Inflammation, Vol. 23, No. 4, 1999

THE ROLE OF SUPEROXIDE DISMUTATION INMALARIA PARASITES

ELI SCHWARTZ,1 AMRAM SAMUNI,2 ILANIT FRIEDMAN,3

ERNEST HEMPELMANN,4 and JACOB GOLENSER3

1Sheba Medical Centre, Tel-Hashomer2Department of Molecular Biology

3The Kuvin Centre for Tropical DiseasesThe Hebrew University-Hadassah Medical School

Jerusalem4Department of Tropical MedicineUniversity of Munchen, Germany

AbstractOxidant stress is associated with the generation of reactive oxygen speciesthat are responsible for the damage of a variety of cellular components. Theprevention of such biological damage can be achieved by dismutation of superoxideto H2O2 which in turn is removed by catalase and GSH peroxidase. However,redox-active iron released during the development of plasmodia in the erythrocyte canmediate the conversion of H2O2 to hydroxyl radical which is more reactive. The rolesof SOD and the nitroxide SOD mimic 4-OH,2,2,6,6,tetramethyl piperidine-N-oxyl(Tempol) were examined in P. falciparum grown in vitro. Both compounds did notprevent the interference with growth inflicted by various inducers of oxidant stress.Moreover, Tempol inhibited parasite growth, in agreement with previous experimentsdepicting accelerated mortality in SOD overexpressing mouse model of malaria.Probably, effective defense against ROS requires balanced increments in antioxidantenzymes and is not necessarily improved by an increase in the activity of one enzyme.

INTRODUCTION

Free radical scavengers, certain enzymes and metal chelators may prevent thedamaging effect of reactive oxygen species (ROS) (1). Superoxide dismutase(SOD), catalase, glutathione reductase and glutathione peroxidase are consid-ered to be the most important protective enzymes. Likewise, catalase can protectPlasmodium falciparum in vitro against ascorbate-induced damage (2).

We investigated the role of superoxide dismutation in malaria parasites. It isa common dogma that biological damage can be prevented by SOD which causesdismutation of the superoxide to H2O2, which in turn is dismutated by catalase

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362 Schwartz et al.

or reduced by GSH peroxidase (3). However, in some organisms excess SODleads to increased oxidant toxicity (4). In addition, redox-active iron releasedduring the development of plasmodia in the erythrocyte may convert the H2O2to the deleterious OH radical via the Fenton reaction (57).

Most of the SOD of P. falciparum is adopted from their host erythrocytes(cyanide sensitive isoenzyme) and a minor part is parasite associated-cyanideresistant (810). There is no endogenous SOD in P. vivax which also concentratesthe SOD from the host cell (11). It was suggested that SOD protects the foodvacuole from radical damage, where lysosomal enzymes degrade hemoglobinand release catalytic redox-active iron (12).

Previously, we examined the effect of elevated intracellular SOD in trans-genie mice with the human expressed SOD gene, on the development of P.berghei. These transgenic mice were more vulnerable to the plasmodial infection(13). The significance of superoxide dismutation was further studied by examin-ing the effect of Tempol, a cell-permeable SOD-mimic, in human erythrocytesparasitized by P. falciparum. The development of P. falciparum in glucose 6phosphate dehydrogenase (G6PD)-deficiency erythrocytes is retarded in com-parison with that in normal cells (due to increased sensitivity of G6PD deficienterythrocytes to oxidant stress, 12). However, as it is possible to adapt the para-sites to grow in G6PD deficient cells and to reduce their sensitivity to oxidantstress (14), the role of SOD in the adaptation was also investigated.

MATERIALS AND METHOD

Experimental Design. The experimental systems consisted of in vitro cultures of P. falci-parum in human erythrocytes. To induce oxidant stress, ascorbate, juglone, plumbagin or paraquatwere included in the medium throughout the experiments. The uptake of radioactive hypoxanthine(HX) served as the parameter for parasite development. Each experiment was repeated at least threetimes and was performed in triplicate cultures. For each individual experiment the deviation of theresults did not exceed 7.5% of the mean value of the triplicate. The figures and tables depict typicalexperiments.

Parasites. Plasmodium falciparum (strain FCR-3) was cultured according to the method ofTrager and Jensen (15). Cultures were synchronized by sorbitol treatment (16).

Blood. Human blood was collected from normal or G6PD deficient volunteers and stored inACD at 4C. The G6PD activity of the deficient blood was determined (17) and was

SOD in Malaria 363

culture. The cells were collected by filtration on glass microfibre filters and radioactivity was counted(using Minaxi, Tri-Carb, Packard). The incorporation of HX in normal non-infected erythrocytesalone did not exceed 3% of the lowest level of uptake by parasitized erythrocytes.

SOD Assessment. Cell extracts were prepared in 0.5% Nonidet p40 and assayed by the spec-trophotometric method of inhibition of nitrite formation from hydroxylammonium chloride (18). Thespecific activity of CuZnSOD in cell extracts was calculated from a standard curve using homoge-neous solution of human CuZnSOD (Biotechnology General, Rehovot, Israel).

Juglone (5 hydroxy 1,4 naphtoquinone), Plumbagin (5 hydroxy 1,4 naphthoquinone) andParaquat were purchased from Sigma. Tempol (4-OH,2,2,6,6-tetramethyl-piperidine-N-oxyl) waspurchased from Aldrich Chemicals.

RESULTS

The role of superoxide dismutation in protecting plasmodia against oxidantstress was investigated using in vitro model. We examined whether SOD alle-viates the effects of oxidant stress which is induced by ascorbate in vitro inerythrocytes parasitized with P. falciparum. The deleterious effect of ascorbateis attributed to reduction of redox-active iron, which in turn induces increasedfluxes of ROS.

Erythrocytes parasitized with the trophozoite stage of P. falciparum weretreated with ascorbate for 8 h during which the incorporation of tritiated HXinto the cells was assessed. Ascorbate inhibited plasmodial development in adose response manner. Addition of up to 100 mg SOD/ml did not protect theparasites against the ascorbate induced damage (Table 1).

Addition of Tempol, a stable nitroxide free radical acting as cell-permeableSOD mimic, did not prevent the oxidative damage inflicted in vitro by paraquator plumbagin, data not shown) on the parasitized erythrocytes. Moreover, Tem-pol alone inhibited parasite development (Table 2).

Table 1. The Effect of SOD on the Inhibition of P. falciparum Development by Ascorbatea

mM Ascorbate

2225

mg/ml SOD

33100

b% inhibition

29313779

aThe control value was about 28,700 cpm for untreated cultures containing 100 ml at 5% hematocritwith 6% parasitemia, pulsed for 8 h.

b% inhibition of incorporation of tritiated HX by untreated plasmodia.

364 Schwartz et al.

Table 2. The Effect of Tempol on P. falciparumDevelopmenta

mM Tempol

0.20.40.8

b% inhibition

11534

aThe control value was about 40,100 cpm for un-treated cultures containing 100 ml at 5% hematocritwith 8% parasitemia, pulsed for 8 h.

b% inhibition of incorporation of tritiated HX byuntreated plasmodia.

The experiments were reproduced in another experimental system includingP. falciparum, normal or G6PD-deficient erythrocytes, juglone (another inducerof oxidant stress) and Tempol. Figure 1 depicts an increased effect of jugloneon the development of P. falciparum in G6PD-deficient erythrocytes, substanti-ating the mechanism of activity of this compound as an inducer of oxidant stress.However, even the moderate damage shown for juglone in normal erythrocytes

Fig. 1. The effect of juglone on the development of P. falciparum trophozoites within normal orG6PD-deficient erythrocytes. Parasitemias were 10%. Juglone and 3[H]-hypoxanthine were presentthroughout the 8 h experiment.

SOD in Malaria 365

Fig. 2. The effects of a combination of juglone and Tempol on P. falciparum development. Para-sitemias were 10%. Juglone, Tempol and 3[H]-hypoxanthine were present throughout the 8 h exper-iment.

could not be prevented by Tempol. Moreover, Tempol further potentiated thejuglone induced damage (Figure 2).

P. falciparum was adapted during a period of 3 months to develop normallyin G6PD-deficient erythrocytes. At this stage the plasmodia were separated fromthe erythrocytes and the activity of SOD was measured. No difference was foundbetween levels of SOD in the adapted and in the nonadapted parasites or parasitesmaintained in normal erythrocytes.

DISCUSSION

Superoxide radical per se is relatively inactive species but may cause dam-age through reactions which lead to the production of more deleterious speciessuch as H2O2, hypochlorite, peroxynitrite and .OH (Scheme 1). SOD, in combi-nation with catalase and GSH-peroxidase, protects cells against the damage, bydismutation of O2. and further removal of H2O2. Plasmodia use these enzymessimilarly to most eukariotic cells but they accumulate most of their SOD fromtheir host cells. This unique phenomenon in cell biology was attributed to thevital role of SOD as a protective enzyme. The parasite has also its own SOD.Both host and plasmodial SODs are active in the parasitized erythrocyte, as canbe proven by examination of SOD activity after electrophoresis of cell extracts

366 Schwartz et al.

(19). The plasmodia are exposed to increased oxidant stress because they releaseredox-active iron which may catalyze the production of deleterious radicals, byhemoglobin degradation. In vivo, the plasmodia must survive in a hostile envi-ronment, especially in the presence of the effector cells of the immune system(6, 7, 20).

In some genetic traits such as G6PD deficiency, in which erythrocytes haveincomplete defense mechanisms, the parasites may encounter unfavorable con-ditions, especially under oxidant stress. The oxidant stress can be induced byimmune responses, drugs or some food constituents (21, 22). Some plasmodialstrains, including the one used in this work, can partially be adapted to oxidantstress (14) while others cannot be adapted (12). It is likely that this adaptationis not due to an increased synthesis of SOD because there were no differencesin the activity of SOD between the adapted and control plasmodia.

Previously, we examined the development of P. berghei in transgenic micehaving the expressed human SOD gene. These mice produce elevated quantitiesof SOD in all their cells. While SOD might be necessary for overcoming oxi-dant stress (providing that H2O2 removal is possible), an excess of SOD mightnot be beneficial to the parasites and even be destructive. Likewise, parasitizederythrocytes from transgenic mice with elevated intracellular SOD were as sen-sitive to oxidant stress as parasitized erythrocytes from normal mice. Moreover,the transgenic mice which were infected by P. berghei died much more quickly(13).

The results with P. falciparum grown in vitro correspond to these obtainedin vivo: the addition of SOD to parasitized human erythrocytes did not protectthem from ascorbate or juglone induced damage. The protective effect of added

Scheme 1.

SOD in Malaria 367

Scheme 2.

SOD might be limited because it does not enter the cell. However, a cell-per-meable SOD-mimic such as Tempol (Scheme 2), did not protect against the oxi-dant stress induced in parasitized erythrocytes by juglone and even potentiatedit. Moreover, Tempol by itself inhibited the growth of the parasites. It shouldbe stressed that in other biological systems Tempol demonstrated an effectiveprotective activity against various kinds of oxidant stress (ascorbate, copper,paraquat and quinones). For instance, Tempol protected Escherichia coli fromjuglone (23). At this stage it is not clear why Tempol inhibited the growth of theplasmodia. Such a compound, that in general is protective against radical dam-age and has a specific antiplasmodial effect, might be considered for antimalarialtherapy.

An excess of SOD can be deleterious to various microorganisms and cells:Meshnick et al. (24) reported that the addition of copper potentiates the anti-malarial activity of diethyldithiocarbamate and that SOD could be an intraeryth-rocytic source of copper. It was found that elevated intracellular SOD alsoincreased the sensitivity of E. coli to radiation (25). An excess of SOD mayincrease reperfusion injury which is mediated by OH radical through the Fentonreaction in the presence of iron. The enhanced production of the radical wasprevented by the inclusion of catalase (26). An overproduction of SOD causesa chronic prooxidant state in mouse epidermal cells because of a lack of par-

368 Schwartz et al.

allel change in catalase (27). Transfection of mouse epidermal cells, which areoverproducing SOD, rendered them more sensitive to oxidant stress. Catalasereversed the toxic effects (28). Mouse L cells and NS20Y neuroblastoma whichexpress human SOD gene also showed increased GSH peroxidase activity (29).

SOD or its mimic (Tempol) are not toxic compounds at the concentrationsthat were used in this research. SOD may play a role as a source of a transitionmetal mediating oxidant stress (24). However, it is more likely that the damag-ing effects were expressed following cellular metabolism, through the produc-tion of H2O2, which is involved in further reactions leading to C1O or .OH(19). Erythrocytes parasitized by P. falcipamm exhibit increased NO synthesis(30). Taylor-Robinson (31) suggests that NO might be released from nitrosyl-hemoglobin of the infected cell. The main target of NO is O2. with which itreacts to yield peroxynitrite. Both oxidative- and nitrosative stress may producesingle strand breaks that activate the nuclear enzyme poly (ADP-ribose) syn-thetase (PARS), which depletes intracellular energetics and increases cell per-meability. This effect which was shown in cardiomyoblasts could be preventedby the PARS inhibitors, 3 aminobenzamide and nicotinamide (3234). Theoreti-cally, SOD as well as SOD-mimics, which remove O2., decrease the productionof toxic ONOO and increase the [NO] steady state. Also it may affect signaltransduction as well as other functions which are associated with cell membrane.However, there is no specific data on nitration of SOD, or on the effects of oxida-tive or nitrosative stress on signal transduction in P. falciparum.

In conclusion, effective defense against ROS requires balanced incrementsin antioxidant enzymes and cannot successfully be improved by increase in theactivity of one enzyme (25). Lack of a parallel respective increase in otherdefense mechanisms would reflect the difference between O2. and the more dele-terious H2O2 and its derivatives.

AcknowledgmentsThis research was supported by grant 9500287 from the USA-IsraelBinational Science Foundation and by grant from the Beryl and Francess Weinstein Foundation.

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