncture this is speculation and future experiments
will address this mechanism. The delayed prevention of
ROS protection is not seen with NAC pretreatment
(data not shown) and this likely reflects the more com-
plex pathway of neuronal GSH enhancement by astro-
cytes (discussed below).
Astrocyte-Mediated GSH Synthesis in Neurons
The above studies illustrate that the ability of astro-
cytes to maintain neuronal GSH homeostasis in response to
a prooxidant may be an important protective mechanism
against ethanol toxicity. Although glutathione is present in
astrocytes and neurons, astrocytes usually contain higher
concentrations than neurons do, both in vivo and in culture
(Cooper, 1997; Dringen, 2000), and emerging evidence
suggests that astrocytes play a central role in maintaining
neuronal GSH homeostasis (Bolanos et al., 1996; Dringen
Fig. 9. A schematic representation of astrocyte synthesis of glutathione,
its export, extracellular processing, and neuron glutathione synthesis. Glu-
tathione is synthesized from its constituent amino acids by a sequential
two-enzyme system. The initial rate-limiting reaction is catalyzed by
g-glutamylcysteine synthetase (gGC), an energy dependent reaction that
produces g-glutamyl-L-cysteine. The second energy-dependent reaction
is catalyzed by GSH synthetase and the product is glutathione. Astrocytes
efflux glutathione into the extracellular space where it is cleaved by
g-glutamyl transpeptidase (gGT) to the dipeptide CysGly and glutamate.
Most of this seems to occur at the astrocyte surface. The dipeptide is
cleaved further by aminopeptidase N (ApN) to cysteine and glycine on
the surface of the neuron. Cysteine is then transported into neurons, pri-
marily by the sodium-dependent ASC system. The availability of cysteine
within the neuron is an important determinant of glutathione synthesis.
Astrocytes Protect Neurons From Apoptosis663
et al., 1999a). The mechanism by which this occurs is illus-
trated in Figure 9. The first step is efflux of GSH from astro-
cytes by a process that may be carrier mediated (Hirrlinger
et al., 2002), although its release is dependent on the con-
centration within the cell (Sagara et al., 1996). Extruded
GSH is subsequently cleaved to the CysGly dipeptide by
the membrane-bound enzyme g-glutamyl transpeptidase
(Dringen et al., 1999a), which is present on the outer sur-
face of the astrocyte, especially on the endfeet that surround
blood vessels (Cambier et al., 2000). The dipeptide is then
cleaved by aminopeptidase N (ApN), a metalloaminopepti-
dase that possesses cysteinylglycinase activity, thereby gener-
ating Cys (Dringen et al., 2001). Cys is transported into the
neuron by the sodium-dependent alanine serine cysteine
(ASC) system, which is reactive towards neutral amino
acids, where it is a controlling factor in neuronal GSH syn-
thesis (Sagara et al., 1993; for review see Lu, 1999). The
present studies illustrate the ability of this pathway to nor-
malize neuron GSH content in the neuron in response to
ethanol exposure (Fig. 7). Interestingly, 4 mg/ml ethanol
concomitantly increased GSH efflux from the astrocyte but
did not deplete the astrocytes of GSH (data not shown).
This could be an important regulatory process whereby the
astrocyte maintains or enhances neuron antioxidant protec-
tion against ethanol-mediated oxidative stress. The means
by which this occurs is under investigation.
In Vivo Consequences of Alterations in GSH
In vivo, ethanol can induce apoptosis in the de-
veloping brain, even in the presence of astrocytes (Mooney
and Miller, 2001; Ramachandran et al., 2001). Gliogenesis
generally follows neurogenesis, however, and persists long
after neurogenesis has ceased (Jacobsen, 1991). The abun-
dance of astrocytes in the developing brain thus may be an
important determinant of the well-documented effects of
timing and duration of ethanol on neurotoxic responses to
the drug (Dexter et al., 1980; Rosett et al., 1983; Smith
et al., 1986; Halmesmaki et al., 1987; Bonthius et al.,
1988; Coles et al., 1991; Heaton et al., 2003). Likewise,
distribution and variations in GSH homeostasis machinery
within populations of astrocytes could be factors in the
regional sensitivity of populations of neurons to ethanol-
induced apoptotic death (West et al., 1990; Hamre and
West, 1993; Marcussen et al., 1994; Goodlett and Eilers,
1997; Maier et al., 1999). Finally, astrocytes are not imper-
vious to ethanol, a factor illustrated in Figure 2. Clearly,
high concentrations of ethanol are more likely than lower
ones to induce neuron damage and a relevant factor may
be damage to astrocytes. Duration of exposure is also a fac-
tor. Studies are currently under way to determine if there
is a connection between ethanol-related damage to astro-
cytes and their ability to protect neurons from ethanol-
In conclusion, these studies illustrate that: (1) astro-
cytes can protect fetal cortical neurons from accelerated
apoptotic death due to acute ethanol exposure; (2) astro-
cyte viability is not strongly affected by ethanol, possibly
due to high GSH content; and (3) a mechanism underlying
astrocyte protection of neurons from ethanol-induced
apoptosis is maintenance of neuron GSH homeostasis.
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