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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 cerebellumare 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

modifies gene expression patterns at the transcriptional and

translational levels (Lopez-Bayghen and Ortega 2010 Lopez-

Bayghen et al 2007) BGC express the GLU transporter

EAAT1GLAST 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 modified in vivo by the

expression of Ca 2thorn-permeable ionotropic glutamate receptorsTherefore 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 GLUtransport because proper function is of paramount importance for

a healthy neuron-glia relationship Our experiments demonstrate

that exposure to iAs downregulates EAAT1GLAST activity and

expression by a mechanism that involves several protein kinases

particularly p38 mitogen-activated protein kinase (p38MAPK)

MATERIALS AND METHODS

Reagents Tissue culture reagents and TRIzol RNA extraction kit

were obtained from GE Healthcare (Carlsbad CA) SB202190 (4-[4-(4-

fluorophenyl)-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 (14-diamino-23-

dicyano-14-bis-(o-aminophenylmercapto) butadiene monoethanolate) DTNB

55-dithiobis(2-nitrobenzoic acid) acetyl-CoA N62-0-dibutyryladenosine

3 5-cyclic monophosphate sodium salt (dbcAMP) wortmannin (Wor)

bisindolylmaleimide I (Bis I) thiobarbituric acid (TBA) ferrous ascorbate

(AscFe2thorn) BSO buthioninesulfoximine TBHP tert-butyl hydroperoxide

TBA phorbol 12-myristate 13-acetate (TPA) Forskolin (7b-acetoxy-813-

epoxy-1a6b9a-trihydroxylabd-14-en-11-one) arsenic acid disodium salt

(Na 2HAsVO4 gt 99 pure) sodium m-arsenite (NaAsIIIO2 gt 99 pure)

and dimethylarsinic acid [DMAsV (CH3)2AsVO(OH) 98 pure] and

dimethyl sulfoxide were all obtained from Sigma-Aldrich Co (San Luis

MO) Methylarsonic acid (MAsV) and disodium salt [CH3AsVO (ONa)2 99

pure] were obtained from Ventron (Danvers MA) Working standards of these

arsenicals which contained 1 lg of Asml were prepared daily from stock

solutions Sodium borohydride (NaBH4) 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 3 106) were plated in plastic culture dishes or well plates

(Corning NY) in Dulbeccorsquos modified Eaglersquos medium (DMEM) containing

10 fetal bovine serum (FBS) 2mM glutamine and gentamicin (50 lgml) (all

obtained from GE Healthcare) and used on the fourth or fifth day after culture

Before any treatment confluent monolayers were switched to low-serum

DMEM media (05 FBS) for 120 min and then treated as indicated in the

figure legends An aqueous sterile stock solution of sodium arsenite (1M

Sigma-Aldrich Co) was prepared and diluted with DMEM05 fetal calf

serum to obtain the desired final 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 figure legends) by theMTT reduction assay [ 3-(4 5-dimethyl-2-thiazolyl)-25-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 CO2

atmosphere After that cells were treated with the MTT reagent (5 mgml) for

approximately 4 h An isopropanolHCl 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 confirmed by the neutral red cytotoxicity assay following the

manufacturer instructions (Sigma-Aldrich Co) Briefly cells were seeded

incubated and exposed to sodium arsenite and were treated with the neutral red

reagent (033 in Dulbeccorsquos PBS from Sigma-Aldrich Co 4 h) Cells were

washed with the fixative solution and lysed after resuspending with the

solubilization solution the optical density at 490 nm was determined using inthe same ELISA plate reader mentioned before

Transporter assays in BGC The uptake of 3H-D-aspartate (used as

a nonmetabolizable analogue of L-GLU) was performed as detailed elsewhere

(Ruiz and Ortega 1995) Briefly the culture medium was exchanged with

solution A [25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Tris

130mM NaCl 54mM KCl 18mM CaCl2 08mM MgCl2 333mM glucose

and 1mM NaHPO4 pH 74] and the cells were preincubated for 30 min at 37C

Subsequently this medium was exchanged with solution A containing 3H-D-

aspartate (04 lCiml) 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 01M 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 (Gonzalezand 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 3H-D-aspartate transport was

analyzed after BGC cultures were incubated with sodium arsenite (15lM)

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



Norwalk CT) equipped with the multiatomizer (Hernandez-Zavala et al 2008)

Before analysis each BGC sample was lysed in 125 ml of 05 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 05 ml aliquots of cysteine-treated samples by

reaction with NaBH4 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 statendashspecific speciation analysis of As under

current operating conditions both above-described procedures determined total

iAs (iAsIII thorn iAsV) MAs (MAsIII thorn MAsV) and DMAs (DMAsIII thorn DMAsV)

For quality control during arsenic speciation analysis sample aliquots spikedwith arsenical standards were used We obtained an accuracy of 996 and

a variation coefficient lt 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 flow cytometry using 5-(and-6)-carboxy-27-

dichlorofluorescein diacetate (DA) method based on the ROS-dependent

oxidation of 2 7-dichlorofluorescin diacetate (DCFH) to fluorescent

dichlorofluorescein (Invitrogen Molecular Probes Gaithersburg MD) (Ali

et al 1992) BGC cells (grown in p60 culture plates) were exposed to sodium

arsenite (15lM) for the indicated times at 37C Then DCFH-DA (15lM) was

added and the incubation extended for 20 min The fluorescence of the cells

was immediately measured by flow 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) Briefly

05 TBA 5 ll of 375 butylated hydroxytoluene (Sigma-Aldrich Co) in

methanol and 5 ll of 15mM 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 Scientific Inc Canada)

Measurements are expressed as a percentage of the control AscFe2thorn

(25mM 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 55-dithiobis-(2-nitrobenzoic acid) (DTNB known as Ellmanrsquos

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 final

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 i ce wi th 1 lg of poly[deoxyinosinic-deoxycytidylic] as nonspecific

competitor (Amersham Biosciences Buckinghamshire UK) and 2 ng of 32P-

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 053 TrisBorate

EDTA buffer The gels were dried and exposed to an autoradiographic film or

scanned with a Typhoon Optical Scanner (Amersham Biosciences) Complex

specificity 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 fig 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 Bur nie MA) as a primer and accor ding to the

manufacturerrsquos 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 profile 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

3H-D-aspartate uptake assays and

transcription reporter assays

iAs (sodium arsenite) 15lM

GLU 1mM

PKC inhibitor Bis I 1lM 30 min before iAsPKC activator TPA 100nM alone 24 h

50nM thorn 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

p38MAPK inhibitor SB202190 1lM 30 min before iAs

Vehicle Dimethyl sulfoxide

GSH determination GSH inhibitor BSO 1mM 30 min before iAs

ARSENITE DOWNREGULATES EAAT1GLAST 541



the following specific 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 plusmn SEs and analyzed by

ANOVA ( p lt 005 was considered statistically significant)

Transient transfections and reporter assays The plasmid p800GLAST-

CAT contains the 5 noncoding region from the chglast (515 to thorn 248 total

763 bp) cloned in the pCAT-BASIC vector (Promega) amplified 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 five SV40 AP-1 sites cloned

upstream for TRE-CAT or five CRE elements for CRE-CAT (Rutberg et al

1999) pGL2-hARE-LUC contains the NQO-hARE site [antioxidant response

element from nicotinamide adenine dinucleotide phosphatequinone oxidore-

ductase gene] cloned in the pGL2-Basic vector was kindly donated by Dr Phil

Jaiswal University of Maryland School of Medicine (Dhakshinamoorthy andJaiswal 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 confluent BGC cultures using a calcium

phosphate protocol with the indicated amount of purified plasmids Under such

conditions the transfection efficacy 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 12000 3 g for 1 min Equal

amounts of protein lysates (80 lg) were incubated with 025 lCi of 14C-

chloramphenicol (50 mCimmol) (Amersham Biosciences) and 08mM acetyl-

CoA (Sigma-Aldrich Co) at 37C Acetylated forms were separated by thin-

layer chromatography and quantified 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-Basicvector 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 Briefly 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) plusmn the SEs A nonparametric one-way ANOVA (Kruskal-Wallis test)

was performed to determine significant differences between conditions When

these analyses indicated significance (at the 005 level) a Dunnrsquos post hoc test

was used to determine which conditions were significantly different from each

other with Prism GraphPad Software

RESULTS

iAs Exposure Impairs GLU Transport

To explore if EAAT1GLAST constitutes a molecular target

for iAs primary cultures of BGC were exposed to iAs and the

EAAT1GLAST transporter activity was evaluated via 3H-D-

aspartate uptake assays A clear decrease in GLU transport was

evident in cells treated with 15lM 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 05lM 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 cellviability being responsible for the diminished transporter

activity As shown in Figure 1C no cell death is associated

with exposure to 15lM 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 EAAT1GLAST 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 Relativeproportions 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) Th is re-

sponse correlates well with a specific 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 quantified the transporterrsquos 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




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 EAAT1GLAST

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 in3H-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) 3H-D-aspartate uptake assays in BGC following exposure to

sodium arsenite (iAs) for the indicated times (hours left) and at the indicated concentrations (range 01ndash5lM 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 thorn-dependent 3H-D-aspartate transport BGC cultures were

incubated with 15lM 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 significant differences p lt 005 and p lt 0001 in panel (A)




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 MEKblockers 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 EAAT1GLAST 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 on3H-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 p38MAPK inhibitor SB2021 at

30 min before iAs prevented the iAs effect by 3H-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 deficiencies associated with exposure to arsenic

have been well documented (Rodrıguez et al 2002)

Glutamatergic transmission is critical for synaptic plasticityand 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 GLUgluta-

mine shuttle by which glial cells clear the synaptic space by

taking up the transmitter transforming it into glutamine and

finally 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 lsquolsquoastrocyteneuron lactate shuttlersquorsquo

(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 first

0

20

40

60

80

100

0

20

40

60

80

100

00

02

04

06

08

10

12

14

16

MMAs

DMAs

0

20

40

80

100

120

0 3 6 12 24 h

A r s e n i c a l s

i n c e l l s

n g A s m g

p r o t e i n

n g A

s m l

0 3 6 12 24 h

A r s e

n i c a l s i n m e d i a ( )

A r s e

n i c a l s

i n c e l l s ( )

0 3 6 12 24 h0 3 6 12 24 h

iAs

A r s e n i c a l s

i n m e d i a

M15iAsM15iAs

M15iAs M15iAs

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 15lM of sodium arsenite for 24 h Error bars SEs of the mean values ( N frac14 3)




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 EAAT1GLAST 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 fiber excitatory postsynaptic currents was

found (Takayasu et al 2005)

The signaling cascade affected by iAs in BGC is PKC

dependent but intracellular Ca 2thorn 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 p38MAPK a known

PKCe substrate (Garczarczyk et al 2009) p38MAPKs 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 (fluorescence relative units TBHP) (B) Lipid peroxidation AscFe2thorn ferrous

ascorbate (C) Intracellular GSH content when indicated 1mM BSO was applied before 15lM iAs In all cases each data point represents the mean of at least

three independent experiments performed in duplicates p lt 005 p lt 001 p lt 0001 and p lt 0001 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 (15lM 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 plusmn SEs from at least four independent experiments p lt 0001 by ANOVA




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 reflected 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 amplified by qRT-PCR (schematic representation)

(B) qRT-PCR amplified 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 plusmn SEs

from at least four independent experiments are shown p lt 001 and p lt 0001 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




aimed at the identification of genes modulated in an Nrf2-

mediated iAs-dependent manner one of which could be

EAAT1GLAST

In summary we demonstrate that glial cells are susceptible

to iAs toxicity and that downregulation of EAAT1GLAST

expression contributes and amplifies 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 15lM iAs for 24 h and then assayed for 3H-D-aspartateuptake 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 plusmn SEs from

at least four independent experiments p lt 001 and p lt 0001 in

a nonparametric one-way ANOVA See Table 1 for experimental details

FIG 6 PKA is activated in iAs-treated BGC (A) 3H-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) Confluent cultures were transfected with 3 lg of p800GLAST-

CAT or CRE-CAT in both cases cells were treated as indicated (Table 1

and lsquolsquoMaterials and Methodsrsquorsquo section) p lt 001 and p lt 0001ANOVA




SUPPLEMENTARY DATA

Supplementary data are available online at httptoxsci

oxfordjournalsorg

FUNDING

CONACyT-Mexico (50414 to ELB 79502 to AO)CONACyT-Mexico fellowship to YCC

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

REFERENCES

Abernathy C O Thomas D J and Calderon R L (2003) Health effects

and risk assessment of arsenic J Nutr 133 1536Sndash1538S

Adams J D Jr Wang B Klaidman L K LeBel C P Odunze I N and

Shah D (1993) New aspects of brain oxidative stress induced by tert-

butylhydroperoxide Free Radic Biol Med 15 195ndash202

Aguirre A Lopez T Lopez-Bayghen E and Ortega A (2000) Glutamateregulates kainate-binding protein expression in cultured chick Bergmann glia

through an activator protein-1 binding site J Biol Chem 275 39246ndash39253

Ali S F LeBel C P and Bondy S C (1992) Reactive oxygen species

formation as a biomarker of methylmercury and trimethyltin neurotoxicity

Neurotoxicology 13 637ndash648

Anthony T E Klein C Fishell G and Heintz N (2004) Radial glia serve

as neuronal progenitors in all regions of the central nervous system Neuron

41 881ndash890

Bak L K Schousboe A and Waagepetersen H S (2006) The glutamate

GABA-glutamine cycle aspects of transport neurotransmitter homeostasis

and ammonia transfer J Neurochem 98 641ndash653

Bardullas U Limon-Pacheco J H Giordano M Carrizales L Mendoza-

Trejo M S and Rodriguez V M (2009) Chronic low-level arsenic

exposure causes gender-specific alterations in locomotor activity dopami-nergic systems and thioredoxin expression in mice Toxicol Appl

Pharmacol 239 169ndash177

Beutler E and Kelly B M (1963) The effect of sodium nitrite on red cell

GSH Experientia 19 96ndash97

Bradford M M (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding Anal Biochem 72 248ndash254

Buege J A and Aust S D (1978) Microsomal lipid peroxidation Methods

Enzymol 52 302ndash310

Calderon J Navarro M E Jimenez-Capdeville M E Santos-Diaz M A

Golden A Rodriguez-Leyva I Borja-Aburto V and Diaz-Barriga F

(2001) Exposure to arsenic and lead and neuropsychological development in

Mexican children Environ Res 85 69ndash76

Catanzaro I Schiera G Sciandrello G Barbata G Caradonna F Proia P

and Di Liegro I (2010) Biological effects of inorganic arsenic on primary

cultures of rat astrocytes Int J Mol Med 26 457ndash462

Chattopadhyay S Bhaumik S Nag Chaudhury A and Das Gupta S

(2002) Arsenic induced changes in growth development and apoptosis in

neonatal and adult brain cells in vivo and in tissue culture Toxicol Lett 128

73ndash84

Chen N Y Ma W Y Huang C Ding M and Dong Z (2000) Activation

of PKC is required for arsenite-induced signal transduction J Environ

Pathol Toxicol Oncol 19 297ndash305

Cuadrado A and Nebreda A R (2010) Mechanisms and functions of p38

MAPK signalling Biochem J 429 403ndash417

Danan M Dally S and Conso F (1984) Arsenic-induced encephalopathy

Neurology 34 1524

Dhakshinamoorthy S and Jaiswal A K (2000) Small maf (MafG and MafK)

proteins negatively regulate antioxidant response element-mediated expres-

sion and antioxidant induction of the NAD(P)H quinone oxidoreductase1

gene J Biol Chem 275 40134ndash40141

Dhar P Jaitley M Kalaivani M and Mehra R D (2005) Preliminary

morphological and histochemical changes in rat spinal cord neurons

following arsenic ingestion Neurotoxicology 26 309ndash320

Espinoza-Rojo M Lopez-Bayghen E and Ortega A (2000) GLAST gene

expression regulation by phorbol esters Neuroreport 11 2827ndash2832

Fauconneau B Petegnief V Sanfeliu C Piriou A and Planas A M

(2002) Induction of heat shock proteins (HSPs) by sodium arsenite in

FIG 7 Involvement of p38MAPK in iAs effect (A) Uptake assays in the

presence of the p38MAPK inhibitor SB202190 prior to iAs treatment (B)

Transfected BGC cultures (3 lg of p800GLASTCAT) treated as indicated p

lt 001 and p lt 0001 in a nonparametric one-way ANOVA See Table 1 for

experimental details




cultured astrocytes and reduction of hydrogen peroxide-induced cell death J

Neurochem 83 1338ndash1348

Garczarczyk D Toton E Biedermann V Rosivatz E Rechfeld F

Rybczynska M and Hofmann J (2009) Signal transduction of

constitutively active protein kinase C epsilon Cell Signal 21 745ndash752

Gegelashvili M Rodriguez-Kern A Sung L Shimamoto K and

Gegelashvili G (2007) Glutamate transporter GLASTEAAT1 directs cell

surface expression of FXYD2gamma subunit of Na K-ATPase in humanfetal astrocytes Neurochem Int 50 916ndash920

Gerr F Letz R Ryan P B and Green R C (2000) Neurological effects of

environmental exposure to arsenic in dust and soil among humans


Gibbs M E Hutchinson D and Hertz L (2008) Astrocytic involvement in

learning and memory consolidation Neurosci Biobehav Rev 32 927ndash944

Gonzalez M I and Ortega A (1997) Regulation of the Na thorn-dependent high

affinity glutamateaspartate transporter in cultured Bergmann glia by phorbol

esters J Neurosci Res 50 585ndash590

Hafeman D M Ahsan H Louis E D Siddique A B Slavkovich V

Cheng Z van Geen A and Graziano J H (2005) Association between

arsenic exposure and a measure of subclinical sensory neuropathy in

Bangladesh J Occup Environ Med 47 778ndash784

Hall A H (2002) Chronic arsenic poisoning Toxicol Lett 128 69ndash72

Hernandez-Zavala A Valenzuela O L Matousek T Drobna Z Dedina J

Garcia-Vargas G G Thomas D J Del Razo L M and Styblo M

(2008) Speciation of arsenic in exfoliated urinary bladder epithelial cells

from individuals exposed to arsenic in drinking water Environ Health

Perspect 116 1656ndash1660

Itoh T Zhang Y F Murai S Saito H Nagahama H Miyate H Saito Y

and Abe E (1990) The effect of arsenic trioxide on brain monoamine

metabolism and locomotor activity of mice Toxicol Lett 54 345ndash353

Jana K Jana S and Samanta P K (2006) Effects of chronic exposure to

sodium arsenite on hypothalamo-pituitary-testicular activities in adult rats

possible an estrogenic mode of action Reprod Biol Endocrinol 4 9

Kobayashi H Yuyama A Ishihara M and Matsusaka N (1987) Effects of

arsenic on cholinergic parameters in brain in vitro Neuropharmacology 26

1707ndash1713

Lin C C Hsu C Hsu C H Hsu W L Cheng A L and Yang C H

(2007) Arsenic trioxide in patients with hepatocellular carcinoma a phase II

trial Invest New Drugs 25 77ndash84

Lopez-Bayghen E Espinoza-Rojo M and Ortega A (2003) Glutamate

down-regulates GLAST expression through AMPA receptors in Bergmann

glial cells Brain Res Mol Brain Res 115 1ndash9

Lopez-Bayghen E and Ortega A (2004) Glutamate-dependent transcrip-

tional regulation of GLAST role of PKC J Neurochem 91 200ndash209

Lopez-Bayghen E and Ortega A (2010) [Glial cells and synaptic activity

translational control of metabolic coupling] Rev Neurol 50 607ndash615

Lopez-Bayghen E Rosas S Castelan F and Ortega A (2007) Cerebellar

Bergmann glia an important model to study neuron-glia interactions Neuron

Glia Biol 3 155ndash167

Lopez-Bayghen E Vega A Cadena A Granados S E Jave L F

Gariglio P and Alvarez-Salas L M (1996) Transcriptional analysis of the 5rsquo-

noncoding region of the human involucrin gene J Biol Chem 271 512ndash520

Malatesta P Hack M A Hartfuss E Kettenmann H Klinkert W

Kirchhoff F and Gotz M (2003) Neuronal or glial progeny regional

differences in radial glia fate Neuron 37 751ndash764

McKinney R A (2010) Excitatory amino acid involvement in dendritic spine

formation maintenance and remodelling J Physiol 588 107ndash116

Mendez J A Lopez-Bayghen E and Ortega A (2005) Glutamate activation

of Oct-2 in cultured chick Bergmann glia cells involvement of NFkappaB J

Neurosci Res 81 21ndash30

Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival

application to proliferation and cytotoxicity assays J Immunol Methods 65

55ndash63

Mukherjee S C Rahman M M Chowdhury U K Sengupta M K Lodh D

Chanda C R Saha K C and Chakraborti D (2003) Neuropathy in arsenic

toxicity from groundwater arsenic contamination in West Bengal India

J Environ Sci Health A Tox Hazard Subst Environ Eng 38 165ndash183

Nagaraja T N and Desiraju T (1993) Regional alterations in the levels of brain biogenic amines glutamate GABA and GAD activity due to chronic

consumption of inorganic arsenic in developing and adult rats Bull Environ

Contam Toxicol 50 100ndash107

Nagaraja T N and Desiraju T (1994) Effects on operant learning and brain

acetylcholine esterase activity in rats following chronic inorganic arsenic

intake Hum Exp Toxicol 13 353ndash356

Opanashuk L A and Finkelstein J N (1995) Relationship of lead-induced

proteins to stress response proteins in astroglial cells J Neurosci Res 42

623ndash632

Ortega A Eshhar N and Teichberg V I (1991) Properties of kainate

receptorchannels on cultured Bergmann glia Neuroscience 41 335ndash349

Pearce L R Komander D and Alessi D R (2010) The nuts and bolts of

AGC protein kinases Nat Rev Mol Cell Biol 11 9ndash22

Pellerin L Bouzier-Sore A K Aubert A Serres S Merle M Costalat Rand Magistretti P J (2007) Activity-dependent regulation of energy

metabolism by astrocytes an update Glia 55 1251ndash1262

Piao F Ma N Hiraku Y Murata M Oikawa S Cheng F Zhong L

Yamauchi T Kawanishi S and Yokoyama K (2005) Oxidative DNA

damage in relation to neurotoxicity in the brain of mice exposed to arsenic at

environmentally relevant levels J Occup Health 47 445ndash449

Qian Y Castranova V and Shi X (2003) New perspectives in arsenic-

induced cell signal transduction J Inorg Biochem 96 271ndash278

Rahman M M Chowdhury U K Mukherjee S C Mondal B K Paul K

Lodh D Biswas B K Chanda C R Basu G K Saha K C et al

(2001) Chronic arsenic toxicity in Bangladesh and West Bengal Indiamdasha

review and commentary J Toxicol Clin Toxicol 39 683ndash700

Rodrıguez V M Carrizales L Jimenez-Capdeville M E Dufour L and

Giordano M (2001) The effects of sodium arsenite exposure on behavioralparameters in the rat Brain Res Bull 55 301ndash308

Rodrıguez V M Carrizales L Mendoza M S Fajardo O R and

Giordano M (2002) Effects of sodium arsenite exposure on development

and behavior in the rat Neurotoxicol Teratol 24 743ndash750

Rosas S Vargas M A Lopez-Bayghen E and Ortega A (2007)

Glutamate-dependent transcriptional regulation of GLASTEAAT1 a role

for YY1 J Neurochem 101 1134ndash1144

Ruiz M and Ortega A (1995) Characterization of an Na(thorn)-dependent

glutamateaspartate transporter from cultured Bergmann glia Neuroreport 6

2041ndash2044

Rutberg S E Adams T L Olive M Alexander N Vinson C and

Yuspa S H (1999) CRE DNA binding proteins bind to the AP-1 target

sequence and suppress AP-1 transcriptional activity in mouse keratinocytes

Oncogene 18 1569ndash1579Schulz H Nagymajtenyi L Institoris L Papp A and Siroki O (2002) A study

on behavioral neurotoxicological and immunotoxicological effects of sub-

chronic arsenic treatment in rats J Toxicol Environ Health A 65 1181ndash1193

Somogyi P Eshhar N Teichberg V I and Roberts J D (1990)

Subcellular localization of a putative kainate receptor in Bergmann glial cells

using a monoclonal antibody in the chick and fish cerebellar cortex

Neuroscience 35 9ndash30

Takayasu Y Iino M Kakegawa W Maeno H Watase K Wada K

Yanagihara D Miyazaki T Komine O Watanabe M et al (2005)

Differential roles of glial and neuronal glutamate transporters in Purkinje cell

synapses J Neurosci 25 8788ndash8793






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 cerebellumare 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

modifies gene expression patterns at the transcriptional and

translational levels (Lopez-Bayghen and Ortega 2010 Lopez-

Bayghen et al 2007) BGC express the GLU transporter

EAAT1GLAST 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 modified in vivo by the

expression of Ca 2thorn-permeable ionotropic glutamate receptorsTherefore 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 GLUtransport because proper function is of paramount importance for

a healthy neuron-glia relationship Our experiments demonstrate

that exposure to iAs downregulates EAAT1GLAST activity and

expression by a mechanism that involves several protein kinases

particularly p38 mitogen-activated protein kinase (p38MAPK)

MATERIALS AND METHODS

Reagents Tissue culture reagents and TRIzol RNA extraction kit

were obtained from GE Healthcare (Carlsbad CA) SB202190 (4-[4-(4-

fluorophenyl)-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 (14-diamino-23-

dicyano-14-bis-(o-aminophenylmercapto) butadiene monoethanolate) DTNB

55-dithiobis(2-nitrobenzoic acid) acetyl-CoA N62-0-dibutyryladenosine

3 5-cyclic monophosphate sodium salt (dbcAMP) wortmannin (Wor)

bisindolylmaleimide I (Bis I) thiobarbituric acid (TBA) ferrous ascorbate

(AscFe2thorn) BSO buthioninesulfoximine TBHP tert-butyl hydroperoxide

TBA phorbol 12-myristate 13-acetate (TPA) Forskolin (7b-acetoxy-813-

epoxy-1a6b9a-trihydroxylabd-14-en-11-one) arsenic acid disodium salt

(Na 2HAsVO4 gt 99 pure) sodium m-arsenite (NaAsIIIO2 gt 99 pure)

and dimethylarsinic acid [DMAsV (CH3)2AsVO(OH) 98 pure] and

dimethyl sulfoxide were all obtained from Sigma-Aldrich Co (San Luis

MO) Methylarsonic acid (MAsV) and disodium salt [CH3AsVO (ONa)2 99

pure] were obtained from Ventron (Danvers MA) Working standards of these

arsenicals which contained 1 lg of Asml were prepared daily from stock

solutions Sodium borohydride (NaBH4) 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 3 106) were plated in plastic culture dishes or well plates

(Corning NY) in Dulbeccorsquos modified Eaglersquos medium (DMEM) containing

10 fetal bovine serum (FBS) 2mM glutamine and gentamicin (50 lgml) (all

obtained from GE Healthcare) and used on the fourth or fifth day after culture

Before any treatment confluent monolayers were switched to low-serum

DMEM media (05 FBS) for 120 min and then treated as indicated in the

figure legends An aqueous sterile stock solution of sodium arsenite (1M

Sigma-Aldrich Co) was prepared and diluted with DMEM05 fetal calf

serum to obtain the desired final 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 figure legends) by theMTT reduction assay [ 3-(4 5-dimethyl-2-thiazolyl)-25-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 CO2

atmosphere After that cells were treated with the MTT reagent (5 mgml) for

approximately 4 h An isopropanolHCl 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 confirmed by the neutral red cytotoxicity assay following the

manufacturer instructions (Sigma-Aldrich Co) Briefly cells were seeded

incubated and exposed to sodium arsenite and were treated with the neutral red

reagent (033 in Dulbeccorsquos PBS from Sigma-Aldrich Co 4 h) Cells were

washed with the fixative solution and lysed after resuspending with the

solubilization solution the optical density at 490 nm was determined using inthe same ELISA plate reader mentioned before

Transporter assays in BGC The uptake of 3H-D-aspartate (used as

a nonmetabolizable analogue of L-GLU) was performed as detailed elsewhere

(Ruiz and Ortega 1995) Briefly the culture medium was exchanged with

solution A [25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Tris

130mM NaCl 54mM KCl 18mM CaCl2 08mM MgCl2 333mM glucose

and 1mM NaHPO4 pH 74] and the cells were preincubated for 30 min at 37C

Subsequently this medium was exchanged with solution A containing 3H-D-

aspartate (04 lCiml) 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 01M 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 (Gonzalezand 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 3H-D-aspartate transport was

analyzed after BGC cultures were incubated with sodium arsenite (15lM)

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































































CAAGC-3 and


CAGG -3



















TABLE 1






GLU 1mM


50nM thorn iAs 24 h































































RESULTS
















































































































mechanism of action

DISCUSSION

























0

20

40

60

80

100

0

20

40

60

80

100

00

02

04

06

08

10

12

14

16

MMAs

DMAs

0

20

40

80

100

120

0 3 6 12 24 h

A r s e n i c a l s

i n c e l l s

n g A s m g

p r o t e i n

n g A

s m l

0 3 6 12 24 h

A r s e


A r s e

n i c a l s

i n c e l l s ( )

0 3 6 12 24 h0 3 6 12 24 h

iAs

A r s e n i c a l s

i n m e d i a

M15iAsM15iAs

M15iAs M15iAs





























































EAAT1GLAST























SUPPLEMENTARY DATA


oxfordjournalsorg

FUNDING


ACKNOWLEDGMENTS





REFERENCES













41 881ndash890

























73ndash84







Neurology 34 1524






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044















































































CAAGC-3 and


CAGG -3



















TABLE 1






GLU 1mM


50nM thorn iAs 24 h































































RESULTS
















































































































mechanism of action

DISCUSSION

























0

20

40

60

80

100

0

20

40

60

80

100

00

02

04

06

08

10

12

14

16

MMAs

DMAs

0

20

40

80

100

120

0 3 6 12 24 h

A r s e n i c a l s

i n c e l l s

n g A s m g

p r o t e i n

n g A

s m l

0 3 6 12 24 h

A r s e


A r s e

n i c a l s

i n c e l l s ( )

0 3 6 12 24 h0 3 6 12 24 h

iAs

A r s e n i c a l s

i n m e d i a

M15iAsM15iAs

M15iAs M15iAs





























































EAAT1GLAST























SUPPLEMENTARY DATA


oxfordjournalsorg

FUNDING


ACKNOWLEDGMENTS





REFERENCES













41 881ndash890

























73ndash84







Neurology 34 1524






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044






































































RESULTS
















































































































mechanism of action

DISCUSSION

























0

20

40

60

80

100

0

20

40

60

80

100

00

02

04

06

08

10

12

14

16

MMAs

DMAs

0

20

40

80

100

120

0 3 6 12 24 h

A r s e n i c a l s

i n c e l l s

n g A s m g

p r o t e i n

n g A

s m l

0 3 6 12 24 h

A r s e


A r s e

n i c a l s

i n c e l l s ( )

0 3 6 12 24 h0 3 6 12 24 h

iAs

A r s e n i c a l s

i n m e d i a

M15iAsM15iAs

M15iAs M15iAs





























































EAAT1GLAST























SUPPLEMENTARY DATA


oxfordjournalsorg

FUNDING


ACKNOWLEDGMENTS





REFERENCES













41 881ndash890

























73ndash84







Neurology 34 1524






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044









































































mechanism of action

DISCUSSION

























0

20

40

60

80

100

0

20

40

60

80

100

00

02

04

06

08

10

12

14

16

MMAs

DMAs

0

20

40

80

100

120

0 3 6 12 24 h

A r s e n i c a l s

i n c e l l s

n g A s m g

p r o t e i n

n g A

s m l

0 3 6 12 24 h

A r s e


A r s e

n i c a l s

i n c e l l s ( )

0 3 6 12 24 h0 3 6 12 24 h

iAs

A r s e n i c a l s

i n m e d i a

M15iAsM15iAs

M15iAs M15iAs





























































EAAT1GLAST























SUPPLEMENTARY DATA


oxfordjournalsorg

FUNDING


ACKNOWLEDGMENTS





REFERENCES













41 881ndash890

























73ndash84







Neurology 34 1524






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044











































































EAAT1GLAST























SUPPLEMENTARY DATA


oxfordjournalsorg

FUNDING


ACKNOWLEDGMENTS





REFERENCES













41 881ndash890

























73ndash84







Neurology 34 1524






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044




















SUPPLEMENTARY DATA


oxfordjournalsorg

FUNDING


ACKNOWLEDGMENTS





REFERENCES













41 881ndash890

























73ndash84







Neurology 34 1524






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044






















































1707ndash1713



























55ndash63













623ndash632



























2041ndash2044


















toxicol. sci. 2011 castro coronel 539 50

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