Neuroprotection by taurine in ethanol-induced apoptosis in the developing cerebellum.

Abstract

Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse cerebellum during the postnatal period.
The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3 h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine (1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase high-performance liquid chromatography (HPLC).
Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group diminished dramatically during the experiment, not being different at 13 h from that in the controls.
We conclude that the neuroprotective action of taurine is not straightforward and seems to be different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in blood plasma.

Full text

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REVIEWOpen Access
Neuroprotection by taurine in ethanol-induced
apoptosis in the developing cerebellum
Andrey G Taranukhin1,2*, Elena Y Taranukhina1†, Pirjo Saransaari1†, Irina M Podkletnova1†, Markku Pelto-Huikko3†,
Simo S Oja1,4†
From 17thInternational Meeting of Taurine
Fort Lauderdale, FL, USA. 14-19 December 2009
Abstract
Background: Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing
central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse
cerebellum during the postnatal period.
Methods: The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol
(20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3
h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine
(1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL
staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours
after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase
high-performance liquid chromatography (HPLC).
Results: Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively
in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive
cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of
activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had
no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group
diminished dramatically during the experiment, not being different at 13 h from that in the controls.
Conclusions: We conclude that the neuroprotective action of taurine is not straightforward and seems to be
different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in
blood plasma.
Background
A moderate alcohol intake may not be harmful and has
even beneficial effects in prevention of cardiovascular
diseases, for example [1]. On the other hand, heavy
alcohol consumption is associated with the reduced
brain mass, neuronal loss, neuropathological changes
and results in the impairment of cognitive functions,
amnesia, dementia and even a significant increase in
mortality [2-4]. In adult rats a short-term increase in
the blood ethanol concentration up to 6 g/l has not
been toxic to the central nervous system [5]. However,
in the developing nervous system the situation is quite
different. The blood ethanol concentration above 0.5 g/l
in mice during their early postnatal life induces mild
apoptotic neurodegeneration [6], which becomes dose-
dependently more severe from the concentration of 2 g/
l upward [7].Intrauterine exposure of the human fetus
to ethanol due heavy drinking or repeated binge drink-
ing of pregnant women causes a wide spectrum of
developmental disorders known as the fetal alcohol syn-
drome [8,9]. The human fetal brain is particularly
* Correspondence: andrey.taranukhin@uta.fi
† Contributed equally
1Brain Research Center, University of Tampere Medical School, Tampere,
Finland
Full list of author information is available at the end of the article
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© 2010 Taranukhin et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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sensitive to the adverse effects of alcohol during the last
trimester of pregnancy, the period of synaptogenesis,
also known as the brain growth spurt period. In rodents,
the same period of increased sensibility to ethanol is
during the early postnatal period [10]. It has been well
documented that acute ethanol exposure to neonatal
mice induces neuronal loss by apoptosis [6,7,11-14].
Prevention of ethanol-induced apoptosis can save a
huge amount of neurons and significantly decrease the
harmful consequences of alcohol intoxication.
Taurine is a simple sulphur-containing free amino
acid abounding in electrically excitable tissues such as
brain, retina, heart and skeletal muscles [15]. It is
involved in a wide range of physiological processes, for
example, in osmoregulation, lipid metabolism, intracel-
lular calcium regulation, neuronal development, neuro-
modulation and cell protection [16-20]. Recent findings
also imply taurine in apoptosis regulation [21-24]. In the
present work we focused on the possible protection of
Purkinje cells and neurons in the internal layer of the
developing cerebellum against apoptosis induced by
acute ethanol administration. Among possible means to
prevent pathological apoptosis taurine is very attractive
since it is a naturally-occurring and non-toxic
compound.
Methods
Animals and treatments
Adult NMRI mice for breeding were purchased from
Harlan, Netherlands. Four (P4) - and seven (P7) - day
old infant male mice were used in the experiments (day
of birth is day 0). The experiments on animals were
carried out in accordance with the European Commu-
nity Council Directive 86/609/EEC. All efforts were
made to reduce their number and suffering. The mice
in each litter were divided into three groups: ethanol-
treated, ethanol+taurine-treated and controls. To induce
acute alcohol intoxication ethanol was mixed in sterile
saline to a 20 % solution and administered subcuta-
neously at a total dose of 5 g/kg (2.5 g/kg at time 1 h
and 2.5 g/kg again at 3 h) to the ethanol and ethanol+
taurine groups. This dosing regimen was well documen-
ted to produce an elevation in the blood alcohol con-
centration above 2 g/l for at least 8 hours and led to
significant and widespread apoptotic neurodegeneration
in the developing brain [7] and cerebellum [25]. The
ethanol+taurine group also received two injections of
taurine (1 g/kg diluted with saline). The first taurine
injection was given one hour before the first ethanol
injection and the second one hour after the second
ethanol injection. The control animals were given saline
subcutaneously. Twelve (P4) and eight hours (P7) after
the first ethanol injection the mice were killed by
decapitation. Blood samples from each animal were
collected separately in lithium-heparin tubes and centri-
fuged at 1750 rpm for 10 min to obtain plasma. The
samples were frozen until HPLC analyses. The cerebella
were rapidly excised and fixed in 4 % paraformaldehyde
in phosphate buffered saline for at least 3 days at 4oC.
They were after the routine histological processing
embedded in paraffin and cut with a microtome into
5-μm thick mid-sagittal sections containing lobules I-X
of the cerebellar vermis.
High Performance Liquid Chromatography (HPLC)
The concentration of taurine in the blood serum was
measured using HPLC with fluorescent detection after
precolumn derivatization with o-phthaldialdehyde
(OPA) using the analysis equipment system of Shimadzu
Scientific Instruments (Kyoto, Japan). The separation
column was 4.6 x 250 mm Ultropac 8 Resin, lithium
form (Farmacia, Denmark). Derivatization of taurine was
performed with the OPA reagent (0.2 % OPA, 0.1 %
mercaptoethanol and 1 % ethanol in 1 M borate buffer,
pH 10.4). The elution was done with lithium citrate buf-
fers in the following order: (1) 0.2 M, pH 2.80, (2) 0.3
M, pH 3.00, (3) 0.5 M, pH 3.15, (4) 0.9 M, pH 3.50, and
(5) 1.6 M, pH 3.30. Fluorescence of taurine derivatives
was measured with an RF-10A detector using the excita-
tion and emission wavelengths set at 340 nm and 450
nm, respectively. The concentrations of taurine were
finally estimated using a commercial amino acid mixture
(Pickering, UK) as an external standard and diamino-n-
butyrate as an internal standard.
Immunohistochemistry
The sections were deparaffinized with xylene and
hydrated in a graded ethanol series to distilled water.
After antigen retrieval by microwave [20 min at 1000 W
in 0.01 M citrate buffer (pH 6.0)], washing in phosphate
buffered saline and blocking with 0.5 % hydrogen perox-
ide in this buffer for 20 min, the specimens were prein-
cubated for 30 min in serum-blocking solution (1 %
bovine serum albumin and 0.3 % Triton X-100 in the
above buffer). The specimens were thereafter incubated
with polyclonal activated caspase-3 antibody [cleaved
caspase-3 (Asp 175) antibody, Cell Signaling Technology
Inc., diluted 1:200 in serum-blocking solution] in moist
chambers overnight at 4°С. After incubation with the
primary antibody, the sections were incubated with bio-
tinylated secondary antibody (goat anti-rabbit 1:500 in
blocking solution) and ABC complex (Vectastain Elite
ABC Kit, Vector Laboratories, Inc.), each for 30 min.
Diaminobenzidine was used as a chromogen to visualize
the sites expressing activated caspase-3 immunoreactiv-
ity. The sections for negative control were incubated
without the primary antibodies to rule out nonspecific
staining. Finally, the sections were counterstained by
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hematoxylin-eosin to better reveal histological details,
dehydrated and mounted.
Detection of cell death in situ
DNA fragmentation is one of the most precise markers
by which apoptotic cells are recognized. In order to
detect DNA fragmentation of cell nuclei, terminal deox-
ynucleotidyl transferase-mediated dUTP nick end-label-
ing (TUNEL) reaction was applied to the paraffin
sections using the In Situ Cell Death Detection Kit,
POD (Roche Applied Science, Germany). After deparaf-
finization, the sections were irradiated with microwaves
in 0.01 M citric acid buffer (pH 6) for 10 min at 750 W.
No inhibition of endogenous peroxidase was performed
because H2O2weakens terminal deoxynucleotidyl trans-
ferase activity [26] and induces DNA breaks [27]. Sec-
tions were incubated with the TUNEL reaction mixture
for 60 min at 37°С. Further incubation with peroxidase-
conjugated antibody was performed for 30 min at 37°С.
The sections were stained with diaminobenzidine for 10
min at the room temperature and then counterstained
with hematoxylin-eosin.
Image analysis and cell counting
The sections were processed in parallel under standar-
dized conditions for immunostaining or TUNEL to
minimize variability in labeling conditions. An image
analysis system comprising of an IBM PC, Nikon Micro-
phot-FXA microscope, SensiCam digital camera (PCO
Computer Optics GmbH), Image-Pro Plus (Media
Cybernetics) was used for analysis of caspase-3 immu-
noreactivity and TUNEL-staining in the histological sec-
tions of the cerebellum. At least five sections cut at the
same level of the cerebellar vermis from every animal
were analyzed. The amount of cells labeled for active
caspase-3 or TUNEL were calculated in every slice in
each lobule and the area of each lobule was also mea-
sured. The data are presented as the average number of
labelled cells per mm2for each experimental group.
Statistical analysis
One-way analysis of variance (ANOVA) was used to
compare the number of activated caspase-3-immunor-
eactive cells and TUNEL-positive cells among the
experimental groups. When ANOVA showed a signifi-
cant difference, the post hoc Bonferroni test was applied
to demonstrate the difference. Each value is expressed
as mean ± standard deviation. Differences were consid-
ered significant when the calculated p value was < 0.05.
Results
In the present work we focused on the Purkinje cells
and neurons in the internal granular cell layer of the
cerebellum. The Purkinje cells were identified by their
size, shape and specific localization in the cerebellar
lobules. Because we did not make specific labeling to
identify different types of neurons and glial cells in the
internal granular cell layer of the cerebellum, we refer to
these cells as internal granular layer (IGL) cells. We stu-
died the possible protective effect of taurine against
ethanol-induced apoptosis in the Purkinje cells on P4
and the IGL cells on P7 mice because of the extremely
high sensitivity of these cell types at these ages to etha-
nol-induced apoptotic neurodegeneration.
Effects of taurine on ethanol-induced caspase-3 activation
Activated caspase-3-immunoreactive (IR) cells in the
IGL found in each cerebellar lobule in the saline-treated
control mice on P7 evidences physiological cell death
which normally occurs at this period of development
(Figure 1A, 1D; Figure 2A, 2D; Figure 3A, 3D). Only a
few activated caspase-3-IR Purkinje cells were found in
the saline-treated control mice on P4.
In the ethanol-treated pups on P4 a large number of
activated caspase-3-IR Purkinje cells were discernible in
all lobules with the highest amount in lobules IX and X
(Figure 4B, 4E). In lobules I-II, III, IV-V, VI-VII and
VIII the number of capase-3-IR Purkinje cells was also
significantly increased when compared to the control
group (Figure 5A). On P7 ethanol administration
induced very little response to caspase-3 activation in
the Purkinje cell layer which hardly differed from the
control group. However, ethanol treatment on P7
induced widespread activation of caspase-3 in the IGL
(Figure 1B, 1E; Figure 2B, 2E; Figure 3B, 3E). The
amount of activated caspase-3-IR IGL cells in each
lobule was markedly greater than in the saline-treated
pups (Figure 5C). The highest immunoreactivity for acti-
vated caspase-3 was found in I-II, III and IV-V vermian
lobules.
In the ethanol+taurine-treated group on P4 the num-
ber of activated caspase-3-IR Purkinje cells in each
lobule was approximately the same as in the pups trea-
ted only with ethanol and significantly higher than in
the saline-treated control group (Figure 4; Figure 5A).
However, in contrast to the Purkinje cells at age P4,
taurine treatment significantly decreased the number of
activated caspase-3-IR cells in the IGL on P7 in each
lobule (Figure 1C, 1F; Figure 2C, 2F; Figure 3C, 3F; Fig-
ure 5C). The amount of rescued cells from caspase-3
activation were different in different lobules and varied
from 34 % to 41 % for lobules VI, VII and X, and from
45 % to 57 % for lobules I-II, III, IV-V, VIII and IX.
Effects of taurine on ethanol-induced DNA fragmentation
A few TUNEL-positive cells in the Purkinje cell layer on
P4 and in the IGL on P7 were detected in the saline-
treated control groups showing physiological cell death
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Figure 1 Immunostaining for activated caspase-3 in lobule II of the cerebellum of 7-day-old mice 8 hours after the first ethanol
injection A, D: control group, B, E: ethanol-treated group, C, F: ethanol+taurine-treated group. The activated caspase-3-immunoreactive cells in
the IGL are indicated by black arrows.
Figure 2 Immunostaining for activated caspase-3 in lobule IV-V of the cerebellum of 7-day-old mice 8 hours after the first ethanol
injection A, D: control group, B, E: ethanol-treated group, C, F: ethanol+taurine-treated group. The activated caspase-3-immunoreactive cells in
the IGL are indicated by black arrows.
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Figure 3 Immunostaining for activated caspase-3 in lobule X of the cerebellum of 7-day-old mice 8 hours after the first ethanol
injection A, D: control group, B, E: ethanol-treated group, C, F: ethanol+taurine-treated group. The activated caspase-3-immunoreactive cells in
the IGL are indicated by black arrows.
Figure 4 Immunostaining for activated caspase-3 in lobule X of the cerebellum of 4-day-old mice 12 hours after the first ethanol
injection A, D: control group, B, E: ethanol-treated group, C, F: ethanol+taurine-treated group. The activated caspase-3-immunoreactive Purkinje
cells are indicated by black arrows.
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Figure 5 Effects of taurine on ethanol-induced apoptosis in the developing cerebellum (A) Number of activated caspase-3
immunoreactive Purkinje cells in the cerebellar lobules of 4-day-old mice in the control (open bars), ethanol-treated (filled bars) and ethanol
+taurine-treated (hatched bars) mice. The results are given per mm2with standard deviations. Number of animals in each group is 5. Ethanol
significantly (P<0.01) increased the number of caspase-3 immunoreactive cells in all lobules. There were no significant differences between the
ethanol and ethanol+taurine groups. (B) Number of apoptotic Purkinje cells (labeled by TUNEL assay) in the cerebellar lobules of 4-day-old mice
in the control (open bars), ethanol-treated (filled bars) and ethanol+taurine-treated (hatched bars) mice. The results are given per mm2with
standard deviations. Number of animals in each group is 5. Ethanol significantly (P<0.01) increased the number of TUNEL-positive Purkinje cells
in all lobules. There were no significant differences between the ethanol and ethanol+taurine groups. (C) Number of activated caspase-3-
immunoreactive cells in the IGL in the cerebellar lobules of 7-day-old mice in the control (open bars), ethanol-treated (filled bars) and ethanol
+taurine-treated (hatched bars) mice. The results are given per mm2with standard deviations. Number of animals in each group is 5. Ethanol
significantly (P<0.01) increased the number of caspase-3 immunoreactive cells in all lobules. The significance of differences between the ethanol
and ethanol+taurine groups: *P<0.05.(D) The number of apoptotic cells (labeled by TUNEL assay) in the IGL in the cerebellar lobules of 7-day-old
mice in the control (open bars), ethanol-treated (filled bars) and ethanol+taurine-treated (hatched bars) mice. The results are given per mm2with
standard deviation. Number of animals in each group is 5. Ethanol significantly (P<0.01) increased the number of TUNEL-positive cells in all
lobules. The significance of differences between the ethanol and ethanol+taurine groups: *P<0.05.
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during normal development. The amount of cells
undergoing physiological cell death in the Purkinje layer
seems to be less than in the IGL and it was in
accordance with our data on activated caspase-3
immunoreactivity.
Ethanol treatment significantly increased the number
of cells with fragmented DNA labeled by the TUNEL
assay in the Purkinje cell layer of P4 mice (Figure 5B).
The largest number of TUNEL-positive Purkinje cells
was found in lobules IX and X (Figure 6B, 6E), although
in the other lobules studied the amount of TUNEL-posi-
tive Purkinje cells was also much greater than in the
control group. At age P7, ethanol administration
induced massive apoptosis in the IGL in all vermian
lobules as indicated by the increased amount of
TUNEL-positive cells (Figure 5D).
Taurine treatment did not change the amount
of TUNEL-positive Purkinje cells on P4 in any lobule
studied when compared to the pups treated only with
ethanol (Figure 5B; Figure 6). The amount of TUNEL-
positive Purkinje cells remained much greater than in
the saline-treated control group. In the ethanol+taur-
ine group at age P7 the number of TUNEL-positive
IGL cells was reduced in comparison to the ethanol
group, the change being statistically significant in all
other lobules except lobules VI and VII (Figure 5D).
Although taurine treatment decreased the number of
TUNEL-positive IGL cells induced by acute alcohol
administration, the number of TUNEL-positive cells in
the ethanol+taurine group remained much larger than
in the control group. Taurine preservation of IGL cells
from DNA fragmentation varied in the lobules from
40 % (lobule I-II) to 68 % (lobule IX).
Dynamics of taurine blood concentration changes during
the experiments
We measured the taurine concentration in blood serum
during the experiments from 4 h onwards after the first
ethanol injection (1 hour after last taurine injection) at
every hour for 12 h and then again at 24 h (Figure 7).
In the saline-treated control group and in the ethanol
group of P7 mice the taurine concentration remained
the same at 4 h, 12 h and 24 h. It was 1.00 ± 0.42
mmol/l in the control group and 0.85 ± 0.24 mmol/l in
the ethanol group. Two subcutaneous injections of taur-
ine each at the dose 1 g/kg (one at 0 h and second at
4 h) markedly increased the taurine level up to 13.36 ±
2.73 mmol/l at 4 h after the first ethanol injection. For
four hours (4 h-7 h) the concentration in the blood was
maintained at about the same high level of 13.20 ± 1.83
mmol/l and then it started to decline gradually and
almost reached the control level by 12 h (1.59 ± 0.83
mmol/l).
Discussion
The central nervous system is extremely sensitive to
alcohol during development and the periods of vulner-
ability are temporally well defined. The time frames of
vulnerability are different for different neuronal popu-
lations. In the developing mouse cerebellum acute
Figure 6 TUNEL staining in lobule X of the cerebellum of 4-day-old mice 12 hours after the first ethanol injection A, D: control group,
B, E: ethanol-treated group, C, F: ethanol+taurine-treated group. TUNEL-positive Purkinje cells are indicated by black arrows.
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alcohol intoxication thus induces massive neuronal
death of Purkinje cells on P2-P6 and of cells in the
IGL on P7-P9 [25]. In our experiments P4 and P7
mice pups were used to induce ethanol-induced degen-
eration of Purkinje and IGL cells, respectively. It is
well established that alcohol induces neuronal death in
the developing brain by apoptosis [11]. Apoptosis is a
type of programmed cell death with specific morpholo-
gic features [28]. Immature neurons dying due to etha-
nol exposure, exhibit biochemical and ultrastructural
features of apoptosis such as activation of caspase-3
[12,13], internucleosomal DNA fragmentation [7,14],
clumping of nuclear chromatin, formation of spherical
chromatin masses and nuclear membrane fragmenta-
tion [7,25,29].
Two major apoptotic pathways have been established:
the death-receptor-mediated and the mitochondrial-
mediated apoptosis [28], also known as the “extrinsic”
and as “intrinsic” apoptotic pathways, respectively [30].
In both pathways, the caspases, a family of cysteine-
dependent aspartate-directed proteases, play an impor-
tant role in initiation, signal transduction and execution
of apoptosis [31,32]. The extrinsic pathway is triggered
by activation of death receptors localized at the cell
membrane surface and it induces caspase-8 processing.
The activated caspase-8 can directly or indirectly acti-
vate effector caspases such as caspases-3, -6 and -7. In
the intrinsic pathway, many factors such as nitric oxide,
oxidants and proapoptotic proteins, e.g. Bax, increase
mitochondrial membrane permeability and release cyto-
chrome C into the cytoplasm. Cytochrome C binds to
Apaf-1 and procaspase-9, forming an apoptosome, lead-
ing to caspase-9 activation [33]. The active caspase-9
cleaves and activates effector caspases, including cas-
pase-3. The activated effector caspases cleave many
structural and functional proteins and activate DNase
which destroys chromosomes and leads to cell death
[31,32]. As shown with Bax-knock-out mice, ethanol-
induced apoptosis in the developing brain is a Bax-
dependent process which requires translocation of the
Bax protein from the cytosol to mitochondria, disrup-
tion of mitochondrial membranes, release of cytochrome
C and activation of caspase-3. All these events and also
the absence of capase-8 activation indicate that alcohol
induces apoptosis in the developing brain via intrinsic
(mitochondrial-mediated) apoptotic pathways [34,35].
In the present experiments we demonstrate that acute
ethanol administration to P4 mice induces activation of
caspase-3 and DNA fragmentation in Purkinje neurons
in all vermian lobules studied. This finding is in concert
with the results of other authors working with mice [25]
and rats [36]. Ethanol administration to mice pups
at age P7 markedly enhanced the number of activated
caspase-3-IR and TUNEL-positive cells in the IGL but
did not markedly affect the Purkinje cells. This time dif-
ference in alcohol sensitivity of different types of neu-
rons is not surprising, being already shown in other
studies [25].
The presence of taurine at high concentrations during
the early ontogenesis is essential for normal develop-
ment [37]. Taurine has been tested in treatment of
many diseases, including cardiovascular disorders, epi-
lepsy, macular degeneration, hepatic disorders, cystic
fibrosis, Alzheimer’s disease and alcoholism [38]. It also
interacts with the effects of ethanol [39]. For instance, it
modulates ethanol-stimulated locomotion [40] and pro-
longs ethanol-induced sedation when given intracerebro-
ventricularly to mice [41,42]. Furthermore, ethanol
administration elicits an increase in extracellular taurine
in the rat cerebral cortex and hippocampus [43]. How-
ever, many findings on taurine and ethanol interactions
have been contradictory. For instance, in behavioural
studies taurine pretreatment has reduced the duration
of ethanol-induced sleep-time and attenuated the loss of
righting reflex [44,45], not altered the ethanol-induced
loss of righting reflex [46] or even enhanced it [41]. It
seems that interactions of taurine and ethanol in the
brain depend largely on the experimental set-up and the
doses of ethanol and taurine administered [19].
Mounting recent evidence indicates that taurine is
involved in apoptosis regulation and protects many cell
types under different pathological conditions such as
ischemia [22,23], high glucose level [47], oxidative stress
[48], and ethanol intoxication [49]. In adult rats intra-
peritoneal taurine injections markedly increase the taur-
ineconcentrationin blood
microdialysates in a dose-dependent manner [50]. After
a single injection of 1 g/kg taurine the maximal plasma
and brain concentrations are reached within 20 min and
they remain significantly higher than in controls for 3.5-
4 h. The present two taurine injections at the dose of
plasmaandbrain
Figure 7 Changes in the taurine concentration in blood plasma
during the experiment in 7-day-old mice Data are presented as
mean values. Standard deviations are shown only for samples at 4
h, 12 h and 24 h after the first ethanol injection.
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1 g/kg each with the 4-h interval keep the level of taur-
ine high for a more prolonged time. The present finding
that in this manner taurine attenuated apoptosis in the
IGL is in accordance with our previous study [49].
However, in P4 mice we got unexpected results that
taurine had no effect on the ethanol-induced activation
of caspase-3 and apoptosis in Purkinje cells. Taurine can
be involved in apoptosis regulation by several ways. First
of all, taurine decreases intracellular free Ca2+by inhi-
biting all types of voltage-gated calcium channels and
the N-methyl-D-aspartate (NMDA) receptor-gated cal-
cium channel [24,51] and by increasing Ca2+buffering
in mitochondria [52]. It prevents in this manner activa-
tion of calpain (calcium-dependent protease) and pro-
tects mitochondrial membranes from disruption [24].
Taurine can also protect cells acting as antioxidants
[15], scavenging at its different physiological concentra-
tions many reactive oxygen and nitrogen species [53],
although ethanol administration to neonatal rats may
not induce oxidative stress to cerebellar granular neu-
rons [54]. For cell survival a balance between the proa-
poptotic protein Bax and the antiapoptotic protein Bcl-2
is very important. A decrease in the Bcl-2 level in cells
leads to translocation of Bax to mitochondria, disruption
of their membranes and a release of cytohrome C from
mitochondria to the cytosol [55]. Taurine application
can restore the pool of Bcl-2 and protect cells against
apoptosis [24]. Since ethanol-induced apoptosis is a
Bax-dependent process [34], we suggest that restoration
of the Bcl-2 level was one possible mechanism of apop-
tosis prevention in our experiments. Taurine may also
be able to rescue cells from apoptosis after the release
of cytochrome C from mitochondria. In ischemic cardi-
omyocytes taurine suppresses the formation of the
Apaf-1/caspase-9 apoptosome, prevents caspase-9 acti-
vation and thereby preserves cells from apoptosis [22].
All these mechanisms may explain why taurine protects
IGL neurons from ethanol-induced apoptosis in P7
mice.
It also seems important to consider another possibility
of taurine protection against the adverse effect of etha-
nol, which is not directly related to apoptosis. Alcohol
alters physical properties of membrane lipids, changing
membrane fluidity and affecting physiologically impor-
tant membrane enzymes, for instance, Na+/K+-ATPase
activity [56-59]. The effects of alcohol on biological
membranes depend on the duration of its administra-
tion. Acute ethanol administration increases the mem-
brane fluidity [60-62], whereas chronic alcohol intake
decreases the fluidity of membranes [60,63,64], which
become more tolerant to the disordering effect of etha-
nol [60,63]. Acute ethanol exposure also decreases Na+/
K+-ATPase activity in the cerebral cortex and brain
stem [65] and kidneys of adult rats [66].
The experimental data on the chronic alcohol effect
on Na+/K+-ATPase activity seem more contradictory.
For instance, chronic alcohol intake has enhanced Na+/
K+-ATPase activity in rat erythrocyte membranes [58]
and in the brain [67]. However, in other studies the
activity of Na+/K+-ATPase was significantly decreased
after chronic alcohol consumption in human erythrocyte
membranes [59] and in the brain and cerebellum of rat
offspring upon chronic alcohol exposure in utero [68].
Taurine also acts as a membrane stabilizer and restores
the depletion of Na+/K+-ATPase activity due to ozone
exposure and prevents the depletion of the enzyme
activity due to cholesterol enrichment [69]. Further-
more, taurine decreases the fluidity of biological mem-
branes when coadministrated with calcium [70]. It
seems thus possible that taurine in addition to its anti-
apoptotic actions can reduce the adverse effects of acute
ethanol administration acting as membrane stabilizer by
the decreasing membrane fluidity and restoring the
Na+/K+-ATPase activity.
Hovewer, the above considerations do not explain why
taurine did not affect apoptosis of Purkinje cells on P4.
An explanation may be the different functional proper-
ties of the neurons studied. The granular neurons in the
IGL are glutamatergic and the Purkinje cells GABAergic.
Ethanol can induce apoptosis in the developing brain by
dual mechanisms, by either blocking NMDA receptors
and or by excessively activating GABAAreceptors [7].
Taurine itself is an inhibitory amino acid which mimics
GABA actions [71,72]. On the other hand, taurine inhi-
bits the glutamate-induced Ca2+elevation [73]. Another
simple explanation for different effects of taurine on
ethanol-induced apoptosis in Purkinje and IGL cells
may be the difference in time when the neuronal popu-
lations were studied. We studied P4 mice at 12 hours
and P7 mice at 8 hours after the first ethanol adminis-
tration. Taurine is readily excreted in urine and its
plasma concentration decreases during the experiments.
At 8 hours the concentration was still relatively high but
at 12 hours almost at the control level; it was not high
enough to protect Purkinje cells.
Conclusions
We show that acute alcohol administration induces
apoptosis in Purkinje cells and in cells in the internal
granule cell layer (IGL) of the cerebellum. The time
frame of sensitivity to ethanol administration is different
for Purkinje and IGL cells. Taurine application was neu-
roprotective against ethanol-induced apoptosis in cells
in the IGL. Prevention of caspase-3 activation and DNA
fragmentation by taurine in IGL cells is likely to be due
to one or several of the following mechanisms, including
the restoration of the pool of Bcl-2, regulation of intra-
cellular Ca2+and inhibition of caspase-9 activation. The
Taranukhin et al. Journal of Biomedical Science 2010, 17(Suppl 1):S12
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failure of prevention of ethanol-induced apoptosis by
taurine in Purkinje cells may result from the different
functional properties of the neurons studied. GABAergic
Purkinje cells are inhibitory and glutamatergic granular
cells excitatory. The decrease in the taurine level during
the experiments may also have an influence since Pur-
kinje cells were studied after a longer interval than IGL
cells.
List of abbreviations used
HPLC: high-performance liquid chromatography; P4: postnatal day 4; P7:
postnatal day 7; TUNEL: terminal deoxynucleotidyl transferase-mediated
dUTP nick end-labeling; OPA: o-phthaldialdehyde; ANOVA: analysis of
variance; IGL: internal granular cell layer; IR: immunoreactive; NMDA:
N-methyl-D-aspartate.
Acknowledgements
The authors are deeply grateful to Mrs. Raija Repo, Mrs. Irma Rantamaa and
Mrs. Ulla M. Jukarainen for excellent technical assistance. This study was
supported by the competitive research funding of the Pirkanmaa Hospital
District and the Finnish Foundation for Alcohol Studies.
This article has been published as part of Journal of Biomedical Science
Volume 17 Supplement 1, 2010: Proceedings of the 17th International
Meeting of Taurine. The full contents of the supplement are available online
at http://www.jbiomedsci.com/supplements/17/S1.
Author details
1Brain Research Center, University of Tampere Medical School, Tampere,
Finland.2Laboratory of Comparative Somnology and Neuroendocrinology,
Sechenov Institute of Evolutionary Physiology and Biochemistry, St.-
Petersburg, Russia.3Department of Developmental Biology, University of
Tampere Medical School and Department of Pathology, Tampere University
Hospital, Tampere, Finland.4Department of Paediatrics, Tampere University
Hospital, Tampere, Finland.
Authors’ contributions
AGT did all laboratory work and drafted the manuscript, EYT performed the
statistical analyses and prepared illustrations, IMP participated in invention of
the idea and design of the study, MPH was the expert in
immunohistochemistry and PS and SSO acted supervisors and composed
the final version of this article. All authors read and approved the
manuscript.
Competing interests
The authors declare that they have no competing interests.
Published: 24 August 2010
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doi:10.1186/1423-0127-17-S1-S12
Cite this article as: Taranukhin et al.: Neuroprotection by taurine in
ethanol-induced apoptosis in the developing cerebellum. Journal of
Biomedical Science 2010 17(Suppl 1):S12.
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