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TOXICOLOGICAL SCIENCES 122(2), 539550 (2011)

doi:10.1093/toxsci/kfr126
Advance Access publication May 19, 2011
Arsenite Exposure Downregulates EAAT1/GLAST Transporter
Expression in Glial Cells
Yaneth Castro-Coronel,*
,
Luz Mar a Del Razo, Miriam Huerta,* Angeles Hernandez-Lopez,* Arturo Ortega,* and
Esther Lo pez-Bayghen*
,1
*Departamento de Genetica y Biolog a Molecular and Departamento de Toxicolog a, Centro de Investigacion y de Estudios Avanzados del IPN,
Mexico, D.F. Mexico
1
To whom correspondence should be addressed at Instituto Politecnico Nacional, 2508, Col. San Pedro Zacatenco, Mexico D.F., CP 07360, Mexico.
Fax: 525-557-473-931. E-mail: ebayghen@cinvestav.mx.
Received March 2, 2011; accepted May 10, 2011
Chronic exposure to inorganic arsenic severely damages the
central nervous system (CNS). Glutamate (GLU) is the major
excitatory amino acid and is highly neurotoxic when levels in the
synaptic cleft are not properly regulated by a family of Na
1
-
dependent excitatory amino acid transporters. Within the
cerebellum, the activity of the Bergmann glia Na
1
-dependent
GLU/aspartate transporter (GLAST) excitatory amino acid
transporter 1 (EAAT1/GLAST) accounts for more than 90%
of GLU uptake. Because exposure to the metalloid arsenite
results in CNS toxicity, we examined whether EAAT1/GLAST
constitutes a molecular target. To this end, primary cultures of
chick cerebellar Bergmann glial cells were exposed to sodium
arsenite for 24 h, and EAAT1/GLAST activity was evaluated via
3
H-D-aspartate uptake. A sharp decrease in GLU transport was
observed, and kinetic studies revealed protein kinase A, protein
kinase C, and p38 mitogen-activated protein kinase-dependent
decreases in K
M
and V
max
concomitant with diminished chglast
transcription. To gain insight into the molecular mechanisms
involved in these phenomena, we investigated the generation of
reactive oxidative species and the lipid peroxidative damage
caused by arsenite exposure. None of these responses were found,
although we did observe an increase in nuclear factor (erythroid-
derived 2)-like 2 DNA-binding activity correlated with a rise in
total glutathione levels. Our results clearly suggest that EAAT1/
GLAST is a molecular target of arsenite and support the critical
involvement of glial cells in brain function and dysfunction.
Key Words: arsenic; glutamate transport; Bergmann glia;
chglast; arsenic metabolism; cerebellum; glutamate signaling.
Arsenic is one of the most important global environmental
toxicants. Humans are exposed to arsenic through the intake of
air, food, and water. Two main forms of inorganic arsenic (iAs)
are found in the environment: arsenite and arsenate (Abernathy
et al., 2003). Most arsenicism is related to drinking tainted
water over a long period of time, resulting in chronic toxicity
associated with a wide range of health problems from skin
cancer to cognitive impairment.
Epidemiological studies have suggested a correlation between
iAs exposure and neurotoxicity (Hall, 2002; Rahman et al., 2001).
Neurotoxic effects from chronic iAs exposure have been reported
in clinical cases and in animal models (Chattopadhyay et al.,
2002; Rodr guez et al., 2001). Acute occupational and environ-
mental exposure to arsenic compounds results in encephalopathy
and impairment of superior neurological functions, such as
learning and memory (Danan et al., 1984), concentration
(Mukherjee et al., 2003), verbal comprehension and attention
(Calderon et al., 2001; Gerr et al., 2000), diminished IQ
(Calderon et al., 2001; Wasserman et al., 2004), as well as
peripheral neuropathy (Hafeman et al., 2005) both in adults and
children. Studies in rodents have shown that arsenic exposure
alters locomotor activity (Bardullas et al., 2009; Chattopadhyay
et al., 2002; Itoh et al., 1990; Rodr guez et al., 2002; Schulz et al.,
2002) and performance in several learning tasks (Nagaraja and
Desiraju, 1994; Rodr guez et al., 2001, 2002).
Arsenic exposure causes alterations in neurotransmitter
systems, including the monoaminergic (Jana et al., 2006; Lin
et al., 2007; Nagaraja and Desiraju, 1993; Rodr guez et al.,
2001), cholinergic (Kobayashi et al., 1987; Nagaraja and
Desiraju, 1994), gamma aminoisobutyric acidergic, and
glutamatergic (Nagaraja and Desiraju, 1993). It also affects
brain development, specically the Purkinje cells of the
cerebellum (Dhar et al., 2005; Piao et al., 2005). Neuronal
damage has long been recognized to play a role in toxin-
induced loss of brain function. However, the mechanisms
underlying the neurotoxic effects of iAs exposure have not
been extensively investigated in vitro.
Glutamate (GLU) is the major excitatory neurotransmitter in
the central nervous system (CNS). A family of sodium-
dependent GLU transporters carries out the neurotransmitter
removal from the synaptic cleft. The bulk of GLU transport is
mediated through the excitatory amino acid transporter 1
(EAAT1/GLU/aspartate transporter [GLAST]) expressed in
Bergmann glial cells (BGC), which is of particular importance
The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: journals.permissions@oup.com

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in the cerebellum. BGC are a special type of radial glia that
extend their processes through the molecular layer of the
cerebellar cortex, enwrapping excitatory and inhibitory synap-
ses (Somogyi et al., 1990). Recent evidence suggests that these
cells might participate in neuronal communication and may
also constitute a neuronal reservoir (Anthony et al., 2004;
Malatesta et al., 2003). BGC cultured from chick cerebellum
are an excellent model for analyzing the molecular and cellular
basis of glial-neuronal signaling (Lopez-Bayghen et al., 2007).
Acting through ionotropic and metabotropic receptors, GLU
modies gene expression patterns at the transcriptional and
translational levels (Lopez-Bayghen and Ortega, 2010; Lopez-
Bayghen et al., 2007). BGC express the GLU transporter
EAAT1/GLAST, and the regulation of transcription and its
activity has been described (Lopez-Bayghen and Ortega, 2004;
Rosas et al., 2007). Plasma membrane GLU transporters are
also involved in the signaling transactions triggered by GLU
(Gegelashvili et al., 2007; Zepeda et al., 2009). Furthermore,
BGC and Purkinje cell morphology is modied in vivo by the
expression of Ca
2
-permeable ionotropic glutamate receptors.
Therefore, strong and continuous communication between
these two cell types is expected (Watanabe, 2002).
Few studies have explored the effects of arsenic exposure in
astrocytes either in vivo or in vitro (Fauconneau et al., 2002;
Opanashuk and Finkelstein, 1995). Exposure of cultured rat
astrocytes to high arsenite concentrations results in cytotoxicity
and DNA damage (Catanzaro et al., 2010). In general, glia cells
and radial glia in particular play a very important role in the
function and structure of glutamatergic synapses. The cellular
basis of arsenic neurotoxicity remains poorly understood;
therefore, in this study, we explored the effects of exposure to
low iAs levels in cultured chick BGC. We focused on GLU
transport because proper function is of paramount importance for
a healthy neuron-glia relationship. Our experiments demonstrate
that exposure to iAs downregulates EAAT1/GLAST activity and
expression by a mechanism that involves several protein kinases,
particularly p38 mitogen-activated protein kinase (p38
MAPK
).
MATERIALS AND METHODS
Reagents. Tissue culture reagents and TRIzol RNA extraction kit
were obtained from GE Healthcare (Carlsbad, CA). SB202190 (4-[4-(4-
uorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol) was obtained from
Tocris-Cookson (Bristol, UK). GLU; H89 (N-[2-(p-bromocinnamylamino)
ethyl]-5-isoquinolinesulfonamide dihydrochloride); PD98059 (2-[2-amino-
3-methoxyphenil]-4H-1-benzopyran-4-one); UO126 (1,4-diamino-2,3-
dicyano-1,4-bis-(o-aminophenylmercapto) butadiene monoethanolate); DTNB,
5,5#-dithiobis(2-nitrobenzoic acid); acetyl-CoA; N6,2#-0-dibutyryladenosine
3#, 5#-cyclic monophosphate sodium salt (dbcAMP); wortmannin (Wor);
bisindolylmaleimide I (Bis I); thiobarbituric acid (TBA); ferrous ascorbate
(Asc/Fe
2
); BSO, buthioninesulfoximine; TBHP, tert-butyl hydroperoxide;
TBA; phorbol 12-myristate 13-acetate (TPA); Forskolin (7b-acetoxy-8,13-
epoxy-1a,6b,9a-trihydroxylabd-14-en-11-one); arsenic acid, disodium salt
(Na
2
HAs
V
O
4
; > 99% pure); sodium m-arsenite (NaAs
III
O
2
; > 99% pure),
and dimethylarsinic acid [DMAs
V
; (CH3)
2
As
V
O(OH); 98% pure]; and
dimethyl sulfoxide were all obtained from Sigma-Aldrich Co. (San Luis,
MO). Methylarsonic acid (MAs
V
) and disodium salt [CH
3
As
V
O (ONa)
2
; 99%
pure] were obtained from Ventron (Danvers, MA). Working standards of these
arsenicals, which contained 1 lg of As/ml, were prepared daily from stock
solutions. Sodium borohydride (NaBH
4
) was obtained from EM Science
(Gibbstown, NJ). Ultrapure phosphoric acid was purchased from J. T. Baker
(Phillipsburg, NJ).
Cell culture and chemical treatment. Primary cultures of cerebellar BGC
were prepared from 14-day-old chick embryos as previously described (Ortega
et al., 1991). Cells (1 3 10
6
) were plated in plastic culture dishes or well plates
(Corning, NY) in Dulbeccos modied Eagles medium (DMEM) containing
10% fetal bovine serum (FBS), 2mM glutamine, and gentamicin (50 lg/ml) (all
obtained from GE Healthcare) and used on the fourth or fth day after culture.
Before any treatment, conuent monolayers were switched to low-serum
DMEM media (0.5% FBS) for 120 min and then treated as indicated in the
gure legends. An aqueous sterile stock solution of sodium arsenite (1M
Sigma-Aldrich Co.) was prepared and diluted with DMEM/0.5% fetal calf
serum to obtain the desired nal concentrations. For signaling analysis,
antagonists or inhibitors were added 30 min before sodium arsenite treatment.
Kinase activators or analogues were added to culture medium in concentrations
and for the times indicated in the Table 1.
Cytotoxicity assessment. Cell viability was measured after treatment with
sodium arsenite (time and concentrations indicated in the gure legends) by the
MTT reduction assay [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide] (Calbiochem, Merck) performed as described by Mosmann
(Mosmann, 1983). Cells were seeded on 24-well plates in 500 ll of culture
media and incubated for 24 h with sodium arsenite at 37C in a 5% CO
2
atmosphere. After that, cells were treated with the MTT reagent (5 mg/ml) for
approximately 4 h. An isopropanol:HCl solution was added to lyse the cells and
solubilize the colored crystals. The optical density of the samples was
determined at 630 nm using an ELISA plate reader Opsys MR (Dynex
Technologies, Frankfurt, Germany). The results obtained by the MTT method
were conrmed by the neutral red cytotoxicity assay, following the
manufacturer instructions (Sigma-Aldrich Co.). Briey, cells were seeded,
incubated, and exposed to sodium arsenite and were treated with the neutral red
reagent (0.33% in Dulbeccos PBS from Sigma-Aldrich Co.; 4 h). Cells were
washed with the xative solution and lysed; after resuspending with the
solubilization solution, the optical density at 490 nm was determined using in
the same ELISA plate reader mentioned before.
Transporter assays in BGC. The uptake of
3
H-D-aspartate (used as
a nonmetabolizable analogue of L-GLU) was performed as detailed elsewhere
(Ruiz and Ortega, 1995). Briey, the culture medium was exchanged with
solution A [25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Tris,
130mM NaCl, 5.4mM KCl, 1.8mM CaCl
2
, 0.8mM MgCl
2
, 33.3mM glucose,
and 1mM NaHPO
4
, pH 7.4], and the cells were preincubated for 30 min at 37C.
Subsequently, this medium was exchanged with solution A containing
3
H-D-
aspartate (0.4 lCi/ml) and incubated for 20 min. Thereafter, the medium was
removed by rapid aspiration and the monolayers were rapidly washed with ice-
cold solution A and solubilized with 0.1M NaOH. Aliquots of the suspension
were used for protein determination and liquid scintillation counting. As a control
for the uptake assay, nontreated cells were incubated with 1mM GLU at 20C for
30 min before the transport assay for an expected diminution in uptake (Gonzalez
and Ortega, 1997). All the uptake data were analyzed using the Sigma Plot
software (Systat Software Inc., Point Richmond, CA) and the Prism, GraphPad
Software (San Diego, CA). In kinetics experiments,
3
H-D-aspartate transport was
analyzed after BGC cultures were incubated with sodium arsenite (1.5lM)
during 24 h. Saturation curves were obtained by the addition of different
concentrations of unlabeled D-aspartate and the kinetic constants (V
max
and K
M
)
determined with a nonlinear regression analysis using Prism GraphPad Software.
Arsenic determination. Arsenic species (iAs and their methylated
metabolites [MAs and DMAs]) present in the culture cells and in the
supernatant media were analyzed by a recently developed HG-CT-AAS
technique using a PerkinElmer Analyst 400 spectrometer (PerkinElmer,
540 CASTRO-CORONEL ET AL.

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Norwalk, CT) equipped with the multiatomizer (Hernandez-Zavala et al., 2008).
Before analysis, each BGC sample was lysed in 1.25 ml of 0.5% solution of
Triton X100 (Sigma-Aldrich Co.) in deionized water. The protein concen-
trations in BGC lysates were determined using the reducing agent compatible
detergent compatible Protein Assay kit (BioRad Laboratories, Hercules, CA);
bovine serum albumin was used for assay calibration. BGC lysates or
supernatant media were digested in 3 ml of 2M ultrapure phosphoric acid at
90C for 4 h; after that, samples were treated with 2% L-cysteine hydrochloride
(EMD Chemicals Inc., Darmstadt, Germany) for 70 min at room temperature.
Treatment with cysteine reduces all pentavalent As species to trivalency.
Hydrides were generated from 0.5 ml aliquots of cysteine-treated samples by
reaction with NaBH
4
in a Tris-HCl (Sigma-Aldrich Co.) buffer (pH 6) as
previously described (Hernandez-Zavala et al., 2008). Although HG-CT-AAS
was developed for the oxidation statespecic speciation analysis of As, under
current operating conditions, both above-described procedures determined total
iAs (iAs
III
iAs
V
), MAs (MAs
III
MAs
V
), and DMAs (DMAs
III
DMAs
V
).
For quality control during arsenic speciation analysis, sample aliquots spiked
with arsenical standards were used. We obtained an accuracy of 99.6% and
a variation coefcient < 10% in samples prepared by triplicate. For BGC
lysates, arsenicals were expressed as ng As per mg of protein.
Reactive oxidative species detection. Reactive oxygen species (ROS)
generation was evaluated by ow cytometry using 5-(and-6)-carboxy-2#,7#-
dichlorouorescein diacetate (DA) method based on the ROS-dependent
oxidation of 2#, 7#-dichlorouorescin diacetate (DCFH) to uorescent
dichlorouorescein (Invitrogen Molecular Probes, Gaithersburg, MD) (Ali
et al., 1992). BGC cells (grown in p60 culture plates) were exposed to sodium
arsenite (1.5lM) for the indicated times at 37C. Then, DCFH-DA (15lM) was
added and the incubation extended for 20 min. The uorescence of the cells
was immediately measured by ow cytometry (FACScalibur, Becton Dick-
inson, Franklin Lakes, NJ). TBHP (100lM for 60 min) was used to generate
ROS (Adams et al., 1993).
Assay for lipid peroxidation. Lipid peroxidation was determined in BGC
based on the formation of TBA-reactive substance (TBARS). Malondialdehyde
(MDA), other aldehydes, and lipid hydroxy-peroxides are able to form adducts
with TBA (Sigma-Aldrich Co.). MDA concentration was used as an index of
lipid peroxidation using the TBARS method (Buege and Aust, 1978). Briey,
0.5% TBA, 5 ll of 3.75% butylated hydroxytoluene (Sigma-Aldrich Co.) in
methanol, and 5 ll of 1.5mM desferoxamine (Sigma-Aldrich Co.) were added
to 1 ml of BGC suspension. Aliquots of the suspension were used for protein
determination. Samples were then heated in a boiling water bath for 20 min and
cooled, and the absorbance was measured at 532 nm using a spectrophotometer
(UV-Vis Lambda-2S PerkinElmer, SpectraLab Scientic Inc., Canada).
Measurements are expressed as a percentage of the control. Asc/Fe
2
(2.5mM for 30 min) was used to induce lipoperoxidative damage.
Determination of glutathione levels. Total glutathione (GSH) was
measured, according to the method described by Beutler (Beutler and Kelly,
1963), using 5,5#-dithiobis-(2-nitrobenzoic acid) (DTNB, known as Ellmans
reagent, Sigma-Aldrich Co.) as key reagent. The absorbance of the yellow-
colored complex was measured at 412 nm. The results were expressed as nmol
per mg of protein. The GSH synthesis inhibitor BSO was added to a nal
concentration of 1mM.
Electrophoretic mobility shift assays. Nuclear extracts were prepared as
described previously (Lopez-Bayghen et al., 1996). All buffers contained
a protease inhibitor cocktail to prevent nuclear factor proteolysis. Protein
concentration was measured by the Bradford method (Bradford, 1976). Nuclear
extracts (approximately 20 lg) from cells (treated as indicated) were incubated
on ice with 1 lg of poly[deoxyinosinic-deoxycytidylic] as nonspecic
competitor (Amersham Biosciences, Buckinghamshire, UK) and 2 ng of
32
P-
end-labeled double-stranded oligonucleotides:
Nrf2: 5#-CTAGGCAGAATGCTGAGTCACGGTGGAA-3#,
activator protein-1 (AP-1) (SV40): 5#-CTAGTTCCGGCTGAGTCAT-
CAAGC-3#, and
nuclear factor kappa B (NFjB): 5#-CTAGTTGAGGGGACTTTCC-
CAGG -3#
The reaction mixtures were incubated for 15 min on ice and electrophoresed
through 6% polyacrylamide gels using a low-ionic strength 0.53 Tris/Borate/
EDTA buffer. The gels were dried and exposed to an autoradiographic lm or
scanned with a Typhoon Optical Scanner (Amersham Biosciences). Complex
specicity for AP-1 (SV40) and NFjB was assessed before by competition
assays and immunoEMSA (Aguirre et al., 2000; Mendez et al., 2005) and for
Nfr2 (Supplementary g. 1).
Quantitative reverse transcription-PCR. Real-time quantitative reverse
transcription-PCR (qRT-PCR) was performed by a two-step method.
Complementary DNA was generated from 1 lg of total RNA by Improm-II
reverse transcription system (Promega, Madison, WI) with oligo(dT) 18
(Fermentas, Glen Burnie, MA) as a primer and according to the
manufacturers instructions. PCR was performed by QuantiTect SYBR Green
PCR Kit (Qiagen, Valencia, CA) in a reaction volume of 20 ll. Triplicate
samples were subjected to quantitative PCR (qPCR) using 7300 Real-Time
PCR System (Applied Biosystems). The qPCR prole consisted of an initial
denaturation step at 95C for 10 min followed by 35 cycles of 95C for 30 s,
60C for 1 min, and 72C for 30 s. A melt curve stage was added. We used
TABLE 1
Signaling Treatments
Assays Action Name Final concentration Time
3
H-D-aspartate uptake assays and
transcription reporter assays
iAs (sodium arsenite) 1.5lM
GLU 1mM
PKC inhibitor Bis I 1lM 30 min before iAs
PKC activator TPA 100nM alone 24 h
50nM iAs 24 h
PI3K inhibitor. Wor 100nM 30 min before iAs
MAPK inhibitor UO126 50lM 30 min before iAs
MAPK inhibitor PD98059 50lM 30 min before iAs
PKA inhibitor H89 20lM 30 min before iAs
PKA activator dbcAMP 500lM 24 h
PKA activator Forskolin (FSK) 100lM 24 h
p38
MAPK
inhibitor SB202190 1lM 30 min before iAs
Vehicle Dimethyl sulfoxide
GSH determination GSH inhibitor BSO 1mM 30 min before iAs
ARSENITE DOWNREGULATES EAAT1/GLAST 541

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the following specic primers to amplify: chglast, GLASTF, 5#-
GGCTGCGGGCATTCCTC-3# and GLASTR, 5#-CGGAGACGATCCAA-
GAACCA-3#. S17 chick ribosomal protein messenger RNA (mRNA) was used
as an internal control. The oligonucleotides used were (1pM, each) S17s, 5#-
CCGCTGGATGCGCTTCATCAG-3# and S17as, 5#-TACACCCGTCTGGG-
CAAC-3#. PCR products were sequenced in order to verify their identity. The
relative abundance of GLAST mRNA is expressed as sample versus a control in
comparison to S17 chick ribosomal mRNA and was calculated using the 2

DDCT
method. Data are presented as mean values SEs and analyzed by
ANOVA (p < 0.05 was considered statistically signicant).
Transient transfections and reporter assays. The plasmid p800GLAST-
CAT contains the 5# noncoding region from the chglast (515 to 248, total
763 bp) cloned in the pCAT-BASIC vector (Promega) amplied by reverse
PCR (Lopez-Bayghen et al., 2003). Michael Gredes from Dr Yuspa laboratory
at NIH kindly donated the reporter vectors TPA-responsive element
chloramphenicol acetyltransferase (TRE-CAT) and cAMP-responsive element
(CRE)-CAT. Both contain the structural gene for CAT under the control of the
Herpes virus thymidine kinase promoter and ve SV40 AP-1 sites cloned
upstream for TRE-CAT or ve CRE elements for CRE-CAT (Rutberg et al.,
1999). pGL2-hARE-LUC contains the NQO-hARE site [antioxidant response
element from nicotinamide adenine dinucleotide phosphate:quinone oxidore-
ductase gene] cloned in the pGL2-Basic vector was kindly donated by Dr Phil
Jaiswal University of Maryland, School of Medicine (Dhakshinamoorthy and
Jaiswal, 2000). Three copies of NFjB element were cloned in pCAT-Promoter
(Promega) to produce NFjB-CAT (Mendez et al., 2005). Transient transfection
assays were performed in 80% conuent BGC cultures using a calcium
phosphate protocol with the indicated amount of puried plasmids. Under such
conditions, the transfection efcacy was close to 50% determined by
a transfection control (b-gal). Treatments were done 4 h posttransfection, and
cell harvesting for reporter assays was performed 24 h posttransfection. Protein
lysates were obtained as follows: cells were harvested in cold PBS buffer, lysed
with one freeze-thaw cycle, and centrifuged at 12,000 3 g for 1 min. Equal
amounts of protein lysates (80 lg) were incubated with 0.25 lCi of
14
C-
chloramphenicol (50 mCi/mmol) (Amersham Biosciences) and 0.8mM acetyl-
CoA (Sigma-Aldrich Co.) at 37C. Acetylated forms were separated by thin-
layer chromatography and quantied using a Typhoon radioactive image
analyzer and the ImageQuant software (GE Healthcare). CAT activities were
expressed as the acetylated fraction corrected for the activity in the pCAT-Basic
vector and are expressed as relative activities to nontreated control cell lysates.
The luciferase activity was determined using the Luciferase Assay System
(Promega). 24 h posttransfection, cells were processed for luciferase
measurement. Briey, protein lysates were obtained from cells harvested in
cold PBS and lysed in 100 ll of Reporter Lysis Buffer (Promega). Equal
amounts of protein lysates (~70 lg) were incubated with luciferase assay
reagent. Light detection was performed in a FluoroSkan Ascent FL 374
(Labsystems), and activity values were normalized to protein content.
Statistical analysis. In all cases, data are expressed as the mean values
(average) the SEs. A nonparametric one-way ANOVA (Kruskal-Wallis test)
was performed to determine signicant differences between conditions. When
these analyses indicated signicance (at the 0.05 level), a Dunns post hoc test
was used to determine which conditions were signicantly different from each
other with Prism, GraphPad Software.
RESULTS
iAs Exposure Impairs GLU Transport
To explore if EAAT1/GLAST constitutes a molecular target
for iAs, primary cultures of BGC were exposed to iAs, and the
EAAT1/GLAST transporter activity was evaluated via
3
H-D-
aspartate uptake assays. A clear decrease in GLU transport was
evident in cells treated with 1.5lM of sodium arsenite (iAs) for
24 and 48 h, a concentration that is epidemiologically relevant
in terms of human exposure (Fig. 1A, left panel). Treatment
with sodium arsenite in concentrations as low as 0.5lM for 24 h
also affected the transporter activity (Fig. 1A, right panel).
Kinetic analysis of the transporter showed an iAs-dependent
decrease in V
max
and K
M
(Fig. 1B).
MTT assays were performed to rule out decreased cell
viability being responsible for the diminished transporter
activity. As shown in Figure 1C, no cell death is associated
with exposure to 1.5lM of sodium arsenite for 24 h. MTT
values were unchanged when cells were iAs treated for 24 h
and changed to iAs-free media for an additional 24 h (Fig. 1C,
right panel). These results were corroborated using the neutral
red method with the same results (data not shown).
To gain insight into the molecular mechanisms of iAs
action on EAAT1/GLAST activity, we analyzed arsenic
metabolism in BGC. Two parameters were taken into
account. Concentration in cells (ng As per mg of protein)
gives potential information for dose-effect relation in tissue
burdens of arsenic species as a function of time. Relative
proportions of arsenic species allow us to assess the capacity
of cells to methylate iAs as a function of time. As clearly
shown in Figure 2, BGC accumulates iAs as well as its
metabolites methylarsenic and DMA, suggesting that
generation of ROS or even lipoperoxidative damages might
be plausible. However, neither an increase in ROS nor
a clear augmentation of lipid peroxidation was detected
(Figs. 3A and 3B). Nevertheless, we were able to detect
a rise in GSH levels, which seems to be dependent on
de novo synthesis as it is sensitive to BSO, an inhibitor of
gamma-glutamylcysteine synthetase (Fig. 3C). This re-
sponse correlates well with a specic increase in the DNA-
binding activity of nuclear factor (erythroid-derived 2)-like
2 (Nrf2), the transcription factor that acts as master regulator
of the antioxidant response. Under the same treatment
conditions, we observed a sevenfold increase in the reporter
vector pGL2-hARE-LUC activity (Figs. 3D and 3E). As
expected, there is an increase in AP-1 complexes, accom-
panied by a clear augmentation of the transcription of
a TRE-driven construct (TRE-CAT) (Figs. 3D and 3E).
There is no change in NFjB protein-DNA complexes from
control or iAs-treated cells (Fig. 3D) and NFjB-CAT did not
indicate any change in activity (Fig. 3E).
Long-term Exposure to iAs Results in chglast Transcriptional
Downregulation
To further explore the mechanism by which iAs impairs
GLU uptake, we quantied the transporters mRNA levels by
real-time PCR (qRT-PCR). The primers shown in panel A of
Figure 4 were used. As depicted in panel C of Figure 4, a sharp
decrease in chglast mRNA was detected. At this stage, we
could not rule out a decrease in chglast mRNA half-life due to
iAs exposure, so we decided to evaluate the transcriptional
activity of the chglast promoter (Rosas et al., 2007). The
542 CASTRO-CORONEL ET AL.

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construct is shown in panel D of Figure 4. Note the presence of
putative DNA-binding sites for several transcription factors.
Exposure of transfected BGC to iAs for 12 and 24 h resulted in
decreased chglast promoter activity. As an experimental
control, cells were exposed to 1mM GLU for 2 h (Rosas
et al., 2007). These results suggest that EAAT1/GLAST
transcription is the molecular target of iAs (Fig. 4).
Signaling Involved in iAs-Dependent Transcriptional Control
Previous work from our group established the pivotal role
of protein kinase C (PKC) in the regulation of EAAT1/
GLAST activity (Gonzalez and Ortega, 1997) and gene
expression (Espinoza-Rojo et al., 2000; Lopez-Bayghen et al.,
2003; Lopez-Bayghen and Ortega, 2004). Furthermore, iAs
activates PKC, phosphatidyl inositol 3 kinase (PI3K), and
extracellular regulated kinase (ERK) (Chen et al., 2000;
Qian et al., 2003; Zhou et al., 2004). Therefore, we decided
to explore if the effects of iAs exposure in BGC involve
PKC activation. To this end, we evaluated the iAs effect in
3
H-D-aspartate uptake in the presence of the PKC inhibitor
Bis I (see Table 1). As shown in panel A of Figure 5, Bis I
treatment prevents iAs-mediated decrease in GLU uptake.
FIG. 1. Exposure to iAs impairs GLU transport in BGC with no effect on cell survival. (A)
3
H-D-aspartate uptake assays in BGC following exposure to
sodium arsenite (iAs) for the indicated times (hours, left) and at the indicated concentrations (range 0.15lM, right panel). Nontreated cells (NT) were incubated
with 1mM GLU for 30 min as control for the uptake assay. (B) Effect of iAs on the kinetics of the Na

-dependent
3
H-D-aspartate transport. BGC cultures were
incubated with 1.5lM of sodium arsenite for 24 h prior to the assay. (C) Cell viability determination after iAs treatment for 24 (left) or 48 h with media change after
24 h of treatment. In all cases, error bars represent SE of the mean from three independent experiments performed in quadruplicate; nonparametric one-way
ANOVA was used to determine signicant differences *p < 0.05 and ***p < 0.001 in panel (A).
ARSENITE DOWNREGULATES EAAT1/GLAST 543

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PKC is a member of the AGC family (Pearce et al., 2010)
and is often activated in a PI3K-dependent manner.
Nevertheless, preincubation with the PI3K blocker Wor
did not prevent the iAs effect, suggesting that the ERK
pathway is not involved. In fact, treatment with the MEK
blockers PD98059 and UO126 did not abolish the iAs effect
(Fig. 5B). The same results were obtained in the correspond-
ing gene reporter assays (Fig. 5C).
Although less characterized, the cAMP pathway is also
involved in EAAT1/GLAST regulation (Espinoza-Rojo et al.,
2000). The protein kinase A (PKA) blocker H89 (20lM, 30
min before iAs) was effective in preventing the iAs effect on
3
H-D-aspartate uptake (Fig. 6A). Similarly, a nonhydrolyzable
cAMP analogue (dbcAMP), as well as a PKA activator
Forskolin (100lM, 24 h), mimicked the iAs effect in both
uptake and gene reporter assays (Fig. 6B). As expected, both
iAs and dbcAMP are capable of increasing the transcription of
a CRE-driven reporter (CRE-CAT in Fig. 6C).
Another member of the mitogen-activated kinases in-
volved in excitotoxic damage and in GLU-dependent
transcriptional control could participate in the iAs effect
(Zepeda et al., 2008). As clearly shown in Figure 7,
treatment with 1lM of the p38
MAPK
inhibitor SB2021 at
30 min before iAs prevented the iAs effect by
3
H-D-aspartate
uptake as well as the transcriptional control level, suggesting
direct involvement of this kinase in the iAs molecular
mechanism of action.
DISCUSSION
Cognitive deciencies associated with exposure to arsenic
have been well documented (Rodr guez et al., 2002).
Glutamatergic transmission is critical for synaptic plasticity,
and the associated molecular transactions correlate with
memory acquisition and formation (Gibbs et al., 2008;
McKinney, 2010). Astrocytes are the most abundant cells in
the CNS, yet have been regarded over the years as supportive
elements. A well-established exception is the functional role
for glia cells in the glutamatergic system. These cells
participate in neurotransmitter recycling via the GLU/gluta-
mine shuttle, by which glial cells clear the synaptic space by
taking up the transmitter, transforming it into glutamine, and
nally releasing it back to the neurons that hydrolyze it to
replenish the synaptic vesicles (Bak et al., 2006). A metabolic
coupling between glia and glutamatergic neurons has been
elegantly described as the astrocyte/neuron lactate shuttle
(Pellerin et al., 2007). In this context, we hypothesized that
astrocytes, rather than neurons, could be the target of iAs
toxicity in the CNS, particularly at very low doses. To
challenge our idea, we decided to explore a fundamental
astrocytic function, GLU uptake.
Exposure of astrocytes to low, but environmentally
relevant, concentrations of iAs impairs GLU transport due
to diminished transcription of the chglast gene. In this
scenario, GLU accumulates in the synaptic space leading rst
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M 1.5 iAs M 1.5 iAs
FIG. 2. Metabolites of arsenite (iAs) in exposed cultured BGC. methylarsenic (MMAs) and DMAs concentrations are shown normalized to cellular protein
content for cells exposed to 1.5lM of sodium arsenite for 24 h. Error bars: SEs of the mean values (N 3).
544 CASTRO-CORONEL ET AL.

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to the spillover of the transmitter and activation of extrasynaptic
receptors (Takayasu et al., 2009) and in the long term to the
transcriptional downregulation of glast, amplifying the effect. In
the absence of EAAT1/GLAST, a mild motor discoordination is
observed along with dramatic increases in cerebellar damage
after brain insults (Watase et al., 1998). Surprisingly, in EAAT1/
GLAST (/) mice, neither a morphological difference in the
cerebellar cortex cytoarchitecture nor a difference in the peak
amplitude of parallel ber excitatory postsynaptic currents was
found (Takayasu et al., 2005).
The signaling cascade affected by iAs in BGC is PKC
dependent but intracellular Ca
2
independent (data not
shown). Therefore, one could imagine that a novel PKC
isoform may be involved. Supporting this interpretation is the
fact that overexpression of the PKCe isoform in BGC
diminishes chglast transcription (Lopez-Bayghen and Ortega,
2004). Interestingly, iAs response involves p38
MAPK
, a known
PKCe substrate (Garczarczyk et al., 2009). p38
MAPK
s have
been reported to phosphorylate a broad range of nuclear
proteins, including transcription factors and regulators of
FIG. 3. Antioxidant response in BGC exposed to iAs. (A) ROS generation (uorescence relative units; TBHP). (B) Lipid peroxidation; Asc/Fe
2
: ferrous
ascorbate. (C) Intracellular GSH content; when indicated, 1mM BSO was applied before 1.5lM iAs. In all cases, each data point represents the mean of at least
three independent experiments performed in duplicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ##p < 0.001 compared with iAs alone; nonparametric one-way
ANOVA. (D and E) Nrf2 DNA-binding and transcriptional activity increases after iAs exposure. (D) Protein-DNA complex patterns obtained with nuclear extracts
from control (nontreated cells [NT]) or iAs-treated cells (1.5lM, 24 h) with the indicated labeled probes. (E) BGC were transfected with 1 lg of each indicated
reporter vector; schematic maps indicate the transcription factor element (and copy number) directing the promoter activity. Activities are expressed relative to
transfected but nontreated cultures. Mean values SEs from at least four independent experiments. ***p < 0.001 by ANOVA.
ARSENITE DOWNREGULATES EAAT1/GLAST 545

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chromatin remodeling (Cuadrado and Nebreda, 2010). We are
currently exploring those that have putative binding sites located
in the chglast promoter (Fig. 4).
The fact that BGC take up and metabolize iAs suggests
that the ROS generation could be a consequence of iAs
exposure. We could not detect ROS generation in the treated
cells. This unexpected result might be explained by the
kinetics of ROS generation, although the fact that iAs
treatment did not increase NFjB DNA-binding or tran-
scriptional activity makes this unlikely. An alternative
explanation for this result is the iAs-induced increase in
GSH levels (Fig. 3C), an effect that is also reected at the
nuclear level with the increase in Nrf2 DNA-binding and
transcriptional activity. Current work in our laboratory is
FIG. 4. Sodium arsenite exposure downregulates chglast gene expression. (A) chglast coding region amplied by qRT-PCR (schematic representation).
(B) qRT-PCR amplied products; S17 chick ribosomal protein mRNA, internal control. (C) qRT-PCR performed with 1 lg of total RNA obtained from cells
treated with iAs or GLU as indicated. (D) Schematic representation of p800GLASTCAT construct: 5# noncoding region of the chglast promoter. Binding sites for
several transcription factors are denoted. Cells were treated 4 h after transfection. Activities are expressed relative to control cells. In all cases, mean values SEs
from at least four independent experiments are shown; **p < 0.01 and ***p < 0.001 by ANOVA. CREB, cAMP-responsive element-binding protein; YY1, Ying
Yang 1; Sp-1, stimulatory protein 1; CEBP, CAAT-binding protein. Arrow indicates the putative transcription starting point.
546 CASTRO-CORONEL ET AL.

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aimed at the identication of genes modulated in an Nrf2-
mediated, iAs-dependent manner, one of which could be
EAAT1/GLAST.
In summary, we demonstrate that glial cells are susceptible
to iAs toxicity and that downregulation of EAAT1/GLAST
expression contributes and amplies neuronal damage. Future
work will address other glial functions affected by iAs
exposure, including metabolic coupling, to fully characterize
the molecular toxicology of iAs in the brain.
FIG. 5. PKC, but not PI3K or MAPK, is involved in iAs signaling in BGC.
BGC were exposed to 1.5lM iAs for 24 h and then assayed for
3
H-D-aspartate
uptake. Kinase inhibitors were added 30 min before treatment. (A) PKC inhibitor
(Bis I) and PKC activator (TPA). (B) PI3K (Wor) or MAPK inhibitors (UO126
and PD98059). Dimethyl sulfoxide: vehicle. (C) BGC cultures were transfected
with 3 lg of p800GLASTCAT and treated as indicated. Mean values SEs from
at least four independent experiments. **p < 0.01 and ***p < 0.001 in
a nonparametric one-way ANOVA. See Table 1 for experimental details.
FIG. 6. PKA is activated in iAs-treated BGC. (A)
3
H-D-aspartate
uptake assays in control or iAs-exposed cells with the PKA inhibitor H89
or the PKA activators, dibutyryl cAMP (dbcAMP) and Forskolin (FSK).
(B) and (C) Conuent cultures were transfected with 3 lg of p800GLAST-
CAT or CRE-CAT; in both cases, cells were treated as indicated (Table 1
and Materials and Methods section).**p < 0.01 and ***p < 0.001,
ANOVA.
ARSENITE DOWNREGULATES EAAT1/GLAST 547

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SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
FUNDING
CONACyT-Mexico (50414 to E.L.B.; 79502 to A.O.);
CONACyT-Mexico fellowship to Y.C.C.
ACKNOWLEDGMENTS
The technical assistance of Luz del Carmen Sanchez Pena,
Blanca Ibarra, and Gerardo Marmolejo is acknowledged. We
thank Bruno Meza for editorial assistance and Dr Amy E.
Cullinan for critical reading of the manuscript.
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