Dringen et al , 1998

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Naunyn-Schmiedeberg’s Arch Pharmacol (1998) 358:616–622 © Springer-Verlag 1998
Abstract Astroglial cells protect neurons against oxidative
damage. The antioxidant glutathione plays a pivotal role in
the neuroprotective action of astroglial cells which is im-
paired following loss of glutathione. Anethole dithioleth-
ione (ADT), a sulfur-containing compound which is used in
humans as a secretagogue, increases glutathione levels in
cultured astroglial cells under “physiological” conditions
and is thought thereby to protect against oxidative damage.
Presently, we report the effect of ADT (3–100 µM) on glu-
tathione content of and efflux from rat primary astroglia-
rich cultures under “pathological” conditions, i.e., extended
deprivation of glucose and amino acids.
Although cellular viability was not affected significantly,
starvation of these cultures for 24 h in a bicarbonate buffer
lacking glucose and amino acids led to a decrease in glu-
tathione and protein content of approximately 43% and
40%, respectively. Although no effect on the protein loss
occurred, the presence of ADT during starvation counter-
acted the starvation-induced loss of intracellular glu-
tathione in a concentration-dependent way. At a concentra-
tion of 100 µM ADT even a significant increase in astroglial
glutathione content was noted after 24 h of starvation. Alike
intracellular glutathione levels, the amount of glutathione
found in the buffer was elevated substantially if ADT was
present during starvation. This ADT-mediated, apparent in-
crease in glutathione efflux was additive to the stimulatory
effect on extracellular glutathione levels of acivicin (100
µM), an inhibitor of extracellular enzymatic glutathione
breakdown. However, the ADT-induced elevation of both
intra- and extracellular glutathione content during starva-
tion was prevented completely by coincubation with bu-
thionine sulfoximine (10 µM), an inhibitor of glutathione
synthesis.
These results demonstrate that, most likely through stimu-
lation of glutathione synthesis, ADT enables astroglial cells
to maintain higher intra- and extracellular levels of glu-
tathione under adverse conditions. Considering the lowered
glutathione levels in neurodegenerative syndromes, we
conclude that further evaluation of the therapeutic potential
of the compound is warranted.
Key words Anethole dithiolethione · Astroglia · 
Glutathione · Primary cultures · Starvation · Oxidative
stress · Neuroprotection
R. Dringen · B. Hamprecht
Physiologisch-Chemisches Institut der Universität Tübingen, 
Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany
B. Drukarch (
)
) 
Graduate School for Neurosciences Amsterdam, Research Institute
Neurosciences Vrije Universiteit, Department of Neurology, 
van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
e-mail: b.drukarch.neurol@med.vu.nl, Fax: +31-20-4448100
ORIGINAL ARTICLE
Ralf Dringen · Bernd Hamprecht · Benjamin Drukarch
Anethole dithiolethione, a putative neuroprotectant, 
increases intracellular and extracellular glutathione levels 
during starvation of cultured astroglial cells
Received: 2 June 1998 / Accepted: 18 September 1998
Introduction
Increased formation of reactive oxygen species (ROS) and
consequent oxidative stress is thought to be involved in the
loss of neurons occurring in chronic (neuro)degenerative
diseases and ischemic brain injury (for review see Ames et
al. 1993). Astroglial cells, amongst other functions, are well
known for their ability to protect brain neurons from oxida-
tive stress, exerted, e.g., in the form of exogenously applied
hydrogen peroxide (Ben-Yoseph et al. 1996; Drukarch et al.
1997a; Langeveld et al. 1995). This phenomenon has been
linked to the greater scavenging capacity of astrocytes as
compared to neurons (Ben-Yoseph et al. 1996). Glu-
tathione, a tripeptide consisting of a glutamate, a cysteine
and a glycine moiety, is the most abundant cellular free thi-
ol and plays an important role in the inactivation of ROS
(Meister and Anderson 1983). Glutathione is synthesized in
large amounts in astroglia. Moreover, loss of glutathione is
accompanied by an enhanced susceptibility of astrocytes to
oxidative damage and impairment of their neuroprotective
action (Ben-Yoseph et al. 1996; Drukarch et al. 1997a).
Thus, considering the importance of glutathione in astro-
cyte-mediated neuroprotection, it is conceivable that com-
pounds which improve glutathione synthesis and/or restore
glutathione levels in astrocytes under pathological condi-
tions will be of therapeutic benefit in neurodegenerative
processes in which ROS are implicated.
Anethole dithiolethione [5-(p-methoxyphenyl)3H-1,2-
dithiole-3-thione; ADT] belongs to a group of cyclic, sul-
fur-containing compounds collectively known as the 1,2-
dithiole-3-thiones of which some are presently being
evaluated on their potential for application in cancer
chemoprevention schemes (for review see Christen 1995).
ADT has been in clinical use for decades as a choleretic and
sialogogue without any major adverse reactions being not-
ed. Interestingly, this highly lipophilic drug has been shown
to increase cellular glutathione levels both in vivo and in
vitro and to protect against (oxidative) damage incurred by
loss of glutathione (Christen 1995; Davies et al. 1987; Man-
suy et al. 1986; Sen et al. 1996), at least outside the nervous
system. Recently, we demonstrated that ADT, in concentra-
tions in which the compound is active in vivo in rodents
(Dansette et al. 1990), also induces a raise in the glu-
tathione content of cultured astroglial cells (Drukarch et al.
1997b). Our data moreover showed that this ADT-mediated
increase in glutathione levels was abolished completely up-
on concomitant treatment of the astrocytes with the glu-
tathione synthesis inhibitor L-buthionine-S,R-sulfoximine
(BSO; Griffith and Meister 1979), thereby indicating that
ADT exerts its effect through stimulation of glutathione
synthesis (Drukarch et al. 1997b). However, to be of thera-
peutic benefit under pathological conditions, ADT should
be able to stimulate synthesis also under adverse circum-
stances in which glutathione levels are decreasing. Expo-
sure to BSO not only blocks glutathione synthesis but even-
tually, through this mode of action, also results in an
extensive reduction of astroglial glutathione content
(Devesa et al. 1993). Unfortunately, for the investigation of
glutathione synthesis in cultured cells during ongoing loss
of glutathione, incubation with BSO is not useful because
the compound is an irreversible inhibitor of g -glutamyl-
cysteine synthetase, the rate-limiting enzyme in glutathione
formation (Griffith and Meister 1979). Also unsuitable in
this context are SH reagents such as diethyl maleate which,
although effective in lowering astroglial glutathione levels,
are known to react not only with glutathione but also with
the thiol groups of enzymes (O’Connor et al. 1995; Yudkoff
et al. 1990). Therefore, in order to study the effect of ADT
on glutathione loss under “pathological conditions,” we re-
verted to our previously established model in which the glu-
tathione content of rat brain-derived astroglia-rich primary
cultures is reduced by extended deprivation of glucose and
amino acids without affecting cellular viability (Dringen
and Hamprecht 1996).
Materials and methods
Materials. Dulbecco’s Modified Eagle’s Medium (DMEM) was ob-
tained from Gibco (Eggenstein, Germany). Cell culture dishes and 96-
well microtiter plates were from Nunc (Wiesbaden, Germany). Fetal
calf serum, reduced glutathione (GSH), glutathione disulfide (GSSG),
and glutathione disulfide reductase from yeast (EC 1.6.4.2) were pur-
chased from Boehringer Mannheim (Mannheim, Germany). NADPH
was from Applichem (Darmstadt, Germany). Acivicin, bovine serum
albumin, BSO, 5,5’-dithio-bis(2-nitrobenzoic acid; DTNB),and 5-
sulfosalicylic acid were obtained from Sigma (Deisenhofen, Germa-
ny). Sodium pyruvate was purchased from Fluka (Neu-Ulm, Germa-
ny). Penicillin G and streptomycin sulfate were obtained from Serva
(Heidelberg, Germany). All other chemicals, of the highest purity
available, were obtained from Merck (Darmstadt, Germany). ADT
was kindly provided by Solvay-Duphar (Weesp, The Netherlands)
through courtesy of Dr. E. Ronken. Stock solutions of ADT were pre-
pared in dimethyl sulfoxide.
Cell culture. Astroglia-rich primary cultures derived from the brains
of neonatal Wistar rats were prepared and cultivated as described
(Hamprecht and Löffler 1985). In short, following preparation the
cells were seeded in plastic culture dishes (50 mm in diameter) and in-
cubated in DMEM containing 10% fetal calf serum, 20 µg/ml strepto-
mycin sulfate, and 20 u/ml penicillin G. The medium was renewed ev-
ery seventh day. Cultures established under these conditions are
widely used for the analysis of astroglial metabolism (Hamprecht and
Dringen 1995). The experiments presented here were carried out us-
ing 14- to 21-day-old cultures. In this range the results did not depend
on the age of the cultures.
Experimental procedures. After removal of the culture medium the
cells were washed twice with 3 ml of a minimal medium (MM; 44
mM NaHCO3, 110 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 0.8 mM
MgSO4, 0.92 mM NaH2PO4, adjusted with CO2 to pH 7.4) and then
incubated for up to 24 h in 3 ml MM containing ADT (0–100 µM) in
the presence or absence of BSO (10 µM) or acivicin (100 µM) in a
Heraeus cell incubator containing a humidified atmosphere of 10%
CO2/90% air. At the time points indicated the incubation buffer was
collected and the cells washed twice with phosphate-buffered saline
(10 mM potassium phosphate buffer, pH 7.4, containing 150 mM
NaCl) and lysed with 1 ml 1% (w/v) sulfosalicylic acid on ice. An ali-
quote part of the incubation buffer was diluted by the same volume of
1% (w/v) sulfosalicylic acid. Incubation buffer and cell lysate super-
natants were analyzed for their content of glutathione (GSx = amount
of GSH plus twice the amount of GSSG) as described (Dringen and
Hamprecht 1996; Dringen et al. 1997) using a modification (Baker et
al. 1990) of the assay described by Tietze (1969). Briefly, 20 µl of the
incubation buffer samples and 10 µl of the cell lysates were mixed
with 80 µl and 90 µl water, respectively, in a well of a 96-well
microtiter plate. After addition of 100 µl reaction mixture (0.1 M so-
dium phosphate buffer, pH 7.5, containing 1 mM EDTA, 0.3 mM
DTNB, 0.4 mM NADPH and 1 u/ml glutathione disulfide reductase)
the increase in absorbance at 405 nm was detected during 15-s inter-
vals over a range of 2.5 min using a microtiter plate reader (Titertek
Plus MS212; ISN Biomedicals, Meckenheim, Germany). Total glu-
tathione content was evaluated with the aid of the software delivered
with the plate reader, by using a calibration curve established with
standard samples in the range of 0–500 pmol GSx per well. The pro-
tein content of the cultured cells was determined according to the
method of Lowry et al. (1951) using bovine serum albumin as a stan-
dard. All experiments complied with the current laws of Germany.
Statistical analysis. Where appropriate, test of variance, homogeneity,
normality and distribution were performed to ensure that the assump-
tions required for standard parametric analysis of variance were satis-
fied. Statistical analysis of the data was performed by ANOVA fol-
lowed by Bonferroni’s post-hoc test to compare group means.
Results
Previously, we reported that astroglia-rich cultures show re-
duced intracellular glutathione content after 24-h incuba-
tion in MM, a bicarbonate-based buffer lacking glucose and
amino acids (Dringen and Hamprecht 1996). However, dur-
ing the first 8 h of starvation in MM the amount of glu-
617
tathione per dish was not reduced but instead slightly but
significantly (P<0.01) elevated (Fig. 1A). Nevertheless,
longer periods of starvation in MM led to a substantial de-
crease in the amount of cellular glutathione, reaching ap-
proximately 57% of the original content after 24 h (Figs.
1A, 2). During incubation in MM glutathione was released
from the cells, yielding a maximal extracellular concentra-
tion of about 10 nmol GSx/3 ml incubation buffer (Fig. 1B),
whilst during starvation for 24 h the amount of cellular pro-
tein decreased to approximately 60% of the original value
(Fig. 1C), without impairment of cellular viability (Dringen
and Hamprecht 1996).
In the presence of ADT (3–100 µM) during starvation
for up to 24 h, the intracellular glutathione content was
higher at all time points than in cells incubated in the ab-
sence of ADT (Fig. 1A). Thus, for instance after 8 h of star-
vation in buffer containing ADT (100 µM) the level of intra-
cellular glutathione was elevated by approximately 34% as
compared to astrocytes incubated in the absence of ADT.
However, during longer incubation in MM also ADT-treat-
ed cultures showed a decline in glutathione levels at an ap-
parently identical rate as observed in control (non-treated)
cultures (Fig. 1A). Nevertheless, during 24 h of starvation,
at concentrations of 12 and 30 µM, ADT was able to pre-
vent a significant drop in glutathione content occurring in
control cells whereas at the highest concentration used (i.e.,
100 µM ADT) glutathione levels were more than twice
those measured in untreated cells and even significantly
higher than in non-starved cultures (Figs. 1A, 2).
In addition to effects on intracellular glutathione con-
tent, during starvation the concentration of glutathione in
the incubation buffer was increased substantially by the
presence of ADT in an apparently concentration-dependent
manner at all time points measured. However, in contrast to
intracellular levels, extracellular glutathione levels contin-
ued to rise for up to 16 h, reaching more than twice that of
control cultures after 24-h incubation with 100 µM ADT
(Fig. 1B). Unlike the intracellular and extracellular glu-
tathione content, the reduction of cellular protein occurring
during starvation was not altered significantly (P>0.05) by
incubation with ADT (Fig. 1C).
618
Fig. 1 Time course of the effect of ADT on A intracellular GSx con-
tent, B GSx content in the incubation buffer and C protein content of
astroglia-rich primary cultures during starvation for up to 24 h. The
cells were incubated for the time periods indicated in 3 ml MM lack-
ing amino acids and glucose in the presence of 0 µM (open circles), 
3 µM (filled circles), 6 µM (open triangles), 12 µM (filled triangles),
30 µM (open squares), or 100 µM (filled squares) ADT, respectively.
For further experimental details see Materials and methods. To sim-
plify the plot in C, only the protein values of the cells incubated with
0 µM (open circles) and 100 µM (filled squares) of ADT are given.
The protein values per dish of the cells incubated with the other con-
centrations of ADT are superimposable to those presented. The data
shown in the figure are those of one representative experiment ex-
pressed as means of triplicates ± SD obtained with cells from replica
plates. In the figure the error bars have been omitted if they were
smaller than the symbols representing the mean values. The experi-
ment was repeated twice on independently prepared cultures with
comparable results
Fig. 2 Intracellular GSx content of astroglia-rich primary cultures
prior to and after a 24-h period of starvation in 3 ml MM lacking ami-
no acids and glucose in the presence or absence of ADT in the indicat-
ed concentrations. For further experimental details see Materials and
methods. The data represent the means – SD of data from n dishes ob-
tained in 3–7 independent experiments. The statistical significance of
the data obtained from starved cellscompared to the glutathione con-
tent of untreated cells (u.c. unstarved cells at t=0) is indicated above
the columns. The statistical significance of the data obtained from
starved cells incubated in the presence of ADT compared to the glu-
tathione content of starved cells incubated in the absence of ADT is
indicated in the columns.*P<0.05; **P<0.01; ***P<0.001
For reasons of limited solubility (Drukarch et al. 1997b)
and supply of the compound, ADT was used in the follow-
up experiments in a concentration of 30 µM, at which con-
centration it had a comparable effect on intracellular and
extracellular glutathione content during starvation to that of
100 µM ADT (Figs. 1, 2). Previously, we demonstrated that
blockade of the breakdown of extracellular GSH by inhibi-
tion of the ectoenzyme g -glutamyl transpeptidase ( g GT)
with acivicin (Stole et al. 1994) strongly increases the
amount of glutathione found in the incubation medium of
unstarved astroglial cells (Dringen et al. 1997). Therefore,
in the present study acivicin (100 µM) was used to test for
the possible involvement of g GT in the positive effect of
ADT on extracellular glutathione levels during starvation.
However, the acivicin-mediated increase in extracellular
glutathione appeared to be additive to the elevation ob-
served during incubation with ADT (Fig. 3B), whilst the
action of acivicin to reduce astroglial glutathione content
occurred to a similar extent in both control and ADT-treat-
ed cultures (Fig. 3A).
The lack of effect of blockade of g GT activity on ADT-
mediated changes in glutathione content during starvation
contrasted sharply with that of inhibition of glutathione
synthesis with the irreversible g -glutamyl cysteine syn-
thetase blocker BSO (Griffith and Meister 1979). In the
presence of BSO (10 µM), astroglial glutathione almost dis-
appeared within the 24-h period of starvation with an esti-
mated half-time of about 5 h (Fig. 4A). More importantly,
this BSO-mediated, accelerated depletion of intracellular
glutathione was not altered to any significant extent by ad-
dition of ADT (Fig. 4A). The loss of intracellular glu-
tathione during incubation in BSO-containing MM was
paralleled by a reduction of extracellular glutathione that
occurred irrespective of treatment with ADT (Fig. 4B).
Discussion
Depriving cultured astroglial cells of amino acids and/or
glucose for protracted periods of time is accompanied by a
decline in glutathione and protein content without simulta-
neously compromising cellular viability (Dringen and
Hamprecht 1996; Papadopoulos et al. 1997). In the absence
of amino acids, protein synthesis will be inhibited due to
lack of precursors. Since protein degradation continues un-
der these circumstances the net result of such starvation
will be a reduction of cellular protein. Indeed, the data pre-
619
Fig. 3 Effect of ADT and/or acivicin on A intracellular GSx levels
and B GSx in the incubation buffer of astroglia-rich primary cultures
during starvation for up to 24 h. The cells were incubated in 3 ml MM
lacking amino acids and glucose with (squares) or without (circles)
100 µM acivicin in the presence (filled symbols) or the absence (open
symbols) of 30 µM ADT. For further experimental details see Materi-
als and methods. The data shown in the figure are those of one repre-
sentative experiment expressed as means of triplicates – SD obtained
with cells from replica plates. In the figure the error bars have been
omitted if they were smaller than the symbols representing the mean
values. The experiment was repeated on an independently prepared
culture with comparable results
Fig. 4 Effect of ADT and/or BSO on A intracellular GSx levels and B
GSx in the incubation buffer of astroglia-rich primary cultures during
starvation for up to 24 h. The cells were incubated in 3 ml MM lack-
ing amino acids and glucose with (filled symbols) or without (open
symbols) 30 µM ADT in the presence (squares) or the absence (cir-
cles) of 10 µM BSO. For further experimental details see Materials
and methods. The data shown in the figure are those of one represen-
tative experiment expressed as means of triplicates – SD obtained
with cells from replica plates. In the figure the error bars have been
omitted if they were smaller than the symbols representing the mean
values. The experiment was repeated on an independently prepared
culture with comparable results
sented here show that during deprivation of amino acids and
glucose for up to 24 h a loss of cellular protein occurred in
cultured astroglial cells which was almost linear with the
time of starvation. However, contrary to expectation, at
least during the first 8 h of starvation cellular glutathione
levels did not decline but were slightly elevated. It has been
reported that the rise in cellular free cysteine concentration
caused by pharmacological blockade of protein synthesis
with cycloheximide is followed by an increase in cellular
glutathione content (Ratan et al. 1994). This may be ex-
plained by assuming that amino acids generated during pro-
teolysis can serve as precursors for glutathione synthesis.
Indeed, the fact that inhibition of glutathione synthesis with
BSO prevented the starvation-induced short-term increase
in astroglial glutathione indicates ongoing glutathione syn-
thesis in the absence of an exogenous supply of precursor
molecules. Furthermore, under these conditions it is to be
expected that not only glutamate, cysteine and glycine will
be used for glutathione synthesis but also those amino acids
that are converted by astroglial cells to one of the three con-
stituent amino acids of glutathione (Dringen and Hamp-
recht 1996; Kranich et al. 1998).
Besides precursor amino acids, glutathione synthesis de-
pends also on the presence of ATP (Meister and Anderson
1983). After starvation for more than 8 h astroglial cells are
no longer able to maintain their high intracellular content of
ATP (Hertz et al. 1995). As a result glutathione synthesis
will be impaired and, due to continued efflux and use of
glutathione, intracellular glutathione levels will fall.
During starvation of astroglial cells for 24 h in the pres-
ence of ADT, at concentrations of the compound ranging
from 6 to 100 µM, the amount of cellular glutathione was
significantly higher than in cells incubated in the absence of
ADT. In fact, at concentrations of 12, 30 and 100 µM, ADT
was able to totally prevent a fall of intracellular glutathione
levels. More detailed analysis of these data shows that ADT
appears to accelerate the increase in intracellular glu-
tathione occurring during the first 8 h of starvation. Since
ADT did not affect the rate of loss of intracellular glu-
tathione during starvation for longer than 8 h, this indicates
that ADT must induce changes in astroglial glutathione me-
tabolism primarily in the initial stages of amino acid and
glucose deprivation. This raises questions as to the possible
underlying mechanism(s).
Recently, we demonstrated that 24-h incubation of non-
starved astroglial cells with ADT induced a concentration-
dependent increase in glutathione content (Drukarch et al.
1997b). This effect of ADT was blocked by coincubation of
the cells with BSO. The present results show that, also dur-
ing starvation, exposure to BSO fully inhibited the ADT-in-
duced elevation of glutathione levels in astroglial cells oc-
curring in the first 8 h. Indeed, in the presence of BSO no
difference was noted between the loss of glutathione from
control cultures and ADT-treated cultures. Elevation of the
activity of g -glutamyl cysteine synthetase has been estab-
lished ex vivo in liver homogenates subsequent to adminis-
tration of ADT (Davies et al. 1987). Moreover, reportedly
another dithiolethione known as oltipraz is able to upregul-
ate g -glutamyl cysteine synthetase mRNA levels in various
tissues (Buetleret al. 1995; O’Dwyer et al. 1996). Unfortu-
nately however, due to technical difficulties, we thus far
failed to measure the activity of this enzyme in our ast-
roglial cultures. Thus, although its exact mode of action re-
mains to be established, it is possible that the ADT-induced
increase in astroglial glutathione content during starvation
is mediated through a quickly initiated stimulation of glu-
tathione synthesis, most likely at the level of g -glutamyl
cysteine synthetase. Since ATP is a substrate of this enzyme
(Meister and Anderson 1983), ATP depletion during long-
term starvation will impair the capacity of ADT-treated ast-
roglial cells to further synthesize glutathione. Consequent-
ly, as illustrated by our data, after the initial increase also in
the presence of ADT astroglial glutathione levels will start
to drop during starvation sustained for more than 8 h. Nev-
ertheless, even after 24 h of glucose and amino acid depri-
vation glutathione levels remain significantly higher in
ADT-treated cells than in astroglial cells starved in the ab-
sence of the compound.
As noted above, glutathione is formed from glutamate,
cysteine and glycine. Thus, ADT could also support glu-
tathione synthesis by increasing the availability of these
precursor amino acids. During starvation only amino acids
present at the onset of the incubation or generated during
starvation can be used for glutathione synthesis (Dringen et
al. 1997). Therefore, under such circumstances an increase
in the intracellular concentration of glutamate, cysteine and
glycine has to be mediated either by inhibition of pathways
involved in the breakdown of these three amino acids or
their precursors (see above), inhibition of release from the
cells of these three amino acids or their precursors (and/)or
increased rates of synthesis of glutamate, cysteine and gly-
cine from their precursors. However, to our knowledge, un-
til now no such mechanism of action has been identified for
ADT or any other dithiolethione compound.
Astroglial cells are able to release large amounts of glu-
tathione (Dringen et al. 1997; Sagara et al. 1996; Yudkoff et
al. 1990). Thus, alternatively, the higher cellular glu-
tathione content during starvation in the presence of ADT
might be caused by inhibition of glutathione efflux. Howev-
er, as our data demonstrate, ADT did not decrease the efflux
but in fact induced a substantial increase in the extracellular
level of glutathione. Furthermore, although acivicin-medi-
ated blockade of g GT in astroglial cells enhances the
amount of extracellular glutathione (Dringen et al. 1997),
our results indicate that ADT does not exert its stimulatory
effect on extracellular glutathione levels via inhibition of
the activity of this (ATP-independent) ectoenzyme which is
involved in the extracellular breakdown of glutathione into
its constituent amino acids (Meister et al. 1981). More like-
ly, the ADT-induced elevation of extracellular glutathione
merely reflects the increase in intracellular glutathione con-
tent of ADT-treated cells since the rate of transporter-medi-
ated glutathione release from astroglial cells is strongly de-
pendent on the intracellular concentration of glutathione
(Sagara et al. 1996). Hence, a rise in the level of intracellu-
lar glutathione as occurring during treatment with ADT
may be expected to be accompanied by a higher efflux from
the cells and a resultant higher accumulation of glutathione
620
in the extracellular compartment, whereas a decrease in in-
tracellular glutathione will have the opposite effect. This
explanation is supported by our observation that extracellu-
lar glutathione levels were reduced considerably, both in
the presence or absence of ADT, following depletion of cel-
lular glutathione with BSO.
Signs of oxidative damage have been detected in most if
not all neurodegenerative syndromes and loss of glu-
tathione has been noted in particular in Parkinson’s disease
and ischemic brain injury (Ames et al. 1993; Bains and
Shaw 1997). As mentioned in the Introduction, a decrease
in glutathione levels, amongst many other effects, will com-
promise the capacity of brain cells to withstand attack by
ROS, such as superoxide radicals and hydrogen peroxide.
Especially astroglial cells are thought to be responsible for
the protection of neurons against oxidative damage, and
glutathione depletion has been shown by us and others to
abolish the neuronal survival-enhancing effect of these
cells, eventually leading to neuronal death (Ben-Yoseph et
al. 1996; Drukarch et al. 1997a). Recently, we provided ev-
idence that this impaired neuroprotective action of glu-
tathione-poor astroglial cells is caused primarily by re-
duced extracellular inactivation of ROS, most probably as a
result of a lowered efflux of glutathione and a consequent
drop in extracellular glutathione levels (Drukarch et al.
1998). Thus, compounds such as ADT which, albeit dem-
onstrated so far only in vitro, are able to maintain or even
increase astroglial glutathione content and release under
adverse conditions over an extended period of time may
prove to be potent neuroprotective agents. Although it
would be attractive to test this assumption in a coculture of
astroglial cells with neurons, this is not feasible since neu-
rons quickly lose their viability during starvation-induced
glutathione depletion (Kranich et al. 1996). Nevertheless,
considering the reported ability of ADT to protect astroglial
cells against oxidative damage also in a glutathione-inde-
pendent manner (Drukarch et al. 1997b) and the fact that it
has been used in humans for a great number of years with-
out major adverse effects, the conclusion appears appropri-
ate that further evaluation of the therapeutic potential of this
drug in named conditions is warranted.
Acknowledgements We would like to thank Dr. J.B. Schulz for his
help with the statistical analysis of our data.
References
Ames BN, Shigenaga MK, Hagen TM (1993) Oxidants, anti-oxidants,
and the degenerative diseases of aging. Proc Natl Acad Sci USA
90:7915–7922
Bains JS, Shaw CA (1997) Neurodegenerative disorders in humans:
the role of glutathione in oxidative stress-mediated neuronal
death. Brain Res Rev 25:335–358
Baker MA, Cerniglia GJ, Zaman A (1990) Microtiter plate assay for
the measurement of glutathione and glutathione disulfide in large
numbers of biological samples. Anal Biochem 190:360–365
Ben-Yoseph O, Boxer PA, Ross BD (1996) Assessment of the role of
the glutathione and pentose phosphate pathways in the protection
of primary cerebrocortical cultures from oxidative stress. J
Neurochem 66:2329–2337
Buetler TM, Gallagher EP, Wang C, Stahl DL, Hayes JD, Eaton DL
(1995) Induction of phase I and phase II drug-metabolizing en-
zyme mRNA, protein and activity by BHA, ethoxyquin, and
oltipraz. Toxicol Appl Pharmacol 135:45–57
Christen MO (1995) Anethole dithiolethione: biochemical consider-
ations. Methods Enzymol 252:316–323
Dansette PM, Sassi A, Deschamps C, Mansuy D (1990) Sulphur con-
taining compounds as antioxidants. In: Emerit I, Packer L, Auclair
C (eds) Antioxidants in therapy and preventive medicine, vol 264.
Plenum Press, New York, pp 209–215
Davies MH, Blacker AM, Schnell RC (1987) Dithiolethione-induced
alterations in hepatic glutathione and related enzymes in male
mice. Biochem Pharmacol 36:568–570
Devesa A, O’Connor JE, Garcia C, Puertes IR, Vina JR (1993) Glu-
tathione metabolism in primary astrocyte cultures: flow
cytometric evidence of heterogeneous distribution of GSH con-
tent. Brain Res 618:181–189
Dringen R, Hamprecht B (1996) Glutathione content as an indicator
for the presence of metabolic pathways of amino acids in ast-
roglial cultures. J Neurochem 67:1375–1382
Dringen R, Kranich O, Hamprecht B (1997) The g -glutamyl
transpeptidase inhibitor acivicin preserves glutathione releasedby
astroglial cells in culture. Neurochem Res 22:727–733
Drukarch B, Schepens E, Jongenelen CAM, Stoof JC, Langeveld CH
(1997a) Astrocyte-mediated enhancement of neuronal survival is
abolished by glutathione deficiency. Brain Res 770:123–130
Drukarch B, Schepens E, Stoof JC, Langeveld CH (1997b) Anethole
dithiolethione prevents oxidative damage in glutathione-depleted
astrocytes. Eur J Pharmacol 329:259–262
Drukarch B, Schepens E, Stoof JC, Langeveld CH, Van Muiswinkel
FL (1998) Astrocyte-enhanced neuronal survival is mediated by
scavenging of extracellular reactive oxygen species. Free Radic
Biol Med 25:217–220
Griffith OW, Meister A (1979) Potent and specific inhibition of glu-
tathione synthesis by buthionine sulfoximine (S-n-butyl homocys-
teine sulfoximine). J Biol Chem 254:7558–7560
Hamprecht B, Dringen R (1995) Energy metabolism. In: Kettenmann
H, Ransom BR (eds) Neuroglia. Oxford University Press, New
York, pp 473–487
Hamprecht B, Löffler F (1985) Primary glial cultures as a model sys-
tem for studying hormone action. Methods Enzymol 109:341–345
Hertz L, Yager JY, Juurlink BHJ (1995) Astrocyte survival in the ab-
sence of exogenous substrate: comparison of immature and ma-
ture cells. Int J Dev Neurosci 13:523–527
Kranich O, Hamprecht B, Dringen R (1996) Different preferences in
the utilization of amino acids for glutathione synthesis in cultured
neurons and astroglial cells derived from rat brain. Neurosci Lett
219:211–214
Kranich O, Dringen R, Sandberg M, Hamprecht B (1998) Utilization
of cysteine and cysteine precursors for the synthesis of glutathione
in astroglial cultures: preference for cystine. Glia 22:11–18
Langeveld CH, Jongenelen CAM, Schepens E, Stoof JC, Bast A, Dru-
karch B (1995) Cultured rat striatal and cortical astrocytes protect
mesencephalic dopaminergic neurons against hydrogen peroxide
toxicity independent of their effect on neuronal development.
Neurosci Lett 192:13–16
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein mea-
surement with the Folin phenol reagent. J Biol Chem
193:265–275
Mansuy D, Sassi A, Dansette PM, Plat M (1986) A new potent inhib-
itor of lipid peroxidation in vitro and in vivo, the hepatoprotective
drug anysyldithiolethione. Biochem Biophys Res Commun
135:1015–1021
Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem
52:711–760
Meister A, Tate SS, Griffith OW (1981) Gamma-glutamyl
transpeptidase. Methods Enzymol 77:237–253
O’Connor E, Devesa A, Garcia C, Puertes IR, Pellin A, Vina JR
(1995) Biosynthesis and maintenance of GSH in primary astrocyte
cultures: role of cystine and ascorbate. Brain Res 680:157–163
O’Dwyer PJ, Szarka CE, Yao K-S, Halbherr TC, Pfeiffer GR, Green
F, Gallo JM, Brennan J, Frucht H, Goosenberg EB, Hamilton TC,
Litwin S, Balshem AM, Engstrom PF, Clapper ML (1996) Modu-
621
lation of gene expression in subjects at risk for colorectal cancer
by the chemopreventive dithiolethione oltipraz. J Clin Invest
98:1210–1217
Papadopoulos MC, Koumenis IL, Dugan LL, Giffard RG (1997) Vul-
nerability to glucose deprivation injury correlates with glutathione
levels in astrocytes. Brain Res 748:151–156
Ratan RR, Murphy TH, Baraban JM (1994) Macromolecular synthe-
sis inhibitors prevent oxidative stress-induced apoptosis in embry-
onic cortical neurons by shunting cysteine from protein synthesis
to glutathione. J Neurosci 14:4385–4392
Sagara J, Makino N, Bannai S (1996) Glutathione efflux from cul-
tured astrocytes. J Neurochem 66:1876–1881
Sen CK, Traber KE, Packer L (1996) Inhibition of NF-kappaB activa-
tion in human T-cell lines by anetholdithiolthione. Biochem
Biophys Res Commun 218:148–153
Stole E, Smith TK, Manning JM, Meister A (1994) Interaction of g -
glutamyl transpeptidase with acivicin. J Biol Chem
269:21435–21439
Tietze F (1969) Enzymic method for quantitative determination of
nanogram amounts of total and oxidized glutathione: applications
to mammalian blood and other tissues. Anal Biochem 27:502–522
Yudkoff M, Pleasure D, Cregar L, Lin Z-P, Nissim I, Stern J, Nissim I
(1990) Glutathione turnover in cultured astrocytes: studies with
[15N]glutamate. J Neurochem 55:137–145
622