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Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa
Molecular Aspects of Medicine 25 (2004) 211–220 www.elsevier.com/locate/mam
Review
Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa Julio F. Turrens
*
Department of Biomedical Sciences, College of Allied Health Professions, University of South Alabama, UCOM 6000, Mobile, AL 36695, USA
Abstract Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries. Although these organisms are eukaryotic cells, some of them present major differences with their mammalian host in selected metabolic pathways. These differences may be exploited as targets for developing better pharmacological agents for the treatment of specific parasitic diseases. This review describes some of the differences in terms of antioxidant defenses between these organisms and their mammalian host, which may provide useful targets for the treatment of these diseases. Some of the potential targets are: (i) iron metabolism in Plasmodium, (ii) the presence of a Fe-containing form of superoxide dismutase in trypanosomatids and malaria-causing parasites, (iii) the unique trypanothionedependent antioxidant metabolism in trypanosomatids, (iv) the ascorbate peroxidase found in Trypanosoma cruzi and perhaps present in other trypanosomatids. 2004 Elsevier Ltd. All rights reserved. Keywords: Malaria; Chagas disease; Leishmania; Trypanosomes; Plasmodium; Ameba; Antioxidant enzymes; Peroxiredoxin; Trypanothione; Ovothiol
*
Tel.: +1-251-380-2714/3343802714; fax: +1-251-380-2711/3343802711. E-mail address: [email protected] (J.F. Turrens).
0098-2997/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2004.02.021
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Contents 1.
Parasitic protozoa and parasitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2.
Oxidative metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
3.
Sources of oxidative species unique to some protozoan parasites . . . . . . . . . . . 214
4.
Antioxidant defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
5.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
1. Parasitic protozoa and parasitosis Parasitic protozoa are responsible for various diseases that affect an enormous proportion of the world’s population (Table 1). Malaria, a disease caused by various species of the Genus Plasmodium, is the most predominant of these serious diseases, infecting approximately 300 million people per year and causing the death of about 1–2 million people, mostly children, every year. Another major group of parasitic diseases is caused by various members of the order Kinetoplastida. These diseases include leishmaniases in their many forms (cutaneous, mucocutaneous and visceral, also know as kala azar) and trypanosomiases (African Sleeping Sickness and Chagas disease, Table 1). In addition, other African trypanosomes (Trypanosoma brucei brucei and T. brucei congolense) target cattle causing a disease called ‘‘nagana’’ which has a major impact in the economy and food supply in African nations. A third group of diseases, amebiases, is found all over the world, but it is difficult to estimate the actual numbers of infected patients because only a small proportion become symptomatic (Walsh, 1986). Finally, the spreading of AIDS has also opened the door for opportunistic diseases responsible for severe complications such as those caused by Toxoplasma gondii and Trypanosoma cruzi (Ferreira and Borges, 2002). Since parasitic protozoa are eukaryotic cells, many metabolic pathways are very similar to those of the mammalian host. As a result, parasitic diseases are more difficult to treat than other infections, and many of the drugs used against them tend to have serious side effects. Thus, identifying metabolic differences between parasite and host could define new targets for pharmacological agents that would not interfere with the host metabolism, leading to more effective chemotherapies.
2. Oxidative metabolism Most parasitic protozoa are aerobic cells and live in oxygenated environments. Aerobic metabolism utilizes molecular oxygen as the electron acceptor in energyproducing oxidations, which in turn becomes reduced by four electrons to two
Disease
Agent
Infection rate per year (millions)
Malaria Leishmaniases (cutaneous, mucocutaneous and visceral) Chagas disease African trypanosomiases Amebiasis
Plasmodium falciparum, P. vivax, P. malariae, and P. ovale Leishmania donovani, L. tropica, L. infantum, L. major, L. amazonensis, L. aethiopica, L. mexicana, L. guyanensis, L. peruviana, L. venezuelensis
300 1.5–2
Trypanosoma cruzi Trypanosoma gambiense, Trypanosoma rhodesiense
0.7–0.8 0.3–0.5
Entamoeba histolyica
Unknowna
Total (millions)
At risk (millions)
Deaths/year
800 12
2000 350
2.7 million 59,000
16–18
100 60
45,000 50,000
500a
–
40,000
The information was collected from the World Health Organization’s website (http://www.who.int) and is also available in various WHO reports. The data concerning Amebiasis was reported by Walsh (1986). a Amebiasis is very common, particularly in developing countries. Only 10% of infected people (40–50 million) become symptomatic and very few develop severe symptoms (Walsh, 1986).
J.F. Turrens / Molecular Aspects of Medicine 25 (2004) 211–220
Table 1 Population (in millions) infected and at risk of infection with some of the diseases caused by parasitic protozoan
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molecules of water. However, molecular oxygen may also be partially reduced to relatively stable species by accepting one, two or three electrons, with the formation of superoxide anion, hydrogen peroxide and hydroxyl radical, respectively (Chance et al., 1979; Turrens, 2003). Some of these species (superoxide anion, hydrogen peroxide) are usually the result of undesirable side reactions in redox processes but may also be produced for specific processes including antimicrobial activity and intracellular signaling (Droge, 2002). Although superoxide anion is not very reactive per se, this radical is a precursor of two strong oxidants. First, superoxide may react with nitric oxide to produce peroxynitrite, an oxidizing and nitrating agent generated by macrophages as a first line of defense during phagocytosis. In addition, superoxide anion is a precursor of hydrogen peroxide and, in the presence of transition metals, the two species are responsible for the formation of hydroxyl radicals. The latter is a strong and non-specific oxidant that may attack and damage all life molecules, including proteins, lipids and nucleic acids. Under normal conditions, all these species are maintained at relatively constant intracellular steady-state concentrations by various enzymes and low molecular antioxidants. An imbalance in this steadystate leads to oxidative stress and may be deleterious and even lethal to all forms of life. In this review we will briefly explore some the major biochemical differences in oxidative metabolism, both in antioxidant defenses and in the sources of oxidative species, between mammalian cells and various disease-causing protozoan parasites, which may potentially be targeted for the treatment of parasitic diseases.
3. Sources of oxidative species unique to some protozoan parasites The life cycle of most members of the genus Plasmodium (among them parasites causing human malaria such as P. falciparum) involves a stage as an intracellular parasite in erythrocytes. During this stage the parasites depend on hemoglobin as their primary nutrient and accumulate vast amounts of iron from heme. As mentioned before, iron is a catalyst in the formation of hydroxyl radicals from superoxide anion and hydrogen peroxide, and therefore it is important for these parasites to be able to sequester the excess iron and heme in a form that is innocuous to the parasite. This is achieved by synthesizing an amorphous brown heme aggregate called hemozoin. Some antimalarials such as chloroquine appear to act by interfering with the formation of this pigment and consequently leaving iron available for reactions that are toxic and lethal for the parasite (Sullivan, 2003). The mitochondrial respiratory chain constitutes one of the primary sources of reactive oxygen species in mammalian cells (Chance et al., 1979; Turrens, 2003). In the mammalian respiratory chain, most of the electrons reducing oxygen come from NADH oxidation through the sequence Complex I fi Complex III fi Complex IV fi oxygen. Alternatively, electrons may enter the chain via other flavoproteins (succinate dehydrogenase or Complex II, glycerol-phosphate dehydrogenase, fatty
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acyl CoA dehydrogenase) and be transferred to oxygen via Complexes III and IV, skipping mitochondrial Complex I (Turrens, 2003). The electron transport chain of several parasitic protozoa presents major differences when compared with the host’s counterpart, and as a result, in the sources of the partially reduced oxygen species. For example, while the insect stage of African trypanosomes (procyclic trypomastigotes) has a functional mitochondrial respiratory chain, the bloodstream forms (trypomastigotes) lack most of the electron transport carriers of the respiratory chain (including all the cytochromes) (Hill, 1976; Turrens et al., 1986). The respiratory chain found in African as well as in all stages of American trypanosomes is also quite different from the mammalian counterpart. Studies carried out using specific inhibitors indicate that the electrons from NADH do not reach cytochrome c via Complex III, since this activity is not inhibited by antimycin (Turrens, 1989; Denicola-Seoane et al., 1992; Santhamma and Bhaduri, 1995). In other words, in trypanosomatids, Complex I is either missing or works in an entirely different way. Furthermore, even the Krebs’ cycle (where most of the mitochondrial NADH is produced) works in a different fashion in trypanosomatids. Some of the Krebs’ cycle enzymes that produce NADH in mammalian cells are missing, for example, a-ketoglutarate dehydrogenase, while other dehydrogenases work in different biochemical ways, for example, isocitrate dehydrogenase reduces NADP instead of NAD (Fairlamb and Opperdoes, 1986). Trypanosomatids and plasmodia also have an enzyme not present in mammalian cells, NADH-fumarate reductase, capable of reducing fumarate to succinate, reverting one of the steps of the Krebs’ cycle (Boveris et al., 1986; Fry and Beesley, 1991; Santhamma and Bhaduri, 1995; Turrens, 1989; Turrens et al., 1992). This enzyme may have an important role in the reoxidation of intracellular NADH. The enzyme NADH-fumarate reductase (Turrens, 1987) and maybe other NADHdehydrogenases unique to trypanosomatids (Fang and Beattie, 2003) can directly transfer electrons to oxygen in the absence of their physiological electron acceptors. Perhaps new drugs could be designed to prevent binding of the physiological oxidant in order to stimulate direct transfer of electrons to oxygen. The main drawback of this approach is that, although these enzymes are unique to trypanosomatids, their substrates are important metabolites in mammalian cells and some of these putative inhibitors could still interfere with the intermediate metabolism in the host. Some drugs capable of redox cycling may also selectively increase oxidative stress in the parasite, as long as they selectively accept electrons from oxidoreductases that are unique to the parasite. In fact, although the electron donors have not been identified, several antitrypanosomal drugs, currently used (nifurtimox (Docampo and Moreno, 1984)) or proposed (b-lapachone (Boveris et al., 1978)) appear to undergo redox cycling selectively killing the parasite as a result of the combination of two processes: (i) the formation of oxygen free radicals, and (ii) the deficient antioxidant defenses found in these parasites. This is particularly relevant in the case of T. cruzi since this organism appears to have a higher intracellular steady-state concentration of hydrogen peroxide as a result of its limited antioxidant defenses (Boveris et al., 1980; Giulivi et al., 1988).
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4. Antioxidant defenses Aerobic organisms evolved two major types of antioxidant defenses in order to minimize the oxidative damage resulting from the presence of oxygen in the environment: enzymatic defenses and low molecular weight antioxidants (Chance et al., 1979). In mammalian cells, oxidative stress is prevented by four main antioxidant enzymes (Fig. 1): (i) Cu, Zn-superoxide dismutase (McCord and Fridovich, 1969; Fridovich, 1995), the enzyme responsible for the conversion of superoxide anion into hydrogen peroxide, that is found in the cytoplasm and in the mitochondrial intermembrane space (Okado-Matsumoto and Fridovich, 2001); (ii) Mn-SOD (Fridovich, 1995), with the same action as Cu, Zn-SOD, but only found in the mitochondrial matrix; (iii) glutathione peroxidase, a peroxidase responsible for the reduction of hydrogen peroxide and hydroperoxides that utilizes reduced glutathione as hydrogen donor (Ursini et al., 1995); and (iv) catalase, the enzyme that catalyzes the dismutation of hydrogen peroxide to oxygen and water (Chance et al., 1979). The activity of glutathione peroxidase requires reduced glutathione which becomes oxidized during the reduction of peroxides. Thus, a separate enzyme, glutathione reductase, is required for the NADPH-dependent reduction of oxidized glutathione in order to maintain a steady-state of GSH. Small molecular weight antioxidants include a variety of molecules that directly react with oxygen radicals and prevent them from oxidizing important biomolecules (vitamin E, vitamin C, uric acid, ubiquinone, and various thiol-containing molecules such as cysteine and glutathione). Parasitic protozoa present major differences in terms of antioxidant defenses, not only when compared to the mammalian host but among themselves. For example, trypanosomatids (Leishmania and Trypanosoma) as well as Plasmodium have an iron-containing isoform of SOD (Meshnick et al., 1983; Becuwe et al., 1996), normally found only in bacteria but absent in other eukaryotic cells.
Fig. 1. Antioxidant enzymatic reactions in mammalian cells. Both forms of superoxide dismutase (Cu, Zn-SOD and Mn-SOD) catalyze the dismutation of superoxide anion. This reaction may also occur spontaneously, although at a slower rate than in the presence of these enzymes. The product, hydrogen peroxide, can be either reduced or dismutated by glutathione peroxidase or by catalase, respectively. Glutathione peroxidase may also reduce other peroxides. Oxidized glutathione is in turn reduced by NADPH in a reaction catalyzed by glutathione reductase.
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The enzyme catalase is missing in trypanosomatids (Boveris et al., 1980) and in Plasmodium. The absence of catalase in Plasmodium has been confirmed after decoding this organism genome (see Plasmod.org in Internet). Yet, other protozoan parasites, such as the opportunistic T. gondii, show catalase activity (Kwok et al., 2004). Various isoforms of the Se-dependent glutathione peroxidase have been detected in Plasmodium (Flohe et al., 2003) but they are absent in all trypanosomatids (Boveris et al., 1980; Flohe et al., 2003). Trypanosomatids, on the other hand, do not have a Se-dependent glutathione peroxidase although a non-Se form of the peroxidase has been detected in both African and American trypanosomes (Boveris et al., 1980; Wilkinson et al., 2000; Wilkinson et al., 2003). This form of glutathione peroxidase reacts with organic peroxides but does not reduce hydrogen peroxide. Instead, the members of the Order Kinetoplastida rely in an entirely different thioldependent antioxidant system to eliminate hydrogen peroxide (Fig. 2). All trypanosomatids synthesize trypanothione, a dithiol derived from glutathione that is unique to the members of the order Kinetoplastida and that has been proposed as a suitable target to develop drugs for the treatment of leishmaniases and trypanosomiases (Fairlamb and Cerami, 1992; Flohe et al., 2003; Krauth-Siegel et al., 2003; Steenkamp, 2003). Trypanothione (bis(glutathiolnyl)spermidine) consists of two molecules of glutathione conjugated with a molecule of spermidine. Although the reduction potential of the thiol groups is the same that of glutathione, their pKa is a whole pH unit lower than that of free glutathione, probably because of electron displacement caused by the extra amino group found in spermidine (Krauth-Siegel et al., 2003). This different pKa may explain trypanothione’s distinct reactivity. For
Fig. 2. Antioxidant metabolism in Kinetoplastida. These cells contain a different form of superoxide dismutase (Fe-SOD) and a unique battery of enzymes involved in peroxide metabolism. These cells do not have either catalase or glutathione peroxidase. Instead, most of their glutathione is converted into trypanothione which in turn may spontaneously reduce tryparedoxin and dehydroascorbate. Reduced tryparedoxin is the hydrogen donor for the tryparedoxin peroxidase reaction. An ascorbate peroxidase has been detected in Trypanosoma cruzi. The enzymes involved in trypanothione metabolism, both in its synthesis and in its reduction, have been proposed as possible targets against the diseases caused by parasites from the genus Leishmania and Trypanosoma.
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example, dehydroascorbate, the oxidation product of ascorbic acid, is reduced by trypanothione but not by glutathione. Similarly, trypanothione directly reduces oxidized tryparedoxin, a thioredoxin found in trypanosomatids which has a central role in peroxide metabolism (see below) (Krauth-Siegel et al., 2003; Steenkamp, 2003). These two non-enzymatic reactions may be vital antioxidant processes in trypanosomatids. First, in T. cruzi dehydroascorbate is produced during the reduction of H2 O2 catalyzed by ascorbate peroxidase (Docampo et al., 1976; Boveris et al., 1980; Wilkinson et al., 2002), trypanothione may regenerate ascorbic acid. Second, the most important peroxidatic system common to all trypanosomatids relies on an enzyme called tryparedoxin peroxidase. Tryparedoxin peroxidase is one of the peroxiredoxins, a family of highly preserved small molecular weight (around 20–30 kDa) cysteine-containing proteins found in most organisms (Rhee et al., 2001; Wilkinson et al., 2002). Most cells, including Plasmodium falciparum (Krnajski et al., 2002), require specific thiol-dependent peroxiredoxin reductases (for example, glutathione-dependent) in order to reduce the thiol groups in peroxiredoxins. In trypanosomatids, however, it has been postulated that a tryparedoxin reductase may not be needed because of the peculiar reactivity of trypanothione as a reductant of tryparedoxin (Schmidt and Krauth-Siegel, 2003). The combined action of trypanothione reductase, tryparedoxin and tryparedoxin peroxidase is central to the maintenance of a low steady-state concentration of H2 O2 (Fig. 2). The vital role of trypanothione, a low molecular weight antioxidant not present in mammalian cells, as an antioxidant in the hydroperoxide metabolism of trypanosomatids provides various potential targets for the treatment of leishmaniases and trypanosomiases. These targets are: (i) trypanothione reductase, the NADPHdependent enzyme that regenerates reduced trypanothione; (ii) the enzymes involved in the synthesis of trypanothione (glutathionylspermidine synthetase and trypanothione synthetase), and (iii) tryparedoxin peroxidase (Flohe et al., 2003; KrauthSiegel et al., 2003; Krieger et al., 2000; Steenkamp, 2003). In this regard, a recent study carried out in T. brucei has shown that a decreased expression of trypanothione reductase decreases infectivity and that a diminution of enzyme activity below 10% of the normal level stops cell growth (Krieger et al., 2000). Another thiol found in parasites but not in mammalian cells is ovothiol A; but its physiological role is still unclear. This compound has been identified in trypanosomes, Leishmania, and amebas (Ariyanayagam and Fairlamb, 2001; Ondarza et al., 2003). Studies in tryapanosomatids show that ovothiol A may be present in very high concentrations in some stages and absent in others (Ariyanayagam and Fairlamb, 2001). It has been proposed that ovothiol A is used as an intracellular antioxidant scavenger although it is less efficient than trypanothione as a reductant.
5. Concluding remarks Parasitic protozoa cause various diseases affecting a significant proportion of the world’s population. The differences and limitations in terms of the biochemical
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systems that afford the antioxidant defenses of the parasites provide a valuable target that should be exploited for the development of an effective chemotherapy.
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Review
Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa Julio F. Turrens
*
Department of Biomedical Sciences, College of Allied Health Professions, University of South Alabama, UCOM 6000, Mobile, AL 36695, USA
Abstract Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries. Although these organisms are eukaryotic cells, some of them present major differences with their mammalian host in selected metabolic pathways. These differences may be exploited as targets for developing better pharmacological agents for the treatment of specific parasitic diseases. This review describes some of the differences in terms of antioxidant defenses between these organisms and their mammalian host, which may provide useful targets for the treatment of these diseases. Some of the potential targets are: (i) iron metabolism in Plasmodium, (ii) the presence of a Fe-containing form of superoxide dismutase in trypanosomatids and malaria-causing parasites, (iii) the unique trypanothionedependent antioxidant metabolism in trypanosomatids, (iv) the ascorbate peroxidase found in Trypanosoma cruzi and perhaps present in other trypanosomatids. 2004 Elsevier Ltd. All rights reserved. Keywords: Malaria; Chagas disease; Leishmania; Trypanosomes; Plasmodium; Ameba; Antioxidant enzymes; Peroxiredoxin; Trypanothione; Ovothiol
*
Tel.: +1-251-380-2714/3343802714; fax: +1-251-380-2711/3343802711. E-mail address: [email protected] (J.F. Turrens).
0098-2997/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2004.02.021
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Contents 1.
Parasitic protozoa and parasitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2.
Oxidative metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
3.
Sources of oxidative species unique to some protozoan parasites . . . . . . . . . . . 214
4.
Antioxidant defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
5.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
1. Parasitic protozoa and parasitosis Parasitic protozoa are responsible for various diseases that affect an enormous proportion of the world’s population (Table 1). Malaria, a disease caused by various species of the Genus Plasmodium, is the most predominant of these serious diseases, infecting approximately 300 million people per year and causing the death of about 1–2 million people, mostly children, every year. Another major group of parasitic diseases is caused by various members of the order Kinetoplastida. These diseases include leishmaniases in their many forms (cutaneous, mucocutaneous and visceral, also know as kala azar) and trypanosomiases (African Sleeping Sickness and Chagas disease, Table 1). In addition, other African trypanosomes (Trypanosoma brucei brucei and T. brucei congolense) target cattle causing a disease called ‘‘nagana’’ which has a major impact in the economy and food supply in African nations. A third group of diseases, amebiases, is found all over the world, but it is difficult to estimate the actual numbers of infected patients because only a small proportion become symptomatic (Walsh, 1986). Finally, the spreading of AIDS has also opened the door for opportunistic diseases responsible for severe complications such as those caused by Toxoplasma gondii and Trypanosoma cruzi (Ferreira and Borges, 2002). Since parasitic protozoa are eukaryotic cells, many metabolic pathways are very similar to those of the mammalian host. As a result, parasitic diseases are more difficult to treat than other infections, and many of the drugs used against them tend to have serious side effects. Thus, identifying metabolic differences between parasite and host could define new targets for pharmacological agents that would not interfere with the host metabolism, leading to more effective chemotherapies.
2. Oxidative metabolism Most parasitic protozoa are aerobic cells and live in oxygenated environments. Aerobic metabolism utilizes molecular oxygen as the electron acceptor in energyproducing oxidations, which in turn becomes reduced by four electrons to two
Disease
Agent
Infection rate per year (millions)
Malaria Leishmaniases (cutaneous, mucocutaneous and visceral) Chagas disease African trypanosomiases Amebiasis
Plasmodium falciparum, P. vivax, P. malariae, and P. ovale Leishmania donovani, L. tropica, L. infantum, L. major, L. amazonensis, L. aethiopica, L. mexicana, L. guyanensis, L. peruviana, L. venezuelensis
300 1.5–2
Trypanosoma cruzi Trypanosoma gambiense, Trypanosoma rhodesiense
0.7–0.8 0.3–0.5
Entamoeba histolyica
Unknowna
Total (millions)
At risk (millions)
Deaths/year
800 12
2000 350
2.7 million 59,000
16–18
100 60
45,000 50,000
500a
–
40,000
The information was collected from the World Health Organization’s website (http://www.who.int) and is also available in various WHO reports. The data concerning Amebiasis was reported by Walsh (1986). a Amebiasis is very common, particularly in developing countries. Only 10% of infected people (40–50 million) become symptomatic and very few develop severe symptoms (Walsh, 1986).
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Table 1 Population (in millions) infected and at risk of infection with some of the diseases caused by parasitic protozoan
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molecules of water. However, molecular oxygen may also be partially reduced to relatively stable species by accepting one, two or three electrons, with the formation of superoxide anion, hydrogen peroxide and hydroxyl radical, respectively (Chance et al., 1979; Turrens, 2003). Some of these species (superoxide anion, hydrogen peroxide) are usually the result of undesirable side reactions in redox processes but may also be produced for specific processes including antimicrobial activity and intracellular signaling (Droge, 2002). Although superoxide anion is not very reactive per se, this radical is a precursor of two strong oxidants. First, superoxide may react with nitric oxide to produce peroxynitrite, an oxidizing and nitrating agent generated by macrophages as a first line of defense during phagocytosis. In addition, superoxide anion is a precursor of hydrogen peroxide and, in the presence of transition metals, the two species are responsible for the formation of hydroxyl radicals. The latter is a strong and non-specific oxidant that may attack and damage all life molecules, including proteins, lipids and nucleic acids. Under normal conditions, all these species are maintained at relatively constant intracellular steady-state concentrations by various enzymes and low molecular antioxidants. An imbalance in this steadystate leads to oxidative stress and may be deleterious and even lethal to all forms of life. In this review we will briefly explore some the major biochemical differences in oxidative metabolism, both in antioxidant defenses and in the sources of oxidative species, between mammalian cells and various disease-causing protozoan parasites, which may potentially be targeted for the treatment of parasitic diseases.
3. Sources of oxidative species unique to some protozoan parasites The life cycle of most members of the genus Plasmodium (among them parasites causing human malaria such as P. falciparum) involves a stage as an intracellular parasite in erythrocytes. During this stage the parasites depend on hemoglobin as their primary nutrient and accumulate vast amounts of iron from heme. As mentioned before, iron is a catalyst in the formation of hydroxyl radicals from superoxide anion and hydrogen peroxide, and therefore it is important for these parasites to be able to sequester the excess iron and heme in a form that is innocuous to the parasite. This is achieved by synthesizing an amorphous brown heme aggregate called hemozoin. Some antimalarials such as chloroquine appear to act by interfering with the formation of this pigment and consequently leaving iron available for reactions that are toxic and lethal for the parasite (Sullivan, 2003). The mitochondrial respiratory chain constitutes one of the primary sources of reactive oxygen species in mammalian cells (Chance et al., 1979; Turrens, 2003). In the mammalian respiratory chain, most of the electrons reducing oxygen come from NADH oxidation through the sequence Complex I fi Complex III fi Complex IV fi oxygen. Alternatively, electrons may enter the chain via other flavoproteins (succinate dehydrogenase or Complex II, glycerol-phosphate dehydrogenase, fatty
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acyl CoA dehydrogenase) and be transferred to oxygen via Complexes III and IV, skipping mitochondrial Complex I (Turrens, 2003). The electron transport chain of several parasitic protozoa presents major differences when compared with the host’s counterpart, and as a result, in the sources of the partially reduced oxygen species. For example, while the insect stage of African trypanosomes (procyclic trypomastigotes) has a functional mitochondrial respiratory chain, the bloodstream forms (trypomastigotes) lack most of the electron transport carriers of the respiratory chain (including all the cytochromes) (Hill, 1976; Turrens et al., 1986). The respiratory chain found in African as well as in all stages of American trypanosomes is also quite different from the mammalian counterpart. Studies carried out using specific inhibitors indicate that the electrons from NADH do not reach cytochrome c via Complex III, since this activity is not inhibited by antimycin (Turrens, 1989; Denicola-Seoane et al., 1992; Santhamma and Bhaduri, 1995). In other words, in trypanosomatids, Complex I is either missing or works in an entirely different way. Furthermore, even the Krebs’ cycle (where most of the mitochondrial NADH is produced) works in a different fashion in trypanosomatids. Some of the Krebs’ cycle enzymes that produce NADH in mammalian cells are missing, for example, a-ketoglutarate dehydrogenase, while other dehydrogenases work in different biochemical ways, for example, isocitrate dehydrogenase reduces NADP instead of NAD (Fairlamb and Opperdoes, 1986). Trypanosomatids and plasmodia also have an enzyme not present in mammalian cells, NADH-fumarate reductase, capable of reducing fumarate to succinate, reverting one of the steps of the Krebs’ cycle (Boveris et al., 1986; Fry and Beesley, 1991; Santhamma and Bhaduri, 1995; Turrens, 1989; Turrens et al., 1992). This enzyme may have an important role in the reoxidation of intracellular NADH. The enzyme NADH-fumarate reductase (Turrens, 1987) and maybe other NADHdehydrogenases unique to trypanosomatids (Fang and Beattie, 2003) can directly transfer electrons to oxygen in the absence of their physiological electron acceptors. Perhaps new drugs could be designed to prevent binding of the physiological oxidant in order to stimulate direct transfer of electrons to oxygen. The main drawback of this approach is that, although these enzymes are unique to trypanosomatids, their substrates are important metabolites in mammalian cells and some of these putative inhibitors could still interfere with the intermediate metabolism in the host. Some drugs capable of redox cycling may also selectively increase oxidative stress in the parasite, as long as they selectively accept electrons from oxidoreductases that are unique to the parasite. In fact, although the electron donors have not been identified, several antitrypanosomal drugs, currently used (nifurtimox (Docampo and Moreno, 1984)) or proposed (b-lapachone (Boveris et al., 1978)) appear to undergo redox cycling selectively killing the parasite as a result of the combination of two processes: (i) the formation of oxygen free radicals, and (ii) the deficient antioxidant defenses found in these parasites. This is particularly relevant in the case of T. cruzi since this organism appears to have a higher intracellular steady-state concentration of hydrogen peroxide as a result of its limited antioxidant defenses (Boveris et al., 1980; Giulivi et al., 1988).
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4. Antioxidant defenses Aerobic organisms evolved two major types of antioxidant defenses in order to minimize the oxidative damage resulting from the presence of oxygen in the environment: enzymatic defenses and low molecular weight antioxidants (Chance et al., 1979). In mammalian cells, oxidative stress is prevented by four main antioxidant enzymes (Fig. 1): (i) Cu, Zn-superoxide dismutase (McCord and Fridovich, 1969; Fridovich, 1995), the enzyme responsible for the conversion of superoxide anion into hydrogen peroxide, that is found in the cytoplasm and in the mitochondrial intermembrane space (Okado-Matsumoto and Fridovich, 2001); (ii) Mn-SOD (Fridovich, 1995), with the same action as Cu, Zn-SOD, but only found in the mitochondrial matrix; (iii) glutathione peroxidase, a peroxidase responsible for the reduction of hydrogen peroxide and hydroperoxides that utilizes reduced glutathione as hydrogen donor (Ursini et al., 1995); and (iv) catalase, the enzyme that catalyzes the dismutation of hydrogen peroxide to oxygen and water (Chance et al., 1979). The activity of glutathione peroxidase requires reduced glutathione which becomes oxidized during the reduction of peroxides. Thus, a separate enzyme, glutathione reductase, is required for the NADPH-dependent reduction of oxidized glutathione in order to maintain a steady-state of GSH. Small molecular weight antioxidants include a variety of molecules that directly react with oxygen radicals and prevent them from oxidizing important biomolecules (vitamin E, vitamin C, uric acid, ubiquinone, and various thiol-containing molecules such as cysteine and glutathione). Parasitic protozoa present major differences in terms of antioxidant defenses, not only when compared to the mammalian host but among themselves. For example, trypanosomatids (Leishmania and Trypanosoma) as well as Plasmodium have an iron-containing isoform of SOD (Meshnick et al., 1983; Becuwe et al., 1996), normally found only in bacteria but absent in other eukaryotic cells.
Fig. 1. Antioxidant enzymatic reactions in mammalian cells. Both forms of superoxide dismutase (Cu, Zn-SOD and Mn-SOD) catalyze the dismutation of superoxide anion. This reaction may also occur spontaneously, although at a slower rate than in the presence of these enzymes. The product, hydrogen peroxide, can be either reduced or dismutated by glutathione peroxidase or by catalase, respectively. Glutathione peroxidase may also reduce other peroxides. Oxidized glutathione is in turn reduced by NADPH in a reaction catalyzed by glutathione reductase.
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The enzyme catalase is missing in trypanosomatids (Boveris et al., 1980) and in Plasmodium. The absence of catalase in Plasmodium has been confirmed after decoding this organism genome (see Plasmod.org in Internet). Yet, other protozoan parasites, such as the opportunistic T. gondii, show catalase activity (Kwok et al., 2004). Various isoforms of the Se-dependent glutathione peroxidase have been detected in Plasmodium (Flohe et al., 2003) but they are absent in all trypanosomatids (Boveris et al., 1980; Flohe et al., 2003). Trypanosomatids, on the other hand, do not have a Se-dependent glutathione peroxidase although a non-Se form of the peroxidase has been detected in both African and American trypanosomes (Boveris et al., 1980; Wilkinson et al., 2000; Wilkinson et al., 2003). This form of glutathione peroxidase reacts with organic peroxides but does not reduce hydrogen peroxide. Instead, the members of the Order Kinetoplastida rely in an entirely different thioldependent antioxidant system to eliminate hydrogen peroxide (Fig. 2). All trypanosomatids synthesize trypanothione, a dithiol derived from glutathione that is unique to the members of the order Kinetoplastida and that has been proposed as a suitable target to develop drugs for the treatment of leishmaniases and trypanosomiases (Fairlamb and Cerami, 1992; Flohe et al., 2003; Krauth-Siegel et al., 2003; Steenkamp, 2003). Trypanothione (bis(glutathiolnyl)spermidine) consists of two molecules of glutathione conjugated with a molecule of spermidine. Although the reduction potential of the thiol groups is the same that of glutathione, their pKa is a whole pH unit lower than that of free glutathione, probably because of electron displacement caused by the extra amino group found in spermidine (Krauth-Siegel et al., 2003). This different pKa may explain trypanothione’s distinct reactivity. For
Fig. 2. Antioxidant metabolism in Kinetoplastida. These cells contain a different form of superoxide dismutase (Fe-SOD) and a unique battery of enzymes involved in peroxide metabolism. These cells do not have either catalase or glutathione peroxidase. Instead, most of their glutathione is converted into trypanothione which in turn may spontaneously reduce tryparedoxin and dehydroascorbate. Reduced tryparedoxin is the hydrogen donor for the tryparedoxin peroxidase reaction. An ascorbate peroxidase has been detected in Trypanosoma cruzi. The enzymes involved in trypanothione metabolism, both in its synthesis and in its reduction, have been proposed as possible targets against the diseases caused by parasites from the genus Leishmania and Trypanosoma.
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example, dehydroascorbate, the oxidation product of ascorbic acid, is reduced by trypanothione but not by glutathione. Similarly, trypanothione directly reduces oxidized tryparedoxin, a thioredoxin found in trypanosomatids which has a central role in peroxide metabolism (see below) (Krauth-Siegel et al., 2003; Steenkamp, 2003). These two non-enzymatic reactions may be vital antioxidant processes in trypanosomatids. First, in T. cruzi dehydroascorbate is produced during the reduction of H2 O2 catalyzed by ascorbate peroxidase (Docampo et al., 1976; Boveris et al., 1980; Wilkinson et al., 2002), trypanothione may regenerate ascorbic acid. Second, the most important peroxidatic system common to all trypanosomatids relies on an enzyme called tryparedoxin peroxidase. Tryparedoxin peroxidase is one of the peroxiredoxins, a family of highly preserved small molecular weight (around 20–30 kDa) cysteine-containing proteins found in most organisms (Rhee et al., 2001; Wilkinson et al., 2002). Most cells, including Plasmodium falciparum (Krnajski et al., 2002), require specific thiol-dependent peroxiredoxin reductases (for example, glutathione-dependent) in order to reduce the thiol groups in peroxiredoxins. In trypanosomatids, however, it has been postulated that a tryparedoxin reductase may not be needed because of the peculiar reactivity of trypanothione as a reductant of tryparedoxin (Schmidt and Krauth-Siegel, 2003). The combined action of trypanothione reductase, tryparedoxin and tryparedoxin peroxidase is central to the maintenance of a low steady-state concentration of H2 O2 (Fig. 2). The vital role of trypanothione, a low molecular weight antioxidant not present in mammalian cells, as an antioxidant in the hydroperoxide metabolism of trypanosomatids provides various potential targets for the treatment of leishmaniases and trypanosomiases. These targets are: (i) trypanothione reductase, the NADPHdependent enzyme that regenerates reduced trypanothione; (ii) the enzymes involved in the synthesis of trypanothione (glutathionylspermidine synthetase and trypanothione synthetase), and (iii) tryparedoxin peroxidase (Flohe et al., 2003; KrauthSiegel et al., 2003; Krieger et al., 2000; Steenkamp, 2003). In this regard, a recent study carried out in T. brucei has shown that a decreased expression of trypanothione reductase decreases infectivity and that a diminution of enzyme activity below 10% of the normal level stops cell growth (Krieger et al., 2000). Another thiol found in parasites but not in mammalian cells is ovothiol A; but its physiological role is still unclear. This compound has been identified in trypanosomes, Leishmania, and amebas (Ariyanayagam and Fairlamb, 2001; Ondarza et al., 2003). Studies in tryapanosomatids show that ovothiol A may be present in very high concentrations in some stages and absent in others (Ariyanayagam and Fairlamb, 2001). It has been proposed that ovothiol A is used as an intracellular antioxidant scavenger although it is less efficient than trypanothione as a reductant.
5. Concluding remarks Parasitic protozoa cause various diseases affecting a significant proportion of the world’s population. The differences and limitations in terms of the biochemical
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systems that afford the antioxidant defenses of the parasites provide a valuable target that should be exploited for the development of an effective chemotherapy.
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