- Original research
- Open Access
In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolenseby iron chelators
Kinetoplastid Biology and Disease volume 5, Article number: 3 (2006)
African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC50) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.
Trypanosoma brucei and T. congolense are the causative agents of sleeping sickness in humans and nagana in cattle, respectively. The protozoan parasites live extracellularly in blood and tissue fluids of mammals and are transmitted by the bite of infected tsetse flies (Glossina spp.). Over 60 million people living in 36 sub-Saharan countries are threatened with sleeping sickness  and 48000 deaths were reported in 2002 . In addition, 46 million cattle are exposed to the risk of contracting nagana and the disease costs an estimated 1340 million USD per year . Chemotherapy of African trypanosomiasis still relies on drugs developed decades ago and some of these display serious toxic side effects [4, 5]. In addition, drug resistance in African trypanosomes is increasing [6, 7]. Thus, new strategies to treat African trypanosomes are required.
In contrast to mammalian cells, bloodstream-form trypanosomes require only small amounts of iron for growth . The reason for this is that bloodstream-form trypanosomes lack cytochromes and contain only four iron-dependent enzymes: aconitase, alternative oxidase, ribonucleotide reductase and superoxide dismutase. Recently, it has been shown that incubation of T. brucei bloodstream forms with the iron chelator deferoxamine results in growth inhibition of the parasite . The compound does not inhibit iron-containing enzymes directly but acts by chelating cellular iron thus preventing its incorporation into newly synthesised apoproteins .
Here we investigated the trypanocidal activity of 13 chelators known to be able to complex iron ions against bloodstream forms of T. brucei and T. congolense in vitro. For comparison, the general cytotoxicity of the compounds was assayed with human myeloid leukaemia HL-60 cells.
The anti-trypanosomal activities and the general cytotoxicities of chelators were evaluated using the Alamar Blue® assay [10, 11]. For each reagent, the 50% inhibitory concentration (IC50) value, i.e. the concentration of a compound necessary to reduce the growth rate of the cells by 50% of that of controls, was determined. With the exception of 5-sulfosalicylic acid and dimethylglyoxime, all other compounds displayed anti-trypanosomal activities, with IC50 values varying 100-fold (Table 1). Generally, T. congolense was somewhat less susceptible to the compounds than T. brucei. A similar observation was recently made for the anti-trypanosomal activities of alkaloids . The most trypanocidal chelators were deferoxamine (Desferal®), 1,10-phenanthroline and its 4,7-diphenyl and 2,9-dimethy-4,7diphenyl (bathocuproine, a Cu1+ chelator) derivatives, and 8-hydroxyquinoline with IC50 values in the micromolar range. Thus, the anti-trypanosomal activities of these compounds are approaching those of commercial drugs used to treat sleeping sickness (suramin: IC50 = 0.4 μM) and nagana (diminazene aceturate (Berenil®): IC50 = 0.5 μM) previously determined for bloodstream forms of T. brucei 427-221a and T. congolense STIB910 under identical experimental conditions .
Except for 5-sulfosalicylic acid, bathocuproine, dimethylglyoxime and quercetin, all other chelators were also active against HL-60 cells, with IC50 values ranging from 6.2 μM to 97 μM (Table 1). However, the IC50 ratios of cytotoxic/trypanocidal activity (selectivity index) were found to be in a modest range for all compounds (Table 1). Only deferoxamine (Desferal®) and 4,7-diphenyl-1,10-phenanthroline gave IC50 ratios between 10 and 30 (Table 1). For comparison, commercially available drugs used for treatment of sleeping sickness and nagana have significant higher selectivity indices (suramin: IC50 ratio = 1944; diminazene aceturate: IC50 = 692 ).
Compounds with a high affinity for iron are common in nature, especially in micro-organisms. Numerous iron chelators, so-called siderophores, have been isolated from bacteria and fungi . Deferoxamine is such an iron chelator produced by the bacterium Streptomyces pilosus. It has been developed into the drug Desferal® which is used for the treatment of acute iron poisoning and chronic iron-overload. In addition, deferoxamine has been shown to exhibit trypanocidal activity against bloodstream forms of T. brucei . Here we demonstrated that deferoxamine is not only active to T. brucei but also to T. congolense bloodstream forms.
The isolation of siderophores in sufficient quantities for clinical applications is difficult and expensive. Therefore, other iron chelators which can be synthetically produced have been investigated in this study. 2,3-Dihydroxybenzoic acid and ethylenediamine-di-o-hydroxyphenylacetic acid are such synthetic iron chelators which have been used in patients with β-thalassaemia major  and to induce iron-deficiency in micro-organisms , respectively. However, both compounds only exhibited a weak trypanocidal effect. 5-Sulfosalicylic acid is another compound which complexes iron ions over a wide pH range but showed no toxic effect for trypanosomes and mammalian cells. The low anti-trypanosomal activities of these compounds may be due to their water solubility. Similarly, a reduced trypanocidal activity was recently observed for water-soluble derivatives of the DNA topoisomerase inhibitor camptothecin .
In contrast to water-soluble chelators, hydrophobic compounds displayed much better anti-trypanosomal activities. Tropolone is a lipid solubilizing agent for cationic metals and has been shown to release iron from cells . It exhibited moderate trypanocidal activities but was also very toxic for mammalian cells. Its 5-methyl derivative showed similar anti-trypanosomal actions but with reduced cytotoxicity. 8-Hydroxyquinoline is a well-known lipophilic iron chelator  and is used as a disinfectant. It was very effective in killing bloodstream forms of T. brucei, however, no prediction can be made regarding its mechanism of action because of the lack of selectivity for metal ions. Quercetin is a flavonoid with metal chelating properties. It was only moderately toxic to trypanosomes. This may be due to its antioxidant properties  counteracting its trypanocidal effect as a chelator depriving the cell of iron. 2,2'-Bipyridine is a membrane-permeant, intracellular Fe2+ chelator and has been shown to inhibit DNA virus replication by blocking the incorporation of iron into viral apoprotein ribonucleotide reductase . However, 2,2'-bipyridine displayed only some trypanocidal activity like the related compound 2,4,6-tris(2-pyridyl)-1,3,5-triazine. On the other hand, 1,10-phenanthroline, a 2,2'-bipyridine derivative with increased lipophilicity and reduced free rotatability, substantially inhibited the growth rate of both trypanosomes and mammalian cells. The 4,7-diphenyl derivative of 1,10-phenanthroline showed a similar trypanocidal activity as its parent compound but was less cytotoxic. However, the anti-trypanosomal action of phenanthrolines seems not to be by depleting the parasites of iron. Against this is the observation that Fe2+ and Cu1+ complexes of 4,7-diphenyl-1,10-phenanthroline display similar trypanocidal activities (data not shown). In addition, bathocuproine (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline), another lipophilic phenanthroline derivative that selectively complexes Cu1+ with no affinity for Fe2+, was also similarly toxic to trypanosomes. It seems that the lipophilicity of the compounds is the crucial factor. It is possible that phenanthrolines incorporate into membranes or intercalate into DNA and induce structural changes. In support of this, it has been shown that Fe2+ complexes involving 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline bind to DNA .
Iron deprivation may be a new strategy for the treatment of African trypanosomiasis. Although the compounds investigated in this study are not suitable for clinical use, our results suggest that lipophilic iron-chelating agents have a potential as novel anti-trypanosomal drugs. This finding may also be exploited in the future by utilizing the wealth of information currently being generated in the development of cell-permeable iron chelators as cancer chemotherapeutic agents [21–23]. In addition, future efforts should also aim at improving the selectivity of iron chelators. Moreover, iron chelators may be of interest for combination therapy with existing anti-trypanosome drugs.
Materials and methods
Alamar Blue® was from BioSource (Camarillo, CA, USA); EMEM with L-glutamine and without phenol red and foetal bovine serum were from Life Technologies (Eggenstein, Germany); RPMI 1640 without L-glutamine and without phenol red was from Bioproducts (Heidelberg, Germany); goat serum was from Boehringer Mannheim (Mannheim, Germany). Desferal was obtained from Ciba-Geigy (Wehr, Germany). All other chelators were from Sigma (Deisenhofen, Germany) or Fluka (Buchs, Switzerland). The Fe2+ complexes of 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline, and 5-methyl-tropolone were kindly provided by Professor Michael Wink (Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-University, Heidelberg, Germany).
Bloodstream forms of T. brucei TC221 was derived from the stock 427  and was obtained from Prof. Peter Overath (Max-Planck Institute für Biologie, Tübingen, Germany). The animal origin of the T. brucei stock 427 cannot be definitely clarified anymore. Bloodstream forms of T. congolense STIB910 was cloned from T. congolense STIB249 which was isolated from a lion in Tansania in 1971 . T. congolense STIB910 was kindly provided by Prof. Reto Brun (Swiss Tropical Institute, Switzerland). Human myeloid leukaemia HL-60 cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).
Bloodstream forms of T. brucei and T. congolense were grown in Baltz medium  (EMEM plus 25 mM HEPES, 2.5 mM glucose, 1 mM Na-pyruvate, 0.05 mM hypoxanthine, 0.001 mM thymidine, 0.04 mM adenosine, 0.025 mM bathocuproindisulfonic acid, pH 7.5, 10 ml/l nonessential amino acids (100×)) supplemented with 16.7% heat-inactivated foetal bovine serum and goat serum, respectively, and 0.2 mM 2-mercaptoethanol. Human myeloid leukaemia HL-60 cells were propagated in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated foetal bovine serum. All cultures were maintained in a humidified atmosphere containing 5% CO2 at 37°C (T. brucei and HL-60 cells) or at 34°C (T. congolense).
Cells were seeded into 96-well tissue culture plates in 200 μl medium containing various concentrations of chelators dissolved in H2O (deferoxamine, 2,3-dihydroxybenzoic acid, ethylenediamine-di-o-hydroxyphenylacetic acid, 5-sulfosalicylic acid, tropolone, 5-methyl-tropolone, 2,2'-bipyridine, 1,10-phenanthroline), 99% ethanol (2,4,6-tris(2pyridyl)-1,3,5-triazine, 4,7-diphenyl-1,10-phenanthroline, 8-hydroxyquinoline, dimethylglyoxime, quercetin), or 100% DMSO (bathocuproine). The controls contained the respective solvent alone. In all experiments, the final solvent concentration was 1% which had no effect on cell growth. To ensure that the cells were in logarithmic growth phase during the entire experiment, they were seeded at an initial density of 1 × 104 T. brucei/ml, 4 × 105 T. congolense/ml, and 1 × 105 HL-60/ml, respectively. After 24 h (trypanosomes) or 43 h (HL-60) incubation, 20 μl of the colorimetric viability indicator Alamar Blue® was added to each well. The cells were incubated for a further 24 h (trypanosomes) or 5 h (HL-60) so that the total incubation time was 48 h. Then, the plates were read on a Dynatech MR5000 ELISA reader (Denkendorf, Germany), using a test wavelength of 550 nm and a reference wave length of 630 nm. Each test was set up in duplicate and repeated three times. The IC50 value was determined by linear interpolation according to the method described in .
World Health Organization: African trypanosomiasis or sleeping sickness. World Health Organ Fact Sheet. 2001, 259: [http://www.who.int/mediacentre/factsheets/fs259/en/]
World Health Organization: The world health report 2004: changing history. Geneva. 2004
Kristjanson PM, Swallow BM, Rowlands GJ, Kruska RL, de Leeuw PN: Measuring the costs of African animal trypanosomiasis, the potential benefits of control and returns to research. Agr Sys. 1999, 59: 79-98. 10.1016/S0308-521X(98)00086-9.
Croft SL: The current status of antiparasitic chemotherapy. Parasitology. 1997, 114 (Suppl): S3-S15.
Fairlamb AH: Chemotherapy of human African trypanosomiasis: current status and future prospects. Trends Parasitol. 2003, 19: 488-494. 10.1016/j.pt.2003.09.002.
Ross CA, Sutherland DV: Drug resistance in trypamosomatids. Trypanosomiasis and Leishmaniasis: Biology and Control. Edited by: Hide G, Mottram JC, Coombs GH, Holmes PH. 1997, Wallingford, Oxon: CAB International, 259-269.
Matovu E, Seebeck T, Enyaru JCK, Kaminsky R: Drug resistance in Trypanosoma brucei spp., the causative agents of sleeping sickness in man and nagana in cattle. Microbes Infect. 2001, 3: 763-770. 10.1016/S1286-4579(01)01432-0.
Steverding D: Bloodstream forms of Trypanosoma brucei require only small amounts of iron for growth. Parasitol Res. 1998, 84: 59-62. 10.1007/s004360050357.
Breidbach T, Scory S, Krauth-Siegel RL, Steverding D: Growth inhibition of bloodstream forms of Trypanosoma brucei by the iron chelator deferoxamine. Int J Parasitol. 2002, 32: 473-479. 10.1016/S0020-7519(01)00310-1.
Räz B, Iten M, Grether-Bühler Y, Kaminisky R, Brun R: The Alamar Blue® assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro. Acta Trop. 1997, 68: 139-147. 10.1016/S0001-706X(97)00079-X.
Merschjohann K, Sporer F, Steverding D, Wink M: In vitro effect of alkaloids on bloodstream forms of Trypanosoma brucei and T. congolense. Planta Med. 2001, 67: 623-627. 10.1055/s-2001-17351.
Winkelmann G: Microbial siderophore-mediated transport. Biochem Soc Trans. 2002, 30: 691-696. 10.1042/BST0300691.
Peterson CM, Graziano JH, Grady RW, Jones RL, Vlassara HV, Canale VC, Miller DR, Cerami A: Chelation studies with 2,3-dihydroxybenzoic acid in patients with β-thalassaemia major. Br J Haematol. 1976, 33: 477-485.
Salamah AA: Effect of ethylenediamine di-o-hydroxyphenylacetic acid and transferrin on the growth of some bacterial strains in vitro. Microbiologica. 1992, 15: 361-366.
Deterding A, Dungey FA, Thompson K-T, Steverding D: Anti-trypanosomal activities of DNA topoisomerase inhibitors. Acta Trop. 2005, 93: 311-316. 10.1016/j.actatropica.2005.01.005.
White GP, Jacobs A, Grady RW, Cerami A: The effect ofchelating agents on iron mobilization in Chang cell cultures. Blood. 1976, 48: 923-929.
Burgess J, Drasdo DN, Patel MS: Solubilities and solvation of aluminum(III), iron(III), and indium(III) 8-hydroxyquinolinates in methanol/water mixtures. J Pharm Sci. 1994, 83: 54-7.
Boadi WY, Iyere PA, Adunyah SE: Effect of quercetin and genistein on copper- and iron-induced lipid peroxidation in methyl linolenate. J Appl Toxicol. 2003, 23: 363-369. 10.1002/jat.933.
Romeo AM, Christen L, Niles EG, Kosman DJ: Intracellular chelation of iron by bipyridyl inhibits DNA virus replication: ribonucleotide reductase maturation as a probe of intracellular iron pools. J Biol Chem. 2001, 276: 24301-24308. 10.1074/jbc.M010806200.
Mudasir , Wijaya K, Yoshioka N, Inoue H: DNA binding of iron(II) complexes with 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline: salt effect, ligand substituent effect, base pair specificity and binding strength. J Inorg Biochem. 2003, 94: 263-271. 10.1016/S0162-0134(03)00007-2.
Buss JL, Greene BT, Turner J, Torti FM, Torti SV: Iron chelators in cancer chemotherapy. Curr Top Med Chem. 2004, 4: 1623-1635. 10.2174/1568026043387269.
Richardson DR: Molecular mechanisms of iron uptake by cells and the use of iron chelators for the treatment of cancer. Curr Med Chem. 2005, 12: 2711-2729. 10.2174/092986705774462996.
Pahl PM, Horwitz LD: Cell permeable iron chelators as potential cancer chemotherapeutic agents. Cancer Invest. 2005, 23: 683-691. 10.1080/07357900500359976.
Hirumi H, Hirumi K, Doyle JJ, Cross GAM: In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei. Parasitology. 1980, 80: 371-382.
Kaminsky R, Schmid C, Grether Y, Holý A, De Clercq E, Naesens L, Brun R: (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA]: a purine analogue with trypanocidal activity in vitro and in vivo. Trop Med Int Health. 1996, 1: 255-263.
Baltz T, Baltz D, Giroud C, Crockett J: Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 1985, 4: 1273-1277.
Huber W, Koella JC: A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop. 1993, 55: 257-261. 10.1016/0001-706X(93)90083-N.
We thank Dr. John Winpenny for critical reading of the manuscript. This work was supported by the Bundesministerium für Forschung und Technologie, Schwerpunkt für tropenmedizische Forschung in Heidelberg (01 KA 9301/3).
The author(s) declare that they have no competing interests.
K.M. carried out the experimental work as part of her VMD. D.S. conceived the study, supervised the execution, and prepared the final draft of the manuscript. Both authors read and approved the final manuscript.