Exposure of Human Leukemic Cells to Direct Electric Current

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ORIGINAL ARTICLE

Copyright 2005 by Humana Press Inc.All rights of any nature whatsoever reserved.1085-9195/05/42:6174/$30.00

Cell Biochemistry and Biophysics 61 Volume 42, 2005

INTRODUCTION

Direct electric current (DC) can influence the

growth and cell biology of several systems (15).Because of such properties, DC has been widely usedfor the treatment of tumors (611). However, the useof DC as an alternative therapy for cancer is limited bythe poor knowledge of the cellular effects generated indifferent conditions in which animal cells are exposedto electric fields (2). In this context, different polarities

of electric current have been shown to induce diverseeffects on animal cells, which is reflected by the vary-ing efficacies of treatments of cancers with DC usingpositive or negative electrodes (8,1214). In suchexperimental systems, cells are exposed to DC as theyare to the soluble products generated after oxidationor reduction of physiologic compounds (1517).Therefore, the products of electrolysis can certainlyinfluence the cells that are exposed to DC in aqueoussystems. Parameters involved in DC treatment such aselectric field strength, electrode polarity, and period of stimulation also influence the type of cell response toan electric stimulus. For instance, the suppression or

Exposure of Human Leukemic Cells to Direct Electric Current

Generation of Toxic Compounds Inducing Cell Death by Different Mechanisms

Venicio F. Veiga, 1 Leonardo Nimrichter, 1 Cesar A. Teixeira, 3 Marcelo M. Morales,2 Celuta S. Alviano,1 Marcio L. Rodrigues, 1 and Carla Holandino 3,*

1Instituto de Microbiologia Professor Paulo de Ges,2 Instituto de Biofsica Carlos Chagas Filho,and 3Departamento de Medicamentos-Faculdade de Farmcia, Centro de Cincias da Sade,

Universidade Federal do Rio de Janeiro, CCS, Bloco K, Segundo Andar, Sala 50, Ilha do Fund ~ao,

21941590, Rio de Janeiro, Brazil

Abstract

Treatment with direct electric current (DC) influences the growth of several cancer cells. In this work, weevaluated the effects of DC treatment on the human leukemic cell line HL60. Human cells were separatelytreated in the presence of the cathode or the anode or without contact with the electrodes. In all systems, DC-treated cells presented an impaired ability to proliferate. Growth inhibition was dependent on the generationof soluble products of electrolysis. Cathodic treatment of HL60 cells predominantly induced lysis, whereastreatment without contact with electrodes did not induce alterations in cell viability. In contrast, cell stimula-tion by the anode resulted in irreversible membrane damage, as demonstrated by trypan blue and 7-aminoactinomycin staining. Analysis of these cells by transmission electron microscopy indicated thatnecrosis is a major mechanism inducing cell death. In addition, apoptotic-like cells were observed under light

microscopy after anodic treatment. Accordingly, DNA from anodic-treated cells presented a typical pattern of apoptosis. Apoptotic cell death was only generated after the treatment of HL60 cells in conditions in whichthe generation of chloride-derived compounds was favored. These results indicate that the nature of the prod-ucts from cathodic or anodic reactions differently influences the mechanisms of cell death induced by DC-derived toxic compounds.

Index Entries: Direct electric current; human leukemic cells; tumor growth; apoptosis; necrosis.

*Author to whom all correspondence and reprint requestsshould be addressed. E-mail: [email protected]


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Flow Cytometry With 7-Aminoactinomycin The progressive increase in membrane permeability

induced by DC treatment was evaluated by 7-aminoactinomycin (7-AAD) incorporation (29,30).Before 7-AAD staining, a cell suspension (2 mL) con-taining 10 6 cells/mL was treated with DC and washed

twice in PBS. DC-treated and control cells were imme-diately incubated with 7-AAD or, alternatively, reintro-duced into the culture medium and incubated for 0, 4,18, or 24 h, as described previously. Control cells werealso introduced into the treatment chambers and incu-

bated as performed for stimulated cells, but withoutany current ow. After these periods, cells were againwashed and incubated in a solution of 7-AAD at 20g/mL (PBS) for 20 min at 4C and protected from light.Cells in this staining solution were then analyzed(10,000 events) in a FACS CALIBUR ow cytometer(Becton Dickinson, Franklin Lakes, NJ). The red uores-cence from 7-AAD was ltered through a 650-long passlter. Multiparameter data analysis was performed withWinmdi software (Salk Flow Cytometry).

Electron Microscopy Ultrastructural alterations in leukemic cells after DC

treatment were evaluated by transmission electronmicroscopy. Cells were first centrifuged and resus-pended in a 2.5% glutaraldehyde solution. After 2 h of incubation in this solution, cells were washed in 0.1 Mcacodylate buffer (pH 7.2) containing 0.2 M sucrose andpostxed in 1% osmium tetroxide for 45 min. After x-ation, they were preincluded in 1.5% agar, dehydrated

with ethanol and acetone, and embedded in Epon (31).Ultrathin sections were prepared with a diamond knifein an KLB ultramicrotome, collected in 300 mesh coopergrids, counterstained with uranyl acetate and lead cit-rate, and examined under a Zeiss 900 transmission elec-tron microscope operating at 80 kV.

Assay for Morphological ChangesThe morphological features of DC-treated cells were

assessed by staining cytocentrifuged preparations bythe May-Grunwald-Giemsa method. At least 200 cells ineach preparation were examined with a light micro-scope. For analysis of cellular alterations, three separateexperiments were performed.

Analysis of DNA Fragmentation HL60 cells were treated with DC as described previ-

ously (DC Stimulation); control systems consisted of exposure of human cells to the chambers under thesame conditions, except for the presence of electric cur-rent (Fig. 1B). A total of 10 7 leukemia cells was obtainedin combined experiments. The cells were washed with

PBS and pelleted by centrifugation. DNA fragmentationwas assayed by a modication of the method of Dukeand Sellins groups (32). Cultured cells were washedtwice with ice-cold PBS and suspended in 100 L lysis

buffer (10 m M Tris HCl/10 m M EDTA/0.5% Triton X-100, pH 8.0), vortex-mixed, sonicated, and incubated on

ice for 20 min. After centrifugation for 20 min at 4C(14,000 g), the supernatant containing fragmented (soluble) DNA was transferred to another tube. Lysis buffer(100 L) was added to the pellet containing insolubleDNA. Both samples were treated with Rnase A (0.5mg/mL) for 1 h at 37C and then with proteinase K(Sigma, 0.4 mg/mL) for 1 h at 37C. After adding 20 L5 M NaCl and 120 L isopropanol, the samples wereincubated overnight at 20C. The soluble fraction of DNA was determined by electrophoresis on 1.5%agarose gel and has a ladder-like appearance.

RESULTSTreatment of HL60 Cells With DCInhibits Proliferation

Treatment with DC has been shown to significantlyinhibit the growth of several tumor cells(2,711,14,17,20,24,3343), which led us to evaluate theinfluence of DC treatment on the proliferation rates of HL60 human leukemic cells. They were treated with aDC intensity of 2 mA for 6 min, according with previ-ously described models of electric stimulation of ani-mal cell lines (16,17,20). Control cells were exposed tothe same conditions of stimulation, except for the pres-ence of electric current. DC-treated cells were washedto remove products of electrolysis and reinoculated(105) into fresh media; the number of cells in culturewas then determined after 0, 4, 18, and 24 h. Cells thatwere treated with DC in anodic or cathodic chambers,as well as without contact with electrodes, were unableto proliferate in normal rates (Fig. 2).

Kinetics of Trypan Blue Incorporationby DC-Treated Cells

The mechanisms by which DC treatment inhibits thegrowth of tumor cells are poorly known. Because the cell

membrane is commonly affected by the action of DC(16,20,44,45), we evaluated the immediate effects of elec-tric treatment on cell lysis and membrane integrity of HL60 cells. Cells were treated for periods varying from 0to 10 min and their viability assayed by the trypan bluemethod. Treatment of HL60 cells in the cathodic cham-

ber (CC) predominantly induced lysis, which wasgreater in higher periods of treatment (Fig. 3). In con-trast, cell stimulation in the anodic chamber (AC)resulted in a progressive augment of permeability to the

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Generation of Toxic Compounds Inducing Cell Death 65


Fig. 2. Treatment of HL60 cells with direct electric current (DC) inhibits proliferation. Cells were treated with DC andinoculated into fresh media. The number of cells was determined after 4, 18, and 24 h. Cells that were treated in an anodicchamber ( ), intermediary chamber ( ), or cathodic chamber ( ) had their growth inhibited. Proliferation rates of cells thatwere not treated with DC ( ) are also shown (for details of control preparation, see Material and Methods). Results of veindependent experiments expressed as mean standard deviation are shown.

Fig. 3. Kinetics of trypan blue incorporation by direct electric current (DC)-treated cell. Cells were treated for periodsvarying from 0 to 10 min and their viability assayed by the trypan blue method. Black bars represent the number of trypan

blue-stained cells, whereas white bars are representative of the number of lysed cells. Indices of stained or lysed cells areshown after their treatment in the cathodic (CC), anodic (AC), intermediary (IC), or control (NS) chambers. Results of veindependent experiments expressed as mean standard deviation are shown.


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trypan blue dye, indicating that membrane integrity wasaffected by these conditions of electric stimulation. Celllysis was also observed, although less intensively than

observed after cathodic treatment. These results areindicative that the cytoplasmic membrane is indeed akey target for electric stimulation in the presence of elec-trodes. Expressive levels of lysis or trypan blue stainingwere not observed when cells were treated in the IC.Exposure of HL60 cells to the chamber of treatment inthe absence of DC resulted in high levels of viability.

To evaluate the concentration of the products of elec-trolysis in viability, HL60 cells were stimulated in thesame exposure conditions, except for the volume of thecell suspension. In this system, the internal volume of the exposure chamber was reduced by the addition of glass slides at the bottom of the chamber, which madepossible the treatment of cell suspensions in the sameconditions but in reduced volumes. This experimentdemonstrated that the lowest viabilities were generatedin smaller volumes (Table 1), indicating that the con-centration of the products of electrolysis directly inu-ences the occurrence of cell death. Cells that weretreated in IC or in the CC presented very similar levelsof viability, which varied from 80% to 95% of viablecells in the different periods and volumes of treatment(data not shown).

Membrane Damage Is Not Reverted by Cultivation of DC-Treated Cells

After the 6-min stimulation, a period of treatment inwhich the most signicant alterations in cell numberand viability are observed in this and others systems(16,17,20), cells were reinoculated into fresh media andcultivated for periods of 4, 18, and 24 h. The cells werethen analyzed by ow cytometry, after staining with 7-AAD (29,30). Control cells (Fig. 4), as well as IC- andcathodic-treated populations (not shown), remainedimpermeable to 7-AAD. AC-treated cells, however, pre-

sented an irreversible and progressive augment in theirpermeability to 7-AAD (Fig. 4, R2), which was accom-panied by the generation of cell bodies with a signi-

cantly reduced size (Fig. 4, R3).

Anodic Treatment Induces Necrosisand Apoptosis in HL-60 Cells

The occurrence of 7-AADstained cells presentingreduced cell sizes could be indicative of differentprocesses leading to cell death (46). In fact, necroticcells are generated after 24 h of cultivation of anodic-treated cells, as shown in Fig. 5. The cells presentedmembrane discontinuity and matrix rarefaction, asobserved for DC-treated P815 and multidrug-resistantK562 cells (16,17).

Previous studies described that apoptosis is trig-gered in human leukemic cell lines after treatment withelectric current (21). Untreated (control), CC-, and IC-stimulated cells presented similar morphological pro-les (not shown). However, Giemsa staining revealedthe occurrence of morphological changes induced byanodic treatment mainly characterized by cell shrink-age and nuclear chromatin clumping (Fig. 6A). Cellularpresentations suggestive of apoptotic bodies were alsoobserved, mainly when anodic-treated cells were culti-vated for prolonged periods. In fact, biochemical fea-tures of apoptosis were detected after DNA analysis bygel electrophoresis (Fig. 6B). DNA extracted from AC-,

but not CC- or IC-stimulated cells, was fragmented intosegments of 180200 base pairs and presented the typi-cal ladder pattern observed in preparations from apop-totic cells (47).

In anodic reactions performed in the presence of chloride, hypochlorous acid (HOCl) is generated as aproduct of electrolysis (15). The latter compound canreact with several amino acids to form chloramines,which are potent inducers of apoptosis (48). To evaluateif such products of electrolysis were in fact the inducers

66 Veiga et al.


Table 1Effect of Chamber Volume on the Viability of HL60 Cells After Different

Periods (min) of DC Treatment

Nonviable Cells a (%)

Chamber volume 2 min 4 min 6 min 8 min 10 min

1.0 mL 60 b 37c 85 b 65c 95 b 95c 99 b 100c 100 b 100c1.5 mL 33 b 33c 58 b 69c 60 b 85c 72 b 99c 90 b 100c2.0 mL 33 b 37c 53 b 65c 57 b 65c 58 b 83c 85 b 94c

aNonviable cells were considered as the addition of the number of lysed and trypan-blue stained cells. bCathodic- and canodic-stimulated cells.


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68 Veiga et al.


Fig. 5. Transmission electron micrographs showing the morphology of control (A) and anodic-treated (B) HL60 cells.Anodic treatment induced membrane discontinuity and matrix rarefaction. Scale bars: 1 m.

Fig. 6. Anodic stimulation induces apoptosis in human cells. (A) Giemsa staining of cytocentrifuged control oranodic-treated cells. HL60-treated cells were inoculated into fresh media and Giemsa-stained after 4, 18, or 24 h of cul-tivation. Morphological changes were mainly characterized by cell shrinkage and nuclear chromatin clumping (inset 24h; scale bar: 20 m). Cellular presentations suggestive of apoptotic bodies were also observed (arrows). Scale bars: 50m. Data from these experiments are representative of three identical assays. (B) Electrophoretic profile of DNAextracted from control (a), cathodic (CC)-treated (b), intermediary (IC)-treated (c) and anodic (AC)-treated (d) cells.After treatment with DC, cells were cultivated for 4, 18, and 24 h, followed by DNA extraction and analysis in agarosegels. DNA from anodic-stimulated cells (d) presented a typical apoptosis pattern. These data are representative of threeidentical assays producing similar profiles.


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of cell death after anodic stimulation, the percentage of nonviable cells was determined by the trypan bluemethod after cultivation of DC-treated cells in differentconditions (Fig. 7). When the cells were cultivated for 24h in the same medium in which they were electricallystimulated, a very low index of viability was observed.In contrast, a lesser number of nonviable cells wasobserved when they were treated with DC and furthercultivated in a fresh medium. In addition, cultivation of nonstimulated cells in a medium in which HL60 cellshave been previously stimulated resulted in a largepopulation of nonviable cells, suggesting that toxic mol-ecules generated during electric treatment were theactive agents generating cell death.

The inuence of chlorine-derived compounds in thegeneration of apoptotic cell death in our system wasevaluated by the anodic treatment of HL-60 cells in PBSsupplemented with glutamine, followed by incubation

in culture medium for 1 and 3 h. During DC treatments,no signicant variations in electric conductivity wereobserved, as determined in a conductivity meter (datanot shown). In control systems, which were treated inphosphate buffer (identical to PBS except for the pres-ence of sodium chloride), no cellular alterations wereobserved (not shown). In this system, electric conduc-tivity also remained constant during DC treatment inthe different conditions. However, leukemic cells thatwere treated in PBS plus glutamine presented a pro-

gressive augment in trypan blue permeability, indicat-ing loss of viability (Fig. 8A). Exposure of HL60 cells tothe same conditions, except for the presence of DC, didnot affect viability. HL60 cells were also evaluatedmicroscopically for the presence of signs of apoptosis,which were in fact observed. Cell shrinkage and nuclear

chromatin clumping, as well as cellular presentationssuggestive of apoptotic bodies, were detected (Fig. 8B).

DISCUSSION

DC can differently inuence the growth of manytumoral cell types (4953). In the present work, wedescribe an inhibitory effect of DC-derived compoundson the proliferation of human leukemic cells. Necrosisand apoptosis were the biologic events associated withcell death in our experimental conditions. In addition,we demonstrate that the mechanism of cell death isdependent on the products of electrolysis and the polar-ity of the electrode, showing for the rst time that pro-grammed cell death is induced by toxic compoundsproduced during anodic stimulation.

Electrode polarity and the products generated dur-ing anodic or cathodic stimulations can be hallmarkedas an important issue influencing tumor inhibition(7,8,12,37,51). Morris and coworkers (52) demon-strated that treatment of tumor in mice yields differentresults depending on whether AC or CC stimulation isused. In their model, cell death in the region of contactwith the anodic electrode is not accompanied by celllysis, whereas cathodic-stimulated cells are disruptedin a great extent. In addition, anodic stimulation has

been shown to be more effective than the cathodictreatment in the clinical use of DC for the treatment of cancer (7,8,24,53). Our studies on the in vitro stimula-tion of tumor cells with DC confirm that differentmechanisms are involved in the growth inhibitioncaused by DC (1517). This and previous studiesdemonstrate that tumor cells can have their growthability impaired even when their morphologies areunaltered, which is a typical feature of IC-treated cells.The mechanisms causing tumor inhibition in theabsence of electrodes are still undefined, but free-float-ing electrons could inactivate ribonucleotide reductase

(54), which is responsible by the conversion of build-ing blocks of RNA into DNA during cell division.However, evident cellular alterations are observed incathodic- and anodic-stimulated cells (1317,37),which confirms that different mechanisms should beinvolved in DC-induced tumor inhibition and, addi-tionally, that they are influenced by the products gen-erated by electrodes with different polarity.

In the present work, an extensive index of cell lysiswas observed when the cells were treated in the cathodic



Fig. 7. Death of HL60 cells after anodic stimulation isinduced by the products of electrolysis. The percentage of nonviable cells was determined after cultivation of directelectric current-stimulated cells for 24 h in the samemedium of electric treatment (A) or in fresh medium (B) .

Alternatively, the cells were removed by centrifugationand the medium of treatment was used to cultivate non-stimulated cells (C) . The viability of nonstimulated cellscultivated in fresh medium for 24 h is shown in (D) .


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chamber, which was accompanied by a progressiveincrease of pH in the medium used for electric treatment.Lysis of cathodic-treated cells in PBS may be explained

by a direct attack of the oxidants generated during DCtreatment on the cell membrane, as previously described(13,55). Superoxide, hydroxyl groups, molecular hydro-gen, and oxygen are major products of cathodic electro-chemical reactions occurring in aqueous solution (14,55).The production of hydroxyl groups in these reactions isprobably a major contributing factor in the pH increase,which, in turn, causes cell lysis (37). Superoxide radicalscan also inuence the occurrence of cell lysis, becausethey are generated by cathodic reactions (55) and are ef-cient agents in the destruction of target cells (56,57).

Electrolysis products actually seem to be involved incell death in our experimental conditions, because theirincreased concentrations resulted in higher levels of celldeath (Table 1). Microscopic analysis of Giemsa-stainedcells revealed that, besides the usual morphologic fea-tures of DC-inhibited cells, an alternative mechanism of death was detected. Although leukemic cells treated inthe presence of the cathode or in the absence of elec-trodes presented morphologies that were similar to con-trol systems, several cells stimulated in the presence of the anode resembled apoptotic bodies. This observationwas conrmed by DNA electrophoresis and indicatedthat apoptosis is in fact an alternative pathway of celldeath in leukemic cells exposed to DC. The induction of

70 Veiga et al.


Fig. 8. Stimulation of HL60 cells in the presence of glutamine.(A)

Human cells were suspended in phosphate-bufferedsaline supplemented with 0.5 m M (squares), 1.0 (circles), or 1.5 (triangles) glutamine and treated with direct electric current(DC) (open symbols). Cell viability was measured immediately after electric stimulation (0 h) or after incubation in freshmedium supplemented with fetal bovine serum for 1 or 3 h. The viability of HL60 cells incubated in the presence of gluta-mine is also shown (closed symbols). (B) Nonstimulated or DC-treated cells that were incubated in the presence of gluta-mine (0.5 m M) for 3 h were also analyzed microscopically after Giemsa staining. Control cells presented their typicalmorphology, whereas DC-stimulated cells resembled apoptotic bodies. Scale bars: 10 m.


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apoptosis by electric and magnetic fields has beendescribed in other systems (21,5860), although this isthe rst report demonstrating that toxic compoundswhose generation is dependent on electrode polarityregulate the induction of this biologic process. UsingDC as the source of electric stimulation, Kurokawa and

coworkers(21)

described that treatment of humanleukemic cell lines had undergone apoptotic cell death, but, in their model, cells were simultaneously treatedwith anodic and cathodic polarities, which makes ananalysis of the products of electrolysis and their inu-ence on the generation of cellular damage difcult.

The occurrence of apoptosis in cells exposed to DCwas apparently derived from electrolysis products fromanodic reactions. Cisplatinum is a well-known DNA-damaging agent and its presence in a DC-stimulatedmedium is expected around the anodic region (61), butnot in the cathode compartment. Therefore, it is possiblethat the generation of cisplatinum complexes con-

tributes to the induction of apoptosis in the anodicchamber. However, experiments using steel electrodesinstead of platinum probes demonstrated that apoptosisstill occurs, which indicates that cisplatinum complexesare not the major inducers of cell death in our system(Holandino et al., unpublished data). We thereforeinvestigated the inuence of chlorine species producedduring anodic stimulations possibly involved in theinduction of apoptosis.

HOCl, which is generated during electric stimulationof aqueous solutions (1315), is known to modify mem-

brane lipids (6264) as well as protein and DNA. Itsreaction with extracellular amino acids results in thegeneration of chloramines, which are less potent oxi-dants (6567). The ability of these reactive oxygenspecies in the generation of apoptosis has been recentlydescribed (48,68).

Most studies of HOCl-induced cytotoxicity have been carried out on cells incubated in buffer solutionsinstead of cell culture media (6769). Although themechanism of cell death was not specifically deter-mined in these studies, results were generally reectiveof necrosis. However, additional studies revealed thattreatment of endothelial cells with HOCl for 15 min in

buffered saline followed by transfer of cells to completemedia resulted in the occurrence of apoptotic cell deathin approx 20% of the population (70). Necrosis was themajor mechanism involved in cell death in the presenceof higher levels of HOCl, which is consistent with ourresults. In our model, leukemic cells were initiallytreated in a buffered system, which means a low avail-ability of amino acids and a consequent low concentra-tion of chloramines. If this hypothesis is true, necrosisand, in lower levels, apoptosis should occur after treat-ment of cells with DC, which is indeed observed in our

experiments. However, treatment of HL-60 cells in PBSsupplemented with glutamine resulted in a predomi-nant occurrence of apoptosis, in accordance with thegeneration of chloramines derived from the reaction

between HOCl and glutamine. When chloride ions wereremoved from the medium of treatment, no cellular

damage occurred (data not shown). This result could beexplained by the facilitated generation of chloraminesfrom the reaction between the amino acid and HOCl,creating an environment more favorable to apoptoticthan necrotic cell death. In this context, the critical taskwill be to identify the specic molecular targets that ini-tiate the apoptotic cascade.

The influence of DC and its soluble products in the biology of animal cells and microorganisms is alargely recognized phenomenon (1,3,7,12,24,25,45).The observation of different cellular DC-inducedeffects contributes with the comprehension of severalphysiologic events, and also raises the possibility of

the use of electric stimulation in the treatment of can-cer (3335,37,4143,52,53,71), wound healing (4,72,73),and fracture repair (74). However, the biologic mecha-nisms whereby DC and their derived compoundsinhibit or stimulate cell growth still require clarifica-tion. In this context, the elucidation of the cellularevents triggered after electric stimulation under con-trolled conditions (e.g., electrical field strength, periodof treatment, electrode polarity, and toxic compoundsgenerated during stimulation) will support the use of in vivo or in vitro models applying DC as a cellgrowthinterfering agent.

ACKNOWLEDGMENTS

We thank Dr. Geraldo A. G. Cidade for helping us inthe physical determinations after DC treatment in theacrylic chamber and Dr. Damijan Miklavcic for helpfuldiscussions. This work was supported by Financiadorade Estudos e Projetos (FINEP), Conselho Nacional deDesenvolvimento Cientfico e Tecnolgico (CNPq),Funda ~ao de Amparo a Pesquisa no Estado do Rio de

Janeiro (FAPERJ), Funda ~ao Jos Bonifcio (FUJB), andPrograma de Apoio a Ncleos de Excelncia (PRONEX).

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Exposure of Human Leukemic Cells to Direct Electric Current

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