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Molecular & Biochemical Parasitology 152 (2007) 192–202

Mitochondrial superoxide mediates heat-induced apoptotic-likedeath in Leishmania infantum

Juan F. Alzate a,b,c, Andres A. Arias a, David Moreno-Mateos a,Alberto Alvarez-Barrientos d, Antonio Jimenez-Ruiz a,∗

a Departamento de Bioquımica y Biologıa Molecular, Campus Universitario, Universidad de Alcala, 28871 Alcala de Henares, Madrid, Spainb Escuela de Bacteriologıa, Universidad de Antioquia, Medellın, Colombia

c Programa de Estudio y control de enfermedades Tropicales, PECET, Universidad de Antioquia, Medellın, Colombiad Fundacion Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain

Received 6 September 2006; received in revised form 22 December 2006; accepted 4 January 2007Available online 9 January 2007

bstract

Previous studies have shown that heat stress triggers a process of programmed cell death in Leishmania infantum promastigotes that resemblespoptosis in higher eukaryotes. Even though this cell death process takes about 40 h to be completed, several early changes in the heat-stressed cellsan be observed. Hyperpolarization of the parasite mitochondrion is the earliest event detected, which correlates with an increase in respiration ratesnd a concomitant increase in superoxide radical production. Induction of oxidative stress seems to mediate the heat-induced cell death process,s indicated by the partial prevention of parasite death observed when cell cultures are supplemented with N-acetyl-cysteine or glutathione. These

ntioxidants are able to diminish the concentration of superoxide radical but they do not prevent mitochondrial hyperpolarization. Treatment ofhe heat stressed parasites with the inhibitors of the mitochondrial respiration TTFA, antimycin A and KCN significantly decreases the productionf superoxide radicals, which confirms the mitochondrial origin of this reactive oxygen species.

2007 Elsevier B.V. All rights reserved.

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eywords: Cellular respiration; Mitochondria; Oxidative stress; Superoxide rad

. Introduction

During mitochondrial respiration, several reactive oxygenpecies (ROS), mainly superoxide anion (O2

•−), are formed asy-products, [1]. Mitochondrial respiratory chain complexes Ind III constitute the main source of superoxide in mammals,hich is then converted to hydrogen peroxide (H2O2) by spon-

aneous dismutation or by superoxide dismutase (SOD) [2,3].

ow concentrations of ROS are then usually present in the cellsnd seem to be implicated in physiological processes. How-ver, overproduction of these toxic oxygen intermediates may

Abbreviations: CMFDA, chloro-methyl-fluorescein-diacetate; DHE, dihy-roethidium; GSH, glutathione; H2DCFDA, 2′,7′-dichlorodihydrofluoresceiniacetate; PI, propidium iodide; NAC, N-acetyl cysteine; TTFA, thenoyltriflu-roacetone; SOD, superoxide dismutase; ROS, reactive oxygen species; PCD,rogrammed cell death∗ Corresponding author. Tel.: +34 91 885 5109; fax: +34 91 885 4585.

E-mail address: antonio.jimenez@uah.es (A. Jimenez-Ruiz).

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166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2007.01.006

Heat shock; Programmed cell death

nduce damage to proteins, lipids and DNA, which eventuallyeads to programmed cell death (PCD) or necrosis [4–7]. Try-anosomatids, the causative agents of African sleeping sicknessTrypanosoma brucei gambiense and T.B. rhodesiense), Naganaattle disease (T. congolense, T.b. brucei), South-American Cha-as’ disease (T. cruzi) and the different forms of Leishmaniasis,ack a typical rotenone-sensitive complex I in their electronransfer chain but, despite this significant difference, respirationn these parasites also leads to the production of ROS [8].

In higher eukaryotes, enzymes such as superoxide dismu-ase [9], catalase [10] and the selenium-containing enzymelutathione peroxidase [11] constitute an effective systemo eliminate these by-products generated during oxidativehosphorylation. Superoxide dismutase is responsible for theismutation of O2

•− into H2O2 and O2, and catalase and glu-

athione peroxidase are responsible for the removal of H2O2 byonverting it into water and molecular oxygen. The latter twonzymes are not found in Trypanosomatids, instead, they con-ain three different classes of peroxidases that use trypanothione

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s the electron donor [12]. Even though the activity of theseeroxidases is lower than that observed in glutathione peroxi-ase, their high concentration (5% of the total soluble protein inome cases [13,14]) allows an efficient control of the oxidativetress associated to the activity of the parasites. On the otherand, as described in higher eukaryotes, superoxide anion elim-nation in these parasites is based on the activity of SOD, forhich two different subtypes have been described (SODA andODB). Overexpression of these SOD in L. chagasi parasitesonferred enhanced protection against the free radical generat-ng agents paraquat and nitroprusside [15], whereas, parasitesontaining a single allele knock out of one of the SODB genesxhibited an important reduction in growth in the presence ofaraquat and a significant decrease in survival within humanacrophages [16]. Similar results have been described for L.

ropica [17]. Because, ROS generation is one of the microbici-al strategies of the macrophage [18], it was not surprising thatesistance to oxidative stress correlated with the ability of thearasite to infect and proliferate within cell culture macrophages15,19].

Recent results from our laboratory indicate that a mild heathock (38 ◦C) induces a process of PCD in Leishmania infan-um promastigotes similar to apoptosis from higher eukaryotes20]. This effect may be relevant during infection because inhis process the parasites are transferred from a temperaturef 25–27 ◦C to 37–38 ◦C (depending on the vertebrate host).t has been postulated that the apoptotic-like death of some par-sites during infection, because of its similarity with apoptosisf the host’s cells, could be involved in restricting the immuneesponse of the host [20]. In an attempt to define the cascade ofrocesses that lead to heat-induced cell death, we searched forarly changes in the parasites during heat exposure. Among theery first alterations, we have found a fast increase in oxygenonsumption by the parasites and an early hyperpolarization ofheir single mitochondrion. The results presented in this paperndicate that the early increase in mitochondrial activity is asso-iated to a rapid increase in O2

•− intracellular concentrationnd that this increase is directly involved in the process of celleath.

. Materials and methods

.1. Chemical and reagents

Dihydroethidium (DHE), chloro-methyl-fluorescein-diace-ate (CMFDA) and 2′,7′-dichlorodihydrofluorescein diacetateH2DCFDA), were obtained from INVITROGEN Molecularrobes (Leiden, The Netherlands). Other reagents of the highestuality were purchased from Sigma (St. Louis, MO) or MerckDarmstadt, Germany).

.2. Cells and culture conditions and heat shock stress

L. infantum promastigotes (M/CAN/ES/96/BCN150 MON-), kindly provided by Dr. Alonso (CBMSO-Universidadutonoma Madrid, Spain), were grown in RPMI-1640 medium

Gibco, Paisley, UK) supplemented with 10% heat-inactivated

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l Parasitology 152 (2007) 192–202 193

oetal calf serum (FCS), antibiotics, and 2 mM glutamine at6 ◦C. Transfected strains were selected at 20 �g/mL GenetecinPROMEGA CAT# 11811-031) in RPMI/10%FCS and thenaintained at 100 �g/mL. In the assays with the transfected

trains, the parasites were maintained 1 week without genetecino avoid interference of this antibiotic with the death process.he absence of any effect on the expression level of the trans-enes was confirmed. Heat treatment of the promastigotes waserformed during logarithmic growth phase at a concentration of.5 × 106 parasites/mL at 38 ◦C. Late stationary promastigotesere obtained after incubation of the parasites for more that 10ays with a starting inoculum of 1 × 106 parasites/mL. In theseonditions, saturation of the culture was achieved after 4 days.

.3. Mitochondrial transmembrane potential (�Ψm)

Parasites in RPMI medium/10%FCS at 1.5 × 106 parasites/L were incubated with 50 nM tetramethylrhodamin methyl

sther perchlorate (TMRM, Sigma CAT# T5428) 15 min prioro their analysis in the cytometer. TMRM loading was carriedut at 26 ◦C in control parasites and at 38 ◦C in heat-stressedarasites and they were kept at these temperatures until theirnalysis in the cytometer. TMRM concentration was adjusted toeasure either hyperpolarization or depolarization of the para-

ite mitochondrion without the need of probe removal. Possiblehanges in the solubility or fluorescence of the dye due to vari-tions in temperature were discarded by using the uncouplerarbonyl cyanide p-trifluoromethoxyphenylhydrazone (100 �MCCP) at both temperatures.

.4. Measure of respiration rates

The overall oxygen consumption by the parasites was mea-ured using the oxygraph (Hansatech-Instruments). Briefly,ogarithmic promastigotes were centrifuged and resuspended inBS 10 mM glucose at a final concentration of 10 × 106/mL.arasite suspension of 1 mL was transferred to the oxygraphhamber and the oxygen consumption rate was measured dur-ng at least 5 min for every treatment. Respiration rates of controlarasites cultured at 26 ◦C were measured. To analyze the effectf HS, the temperature of these parasites was raised from 26 ◦Co 38 ◦C with a constant flow of water at 38 ◦C over the exteriorompartment of the measuring chamber. As a control, oxy-en consumption was completely inhibited by the addition ofotassium cyanide (KCN) 3 mM at both temperatures (26 ◦C or8 ◦C).

.5. ROS production detection and antioxidants treatment

Before each experiment the parasites were preloaded 45 minrior to the analysis with dihydroethidium (DHE) or 2′,7′-ichlorodihydrofluorescein diacetate (H2DCFDA) at 1 �M anduorescence emission was measured at the indicated times using

FACScalibur flow cytometer (Beckton-Dickinson, BD). Glu-

athione (GSH) and N-acetyl-cysteine (NAC) were dissolved interile water and added to 1 mM final concentration 1 h prior tohe heat challenge.

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The analysis of the production of superoxide under oxidativehosphorylation uncoupling conditions was carried out with theonophores FCCP and CCCP: parasites were loaded with theHE probe as described before, 35 min later the ionophoresCCP or CCCP were added at the concentrations indicated ingures; then the DHE fluorescence was measured with the flowytometer after 60 min of incubation at 26 ◦C or 38 ◦C.

For the induction of oxidative stress with menadione (dis-olved 50 mM in ethanol), this compound was supplemented atconcentration of 10 �M to the parasite culture and incubated

or the time indicated on each experiment.

.6. Intracellular thiol levels measures

Intracellular thiol levels were measured with the thiol reactiverobe chloro-methyl-fluorescein-diacetate (CMFDA) [21,22].fter 1 h of treatment in the different conditions, aliquots of00 �L of the treated parasites (26 ◦C, 26 ◦C + Menadione or8 ◦C) were supplemented with 100 nM of the probe CMFDA,ncubated for 30 min at 26 ◦C and protected from light. Then, thearasites were centrifuged at 800 × g for 5 min, the medium withhe probe was removed and the parasites were resuspended in00 �L of PBS. Subsequently, the parasites were re-centrifuged,he PBS was removed and the parasites were resuspended in00 �L of PBS and then immediately analyzed with the flowytometer.

.7. DNA content analysis by flow cytometry

One and a half million parasites were centrifuged at 1000 × gor 5 min, the pellet was resuspended in 100 �L of PBS, 600 �Lf −20 ◦C cold 70% ethanol were added and then parasitesere incubated on ice for 30 min. After incubation, the para-

ites were washed with 1 mL of PBS-EDTA 50 mM, pelletedt 1000 × g and then resuspended in 400 �L of PBS/50 mM

DTA/50 �g/mL RNase and incubated for 30 min at 37 ◦C.hen, propidium iodide (PI) was added to a final concentra-

ion of 5 �g/mL and the parasites were immediately analyzedor PI fluorescence.

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ig. 1. Analysis of mitochondrial membrane potential (�Ψm) and oxygen consumpnfantum promastigotes. Parasites grown at 26 ◦C were exposed to 38 ◦C for 90 min.t 26 ◦C and 38 ◦C during the same period of time. Fluorescence was analyzed by flncubated at 26 ◦C and 38 ◦C. Respiration of the parasites was abolished at both temphe asterisks indicate a significant difference between mean values of fluorescence (A-Values are indicated under each panel. Error bars represent standard deviation.

al Parasitology 152 (2007) 192–202

.8. Measure of parasite death after HS challenge

DNA content of the parasites and plasma membrane per-eability to propidium iodide (PI) were analyzed as described

reviously [20].

.9. Inhibition of cellular respiration

Exponentially growing parasites were pre-incubated withCN 10 �M, antimycin A 10 �M or thenoyltrifluoroacetone

TTFA) 500 �M during 30 min before the heat treatment.

.10. Flow cytometry data acquisition and analysis

Parasites treated with the procedures described above werenalyzed in a FACScalibur flow cytometer (BD, san Jose, CA)quipped with a 488 nm Ar-laser. All the analyses and graphicalepresentations were carried out with the package CELLQUESTRO (BD) version 4.

.11. Fluorescence microscopy

Logarithmically growing parasites were labelled with 1 �MHE in RPMI as described above, then incubated at 38 ◦C or6 ◦C during 1 h and labelled with 10 �g/mL Hoechst 33342.ne drop of each cell culture was deposited on a glass slide,

overed with a coverslip and immediately photographed in auorescent microscope (NIKON Diaphot 300). Excitation wasone with an UV-filtered Hg lamp.

.12. Determination of SOD activity

Either logarithmic or late stationary parasites were lysedn 100 �L of ice cold lysis buffer (20 mM HEPES, pH 7.2,

ion of 160 × 106 parasites/mL. After sonication, lysates wereentrifuged at 1500 × g for 5 min at 4 ◦C. SOD activity wasetermined in the supernatants as indicated by the kit manufac-urer (superoxide dismutase assay kit; Cayman Chemical).

tion in heat-treated parasites. (A) Relative fluorescence due to TMRM of L.As a control, parasites were treated with the uncoupler FCCP (100 �M) bothow cytometry (n = 3). (B) Oxygen consumption of L. infantum promastigoteseratures by addition of KCN at 3 mM (26 ◦C + KCN and 38 ◦C + KCN) (n = 5).) or oxygen consumption (B) of the parasites incubated at 38 ◦C and at 26 ◦C.

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cbdoat 26 ◦C. Differences in the intensity of fluorescence are statisti-cally significant after 20 min of HS treatment and continue to beso even after 16 h of heat treatment, when the presence of deadparasites is already evident (data not shown). Pre-incubation of

Fig. 2. (A) Analysis of O2•− intracellular concentration in heat-treated par-

asites. L. infantum parasites were incubated at the temperatures indicated infigure (26 ◦C and 38 ◦C). Parasites incubated at 38 ◦C were also supplementedwith GSH 1 mM (38 ◦C + GSH) or NAC 1 mM (38 ◦C + NAC) 1 h before heattreatment. DHE-derived fluorescence was evaluated by flow cytometry at thetime points indicated in figure. Error bars represent standard deviation. Aster-isks indicate a significant difference between mean values of fluorescence ofthe parasites incubated at 38 ◦C and at 26 ◦C. *p < 0.05; **p < 0.001; n = 3. (B)Analysis of intracellular peroxide concentration in heat-treated parasites. L.infantum parasites were incubated at the temperatures indicated in figure (26 ◦Cand 38 ◦C). H2DCFDA-derived fluorescence was evaluated by flow cytometryat the time points indicated in figure. Error bars represent standard deviation(n = 3). (C) Analysis of mitochondrial membrane potential (�Ψm) in the pres-ence of the antioxidants GSH and NAC. L. infantum parasites were incubated atthe temperatures indicated in the figure (26 ◦C and 38 ◦C). Parasites incubatedat 38 ◦C were also supplemented with GSH 1 mM (38 ◦C + GSH) or NAC 1 mM

J.F. Alzate et al. / Molecular & Bioch

.13. Statistics

All the analyses were carried out with a minimum of threendependent experiments. Statistical significance of the dif-erences between treatments were evaluated by a two-tailednpaired t-test.

. Results

.1. Heat shock induces a rapid mitochondrialyperpolarization and increases O2 consumption rates

The highly sensitive probe TMRM was used to measurehanges in the mitochondrial transmembrane potential (�Ψm)f logarithmically growing L. infantum promastigotes exposedo 38 ◦C. Previous studies in our group have already shown thebility of this probe to specifically detect changes in this poten-ial in Leishmania parasites [20]. The results presented in Fig. 1Andicate a rapid increase in �Ψm when the parasites are incu-ated at 38 ◦C, as shown by the significant and sustained increasen TMRM-derived fluorescence observed after 30 min of heatxposure. The uncoupler FCCP (100 �M) was added as a con-rol both at 26 ◦C and 38 ◦C. The similar pattern of fluorescencen the presence of the uncoupler at both temperatures discardsny possible effect of the temperature in the fluorescence of theMRM-labelled parasites.

To assess whether the early mitochondrial hyperpolarizationould be due to an increase in mitochondrial activity, oxygenonsumption of the promastigotes was measured under basalnd heat-stressing conditions (26 ◦C and 38 ◦C, respectively).s shown in Fig. 1B, promastigotes double their oxygen con-

umption rates early after transferring to 38 ◦C, which suggesthat hyperpolarization could be the consequence of an increasedow of electrons across the respiratory chain when the para-ites are exposed to 38 ◦C. As expected, oxygen consumption atoth 26 ◦C and 38 ◦C is almost abolished when the parasites arencubated with the respiratory chain complex IV inhibitor KCN.

.2. HS induces oxidative stress in Leishmaniaromastigotes

As indicated before, it has been described that the electronransport that occurs during mitochondrial oxidative phospho-ylation generates a significant amount of ROS. Accordingly,t would not be surprising that the described increase in mito-hondrial respiration rates and mitochondrial hyperpolarizationere associated to higher rates of ROS production and, conse-uently, to higher concentration of this species into the cell. Therobes dihydroethidium (DHE) [23] and dihydrodichlorofluo-escein (H2DCFDA) [24] were used to detect changes in thentracellular concentration of O2

•− and peroxides, respectively.oth of them were used at 1 �M concentration and the parasitesere exposed to them 45 min prior to any change in temper-

ture conditions. In both cases, the probe is modified by theresence of its specific ROS and transforms in a fluorescentolecule that accumulates inside the cell. Accordingly, a pro-

ressive increase in fluorescence is expected with time, even in

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l Parasitology 152 (2007) 192–202 195

ontrol conditions, due to the physiological production of ROSy the mitochondrion. As shown in Fig. 2A, an increase in DHE-erived fluorescence is observed immediately after incubationf the parasites at 38 ◦C when compared with control parasites

38 ◦C + NAC) 1 h before heat treatment. Relative fluorescence due to TMRMas evaluated by flow cytometry at 30 and 120 min of treatment. Error bars repre-

ent standard deviation. Asterisks indicate a significant difference between meanalues of fluorescence of the parasites incubated at 38 ◦C (38 ◦C; 38 ◦C + GSH;8 ◦C + NAC) and at 26 ◦C. *p < 0.05; n = 3.

196 J.F. Alzate et al. / Molecular & Biochemical Parasitology 152 (2007) 192–202

Fig. 3. Intracellular thiol levels in heat-treated parasites. (A) L. infantum promastigotes were incubated at 26 ◦C either in the presence or in the absence of 10 �Mmenadione. The oxidation rate of the DHE was measured by flow cytometry during the first 90 min of treatment. (B) Cellular levels of thiols were estimated witht d in parasites incubated for 1 h in the following conditons: 26 ◦C; 26 ◦C with 10 �Mm tion. Asterisks indicate a significant difference between mean values of fluorescence(

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Fig. 4. Analysis of O2•− intracellular concentration in heat-treated para-

sites exposed to respiratory chain inhibitors. L. infantum promastigotes werepreloaded with Dihydroethidium 1 �M (DHE) as described in materials andmethods, exposed at the temperatures indicated in figure and treated with therespiratory chain inhibitors: (A) TTFA (Complex II); (B) antimycin A (Complex

he probe CMFDA. The fluorescence corresponding to this probe was measureenadione (26 ◦C + menadione) and 38 ◦C. Error bars represent standard devia

n = 3).

eat-treated parasites with the antioxidants Glutathione (GSH)r N-acetyl-cysteine (NAC) restores the fluorescent signal tohe basal levels observed in control (26 ◦C) parasites (Fig. 2A).nexpectedly, H2DCFDA-derived fluorescence does not show

ny increase in heat-stressed parasites (Fig. 2B), which indicateshat peroxide concentration is not affected during the heat shock.

Since treatment of the parasites with the antioxidants GSHnd NAC restores O2

•− concentration of heat-stressed parasiteso basal levels, mitochondrial transmembrane potential of theeat-treated parasites in the presence of GSH and NAC wasvaluated, in an attempt to determine whether mitochondrialyperpolarization could be a consequence of O2

•− accumu-ation. The results presented in Fig. 2C demonstrate that theitochondrial hyperpolarization associated to HS is maintained

n the presence of the antioxidants, which indicates that thebserved alterations in the mitochondria are not a consequencef ROS production.

Increased cellular levels of ROS can lead the cell to aore oxidized state termed oxidative stress, characterized by

he oxidation of several intracellular molecules and/or groups,uch as the intracellular thiol groups. In order to determinehether the increased production of superoxide radical underS induces oxidative stress in the promastigotes, we treated thearasites with chloro-methyl-fluorescein-diacetate (CMFDA), aell-permeant thiol-reactive probe [21,22]. This molecule reactsith thiol groups, labelling the cell with a green fluorescence

hat is proportional to the thiol levels of the cell, allowing us toompare the thiol levels at basal conditions (26 ◦C) or after heattress (38 ◦C). Menadione, a well-known inducer of oxidativetress, was used as a positive control. The results presented inig. 3A demonstrate that menadione induces an accelerated oxi-ation of the probe DHE when compared to the control parasites,s an indication of its ability to increase the intracellular con-entration of O2

•−. As expected, treatment of the parasites withenadione for 1 h induced a significant reduction in the fluores-

ent signal associated to CMFDA (Fig. 3B), as an indication ofhe decrease in the concentration of thiol groups. A significant

eduction of the fluorescence was also observed in the parasitesfter incubation at 38 ◦C for 1 h, which demonstrates a reductionn the thiol levels in the heat-treated cells that is indicative ofxidative stress (Fig. 3B).

III) and (C) KCN (Complex IV). Fluorescence was evaluated by flow cytometryat the time points shown in each figure. Error bars represent standard deviation.Asterisks indicate a significant difference between mean values of fluorescenceof the parasites incubated at 38 ◦C in the absence or in the presence of respiratorychain inhibitors. The p-value is indicated in each panel (n = 3).

J.F. Alzate et al. / Molecular & Biochemical Parasitology 152 (2007) 192–202 197

Fig. 5. Superoxide intracellular distribution in heat-treated parasites. Parasites were preloaded with DHE 1 �M, then exposed for 1 h to 26 ◦C or 38 ◦C, stainedw . (A) Ps m bar4 tainin

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b3the kinetoplast, the two DNA-containing structures present inthe parasites. The kinetoplast is a structure composed by a cate-nated network of DNA maxicircles and minicircles located intothe mitochondrion, close to the base of the flagellum that consti-

Fig. 6. Effect of mitochondrial uncouplers on the O2•− production. Promastig-

otes were preloaded with 1 �M DHE and then supplemented with the uncouplingagents FCCP or CCCP at the concentrations indicated in figure. After 60 minof incubation at 26 ◦C or 38 ◦C, the DHE oxidation was measured by flow

ith Hoechst33342 and observed at the microscope at a 1000× magnificationame parasites were observed with a 330–380 nm excitation filter and a 435 n50–490 nm excitation filter and a 520 nm barrier filter for 2-hydroxyethidium s

.3. Inhibitors of cellular respiration diminish the O2•−

roduction

In order to determine the participation of the mitochondrialespiratory complexes in the HS-induced O2

•− production ineishmania promastigotes, specific inhibitors of the electron

ransfer chain were added to the parasite cell cultures beforehe HS challenge, and O2

•− production was then analyzed. Ashown in Fig. 4A, a significant reduction in O2

•− productionan be observed when the promastigotes are incubated with thenhibitor of the complex II TTFA. Similarly, treatment of thearasites either with the inhibitor of complex III, antimycin AFig. 4B), or with the inhibitor of complex IV, cyanide (Fig. 4C),ignificantly decreases the steady state concentration of O2

•−nder HS conditions. Rotenone, the usual inhibitor of complex, was not tested because of the differences already described inhe complex I of the respiratory chain in Leishmania parasites8]. The results obtained with the inhibitors of complex II, III andV indicate that a partial blockage of the electron transport sig-ificantly diminishes the rate of heat-induced O2

•− production.t must be pointed out that the concentrations of these inhibitorsad to be adjusted to diminish but not abolish mitochondrial O2onsumption because a strong inhibition of respiration duringhe heat treatment caused a massive death of the parasites after

few minutes. The significant inhibition of O2•− production

bserved when the parasites are treated with respiratory chainnhibitors confirms the major role of mitochondrial respiration

n the production of this radical under HS conditions.

Mitochondrial localization of the O2•− production was

urther confirmed by fluorescence microscopic analysis of heat-reated parasites stained with DHE and Hoechst 33342. A clear

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hase-contrast image of the parasites incubated at both temperatures. (B) Therier filter for Hoechst33342 staining. (C) The same parasites observed with ag.

lue signal due to Hoechst labelling of DNA (Fig. 5B; 26 ◦C and8 ◦C) can be observed both at 26 ◦C and 38 ◦C in the nuclei and

ytometry. Control parasites without uncouplers were also analyzed at 26 ◦Cnd 38 ◦C. Bars represent the mean of DHE fluorescence. Error bars representtandard deviation. Asterisks indicate a significant difference of the mean val-es of fluorescence vs. the fluorescence of the control parasites incubated at theame temperature (26 ◦C or 38 ◦C) (n = 3).

198 J.F. Alzate et al. / Molecular & Biochemical Parasitology 152 (2007) 192–202

Fig. 7. Effect of the antioxidants GSH and NAC on the parasite heat-induced cell death. L. infantum parasites were supplemented with GSH 1 mM or NAC 1 mMprior to the heat-treatment and compared with heat-treated non-supplemented parasites. Heat treatment was extended for 24 h. Apoptotic parasites were identifiedeither by their low �Ψm detected by low TMRM-derived fluorescence (A) or by the presence of degraded DNA (B). The histogram (C) shows the percentage ofpositive parasites for both death-related criteria: low TMRM staining (low �Ψm) and DNA degradation (Hypodiploids). Error bars represent standard deviation.Comparison between the antioxidant-supplemented and the non-supplemented parasites was done by a two-tailed unpaired t-test. *p < 0.0001; n = 6.

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utes the genome of this organelle. On the other hand, only DNArom the kinetoplast is labelled with the 2-hydroxyethidium gen-rated after oxidation of DHE (Fig. 5C; 38 ◦C). This labelling isbserved in the parasites treated at 38 ◦C but not in those treatedt 26 ◦C. The labelling of DNA from kinetoplast but not from theucleus that is specifically observed in the heat-treated parasitesndicates that heat-induced production of O2

•− must be mainlyocated in the mitochondrial matrix.

.4. Heat shock induced superoxide formation isndependent of �Ψm

It is generally accepted that mitochondrial hyperpolariza-ion in mammals leads to an increase in the O2

•− productionnd, consequently, the use of mitochondrial uncouplers usually

ecreases ROS formation in these cells [1]. The results pre-ented demonstrate that HS induces in Leishmania both events:itochondrial hyperpolarization and mitochondrial ROS pro-

uction. To test whether the heat-induced superoxide formation

sidt

ig. 8. Comparison of the heat-shock effect in logarithmic and late stationary parasiteromastigotes. Error bars represent standard deviation. The asterisk indicates a signifiogarithmic parasites. p < 0.0001. Error bars represent standard deviation (n = 6). (B)8 ◦C for 24 h. Dead parasites are identified by their positive PI staining. Fluorescence whe mean values of PI-derived fluorescence between logarithmic and late stationary pauorescence due to TMRM of L. infantum promastigotes. Parasites grown at 26 ◦C wehe asterisks indicate a significant difference between mean values of fluorescencequares indicate a significant difference between mean values of fluorescence of the lepresent standard deviation (n = 9). (D) Effect of acidification of the culture media in hrowing promastigotes were transferred to fresh media at pH 5.4 and maintained in tht the same temperatures but in fresh media at pH 7.2. The asterisk indicates a signifit 38 ◦C and at 26 ◦C (p < 0.005). Error bars represent standard deviation (n = 3).

l Parasitology 152 (2007) 192–202 199

s dependent on the �Ψm, we measured the DHE oxidation ratest 26 ◦C and at 38 ◦C under uncoupling conditions (Fig. 6). Ifhe observed increase in O2

•− production at 38 ◦C were dueo the increase in �Ψm, DHE oxidation should be equivalentt both temperatures in the presence of mitochondrial uncou-lers. Unexpectedly, the already described increase in DHExidation under heat-shock conditions is maintained even inhe presence of the uncouplers FCCP or CCCP, which indicateshat heat-induced O2

•− production is not due to the increasen �Ψm.

.5. Antioxidants prevent heat-induced apoptotic-like death

The evidence that O2•− concentration in the heat-stressed

arasites is considerably higher than that of control para-

ites led us to test the possible role of oxidative stress in thenduction of the already described heat-induced apoptotic-likeeath that occurs after a prolonged exposition of the parasiteso 38 ◦C (24 h) [20]. To address this question, we analyzed

s. (A) SOD activity was analyzed in logarithmic and late stationary L. infantumcant difference of the mean values of SOD activity between late stationary andLogarithmic and late stationary L. infantum promastigotes were incubated atas analyzed by flow cytometry. The asterisk indicates a significant difference of

rasites (p < 0.0001). Error bars represent standard deviation (n = 6). (C) Relativere exposed to 38 ◦C for 90 min. Fluorescence was analyzed by flow cytometry.

of the logarithmic parasites incubated at 38 ◦C and at 26 ◦C (p < 0.0001). Theate stationary parasites incubated at 38 ◦C and at 26 ◦C (p < 0.0001). Error barseat-induced cell death of logarithmic L. infantum promastigotes. Exponentiallyat media for 12 h prior to heat shock at 38 ◦C. Control parasites were incubatedcant difference between mean values of fluorescence of the parasites incubated

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hether the observed antioxidant-mediated reduction in O2•−

oncentrations is able to protect the promastigotes against heat-nduced apoptotic-like death. Cell death was quantified eithery analyzing the percentage of parasites that have lost theiritochondrial transmembrane potential (low �Ψm) or the per-

entage of parasites that have partially degraded their DNAHypodiploids), both processes already described as character-stic of an apoptotic-like death in Leishmania parasites [20].s shown in Fig. 7A and C, after 24 h of heat treatment, theercentage of parasites with low �Ψm decreases from 85% ineat-stressed control parasites to less than 60% in heat-stressedarasites supplemented with GSH or NAC. A similar reductionrom 80% to less than 50% is observed when the percentage ofypodiploid cells is used as the criteria for cell death (Fig. 7Bnd C). These results indicate that oxidative stress is a major,lthough probably not exclusive, determinant in HS-inducedpoptotic-like death in L. infantum.

.6. Stationary promastigotes express higher levels of SODnd are more resistant to heat-induced death

It has been previously reported that late stationary promastig-tes express higher levels of SOD than those growing at theogarithmic phase [15]. Higher levels of SOD activity shouldecrease the steady state concentration of O2

•− inside the para-ites and, according to the results shown above, these parasiteshould be more resistant to heat-induced cell death. The resultshown in Fig. 8 indicate that SOD activity in late stationary par-sites is 10-fold higher that that observed in logarithmic onesFig. 8A) which, as expected, correlates with a higher resistanceo heat-induced death as revealed by a decrease in the numberf PI positive cells (Fig. 8B). It must be pointed out that deathaused by incubation at 38 ◦C is diminished but not abolishedn late stationary parasites.

We have already shown that mitochondrial hyperpolarizationt 38 ◦C does not depend on O2

•− concentrations. Consistently,he higher level of SOD activity in late stationary promastigotesoes not affect the heat-induced hyperpolarization of the inneritochondrial membrane, as revealed by a similar increase in

he relative TMRM-derived fluorescence in both promastigoteopulations (Fig. 8C).

To test whether acidic conditions, associated to both latetationary promastigotes and promastigotes invading the hostacrophages, could affect the heat-induced cell death process,e analyzed the percentage of PI positive cells after heat shock of

ogarithmic parasites maintained for 12 h at pH 5.4. The resultshown in Fig. 8D indicate that parasites incubated at acidiconditions are slightly more prone to death after heat shock, pre-umably as a consequence of the addition of the two stressingtimuli.

. Discussion

Trypanosomatids are among the present-day nucleated cellshat earlier diverged during evolution from the branch that hased to higher eukaryotes. This is probably the reason why they

tcod

al Parasitology 152 (2007) 192–202

iffer significantly from most eukaryotic cells in many molec-lar aspects, while simultaneously retaining features usuallyonsidered characteristic of plants, yeasts or even bacteria.he organization of their genes in large clusters of repeatedequences whose transcription begins in a few specific positionsn every chromosome, the lack of RNA polymerase II promotersr the fact that all their mRNAs contain the same sequence addedo the coding region by a trans-splicing event are some examplesf their special peculiarities. Their single large mitochondrionnd their atypical respiratory chain, which lacks a typical com-lex I and oxidizes NADH by an alterative route [8], by no meansscape from this pattern of molecular and cellular uniqueness.owever, as in higher eukaryotic cells, ROS production dur-

ng electron transfer has been demonstrated in these parasites8]. The low concentration of ROS generated during normaletabolism of eukaryotic cells is involved in several cellular

rocesses such as cell signaling [25,26]. However, overproduc-ion of such respiration by-products may lead to the modificationf macromolecules inside the cells, which eventually causes celleath by PCD or necrosis.

Our results indicate that both O2•− anion and peroxides are

roduced in Leishmania promastigotes under in vitro standardrowth conditions, as revealed by the constant degradation of thepecific probes DHE and H2DCFDA, respectively. This basalOS production seems to be perfectly controlled by scavengerystems in the cell, which maintain the steady-state concentra-ion of these species at safe and constant levels.

Parasites subjected to 38 ◦C are still able to control peroxideoncentrations in the cell during a period of several hours, but2•− concentration rapidly increases after the change in tem-

erature, probably as an indication of an overproduction of thisoxic radical that cannot be degraded at a sufficient rate underhese conditions. It would be expected that some of the O2

•−roduced was dismutated, either enzymatically or chemically, toeroxide in this cells. However, we are not able to detect a sig-ificant increase in the concentration of peroxides, which mighte a consequence of the high peroxiredoxin activity alreadyescribed for these parasites [27]. It has been estimated thatn the trypanosomatid Crithidia fasiculata, peroxidases accountor 5% of the total soluble protein [13,14].

Simultaneously, the single mitochondrion in the heat-stressedarasites seems to be working at a higher rate as indicated byoth its higher rate of O2 consumption and its observed hyper-olarization. To test whether this higher mitochondrial activityight be responsible for the increased levels of O2

•− pro-uction, several respiratory chain inhibitors were tested undereat-stressing conditions. Our results demonstrate that three ofhese inhibitors: TTFA, antimycin A and cyanide are able toower O2

•− intracellular concentrations under heat-shock condi-ions, as an indication of the mitochondrial origin of this reactivepecies. Traditionally, in mammalian models of mitochondrialOS generation, the production of these oxidant is not revertedy the use of inhibitors of complexes III and IV because normally

hese treatments result in the reverse transfer of electrons fromomplex II to I and a concomitant generation of more super-xide radical. As indicated before, complex I in Leishmaniaiffers from that present in mammalians, which might explain

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hy the use of inhibitors of complex III and IV does not increase2•− production. In fact, our evidences indicate that incubation

f the promastigotes with inhibitors of complexes II, III or IVignificantly decreases the steady state concentration of O2

•−.ur results indicate that any decrease in the flow of electrons

hrough the mitochondrial complexes decreases the superoxideroduction, suggesting a direct relationship between respirationates and ROS generation. A decrease in mitochondrial super-xide generation after treatment with antimycin A and KCNas also been proved to occur in some specific cases in mam-als. When porcine renal tubular cells or human lung cancer

ells were, respectively, treated with cisplatin or bortezomib (aroteasome inhibitor), the researchers observed a mitochondrialyperpolarization accompanied of ROS production. Under theseircumstances, treatment of the cells with antimycin A loweredhe ROS production [28,29]. Furthermore, the treatment of theorcine tubular cells with KCN also led to a decline in ROS pro-uction [28]. The evidences presented by these authors support,ike our results, that the decrease in the electron transport ratesn the respiratory chain diminishes O2

•− formation, probably asconsequence a decrease in electron leaking.

The microscopic analysis of heat-stressed parasites stainedith DHE revealed that the 2-Hydroxyethidium produced afterxidation of this probe with O2

•− is mainly located inside theingle mitochondrion of the parasite, as indicated by the fluo-escence associated with the DNA in the kinetoplast but not inhe nucleus. This result confirms that the single mitochondrions probably the source of most of the O2

•− generated during theeat treatment of the parasites.

In mammalian cells, it has been described that hyperpolar-zation of the inner mitochondrial membrane leads to O2

•−roduction [1]. Consequently, treatment of mammalian cellsith mitochondrial uncouplers usually ablates O2

•− formation30]. Surprisingly, our results demonstrate that mitochondrialyperpolarization is not responsible for the increase in superox-de generation under heat stress conditions, as indicated by theact that this increase is not abolished when �Ψm is decreaseds a consequence of the presence of the uncoupling agents FCCPr CCCP. This result, together with the increase in O2 con-umption rates observed at 38 ◦C, suggests that incubation ofhe parasites at this temperature increases the electron transferate along the mitochondrial complexes, which probably aug-ents the process of electron leaking associated to the movement

f electrons through the respiratory complexes and, in conse-uence, increases the rate of O2

•− generation.The molecular response to heat shock has been extensively

tudied in Leishmania. It has been demonstrated that upon heatreatment the translational machinery is mainly oriented to theynthesis of HSP70 and HSP83 [31], although some other heathock proteins are also translated [32]. However, despite thereferential translation of the HSP70 and HSP83, the steadytate level of these proteins is not significantly affected afterhe heat treatment as a consequence of their high concentrations

nder normal growing conditions [33]. Accordingly, becausehe concentration of HSPs does not change significantly duringeat shock in these parasites, it is not expected that HSPs maye involved in the process of ROS generation at 38 ◦C.

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l Parasitology 152 (2007) 192–202 201

Published results have already shown that Leishmania pro-astigotes die after a mild heat-shock treatment (38 ◦C)

ollowing a process similar to the apoptotic death in higherukaryotes [20]. The data presented in this paper demonstratehat treatment of the parasites at 38 ◦C causes a rapid increasen the O2

•− anion concentration and a subsequent induction ofxidative stress, which suggests that this reactive species mighte involved in the apoptotic cell death process already described.ur data indicating that GSH or NAC are able to maintain a low2•− concentration both in control and in heat-stressed parasites

nd that these antioxidants simultaneously reduce the percent-ge of dead parasites after HS strongly support this hypothesis.he effect of these two antioxidants could be due to two differentechanisms: first, both molecules can enter in the biosynthetic

athway of trypanothione, the major intracellular thiol in Leish-ania; consequently, they are able to increase the intracellular

evels of this molecule, rendering the cell more resistant toxidative stress; second, both GSH and NAC are direct scav-ngers of the superoxide radical, and hence, are able to reducets intracellular levels.

The relationship between HS and oxidative stress in Leish-ania parasites has already been suggested by Miller et al. [34],ho showed that HS can induce resistance to H2O2-mediatedeath. It is also interesting to point out that over-expressionf SOD in promastigotes increases their ability to infectacrophages in vitro [16]. It is tempting to speculate that, in

hese genetically modified parasites, over-expression of SODight exert a protective effect during in vitro macrophage infec-

ion not only by its action against the ROS cocktail delivered byhe infected macrophages (as it had been proposed previously)ut also against the endogenous O2

•− generated during the heathock associated to infection. In fact, due to the short life ofhis radical and to its low capacity to diffuse through the cell

embranes, the most likely explanation of the SOD-inducednfectivity would be the ability of this enzyme to eliminate thentracellular O2

•− generated in the parasite’s mitochondrionfter the heat shock associated to infection. It must be pointedut that even though over-expression of SOD increases the infec-ivity of the parasites, wild type parasites are obviously able tonfect the vertebrate host. This means that the enzymes presentn wild type parasites are able to protect a significant proportionf the population against the restrictive conditions found in theew host. In fact, the results presented in this paper, together withhose reported by other authors [15], clearly demonstrate that latetationary parasites, which are similar to the metacyclic infec-ive forms, express a ten fold higher SOD activity that correlatesith a higher resistance to heat stress. However, a significantroportion of this parasite population is still clearly suscepti-le to heat-induced death. In this context, we have postulatedefore that the co-existence of dying and surviving parasites dur-ng infection might be relevant in order to escape the immuneystem of the vertebrate, possibly because the parasites dyingy an apoptotic-like pattern present molecular signals to the

acrophages that would impair their activation [20]. Obviously,

he transformation of promastigotes in amastigotes that occursnto the host macrophages during the first hours of infection,resumably due to a combination of high temperature, low pH

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nd probably some other signals, is associated to an adapta-ion to the new temperature, which now becomes the optimalrowing temperature for the differentiated parasite. This pro-ess of adaptation is not easily reproducible in the laboratory,here acidification of the media must be accompanied by slow

ncreases in temperature in order to obtain replicating axenicmastigotes.

cknowledgements

We thank Isabel Trabado and Gema Cervantes for their assis-ance with cell culture and flow cytometry data acquisition. Wehank Dr. Gago for his helpful discussions. We also thank Gemaerez for her useful suggestions about SOD experiments. Thisork was supported by grants PI021052 from Instituto de Saludarlos III and SAF2001-0989 from Ministerio de Ciencia yecnologıa.

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