Mechanism of metalloid-induced death in Leishmania spp.: Role of iron, reactive oxygen species,...

Free Radical Biology & Medicine 40 (2006) 1857–1868www.elsevier.com/locate/freeradbiomed

Original Contribution

Mechanism of metalloid-induced death in Leishmania spp.: Role of iron,reactive oxygen species, Ca2+, and glutathione

Ashish Mehta, Chandrima Shaha ⁎

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India

Received 9 December 2005; accepted 20 January 2006Available online 17 February 2006

Abstract

There is growing evidence that metalloid-induced cell death in protozoan parasites is due to oxidative injury; however, the biochemicalchanges related to this event are not fully understood. Leishmania spp. demonstrated cross-resistance to two related metalloids, arsenic andantimony, and both metalloids induced cell death accompanied by cell shrinkage and DNA fragmentation that was preceded by an increase inreactive oxygen species. Both drugs caused mitochondrial dysfunction in terms of loss of membrane potential and a drop in ATP levels. Arsenictreatment resulted in an elevation of intracellular Ca2+ levels that did not occur with antimony exposure. Cellular glutathione level was reducedafter antimony treatment but arsenic did not affect glutathione. Inhibition of Ca2+ influx during arsenic treatment reduced cell death, whereassupplementation of glutathione during antimony treatment rescued cell loss. Under iron-depleted conditions, the cytotoxic effects of arsenic andantimony did not occur and cell survival increased; in contrast, the presence of excess iron resulted in higher cell death. Therefore, this studyprovides a new possibility that iron can potentiate parasite death induced by metalloids like arsenic and antimony. In addition, an importantobservation is that the two similar metalloids produce toxicity by very different mechanisms.© 2006 Elsevier Inc. All rights reserved.

Keywords: Leishmania; ROS; Calcium; Iron; Antimony; Arsenic; Glutathione; Free radicals

Reactive oxygen species (ROS) are generated by pathogen-infected cells to combat infection. The same principle works forcertain anti-pathogen drugs to kill the organisms in an infectedcell. This property of a drug, to generate ROS to causedestruction of cellular macromolecular components, is impor-tant because this action can be modulated to derive appropriateaction. For this, an understanding of the cellular consequencesof drug treatment is necessary. Metalloids are the main line oftreatment for diseases like Leishmaniasis caused by the

Abbreviations: ROS, reactive oxygen species; SbIII, potassium antimonytartarate; GSH, glutathione; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylben-zimidazolylcarbocyanine iodide; CM-H2DCFDA, 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; fluo-3AM, fluo-3 acetoxymethylester; AsIII, sodium arsenite; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; DFO, deferoxamine; BSO, butathione sulfoximine;TUNEL, terminal deoxynucleotidyltransferase enzyme-mediated dUTP nick-end labeling; PI, propidium iodide; FIU, fluorescence intensity units; ΔΨm,mitochondrial membrane potential.⁎ Corresponding author. Fax: +11 2616 2125.E-mail address: [email protected] (C. Shaha).

0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2006.01.024

kinetoplastid parasites [1,2]. Although metalloids have longbeen used to treat diseases caused by the kinetoplastid parasites,the biochemistry of the process is not fully understood and gapsin our understanding of the metalloid mechanism of actionremain. Two closely related metalloids like arsenic andantimony are the drugs of choice to treat diseases caused bythe kinetoplastid parasites, and interestingly, arsenic-resistantLeishmania spp. show cross-resistance to antimony, and thesestrains are used as models to study various aspects of parasitebiology, including drug resistance [3–8]. Antimony and arsenicare members of the same group, XV, of the periodic table andare transported in cells by the same channels, carriers [9], andpumps [10]. Leishmania spp. are obligate intracellular parasitesof the vertebrate reticuloendothelial system, existing as free-swimming forms in the gut of the insect host and as immotileintracellular amastigotes in the macrophages of the vertebratehost [11]. Our earlier studies demonstrated that the metalloidantimony generates ROS to kill Leishmania donovani amasti-gotes inside the phagolysosomes of macrophages [12], but earlycellular changes induced by the drug are difficult to trace in

mailto:[email protected]

http://dx.doi.org/10.1016/j.freeradbiomed.2006.01.024

1858 A. Mehta, C. Shaha / Free Radical Biology & Medicine 40 (2006) 1857–1868

these forms because a purification from phagolysosomes isnecessary, which itself could induce changes.

Arsenic is used as a drug for acute promyelocytic leukemia[13], by which arsenate can uncouple oxidative phosphorylationor it can stimulate mitochondrial respiration contributing todecreased ATP levels and inflict cellular injury via thegeneration of ROS [14]. Pentavalent antimony, the formgenerally used for treatment of visceral leishmaniasis, isreduced to trivalent antimony (SbIII), the active form inbiological systems [15], and SbIII is reported to exertcytotoxicity through the inhibition of glucose metabolism andATP formation [16] or through its interactions with keysulfhydryl groups of Leishmania proteins [17]. SbIII decreasesintracellular thiol buffering capacity by promoting loss oftrypanothione, a glutathione (GSH)–spermidine compound [4],thereby profoundly perturbing the thiol redox potential of thecell [7].

Leishmania spp. are hemoflagellate parasites that requireiron for growth of both the mammalian and the insect stages[18,19]; however, there is little information regarding the role ofiron during drug-induced death. There is evidence thatdisruption of Leishmania iron metabolism by host-generatednitric oxide is a strategy used by the mammalian host to kill theparasite [20] and therefore it is important to understand therelationship between the drugs and iron because modificationsin these can modulate drug action. The electronic structure ofiron and its capacity to drive one-electron reactions predeter-mine iron as a major component in the production andmetabolism of ROS in biological systems and therefore thistransition metal is potentially important for drug-inducedcytotoxic effects [21].

Using L. donovani promastigotes as a model system toinvestigate the mechanisms of metalloid toxicity in lowereukaryotes, we demonstrate that the early cellular responseto both arsenic and antimony are similar in terms ofinducing mitochondrial dysfunction. We present the novelfinding that iron is required for the cytotoxic effects ofarsenic and antimony. In addition, we make the importantobservation that these two very similar metalloids producetoxicity by very different mechanisms; whereas ROS are theprimary cytotoxic agents for SbIII, Ca2+ is responsible forcell death caused by arsenic treatment.

Materials and methods

Materials

The apoptosis detection system was procured from Promega(Madison, WI, USA). ATP assay kit, JC-1 (5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide),fluo-3AM (fluo-3 acetoxymethyl ester), and CM-H2DCFDA (5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diace-tate, acetyl ester) were obtained from Molecular Probes(Eugene, OR, USA). Sodium arsenite (AsIII) was obtainedfrom Fluka Chemie GmbH (Switzerland). EGTA (ethyleneglycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid),GSH, deferoxamine (DFO), butathione sulfoximine (BSO),

pimozide, ferric chloride, SbIII, O-phthalaldehyde and medium199 and all other chemicals unless otherwise mentioned wereobtained from Sigma Chemical Co. (St. Louis, MO, USA).

Cell culture

Promastigotes of L. donovani UR6 strain were cultured inblood-agar slants as described previously [22–24]. Briefly, thecultures were grown on agar slants containing 1% glucose, 5.2%brain-heart infusion agar extract, and rabbit blood (6% v/v) at25°C with gentamycin at a final concentration of 1.5 mg ml−1.For experimental purposes, promastigotes in the log phase ofgrowth were recovered from 3-day-old blood agar slants andsuspended in medium 199 containing 0.72 mg/L ferric nitrateand supplemented with 10% fetal calf serum, centrifuged, andresuspended in the same medium to achieve a culture density of1 × 107 cells ml−1.

Treatments

Promastigotes harvested from blood-agar slants and resus-pended in medium as mentioned above were incubated withvarious doses of SbIII (30–300 μg ml−1) and AsIII (5–30 μgml−1) and one dose of each was selected for furtherinvestigations. GSH and BSO were used at concentrations of10 and 1 mM, respectively, with 1 h preincubation before drugtreatments. Similarly, in Ca2+ related studies, Ca2+ channelblockers bepridil (50 μM), benzamil (50 μM), nifedipine(10 μM), verapamil (20 μM), and pimozide (20 μM) were usedfor blocking Na+/Ca2+ exchanger and voltage-gated L-type andT-type Ca2+ channels. The Ca2+ chelator EGTA and the ironchelator DFO were used at concentrations of 3 mM and500 μM, respectively. All Ca2+ channel blockers were added30 min before addition of the drugs to the cells. Ferric chloridewas used at 100 (Fe-1), 200 (Fe-2), and 400 μM (Fe-3)concentrations.

Microscopy

Twenty-four hours after various treatments, cells werevisualized under a Nikon confocal microscope C1 (Nikon,Inc., Japan), using an argon laser (488 nm) for recording ofNomarski images using an oil immersion 60× DIC objective inthe transmission mode. The ratio of length and breadth of thecells was measured using Image Pro Plus version 5.0 software(Media Cybernetics, MD, USA) and the ratio of length vsbreadth was subsequently calculated. JC-1-stained cells werevisualized using an argon-ion laser (488 nm) and a red HeNelaser (543 nm) with the above microscope. Cells stained forROS with CM-H2DCFDA were scanned with the argon laser(488 nm).

Detection of DNA fragmentation and cell viability

Terminal deoxynucleotidyltransferase enzyme-mediateddUTP nick-end labeling (TUNEL) detection of DNA fragmen-tation was carried out using a TUNEL assay kit according to the

1859A. Mehta, C. Shaha / Free Radical Biology & Medicine 40 (2006) 1857–1868

manufacturer’s instructions as described previously [24].Briefly, cells were fixed in 4% formaldehyde, and postfixationpermeabilization was carried out with 0.2% (v/v) Triton X-100for 10 min at room temperature followed by incubation withbuffer containing nucleotide mix (50 μM fluorescein-12–dUTP,100 μM dATP, 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 7.6)for 1 h at 37°C. Cell viability was checked by staining the cellswith propidium iodide (PI) at 1 μg ml−1. Terminal deoxynu-cleotidyltransferase-labeled and PI-labeled cells were analyzedby flow cytometry.

Flow cytometry

Cells (107) from different experimental groups were run ona BD-LSR flow cytometer (Becton–Dickinson, San Jose, CA,USA) equipped with a 20-mV, 488 nm air-cooled argon-ionlaser. All signals were detected using the following settings:for fluorescence 1 (green), a 530/28 bandpass filter was usedand fluorescence was measured in the log mode, and forfluorescence 3 (red), a 575/26 bandpass filter was used andfluorescence was measured in the log mode. Cells wereisolated from fragments by gating on the forward-and side-scatter signals, and subsequently promastigotes were detectedand analyzed according to their relative fluorescenceintensities compared with unstained promastigotes. Analyseswere performed on 100,000 gated events, and numeric datawere processed using WinMDI shareware. All plots arerepresentative of three or four experiments.

Measurement of ROS

To monitor the level of ROS, the cell-permeant probe CM-H2DCFDA was used as described previously [22–24]. CM-H2DCFDA is a nonpolar compound that readily diffuses intocells, where it is hydrolyzed to the nonfluorescent derivativedichlorodihydrofluorescein and trapped within the cells. In thepresence of a proper oxidant, dichlorodihydrofluorescein isoxidized to the highly fluorescent 2,7-dichlorofluorescein.Briefly, 107 cells were suspended in medium 199 andincubated with 2 μg ml−1 CM-H2DCFDA for 15 min in thedark; subsequently, treatments were carried out, and fluores-cence was monitored. Fluorescence was measured at anexcitation wavelength of 488 nm and emission wavelength of530 nm. For each experiment, fluorometric measurementswere performed in triplicate and expressed as fluorescenceintensity units (FIU). All measurements were carried out in aFluostarOptima spectrofluorometer (BMG Technologies,Offenburg, Germany).

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨm) was measuredusing the JC-1 probe as described previously [23,24]. JC-1 is acationic mitochondrial vital dye that is lipophilic and becomesconcentrated in the mitochondria in proportion to the membranepotential; more dye accumulates in mitochondria with greaterΔΨm and ATP-generating capacity. Therefore, the fluorescence

of JC-1 can be considered as an indicator of relativemitochondrial energy state. The dye exists as a monomer atlow concentrations (emission, 530 nm, green fluorescence) butat higher concentrations forms J aggregates (emission, 590 nm,red fluorescence). Briefly, cells were labeled for 10 min with10 μM JC-1 at 37°C, washed, and resuspended in medium, andbaseline fluorescence was measured. Subsequently, the com-pounds were added and the changes in fluorescence weremonitored at two different wavelengths as mentioned above.The ratio of the reading at 590 nm to the reading at 530 nm wasconsidered as the relative ΔΨm value.

Measurement of cytosolic free Ca2+concentrations

Changes in intracellular Ca2+concentration were monitoredwith the fluorescent probe fluo-3AM as described previously[24]. Briefly, cells were loaded for 30 min at 25°C with 5 μMfluo-3AM containing 1 μM pluronic acid F-127 for properdispersal and 0.25 mM sulfinpyrazone, an organic aniontransport inhibitor to reduce leakage of the fluo-3 dye. Justbefore use, cells were washed with medium to removenonhydrolyzed fluo-3AM. Fluorescence measurements wereperformed at 25°C at an excitation of 488 nm and emission of522 nm. To convert fluorescence values into absolute [Ca2+]i,calibration was performed at the end of each experiment. [Ca2+]iwas calculated using the equation

½Ca2þ�i ¼ KdðF � FminÞ=ðFmax � FÞ;

where Kd is the dissociation constant of the Ca2+–fluo-3complex (400 nM), and F represents the fluorescence intensityof the cells. Fmax represents the maximum fluorescence(obtained by treating cells with 10 μM calcium ionophoreA23187), and Fmin corresponds to the minimum fluorescence(obtained from ionophore-treated cells in the presence of 3 mMEGTA). Fluorescence intensities were expressed as the increasein fluorescence with respect to baseline fluorescence intensitybefore stimulation.

Reduced glutathione

Reduced glutathione was measured as described previouslywith minor modifications [24]. Briefly, 107 cells weresuspended in phosphate–EDTA buffer, to which 25% phos-phoric acid was added to precipitate the proteins. The sampleswere centrifuged at 10,000g for 30 min at 4°C. For GSHestimation, 0.1 ml of the supernatant was mixed withphosphate–EDTA buffer (pH 8.0) and 1 mg ml−1 O-phthalaldehyde. Fluorescence was determined with an excita-tion wavelength of 350 nm and an emission wavelength of420 nm.

ATP assay

ATP was measured by a bioluminescence assay using anATP determination kit as described previously [23,24]. Theassay is based on luciferase’s requirement for ATP in producing


light (emission maximum ∼560 nm at pH 7.8). Briefly, cells(∼1 × 106) after different treatments were resuspended inreaction buffer containing 1 mM dithiothreitol, 0.5 mMluciferin, and 12.5 μg ml−1 luciferase and gently mixed, afterwhich readings were taken in a Fluostar Optima multilabeldetection system (BMG Technologies, Offenburg, Germany).ATP standard curves were run in all experiments with differentconcentrations of ATP and calculations were made against thecurve, and cellular ATP levels were expressed as nmol/107 cells.

Statistics

Paired comparisons were conducted using a paired t test andall data are presented as means ± SE. Differences wereconsidered significant at the 0.05 level of confidence.

Results

Mitochondrial function in the presence of antimony andarsenic

For survival of any cell, mitochondria are essential cellularorganelles that play central roles in energy metabolism andapoptosis. During oxidative phosphorylation, electrons aredelivered through the mitochondrial respiratory complexesand a proton gradient is established across the innermitochondrial membrane as the energy source for ATPproduction. One important biochemical event vital for cellsurvival associated with this metabolic process is the mainte-nance of ΔΨm [25]. Studies from this laboratory have shownthat variations in ΔΨm induced by drugs or oxidative stress areassociated with cell survival in L. donovani [22,24]. In thisinvestigation, we used loss of ΔΨm as a parameter to selecteffective doses of arsenic and antimony for investigations intothe processes associated with metalloid-induced cell death.Several doses of both SbIII (30–300 μg ml−1) and AsIII (5–30 μg ml−1) were used and a significant fall in ΔΨm wasobserved with 300 μg ml−1 SbIII and 30 μg ml−1 AsIII (Fig. 1A).With both the metalloids, a 30–45% fall in ΔΨm occurredwithin the first 30 min; however, a further significant dropoccurred between 120 and 150 min post-drug exposure. Fig. 1Bshows the microscopic view of cells labeled with thepotentiometric probe JC-1 at 1 and 3 h. Note that a number ofred-labeled mitochondria with higher ΔΨm are visible incontrols, whereas green-labeled mitochondria with lower ΔΨm

appear in the treated groups. The ΔΨm is related to ATPproduction and therefore ATP level is another importantparameter for mitochondrial function. A significant decline inATP levels was observed within the first hour post-drugtreatment in both SbIII-and AsIII-treated groups (Fig. 1C).Therefore, it was clear that both SbIII (300 μg ml−1) and AsIII

(30 μg ml−1) were effective in disrupting mitochondrialfunction. Henceforth, the two doses are referred to as AsIII

and SbIII only.Changes in morphology are an inherent feature of parasitic

lifestyle in addition to being a characteristic under various stresssituations [26]. Based on prior knowledge that antileishmanial

agents do cause morphological changes [24], we checked themorphology of the promastigotes after drug treatment. The L.donovani in culture is a free-swimming form with an elongatedstructure and a beating flagellum (Fig. 1D, a). The capacity toswim and also the cell shape changed in SbIII-and AsIII-treatment groups, with the slender forms being reduced tostumpy or round forms (Fig. 1D, b and c) in comparison tocontrols (Fig. 1D, a). The cell sizes were decreased in the drug-treated groups, indicating cell shrinkage (length vs breadthratio, control 4.53 ± 0.492, SbIII 1.72 ± 0.021, AsIII

1.63 ± 0.014; mean ± SE, n = 3). Therefore, the above dataprovide evidence for the ability of both the metalloids to inducecell shrinkage and mitochondrial dysfunction, which ismanifested by a fall in ΔΨm and ATP loss.

Role of iron during metalloid treatment

The purpose of this part of the study was to focus on therequirement for iron during drug-induced mitochondrialchanges. For this, we used DFO, an iron chelator used iniron-deprivation studies. Presence of DFO during drugtreatment was able to inhibit the fall in ΔΨm induced by SbIII

(Fig. 2A) as well as by AsIII (Fig. 2B), suggesting that thepresence of iron was a prerequisite for drug-induced alterationsin mitochondrial function. To further confirm the involvementof iron, excess iron was added along with DFO to saturate itsbinding sites for iron. This was able to inhibit the ability of DFOto reduce metalloid-inducedΔΨm and ATP loss (Figs. 2A–2D).Therefore, this was evidence that the protective action of DFOon the mitochondria was mediated by its iron-chelating action.To asses if the iron-chelating effect of DFO translated intoincreased cell survival, experiments on cell viability werecarried out with cells preincubated with DFO before treatmentwith the drugs. Presence of DFO during metalloid exposurecould decrease cell death and excess iron added during thesetreatments could increase the cell death percentages (Fig. 3A).This suggested a clear requirement for the presence of iron forthe cytotoxic effect of both the drugs on mitochondrial functionand consequently cell death.

Changes in ROS in response to arsenic and antimony

Mitochondrial dysfunction is capable of inducing severecellular injury through the reduction of molecular oxygen in aone-electron fashion to yield a series of ROS such as superoxideradical, hydrogen peroxide, and hydroxyl radical that arecapable of causing damage to the components of the electrontransport apparatus, disrupting mitochondrial functioning,limiting cellular ATP levels, and ultimately resulting in celldeath [25]. ROS levels were measured with the cell-permeabledye CM-H2DCFDA [27]. Within a period of 6 h, it wasobserved that ROS generation increased post-treatment withSbIII and AsIII, SbIII-induced generation being comparativelyhigher in amount (Fig. 3B). Inset of Fig. 3B showsphotomicrographs of fluorescent cells labeled with CM-H2DCFDA. It is notable that ROS generation occurred after180 min, whereas the sharp decrease in ΔΨm was visible at

Fig. 1. Antimony and arsenic induce mitochondrial dysfunction. (A) Promastigotes (107 cell ml−1) were exposed to SbIII (300 μg ml−1) and AsIII (30 μg ml−1) in vitrofor the time points indicated and monitored for the status of the mitochondrial potential with the potentiometric probe JC-1 (10 μM). Time-dependent changes inrelative ΔΨm values are expressed as the ratio of the reading at 590 nm (aggregate) to the reading at 530 nm (monomer). (B) Photomicrographs of promastigotesstained with JC-1 probe (10 μM). a, c, e, g, i, and k, represent the green channel (monomeric JC-1, 530 nm), whereas b, d, f, h, j, and l represent the red channel(aggregated JC-1, 590 nm). a–f are the images after 1 h of drug treatment, whereas g–l show images after 3 h treatment. Controls: a, b (1 h), g, h (3 h). c, d (SbIII, 1 h); i,j (SbIII, 3 h); e, f (AsIII, 1 h); k, l (AsIII, 3 h). Note the increase in the monomeric form of the dye in the AsIII-and SbIII-treated groups at 3 h, i and k. (C) Decrease in ATPlevels monitored for 6 h after treatment with the two metalloids. Data represent the means ± SE (n = 3). (D) Nomarski images showing cell shape after treatment withthe two metalloids. Note the change in morphology of the promastigotes in groups treated with SbIII and AsIII. a, control; b, SbIII (300 μg ml−1); c, AsIII (30 μg ml−1).


120 min, showing that ROS increase was a postmitochondrialevent.

GSH and metalloid treatment

Having found that a significant generation of ROS took placeafter 180 min, it was pertinent to check GSH levels becauseredox molecules like GSH are important for detoxification ofxenobiotics from cells [28]. In the kinetoplastid parasites, GSHis a component of trypanothione, the primary detoxificationsystem in these cells [29]. Interestingly, a fall in GSH level wasrecorded with SbIII treatment within the first 30 min, whichdecreased further with time (Fig. 3C). In contrast, the AsIII groupshowed virtually no change (Fig. 3C). This suggested aninvolvement of GSH in SbIII detoxification. To furtherilluminate the mechanistic imperatives of GSH action, it wasexplored if addition or depletion of GSH during drug treatmentaffected cell survival. GSH supplementation prevented cell

death as was evident from cell counts in DNA fragmentationassays (Fig. 3D) and membrane permeability studies (Fig. 3E).For depletion of GSH, we used BSO, a γ-glutamylcysteinesynthetase inhibitor [30], during drug treatment, and thisresulted in a significant increase in death percentages in theSbIII group, but not in the AsIII group (Fig. 3F). Clearly, acompromised level of GSH made the cells more susceptible toSbIII. Given the differences in the two responses, the above datademonstrate that GSH is important inmodulating SbIII action butnot AsIII effects on cells.

Intracellular Ca2+ changes during metalloid treatment

Our earlier studies demonstrate the involvement of Ca2+ inLeishmania cell survival in a variety of stress situations.Because there was no decline in GSH levels induced by AsIII

but there was an increase in ROS levels, it was explored if therewas a change in cellular Ca2+. Interestingly, AsIII induced an

Fig. 2. DFO inhibits ΔΨm loss and decline in ATP levels. (A) Preincubation of promastigotes with DFO (500 μM) before exposure to SbIII inhibits the loss of ΔΨm.When excess iron was present, Fe-1 (100 μM), Fe-2 (200 μM), and Fe-3 (400 μM), there was a dose-dependent neutralization of the effect of DFO onΔΨm. (B) Whenpromastigotes were incubated with DFO during exposure to AsIII the loss of ΔΨm was inhibited. When excess iron was present, Fe-1 (100 μM), Fe-2 (200 μM), andFe-3 (400 μM), there was a dose-dependent neutralization of the effect of DFO on ΔΨm. (C) Incubation of promastigotes with DFO during exposure to SbIII inhibitsthe decline in ATP levels. When excess iron was present, Fe-1 (100 μM), Fe-2 (200 μM), and Fe-3 (400 μM), there was a dose-dependent neutralization of the effect ofDFO on the decline in ATP levels. (D) When promastigotes were preincubated with DFO before exposure to AsIII the decline in ATP levels induced by the drug wasinhibited. When excess iron was present, Fe-1 (100 μM), Fe-2 (200 μM), and Fe-3 (400 μM), there was a dose-dependent neutralization of the effect of DFO on thedecline in ATP levels. Data represent the means ± SE (n = 3). *p < 0.05 compared with controls. §p < 0.05 compared with the SbIII-or AsIII-treated groups. ¶p < 0.05compared with the SbIII + DFO group or AsIII + DFO group.


increase in intracellular Ca2+ levels, whereas SbIII was unable todo so (Fig. 4A). A possible way to prove if this elevated Ca2+ hadany effect on cell functioning was to inhibit the Ca2+ increaseand look at biochemical parameters and cell viability. This couldbe accomplished by blocking the Ca2+ pooling mechanisms. Toidentify themechanism of intracellular Ca2+ increase, we neededto distinguish if Ca2+ increase occurred by pooling from internalCa2+ pools or entry was through Ca2+ channels or exchangersfrom extracellular sources. We used EGTA, a Ca2+ chelator, toblock entry of external Ca2+ into the cells, so that any increasefrom internal sources could be visualized. EGTA completelylowered the drug-induced increase in Ca2+ (Fig. 4A),implicating external Ca2+ as the primary source of elevatedinternal Ca2+. Because EGTA is a relatively nonspecific ionchelator and its effect could therefore be due to chelation ofother ions than Ca2+, we needed to identify a more specificway to block Ca2+ selectively. The observation that Ca2+

entered from external sources implied that the entry was via

some ion channel or exchanger; therefore, Ca2+ channelblockers and Ca2+ exchanger inhibitors were tested for theirability to restrict metalloid-induced Ca2+ entry. It was found thatNa+/Ca2+ exchanger inhibitors like bepridil and benzamil and L-type Ca2+ channel blockers like nifedipine and verapamil had noeffect on the influx of Ca2+ (at 6 h, control 60.4 ± 4.6, AsIII

309.6 ± 15.5, AsIII + bepridil 320.4 ± 12.5, AsIII + benzamil304.2 ± 10.2, AsIII + nifedipine 325.1 ± 10.2, AsIII + verapamil295.1 ± 12.4; mean ± SE, n = 4), whereas pimozide, a T-typechannel blocker, could inhibit the influx of Ca2+ (Fig. 4B). It isnoteworthy that the Ca2+ increase occurred well after the lossof mitochondrial function, but was preceded by a ROSincrease.

Effect of iron modulation on ROS and Ca2+ levels

Iron deprivation and addition of excess iron were modulatingdrug effects on mitochondria; therefore, we sought to determine

Fig. 3. Effects of AsIII and SbIII on cell death, ROS generation, and glutathione levels. (A) Flow-cytometric analysis of cell viability by analysis of PI-labeled cells at 24 h after preincubation with 500 μMDFO or 200 μMiron followed by treatment with AsIII and SbIII. Histograms represent three experiments with at least 100,000 cells analyzed/group/experiment. Data represent the means ± SEM (n = 3). *p < 0.05 compared with controls.§p < 0.05 compared with the respective treated groups. (B) Promastigotes labeled with fluorescent dye CM-H2DCFDA show the level of ROS generated by the drugs. Note the increase in ROS levels after treatment withSbIII and AsIII. Data represent the means ± SE (n = 3). Inset: Photomicrographs of cells labeled with CM-H2DCFDA after 4 h of treatment with the two metalloids. a, control; b, SbIII (300 μg ml−1); c, AsIII (30 μg ml−1).(C) Changes in the GSH levels at different time points after treatment of the promastigotes with SbIII and AsIII. Note that SbIII causes a significant drop in GSH levels after treatment by 2 h. Data represent the means ± SE(n = 3). (D and E) Flow-cytometric analysis of TUNEL-labeled and PI-labeled cells at 24 h, percentages of which are represented by bars. Promastigotes were preincubated with 10 mMGSH for 1 h followed by treatmentwith the two metalloids. Histograms are means of three experiments with at least 100,000 cells analyzed/group/experiment. Note the increase in cell viability in SbIII-treated groups preincubated with GSH compared withSbIII-only groups. Data are means ± SE (n = 3). *p < 0.05 compared with controls. §p < 0.05 compared with the respective treated group. (F) Effects of BSO preincubation on cell death in SbIII-treated groups, which wereanalyzed by flow-cytometric analysis of PI-labeled cells at 24 h. Note the increase in cell death in the presence of SbIII under GSH-depleted conditions. Histograms are means of three experiments with at least 100,000cells analyzed/group/experiment. *p < 0.05 compared with controls. §p < 0.05 compared with the respective treated group.

1863A.Mehta,

C.Shaha

/Free

Radical

Biology

&Medicine

40(2006)

1857–1868

Fig. 4. Arsenic induces Ca2+ influx. (A) The changes in intracellular Ca2+ levels after treatment with the twometalloids. Cells (107ml−1) were loaded with the fluorescent dye fluo-3AM tomeasure the cytosolic Ca2+ levelsat various time points as indicated. Note that AsIII treatment causes an increase in the Ca2+ levels, whereas SbIII is unable to do so. Preincubation for 30 min with a calcium chelator, EGTA, decreased the calcium increaseafter AsIII treatment. Data represent the means ± SE (n = 3). (B) Promastigotes were preincubated for 30 min with or without 20 μMpimozide before treatment with AsIII. Inset: Photomicrographs of cells stained with fluo-3AMafter 3 h of treatment. a, control; b, AsIII, c, AsIII + pimozide. (C) Intracellular Ca2+ levels increase after the addition of Fe (200μM) alongwith AsIII, whereas this increase was inhibited after addition of DFO (500μM)before AsIII treatment. Data represent the means ± SE (n = 3). (D and E) Changes in ROS levels of promastigotes after preincubation for 1 h with or without 500 μMDFO or 200 μMFe. Note that preincubation with DFOwas able to reduce the ROS increase in AsIII-as well as in SbIII-treated groups, whereas Fe (200 μM) along with the metalloids was able to increase the ROS levels. Data represent the means ± SE (n = 3).

1864A.Mehta,

C.Shaha

/Free

Radical

Biology

&Medicine

40(2006)

1857–1868

Fig. 5. Effects of delayed inhibition of ROS and Ca2+. (A) Changes in ROS levels measured by CM-H2DCFDA after addition of GSH at different time points (0, 60,120, and 180 min) post-SbIII treatment. Note that addition of GSH was able the reduce ROS levels at all time points. Data represent the means ± SE (n = 3). (B) Flow-cytometric analysis of PI-labeled cells at 24 h after addition of GSH at different time points as indicated (0–180 min) along with SbIII treatment. Histograms are meansof three experiments with at least 100,000 cells analyzed/group/experiment. Note the increase in cell viability in SbIII-treated groups incubated with GSH comparedwith SbIII-only groups. Data are means ± SE (n = 3). *p < 0.05 compared with controls. §p < 0.05 compared with the SbIII-treated group. (C) Changes in intracellularCa2+ levels after addition of pimozide at different time points (0–120 min) post-AsIII treatment. Note that the addition of pimozide was able the reduceintracellular Ca2+ levels. Data represent the means ± SE (n = 3). (D) Flow-cytometric analysis of PI-labeled cells at 24 h after addition of pimozide at differenttime points (as indicated) along with AsIII treatment. Histograms are means of three experiments with at least 100,000 cells analyzed/group/experiment. Note theincrease in cell viability in AsIII-treated groups incubated with pimozide compared with AsIII-only groups. Data are means ± SE. *p < 0.05 compared withcontrols. §p < 0.05 compared with the AsIII-treated group.


if these effects resulted in interference with the increase in ROSand Ca2+ [31]. Preincubation with DFO before AsIII treatmentcould reduce the Ca2+ increase induced by AsIII and addition ofexcess iron increased this Ca2+ level further (Fig. 4C). Fig. 4Dshows the ability of DFO to reduce AsIII-induced ROS increaseand the ability of iron to increase AsIII-induced ROS. DFO wasable to reduce SbIII-induced ROS level increase as well, andexcess iron was able to increase SbIII-induced ROS (Fig. 4E).

Effects of blockade of ROS and Ca2+ at different time pointsduring drug treatment on cell viability

Because after mitochondrial changes there was an increase inCa2+ and an increase in ROS, it was of interest to see if thesewere the effector molecules in bringing about cell death or weremerely consequences of ΔΨm changes without having any

effect on cell viability. Fig. 5A shows that ROS were inhibitedby GSH when it was added at different time points (Fig. 5A).Cell survival was measured consequent to addition of GSH atdifferent time points and GSH was able to salvage SbIII-induceddeath when added at early time points (Fig. 5B). However, GSHadded after 300 min rescued a much smaller number of cellsfrom death (Fig. 5B). Pimozide supplementation at differenttime points during AsIII treatment successfully inhibited Ca2+

entry (Fig. 5C) and Ca2+ levels were reduced by pimozidetreatment, which resulted in increased cell survival (Fig. 5D).However, pimozide added after 300 min had a reduced effect onthe salvage of cell death induced by AsIII. From these data it wasclear that later addition of GSH or pimozide during SbIII or AsIII

treatment, respectively, had reduced salvaging effect on celldeath. Therefore, cell damage initiated by ROS and Ca2+ withinthe first 300 min seems to be lethal.


Discussion

Metalloids are vital for the treatment of diseases caused bykinetoplastid parasites. The primary aim of this study was toinvestigate the mechanisms associated with metalloid-inducedcell death in L. donovani promastigotes. The novel findings arethat iron is essential for parasite death caused by SbIII and AsIII

and that these two very similar metalloids produce toxicity bydifferent mechanisms. By synthesizing the total data obtained, itis evident that (i) both AsIII and SbIII are able to induce celldeath, (ii) both the metalloids interfere with mitochondrialfunction, (iii) iron seems to play a pivotal role in mediating drugeffects, (iv) SbIII but not AsIII lowers GSH levels and GSHsupplementation is able to increase cell survival in SbIII-but notin AsIII-exposed promastigotes, (vi) AsIII but not SbIII increasesintracellular Ca2+ levels and inhibition of Ca2+ influx reducescell death in AsIII-treated cells.

The promastigotes of L. donovani were an ideal system tostudy the effects of the two related metalloids arsenic andantimony because cross-resistance to antimony in arsenic-resistant forms has been reported in this particular stage of thelife cycle [3–6]. For selection of drug doses, the mitochondrialmembrane potential was chosen as a parameter because ourearlier studies as well as studies from other laboratories haveshown that the single mitochondrion of the kinetoplastidparasite is a good indicator of cellular dysfunction [23,32].Because both metalloids generated unfavorable conditions inthe cells within a comparable time frame, inducing mitochon-drial dysfunction, both drugs may have the ability to affect themitochondria. It is important to note that the dose required toinduce mitochondrial dysfunction for AsIII was 10 times lower(30 μg ml−1) than that of SbIII (300 μg ml−1) in inducing thecytotoxic effects, suggesting greater efficacy of AsIII indisrupting cellular function in these parasitic forms.

If we consider the early changes, it is clear that bothmetalloids require iron to induce mitochondrial dysfunction.The observations on the role of iron are important in the contextof survival of the kinetoplastid parasites because their growth issupported by iron in mammalian cells [33,34] and irondepletion has been suggested as a possible therapy againstLeishmaniasis [33]. The importance of iron in mediating drugeffects was substantiated by the inhibition of drug-inducedΔΨm and ATP loss when iron was chelated with DFO in thepresence of the metalloids. The specificity of DFO action wassubstantiated from studies in which DFO saturated with excessiron was unable to provide protective action against drug-induced ΔΨm or ATP loss. The mitochondrial dysfunctionseemed to be the primary cause of cell death because when DFOwas used to prevent ΔΨm loss, cell survival increased. Incontrast, the presence of excess iron during drug exposureincreased cell death compared to only metalloid exposure,showing that the presence or absence of iron was related to drugefficacy in terms of cell killing. This death was accompanied byDNA fragmentation and cell shrinkage, showing an apoptosis-like death phenotype, and possibly explains why minimal ATPgeneration was maintained, as this form of death requires about10-15% ATP to complete the death process [35].

The next question was how this mitochondrial dysfunctioninduced by the drugs led to cell killing. The effector moleculesthat induce cell death could be several. In this case both themetalloids were able to generate ROS that by themselves candamage cellular macromolecules or induce a Ca2+ influx.Interestingly, with SbIII, ROS were the primary effectormolecules, because scavenging of ROS by GSH led to a fallin cell death percentages. This was further substantiated whendepletion of GSH during SbIII treatment resulted in a higher celldeath. In the absence of this cellular antioxidant, the ROS-induced damage would therefore be more pronounced. Studiesin L. tarentolae show that GSH is necessary for antimonyremoval from the cell and trypanothione is the main target forarsenicals in Trypanosoma [29]. The observation that ROSgenerated by SbIII could be prevented if cellular thiol levelswere increased by incubation of the cells with GSH, unlikearsenic-treated cells, in which GSH could not reduce ROS,demonstrates that the handling of ROS generated by the twodrugs is different. This is possible because the binding ofarsenic to GSH is known to reduce the capacity of GSH toeliminate ROS [36,37]. It is likely because the sulfhydryl groupof the cysteine moiety of GSH has a high affinity for metals,forming thermodynamically stable mercaptides with severalmetals, including mercury and arsenic [37], and therefore GSHbecomes unavailable for other reactions. Our data support theobservation that SbIII induces GSH loss in Leishmania spp. [7];however, the involvement of GSH during arsenic treatmentseems not to be valid in L. donovani as reported for Africantrypanosomes [29].

AsIII cytotoxicity could be attributed to Ca2+ becauseinhibition of Ca2+ increase by blocking the relevant Ca2+

channel prevented ΔΨm fall and cell death. The success ofEGTA and pimozide in preventing Ca2+ influx shows that thesource of increased intracellular Ca2+ was solely fromextracellular sources. Because L-type channel and Na+/Ca2+

exchanger blockers did not interfere with Ca2+ entry, but a T-type channel blocker did, Ca2+ was probably entering the cellthrough T-type channels. It is known that ROS are able toinduce opening of T-type Ca2+ channels in vascular smoothmuscle cells [38]; however, whether that was occurring hereremains an open question although experiments suggests such amechanism. Emptying of intracellular Ca2+ stores and/oralteration in intracellular Ca2+ levels can modulate cell deathin almost all cell types and our earlier studies with L. donovanishowed that during oxidative stress Ca2+ was able to modulatecell death [23]. The fact that the Ca2+ increase occurring post-drug treatment was preceded by an elevation of ROS levelssuggested that ROS elevation was responsible for the Ca2+

increase observed. This hypothesis is likely because ironchelation leads to a decrease in ROS resulting in a decrease inCa2+.

Because cell death with both AsIII and SbIII was accompa-nied by cell shrinkage and DNA fragmentation, it seems that thecells express an apoptotic phenotype. Studies from thislaboratory and others have shown that under oxidative stress,treatment with drugs leads to apoptotic death in Leishmaniaspp. [23,32]. This study therefore provides evidence for a


connection between iron, ROS increase, and Ca2+ duringarsenic treatment and between iron, ROS increase, and GSHduring antimony treatment. Interpreting the above results, wepropose a new possibility of iron as an important component inmodulating the effects of the metalloid drugs in the lowereukaryotic organism L. donovani, an observation previouslyunknown. The other important findings of interest are the verydifferent cellular effects induced by the two related metalloidswhile inducing cell death. Application of such knowledge couldprovide opportunities to develop efficient drugs by increasingiron overload or targeting depletion of GSH.

Acknowledgments

This work was supported by the Indian Council of MedicalResearch and Department of Biotechnology, Government ofIndia.

References

[1] Berman, J. Current treatment approaches to leishmaniasis. Curr. Opin.Infect. Dis. 16:397–401; 2003.

[2] Singh, S.; Sivakumar, R. Challenges and new discoveries in the treatmentof leishmaniasis. J. Infect. Chemother. 10:307–315; 2004.

[3] Lee, S. T.; Tarn, C.; Chang, K. P. Characterization of the switch ofkinetoplast DNA minicircle dominance during development and reversionof drug resistance in Leishmania. Mol. Biochem. Parasitol. 58:187–203;1993.

[4] Mukhopadhyay, R.; Dey, S.; Xu, N.; Gage, D.; Lightbody, J.; Ouellette,M.; Rosen, B. P. Trypanothione overproduction and resistance toantimonials and arsenicals in Leishmania. Proc. Natl. Acad. Sci. USA93:10383–10387; 1996.

[5] Rosen, B. P. Resistance mechanisms to arsenicals and antimonials. J. BasicClin. Physiol. Pharmacol. 6:251–263; 1995.

[6] Zhou, Y.; Messier, N.; Ouellette, M.; Rosen, B. P.; Mukhopadhyay, R.Leishmania major LmACR2 is a pentavalent antimony reductase thatconfers sensitivity to the drug pentostam. J. Biol. Chem. 279:37445–37451; 2004.

[7] Wyllie, S.; Cunningham, M. L.; Fairlamb, A. H. Dual action of antimonialdrugs on thiol redox metabolism in the human pathogen Leishmaniadonovani. J. Biol. Chem. 279:39925–39932; 2004.

[8] Gull, K. The biology of kinetoplastid parasites: insights and challengesfrom genomics and post-genomics. Int. J. Parasitol. 31:443–452;2001.

[9] Meng, Y. L.; Liu, Z.; Rosen, B. P. As(III) and Sb(III) uptake by GlpF andefflux by ArsB in Escherichia coli. J. Biol. Chem. 279:18334–18341;2004.

[10] Dey, S.; Ouellette, M.; Lightbody, J.; Papadopoulou, B.; Rosen, B. P. AnATP-dependent As(III)-glutathione transport system in membrane vesiclesof Leishmania tarentolae. Proc. Natl. Acad. Sci. USA 93:2192–2197; 1996.

[11] Guerin, P. J.; Olliaro, P.; Sundar, S.; Boelaert, M.; Croft, S. L.; Desjeux, P.;Wasunna, M. K.; Bryceson, A. D. Visceral leishmaniasis: current status ofcontrol, diagnosis, and treatment, and a proposed research and develop-ment agenda. Lancet Infect. Dis. 2:494–501; 2002.

[12] Sudhandiran, G.; Shaha, C. Antimonial-induced increase in intracellularCa2+ through non-selective cation channels in the host and the parasite isresponsible for apoptosis of intracellular Leishmania donovani amasti-gotes. J. Biol. Chem. 278:25120–25132; 2003.

[13] Douer, D.; Tallman, M. S. Arsenic trioxide: new clinical experience withan old medication in hematologic malignancies. J. Clin. Oncol.23:2396–2410; 2005.

[14] Liu, J.; Shen, H. M.; Ong, C. N. Role of intracellular thiol depletion,mitochondrial dysfunction and reactive oxygen species in Salvia

miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells. LifeSci. 69:1833–1850; 2001.

[15] Shaked-Mishan, P.; Ulrich, N.; Ephros, M.; Zilberstein, D. Novelintracellular SbV reducing activity correlates with antimony suscep-tibility in Leishmania donovani. J. Biol. Chem. 276:3971–3976;2001.

[16] Berman, J. D.; Hanson, W. L.; Chapman, W. L.; Waits, V. B.; Cox, R. H.Toxicity of formycin B to hamster and dog. J. Parasitol. 73:1267–1268;1987.

[17] Roberts, W. L.; Berman, J. D.; Rainey, P. M. In vitro antileishmanialproperties of tri-and pentavalent antimonial preparations. Antimicrob.Agents Chemother. 39:1234–1239; 1995.

[18] Wilson, M. E.; Vorhies, R. W.; Andersen, K. A.; Britigan, B. E.Acquisition of iron from transferrin and lactoferrin by the protozoanLeishmania chagasi. Infect. Immun. 62:3262–3269; 1994.

[19] Borges, V. M.; Vannier-Santos, M. A.; de Souza, W. Subverted transferrintrafficking in Leishmania-infected macrophages. Parasitol. Res. 84:811–822; 1998.

[20] Lemesre, J. L.; Sereno, D.; Daulouede, S.; Veyret, B.; Brajon, N.;Vincendeau, P. Leishmania spp.: nitric oxide-mediated metabolic inhibi-tion of promastigote and axenically grown amastigote forms. Exp.Parasitol. 86:58–68; 1997.

[21] Valko, M.; Morris, H.; Cronin, M. T. Metals, toxicity and oxidative stress.Curr. Med. Chem. 12:1161–1208; 2005.

[22] Das, M.; Mukherjee, S. B.; Shaha, C. Hydrogen peroxide inducesapoptosis-like death in Leishmania donovani promastigotes. J. Cell Sci.114:2461–2469; 2001.

[23] Mukherjee, S. B.; Das, M.; Sudhandiran, G.; Shaha, C. Increase incytosolic Ca2+levels through the activation of non-selective cationchannels induced by oxidative stress causes mitochondrial depolarizationleading to apoptosis-like death in Leishmania donovani promastigotes.J. Biol. Chem. 277:24717–24727; 2002.

[24] Mehta, A.; Shaha, C. Apoptotic death in Leishmania donovanipromastigotes in response to respiratory chain inhibition: complex IIinhibition results in increased pentamidine cytotoxicity. J. Biol. Chem.279:11798–11813; 2004.

[25] Ly, J. D.; Grubb, D. R.; Lawen, A. The mitochondrial membrane potential(deltapsi(m)) in apoptosis; an update. Apoptosis 8:115–128; 2003.

[26] Saraiva, E. M.; Pinto-da-Silva, L. H.; Wanderley, J. L.; Bonomo, A. C.;Barcinski, M. A.; Moreira, M. E. Flow cytometric assessment ofLeishmania spp metacyclic differentiation: validation by morphologicalfeatures and specific markers. Exp. Parasitol. 110:39–47; 2005.

[27] Duranteau, J.; Chandel, N. S.; Kulisz, A.; Shao, Z.; Schumacker, P. T.Intracellular signaling by reactive oxygen species during hypoxia incardiomyocytes. J. Biol. Chem. 273:11619–11624; 1998.

[28] Fairlamb, A. H.; Cerami, A. Metabolism and functions of trypanothione inthe Kinetoplastida. Annu. Rev. Microbiol. 46:695–729; 1992.

[29] Fairlamb, A. H.; Henderson, G. B.; Cerami, A. Trypanothione is theprimary target for arsenical drugs against African trypanosomes. Proc.Natl. Acad. Sci. USA 86:2607–2611; 1989.

[30] Nicole, A.; Santiard-Baron, D.; Ceballos-Picot, I. Direct evidence forglutathione as mediator of apoptosis in neuronal cells. Biomed. Pharmac-other. 52:349–355; 1998.

[31] Brookes, P. S.; Yoon, Y.; Robotham, J. L.; Anders, M. W.; Sheu, S. S.Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J.Physiol., Cell Physiol. 287:C817–C833; 2004.

[32] Luque-Ortega, J. R.; Rivero-Lezcano, O. M.; Croft, S. L.; Rivas, L.In vivo monitoring of intracellular ATP levels in Leishmaniadonovani promastigotes as a rapid method to screen drugs targeting bio-energetic metabolism. Antimicrob. Agents Chemother. 45:1121–1125;2001.

[33] Soteriadou, K.; Papavassiliou, P.; Voyiatzaki, C.; Boelaert, J. Effect of ironchelation on the in-vitro growth of Leishmania promastigotes.J. Antimicrob. Chemother. 35:23–29; 1995.

[34] Weinberg, E. D. The role of iron in protozoan and fungal infectiousdiseases. J. Eukaryotic Microbiol. 46:231–238; 1999.

[35] Lemasters, J. J.; Qian, T.; Bradham, C. A.; Brenner, D. A.; Cascio, W. E.;Trost, L. C.; Nishimura, Y.; Nieminen, A. L.; Herman, B. Mitochondrial


dysfunction in the pathogenesis of necrotic and apoptotic cell death.J. Bioenerg. Biomembr. 31:305–319; 1999.

[36] Hour, T. C.; Huang, C. Y.; Lin, C. C.; Chen, J.; Guan, J. Y.; Lee, J. M.; Pu,Y. S. Characterization of molecular events in a series of bladder urothelialcarcinoma cell lines with progressive resistance to arsenic trioxide.Anticancer Drugs 15:779–785; 2004.

[37] Wang, W.; Ballatori, N. Endogenous glutathione conjugates: occurrenceand biological functions. Pharmacol. Rev. 50:335–356; 1998.

[38] Tabet, F.; Savoia, C.; Schiffrin, E. L.; Touyz, R. M. Differential calciumregulation by hydrogen peroxide and superoxide in vascular smoothmuscle cells from spontaneously hypertensive rats. J. Cardiovasc.Pharmacol. 44:200–208; 2004.

Mechanism of metalloid-induced death in Leishmania spp.: Role of iron, reactive oxygen species,...

Documents

Transcript of Mechanism of metalloid-induced death in Leishmania spp.: Role of iron, reactive oxygen species,...