Anti Ox Id Ante

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Original Contribution

Bilirubin inhibits Plasmodium falciparum growth through

the generation of reactive oxygen species

Sanjay Kumara, Mithu Guha b, Vinay Choubey a, Pallab Maity b, Kumkum Srivastava c,Sunil K. Puri c, Uday Bandyopadhyay b,

aDrug Target Discovery and Development Division, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg,

Lucknow 226001, Uttar Pradesh, Indiab Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, West Bengal, India

c Parasitology Division, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India

Received 3 July 2007; revised 28 September 2007; accepted 17 October 2007Available online 17 November 2007

Abstract

Free heme is very toxic because it generates highly reactive hydroxyl radicals (U

OH) to cause oxidative damage. Detoxification of free heme by

the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product.

Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we

report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in

glutathione content, and increased formation of H2O2 andU

OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results

indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein

(Gene ID PFI0450c), and reduces the mitochondrial membrane potential.U

OH scavengers such as mannitol, as well as the spin trap -phenyl-n-

tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that

bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probablyto protect itself from bilirubin-induced cell death as a second line of defense.

2007 Elsevier Inc. All rights reserved.

Keywords: Bilirubin; Hydroxyl radical; Apoptosis; Malaria parasite; Hemozoin; Free radicals

The malaria parasite (Plasmodium spp.), during intraery-

throcytic stages, digests huge quantities of hemoglobin and

releases a large amount of highly toxic redox-active free heme

[1]. To overcome the free heme toxicity, the malaria parasite is

equipped with unique heme detoxification systems. Amongthese, hemozoin formation is considered to be the main heme

detoxification system and the inhibition of hemozoin formation

leads to parasite death [1,2]. In contrast, detoxification of free

heme by the heme oxygenase-1 (HO-1) system is a very

common process in higher eukaryotes, including mammals [3].

To degrade heme, HO-1 requires the microsomal NADPH-

cytochrome P-450 reductase [4] and shunts reducing equivalents

from NADPH-cytochrome P-450 reductase to the -methene

bridge and cleaves the tetrapyrrolic ring of heme, causing the

liberation of CO plus an equimolar amount of biliverdin.

Biliverdin is converted into bilirubin by biliverdin reductase [5].Interestingly, the malaria parasite lacks this common heme

oxygenase (www.plasmodb.org) to catabolize free heme.

Bilirubin has both antioxidant and pro-oxidant properties [6].

Bilirubin, at low physiological concentrations (0.0110 M),

scavenges reactive oxygen species (ROS), reduces oxidant-

induced cellular injury, and attenuates oxidative stress [7,8].

Unconjugated bilirubin (UCB) is a scavenger of ROS such as

hydrogen peroxide, peroxynitrite, and peroxyl radicals, both in

vivo and in vitro, and plays a key physiological role in cyto-

protection against oxidant-mediated cell damage [9,10]. In

contrast, elevated concentrations (N20 M) of UCB have

Available online at www.sciencedirect.com

Free Radical Biology & Medicine 44 (2008) 602613www.elsevier.com/locate/freeradbiomed

Abbreviations: HO, Heme oxygenase; UOH, Hydroxyl radical; PBN,

-phenyl-tert-butylnitrone. Corresponding author. Fax: +91 33 24730284.

E-mail address: [email protected] (U. Bandyopadhyay).

0891-5849/$ - see front matter 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2007.10.057
http://www.plasmodb.org/mailto:[email protected]://dx.doi.org/10.1016/j.freeradbiomed.2007.10.057http://dx.doi.org/10.1016/j.freeradbiomed.2007.10.057mailto:[email protected]://www.plasmodb.org/


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deleterious effects in both neuronal and nonneuronal tissues

[11]. It develops oxidative stress by generating intracellular

ROS in Hepa1c1c7 cells and causes lipid peroxidation [12]. It

causes brain damage in newborn piglets by increasing lipid

peroxidation [13]. Oxidative stress is frequently associated with

the induction of apoptosis [14,15]. UCB induces apoptosis in

astrocytes at moderately elevated concentrations [16]. UCB-mediated programmed cell death was also reported in cultured

rat aortic smooth muscle cells and bovine brain endothelial cells

[17,18]. Thus the concentration as well as the type of cell is vital

for bilirubin to exert when or where it will be pro-oxidant or

antioxidant. Here, we report that bilirubin develops oxidative

stress in Plasmodium falciparum and inhibits parasite growth.

Further, we give evidence that bilirubin-induced oxidative

stress-mediated parasite death is associated with the induction

of caspase-like protease activity, up-regulation of the expression

of apoptosis-related protein (Gene ID PFI0450c), and reduction

of the mitochondrial membrane potential (m).

Materials and methods

Hemin, RPMI 1640, saponin, glutathione (GSH), caspase-3

assay kit, Triton X-100, proteinase K, mannitol, -phenyl-n-

tert-butylnitrone (PBN), Nonidet P-40, dichlorofluorescein

diacetate, thiobarbituric acid, trichloroacetic acid (TCA),

tetraethoxypropane, antimycin A, Hoechst 33342, and bilirubin

were purchased from Sigma (St. Louis, MO, USA). Albumax II

was procured from Gibco BRL (Grand Island, NY, USA).

Giemsa stain was purchased from Qualigens Fine Chemicals

(India). [3H]Hypoxanthine and Ready to Go RT-PCR beads

were purchased from Amersham Biosciences (Arlington

Heights, IL, USA). RNeasy kit was purchased from Qiagen(Valencia, CA, USA). 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-

benzimidazolcarbocyanine iodide (JC1) was purchased from

Molecular Probes (Eugene, OR, USA). All other chemicals

were of analytical grade purity. Bilirubin (Sigma Chemical Co.)

was further purified to remove biliary lipids as described earlier

[19]. The purified bilirubin was dissolved in 0.01 N NaOH

followed by dilution with triple-distilled water to make 1 ml

of a 1 mM stock and prepared fresh each time before use.

Immediately after the preparation, the bilirubin stock solution

was used. The stock solution was kept on ice protected from

light and all incubations containing bilirubin were carried out in

the dark to avoid degradation.

Parasite culture

The ring-synchronized P. falciparum (clone NF-54) was

grown as described [20] at a hematocrit level of 5% in complete

RPMI medium (CRPMI; RPMI 1640 medium supplemented

with 25 mM Hepes, 50 g ml1 gentamycin, 370 M hypo-

xanthine, and 0.5% (w/v) Albumax II) in tissue-culture flasks

(25 and 75 cm2) with loose screw caps. Used medium was

replaced with fresh medium once in 24 h and the culture was

routinely monitored through Giemsa staining of thin smears.

The ring-synchronized P. falciparum was usually cultured for

48 h to complete one cycle from ring to schizont stage [21]. As

the effect of drug or agent may be stage specific, to see the effect

of bilirubin on the growth of the parasite at any stage, it was

cultured for 48 h in the presence or absence of different con-

centrations of bilirubin.

Free (unbound) bilirubin in culture medium containing Albu-

max II (0.5% (w/v); a lipid-rich albumin from bovine serum,

generally used to culture P. falciparum) [22] was measured atdifferent total bilirubin levels using the peroxidase method as

described [23] in a Shimadzu UV/Vis 1700 spectrophotometer.

Isolation of parasites from infected erythrocytes and

preparation of parasite lysate

Parasites were isolated as described previously [24]. Briefly,

erythrocytes with10% parasitemia were centrifuged at 800 g

for 5 min, washed, and resuspended in cold phosphate-buffered

saline (PBS) (137 mM NaCl, 2.7 mM KCl, 5.3 mM Na 2HPO4,

and 1.8 mM KH2PO4). An equal volume of 0.5% saponin in

PBS (final concentration 0.25%) was added to the erythrocytesuspension and kept on ice for 15 min. It was centrifuged at

1300 gfor 5 min to get the parasite pellet, and finally the pellet

was washed with PBS and the isolated parasites were lysed in

PBS by mild sonication (30-s pulse, bath-type sonicator) at 4C.

The whole lysate was stored at 20C for future use. The

protein concentration in the parasite lysate was estimated as

described [25].

Measurement of lipid peroxidation as an index of oxidative

damage

P. falciparum culture (4% parasitemia, ring synchronized)

was incubated in the absence and presence of differentconcentrations of bilirubin for 48 h. After incubation, parasites

were isolated and resuspended in PBS (500 l) to prepare

parasite lysate as described above and the lipid peroxidation

product from the lysate was measured as described earlier

[26,27]. In brief, an aliquot (50 l) of the parasite lysate was

allowed to react with 100 l of trichloroacetic acidthiobarbi-

turic acidHCl reagent containing 0.01% butylated hydroxyto-

luene, heated in a boiling water bath for 15 min, cooled, and

centrifuged, and the supernatant was used for thiobarbituric

acid-reactive substances determination at 535 nm using tetra-

ethoxypropane as standard and expressed as nanomoles of lipid

peroxide/milligram of lysate protein.

Measurement of reduced glutathione

P. falciparum (4% parasitemia) was cultured in the presence

or absence of different concentrations of bilirubin. After 48 h of

treatment, the culture was washed twice with PBS and the

parasite was isolated from the infected erythrocytes as described

above. GSH content from control and bilirubin-treated parasites

was determined as described earlier[15,27,28]. In brief, parasite

lysate (50 l) was mixed with an equal volume of 10% TCA and

the protein precipitate was removed by centrifugation. The

supernatant was added to an equal volume of 0.8 M TrisCl, pH

9, containing 20 mM 5,5-dithionitrobenzoic acid to yield the

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yellow chromophore of thionitrobenzoic acid, which was

measured at 412 nm. GSH content was measured as nmol/mg

parasite lysate protein.

Measurement of intraparasitic H2O2

P. falciparum (2% parasitemia) was cultured in the presenceor absence of different concentrations of bilirubin for a period

of 48 h and intraparasitic H2O2 was measured as described [29].

In brief, the culture (after bilirubin treatment) was further

incubated for 30 min in CRPMI containing 2,7-dichlorofluor-

escein diacetate (10 M). The culture was then washed twice

with PBS and the parasites were isolated from control and

treated groups and isolated parasites were lysed as described

above. H2O2 was measured in control or bilirubin-treated

parasites by measuring the fluorescent dichlorofluorescein

formed. Fluorescence intensities were recorded from the lysates

in a PerkinElmer LS 50B spectrofluorimeter in a 5-mm path-

length quartz cell in a total volume of 1 ml at wavelengths 502and 523 nm for excitation and emission, respectively. H2O2content was expressed as fluorescence intensity/milligram of

parasite lysate protein.

Measurement of hydroxyl radical (UOH) generation

UOH generated in the P. falciparum after bilirubin treatment

at various concentrations was measured using dimethyl

sulfoxide (DMSO) as a UOH scavenger [27,30]. In brief,

P. falciparum culture (200 l) (2% parasitemia, ring+ early

trophozoites stage) was grown in multiwell plates in the

presence or absence of different concentrations of bilirubin

containing 20 l of 25% DMSO for 48 h. DMSO (20 l) wasadded along with the stipulated concentrations of bilirubin each

time when the medium was changed (once in 24 h). A negative

control (parasite only) was made without DMSO and bilirubin

as a reagent blank. After 48 h, the culture was centrifuged at

800 g for 5 min, washed, and resuspended in cold PBS. The

parasites were isolated as described above and the isolated

parasites were lysed in triple-distilled water and processed for

the extraction of methanesulfinic acid formed by the reaction ofUOH with DMSO. Methanesulfinic acid formed was allowed to

react with Fast Blue BB salt and the intensity of the resulting

yellow chromophore was measured at 425 nm using benzene-

sulfinic acid as standard.

Effect of bilirubin on hemozoin formation

Hemozoin (-hematin) formation catalyzed by parasite

lysate in vitro was assayed in the presence or absence of

different concentrations of bilirubin by following the method

described earlier[31,32]. The assay mixture contained in a final

volume of 1 ml: 100 mM sodium acetate buffer, pH 5.2, 100 M

hemin, and parasite lysate (20 l) in the absence or presence of

various concentrations of bilirubin. The reaction was initiated

by the addition of hemin and incubated for 12 h at 37C. The

reaction was terminated by centrifugation at 15,000g for

10 min at room temperature. The pellet was washed twice with

100 mM TrisHCl buffer, pH 7.8, containing 2.5% SDS and

finally with 100 mM bicarbonate buffer, pH 9.2. The insoluble

pellet (hemozoin) was solubilized in 50 l of 2 N NaOH

and diluted further to 1 ml with 2.5% SDS. The absorbance

of the solution was recorded at 400 nm and an extinction

coefficient of 91 mM1 cm1 [33] was used to quantitate hemo-

zoin formation. In P. falciparum, the amount of hemozoinformed in the presence or absence of bilirubin was also measured

as described earlier [34].

Measurement of heme content

Heme content in control and bilirubin-treated P. falciparum

was measured as described earlier [35]. In brief, P. falciparum

was cultured in the presence or absence of varying concentra-

tions of bilirubin for 48 h. Then the culture was centrifuged

to pellet the cells and the cell pellet was washed in PBS to

remove the bilirubin. Then concentrated formic acid (1 ml)

was added to solubilize each pellet and the heme concentrationof the formic acid solution was determined in a Shimadzu

UV/Vis 1700 spectrophotometer at 398 nm (extinction coeffi-

cient 1.56105 M1 cm1). Heme content was expressed as

nmol/mg of cell protein.

In vivo growth of P. yoelii and isolation of trophozoite-infected

red cells

Mice (Swiss albino, 1820 g) were infected with P. yoelii

by intraperitoneal passage of 1 106 infected erythrocytes [36]

and parasitemia was monitored by microscopic examination of

Giemsa-stained thin blood smear. After 4 days of inoculation,

blood was collected in acid citrate dextrose (0.0347 M citricacid, 0.0748 M sodium citrate, 0.1359 M dextrose) at

approximately 50% parasitemia. Infected blood was passed

through CF-11 cellulose (Whatman) to remove white blood

cells [37]. The collected red blood cells (RBC) were washed and

trophozoite-rich infected RBC were isolated as described [38].

Measurement of mitochondrial transmembrane potential

Isolated trophozoite-rich infected RBC at a concentration of

5106/ml in RPMI 1640 medium containing 1% fetal bovine

serum were incubated in the presence or absence of bilirubin

(40 M) for 1 h at room temperature (30C). For positivecontrol, the same number of cells were incubated with anti-

mycin A. The infected RBC were washed (three times) in PBS

to remove excess bilirubin or antimycin A and the cell pellet

was suspended in 1 ml of CRPMI. Then JC1 (153 nM) was

added to each cell suspension and incubated for 10 min in the

dark at 25C. At the end of the incubation, the fluorescence was

recorded in a PerkinElmer Life Sciences Lambda L.S 50B

spectrofluorimeter (excitation 490; emission 590 nm) [39]. The

JC1 uptake (J-aggregate formation) was also analyzed by

fluorescence microscopy using JC1-treated cell suspension. In

brief, the JC1-treated cell suspension was washed (three times)

in CRPMI and the resulting infected cell pellet was suspended

in 100 l CRPMI. Twenty microliters of this cell suspension

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was used to analyze the formation of J-aggregate (emission

590 nm, TX2 green filter) as a measure of mitochondrial uptake

and monomer (emission 530 nm, I3 blue filter) quickly under

100 oil immersion lens in a Leica DM LB 2 fluorescence

microscope.

Assay of caspase-like (DEVDase) activity

To study the activation of caspase, caspase-3-like activity

was measured in whole P. falciparum lysate, using commer-

cially available kits and according to the manufacturer's pro-

tocol (Sigma) as described earlier [15]. In brief, P. falciparum

culture (4% parasitemia) was incubated in the absence or

presence of bilirubin (40 M) for 48 h at 37C in the dark.

After incubation, parasites were isolated and sonicated in

PBS (100 l) to prepare parasite lysate. Parasite lysate (10 l

containing 30 g protein) was mixed with assay buffer and

caspase-3 substrate (Ac-DEVD-pNA) in a microtiter plate.

After 24 h of incubation at 37C, the absorbance of pNAreleased was measured at 405 nm in a microtiter plate reader.

Caspase-3-like activity was confirmed using the caspase-3

inhibitor provided in the kit.

Semiquantitative RT-PCR for apoptosis-related protein

P. falciparum culture (2% parasitemia) was incubated in

the absence or presence of bilirubin (40 M) for 48 h. After

incubation, the culture was pelleted and washed twice with

PBS. The parasite pellet was used for RNA isolation using the

RNeasy kit (Qiagen). Freshly isolated parasites were immedi-

ately suspended in RNA Later solution and RNA was extracted

from the parasites using the Qiagen RNeasy Protect kit ac-cording to the manufacturer's instructions. Nucleic acid bound

to the RNeasy column was incubated with 5 kunitz units of

RNase-free DNase in 50 mM TrisHCl (pH 7.5) and 10 mM

MgCl2 for 20 min at 37C to remove DNA contamination in the

RNA preparation. The purity of the RNA was checked in 1%

agarose gel and quantitated by measuring the OD at 260 nm.

An equal amount of RNA (1.5 g) was used for RT-PCR of

apoptosis-related protein (Gene ID PFI0450c) using the

following sets of primers: forward primer (1 M final con-

centration), 5-ATGAATATTGAAAAAGCCG-3; reverse pri-

mer (1 M final concentration), 5-CATATAATCTTCTTCG-

TTGAAATC-3 [40]. RT-PCR was performed using Ready toGo RT-PCR beads (Amersham Pharmacia) with the following

PCR program: cDNA synthesis at 42C for 30 min; 94C for

2 min for initial denaturation; then 35 cycles of denaturation at

94C for 1 min, annealing at 55C for 1 min, extension at 72C

for 1.5 min; and then 72C for 7 min. Simultaneously, positive

control primers for seryl-tRNA synthetase were added in each

RT-PCR. Seryl-tRNA synthetase is expressed equally in each

stage of the P. falciparum [41]. Primer sequences used for seryl-

tRNA synthetase were 5-GAGGAATTTTACGTGTTCAT-

CAA-3 (forward) and 5-GATTACTTGTAGGAAAGAATCC-

TTC-3 (reverse). RT-PCR products were analyzed through

electrophoresis on 1% agarose gel in TAE buffer at 10 V/cm and

documented.

Nuclear morphology by Hoechst staining

P. falciparum culture (2% parasitemia, ring stage) was

incubated in the absence or presence of bilirubin (40 M)

for 48 h. After incubation, P. falciparum culture (control and

bilirubin (40 M)-treated) was centrifuged at 2500 rpm for

5 min to remove the medium and the pellet containing parasite-infected red cells was suspended in PBS. Hoechst 33342 (10 g/

ml) was added to it and kept for 10 min in the dark at 37C.

Then the cells were washed three times in PBS to remove

the excess fluorescent stains and fixed with 3.7% paraformal-

dehyde in PBS and the parasite was then visualized at 100

with oil using a UV filter under a Leica DM LB 2 fluorescence

microscope.

Assay of antimalarial activity of bilirubin

Inhibition ofP. falciparum growth was studied by following

[

3

H]hypoxanthine uptake as described earlier [42]. Synchroni-zation of the parasites to uniform ring stage was achieved using

5% aqueous D -sorbitol as described earlier [43]. To see the

effect of bilirubin, the ring-synchronized P. falciparum (para-

sitemia 0.51%) was cultured in multiwell (200 l/well) plates

in the presence or absence of different concentrations of

bilirubin. After 48 h [3H]hypoxanthine (0.7 Ci/well) was

added to each well and further cultured for 48 h to monitor

parasite viability by measuring incorporation of [3H]hypox-

anthine in parasite nucleic acids. P. falciparum culture was

harvested and washed twice in PBS. The parasite pellet was

dissolved in 100 l of 3 N NaOH by keeping it at 37C for 6 h

followed by scintillation counting for [3H]hypoxanthine uptake

measurement.

Statistical analysis

Data shown are means SEM. Statistical analysis for

parametric data was calculated using Student's t test or

ANOVA wherever applicable. The ANOVA was followed by

post hoc analysis (multiple comparison ttest) for the evaluation

of the difference between individual groups. For nonparametric

data analysis, the MannWhitney U test and KruskalWallis

ANOVA (wherever applicable) were performed. Kruskal

Wallis ANOVA was followed by multiple comparison test

(Holm-Sidak methods). A pb0.05 was considered statisticallysignificant.

Results

Bilirubin induces oxidative stress in P. falciparum

GSH level and the formation of lipid peroxidation product

were measured after incubation of P. falciparum with various

concentrations of bilirubin (Fig. 1). Bilirubin decreased GSH

levels (Fig. 1A) in a concentration-dependent manner. GSH

levels decreased 50 and 80% at 10 and 30 M bilirubin,

respectively (Fig. 1A, inset). The decrease in GSH level was

also associated with the increased formation of lipid peroxide in



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the parasite (Fig. 1B). Bilirubin increased lipid peroxidation by

65% at 10 M and 150% at 30 M compared with the control

value (Fig. 1B, inset). In order to assess whether increased lipid

peroxidation and GSH depletion, the indicators of oxidative

stress, are due to generation of H2O2, the intraparasitic H2O2

level was measured after incubation with various concentrationsof bilirubin. Interestingly, bilirubin increased the generation of

intraparasitic H2O2 in a concentration-dependent manner (Fig.

2A), causing a 200% increase at 10 M and 400% increase at

30 M bilirubin (Fig. 2A, inset). Furthermore, the parasite,

when incubated with bilirubin, showed gradual increased

generation of UOH as a function of bilirubin concentration

(Fig. 2B and inset).

Bilirubin inhibits hemozoin formation

As free heme is one of the major sources of ROS generation,

we expected that it should accumulate to cause oxidative stress

if hemozoin formation is inhibited in the parasite in the presence

of bilirubin. Hemozoin formation was followed using lysates of

the multidrug-resistant P. yoelii (Fig. 3A) and chloroquine-

sensitive P. falciparum (Fig. 3B). The results clearly indicate

that bilirubin prevents hemozoin formation in a concentration-

dependent manner in both cases (Figs. 3A and B). IC50 values

were found to be 14 M forP. yoelii (Fig. 3A, inset) and 10 M

forP. falciparum (Fig. 3B, inset) lysates. Bilirubin also inhibitshemozoin formation in P. falciparum in culture with an IC50 of

8 M (Fig. 3C and inset).

The inhibition of hemozoin formation in the parasite may

lead to the accumulation of heme. The data indicate that

bilirubin concentration-dependently increases the heme content

in the parasite (Fig. 4). Thus, the pro-oxidant effect of bilirubin

is probably due to the inhibition of hemozoin formation and

subsequent accumulation of toxic heme.

It is accepted that the free or unbound form of bilirubin

mainly mediates the biological effect. Because P. falciparum

was cultured for 48 h (for one full intraerythrocytic cycle from

ring to schizont stage) in the presence of various concentrationsof bilirubin to follow oxidative stress and hemozoin formation

in the parasite, the free bilirubin (Bf) concentration may likely

Fig. 1. Bilirubin develops oxidative stress in P. falciparum. (A) GSH and (B)

lipid peroxide were measured in P. falciparum in the presence or absence of

various concentrations of bilirubin. The data presented are meansSEM (n =6).

*pb0.05 vs control, ***pb0.001 vs control.

Fig. 2. Bilirubin stimulates the generation of intraparasitic H2O2 and hydroxyl

radical (U

OH). (A) H2O2 and (B)U

OH were measured in P. falciparum in the

presence or absence of various concentrations of bilirubin as described

under Materials and methods. The data presented are means SEM (n =6).***pb0.001 vs control.



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decline during the 48 h of incubation due to oxidative

degradation. Therefore, the Bf level was measured after 48 h.

The data indicated that the Bf level was decreased significantly

and the Bfconcentration, which was initially 40 M, was found

to be 9 M (Fig. 5). Because bilirubin offers maximum in-

hibition of hemozoin formation in the parasite in culture at

40 M (initial level), we used this concentration in most of the

experiments later on.

Bilirubin reduces mitochondrial transmembrane potential of

the malaria parasite

The generation of ROS and the associated oxidative stress

activate the mitochondrial pathway of apoptosis [14] or cause

mitochondrial dysfunction [44]. In order to investigate whether

oxidative stress induced by bilirubin can lead to mitochondrial

dysfunction in the malaria parasite, alteration of the mitochon-

drial m, a marker for dysfunction, was measured. The mwas measured by the fluorescence change in the membrane-

potential-sensitive dye, JC1. In intact healthy mitochondria

with higherm, JC1 would accumulate in the mitochondrial

matrix to form the J-aggregate, showing intense fluorescence

at 590 nm. Mitochondria with open transition pores have low

m and would accumulate less JC1, leading to lower for-

mation of aggregates, thereby showing weak fluorescence at590 nm. Bilirubin can effectively decrease m as indicated by

spectrofluorimetric analysis (Fig. 6A) and fluorescence micro-

scopic studies (Fig. 6B). Antimycin A, a known probe used to

decrease mitochondrial membrane potential [45], was used as a

positive control. Fluorescence microscopic analysis clearly

indicates that the formation of J-aggregates (red fluorescence,

590 nm) (Fig. 6B, images a) or the ratio of 590 nm/530 nm

(Fig. 6B, images c) in the trophozoite-infected red cell (control)

was higher than in bilirubin-or antimycin A-treated trophozoite-

infected red cells. In contrast, the green fluorescence (JC1Fig. 3. Inhibition of hemozoin formation by bilirubin. Hemozoin formation was

followed in the presence or absence of different concentrations of bilirubin as

indicated. (A) Formation of hemozoin using P. yoelii lysate. (B) Formation ofhemozoin using P. falciparum lysate. (C) Hemozoin content ofP. falciparum in

culture in the presence or absence of various concentrations of bilirubin as

indicated. The data presented are meansSEM (n =6). *pb0.05, **pb0.01,

***pb0.001 vs control.

Fig. 4. Bilirubin increases heme content in the parasite. Heme content was

measured as described under Materials and methods. The data presented are

meansSEM (n =6). **pb0.01, ***pb0.001 vs control.

Fig. 5. Measurement of free bilirubin in culture after 48 h of incubation. Free

bilirubin concentration was measured as described under Materials and methods.The data presented are meansSEM (n =6). *pb0.05, ***pb0.001.



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monomer, 530 nm) was much lower in the control compared

with bilirubin-or antimycin A-treated cells (Fig. 6B, images b).

Thus, bilirubin alters mitochondrial membrane potential of the

malaria parasite.

Bilirubin induces an apoptosis-like phenomenon in the malaria

parasite

Oxidative stress may lead to the induction of apoptosis in

many cells. In order to investigate whether bilirubin can induce

apoptosis or pseudoapoptosis [46] or an apoptosis-likephenomenon in the malaria parasite via the development of

oxidative stress, caspase-3-like protease (DEVDase) activity,

expression of a putative apoptosis-related protein (Gene ID

PFI0450c), and nuclear morphology were measured. Although

the P. falciparum genome lacks classical caspase-3 (www.

plasmodb.org), the cytosolic fraction of bilirubin-treated

parasites significantly cleaved the caspase-3 substrate, DEVD-

pNA, whereas the same fraction of control parasite showed very

little activity (Fig. 7A). The result showed that bilirubin caused

a sixfold activation of caspase-3-like proteases over the control

value. Moreover, the activity of caspase-3 was significantly

inhibited in the presence of Ac-DEVD-CHO, a potent inhibitorof caspase-3, indicating that P. falciparum contains a protease

having caspase-3-like activity. In the P. falciparum genome, a

gene for a putative apoptosis-related protein (PfARP) was

predicted and annotated in chromosome 9 (Gene ID PFI0450c;

www. plasmodb.org). RT-PCR analysis indicates that bilirubin

causes significant up-regulation of the expression of the PfARP

gene (Figs. 7B and C) over the control. The expression pattern

of the seryl-tRNA synthetase (internal control) was, however,

not affected (Figs. 7B and C). To check apoptosis-like events in

the parasite, the nuclear morphology of the parasite was

observed by Hoechst staining. The result indicated that bilirubin

caused the condensation of parasite chromatin as evident from

intense fluorescence compared to the control parasite (Fig. 8).

Fig. 6. Effect of bilirubin on mitochondrial transmembrane potential of Plasmodium. (A) Measurement of mitochondrial transmembrane potential in control and

bilirubin-(40M) and antimycin A-(10 M) treated trophozoite-infected red cells by spectrofluorimetry as described under Materials and methods. (B) Fluorescence

microscopic analysis of transmembrane potential in control and bilirubin-(40 M) and antimycin A-(10 M) treated trophozoite-infected red cells. (a) J-aggregate

formation (emission, 590 nm), (b) JC1 monomer (emission 530 nm), and (c) 590 nm/530 nm ratio (merged).

Fig. 7. Effect of bilirubin on caspase-like (DEVDase) activity, expression of

PfARP, and DNA damage in P. falciparum. (A) The caspase-3-like activity was

measured in the cytosolic fraction (30g protein) frombilirubin-(40M) treated

or control parasites. (B) RT-PCR analysis to follow the expression of PfARP. (C)

Histogram representing the densitometric analysis of PfARP and seryl-tRNA

synthetase expression (fold of PfARP expression relative to seryl-tRNA

synthetase expression) in P. falciparum in the presence or absence of bilirubin.***pb0.01 vs control, **pb0.01 vs control, *pb0.01 vs bilirubin.

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Thus, bilirubin induces an apoptosis-like phenomenon in the

malaria parasite.

Effect of bilirubin on P. falciparum growth in culture

Now the question arises whether bilirubin-induced oxidative

stress or the associated apoptosis-like phenomenon is linked

to parasite death? The data show that bilirubin inhibits

P. falciparum growth in vitro in a concentration-dependent

manner as evident from the decreased hypoxanthine uptake

(Table 1). Bilirubin inhibits P. falciparum growth by 18 and

80% at 10 and 80 M, respectively.

Antioxidant treatment protects P. falciparum from

bilirubin-induced oxidative stress and growth inhibition

In order to assess whether the oxidative stress induced by

bilirubin is responsible for the inhibition of parasite growth, the

effects of antioxidants or ROS scavengers (UOH scavengers,

such as mannitol and spin traps like PBN) were studied on

lipid peroxidation, H2O2 generation, and P. falciparum growth.

Results indicate that these antioxidants significantly decreased

bilirubin-induced lipid peroxidation (Fig. 9A) and the genera-

tion of intraparasitic H2O2 (Fig. 9B). Moreover, the well-knownUOH scavenger, mannitol, protected against the fall in JC1

uptake (mitochondrial potential) in P. falciparum by bilirubin

(Fig. 10A). Again, both mannitol and PBN significantly prevent

bilirubin-induced activation of caspase-like activity (Fig. 10B)

as well as induction of ARP expression (Figs. 10C and D). The

expression pattern of the seryl-tRNA synthetase (internal

control) was, however, not affected. Finally, we monitored

parasite growth by following [3H]hypoxanthine uptake in the

presence or absence of mannitol or caspase-3 inhibitor (DEVD-

CHO) to test whether bilirubin-induced oxidative stress is

mainly responsible for the growth inhibition of P. falciparum

via the induction of apoptosis-like events. Interestingly, both

mannitol and DEVD-CHO significantly protected P. falciparumfrom bilirubin-induced growth inhibition (Fig. 11).

Discussion

Evidence has been presented to show that bilirubin

effectively induces oxidative stress, reduces mitochondrial

membrane potential, up-regulates the expression of PfARP in

the malaria parasite, and finally inhibits P. falciparum growth.

The possible mechanism by which bilirubin develops

oxidative stress and parasite death is mediated through its

inhibitory effect on hemozoin formation, leading to excess

accumulation of free heme in the parasite [47,48], which

may stimulate the generation of ROS. Inhibition of heme

Fig. 8. Effect of bilirubin on P. falciparum nuclear morphology. Hoechst

staining was done as described under Materials and methods.

Table 1

Effect of bilirubin on P. falciparum growth in culture

[3H]Hypoxanthine uptake

(% inhibition, mean SEM)

Control 0

+Bilirubin (1.0 M) 4 1

+Bilirubin (10 M) 18 2





pb0.05 vs control.

Fig. 9. Effects of antioxidants on bilirubin-induced oxidative stress and

P. falciparum growth. (A) Lipid peroxide and (B) H2O2 were measured in the

presence or absence ofU

OH scavengers during bilirubin (40 M) treatment as

described under Material and methods. P. falciparum culture (4% parasitemia)

was treated with bilirubin along with PBN (40 mM) or mannitol (10 mM) for

48 h. The data presented are meansSEM (n =6). ***pb0.001 vs control,###pb0.01 vs bilirubin.



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detoxification function is known to kill the parasite through

membrane lysis and interference of other vital functions[2,49,50]. It is already known that bilirubin can prevent heme

crystal growth in vitro by forming complexes with heme [51].

Moreover, different heme catabolic products and protoporphyr-

ins are known to inhibit hemozoin formation by coordinating

with hematin via cofacial links [52] and offer antimalarial

activity [53]. Bilirubin may inhibit hemozoin formation by

following the same mechanism of noniron porphyrins, but

further studies are required to confirm it. The malaria parasite isvery much susceptible to oxidative stress [54,55] and bilirubin,

by inhibiting hemozoin formation, may develop oxidative stress

and inhibit parasite growth. However, bilirubin can generate

ROS in many mammalian cells, in which mitochondria are a

major source of ROS [11,12] and in which hemozoin formation

is completely absent. Therefore, we cannot exclude other

possible routes for the generation of ROS in the parasite.

Jaundice in malaria is mostly due to hemolysis, and the range of

total serum bilirubin is 348.2 mg% (50830 M) [56].

Antimalarial treatment brings the elevated level of bilirubin in

malaria to normal range [0.51.5 mg%] [57,58]. Very high

concentrations of bilirubin formed during malaria may be pro-oxidant in nature. Although at this concentration it can damage

various host cells, it may simultaneously protect the host from

parasite burden by inhibiting its growth. We kept total bilirubin

concentration to between 10 and 40 M (which allowed a

moderate parasitemia) just to see the effect of bilirubin on

P. falciparum growth, avoiding its nonspecific effects at a very

high concentration.

Our studies indicate that bilirubin-induced oxidative stress

is associated with the reduction of m. The loss of mis considered one of the most significant events in oxidative

stress-mediated activation of mitochondrial pathway of apop-

tosis [11,59]. Bilirubin is known to induce apoptosis through

the mitochondrial pathway by developing oxidative stress

Fig. 11. Effects of antioxidant and DEVD-CHO on bilirubin-induced

P. falciparum growth inhibition. P. falciparum culture (4% parasitemia) was

treated with bilirubin along with mannitol (10 mM) or DEVD-CHO (100 M)

for 48 h. P. falciparum growth was monitored by following [3H]hypoxanthine

uptake as described under Material and methods. ***pb

0.05 vs control,#pb0.01 vs bilirubin, ##pb0.05 vs bilirubin.

Fig. 10. Effects of antioxidants on bilirubin-induced changes in mitochondrial potential, caspase-like activity, and expression of apoptosis-related protein (ARP).

P. falciparum culture (4% parasitemia) was treated with bilirubin along with PBN (40 mM) or mannitol (10 mM) for 48 h. Then (A) JC1 uptake, (B) caspase-like

activity, and (C, D) ARP expression were measured as described under Material and methods. ***pb0.01 vs control, #pb0.01 vs bilirubin, ##pb0.05 vs bilirubin.



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[11,12,59,60]. Our studies indicate that bilirubin induces an

apoptosis-like phenomenon in P. falciparum probably by up-

regulating the expression of PfARP and activating a caspase-

like protease. Hoechst staining also indicates the occurrence of

chromatin condensation in the parasite by bilirubin. The role of

PfARP in P. falciparum apoptosis is not known; nevertheless,

from the evidence presented it can be proposed that bilirubininduces apoptosis-like events in the parasite to cause cell death.

Recently, by measuring the conventional parameters for

apoptosis, it has been claimed that death caused by most of

the antimalarials is not associated with classical apoptosis [61],

except for atovaquone, which reduces mitochondrial membrane

potential and respiration [45,61], vital parameters for mitochon-

drial pathway of apoptosis. Apoptosis in any form in the malaria

parasite is not well documented. However, it has been reported

that chloroquine causes apoptosis in a chloroquine-sensitive

strain of P. falciparum by DNA fragmentation [62,63].

Caspase-like activity has also been observed in Plasmodium

berghei ookinetes, which results in the apoptosis-like death ofmore than 50% of the parasites at this stage in the mosquito

midgut [64]. The parasite i tself dies by a process of

programmed cell death in the lumen of the midgut before

invasion has occurred. Caspase-like activity was detected in the

cytoplasm of the ookinetes, despite the absence of genes

homologous to caspases in the genome of Plasmodium or any

unicellular eukaryote [65]. Interestingly, P. falciparum contains

metacaspase (Gene ID PF14_0363) and various cysteine

proteases (PFB0325c, PFB0330c, PFB0335c, PFB0340c,

PFB0345c). Oxidative stress is known to induce apoptosis in

the unicellular parasite Leishmania donovani, which lacks

classical caspases and in which the activation of caspase-3-like

proteases has been reported [66]. However, further studies arenecessary to confirm whether the observed caspase activity

originates from metacaspase (Gene ID PF14_0363) or from any

cysteine protease. In the P. falciparum genome, a putative gene

for PfARP (Gene ID PFI0450c) is annotated on chromosome 9

(www.plasmodb.org). PfARP has the highest degree of

homology to mammalian TF-1 cell apoptosis-related gene-19

(TFAR-19). The growth factor or serum deprivation leads to

overexpression of TFAR-19 in TF-1 cells, which results in

apoptosis in those cells. Moreover, PfARP also has a close

resemblance to PDCD5, which has been reported to have a

regulatory role in paraptosis, a form of programmed cell death

distinct from apoptosis [67]. Similarity search results pointtoward diverse possibilities for the functional roles of PfARP in

P. falciparum, such as some role in classical apoptosis or in

nonclassical programmed cell death. Interestingly, bilirubin up-

regulates the expression of PfARP in P. falciparum. But it

remains to be established whether this protein functions as a

helper in the death process. If bilirubin inhibits P. falciparum

growth through the induction of oxidative stress, antioxidant

treatment should protect the parasite from bilirubin-induced cell

death. UOH scavengers and spin traps remarkably inhibited

bilirubin-induced oxidative stress in the parasite and both

antioxidant and caspase inhibitor (DEVD-CHO) significantly

protected P. falciparum from bilirubin-induced growth inhibi-

tion. This suggests that bilirubin-induced oxidative stress is

responsible for the inhibition ofP. falciparum growth. We thus

conclude that bilirubin induces oxidative stress, which stimu-

lates an apoptosis-like death in the malaria parasite.

It can be suggested that the increased formation of bilirubin

by the host during malaria, due to hemolysis, may aggravate the

oxidative damage to the host red cells and neurons, but may also

be protective, in part, by enhancing the oxidative destruction ofthe parasites. Moreover, this study provides a logical explana-

tion for why this parasite does not have an HO-1 system. As

bilirubin is toxic to the malaria parasite, it lacks a conventional

HO-1 system to protect itself from bilirubin-induced cell death

as a second line of defense.

Acknowledgment

Sanjay Kumar gratefully acknowledges the Council of

Scientific and Industrial Research, New Delhi, for providing a

Senior Research Fellowship to carry out this work and

providing fund from Suprainstitutional project (SIP0007).

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