Molecular events associated with arsenic-induced malignant transformation of human prostatic epithelial cells: aberrant genomic DNA methylation and K-ras oncogene activation

Numerous studies link arsenic exposure to human cancers in a variety of tissues, including the prostate. Our prior work showed that chronic arsenic exposure of the non-tumorigenic, human prostate epithelial cell line, RWPE-1, to low levels of (5 microM) sodium arsenite for 29 weeks resulted in malignant transformation and produced the tumorigenic CAsE-PE cell line. The present work focuses on the molecular events occurring during this arsenic-induced malignant transformation. Genomic DNA methylation was significantly reduced in CAsE-PE cells. A time course experiment showed that during malignant transformation DNA methyltransferase activity was markedly reduced by arsenic. However, DNA methyltransferase mRNA levels were not affected by arsenic exposure. Microarray screening showed that K-ras was highly overexpressed in CAsE-PE cells, a result further confirmed by Northern blot and Western blot analyses. Since ras activation is thought to be a critical event in prostate cancer progression, further detailed study was performed. Time course experiments also showed that increased K-ras expression preceded malignant transformation. Mutational analysis of codons 12, 13, and 61 indicated the absence of K-ras mutations. The K-ras gene can be activated by hypomethylation, but our study showed that CpG methylation in K-ras promoter region was not altered by arsenic exposure. Arsenic metabolism studies showed RWPE-1, CAsE-PE, and primary human prostate cells all had a very poor capacity for arsenic methylation. Thus, inorganic arsenic-induced transformation in human cells is associated with genomic DNA hypomethylation and K-ras overexpression. However, overexpression of K-ras occurred without mutations and through a mechanism other than promoter region hypomethylation.     

Effect of whey protein isolate on intracellular glutathione and oxidant-induced cell death in human prostate epithelial cells.

Cysteine is the rate-limiting amino acid for synthesis of the ubiquitous antioxidant glutathione (GSH). Bovine whey proteins are rich in cystine, the disulfide form of the amino acid cysteine. The objective of this study was to determine whether enzymatically hydrolyzed whey protein isolate (WPI) could increase intracellular GSH concentrations and protect against oxidant-induced cell death in a human prostate epithelial cell line (designated RWPE-1). Treatment of RWPE-1 cells with hydrolyzed WPI (500 microg/ml) significantly increased intracellular GSH by 64%, compared with control cells receiving no hydrolyzed WPI (P<0.05). A similar increase in GSH was observed with N-acetylcysteine (500 microM), a cysteine-donating compound known to elevate intracellular GSH. In contrast, treatment with hydrolyzed sodium caseinate (500 microg/ml), a cystine-poor protein source, did not significantly elevate intracellular GSH. Hydrolyzed WPI (500 microg/ml) significantly protected RWPE-1 cells from oxidant-induced cell death, compared with controls receiving no WPI (P<0.05). The results of this study indicate that WPI can increase GSH synthesis and protect against oxidant-induced cell death in human prostate cells.     

Chronic exposure to arsenite enhances influenza virus infection in cultured cells

Arsenic is a ubiquitous environmental toxicant that has been associated with human respiratory diseases. In humans, arsenic exposure has been associated with increased risk of respiratory infection. Considering the existing epidemiological evidence and the well-established impact of arsenic on epithelial cell biology, we posited that the effect of arsenic exposure in epithelial cells could enhance viral infection. In this study, we characterized influenza virus A/WSN/33 (H1N1) infection in Madin-Darby Canine Kidney (MDCK) cells chronically exposed to low levels of sodium arsenite (75 ppb). We observed a 27.3-fold increase in viral matrix (M2) protein (24 hours postinfection [p.i.]), a 1.35-fold increase in viral mRNA levels, and a 126% increase in plaque area in arsenite-exposed MDCK cells (48 hours p.i.). Arsenite exposure resulted in 114% increase in virus attachment-positive cells (2 hours p.i.) and 224% increase in α-2,3 sialic acid-positive cells. Interestingly, chronic exposure to arsenite reduced the effect of the antiviral drug, oseltamivir in MDCK cells. We also found that exposure to sodium arsenite resulted in a 4.4-fold increase in viral mRNA levels and significantly increased cytotoxicity in influenza A/Udorn/72 (H3N2) infected BEAS-2B cells. This study suggests that chronic arsenite exposure could result in enhanced influenza infection in epithelial cells, and that this may be mediated through increased sialic acid binding. Finally, the decreased effectiveness of the anti-influenza drug, oseltamivir, in arsenite-exposed cells raises substantial public health concerns if this effect translates to arsenic-exposed, influenza-infected people.     

Inhibition of cellular enzymes by equine catechol estrogens in human breast cancer cells: specificity for glutathione S-transferase P1-1

Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the catechol estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another catechol estrogen, 4,17beta-hydroxyequilenin (4,17beta-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo.     

Effects on levels of glutathione and some related enzymes in tissues after an acute arsenic exposure in rats and their relationship to dietary protein deficiency

Arsenic is a potent toxin, carcinogen and modulator of antioxidant defense system. In this study, male rats of Wistar strain, maintained on either 18% or 6% protein (casein) diet, received an acute i.p. exposure to sodium arsenite (As3+) at its LD50 dose (15.86 mg/kg body weight). One hour after the arsenic exposure, glutathione (GSH) concentration was significantly depleted and lipid peroxidation was increased. A relationship between any two of tissue arsenic concentrations, GSH levels and lipid peroxidation values was observed only for liver when the proportional changes of respective parameters in either of the dietary groups of animals were compared. This suggests that, in liver, arsenic metabolism appears dependant upon the GSH concentration. Acute arsenic exposure significantly increased the glutathione peroxidase (GPx) activity in liver of both dietary groups and in kidney of only the 18% protein-fed group of animals. The glutathione-S-transferase (GST) activity significantly decreased in liver of the 18% protein-fed animals while GST increased in kidney of both the 18% and the 6% protein-fed groups. No significant change in glutathione reductase (GR) or glucose-6-phosphate dehydrogenase (G6PDH) activity was observed. In the present investigation, liver as a whole seems to be more affected in terms of GSH level and GST activity. The mode of responses of GPx and GR activities as well as the unaltered G6PDH activity might result in arsenic-induced GSH depletion and increase in lipid peroxidation. The animals of the 6% protein-fed group, appeared to be affected less in terms of tissue arsenic concentration, GSH level and GST activity. lipid peroxidation,     

Glutathione S-transferases and glutathione peroxidases in doxorubicin-resistant murine leukemic P388 cells

Energy-dependent rapid drug efflux is believed to be a major factor in cellular resistance to doxorubicin (DOX). However, several recent studies have demonstrated that cellular DOX retention alone does not always correlate with its cytotoxicity and suggest that mechanisms other than rapid drug efflux may also be important. In the present study, we have compared glutathione (GSH) S-transferase (GST), selenium-dependent GSH peroxidase and selenium-independent GSH peroxidase II activities in DOX-sensitive (P388/S) and resistant (P388/R) mouse leukemic cells. The GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) was markedly higher in P388/R cells compared to P388/S cells. Purification of GST by GSH-affinity chromatography from an equal number of P388/S and P388/R cells revealed an increased amount of GST protein in P388/R cells. Immunological studies indicated that alpha and pi type GST isoenzymes were 1.27- and 2.2-fold higher, respectively, in P388/R cells compared to P388/S cells. Selenium-dependent GSH peroxidase activity was similar in both the cell lines, whereas selenium-independent GSH peroxidase II activity was approximately 1.36-fold higher in P388/R cells compared to P388/S cells. These results suggest that increased GSH peroxidase II activity in P388/R cells may contribute to cellular DOX resistance by enhancing free radical detoxification in this cell line.     

Role of glutathione and glutathione S-transferase in chlorambucil resistance.

A chlorambucil (CLB)-resistant cell line, N50-4, was developed from the established mouse fibroblast cell line NIH 3T3, by multistep drug selection. The mutant cells exhibited greater than 10-fold resistance to CLB. Alterations in GSH and glutathione S-transferase (GST) were found in CLB-resistant variants. A 7-10-fold increase in cellular GSH content and a 3-fold increase in GST activity were detected in N50-4 cells, compared with parental cells, as determined by enzymatic assays. An increase in steady state levels of the GST-alpha isozyme mRNA was found in the CLB-resistant cells, as analyzed by Northern blotting. No GST gene amplification or rearrangement was shown by Southern blot analysis. To test the relative roles of GSH and GST in CLB resistance, a number of GSH- and GST-blocking agents were used. The CLB toxicity was significantly enhanced in N50-4 cells by administration of either the GSH-depleting agent buthionine sulfoximine or the GST inhibitors ethacrynic acid or indomethacin. The resistance to CLB cytotoxicity in N50-4 cells, however, was still significantly higher than that of parental cells. The resistance of N50-4 cells to CLB was almost completely abolished by combination pretreatment yielding both GSH depletion and GST inhibition. The results indicate that both increased cellular GSH content and increased GST activity play major roles in CLB resistance in N50-4 mutant cells.     

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase mu.

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.     

Cellular glutathione prevents cytolethality of monomethylarsonic acid

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, forming compounds such as monomethylarsonic acid (MMAs(V)) and dimethylarsinic acid (DMAs(V)). However, much less information is available on the in vitro toxic potential or mechanisms of these methylated arsenicals, especially MMAs(V). We studied the molecular mechanisms of in vitro cytolethality of MMAs(V) using a rat liver epithelial cell line (TRL 1215). MMAs(V) was not cytotoxic in TRL 1215 cells even at concentrations exceeding 10 mM, but it became weakly cytotoxic and induced both necrotic and apoptotic cell death when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor, l-buthionine-[S,R]-sulfoximine (BSO), or the glutathione reductase inhibitor, carmustine. Similar results were observed in the other mammalian cells, such as human skin TIG-112 cells, chimpanzee skin CRT-1609 cells, and mouse metallothionein (MT) positive and MT negative embryonic cells. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyses GSH-substrate conjugation, also enhanced the cytolethality of MMAs(V), but aminooxyacetic acid (AOAA), an inhibitor of beta-lyase that catalyses the final breakdown of GSH-substrate conjugates, had no effect. Both the cellular GSH levels and the cellular GST activity were increased by the exposure to MMAs(V) in TRL 1215 cells. On the other hand, the addition of exogenous extracellular GSH enhanced the cytolethality of MMAs(V), although cellular GSH levels actually prevented the cytolethality of combined MMAs(V) and exogenous GSH. These findings indicate that human arsenic metabolite MMAs(V) is not a highly toxic compound in mammalian cells, and the level of cellular GSH is critical to its eventual toxic effects.     

Studies on the mechanisms of arsenic-induced self tolerance developed in liver epithelial cells through continuous low-level arsenite exposure.

Arsenic (As) is a human carcinogen. Our prior work showed that chronic (>18 weeks) low level (500 nM) arsenite (As3+) exposure induced malignant transformation in a rat liver epithelial cell line (TRL 1215). In these cells, metallothionein (MT) is hyper-expressible, a trait often linked to metal tolerance. Thus, this study examined whether the adverse effects of arsenicals and other metals were altered in these chronic arsenite-exposed (CAsE) cells. CAsE cells, which had been continuously exposed to 500 nM arsenite for 18 to 20 weeks, and control cells, were exposed to As3+, arsenate (As5+), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), antimony (Sb3+), cadmium (Cd2+), cisplatin (cis-Pt), and nickel (Ni2+) for 24 h and cell viability was determined by metabolic integrity. The lethal concentration for 50% of exposed cells (LC50) for As3+ was 140 microM in CAsE cells as compared to 26 microM in control cells, a 5.4-fold increase in tolerance. CAsE cells were also very tolerant to the acute toxic effects of As5+ (LC50 > 4000 microM) compared to control (LC50 = 180 microM). The LC50 for DMA was 4.4-fold higher in CAsE cells than in control cells, but the LC50 for MMA was unchanged. There was a modest cross-tolerance to Sb3+, Cd2+, and cis-Pt in CAsE cells (LC50 1.5-2.0-fold higher) as compared to control. CAsE cells were very tolerant to Ni2+ (LC50 > 8-fold higher). Culturing CAsE cells in As(3+)-free medium for 5 weeks did not alter As3+ tolerance, implicating an irreversible phenotypic change. Cellular accumulation of As was 87% less in CAsE cells than control and the accumulated As was more readily eliminated. Although accumulating much less As, a greater portion was converted to DMA in CAsE cells. Altered glutathione (GSH) levels were not linked with As tolerance. A maximal induction of MT by Zn produced only a 2.5-fold increase in tolerance to As3+ in control cells. Cell lines derived from MT normal mice (MT+/+) were only slightly more resistant (1.6-fold) to As3+ than cells from MT null mice (MT-/-). These results show that CAsE cells acquire tolerance to As3+, As5+, and DMA. It appears that this self-tolerance is based primarily on reduced cellular disposition of the metalloid and is not accounted for by changes in GSH or MT.     

Role of glutathione in dimethylarsinic acid-induced apoptosis

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethylarsinic acid (DMAs(V)). Recent evidence indicates that DMAs(V) is a complete carcinogen in rodents although evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMAs(V) using a rat liver epithelial cell line (TRL 1215). DMAs(V) selectively induced apoptosis in TRL 1215 cells; its LC(50) value after 48 h exposure was 4.5 mM. The addition of a glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO), actually decreased DMAs(V)-induced apoptosis. DMAs(V) exposure temporarily decreased cellular reduced glutathione (GSH) levels and enhanced cellular glutathione S-transferase (GST) activity from 6 h after the exposure when the cells were still alive. Also, DMAs(V) exposure activated cellular caspase 3 activity with a peak at 18 h after the exposure when apoptosis began, and BSO treatment completely inhibited this enzyme activity. The additions of inhibitors of caspase 3, caspase 8, and caspase 9 significantly reduced DMAs(V)-induced apoptosis. Taken together, these data indicate that cellular GSH was required for DMAs(V)-induced apoptosis to occur, and activation of cellular caspases after conjugation of DMAs(V) with cellular GSH appears to be of mechanistic significance. Further research will be required to determine the role of intracellular GSH and methylation in the toxicity of arsenicals in chronic arsenic poisoning or in cases where arsenicals are used as chemotherapeutics.     

低剂量砷长期诱导人肝细胞获得耐砷性的实验研究

目的：培养人肝细胞（L-02）耐砷细胞株,为生物体对砷的耐受机制的研究奠定基础。方法：采用人肝细胞（L-02）在含有低剂量亚砷酸钠（NaAsO2）的培养基中长期培养,并设同步对照细胞组,利用噻唑兰（MTT）检测法计算细胞生存率、半数致死量（LDso）及细胞内砷浓度作为反映细胞对砷耐受性改变的指标。结果：细胞经砷诱导6周后,在24h急性砷中毒试验中．实验组细胞对急性染砷表现出明显的耐受性提高,实验组在各浓度下细胞生存率都明显高于同步对照组,实验组LD。为23．1μmol／L,对照组LD50为10．2μmol/L。实验组各浓度下的砷浓度都明显低于同步对照组（P〈0．001）。结论：人正常肝细胞与细菌、真菌、哺乳动物及人前列腺细胞-样在长期低剂量砷诱导下具有可诱导的对砷的耐受性。     

Overexpression of glutathione S-transferase and elevation of thiol pools in a multidrug-resistant human colon cancer cell line.

A human colon cancer cell line with acquired multidrug resistance (MDR) was assayed for the intracellular GSH level and the activity of GSH-S-transferase (GST), which catalyzes the conjugation reaction of electrophilic drugs with GSH. The GSH level and GST activity (as measured with 1-chloro-2,4-dinitrobenzene) were elevated in the resistant cells by 1.7-fold and 2-fold, respectively. This elevated catalytic activity of the resistant cells was reflected in a 2-fold increase in GST-pi mRNA, which was not the result of gene amplification. In addition, buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased Adriamycin sensitivity in both the MDR and the parental cells, affecting the former more than the latter. The effects seen with buthionine sulfoximine were not seen with puromycin and actinomycin D. A dramatic overexpression of mdr1, a P-glycoprotein gene responsible for the MDR phenotype, was also observed in the MDR cells. In contrast, none of these products (i.e., mdr P-glycoprotein, GSH level, total GST activity, GST-pi gene copy, and GST-pi mRNA level) was elevated in HeLa cells resistant to cisplatin and some alkylating agents, supporting the notion that the acquisition of cisplatin resistance differs from the mechanism of MDR. These results indicate that the intrinsic GSH level and GST-pi activity affect anthracycline resistance per se and not MDR in the human colon cancer cells.     

In vitro effect of arsenical compounds on glutathione-related enzymes

The mechanism of arsenic toxicity is believed to be due to the ability of arsenite (As(III)) to bind protein thiols. Glutathione (GSH) is the most abundant cellular thiol, and both GSH and GSH-related enzymes are important antioxidants that play an important role in the detoxification of arsenic and other carcinogens. The effect of arsenic on the activity of a variety of enzymes that use GSH has been determined using purified preparations of glutathione reductase (GR) from yeast and bovine glutathione peroxidase (GPx) and equine glutathione S-transferase (GST). The effect on enzyme activity of increasing concentrations (from 1 microM to 100 mM) of commercial sodium arsenite (As(III)) and sodium arsenate (As(V)) and a prepared arsenic(III)-glutathione complex [As(III)(GS)(3)] and methylarsenous diiodide (CH(3)As(III)) has been examined. GR, GPx, and GST are not sensitive to As(V) (IC(50) > 50 mM), and none of the enzymes are inhibited or activated by physiologically relevant concentrations of As(III), As(III)(GS)(3), or CH(3)As(III), although CH(3)As(III) is the most potent inhibitor (0.3 mM < IC(50) < 1.5 mM). GPx is the most sensitive to arsenic treatment and GST the least. Our results do not implicate a direct interaction of As with the glutathione-related enzymes, GR, GPx, and GST, in the mechanism of arsenic toxicity. CH(3)As(III) is the most effective inhibitor, but it is unclear whether this product of arsenic metabolism is produced at a sufficiently high concentration in critical target tissues to play a major role in either arsenic toxicity or carcinogenesis.     

A keratin 18 transgenic zebrafish Tg(k18(2.9):RFP) treated with inorganic arsenite reveals visible overproliferation of epithelial cells

Inorganic arsenic has strong human carcinogenic potential, but the availability of an animal model to study toxicity is extremely limited. Here, we used the transgenic zebrafish line Tg(k18(2.9):RFP) as an animal model to study arsenite toxicity. This line was chosen because the red fluorescent protein (RFP) is expressed in stratified epithelia (including skin), due to the RFP reporter driven by the promoter of the zebrafish keratin 18 gene. We titrated doses of inorganic arsenite for zebrafish embryos and found that arsenite exposure at 50 microM for 120 h was suitable for mimicking a long-term, chronic effect. When embryos derived from Tg(k18(2.9):RFP) adults were treated with this arsenite dose and time of exposure, abnormal phenotypes were not noticeable under the light microscope. However, arsenic keratosis was visible in the epithelial cells under the fluorescent microscope. Morphological defects became more severe with increased dose and exposure duration, suggesting that the severity of skin lesions was dose- and time-dependent. Histochemical examination of keratosis after 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the epithelial cells overproliferated after treatment with arsenite. Therefore, this Tg(k18(2.9):RFP) zebrafish line is an excellent model for studying toxicity induced by inorganic arsenite and may have potential for studying other environmental pollutants.     

Helper T cell subpopulations from women are more susceptible to the toxic effect of sodium arsenite in vitro

Arsenic is known to produce inhibition as well as induction of proliferative responses in animal and human cells depending on the doses. Despite the amount of information on the immunotoxic effects of arsenic exposure in different animal models, little is known in humans. Arsenic susceptibility of lymphocyte subpopulations (T helper (Th), CD4+; T cytotoxic (Tc), CD8+) and whether arsenic effects are gender related are still to be determined. This work evaluated the in vitro toxicity of sodium arsenite on human T lymphocyte subpopulations from men and women. Peripheral blood mononuclear cells (PBMC) obtained from healthy young men and women were treated with sodium arsenite (0.01, 0.1, and 1 microM). We assessed cell viability, cell proliferation, and the proportion of Th and Tc cells after 48 or 72h of arsenic exposure in resting and phytohemagglutinin M (PHA)-activated PBMC. We observed that sodium arsenite at 1 microM was more toxic for Th than for Tc cells in PBMC from women. Besides, T lymphocytes from women were more affected by the cell proliferation inhibition induced by arsenic, suggesting that women could be more susceptible to the toxic and immunotoxic effects caused by arsenic exposure.