Arch Toxicol (2007) 81:251–259

DOI 10.1007/s00204-006-0152-7

INORGANIC COMPOUNDS

Sodium arsenite-induced inhibition of cell proliferation is related to inhibition of IL-2 mRNA expression in mouse activated T cells

Patricia Conde · Leonor C. Acosta-Saavedra · Raquel C. Goytia-Acevedo · Emma S. Calderon-Aranda

Received: 22 June 2006 / Accepted: 28 August 2006 / Published online: 29 September 2006© Springer-Verlag 2006

Abstract A proposed mechanism for the As-inducedinhibition of cell proliferation is the inhibition of IL-2secretion. However, the eVects of arsenite on IL-2mRNA expression or on the ERK pathway in acti-vated-T cells have not yet been described. We exam-ined the eVect of arsenite on IL-2 mRNA expression,cell activation and proliferation in PHA-stimulatedmurine lymphocytes. Arsenite (1 and 10 �M)decreased IL-2 mRNA expression, IL-2 secretion andcell proliferation. Arsenite (10 �M) strongly inhibitedERK-phosphorylation. However, the partial inhibition(50%) of IL-2 mRNA produced by 1 �M, consistentwith the eVects on IL-2 secretion and cell proliferation,could not be explained by the inhibition of ERK-phos-phorylation, which was not aVected at this concentra-tion. The inhibition of IL-2 mRNA expression causedby 1 �M could be associated to eVects on pathwayslocated downstream or parallel to ERK. Arsenite alsodecreased early activation (surface CD69+ expression)in both CD4+ and CD8+, and decreased total CD8+

count without signiWcantly aVecting CD4+, supportingthat the cellular immune response mediated by cyto-toxic T cells is an arsenic target. Thus, our resultssuggest that arsenite decreases IL-2 mRNA levels and

T-cell activation and proliferation. However, furtherstudies on the eVects of arsenite on IL-2 gene transcrip-tion and IL-2 mRNA stability are needed.

Keywords Sodium arsenite · IL-2 mRNA · T-cell activation · Cell proliferation · Phosphorylated ERKs

Introduction

Inorganic arsenic (As) is a contaminant distributedworldwide which exists in two valence states: arsenite(As3+) and arsenate (As5+). Arsenite is considered tobe the most toxic form and chronic ingestion ofarsenic-contaminated drinking water has been associ-ated with immunotoxicity (Burns and Munson 1993;Ostrosky-Wegman et al. 1991). Gallium arsenide sup-pressed primary antibody response and T cell prolifer-ation induced by mitogens or anti-CD3 plusinterleukin-2 (IL-2) (Burns and Munson 1993). Studieson murine and human lymphocytes stimulated withphytohemaglutinin (PHA) have shown that arseniteinhibits cell proliferation (Ostrosky-Wegman et al.1991; Goytia-Acevedo et al. 2003) and induces cellulararrest in S and G2 phases (Petres et al. 1977). Accord-ingly, a decrease in the mitogenic-induced response ofperipheral mononuclear cells was reported in humanssuVering arsenic-induced Bowen’s disease (Yu et al.1998). However, the mechanisms of As-induced inhibi-tion of cell proliferation have not yet been elucidated.Some mechanisms proposed, among others, are theinhibition of IL-2 secretion (Burns and Munson 1993)or a delay in T cell activation (Galicia et al. 2003).Indeed, in vitro studies have shown that As exposure:

P. Conde · L. C. Acosta-Saavedra · E. S. Calderon-Aranda (&)Sección Toxicología, Centro de Investigación y de Estudios Avanzados, CINVESTAV, P.O. Box 14-740, Mexico, D.F., 07360, Mexicoe-mail: scalder@cinvestav.mx

R. C. Goytia-AcevedoFacultad de Medicina, Universidad Juarez del Estado de Durango, Gomez Palacio, Durango, 03500, Mexico

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252 Arch Toxicol (2007) 81:251–259

(1) reduced IL-2 secretion without aVecting IL-2mRNA in human mononuclear cells (Vega et al. 1999);(2) delayed IL-2 production and secretion; and (3)arrested T cells in G0 (Galicia et al. 2003).

T cells play a central role in the initiation and regula-tion of cellular immune response, and several lines of evi-dence indicate that regulation of IL-2 expression takesplace largely at the transcription level (Crabtree andClipstone 1994; Modiano et al. 1999). IL-2 gene expres-sion and secretion are critical steps for proliferation, earlyT cell clonal expansion and diVerentiation of activated-Tlymphocytes. It is well known that T cell activationrequires both IL-2 and IL-2 receptor expression for pro-gression from G0 to G1 (Crabtree and Clipstone 1994;Modiano et al. 1999; Li et al. 1999; Koike et al. 2003). Tcell-activation normally requires recognition of speciWcantigens through T cell receptor (TCR) or cross-linkingof the complex-TCR with speciWc antibodies or mitogenssuch as PHA or concanavalin A. The engagement ofcomplex TCR starts a series of phenotypic changes,resulting in suboptimal activation of several second mes-senger pathways such as calcium mobilization, proteinkinase C (PKC) activation and phosphorylation of mito-gen-activated protein kinase (MAPK). Recently,Hughes-Fulford et al. (2005) provided evidence that Tcell activation is mediated by extracellular signal-regu-lated kinases (ERKs), PKC and by transient PKA signal-ing during early stages of activation. In addition to p21ras-mediated signaling, PKC/Raf-1/MEK-1/ERK2 phos-phorylation cascade is necessary (Cobb and Goldsmith1995) but not enough for IL-2 gene transcription(Izquierdo et al. 1993). It has also been shown, by usingMEK-1 inhibitors, that ineVective ERKs phosphoryla-tion results in unsuccessful trans-activation of IL-2 gene(Musgrave et al. 2004; Hughes-Fulford et al. 2005).

The promoter of the IL-2 gene contains binding sitesfor transcriptional factors such as NF-AT, NF-�B, AP-1 and OCT-binding proteins, whose cooperative bind-ing determines the balance of transcriptional activationof IL-2 (SerXing et al. 1989). In addition to transcrip-tional regulation of IL-2 expression, mRNA stability isan important post-transcriptional control point (Efratand Kaempfer 1984; Seko et al. 2004). The mRNA IL-2degradation rates depend on factors that controlmRNA stability, although the precise mechanismsremain unclear (Musgrave et al. 2004; Meisner et al.2004). In fact, CD28 and CD2 pathways participate inT cell activation, as co-stimulatory signals by stabilizingIL-2 mRNA (Seko et al. 2004).

It has been shown that arsenite modulates ERKactivation in unstimulated-lymphocytes (Hossain et al.2000). However, in activated T lymphocytes, the eVectsof arsenite on IL-2 mRNA expression and ERK, criti-

cal pathways involved in IL-2 transcription, have notyet been described, neither their relationship with theantiproliferative eVect.

Therefore, this study was carried out to examine theeVect of arsenite on IL-2 mRNA expression and cellactivation. For this purpose, IL-2 secretion, cell prolif-eration, ERK1/2 phosphorylation and surface CD69expression in CD8+ and CD4+ cells were determined inPHA-stimulated murine lymphocytes.

Materials and methods

Mice

Female C57BL/6 mice (6–8 weeks old) were obtainedfrom the animal facility at CINVESTAV and main-tained in sterile and isolated polycarbonate cages withcertiWed diet and sterile tap water ad libitum.

Mononuclear cell isolation

Mice were sacriWced by cervical dislocation; thenspleens were removed in sterile conditions and a sus-pension of splenocytes was obtained. Mononuclearcells were separated on Ficoll-Hypaque (NycomedPharma, Oslo, NW). Cells were resuspended in com-plete media consisting of RPMI 1640 (Gibco, GrandIsland, NY) supplemented with 20 mM HEPES and100 U/ml penicillin, 100 �g/ml streptomycin (SigmaChem. Co., St Louis, MO), 2 mM L-glutamine, 5% fetalbovine serum (Gibco, Grand Island, NY), 50 �M �-mercaptoethanol (Bio-Rad Laboratories, Hercules,CA) and incubated at 37°C and 5% CO2.

Mononuclear cell treatments

Mononuclear cells (1 £ 106 cells/ml) were cultured inRPMI 1640 complete media and at time zero, 10 �g/mlof PHA plus sodium arsenite (arsenite) at 1 or 10 �MWnal concentration were added. Cultures activated with10 �g/ml of PHA were used as positive control. The neg-ative control consisted of cells neither PHA-activatednor exposed to arsenite. The evaluated parameters wereat the times indicated for each experimental condition.

Arsenite concentrations were chosen consideringthat: (1) 1 �M was used previously to study its antipro-liferative eVects both in mouse and human mononu-clear cells (Goytia-Acevedo et al. 2003; Vega et al.1999; Galicia et al. 2003) and (2) this concentrationinhibited T cell proliferation by 50% in mouse cells(Goytia-Acevedo et al. 2003). In contrast, 10 �Marsenite almost completely inhibited mouse T cell

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Arch Toxicol (2007) 81:251–259 253

proliferation. Both concentrations are within the rangeof urinary As reported in children environmentallyexposed (Soto-Peña et al. 2006).

T cell proliferation assay

To study arsenite eVects on lymphocyte proliferation,cells (1 £ 106 cells/ml) in complete media (200 �l/well)were exposed simultaneously to arsenite (1 or 10 �M)and 10 �g/ml PHA and incubated for 48 h. Prolifera-tion was determined by incorporation of [3H]-thymi-dine. Cultures were pulsed with 0.5 �Ci/well of [3H]-thymidine (2 Ci/mmol; Amersham Life Science) 6 hbefore harvesting time. Cells were harvested on glass–Wber Wlters by using a Skatron combi cell harvester(Skatron Inst. Lier, Norway). The incorporation of[3H]-thymidine was measured in a scintillation counter(Beckman, LS 6500). Three independent experimentswere done by triplicate.

T cell viability assay

We tested arsenite (1 and 10 �M) cytotoxic eVects inPHA-stimulated T cells at 12, 24, 48 h by using the col-orimetric MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-bi-phenyl tetrazolium) assay previously reported by Car-michael et al. (1987). Three independent experimentswere done by triplicate.

Detection of secreted IL-2

Cells (1 £ 106 cells/ml) were exposed simultaneously toarsenite (1 or 10 �M) and PHA (10 �g/ml) and incu-bated for the times indicated. Supernatants were col-lected and stored at ¡20°C until use for cytokinedetermination by ELISA. Three independent experi-ments were done by triplicate.

IL-2 expression by RT-PCR

Total cellular RNA was extracted with guanidiniumisothiocyanate-phenol-chloroform (Chomczynski andSacchi 1987). Two micrograms of total RNA was usedto obtain cDNA and the expression of IL-2 mRNA wasevaluated for semiquantitative PCR. Housekeepinggene control was Glyceraldehyde-3-phosphate dehydro-genase (G3PDH) expression. Primers sequences forconventional PCR were: IL-2 sense-GTCACATTGACACTTGTGCTCC, IL-2 antisense-AGTCAAATCCAGAACATGCCG (Gao et al. 2001); G3PDH sense-CATGTAGGCCATGAGGTCCACCAC, G3PDHantisense-TGAAGGTCGGTGTGAACGGATTTGGC. The cDNA was ampliWed using a program of

30 cycles (94°C for 45 sec; 56°C for 45 sec; 72°C for 45sec) and ended with incubation for 7 min at 72°C.AmpliWcation products were 293 bp for IL-2 and 451 bpfor G3PDH, respectively. PCR products were separatedin 2% agarose gel and visualized by ethidium bromidestaining; densitometric analysis was made with a Gene-Genius imager processor (Syngene, Cambridge, UnitedKingdom). As an additional positive control for IL-2expression, we used cells stimulated with 25 ng/ml phor-bol 12-myristate 13-acetate (PMA) plus ionomicyn(1 �g/ml). Before evaluating the eVects of arsenite onIL-2 mRNA, the kinetics of IL-2 gene expression wasperformed in cells stimulated with 10 �g/ml PHA. Themaximum expression level was found at 24 h. Two inde-pendent experiments were done by triplicate.

CD69, CD4 and CD8 detection by Xow cytometry

Samples were triple stained with PerCP anti-mouseCD69 antibodies, FITC anti-mouse CD4 and PE anti-mouse CD8 antibodies (BD PharMingen, San Jose,CA, USA) for 30 min in the dark at room temperatureand Wxed with 0.5% paraformaldehyde. Cells wereanalyzed using a FACSCalibur (Becton Dickson, SanJose, CA, USA) with a 488-nm argon laser. Analysisgates were set for lymphocytes according to forwardand side scatter properties. Three independent experi-ments were done by triplicate.

ERK phosphorylation

Cells (10 ml, 1 £ 106 cells/ml) were treated for 20 minwith or without arsenite (1 or 10 �M) and simulta-neously stimulated with 10 �g/ml of PHA. As negativecontrol we used cells neither PHA-stimulated norexposed to arsenic. Cells were washed with cold TD(12 mM Tris-Base, 68 mM NaCl, 2.5 mM KCl, 0.4 mMmonobasic sodium phosphate, pH 7.4), and cellular pel-let was obtained by centrifugation at 13,000 rpm at 4°Cfor 5 min and then was suspended in two volumes of icecold buVer A [10 mM HEPES, 10 mM KCl, 0.1 mMEDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF,10 �g/ml Leupeptin, 5 �g/ml antipain, 5 �g/ml quimosta-tin, 1 �g/ml pepstatin, 5 �g/ml benzamidine (SigmaChem. Co., St Louis, MO)]. After incubation (15 min at4°C) one volume of 10% NP-40 was added. Aliquotswere stored at ¡87°C until use. Total protein concentra-tion was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). An equal amount ofprotein from each sample was separated by PAGE-SDS(polyacrylamide gel electrophoresis-sodium dodecylsulphate; 12.5% acrylamide) at 80V constant voltage.Proteins were transferred to PVDF membranes (Milli-

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pore Corporation, Bedford, MA, USA) by a HoeVersemidry system (Amersham Biosciences, Buckingham-shire, UK). Blots were blocked 1 h with 3% fat-freemilk, in phosphate saline buVer (PBS) pH 7.4 and spe-ciWc proteins were detected by using rabbit speciWc anti-bodies against phosphorylated-ERK1/2 (Cell SignalingTechnology, Inc, Beverly, MA, USA), followed by goatanti-rabbit-HRP (Zymed, Laboratories Inc., Carlsbad,CA). Protein bands were visualized by means of theECL detection kit (Amersham Biosciences, Bucking-hamshire, UK) and using X-ray Wlms (Kodak, Palo Alto,CA, USA). ERK phosphorylation levels were normal-ized by ERK2 content. The same membrane to detectedp-ERK, after stripped was re-probed to total ERK2immunodetection by rabbit speciWc antibody (Cell Sig-naling Technology, Inc, Beverly, MA, USA). Data fromtwo independent experiments were performed.

Statistical analysis

The diVerence between the variables within the treat-ments was compared by using a one-way analysis ofvariance (randomized block design) followed by Dun-

nett’s test. DiVerences were considered statistically sig-niWcant when P < 0.05.

Results

EVect of arsenite on IL-2 mRNA expression

To determine whether arsenite aVects IL-2 geneexpression, we examined IL-2 mRNA expression inPHA-stimulated cells treated with arsenite for 24 h(Fig. 1). As expected, IL-2 expression was induced inPHA-stimulated cells and reached its maximum eVectat 24 h. In PHA-stimulated cells, 1 �M arsenitedecreased IL-2 mRNA by 60%, whereas at 10 �M IL-2mRNA was undetectable. In contrast, arsenite (1, 10,and 100 �M) did not modify IL-2 mRNA levels in non-stimulated lymphocytes (Fig. 1).

EVect of arsenite on IL-2 secretion

To study the consequences of arsenite-induced inhibi-tion of IL-2 mRNA on IL-2 secretion, the amount of

Fig. 1 Arsenite decreased IL-2 mRNA expression in PHA-stim-ulated lymphocytes. Cells were stimulated with PHA (10 �g/ml)and exposed to arsenite (1 or 10 �M). After 24 h of treatment to-tal RNA was obtained, equal concentrations from each experi-mental condition were used to cDNA obtention and mRNA IL-2expression was evaluated by conventional RT-PCR. a Image ofIL-2 and G3PDH PCR ampliWcation products separated in a 2%agarose gel corresponding to results of a representative experi-ment from two independents. b IL-2 mRNA expression is shownas percentage (%) of the IL-2 band intensity from two indepen-

dent experiments shown in (a), after normalization relative to theG3PDH band density and considering IL-2 mRNA obtained inPHA-stimulated cells as 100%. Treatments were control, non-treated and unstimulated cells, PHA-stimulated cells, cells ex-posed to 1 �M arsenite, cells exposed to 10 �M arsenite, PHA-stimulated cells and exposed to 1 �M arsenite. PHA-stimulatedcells and exposed to 10 �M arsenite. Cells exposed to 100 �M so-dium arsenite. PMA + Ionomycin corresponds to a positive con-trol of IL-2 gene expression. MW is the molecular weight ladder

B

MW

A

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8

IL-2

exp

ress

ion

(%

)

PHA 10 µg/ml + - - + + - PMA/IonomycinSodium arsenite ( M) Control - 1 10 1 10 100

1 2 3 4 5 6 7 8

)

PHA g/ml + - - + + nSodium arsenite (µM) - 1 10 1 10 100

PHA 10 µg/ml + - - + + -

Sodium arsenite (µM) Control - 1 10 1 10 100 PMA/Ionomycin

G3PDH

IL-2290 bp

240 bp

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Arch Toxicol (2007) 81:251–259 255

IL-2 protein in supernatants was determined. Asexpected, PHA induced IL-2 secretion but 1 �M arse-nite decreased 50% the amount of IL-2 secreted inPHA-stimulated cells and a stronger suppression wasinduced by 10 �M (Fig. 2). The time course (72 h) ofarsenite (10 �M) on IL-2 secretion was followed andIL-2 remained undetectable in PHA-stimulated cellsfor the duration of the experiment. At 72 h, there wereno statistical diVerences in IL-2 secretion betweenPHA-stimulated control and PHA-stimulated cellsplus arsenite (1 �M), and this could be probably due toa decreased response of stimulated cells (Fig. 2).

EVect of arsenite on ERK1/2 phosphorylation

We examined the status of ERK1/2 phosphorylation toevaluate whether the negative eVects on IL-2 mRNAexpression and IL-2 secretion were related to altera-tions in ERK1/2 activation. The results showed aninduction of ERK1/2 phosphorylation in PHA-stimu-lated cells (Fig. 3). Arsenite (10 �M) almost completelyinhibited PHA-induced phosphorylation whereas 1 �Mfailed to aVect PHA-induced ERK1/2 phosphorylation(Fig. 3).

EVect of arsenite on surface CD69 expression and CD4+, CD8+ subsets and cell proliferation

In order to determine the eVect of arsenite on other Tcell activation parameters, the eVects of arsenite onsurface CD69 expression, an early marker of T cellactivation, on both total and activated T helper (CD4+)

and T cytotoxic (CD8+), as well as cell proliferationand viability of PHA-stimulated cells, were studied.

The surface CD69+ expression induced at 4 h inPHA-stimulated cells was markedly decreased (60%)by both concentrations of arsenite, as compared withcells uniquely stimulated with PHA (Fig. 4). Therewere no signiWcant eVects of arsenite (1 or 10 �M) onsurface CD69+ expression in both T unstimulated cellsubsets (Table 1). Arsenite, at both concentrations,decreased total CD8+ cells, without signiWcant eVectson total CD4+ (Table 1).

Arsenite eVects on proliferation were also evaluatedand, as expected, a concentration-dependent inhibitionof PHA-induced proliferation was observed (Table 2).Indeed, at 48 h arsenite (1 �M) decreased cell prolifer-ation by 50%, whereas 10 �M inhibited proliferationby 89%. Cytotoxicity determined in parallel showed asigniWcant reduction in cells exposed to 1 and 10 �Marsenite, as compared to PHA-stimulated cells(Table 2). Arsenite 1 �M decreased cell viability by15%, whereas arsenite 10 �M decrease by approxi-mately 32% from 12 h of treatment (Table 2).

Discussion

Apart from the implications discussed below, our datasuggest that the inhibitory eVects of arsenite on cellproliferation and IL-2 secretion are a consequence, atleast partly of the inhibition of IL-2 mRNA expressionand/or alteration of IL-2 mRNA stability. Our Wndingsare in agreement with previous studies reporting thatarsenite inhibits cell proliferation and IL-2 secretion(Goytia-Acevedo et al. 2003; Ostrosky-Wegman et al.1991; Vega et al. 1999; Galicia et al. 2003). Our dataare in agreement with recent data, which showed thatIL-2 secretion was negatively associated to urinary Asin children environmentally exposed to arsenic (Soto-Peña et al. 2006), and correlated with IL-2 mRNAexpression which was also negatively associated to Asexposure (Luna et al. 2003). We have shown that arse-nite decreased IL-2 mRNA expression in a concentra-tion-dependent manner which correlated with theinhibition of IL-2 secretion and cell proliferation.These Wndings are consistent with evidence concerningthe critical role of IL-2 gene expression for early T cellactivation (Modiano et al. 1999), where the ERK path-way plays a central role (Musgrave et al. 2004). Theyare also in agreement with previous studies showingthat arsenite modulates the expression of other genessuch as GM-CSF and TGF-� (Germolec et al. 1996).The inhibition of IL-2 mRNA found here is consistentwith the arrest of T cells in G0 induced by arsenite

Fig. 2 Arsenite inhibited IL-2 secretion in PHA-stimulated lym-phocytes. Cells were stimulated with PHA (10 �g/ml) and ex-posed to arsenite 1 or 10 �M. IL-2 secretion was evaluated byELISA at 24, 48 and 72 h. Each bar represents mean § SD of trip-licates from three independent experiments. The asterisk indi-cates P < 0.05 as compared to PHA-stimulated cells and symbolmeans P < 0.05 as compared to PHA-stimulated cells treated with1 �M arsenite

0

5

10

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24 h 48 h 72 h

IL-2

Co

nce

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PHA 10 µg/mlPHA+Sodium arsenite 1µMPHA+ Sodium arsenite 10 µM

*& *&

*

*

*&

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256 Arch Toxicol (2007) 81:251–259

(1.0 �M) in human mononuclear cells exposed in vitro(Galicia et al. 2003). Since IL-2 mRNA expression iscritical for cell cycle activation, the inhibition of itsexpression contributes to explain the arrest in G0.

However, our Wndings on IL-2 mRNA expression werenot in agreement with those of Vega et al. (1999) whoreported that IL-2 gene expression in human periph-eral blood mononuclear cells (PBMC) exposed in vitroto arsenite (0.01–1.0 �M) was unaVected in most of thesamples collected from seven adult individuals.

The strong inhibitory eVects of 10 �M arsenite onERK phosphorylation could be related to the inhibi-tion of IL-2 mRNA since previous studies have shownthe critical role played by ERK in the transactivationof IL-2 gene and the early T cell activation and prolif-eration (Crabtree and Clipstone 1994; Modiano et al.1999). In fact, the essential role of the ERK pathwayfor T cell activation has been demonstrated usinginhibitors of MEK-1 resulting in unsuccessful IL-2gene transactivation (Musgrave et al. 2004; Hughes-Fulford et al. 2005). To the best of our knowledge, thisis the Wrst study showing the inhibitory eVect of arse-nite (10 �M) on ERK phosphorylation in activated Tcells. Our Wndings are in agreement with data reportedby Doza et al. (1998) who also found that arsenite(500 �M) suppressed MAP kinase kinase (MAPKK)and MAP kinase (MAPK) activation in EGF-stimu-lated Swiss 3T3 and A431 cell lines. In marked con-trast, arsenite (3.2–200 �M) induced ERK activation innon-stimulated C141 (Huang et al. 1999), whereas

Fig. 3 EVects of arsenite on ERK1/2 phosphorylation induced byPHA. a The activation of ERK1/2 was determined by the level ofphosphoylated-ERK1/2 (p-ERK1/2) by Western blot in cellsstimulated 20 min with PHA (10 �g/ml), with or without arsenite1 or 10 �M. Total ERK2 level detected in the same membraneused for p-ERK1/2 is shown also in (a). The negative control con-

sisted of cells neither activated nor exposed to arsenite. Right lanecorresponds to a positive control for detection of p-ERK2 (com-mercially puriWed p-ERK2). b p-ERKs level is shown as the ratioof p-ERK/total ERK2. PHA-stimulated level of p-ERK1/2 wasconsidered as 100%. The images and data were representative ofan experiment from two independents

A

p-Erk2 (44kD)

PHA 10 µg/ml + Control + +Sodium arsenite (µM) - 1 10

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K1/

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/ER

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PHA 10 µg/ml + +Sodium arsenite (µM) - Control 1 10 p-ERK2

p-Erk1 (42kD)

Erk2

+

Fig. 4 Arsenite inhibited PHA-activation in T lymphocytes.Activation of T lymphocytes was evaluated through CD69expression by Xow cytometry at 4 h in the control (cells neitheractivated nor exposed to arsenite), PHA-stimulated cells (10 �g/ml PHA), and PHA-stimulated cells simultaneously treated witharsenite (1 or 10 �M). Results correspond to the sum of cellsCD69+CD4+ plus CD69+CD8+, expressed as percentage. The cellpercentage obtained in PHA-stimulated cells was considered as100%. Data correspond to mean § SD of three independentexperiments. The asterisk indicates P < 0.05 as compared toPHA-stimulated cells

CD

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tive

cells

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PHA 10 µg/ml Control + + + Sodium arsenite --- 1µM 10µM

* *

ontrol + + + ---

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Chevalier et al. (2000) did not Wnd eVect of 5-min treat-ment with sodium arsenite (0.2 mM) on basal levels ofERK1/2 phosphorylation of vascular smooth musclecells. Thus, it appears that the eVects of arsenite aredependent on the activation status. On the other hands,Tanaka-Kagawa et al. (2003) showed that arsenite(100 �mol/l) and arsenate (800 �mol/l) in normal humanepidermal keratinocytes induced activation of ERK1/2,JNK and p38; however, ERK1/2 phosphorylation

occurred prior than other MAPK and dependent atleast partially of phosphorylation of the epidermalgrowth factor receptor.

The partial inhibition (»50%) of IL-2 mRNA pro-duced by 1 �M, consistent with the eVects on IL-2secretion and cell proliferation, could not be explainedby the inhibition of ERK-phosphorylation, since thisconcentration was unable to aVect ERK-phosphoryla-tion. Consistent with this view, earlier reports haveshown that ERK phosphorylation is necessary but notsuYcient for activating IL-2 gene transcription (Cobband Goldsmith 1995). It has been recognized thatbesides transcription regulation of the IL-2 gene, post-transcriptional mechanisms involved in mRNA stabil-ity are also important for IL-2 expression (Seko et al.2004). The inhibition of IL-2 mRNA expression causedby 1 �M, could be associated to modulation on path-ways downstream or parallel to ERK´s, such as CD28or CD2, which are also important for IL-2 mRNA sta-bility and participate in T cell activation as co-stimula-tory signals (Seko et al. 2004). This association isexpected since previous studies on diVerent cell modelshave shown that arsenite aVects intracellular pathwaysand transcription factors involved in IL-2 gene expres-sion. Hu et al. (2002) reported that chronic exposure ofhuman Wbroblasts to arsenite (100 and 500 nM)decreased c-Jun, c-Fos, and Ref-1 protein levels,besides decreasing AP-1 and NF-�B activity. Arsenite(500 �M) also inhibited AP-1 transcriptional activityand CRE (cyclic AMP responsive element) bindingproteins in IL-1�-stimulated Caco-2 cells (Hershkoet al. 2003).

In addition, we evaluated if the decrease of IL-2mRNA could be explained by the citotoxicity of arse-nite. Cell death could not explained the eVect on IL-2mRNA, since arsenite induced citotoxicity by 20 and32% (in cells exposed to arsenite 1 and 10 �M, respec-tively) whereas IL-2 mRNA decreased by approxi-mately 50% (1 �M arsenite) and 100% (10 �Marsenite). Despite these diVerences on cell viability,and to avoid misinterpretation of IL-2 mRNA results,total RNA from cells upon the diVerent experimentalschedule, was adjusted to equal concentration forcDNA preparation and a housekeeping gene(G3PDH) was included as control. In our opinion,these arguments may suggest that arsenite-induceddecreasing of IL-2 mRNA levels was not due to celldeath.

Although we did not determine the mechanism ofIL-2 mRNA inhibition, the eVect could be associatedto the capability of arsenite to induce free radical oxy-gen species (ROS) through interaction with thiolgroups and arsenate acting as an uncoupler of oxidative

Table 1 EVects of arsenite on surface CD69 expression andCD4+, CD8+ subsets

The samples were stained with PerCP-anti-mouse CD69 antibod-ies, FITC-anti-mouse CD4 and PE-anti-mouse CD8 and analyzedby Xow cytometry. The percentage obtained in PHA-stimulatedcells was considered as 100%. Data correspond to mean § SDfrom three independent experiments by triplicate

*Statistically signiWcant (P < 0.05) compared to PHA-stimulatedcellsa Sodium arsenite

CD69+

CD4+ (%)

CD8+ (%)

CD4+ (%)

CD8+ (%)

PHA 10 �g/ml 100 § 16 100 § 29 100 § 2.8 100 § 1.8 PHA 10 �g/ml Arsenitea 1 �M 38 § 15* 28 § 10* 69 § 12 37 § 8*Arsenitea 10 �M 47 § 19* 28 § 10* 69 § 15 69 § 8*

Table 2 EVects of arsenite on cell proliferation and viability

Values represent mean § SD of triplicates from three indepen-dent experiments. Results are expressed as percentage (%) ofthymidine incorporation or viability in PHA-stimulated cells thatwas considered 100%

*SigniWcantly diVerent (P < 0.05) comparing with PHA-stimu-lated cellsa Proliferation was evaluated by [3 H]-Thymidine incorporationb Viability was determined by the MTT assayc Sodium arsenite

Time (h) Percentage

Proliferationa Viabilityb

PHA 10 �g/ml6 100 § 7 100 § 10 12 100 § 14 100 § 1124 100 § 8 100 § 3248 100 § 15 100 § 4

Arsenitec 1 �M6 80 § 11 94 § 2 12 36 § 13 85 § 4*24 82 § 7* 73 § 6*48 52 § 16* 85 § 5*

Arsenitec 10 �M6 25 § 14 98 § 612 32 § 4 68 § 6*24 7 § 2* 65 § 1*48 11 § 2* 67 § 2*

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phosphorylation (Snow 1992; Thomas et al. 2001; Linet al. 2001). In addition, it is well known that endoge-nous ROS modulate intracellular signals, such asprotein tyrosine phosphorylation participating in lym-phocyte activation and mitogenesis, and recently it hasbeen reported that a precise balance of ROS is impor-tant to determine the fate of signal activation (Paniet al. 2000; Hardy and Hunt 2004).

The results showing surface CD69+ expressiondecreased with both arsenite concentrations by 60%.NFAT and AP-1 regulate the eYcient transactivationof both CD69+ and IL-2 (Del Rio et al. 2004), thus thedecrease observed in CD69+ expression may beinvolved in the reduction of IL-2 expression and secre-tion. Our results are in agreement with data fromTenorio and Saavedra (2005) that found a downregula-tion in CD69+ surface expression due to arsenite expo-sure. Our Wndings that arsenite decreased earlyactivation (surface CD69+ expression) in both CD4+

and CD8+, and in total CD8+ count without signiW-cantly aVecting CD4+, are also in agreement with datarecently reported by Tenorio and Saavedra (2005), inCD4+ and CD8+ puriWed cells from PBMC. Indeed, ourstudy supports the idea that the cellular immuneresponse regulated by activated T cells, particularlythat mediated by cytotoxic T cells (CD8+), is a targetfor arsenic. These results may be related with theimmune depression, particularly cell-mediated protec-tion, a mechanism potentially underlying the develop-ment of cancer and intracellular infection associated toarsenic exposure.

In conclusion, our results suggest that arsenitedecreases IL-2 mRNA levels and consequently T-cellactivation. However, further studies on the eVects ofarsenite on IL-2 mRNA stability, which fall beyond thescope of the present study, are needed.

Acknowledgments We thank Dr. Mariano E. Cebrian forhelpful discussion and critical reading of the manuscript. Weappreciate the technical assistance of VH. Rosales and V. Nu-ñez. This work was partially supported by the Mexican Councilfor Science and Technology (Conacyt-34508-M and Conacyt-42297-M).

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