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International Immunopharmacology 3 (2003) 671–682

Sodium arsenite retards proliferation of PHA-activated T cells

by delaying the production and secretion of IL-2

Georgina Galiciaa, Rosario Leyvaa, Eda Patricia Tenorioa,Patricia Ostrosky-Wegmanb, Rafael Saavedraa,*

aDepartamento de Inmunologıa y Enfermedades Infecciosas, Instituto de Investigaciones Biomedicas,

Universidad Nacional Autonoma de Mexico, Apartado Postal 70228, CU, Mexico City CP 04510, MexicobDepartamento de Medicina Genomica y Toxicologıa Ambiental, Instituto de Investigaciones Biomedicas,

Universidad Nacional Autonoma de Mexico, Mexico City, Mexico

Received 1 October 2002; received in revised form 15 January 2003; accepted 13 February 2003

Abstract

Arsenic is a metalloid that commonly contaminates drinking water, and is a known human carcinogen. It has been shown that

peripheral blood mononuclear cells (PBMCs) from healthy donors treated in vitro with NaAsO2 and stimulated with

phytohemagglutinin (PHA) show a lower proliferation than nontreated cells.We reported previously a reduction in the secretion of

IL-2 in NaAsO2-treated PBMCs stimulated with PHA, an observation that might explain, in part, the reduction in proliferation.

Since arsenic induces cystoskeleton alterations, which in turn may affect protein transport of the cell, we assumed that NaAsO2

induced an accumulation of IL-2 inside the cells, and thus a reduction in the secretion of IL-2. In order to demonstrate this

hypothesis, we assessed the intracellular IL-2 at the single cell level by flow cytometry, and unexpectedly found a reduction in the

percentage of IL-2 producing T cells in the presence of NaAsO2. We tracked the proliferation of T cells by using the 5,6-

carboxyfluorescein diacetate succinimidyl ester (CFSE) dye and found that NaAsO2 slows down the entrance to cell division and

delays the proliferation of cells that have already entered the cell cycle. Nevertheless, the expression of the activation molecules,

CD25 and CD69, was unaltered. Assessment of the intracellular and secreted IL-2 in kinetic experiments showed that in fact,

NaAsO2 delays the production of IL-2, given that a recovery of both intracellular and secreted IL-2 was detected at 72 h.

Evaluation of the cell cycle showed a higher proportion of cells in G0/G1 and a lower proportion in G2/M in the presence of

NaAsO2. We thus conclude that NaAsO2 reduces proliferation of T cells by delaying the production and secretion of IL-2, thus

blocking T cells in G1; as a consequence, the entry to cell cycle and the rounds of cell division are retarded, and a lower

proliferation of T cells is hence observed.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Arsenic; IL-2; T cells; CFSE; Cell cycle; Proliferation

1567-5769/03/$ - see front matter D 2003 Elsevier Science B.V. All right

doi:10.1016/S1567-5769(03)00049-3

* Corresponding author. Tel.: +52-55-56-22-33-68; fax: +52-

55-56-22-33-69.

E-mail address: saavedra@servidor.unam.mx (R. Saavedra).

1. Introduction

Arsenic is a known human carcinogen [1]. It is

found in contaminated groundwater as a result of

weathering of rocks, industrial waste discharges, and

agricultural use of arsenical herbicides and pesticides

s reserved.

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682672

[2]. Inorganic arsenic may contaminate drinking

water, causing chronic exposure for millions of people

worldwide [3], and inducing skin lesions, hyperker-

atosis, dermatitis, polyneuropathy, and cancer. It has

been demonstrated that arsenic induces chromosomal

aberrations, micronuclei, sister chromatid exchanges

in human T cells, and interferes with DNA-repairing

enzymes [4–7]. As3 + alters the cytoskeleton structure

in different mammalian cell types [6,8], and regulates

the activity of some transcription factors [9–11].

Alterations by arsenic in the expression of oxidative

stress genes [9,12–14] and in growth factor genes

have also been reported in different cell lines [15,16].

Peripheral blood mononuclear cells (PBMCs) ob-

tained from chronically exposed individuals show a

lower replicating activity than nonexposed individu-

als, when stimulated with phytohemagglutinin (PHA)

[5]. This effect is also observed in PBMCs from

nonexposed donors when treated in vitro with

NaAsO2 [17]. In a previous work, we started to study

the mechanism by which arsenic induces a reduction

of proliferation of PBMCs stimulated with PHA, and

we found a reduction in the levels of secreted IL-2 by

those cells [17]; this reduction may explain, in part,

the decrease in proliferation, since IL-2 is an essential

growth factor for T cells [8,18–20].

Given that arsenic induces cytoskeleton alterations

[8,19], which, in turn, may affect the protein transport

of the cell [21], we hypothesized that the synthesized

IL-2 would accumulate inside the cell, which would

decrease the secretion of the lymphokine, and there-

fore reduce the proliferation of T cells. In this paper,

we tried to verify this hypothesis and to extend our

previous observations by evaluating the presence of

the IL-2 intracellularly by flow cytometry, a method

that allows the detection of cytokines at the single cell

level. We also tracked the proliferation of T cells in

the presence of NaAsO2 using the fluorescein-based

dye 5,6-carboxyfluorescein diacetate succinimidyl

ester (CFSE), a method that allows to study the

division history of individual cells by flow cytometry.

2. Materials and methods

2.1. Culture medium

All reagents were of cell culture grade, ob-

tained from GIBCO (Rockville, MD) or Sigma

(St. Louis, MO). PBMCs were cultured in RPMI

1640 supplemented with L-glutamine (2 mM),

nonessential amino acids (10 mM), HEPES (25

mM), and 10% (vol/vol) heat-inactivated fetal calf

serum (FCS). For cultures of CTLL-2 cells,

media were the same as above but supplemented

with 2-mercaptoethanol (5� 10� 5 M), sodium

pyruvate (1 mM), and human recombinant IL-2

(rIL-2) (4 IU/ml; Boehringer, Mannheim, Ger-

many).

2.2. Isolation of PBMCs

Blood was obtained from healthy male labora-

tory staff members, nonsmokers, 22–40 years old,

and unexposed to arsenic-contaminated water.

PBMCs were isolated from heparinized blood using

Ficoll–Paque gradient (Pharmacia, Uppsala, Swe-

den) as described previously [17]; they were

washed twice with Dulbecco’s phosphate-buffered

saline (DPBS), resuspended in culture medium, and

used immediately.

2.3. Cell proliferation assay by [3H]thymidine

incorporation

One hundred thousand PBMCs were incubated

for 24 h with NaAsO2 (0.01, 0.1, and 1.0 AM, final

concentrations; Sigma) in 100 Al of culture medium

in triplicate wells of a 96-well flat-bottom plate

(Costar, Cambridge, MA), at 37 jC in a humidified

atmosphere containing 5% CO2 in air. After incu-

bation, cells were stimulated with PHA (5 Al of

stock solution provided by the manufacturer; Sigma)

in 100 Al of culture medium containing the same

NaAsO2 concentration, for a further 48 h. Cells

were pulsed with 0.5 ACi of [3H]thymidine (Amer-

sham Pharmacia Biotech, Uppsala, Sweden) in 20

Al of culture medium for the last 18 h and har-

vested onto glass fiber filters by an automatic cell

harvester (Skatron, Sterling, VA). [3H]thymidine

incorporation was assessed by liquid scintillation

spectroscopy on a Betaplate counter (Wallac, Turku,

Finland), and results were expressed as percent

[3H]thymidine incorporation [mean counts per mi-

nute (cpm) in the presence of NaAsO2 divided by

mean cpm in the absence of NaAsO2 and multiplied

by 100].

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682 673

2.4. Production of supernatants for detection of IL-2

One million PBMCs were incubated for 24 h with

NaAsO2 (0.01, 0.1, and 1.0 AM, final concentra-

tions) in 1 ml of culture medium in wells of a 24-

well flat-bottom plate (Costar), as described above.

After incubation, cells were stimulated with PHA

(10 Al of stock solution provided by the manufac-

turer) in 1 ml of culture medium containing the same

NaAsO2 concentration, and incubated for a further

48 h. Cells were centrifuged (490� g, 5 min), and

supernatants were filtered through a 0.22-Am pore

membrane and stored at � 20 jC until use for the

detection of IL-2.

2.5. Cell culture for flow cytometry analysis

One million PBMCs were incubated with 1.0 AMNaAsO2 (final concentration) in 1 ml of culture

medium for 24 h in a 24-well cell culture plate

(Costar); after incubation, cells were stimulated with

PHA (10 Al of stock solution provided by the manu-

facturer) in 1 ml of culture medium containing 1.0 AMNaAsO2, and incubated for another 48 h; as control

for inhibition of cellular transport, some wells were

incubated for the last 6 h with Golgi Stop solution

(Pharmingen, San Diego, CA). Cells were harvested

by centrifugation (490� g, 5 min), resuspended in

DPBS, washed twice, and used immediately for

immunofluorescence assays and viability, as described

below.

2.6. Determination of cell division by CFSE staining

CFSE (Molecular Probes, Eugene, OR) was dis-

solved at 5 mM in DMSO, and stored in aliquots at

� 20 jC. PBMCs (5� 106 cells/ml in DPBS) were

stained with 5 AM CFSE in the same buffer (15 min,

37 jC, in darkness) with occasional stirring. Staining

was stopped by adding culture medium, centrifuged

(490� g, 5 min), and resuspended in culture medium

(106 cells/ml). Stained cells were cultured for 96 h

with PHA, in the presence or absence of NaAsO2, as

described for [3H]thymidine incorporation experi-

ments. At the end of the incubation, cells were washed

twice with DPBS and analyzed by FACS. Percentages

of divided cells were calculated according to Lyons

[22].

2.7. Immunofluorescence

DPBS-washed cells were resuspended in 100 Alof wash buffer (DPBS + 1% FCS + 0.1% NaN3)

containing the antibodies (1 Ag/106 cells), and

incubated for 30 min (4 jC, in darkness); they

were washed twice, fixed with p-formaldehyde (1%

in DPBS), resuspended in DPBS, and analyzed on

a FACScan. Antibodies were anti-CD3-FITC- or

anti-CD3-CyChrome- (clone UCHT1; Pharmingen),

anti-CD69-FITC (clone FN50; Pharmingen), anti-

IL-2-PE (clone MQ1-17H12; Pharmingen), and

anti-CD25-PE (clone 2A3; Becton Dickinson,

Mountain View, CA), and used as indicated by

the manufacturers.

2.8. Assessment of intracellular IL-2

One million PHA-stimulated cells were stained

with anti-CD3-FITC mAb as described above, and

washed three times with DPBS. They were resus-

pended in 250 Al of Cytofix/Cytoperm buffer (Phar-

mingen) and incubated for 20 min at 4 jC; they were

washed twice with 1 ml of the Perm/Wash buffer

(Pharmingen) and resuspended in 100 Al of Cytofix/Cytoperm buffer + 100 Al DPBS containing 1 Ag of

anti-IL-2-PE mAb; after incubation (30 min, 4 jC, indarkness), they were washed three times with Perm/

Wash buffer, resuspended in DPBS (500 Al), and

analyzed in a FACScan.

2.9. Viability test

For determination of viability, we used the propi-

dium iodide (PI) exclusion test, according to Ronot et

al. [23]. Activated or nonactivated PBMCs, treated

with or without NaAsO2 at different time points, were

washed twice with DPBS, resuspended in the same

buffer, stained with 1 Ag/ml PI (Sigma), incubated for

5 min at room temperature, and analyzed immediately

by FACS.

2.10. Cell cycle analysis

PBMCs were stimulated with PHA in the presence

or absence of NaAsO2 as described above, for 72 h.

Cells were washed with DPBS and 106–107 cells

were perfectly resuspended in 500 Al of the same

Fig. 1. Inhibition by NaAsO2 of proliferation of human PBMCs

stimulated with PHA. PBMCs (105 cells/well) from seven donors

were incubated with 0.01, 0.1, or 1.0 AM NaAsO2 for 24 h, and

stimulated with PHA for a further 48 h. Proliferation was assessed

by [3H]thymidine incorporation. Results are expressed as percent-

age of [3H]thymidine incorporation [(cpm in the presence of

NaAsO2/cpm in the absence of NaAsO2)� 100].

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682674

buffer. They were fixed in 4.5 ml of 70% ethanol and

incubated at 4 jC for 2 h. They were washed with

DPBS and resuspended in fresh 0.2 M Tris–HCl (pH

7.5) + 4 mM MgCl2 + 0.5% (vol/vol) Triton X-100,

containing PI (50 Ag/ml) and RNase (50 Ag/ml),

incubated for 5 min, and analyzed by FACS.

2.11. IL-2 assay

Determination of IL-2 in supernatants was deter-

mined using the bioassay described by Gillis et al.

[24], using the CTLL-2 cells and human recombinant

IL-2 (Boehringer) as standard, as described previously

[17].

2.12. Flow cytometry

Samples were analyzed on a FACScan flow cytom-

eter (Becton Dickinson) equipped with a 488-nm argon

laser. CFSE and FITC were detected on the FL1

channel, PI and PE on the FL2 channel, and CyChrome

on the FL3 channel. For immunofluorescence, viability

test, and cell cycle analysis, 10,000 events of each

sample were captured; for the determination of cell

proliferation of CFSE-stained cells, 20,000 events were

captured. Samples were analyzed using the Cell Quest

software (Becton Dickinson). Lymphocytes and blasts

were identified by forward scatter characteristics (FSC)

and side scatter characteristics (SSC).

2.13. Statistical analysis

The Student’s t test was used for comparison

between samples, using the PRISM software (Graph-

Pad, San Diego, CA).

3. Results

We first tested the effect of NaAsO2 on the

proliferation of T cells by the [3H]thymidine incorpo-

ration assay. We only tested male donors because we

had found a high variability in the response of PBMCs

from female donors, as it has been reported [25]. A

representative panel of donors is shown in Fig. 1. As

can be observed, a reduction in proliferation is

observed in most of the donors, in a dose-dependent

manner, at concentrations ranging from 0.01 to 1.0

AM NaAsO2. Reduction in proliferation was obtained

with the lowest concentration of NaAsO2 in some

donors, while in others, the reduction was observed

only with the highest concentration tested, or not

reduced (Fig. 1). Percentage of inhibition was be-

tween 20% and 98%, with the higher NaAsO2 con-

centration tested (1.0 AM). These results agree with

previous reports showing the inhibitory effect of

NaAsO2 on the proliferation of human T cells stimu-

lated with PHA [5].

In order to obtain more information about the effect

of NaAsO2 in the proliferative capability of the cells,

PBMCs were labeled with the fluorescein-based dye

CFSE, and the cell proliferation was tracked by flow

cytometry. Results from a typical experiment are

shown in Fig. 2. As can be seen, cells stimulated in

the absence of NaAsO2 for 96 h showed six rounds of

division (M2–M7), while in the presence of 1.0 AMNaAsO2, cells showed only five rounds of cell divi-

sion (Fig. 2)—an observation that suggests a delay in

the proliferation of T cells. Furthermore, a higher

number of cells in M1 region (28%), which corre-

spond to nondividing cells, was observed in the

presence of NaAsO2 when compared with nontreated

cells (10.5%); these results suggest that arsenic delays

the division of T cells and/or the entrance to mitosis.

Fig. 2. NaAsO2 reduces the rounds of cell divisions. PBMCs stained with CFSE were stimulated with PHA in the absence (straight line) or the

presence (dotted line) of 1.0 AM NaAsO2 for 96 h, and proliferation was analyzed in FACS as described in Materials and Methods. The

lymphocyte region was first identified by FSC and SSC, gated, and analyzed for the CFSE fluorescence. Results shown are representative of

those obtained from eight different donors.

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682 675

Using the results obtained from eight donors by

FACS analysis with the CFSE technique, as described

by Lyons [22], we calculated the proportion of starting

cells that entered cell division (divided cells) in the

presence and absence of NaAsO2. As shown in Table

1, the mean of divided cells from untreated PBMCs

was 46.1F16.3, but in the presence of 1.0 AMNaAsO2, a mean of 33.6F 16.4 was obtained (mean

reduction of 30%). Comparison of reduction between

Table 1

Percentage of divided cells in PBMCs stimulated with PHA in the

presence or absence of NaAsO2

Donor Percentage of divided cells Reduction

0 AM NaAsO2 1.0 AM NaAsO2

(%)

1 44.7 26.1 41.6

2 40.4 31.1 23.0

3 26.0 12.0 53.8

4 46.1 37.5 18.7

5 26.6 17.8 33.0

6 47.3 31.3 33.8

7 69.7 61.1 12.0

8 68.2 51.8 24.1

MeanF S.D. 46.1F16.3 33.6F 16.4* 30.0F 4.7

Percentage of divided cells was calculated according to Lyons [22].

*P< 0.0001.

both groups showed that this difference is statistically

significant (P < 0.0001). All these results suggest that

NaAsO2 has two effects on T cells: prevention of

entrance to cell division, and a delay in proliferation

of the cells that succeeded to enter the cell cycle.

Since it can be argued that the reduction in prolif-

eration could be due to a toxic effect of NaAsO2 and

although we have previously determined the absence of

a toxic effect of 1.0 AM NaAsO2 by the trypan blue

assay [17], we confirmed this observation by using the

PI exclusion test by FACS analysis [23], a more

sensitive technique. First, we tested whether NaAsO2

killed the cells during the 24-h incubation period before

PHA stimulation; as shown in Table 2, no significant

differences in the mean percentage of viability of

PBMCs from eight donors are observed between

untreated and NaAsO2-treated cells (97.8F 1.2 vs.

97.4F 1.9). Next, the same cells were stimulated with

PHA for 48 h in the presence of NaAsO2 and the

viability was determined at the end of the experiment;

as observed in Table 2, the mean percentage of viability

was also nearly unchanged when cells were stimulated

in the absence or presence of NaAsO2 (92.9F 4.1 vs.

91.6F 4.2). Thus, all these data confirm that the

reduction in the proliferation of T cells is not due to a

toxic effect by NaAsO2.

Table 2

Percentage of viability of PBMCs treated with NaAsO2

Donor Viability (%)

A B

0 AMNaAsO2

1.0 AMNaAsO2

0 AMNaAsO2

1.0 AMNaAsO2

1 97 97.5 85 87

2 97 95 94 90

3 96 95.5 88 86

4 97 95 95 89

5 99 99 96 97

6 99.2 99.1 96 97

7 99 99 93.5 93

8 98 99 96 94

MeanF S.D. 97.8F 1.2 97.4F 1.9 92.9F 4.1 91.6F 4.2

PBMCs were treated with NaAsO2 (1 AM) for 24 h and viability

was assessed (A). Cells were further stimulated with PHA and

viability was determined after 48 h (B). Viability is expressed as

percentage of PI-negative cells from the lymphocyte gate.

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682676

Since the reduced proliferation of T cells was not

caused by a toxic effect of NaAsO2, we tested whether

the T cells were activated correctly, by analyzing the

expression of CD69 and CD25 molecules, whose

expression is characteristic of the activation of T cells.

Determination of both CD69 (Fig. 3A) and CD25

(Fig. 3B) in the CD3+ population of PHA-stimulated

cells showed that both markers remain unaffected in

the presence of 1.0 AM of NaAsO2, suggesting that

Fig. 3. Expression of CD69 and CD25 in PBMCs stimulated with PHA in t

or without 1.0 AM NaAsO2 for 24 h, and stimulated with PHA for a furthe

(A), or an anti-CD25-PE (B) mAb, as described in Materials and Methods,

line); cells stimulated with 1.0 AM NaAsO2 (thick line); and cells stimulate

representative of four donors, each one repeated twice.

the activation of the T cells is not altered by this

metalloid.

Previous studies by our group showed that T cells

stimulated in the presence of NaAsO2 secreted lower

levels of IL-2 [17], an observation that could explain,

in part, the reduced proliferation of T cells in culture.

No reduction in the expression of the IL-2 gene was

reported, thus suggesting that NaAsO2 does not

affect the transcription of the IL-2 gene [17]. Since

NaAsO2 alters polymerization of microtubules [8,19],

which, in turn, are involved in the transport of

proteins, we hypothesized that NaAsO2 blocks the

intracellular transport of IL-2, leading to an accumu-

lation of this lymphokine in cells treated with

NaAsO2. In order to demonstrate this hypothesis,

we evaluated the intracellular IL-2 expression at the

single cell level by FACS analysis at 48 h poststi-

mulation with PHA. The results of a representative

experiment are shown in Fig. 4. As can be observed,

38% of the CD3+ cells are positive for IL-2 in

nontreated PBMCs (Fig. 4A), while only 18% of

the CD3+ cells are positive for IL-2 when treated

with 1.0 AM NaAsO2 (Fig. 4B); thus, a 53% reduc-

tion in T cells producing IL-2 was detected in

stimulated PBMCs incubated with NaAsO2—a result

that was unexpected. As a positive control, we

included PHA-stimulated PBMCs incubated for the

last 6 h with monensin; as anticipated, an increase in

CD3+IL-2+ cells was observed due to a block in the

he presence of NaAsO2. PBMCs (106 cells/well) were incubated with

r 48 h. After washing, cells were incubated with an anti-CD69-FITC

and analyzed in a FACScan. Cells stimulated without NaAsO2 (thin

d and incubated with isotype control (dotted line). Results shown are

Fig. 4. FACS analysis of intracellular IL-2 in PBMCs treated with NaAsO2. PBMCs (106 cells/well) were incubated with or without 1.0 AMNaAsO2 for 24 h, and stimulated with PHA for a further 48 h. Cells were collected and stained for surface CD3 and intracellular IL-2 as

described in Materials and Methods. The lymphocyte region was first identified by FSC and SSC, gated, and analyzed for the CD3 and IL-2

expression. (A) Stimulated cells nontreated with NaAsO2. (B) Stimulated cells treated with 1.0 AM NaAsO2. (C) Stimulated cells nontreated

with NaAsO2, but incubated for the last 6 h with monensin, and further incubated with the antibodies. Results shown are representative of those

obtained from seven donors, repeated at least twice.

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682 677

intracellular protein transport (Fig. 4C). Therefore,

these results suggest that NaAsO2 does not hamper

the transport of IL-2, but seems to inhibit or down-

regulate its production.

The results obtained from seven donors tested are

summarized in Table 3. As can be observed, a

reduction in the percentage of CD3+IL-2+ cells was

found in all donors, ranging from 25% to 87%

reduction in IL-2-producing T cells. Comparison of

the mean percentageF S.D. of CD3+IL-2+ cells from

untreated (21.7F 7.6) vs. NaAsO2-treated cells

(9.78F 7.8), determined by statistical analysis,

showed that the difference observed between the

two groups is statistically significant (P= 0.0009).

Table 3

Percentage of CD3+IL-2+ cells in PBMCs treated or nontreated with

NaAsO2 and stimulated with PHA

Donor Percentage of CD3+IL-2+ cells Reduction

0 AM NaAsO2 1.0 AM NaAsO2

(%)

1 25 11 56.0

2 21 4 81.0

3 23 3 87.0

4 16 5 68.8

5 19 7 63.2

6 36 26 27.8

7 12 9 25.0

MeanF S.D. 21.7F 7.6 9.78F 7.8* 58.4F 24.2

*P< 0.0009.

These results indicate that NaAsO2 reduces the pro-

duction of IL-2 and does not hamper the intracellular

transport of the lymphokine as we initially proposed.

One possible explanation for the results obtained

above is that NaAsO2 would hinder the binding of the

antibodies to their epitopes in the IL-2 and/or the CD3

molecule. To explore this possibility, we stimulated

cells with PHA for 48 h, as described in Materials and

Methods, NaAsO2 (1.0 AM) was added, incubated for

the last 2 h, and the immunofluorescence for CD3 and

intracellular IL-2 was then carried out; we also

included a control that consisted of PHA-stimulated

cells without NaAsO2, but 1 AM NaAsO2 was

included in all incubation and washing buffers during

the immunofluorescence assay. In neither case did we

observe a reduction in IL-2+CD3+ cells (data not

shown) when compared to nontreated cells, thus

indicating that the reduction of IL-2-producing T cells

described above was not due to an inhibition of the

binding of antibodies to their epitopes by NaAsO2.

All the experiments described above were carried

out only at 48 h poststimulation. We analyzed IL-2

secretion and intracellular IL-2 at different time points

to test if a recovery could be observed. We thus

measured the percentage of IL-2-producing cells and

also the presence of IL-2 in the supernatants at 24, 48,

and 72 h after stimulation, in the presence and absence

of NaAsO2. A representative experiment is shown in

Fig. 5. Kinetics of appearance of CD3+IL-2+ cells and production of IL-2 in PBMCs stimulated with PHA in the presence of NaAsO2. PBMCs

(106 cells/well) were incubated with 1.0 AM NaAsO2 (black bars) or left untreated (white bars) for 24 h, and stimulated with PHA. Cells and

supernatants were harvested at 24, 48, and 72 h after stimulation. (A) Percentage of CD3+IL-2+ cells. (B) Determination of IL-2 in supernatants.

Results shown are representative of those obtained from seven donors.

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682678

Fig. 5A. As can be observed, in the absence of

NaAsO2, IL-2-producing T cells increased over time,

reaching a maximum at 72 h; in the case of PBMCs

incubated with NaAsO2, IL-2-producing T cells also

increased with time and reached a maximum at 72 h,

but the percentage of IL-2-producing T cells was

lower that in nontreated cells. IL-2 levels in the

supernatants, however (Fig. 5B), were undetectable

at 24 h poststimulation in the presence of NaAsO2,

while higher levels were present in the absence of

Fig. 6. Cell cycle determination of PBMCs stimulated with PHA in the pre

were harvested at 72 h after stimulation, and treated as described in Materia

treated with 1.0 AM NaAsO2.

NaAsO2, but at 48 h, IL-2 was detected in the

presence of NaAsO2, although the levels were lower

than in the nontreated cells; at 72 h, IL-2 levels

detected in the presence of NaAsO2 were increased

and, in the case of control cells, levels were lower than

those detected at 24 and 48 h. All these data show that

IL-2 production by T cells is retarded when stimulated

with PHA in the presence of NaAsO2, but a recovery

over time is observed. These data clearly show a delay

in the kinetics of IL-2 production, and suggest that the

sence of NaAsO2. PBMCs were treated as described in Fig. 4, cells

ls and Methods for cell cycle analysis. (A) Untreated cells. (B) Cells

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682 679

cell cycle of T cells exposed to NaAsO2 could be

retarded.

In order to verify that the cell cycle of cells was

indeed delayed, we carried out a cell cycle analysis in

cells treated with NaAsO2. As shown in Fig. 6, a

lower proportion of cells in G2/M and S, and a higher

proportion in G0/G1, were observed in cells treated

with NaAsO2, demonstrating that this compound

stops the cells in G0/G1 and hence delays the mitosis

of T cells.

4. Discussion

It has previously been reported that cells from

normal donors treated with NaAsO2 in vitro [5] and

from donors chronically exposed to arsenic-contain-

ing water [26] showed a reduced proliferation of

PBMCs when stimulated with PHA. However, the

mechanism of these effects has been only partially

elucidated.

In the present work, we found that NaAsO2

reduces the proliferation of cells in a dose-dependent

manner at the concentrations tested (0.01, 0.1, and 1.0

AM), when proliferation was evaluated by [3H]thymi-

dine incorporation, as it has been reported previously

[5,17]. We also observed a difference in susceptibility

among donors; that is, although a decrease in prolif-

eration was observed for most individuals, the degree

of this reduction was highly variable (20–98%). This

difference in susceptibility has been reported, and it

has been explained by differences in the metabolism

of arsenic among individuals [27–29].

To obtain more information about the effect of

NaAsO2 on the proliferation of T cells, the PBMCs

were labeled with the fluorescein-based dye CFSE,

and cell proliferation was tracked by flow cytometry.

As reported earlier, this method is based on the

sequential halving of the cell fluorescence after each

division, which allows to study the division history of

individual cells, specifically to record the number of

rounds of cell division [22]. Using this technique, we

observed that cells incubated with NaAsO2 and stimu-

lated with PHA showed one to two rounds of cell

division less than control cells, suggesting that As3 +

delays the proliferation of T lymphocytes. In order to

calculate the number of the initial cells that succeeded

to enter to the cell cycle (divided cells [22]), we

analyzed the FACS results and found that NaAsO2

reduces the number of initial cells entering the cell

cycle (Table 1). We thus conclude that NaAsO2

retards proliferation of human T cells stimulated with

PHA.

We verified that the reduction in proliferation of T

cells was not due to a toxic effect of the NaAsO2, by

detecting the percentage of dead cells by flow cytom-

etry using the fluorescent dye, propidium iodide. The

use of this technique offers several advantages over

conventional microscopic exclusion methods, like the

greater number of cells analyzed and the multipara-

metric analysis [23], which make it more reliable,

reproducible, sensitive, and faster.

We thus assumed that the reduction in the number

of initial T cells entering the cell cycle when exposed

to NaAsO2 could be due to an interference of some

molecules involved in the activation of T lymphocytes

[17,30]. However, when the expression of CD69 and

CD25 molecules was analyzed, we did not find

alterations in the T lymphocytes incubated with

NaAsO2, suggesting that some of the early events

important for T cell activation are not affected by

As3 +. The effect of NaAsO2 on other early events of

the activation of T cells, however, still needs to be

further studied.

As3 + has an affinity to SH groups, mainly to vic-

inal dithiols [31–33]. Since proteins composing cys-

toskeleton contain abundant cysteine residues [19], it

is thus a cellular target of arsenic. In fact, it has been

reported that As3 + alters cytoskeletal morphology,

decreases cytoskeletal protein synthesis [19], disrupts

microtubule assembly, and inhibits tubulin polymer-

ization and spindle formation [8].

In addition, microtubules are required in some of

the steps of membrane trafficking and are also respon-

sible for the stability of the Golgi apparatus [21].

Since it was reported that IL-2 secretion in human T

lymphocytes exposed to arsenic is reduced [17], we

assumed that IL-2 secretion was inhibited due to some

of the arsenic effects on cytoskeleton and, as a result,

an accumulation of this lymphokine inside the T cells

should been observed.

The determination of the intracellular IL-2 in cells

exposed to NaAsO2 by flow cytometry showed a

reduction of CD3+ cells producing IL-2, a result

which is opposite to our initial hypothesis. In addition,

quantification of the lymphokine in the supernatants

G. Galicia et al. / International Immunopharmacology 3 (2003) 671–682680

showed that the IL-2 levels remained unchanged in

the presence of NaAsO2. These results seemed contra-

dictory to those previously reported by our group [17],

in which IL-2 levels in supernatants were found to be

reduced in the presence of NaAsO2; however, in that

study, the determination was carried out at 24 h

poststimulation only.

One possibility to explain the observed differences

is that NaAsO2 delays the production of IL-2. If this

hypothesis were true, a recovery should have been

observed at later time points. We thus measured the

production of IL-2 and determined the percentage of

IL-2-producing cells at 24, 48, and 72 h after stim-

ulation with PHA. This experiment (Fig. 5) showed

that after an initial reduction, both the proportion of

IL-2-producing T cells as well as the secreted IL-2

recovered after 48 and 72 h, thus suggesting that in

the presence of NaAsO2, the synthesis and secretion

of the lymphokine are, in fact, retarded.

It is not clear how IL-2 secretion is delayed. How-

ever, it has been reported that arsenic blocks the

activation of the transcription factor NF-nB [11], and

alters IL-1a production [34], which could be related to

the observed effects in our experiments. The role of

these events, though, remains to be established.

The data obtained in the proliferation assays by the

[3H]thymidine incorporation assay and by the CFSE

technique suggested that the cell cycle of the cells

treated with NaAsO2 was also retarded, and the results

of the kinetic experiment for the detection of IL-2

pointed to the same hypothesis. The determination of

the cell cycle of these cells by flow cytometry did

demonstrate that the cells exposed to NaAsO2 present a

delay in the cell cycle, since a higher number of cells

are found in the G0/G1 phase and cell percentage in S

and G2/M phases is reduced. These results agree with

those previously reported in different cell lines [35,36].

Antigen- or mitogen-induced activation of T cells

triggers a signal transduction cascade, which includes

phosphorylation of several proteins, activation of

phospholipase C, increase of intracellular free Ca2 +,

activation of several transcription factors and lympho-

kine genes, and production of IL-2 [37]. The transition

from G1 into the replicative phases of the cell cycle is

mediated by IL-2 [38,39]; the magnitude and extent of

T cell proliferation are influenced by the concentration

of IL-2 produced and the period during which it is

available [38]. Thus, since no G1 progression factor is

present (IL-2) during the first time points of the

NaAsO2-treated cells (Fig. 5), the T lymphocytes are

blocked in the G1 phase (Fig. 6). However, at later time

points, IL-2 is synthesized and secreted, although at

lower levels, and the T cells can progress from the G1

to the S phase of the cell cycle. Therefore, it can be

concluded that the absence and reduction in IL-2 levels

in NaAsO2-treated cells are the main events respon-

sible for the decreased proliferation in our system.

Nevertheless, it has been reported that arsenic induces

DNA damage [36], interferes with DNA repair

enzymes [40], alters phosphorylation of proteins

involved in proliferation [15], alters p53 expression

[41,42], and blocks DNA repair [43]. These effects

should also contribute to the cell cycle delay.

In conclusion, in this paper, we demonstrate that

NaAsO2 retards the kinetics of production and secre-

tion of IL-2 of T cells stimulated with PHA. As a

consequence, T cells show a delay in entry to cell

cycle, and those cells that succeed to enter proliferate

slower.

Acknowledgements

This work was partially supported by grant IN-

216997 from the DGAPA, UNAM.

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