- Email: [email protected]
Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and Crotoxin B
Acta Tropica 124 (2012) 126–132
Contents lists available at SciVerse ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and Crotoxin B J.C. Quintana a,d,∗ , A.M. Chacón a , L. Vargas a , C. Segura b , J.M. Gutiérrez c , J.C. Alarcón a a
Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín, Colombia Grupo Malaria, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica d Director de Desarrollo de Programas e Investigación, Dirección Nacional de Posgrados, Universidad Cooperativa de Colombia, Medellín, Colombia b c
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 8 July 2012 Accepted 12 July 2012 Available online 31 July 2012 Keywords: Plasmodium falciparum Malaria Snake venom Crotalus durissus cumanensis Phospholipase A2 Crotoxin B Antimalarial activity
a b s t r a c t The antiplasmodial activity of phospholipases A2 (PLA2 ) isolated from different animals has been studied. We explored the in vitro anti Plasmodium falciparum effect of a fraction containing crotoxin, Crotoxin B and whole venom of the rattlesnake Crotalus durissus cumanensis. Fraction II (crotoxin complex) was obtained by size exclusion chromatography, whereas Crotoxin B was purified by RP-HPLC. The whole venom is active against the parasite at concentrations of 0.17 ± 0.03 g/ml, fraction II at 0.76 ± 0.17 g/ml and Crotoxin B at 0.6 ± 0.04 g/ml. Differences were observed in the cytotoxic activity against peripheral mononuclear cells, with Crotoxin B exhibiting the highest cytotoxicity. The concentration of Crotoxin B required to exert cytotoxic activity was higher than that required to exert antiplasmodial activity. Lethality in mice confirmed the higher toxicity and neurotoxicity of whole venom and fraction II, whereas Crotoxin B was not lethal at the doses tested. These results suggest the potential of Crotoxin B as a lead compound for antimalarial activity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Malaria is caused by parasites of the genus Plasmodium and is a public health problem in tropical and sub-tropical regions of the world. Annually malaria provokes approximately 1.5 million deaths. Over 85% of them occur in Africa with P. falciparum as the leading species involved in most fatalities (Breman et al., 2004; WHO, 2008). The W.H.O. report confirmed almost 1 million deaths during the previous year (WHO, 2010). The most widely used treatment consists of artemisinin-based combined therapies (WHO, 2010). High rates of antimalarial treatment failure have led to the investigation of possible therapeutic alternatives, among which toxins and poisons derived from animals and plants are promising candidates (Abdel-Sattar et al., 2010; Ayuko et al., 2009; Gao et al., 2010; Karunamoorthi et al., 2010; Milhous and Weina, 2010; Muller et al., 2010).
∗ Corresponding author at: Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín, Colombia. Tel.: +57 4 2196536; fax: +57 4 2631914. E-mail addresses: [email protected], [email protected] (J.C. Quintana). 0001-706X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2012.07.003
Rattlesnakes (genus Crotalus, family Viperidae) are pit vipers with widespread distribution in the Americas (from North America to northern Argentina), being classified into many species and subspecies. Among them, Crotalus durissus cumanensis is distributed in Colombia and Venezuela (Campbell and Lamar, 2004). The venoms of South American subspecies of C. durissus are composed of a complex mixture of peptides, enzymes and toxins, such as crotamine, gyroxin, convulxin, thrombin-like serine proteinase (Bucaretchi et al., 2002; Oshima-Franco et al., 1999) and the crotoxin complex (a heterodimer composed of two subunits, A and B). Crotoxin complex is responsible for the high toxicity of the venom due to neurotoxic, nephrotoxic and myotoxic activities (AzevedoMarques et al., 1985; Martins et al., 2002; Oshima-Franco et al., 1999), which may provoke death by neurotoxic paralysis or acute renal failure. Phospholipases A2 -type (PLA2 , EC 3.1.1.4) are a superfamily characterized by their ability to hydrolyze phospholipids at the sn-2 ester bond of fatty acids to produce lysophospholipids and fatty acids. Secreted phospholipases A2 (sPLA2 ) share several characteristics: low molecular mass (13–18 kDa), numerous disulfide bridges, histidyl and aspartyl catalytic residues and a highly conserved calcium binding region (Ca2+ ) (Six and Dennis, 2000; Talvinen and Nevalainen, 2002). PLA2 s from snake venoms
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
127
Fig. 1. (A) Chromatographic elution profile on Sephacryl S-200 of the venom of C. d. cumanensis. The shaded fractions correspond to the fractions used, (B) SDS-PAGE (12%) separation of crude C. d. cumanensis venom (lane 1), fraction II from gel filtration chromatography (lane 2), Crotoxin B (lane 3), and fraction I from gel filtration chromatography (lane 4). MW correspond to molecular weight markers, (C) reverse-phase HPLC separation on a C-18 column of fraction II from gel filtration chromatography. The shaded area corresponds to Crotoxin B, (D) molecular mass determination of Crotoxin B by mass spectrometry.
exhibit a variety of toxicological and pharmacological activities, such as myotoxicity, neurotoxicity, anticoagulant activity, edema inducing-activity, cardiotoxicity, bactericidal activity, antiparasitic effect, and various effects on platelet aggregation (Andriao-Escarso et al., 2000; Barbosa et al., 2005; Costa Torres et al., 2010; Evangelista et al., 2010; Gutierrez and Lomonte, 1995; Harris et al., 2000; Kini, 2003; Kini and Evans, 1987; Landucci et al., 2000; Murakami et al., 2005). In recent years, several pharmacological applications for PLA2 have been described, including a potential activity against parasites (Costa Torres et al., 2010; Deregnaucourt and Schrevel, 2000; Guillaume et al., 2004; Passero et al., 2008). Owing to the high concentration of Crotoxin B, a PLA2 , in the venom of C. d. cumanensis, this venom constitutes a potential source of antimalarial activity. The aim of this study was to characterize the Crotoxin B from the venom of C. d. cumanensis, comparatively with a fraction containing the whole crotoxin complex and with the crude venom, for its in vitro antiplasmodial activity against P. falciparum, as well as its cytotoxicity on a human cell line and its acute toxicity in mice.
2. Materials and methods 2.1. Venom and reagents The venom was obtained by manual milking of 15 specimens from different regions of Colombia held in captivity at the Serpertarium of the University of Antioquia (Medellín, Colombia). Venoms were pooled, centrifuged (3000 rpm, 15 min), and the resulting supernatants were lyophilized and stored at −20 ◦ C until use. Acetonitrile (CH3 CN) and trifluoroacetic acid (CF3 COOH) HPLC grade were purchased from Fisher Scientific (Loughborough, UK), Histopaque® -1077, RPMI-1640 medium culture, Thiazolyl Blue Tretrazolium Bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (Sigma–Aldrich, St. Louis, USA). Water for HPLC was deionised to a degree of purity of 17 . 2.2. Venom fractionation Crotoxin B was purified from the venom of C. d. cumanensis using size molecular exclusion chromatography on a BioRad
128
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
Fig. 2. Results of BLAST alignments for identified peptides. The boxes represent conserved amino acids, a Crotalus durissus ruruima Crotoxin B Swiss Protein ID: P86169.1, b Crotalus durissus terrificus Crotoxin B Protein Data Bank ID: 2QOG, c Crotalus durissus collilineatus Crotoxin B Swiss protein ID: P0CAS2.1, d Crotalus durissus terrificus Crotoxin B Swiss protein ID: P0CAS2.1, e Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS3.1, f Crotalus durissus terrificus Crotoxin B Swiss protein ID: P0CAS7.1, g Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS4.1, h Crotalus durissus cumanensis Crotoxin B Swiss protein ID: P86806.1, iCrotalus durissus ruruima Crotoxin B Swiss protein ID: P86805.1.
chromatography system (Econo Model) and reverse phase HPLC (RP-HPLC) (Shimadzu, Model Prominence, Shimadzu Corporation, Kyoto, Japan). For this, 50 mg of venom was dissolved in phosphatebuffered saline (PBS), pH 7.2, and passed through Sephacryl S-200 (120 cm × 1.8 cm) at the flow rate 1.0 ml/min. The resulting fractions were analyzed for PLA2 activity. Further separation of the PLA2 fractions was carried out using RP-HPLC in a column C-18 (pore 5 m, 250 mm × 4.6 mm mark RESTEK Bellefonte, Pensilvania, USA), after elution in a linear gradient (0–100%) acetonitrile (v/v) in 0.1% (v/v) trifluoroacetic acid at a flow rate 1.0 ml/min. Finally, fractions were lyophilized and stored at −20 ◦ C until use. Proteins in each fraction were separated under non-reducing conditions by electrophoresis in SDS-polyacrylamide gel (SDSPAGE) 15% (Laemmli, 1970). Their molecular weight was estimated according to molecular weight markers (range 97.4–14.4 kDa, BioRad, Philadelphia, PA, USA). The gels were stained with Coomassie Brilliant Blue G-250. The molecular mass of Crotoxin B was confirmed by direct infusion into a mass spectrometer nanoESI/MS IonTrap series (model 6310 Agilent Technologies).
2.3. Identification of proteins by HPLC-nESI-MS/MS Crotoxin B isolated from C. d. cumanensis venom (fraction II, Fig. 1A and C) was digested with trypsin (0.1 ng) at 30 ◦ C (Agilent Technologies) overnight and injected into LC/MS/MS (1200 series, Agilent Technologies) on a nano column C-18 (Agilent Zorbax 300SB-C18, 150 mm × 0.075 mm, 3.5 m) coupled to a mass spectrometer IonTrap MSD (Agilent Technologies 6310 series).
2.5. BLAST search of the identified peptides The identified peptides were subjected to a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) to determine the identity with other PLA2 family proteins. This identity was performed in BLASTP and search parameters having as nonredundant protein sequence (nr) and a snake organism. 2.6. Acute toxicity of the venom and its fractions The Median Lethal Dose (LD50 ) was determined by the Spearman–Karber method (World Health Organization, 1981), using groups of four mice (Swiss–Webster mice strain) injected intraperitoneally (IP) with varying doses of either fractions or whole C. d. cumanensis venom, previously dissolved in 0.5 ml PBS, pH 7.2. Fatalities were recorded within 48 h and the results are expressed as the mean of three repetitions. 2.7. Cytotoxic activity Peripheral blood mononuclear cells (PBMC) were separated by centrifugation of citrated human blood (400 g, 30 min) with Histopaque® -1077 (Sigma–Aldrich, St Louis, USA), washed with PBS, and transferred to 96 well plates at a concentration of 3 × 105 cells/well. Cells were incubated with different concentrations of toxins (37 ◦ C, 5% CO2 ) for 24 h. After this time, 40 l of MTT was added and incubated for 3 h (same conditions as described). The reaction was halted by adding 130 l of dimethyl sulfoxide (DMSO) and readings were performed in a microplate reader at 420 nm. The 50% cytotoxic dose was calculated by linear regression (Lomonte et al., 1993).
2.4. Search database
2.8. Indirect hemolysis
The resulting spectra were deconvoluted and processed by using the program Spectrum Mill (Agilent Technology) in the NCBInr database and online using Mascot (MatrixScience). The parameters of search including digestion with trypsin as fixed modification indicated Carbamidomethylation modified (C). The minimum score for the intensity of each peak was 50%, monoisotopic mass, mass tolerance of 2.5 Da and a way to search for identity.
This was evaluated following the method that uses agarose gel–erythrocyte–egg yolk as substrate (Gutierrez et al., 1988; Habermann and Hardt, 1972). We estimated the minimum indirect hemolytic dose (MIHD), defined as the dose producing a hemolytic halo of 20 mm in diameter after 20 h of incubation. In addition, different doses of venom, and fractions I, II and Crotoxin B were incubated with fresh human red blood cells for 30 min at 37 ◦ C in the
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
129
presence of 250 l of either inactivated human serum, inactivated human plasma, egg yolk or PBS. Afterwards, samples were centrifuged and the percentage of lysis was determined by assessing the release of hemoglobin by recording the absorbance at 540 nm. As a control of 100% lysis, 2% Triton X-100 was used. The results were expressed as percentage of lysis. 2.9. Culture of Plasmodium falciparum Based on the procedure described by Trager and Jenson (1978), parasites were grown at 37 ◦ C in A+ human erythrocytes to a hematocrit of 2% and 3–6% parasitemia under an atmosphere of 3% CO2 , 6% O2 and 91% N2 . 2.10. Determination of growth inhibition of P. falciparum and antiplasmodial activity of venom, fractions and Crotoxin B Increasing concentrations of whole venom (0.05–0.5 g/ml), fraction I (1–100 g/ml), crotoxin complex (fraction II) (0.1–1.0 g/ml) or Crotoxin B (0.1–1.0 g/ml) in complete medium were added in 96-well plates (100 l/well) and incubated with asynchronous P. falciparum FCB1 (Colombia) (1.5% parasitemia, 4% haematocrit, 100 l/well). Parasites were incubated as previously described (Trager and Jenson, 1978). After 24 h 0.5 mCi of 3H-hypoxanthine was added to the culture and parasites were cultured for further 24 h at the same conditions. Finally, the plates were freeze-thawed and parasites were harvested onto filter paper, followed by the addition of liquid scintillation cocktail. The incorporation of 3H-hypoxanthine was determined in a Microbeta counter 1450 (Wallac, Perkin Elmer). The percentage of growth inhibition was calculated based on 100% uptake of the 3H-hypoxanthine of controls (parasites in culture medium, incomplete RPMI). The IC50 values, maximum IC50 (IC50 ) and minimum IC100 (IC100 min ) were determined from dose–response curves according to Desjardins et al. (1979), establishing IC100 min as the minimum concentration required to kill 100% of the parasites within 48 h, IC50 max , as the maximum concentration without effect on parasite growth under equal conditions. Nonlinear regression logistic dose–responses model was used to estimate the IC50 and IC100 ± CI 95% values for each compound. 2.11. Statistical analysis The results are presented as mean ± SD of three replicates. The significance of the differences between means were determined by analysis of variance followed by Dunnett’s test for intragroup comparisons, and differences were considered significant when p < 0.05. 3. Results 3.1. Isolation of fractions and Crotoxin B Out of the four major fractions separated by gel filtration, fraction II showed PLA2 activity. Therefore this fraction was selected for the tests. In addition, a fraction with l-amino acid oxidase (fraction I) was also included in the evaluation of antiplasmodial activity (data not shown) (see Fig. 1A). Fraction II was subjected to separation by RP-HPLC on a C-18 column. This separation revealed the presence of 10 subfractions. Only subfraction IV showed activity of PLA2 (see graph 1C shaded area).
Fig. 3. Indirect hemolytic activity in solution. (A) Hemolysis when using either eggyolk, (B) inactivated human plasma or (C) inactivated human serum as substrates.
up to 14 kDa. Lane three corresponds to the Crotoxin B subfraction obtained by RP-HPLC, showing a single band (Fig. 1B).
3.2. SDS-PAGE
3.3. Identification of the protein
Electrophoresis showed that fraction I (lane 4) proteins have molecular weights ranging from 25 kDa to 80 kDa, whereas fraction II (lane 2) showed bands at molecular weights from 21 kDa
The analysis of tandem mass MS/MS indicates that the isolated PLA2 corresponds to Crotoxin B (see Table 1). Additionally; the identified peptides were subjected to BLAST program to determine their
130
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
Table 1 Protein identification results for Crotalus durissus cumanensis-PLA2 by ESI-MS/MS. Peptide sequence obtain for MS/MS, monoisotopic mass and charge for each peptide. (a) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P86169.1, (b) Crotalus durissus terrificus Crotoxin B Protein Data Bank ID: 2QOG, (c) Crotalus durissus collilineatus Crotoxin B Accession number SP|P0CAS2.1, (d) Crotalus durissus terrifics Crotoxin B Swiss protein ID: P0CAS2.1, (e) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS3.1, (f) Crotalus durissus terrificus Crotoxin B Swiss protein ID: P0CAS7.1, (g) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS4.1, (h) Crotalus durissus cumanensis Crotoxin B Swiss protein ID: P86806.1, (i) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P86805.1. MS/MS-derived sequence
MH+ (monoisotopic)
z
Scorea
CNTKWDIYPYSLK SLSTYKYGYMFYPDSR KNAIPFYAFYGCYCGWGGR GTWCEEQICECDR CCFVHDCCYGK CRGPSETC SGYITCGK
1687.815 1977.905 2288.155 1742.657 1505.543 966.377 885.414
2+ 3+ 3+ 2+ 2+ 2+ 2+
18.49 17.84 17.53 15.23 11.98 9.61 8.61
a
Score according to Spectrum Mill (Agilent Technologies).
Table 2 Activities of venom, fraction and Crotoxin B. Antiplasmodial activity (IC50 ), lethality (LD50 ), Cytotoxicity (CC50 ) and neurotoxicity. Compound
Antimalarial activity IC50 (g/ml)
Venom C. d. cumanensis Peak I Peak II Crotoxin B CQa
0.17 48.36 0.76 0.6 323.35
± ± ± ± ±
0.03 16.16 0.17 0.04 6.97
Lethal dose LD50 (g/kg)
Cytotoxicity CC50 (g/ml)
Neurotoxicity
117 (84–168.57) ND 12.72 (10.49–14.56) 700 ND
38.59 ± 0.57 57.56 ± 1.77 33.60 ± 1.09 18.23 ± 0.57 ND
Yes ND Yes No –
ND: not determined, no deaths were recorded. a This result corresponds to a nM concentration. The results of CC50 are presented as mean ± SEM.
identity with other PLA2 s (see Fig. 2). Further analysis revealed that several peptides have homology with the PLA2 domain common to these proteins. The molecular mass of Crotoxin B by mass spectrometry was 14,197.6 Da (see Fig. 1D). 3.4. Haemolytic activity of PLA2 from C. d. cumanensis Fraction II showed a minimum indirect hemolytic dose of 25 g, while fraction I was inactive. Similarly, Crotoxin B isolated by RPHPLC showed a minimum indirect hemolytic dose of 12 g (data not shown). The hemolysis test with different substrates showed similar results regardless of the substrate used (egg yolk, plasma or human serum). Moreover, Crotoxin B showed the highest activity, as compared to the venom and fraction II (see Fig. 3).
4. Discussion A number of reports have described the ability of snake venom PLA2 to inhibit bacteria, fungi and parasites, such as Leishmania spp, Giardia duodenalis, Trypanosoma cruzi and Plasmodium falciparum (Adade et al., 2010; Costa Torres et al., 2010; Guillaume et al., ˜ et al., 2004; Santamaria et al., 2005; Shinohara et al., 2004; Nunez 2006), evidencing that venom PLA2 s represent a valuable source of molecules with anti-microbial and anti-parasite activity. Owing to the public health relevance of P. falciparum and to the increasing appearance of resistance to antimalarial agents, the search for
3.5. Antiplasmodial activity of fractions and Crotoxin B Fractions I and II resulting from venom fractionation showed antiplasmodial activity on FCB1 strain of P. falciparum. Fraction II was more active than fraction I (see Table 2). Likewise, Crotoxin B proved to be more active than fraction II from which it was isolated. 3.6. Cytotoxic activity Analysis of the cytotoxic effect of the whole venom, fractions I, II, and Crotoxin B on PBMC cells showed that Crotoxin B was more cytotoxic than the complete venom and fraction II, while fraction I showed low cytotoxicity on PBMC cells (see Fig. 4). The doses required to exert cytotoxic activity on PBMC cells were higher than those required to exert anti-plasmodial activity (Table 2), thus conferring a selectivity index. 3.7. Acute toxicity LD50 of C.d. cumanensis venom was 117 g/kg (C.I. 84–168.5), whereas the LD50 of fraction II was 12.7 g/kg (C.I. 10.49–14.56). Crotoxin B isolated by RP-HPLC was devoid of toxicity since fatalities were not observed at doses as high as 700 g/kg (see Table 2).
Fig. 4. Dose–response of cytotoxicity of whole venom, fractions and Crotoxin B on peripheral blood mononuclear cells. Fractions I and II were obtained by size exclusion chromatography on Sephacryl S-200, whereas Crotoxin B was obtained by RP-HPLC on a column C-18.
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
novel therapeutic alternatives is highly relevant. In this study we assessed the antiplasmodial activity of Crotoxin B from the venom of C. d. cumanensis. Alignment results showed that Crotoxin B of C. d. cumanensis of Colombia is similar to the isoforms of Crotoxin B from other South American subspecies of rattlesnakes, but the peptides aligned with the previously characterized Crotoxin B of C. d. cumanensis of Venezuela revealed differences in some amino acids. These differences are probably due to the presence of different isoforms of the enzyme among different venoms, or in the same venom, as previously described (Faure and Bon, 1988; Faure et al., 1993, 1994). Guillaume et al. (2004) showed that the removal of phospholipids from cultures of P. falciparum reduced the antiplasmodial activity of PLA2 , indicating that most of the antiparasitic effect was due to enzymatic activity, with release of fatty acids and lysophospholipids, which exert an action on the parasite probably due to their detergent activity. Our data showed that the venom of C. d. cumanensis, as well as fraction II and Crotoxin B induced indirect hemolysis when the source of phospholipids was either egg yolk, plasma or serum inactivated in the presence of calcium. This ability to release fatty acids and lysophospholipids by enzymatic action, when the parasites are incubated in the presence of serum or plasma, may be responsible for the antiplasmodial activity observed, in agreement with previous studies (Deregnaucourt and Schrevel, 2000; Guillaume et al., 2004). During the intraerythrocytic development of Plasmodium, structural and functional changes in erythrocyte membrane functions occur. In addition, changes in the permeability of the membrane, the expression of the parasite proteins and changes in the lipid composition of the bilayer have been reported (Hill and Desai, 2010; Vial and Ancelin, 1998). Increased erythrocyte membrane permeability may also be responsible for the activity of PLA2 , as demonstrated by PLA2 induced lysis in the absence of serum in the in vitro culture system (Moll et al., 1990). Both crude venom and fraction II C.d. cumanensis are highly neurotoxic, owing to the action of the crotoxin complex. Nevertheless, the PLA2 subunit of crotoxin, i.e. Crotoxin B, shows negligible neurotoxicity, i.e. lethality, even when given at doses as high as 700 g/kg ˜ et al., in mice, consistent with results from other authors (Pereanez 2009). This low toxicity may be useful in future in vivo studies in experimental animals and in the search for potential pharmacological activities of this PLA2 . The cytotoxic activity of venoms and PLA2 on mammalian cells appears to be an obvious problem when attempting to use them in biomedical applications. However, in our case, the dose required to exert cytotoxic activity on human cells is much higher than the dose required to exert antimalarial activity. Crude venom, Crotoxin B and fraction II were 227 times, 44 times and 30 times more toxic, respectively, on P. falciparum than on human cells. Moreover, antimalarial activity was also higher than the indirect hemolytic effect. While other authors have shown that cytotoxic activity on tumor cells and erythrocytes is dependent on serum (Vadas, 1997), in our study cells were cultured with fetal bovine serum (FBS) and 2% inactivated serum or plasma. Even in these experimental conditions, the antimalarial activity was higher than the cytotoxic or indirect hemolytic activities. It is necessary to extend the analysis of cytotoxicity to other human cell types, to corroborate the low cytotoxicity of PLA2 . In conclusion, Crotoxin B, constitutes an interesting molecule with antimalarial action, exerting antiplasmodial activity at doses which are not lethal to mice and which are not cytotoxic to human blood mononuclear cells. Further studies are needed to confirm and extend the possible application of Crotoxin B as a lead molecule for the development of new antimalarial compounds.
131
Acknowledgements The authors thank Carlos Augusto Uribe, for his help in the mass spectrometer. This work was supported by the Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) project number 111540820526 and Universidad de Antioquia. This study was performed as partial requirement for the PhD degree of Juan Carlos Quintana Castillo at Universidad de Antioquia. References Abdel-Sattar, E., Maes, L., Salama, M.M., 2010. In vitro activities of plant extracts from Saudi Arabia against malaria, leishmaniasis, sleeping sickness and Chagas disease. Phytotherapy Research 24, 1322–1328. Adade, C.M., Cons, B.L., Melo, P.A., Souto-Padron, T., 2010. Effect of Crotalus viridis viridis snake venom on the ultrastructure and intracellular survival of Trypanosoma cruzi. Parasitology 138, 46–58. Andriao-Escarso, S.H., Soares, A.M., Rodrigues, V.M., Angulo, Y., Diaz, C., Lomonte, B., Gutierrez, J.M., Giglio, J.R., 2000. Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie 82, 755–763. Ayuko, T.A., Njau, R.N., Cornelius, W., Leah, N., Ndiege, I.O., 2009. In vitro antiplasmodial activity and toxicity assessment of plant extracts used in traditional malaria therapy in the Lake Victoria Region. Memorias do Instituto Oswaldo Cruz 104, 689–694. Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., 1985. Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil. Toxicon 23, 631–636. Barbosa, P.S., Martins, A.M., Havt, A., Toyama, D.O., Evangelista, J.S., Ferreira, D.P., Joazeiro, P.P., Beriam, L.O., Toyama, M.H., Fonteles, M.C., Monteiro, H.S., 2005. Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom. Toxicon 46, 376–386. Breman, J.G., Alilio, M.S., Mills, A., 2004. Conquering the intolerable burden of malaria: what’s new, what’s needed: a summary. American Journal of Tropical Medicine and Hygiene 71, 1–15. Bucaretchi, F., Herrera, S.R., Hyslop, S., Baracat, E.C., Vieira, R.J., 2002. Snakebites by Crotalus durissus ssp in children in Campinas, Sao Paulo, Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo 44, 133–138. Campbell, J.A., Lamar, W., 2004. The Venomous Reptiles of the Western Hemisphere. Cornell University Press, Ithaca, p. 870. Costa Torres, A.F., Dantas, R.T., Toyama, M.H., Diz Filho, E., Zara, F.J., Rodrigues de Queiroz, M.G., Pinto Nogueira, N.A., Rosa de Oliveira, M., de Oliveira Toyama, D., Monteiro, H.S., Martins, A.M., 2010. Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: phospholipase A2 and l-amino acid oxidase. Toxicon 55, 795–804. Deregnaucourt, C., Schrevel, J., 2000. Bee venom phospholipase A2 induces stagespecific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components. Journal of Biological Chemistry 275, 39973–39980. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710–718. Evangelista, I.L., Martins, A.M., Nascimento, N.R., Havt, A., Evangelista, J.S., de Noroes, T.B., Toyama, M.H., Diz-Filho, E.B., Toyama Dde, O., Fonteles, M.C., Monteiro, H.S., 2010. Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2 . Toxicon 55, 1061–1070. Faure, G., Bon, C., 1988. Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities. Biochemistry 27, 730–738. Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., 1994. The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus. European Journal of Biochemistry 223, 161–164. Faure, G., Harvey, A.L., Thomson, E., Saliou, B., Radvanyi, F., Bon, C., 1993. Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action. European Journal of Biochemistry 214, 491–496. Gao, B., Xu, J., Rodriguez Mdel, C., Lanz-Mendoza, H., Hernandez-Rivas, R., Du, W., Zhu, S., 2010. Characterization of two linear cationic antimalarial peptides in the scorpion Mesobuthus eupeus. Biochimie 92, 350–359. Guillaume, C., Deregnaucourt, C., Clavey, V., Schrevel, J., 2004. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serumdependent. Toxicon 43, 311–318. Gutierrez, J.M., Chaves, F., Rojas, E., Elizondo, J., Avila, C., Cerdas, L., 1988. Production of monovalent anti-Bothrops asper antivenom: development of immune response in horses and neutralizing ability. Revista de Biologia Tropical 36, 511–517. Gutierrez, J.M., Lomonte, B., 1995. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon 33, 1405–1424. Habermann, E., Hardt, K.L., 1972. A sensitive and specific plate test for the quantitation of phospholipases. Analytical Biochemistry 50, 163–173.
132
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
Harris, J.B., Grubb, B.D., Maltin, C.A., Dixon, R., 2000. The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin. Experimental Neurology 161, 517–526. Hill, D.A., Desai, S.A., 2010. Malaria parasite mutants with altered erythrocyte permeability: a new drug resistance mechanism and important molecular tool. Future Microbiology 5, 81–97. Karunamoorthi, K., Ilango, K., Murugan, K., 2010. Laboratory evaluation of traditionally used plant-based insect repellent against the malaria vector Anopheles arabiensis Patton (Diptera: Culicidae). Parasitology Research 106, 1217–1223. Kini, R.M., 2003. Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon 42, 827–840. Kini, R.M., Evans, H.J., 1987. Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2 . Journal of Biological Chemistry 262, 14402–14407. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Landucci, E.C., de Castro, R.C., Toyama, M., Giglio, J.R., Marangoni, S., De Nucci, G., Antunes, E., 2000. Inflammatory oedema induced by the lys-49 phospholipase A(2) homologue piratoxin-i in the rat and rabbit. Effect of polyanions and pbromophenacyl bromide. Biochemical Pharmacology 59, 1289–1294. Lomonte, B., Gutierrez, J.M., Romero, M., Nunez, J., Tarkowski, A., Hanson, L.A., 1993. An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies. Journal of Immunological Methods 161, 231–237. Martins, A.M., Toyama, M.H., Havt, A., Novello, J.C., Marangoni, S., Fonteles, M.C., Monteiro, H.S., 2002. Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys. Toxicon 40, 1165–1171. Milhous, W.K., Weina, P.J., 2010. Plant science. The botanical solution for malaria. Science 327, 279–280. Moll, G.N., Vial, H.J., van der Wiele, F.C., Ancelin, M.L., Roelofsen, B., Slotboom, A.J., de Haas, G.H., van Deenen, L.L., Op den Kamp, J.A., 1990. Selective elimination of malaria infected erythrocytes by a modified phospholipase A2 in vitro. Biochimica et Biophysica Acta 1024, 189–192. Muller, G.C., Beier, J.C., Traore, S.F., Toure, M.B., Traore, M.M., Bah, S., Doumbia, S., Schlein, Y., 2010. Successful field trial of attractive toxic sugar bait (ATSB) plantspraying methods against malaria vectors in the Anopheles gambiae complex in Mali, West Africa. Malaria Journal 9, 210. Murakami, M.T., Arruda, E.Z., Melo, P.A., Martinez, A.B., Calil-Elias, S., Tomaz, M.A., Lomonte, B., Gutierrez, J.M., Arni, R.K., 2005. Inhibition of myotoxic activity of
Bothrops asper myotoxin II by the anti-trypanosomal drug suramin. Journal of Molecular Biology 350, 416–426. ˜ V., Arce, V., Gutierrez, J.M., Lomonte, B., 2004. Structural and functional charNunez, acterization of myotoxin I, a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops atrox. Toxicon 44, 91–101. Oshima-Franco, Y., Hyslop, S., Prado-Franceschi, J., Cruz-Hofling, M.A., RodriguesSimioni, L., 1999. Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin. Toxicon 37, 1341–1357. Passero, L.F., Laurenti, M.D., Tomokane, T.Y., Corbett, C.E., Toyama, M.H., 2008. The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection. Parasitology Research 102, 1025–1033. ˜ ˜ Pereanez, J.A., Nunez, V., Huancahuire-Vega, S., Marangoni, S., Ponce-Soto, L.A., 2009. Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis. Toxicon 53, 534–542. Santamaria, C., Larios, S., Quiros, S., Pizarro-Cerda, J., Gorvel, J.P., Lomonte, B., Moreno, E., 2005. Bactericidal and antiendotoxic properties of short cationic peptides derived from a snake venom Lys49 phospholipase A2 . Antimicrobial Agents and Chemotherapy 49, 1340–1345. Shinohara, L., de Freitas, S.F., da Silva, R.J., Guimaraes, S., 2006. In vitro effects of Crotalus durissus terrificus and Bothrops jararaca venoms on Giardia duodenalis trophozoites. Parasitology Research 98, 339–344. Six, D.A., Dennis, E.A., 2000. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochimica et Biophysica Acta 1488, 1–19. Talvinen, K.A., Nevalainen, T.J., 2002. Cloning of a novel phospholipase A2 from the cnidarian Adamsia carciniopados. Comparative Biochemistry and Physiology B: Biochemistry and Molecular Biology 132, 571–578. Trager, W., Jenson, J.B., 1978. Cultivation of malarial parasites. Nature 273, 621–622. Vadas, P., 1997. Group II phospholipases A2 are indirectly cytolytic in the presence of exogenous phospholipid. Biochimica et Biophysica Acta 1346, 193–197. Vial, H.J., Ancelin, M.L., 1998. Malarial lipids. In: Sherman, I.W. (Ed.), Malaria: Parasite Biology, Pathogenesis, and Protection. ASM Press, Washington, DC, pp. 159–175. World Health Organization, 1981. Progress in the characterization of venoms and standardization of antivenoms. W.H.O. Offset Publication 58, 1–44. WHO, 2008. World Malaria Report 2008. World Health Organization, Geneva, http://malaria.who.int/wmr2008/malaria2008.pdf. WHO, 2010. World Malaria Report 2010. WHO, Genova, http://www.who.int/ malaria/publications/atoz/artemisinin resistance containment 2011.pdf.
Contents lists available at SciVerse ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and Crotoxin B J.C. Quintana a,d,∗ , A.M. Chacón a , L. Vargas a , C. Segura b , J.M. Gutiérrez c , J.C. Alarcón a a
Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín, Colombia Grupo Malaria, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica d Director de Desarrollo de Programas e Investigación, Dirección Nacional de Posgrados, Universidad Cooperativa de Colombia, Medellín, Colombia b c
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 8 July 2012 Accepted 12 July 2012 Available online 31 July 2012 Keywords: Plasmodium falciparum Malaria Snake venom Crotalus durissus cumanensis Phospholipase A2 Crotoxin B Antimalarial activity
a b s t r a c t The antiplasmodial activity of phospholipases A2 (PLA2 ) isolated from different animals has been studied. We explored the in vitro anti Plasmodium falciparum effect of a fraction containing crotoxin, Crotoxin B and whole venom of the rattlesnake Crotalus durissus cumanensis. Fraction II (crotoxin complex) was obtained by size exclusion chromatography, whereas Crotoxin B was purified by RP-HPLC. The whole venom is active against the parasite at concentrations of 0.17 ± 0.03 g/ml, fraction II at 0.76 ± 0.17 g/ml and Crotoxin B at 0.6 ± 0.04 g/ml. Differences were observed in the cytotoxic activity against peripheral mononuclear cells, with Crotoxin B exhibiting the highest cytotoxicity. The concentration of Crotoxin B required to exert cytotoxic activity was higher than that required to exert antiplasmodial activity. Lethality in mice confirmed the higher toxicity and neurotoxicity of whole venom and fraction II, whereas Crotoxin B was not lethal at the doses tested. These results suggest the potential of Crotoxin B as a lead compound for antimalarial activity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Malaria is caused by parasites of the genus Plasmodium and is a public health problem in tropical and sub-tropical regions of the world. Annually malaria provokes approximately 1.5 million deaths. Over 85% of them occur in Africa with P. falciparum as the leading species involved in most fatalities (Breman et al., 2004; WHO, 2008). The W.H.O. report confirmed almost 1 million deaths during the previous year (WHO, 2010). The most widely used treatment consists of artemisinin-based combined therapies (WHO, 2010). High rates of antimalarial treatment failure have led to the investigation of possible therapeutic alternatives, among which toxins and poisons derived from animals and plants are promising candidates (Abdel-Sattar et al., 2010; Ayuko et al., 2009; Gao et al., 2010; Karunamoorthi et al., 2010; Milhous and Weina, 2010; Muller et al., 2010).
∗ Corresponding author at: Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín, Colombia. Tel.: +57 4 2196536; fax: +57 4 2631914. E-mail addresses: [email protected], [email protected] (J.C. Quintana). 0001-706X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2012.07.003
Rattlesnakes (genus Crotalus, family Viperidae) are pit vipers with widespread distribution in the Americas (from North America to northern Argentina), being classified into many species and subspecies. Among them, Crotalus durissus cumanensis is distributed in Colombia and Venezuela (Campbell and Lamar, 2004). The venoms of South American subspecies of C. durissus are composed of a complex mixture of peptides, enzymes and toxins, such as crotamine, gyroxin, convulxin, thrombin-like serine proteinase (Bucaretchi et al., 2002; Oshima-Franco et al., 1999) and the crotoxin complex (a heterodimer composed of two subunits, A and B). Crotoxin complex is responsible for the high toxicity of the venom due to neurotoxic, nephrotoxic and myotoxic activities (AzevedoMarques et al., 1985; Martins et al., 2002; Oshima-Franco et al., 1999), which may provoke death by neurotoxic paralysis or acute renal failure. Phospholipases A2 -type (PLA2 , EC 3.1.1.4) are a superfamily characterized by their ability to hydrolyze phospholipids at the sn-2 ester bond of fatty acids to produce lysophospholipids and fatty acids. Secreted phospholipases A2 (sPLA2 ) share several characteristics: low molecular mass (13–18 kDa), numerous disulfide bridges, histidyl and aspartyl catalytic residues and a highly conserved calcium binding region (Ca2+ ) (Six and Dennis, 2000; Talvinen and Nevalainen, 2002). PLA2 s from snake venoms
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
127
Fig. 1. (A) Chromatographic elution profile on Sephacryl S-200 of the venom of C. d. cumanensis. The shaded fractions correspond to the fractions used, (B) SDS-PAGE (12%) separation of crude C. d. cumanensis venom (lane 1), fraction II from gel filtration chromatography (lane 2), Crotoxin B (lane 3), and fraction I from gel filtration chromatography (lane 4). MW correspond to molecular weight markers, (C) reverse-phase HPLC separation on a C-18 column of fraction II from gel filtration chromatography. The shaded area corresponds to Crotoxin B, (D) molecular mass determination of Crotoxin B by mass spectrometry.
exhibit a variety of toxicological and pharmacological activities, such as myotoxicity, neurotoxicity, anticoagulant activity, edema inducing-activity, cardiotoxicity, bactericidal activity, antiparasitic effect, and various effects on platelet aggregation (Andriao-Escarso et al., 2000; Barbosa et al., 2005; Costa Torres et al., 2010; Evangelista et al., 2010; Gutierrez and Lomonte, 1995; Harris et al., 2000; Kini, 2003; Kini and Evans, 1987; Landucci et al., 2000; Murakami et al., 2005). In recent years, several pharmacological applications for PLA2 have been described, including a potential activity against parasites (Costa Torres et al., 2010; Deregnaucourt and Schrevel, 2000; Guillaume et al., 2004; Passero et al., 2008). Owing to the high concentration of Crotoxin B, a PLA2 , in the venom of C. d. cumanensis, this venom constitutes a potential source of antimalarial activity. The aim of this study was to characterize the Crotoxin B from the venom of C. d. cumanensis, comparatively with a fraction containing the whole crotoxin complex and with the crude venom, for its in vitro antiplasmodial activity against P. falciparum, as well as its cytotoxicity on a human cell line and its acute toxicity in mice.
2. Materials and methods 2.1. Venom and reagents The venom was obtained by manual milking of 15 specimens from different regions of Colombia held in captivity at the Serpertarium of the University of Antioquia (Medellín, Colombia). Venoms were pooled, centrifuged (3000 rpm, 15 min), and the resulting supernatants were lyophilized and stored at −20 ◦ C until use. Acetonitrile (CH3 CN) and trifluoroacetic acid (CF3 COOH) HPLC grade were purchased from Fisher Scientific (Loughborough, UK), Histopaque® -1077, RPMI-1640 medium culture, Thiazolyl Blue Tretrazolium Bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (Sigma–Aldrich, St. Louis, USA). Water for HPLC was deionised to a degree of purity of 17 . 2.2. Venom fractionation Crotoxin B was purified from the venom of C. d. cumanensis using size molecular exclusion chromatography on a BioRad
128
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
Fig. 2. Results of BLAST alignments for identified peptides. The boxes represent conserved amino acids, a Crotalus durissus ruruima Crotoxin B Swiss Protein ID: P86169.1, b Crotalus durissus terrificus Crotoxin B Protein Data Bank ID: 2QOG, c Crotalus durissus collilineatus Crotoxin B Swiss protein ID: P0CAS2.1, d Crotalus durissus terrificus Crotoxin B Swiss protein ID: P0CAS2.1, e Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS3.1, f Crotalus durissus terrificus Crotoxin B Swiss protein ID: P0CAS7.1, g Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS4.1, h Crotalus durissus cumanensis Crotoxin B Swiss protein ID: P86806.1, iCrotalus durissus ruruima Crotoxin B Swiss protein ID: P86805.1.
chromatography system (Econo Model) and reverse phase HPLC (RP-HPLC) (Shimadzu, Model Prominence, Shimadzu Corporation, Kyoto, Japan). For this, 50 mg of venom was dissolved in phosphatebuffered saline (PBS), pH 7.2, and passed through Sephacryl S-200 (120 cm × 1.8 cm) at the flow rate 1.0 ml/min. The resulting fractions were analyzed for PLA2 activity. Further separation of the PLA2 fractions was carried out using RP-HPLC in a column C-18 (pore 5 m, 250 mm × 4.6 mm mark RESTEK Bellefonte, Pensilvania, USA), after elution in a linear gradient (0–100%) acetonitrile (v/v) in 0.1% (v/v) trifluoroacetic acid at a flow rate 1.0 ml/min. Finally, fractions were lyophilized and stored at −20 ◦ C until use. Proteins in each fraction were separated under non-reducing conditions by electrophoresis in SDS-polyacrylamide gel (SDSPAGE) 15% (Laemmli, 1970). Their molecular weight was estimated according to molecular weight markers (range 97.4–14.4 kDa, BioRad, Philadelphia, PA, USA). The gels were stained with Coomassie Brilliant Blue G-250. The molecular mass of Crotoxin B was confirmed by direct infusion into a mass spectrometer nanoESI/MS IonTrap series (model 6310 Agilent Technologies).
2.3. Identification of proteins by HPLC-nESI-MS/MS Crotoxin B isolated from C. d. cumanensis venom (fraction II, Fig. 1A and C) was digested with trypsin (0.1 ng) at 30 ◦ C (Agilent Technologies) overnight and injected into LC/MS/MS (1200 series, Agilent Technologies) on a nano column C-18 (Agilent Zorbax 300SB-C18, 150 mm × 0.075 mm, 3.5 m) coupled to a mass spectrometer IonTrap MSD (Agilent Technologies 6310 series).
2.5. BLAST search of the identified peptides The identified peptides were subjected to a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) to determine the identity with other PLA2 family proteins. This identity was performed in BLASTP and search parameters having as nonredundant protein sequence (nr) and a snake organism. 2.6. Acute toxicity of the venom and its fractions The Median Lethal Dose (LD50 ) was determined by the Spearman–Karber method (World Health Organization, 1981), using groups of four mice (Swiss–Webster mice strain) injected intraperitoneally (IP) with varying doses of either fractions or whole C. d. cumanensis venom, previously dissolved in 0.5 ml PBS, pH 7.2. Fatalities were recorded within 48 h and the results are expressed as the mean of three repetitions. 2.7. Cytotoxic activity Peripheral blood mononuclear cells (PBMC) were separated by centrifugation of citrated human blood (400 g, 30 min) with Histopaque® -1077 (Sigma–Aldrich, St Louis, USA), washed with PBS, and transferred to 96 well plates at a concentration of 3 × 105 cells/well. Cells were incubated with different concentrations of toxins (37 ◦ C, 5% CO2 ) for 24 h. After this time, 40 l of MTT was added and incubated for 3 h (same conditions as described). The reaction was halted by adding 130 l of dimethyl sulfoxide (DMSO) and readings were performed in a microplate reader at 420 nm. The 50% cytotoxic dose was calculated by linear regression (Lomonte et al., 1993).
2.4. Search database
2.8. Indirect hemolysis
The resulting spectra were deconvoluted and processed by using the program Spectrum Mill (Agilent Technology) in the NCBInr database and online using Mascot (MatrixScience). The parameters of search including digestion with trypsin as fixed modification indicated Carbamidomethylation modified (C). The minimum score for the intensity of each peak was 50%, monoisotopic mass, mass tolerance of 2.5 Da and a way to search for identity.
This was evaluated following the method that uses agarose gel–erythrocyte–egg yolk as substrate (Gutierrez et al., 1988; Habermann and Hardt, 1972). We estimated the minimum indirect hemolytic dose (MIHD), defined as the dose producing a hemolytic halo of 20 mm in diameter after 20 h of incubation. In addition, different doses of venom, and fractions I, II and Crotoxin B were incubated with fresh human red blood cells for 30 min at 37 ◦ C in the
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
129
presence of 250 l of either inactivated human serum, inactivated human plasma, egg yolk or PBS. Afterwards, samples were centrifuged and the percentage of lysis was determined by assessing the release of hemoglobin by recording the absorbance at 540 nm. As a control of 100% lysis, 2% Triton X-100 was used. The results were expressed as percentage of lysis. 2.9. Culture of Plasmodium falciparum Based on the procedure described by Trager and Jenson (1978), parasites were grown at 37 ◦ C in A+ human erythrocytes to a hematocrit of 2% and 3–6% parasitemia under an atmosphere of 3% CO2 , 6% O2 and 91% N2 . 2.10. Determination of growth inhibition of P. falciparum and antiplasmodial activity of venom, fractions and Crotoxin B Increasing concentrations of whole venom (0.05–0.5 g/ml), fraction I (1–100 g/ml), crotoxin complex (fraction II) (0.1–1.0 g/ml) or Crotoxin B (0.1–1.0 g/ml) in complete medium were added in 96-well plates (100 l/well) and incubated with asynchronous P. falciparum FCB1 (Colombia) (1.5% parasitemia, 4% haematocrit, 100 l/well). Parasites were incubated as previously described (Trager and Jenson, 1978). After 24 h 0.5 mCi of 3H-hypoxanthine was added to the culture and parasites were cultured for further 24 h at the same conditions. Finally, the plates were freeze-thawed and parasites were harvested onto filter paper, followed by the addition of liquid scintillation cocktail. The incorporation of 3H-hypoxanthine was determined in a Microbeta counter 1450 (Wallac, Perkin Elmer). The percentage of growth inhibition was calculated based on 100% uptake of the 3H-hypoxanthine of controls (parasites in culture medium, incomplete RPMI). The IC50 values, maximum IC50 (IC50 ) and minimum IC100 (IC100 min ) were determined from dose–response curves according to Desjardins et al. (1979), establishing IC100 min as the minimum concentration required to kill 100% of the parasites within 48 h, IC50 max , as the maximum concentration without effect on parasite growth under equal conditions. Nonlinear regression logistic dose–responses model was used to estimate the IC50 and IC100 ± CI 95% values for each compound. 2.11. Statistical analysis The results are presented as mean ± SD of three replicates. The significance of the differences between means were determined by analysis of variance followed by Dunnett’s test for intragroup comparisons, and differences were considered significant when p < 0.05. 3. Results 3.1. Isolation of fractions and Crotoxin B Out of the four major fractions separated by gel filtration, fraction II showed PLA2 activity. Therefore this fraction was selected for the tests. In addition, a fraction with l-amino acid oxidase (fraction I) was also included in the evaluation of antiplasmodial activity (data not shown) (see Fig. 1A). Fraction II was subjected to separation by RP-HPLC on a C-18 column. This separation revealed the presence of 10 subfractions. Only subfraction IV showed activity of PLA2 (see graph 1C shaded area).
Fig. 3. Indirect hemolytic activity in solution. (A) Hemolysis when using either eggyolk, (B) inactivated human plasma or (C) inactivated human serum as substrates.
up to 14 kDa. Lane three corresponds to the Crotoxin B subfraction obtained by RP-HPLC, showing a single band (Fig. 1B).
3.2. SDS-PAGE
3.3. Identification of the protein
Electrophoresis showed that fraction I (lane 4) proteins have molecular weights ranging from 25 kDa to 80 kDa, whereas fraction II (lane 2) showed bands at molecular weights from 21 kDa
The analysis of tandem mass MS/MS indicates that the isolated PLA2 corresponds to Crotoxin B (see Table 1). Additionally; the identified peptides were subjected to BLAST program to determine their
130
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
Table 1 Protein identification results for Crotalus durissus cumanensis-PLA2 by ESI-MS/MS. Peptide sequence obtain for MS/MS, monoisotopic mass and charge for each peptide. (a) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P86169.1, (b) Crotalus durissus terrificus Crotoxin B Protein Data Bank ID: 2QOG, (c) Crotalus durissus collilineatus Crotoxin B Accession number SP|P0CAS2.1, (d) Crotalus durissus terrifics Crotoxin B Swiss protein ID: P0CAS2.1, (e) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS3.1, (f) Crotalus durissus terrificus Crotoxin B Swiss protein ID: P0CAS7.1, (g) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P0CAS4.1, (h) Crotalus durissus cumanensis Crotoxin B Swiss protein ID: P86806.1, (i) Crotalus durissus ruruima Crotoxin B Swiss protein ID: P86805.1. MS/MS-derived sequence
MH+ (monoisotopic)
z
Scorea
CNTKWDIYPYSLK SLSTYKYGYMFYPDSR KNAIPFYAFYGCYCGWGGR GTWCEEQICECDR CCFVHDCCYGK CRGPSETC SGYITCGK
1687.815 1977.905 2288.155 1742.657 1505.543 966.377 885.414
2+ 3+ 3+ 2+ 2+ 2+ 2+
18.49 17.84 17.53 15.23 11.98 9.61 8.61
a
Score according to Spectrum Mill (Agilent Technologies).
Table 2 Activities of venom, fraction and Crotoxin B. Antiplasmodial activity (IC50 ), lethality (LD50 ), Cytotoxicity (CC50 ) and neurotoxicity. Compound
Antimalarial activity IC50 (g/ml)
Venom C. d. cumanensis Peak I Peak II Crotoxin B CQa
0.17 48.36 0.76 0.6 323.35
± ± ± ± ±
0.03 16.16 0.17 0.04 6.97
Lethal dose LD50 (g/kg)
Cytotoxicity CC50 (g/ml)
Neurotoxicity
117 (84–168.57) ND 12.72 (10.49–14.56) 700 ND
38.59 ± 0.57 57.56 ± 1.77 33.60 ± 1.09 18.23 ± 0.57 ND
Yes ND Yes No –
ND: not determined, no deaths were recorded. a This result corresponds to a nM concentration. The results of CC50 are presented as mean ± SEM.
identity with other PLA2 s (see Fig. 2). Further analysis revealed that several peptides have homology with the PLA2 domain common to these proteins. The molecular mass of Crotoxin B by mass spectrometry was 14,197.6 Da (see Fig. 1D). 3.4. Haemolytic activity of PLA2 from C. d. cumanensis Fraction II showed a minimum indirect hemolytic dose of 25 g, while fraction I was inactive. Similarly, Crotoxin B isolated by RPHPLC showed a minimum indirect hemolytic dose of 12 g (data not shown). The hemolysis test with different substrates showed similar results regardless of the substrate used (egg yolk, plasma or human serum). Moreover, Crotoxin B showed the highest activity, as compared to the venom and fraction II (see Fig. 3).
4. Discussion A number of reports have described the ability of snake venom PLA2 to inhibit bacteria, fungi and parasites, such as Leishmania spp, Giardia duodenalis, Trypanosoma cruzi and Plasmodium falciparum (Adade et al., 2010; Costa Torres et al., 2010; Guillaume et al., ˜ et al., 2004; Santamaria et al., 2005; Shinohara et al., 2004; Nunez 2006), evidencing that venom PLA2 s represent a valuable source of molecules with anti-microbial and anti-parasite activity. Owing to the public health relevance of P. falciparum and to the increasing appearance of resistance to antimalarial agents, the search for
3.5. Antiplasmodial activity of fractions and Crotoxin B Fractions I and II resulting from venom fractionation showed antiplasmodial activity on FCB1 strain of P. falciparum. Fraction II was more active than fraction I (see Table 2). Likewise, Crotoxin B proved to be more active than fraction II from which it was isolated. 3.6. Cytotoxic activity Analysis of the cytotoxic effect of the whole venom, fractions I, II, and Crotoxin B on PBMC cells showed that Crotoxin B was more cytotoxic than the complete venom and fraction II, while fraction I showed low cytotoxicity on PBMC cells (see Fig. 4). The doses required to exert cytotoxic activity on PBMC cells were higher than those required to exert anti-plasmodial activity (Table 2), thus conferring a selectivity index. 3.7. Acute toxicity LD50 of C.d. cumanensis venom was 117 g/kg (C.I. 84–168.5), whereas the LD50 of fraction II was 12.7 g/kg (C.I. 10.49–14.56). Crotoxin B isolated by RP-HPLC was devoid of toxicity since fatalities were not observed at doses as high as 700 g/kg (see Table 2).
Fig. 4. Dose–response of cytotoxicity of whole venom, fractions and Crotoxin B on peripheral blood mononuclear cells. Fractions I and II were obtained by size exclusion chromatography on Sephacryl S-200, whereas Crotoxin B was obtained by RP-HPLC on a column C-18.
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
novel therapeutic alternatives is highly relevant. In this study we assessed the antiplasmodial activity of Crotoxin B from the venom of C. d. cumanensis. Alignment results showed that Crotoxin B of C. d. cumanensis of Colombia is similar to the isoforms of Crotoxin B from other South American subspecies of rattlesnakes, but the peptides aligned with the previously characterized Crotoxin B of C. d. cumanensis of Venezuela revealed differences in some amino acids. These differences are probably due to the presence of different isoforms of the enzyme among different venoms, or in the same venom, as previously described (Faure and Bon, 1988; Faure et al., 1993, 1994). Guillaume et al. (2004) showed that the removal of phospholipids from cultures of P. falciparum reduced the antiplasmodial activity of PLA2 , indicating that most of the antiparasitic effect was due to enzymatic activity, with release of fatty acids and lysophospholipids, which exert an action on the parasite probably due to their detergent activity. Our data showed that the venom of C. d. cumanensis, as well as fraction II and Crotoxin B induced indirect hemolysis when the source of phospholipids was either egg yolk, plasma or serum inactivated in the presence of calcium. This ability to release fatty acids and lysophospholipids by enzymatic action, when the parasites are incubated in the presence of serum or plasma, may be responsible for the antiplasmodial activity observed, in agreement with previous studies (Deregnaucourt and Schrevel, 2000; Guillaume et al., 2004). During the intraerythrocytic development of Plasmodium, structural and functional changes in erythrocyte membrane functions occur. In addition, changes in the permeability of the membrane, the expression of the parasite proteins and changes in the lipid composition of the bilayer have been reported (Hill and Desai, 2010; Vial and Ancelin, 1998). Increased erythrocyte membrane permeability may also be responsible for the activity of PLA2 , as demonstrated by PLA2 induced lysis in the absence of serum in the in vitro culture system (Moll et al., 1990). Both crude venom and fraction II C.d. cumanensis are highly neurotoxic, owing to the action of the crotoxin complex. Nevertheless, the PLA2 subunit of crotoxin, i.e. Crotoxin B, shows negligible neurotoxicity, i.e. lethality, even when given at doses as high as 700 g/kg ˜ et al., in mice, consistent with results from other authors (Pereanez 2009). This low toxicity may be useful in future in vivo studies in experimental animals and in the search for potential pharmacological activities of this PLA2 . The cytotoxic activity of venoms and PLA2 on mammalian cells appears to be an obvious problem when attempting to use them in biomedical applications. However, in our case, the dose required to exert cytotoxic activity on human cells is much higher than the dose required to exert antimalarial activity. Crude venom, Crotoxin B and fraction II were 227 times, 44 times and 30 times more toxic, respectively, on P. falciparum than on human cells. Moreover, antimalarial activity was also higher than the indirect hemolytic effect. While other authors have shown that cytotoxic activity on tumor cells and erythrocytes is dependent on serum (Vadas, 1997), in our study cells were cultured with fetal bovine serum (FBS) and 2% inactivated serum or plasma. Even in these experimental conditions, the antimalarial activity was higher than the cytotoxic or indirect hemolytic activities. It is necessary to extend the analysis of cytotoxicity to other human cell types, to corroborate the low cytotoxicity of PLA2 . In conclusion, Crotoxin B, constitutes an interesting molecule with antimalarial action, exerting antiplasmodial activity at doses which are not lethal to mice and which are not cytotoxic to human blood mononuclear cells. Further studies are needed to confirm and extend the possible application of Crotoxin B as a lead molecule for the development of new antimalarial compounds.
131
Acknowledgements The authors thank Carlos Augusto Uribe, for his help in the mass spectrometer. This work was supported by the Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) project number 111540820526 and Universidad de Antioquia. This study was performed as partial requirement for the PhD degree of Juan Carlos Quintana Castillo at Universidad de Antioquia. References Abdel-Sattar, E., Maes, L., Salama, M.M., 2010. In vitro activities of plant extracts from Saudi Arabia against malaria, leishmaniasis, sleeping sickness and Chagas disease. Phytotherapy Research 24, 1322–1328. Adade, C.M., Cons, B.L., Melo, P.A., Souto-Padron, T., 2010. Effect of Crotalus viridis viridis snake venom on the ultrastructure and intracellular survival of Trypanosoma cruzi. Parasitology 138, 46–58. Andriao-Escarso, S.H., Soares, A.M., Rodrigues, V.M., Angulo, Y., Diaz, C., Lomonte, B., Gutierrez, J.M., Giglio, J.R., 2000. Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie 82, 755–763. Ayuko, T.A., Njau, R.N., Cornelius, W., Leah, N., Ndiege, I.O., 2009. In vitro antiplasmodial activity and toxicity assessment of plant extracts used in traditional malaria therapy in the Lake Victoria Region. Memorias do Instituto Oswaldo Cruz 104, 689–694. Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., 1985. Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil. Toxicon 23, 631–636. Barbosa, P.S., Martins, A.M., Havt, A., Toyama, D.O., Evangelista, J.S., Ferreira, D.P., Joazeiro, P.P., Beriam, L.O., Toyama, M.H., Fonteles, M.C., Monteiro, H.S., 2005. Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom. Toxicon 46, 376–386. Breman, J.G., Alilio, M.S., Mills, A., 2004. Conquering the intolerable burden of malaria: what’s new, what’s needed: a summary. American Journal of Tropical Medicine and Hygiene 71, 1–15. Bucaretchi, F., Herrera, S.R., Hyslop, S., Baracat, E.C., Vieira, R.J., 2002. Snakebites by Crotalus durissus ssp in children in Campinas, Sao Paulo, Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo 44, 133–138. Campbell, J.A., Lamar, W., 2004. The Venomous Reptiles of the Western Hemisphere. Cornell University Press, Ithaca, p. 870. Costa Torres, A.F., Dantas, R.T., Toyama, M.H., Diz Filho, E., Zara, F.J., Rodrigues de Queiroz, M.G., Pinto Nogueira, N.A., Rosa de Oliveira, M., de Oliveira Toyama, D., Monteiro, H.S., Martins, A.M., 2010. Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: phospholipase A2 and l-amino acid oxidase. Toxicon 55, 795–804. Deregnaucourt, C., Schrevel, J., 2000. Bee venom phospholipase A2 induces stagespecific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components. Journal of Biological Chemistry 275, 39973–39980. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710–718. Evangelista, I.L., Martins, A.M., Nascimento, N.R., Havt, A., Evangelista, J.S., de Noroes, T.B., Toyama, M.H., Diz-Filho, E.B., Toyama Dde, O., Fonteles, M.C., Monteiro, H.S., 2010. Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2 . Toxicon 55, 1061–1070. Faure, G., Bon, C., 1988. Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities. Biochemistry 27, 730–738. Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., 1994. The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus. European Journal of Biochemistry 223, 161–164. Faure, G., Harvey, A.L., Thomson, E., Saliou, B., Radvanyi, F., Bon, C., 1993. Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action. European Journal of Biochemistry 214, 491–496. Gao, B., Xu, J., Rodriguez Mdel, C., Lanz-Mendoza, H., Hernandez-Rivas, R., Du, W., Zhu, S., 2010. Characterization of two linear cationic antimalarial peptides in the scorpion Mesobuthus eupeus. Biochimie 92, 350–359. Guillaume, C., Deregnaucourt, C., Clavey, V., Schrevel, J., 2004. Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serumdependent. Toxicon 43, 311–318. Gutierrez, J.M., Chaves, F., Rojas, E., Elizondo, J., Avila, C., Cerdas, L., 1988. Production of monovalent anti-Bothrops asper antivenom: development of immune response in horses and neutralizing ability. Revista de Biologia Tropical 36, 511–517. Gutierrez, J.M., Lomonte, B., 1995. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon 33, 1405–1424. Habermann, E., Hardt, K.L., 1972. A sensitive and specific plate test for the quantitation of phospholipases. Analytical Biochemistry 50, 163–173.
132
J.C. Quintana et al. / Acta Tropica 124 (2012) 126–132
Harris, J.B., Grubb, B.D., Maltin, C.A., Dixon, R., 2000. The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin. Experimental Neurology 161, 517–526. Hill, D.A., Desai, S.A., 2010. Malaria parasite mutants with altered erythrocyte permeability: a new drug resistance mechanism and important molecular tool. Future Microbiology 5, 81–97. Karunamoorthi, K., Ilango, K., Murugan, K., 2010. Laboratory evaluation of traditionally used plant-based insect repellent against the malaria vector Anopheles arabiensis Patton (Diptera: Culicidae). Parasitology Research 106, 1217–1223. Kini, R.M., 2003. Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon 42, 827–840. Kini, R.M., Evans, H.J., 1987. Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2 . Journal of Biological Chemistry 262, 14402–14407. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Landucci, E.C., de Castro, R.C., Toyama, M., Giglio, J.R., Marangoni, S., De Nucci, G., Antunes, E., 2000. Inflammatory oedema induced by the lys-49 phospholipase A(2) homologue piratoxin-i in the rat and rabbit. Effect of polyanions and pbromophenacyl bromide. Biochemical Pharmacology 59, 1289–1294. Lomonte, B., Gutierrez, J.M., Romero, M., Nunez, J., Tarkowski, A., Hanson, L.A., 1993. An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies. Journal of Immunological Methods 161, 231–237. Martins, A.M., Toyama, M.H., Havt, A., Novello, J.C., Marangoni, S., Fonteles, M.C., Monteiro, H.S., 2002. Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys. Toxicon 40, 1165–1171. Milhous, W.K., Weina, P.J., 2010. Plant science. The botanical solution for malaria. Science 327, 279–280. Moll, G.N., Vial, H.J., van der Wiele, F.C., Ancelin, M.L., Roelofsen, B., Slotboom, A.J., de Haas, G.H., van Deenen, L.L., Op den Kamp, J.A., 1990. Selective elimination of malaria infected erythrocytes by a modified phospholipase A2 in vitro. Biochimica et Biophysica Acta 1024, 189–192. Muller, G.C., Beier, J.C., Traore, S.F., Toure, M.B., Traore, M.M., Bah, S., Doumbia, S., Schlein, Y., 2010. Successful field trial of attractive toxic sugar bait (ATSB) plantspraying methods against malaria vectors in the Anopheles gambiae complex in Mali, West Africa. Malaria Journal 9, 210. Murakami, M.T., Arruda, E.Z., Melo, P.A., Martinez, A.B., Calil-Elias, S., Tomaz, M.A., Lomonte, B., Gutierrez, J.M., Arni, R.K., 2005. Inhibition of myotoxic activity of
Bothrops asper myotoxin II by the anti-trypanosomal drug suramin. Journal of Molecular Biology 350, 416–426. ˜ V., Arce, V., Gutierrez, J.M., Lomonte, B., 2004. Structural and functional charNunez, acterization of myotoxin I, a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops atrox. Toxicon 44, 91–101. Oshima-Franco, Y., Hyslop, S., Prado-Franceschi, J., Cruz-Hofling, M.A., RodriguesSimioni, L., 1999. Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin. Toxicon 37, 1341–1357. Passero, L.F., Laurenti, M.D., Tomokane, T.Y., Corbett, C.E., Toyama, M.H., 2008. The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection. Parasitology Research 102, 1025–1033. ˜ ˜ Pereanez, J.A., Nunez, V., Huancahuire-Vega, S., Marangoni, S., Ponce-Soto, L.A., 2009. Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis. Toxicon 53, 534–542. Santamaria, C., Larios, S., Quiros, S., Pizarro-Cerda, J., Gorvel, J.P., Lomonte, B., Moreno, E., 2005. Bactericidal and antiendotoxic properties of short cationic peptides derived from a snake venom Lys49 phospholipase A2 . Antimicrobial Agents and Chemotherapy 49, 1340–1345. Shinohara, L., de Freitas, S.F., da Silva, R.J., Guimaraes, S., 2006. In vitro effects of Crotalus durissus terrificus and Bothrops jararaca venoms on Giardia duodenalis trophozoites. Parasitology Research 98, 339–344. Six, D.A., Dennis, E.A., 2000. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochimica et Biophysica Acta 1488, 1–19. Talvinen, K.A., Nevalainen, T.J., 2002. Cloning of a novel phospholipase A2 from the cnidarian Adamsia carciniopados. Comparative Biochemistry and Physiology B: Biochemistry and Molecular Biology 132, 571–578. Trager, W., Jenson, J.B., 1978. Cultivation of malarial parasites. Nature 273, 621–622. Vadas, P., 1997. Group II phospholipases A2 are indirectly cytolytic in the presence of exogenous phospholipid. Biochimica et Biophysica Acta 1346, 193–197. Vial, H.J., Ancelin, M.L., 1998. Malarial lipids. In: Sherman, I.W. (Ed.), Malaria: Parasite Biology, Pathogenesis, and Protection. ASM Press, Washington, DC, pp. 159–175. World Health Organization, 1981. Progress in the characterization of venoms and standardization of antivenoms. W.H.O. Offset Publication 58, 1–44. WHO, 2008. World Malaria Report 2008. World Health Organization, Geneva, http://malaria.who.int/wmr2008/malaria2008.pdf. WHO, 2010. World Malaria Report 2010. WHO, Genova, http://www.who.int/ malaria/publications/atoz/artemisinin resistance containment 2011.pdf.