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Oxidant and Antioxidants

Key issues 1. Definitions 2. Range of axidants and antioxidants in parasitesm 3. Therapeutical applications Some definitions An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidative stress refers to an imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to damage.

Introduction
Oxidation reactions can produce free radicals which can in turn start chain reactions. Free radicals are very reactive and can propagate by getting involved in chain reactions with less reactive types (Lavon J Dunne, 2001). Such chain reactions can damage biomolecules of the pathogen such as enzymes and DNA. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols (Sies, Helmut, 1997). Known free radicals are hydroxyl, superoxide and peroxide. Antioxidant defence involves several strategies, both enzymatic and non-enzymatic. Low levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Because they live in oxygen-poor environments, parasites are particularly sensitive to oxidative stress. Catalase is absent in many. Glutathione peroxidase activities are very low or absent, and in some cases, such as in trypanosomatids, replaced by trypanothione-dependent peroxidase activities. In any case, peroxidase activities are quite low when compared with the glutathione peroxidase activities in mammalian organs. Superoxide dismutases, however, are present in most parasites, and these are features typical of facultative aerobes. Since superoxide dismutase catalyzes the dismutation of superoxide anion to form hydrogen peroxide and there is a deficiency in hydrogen peroxidemetabolizing systems, this explains why superoxide anion is so toxic to parasites. Parasites may tolerate a slow endogenous rate of hydrogen peroxide generation in their natural habitats but they are quite sensitive to an increased steady-state concentration of hydrogen peroxide as a result of drug metabolism or phagocytic cell attack. Reactive oxygen species are important mediators of several forms of cell damage in parasites and this discussion will focus on the defense mechanisms of parasites against these species.

Cellular mediators of innate immune system are phagocytes (neutrophils, monocytes or macrophages). Fusion of phagosome with lysosome causes release of toxic macromolecules into the phagosome. The mechanisms phagocytes use to kill pathogens are diverse and complex. They divided into oxygen-dependent and oxygen-independent mechanisms. Killing mechanisms of macrophages

Generation of free radicals and toxicity of oxygen in parasites Binding of Fc receptors on phagocytes causes increase in its oxygen uptake called the respiratory burst. This influx of oxygen uses oxidative mechanisms to damage microbes within phagolysosome. Binding of Fc receptors activates an NADPH oxidase that reduces O 2 to O2- (superoxide). Superoxide can decay to hydroxide radical (OH.) or be converted into hydrogen peroxide (H2O2) by the enzyme superoxide dismutase. 2H + + O2-+ O2- H2O2 + O2 In neutrophils, H2O2 and chloride ion (Cl-) are catalysed by myeloperoxidase to singlet oxygen (1O2), (another reactive oxygen species) thru hypochlorous acid (HOCl). Parasites are highly sensitive to reagent H2O2, to different O2- (such as autoxidation of thiols, xanthine-xanthine oxidase, dihydro-orotate-dihydro-orotate oxidase) and H2O2-generating systems (such as glucose-glucose oxidase), and organic peroxides (such as tert-butyl hydroperoxide) (Docampo, R. et al, 1984) They are sensitive to both drug-generated and phagocyte-derived oxygen reactive species. H2O2 and O2- are damaging because they can give rise to the extremely reactive hydroxyl radical (OH-) in the presence of transition metal ions (Fenton reaction)

O2- + M n+1 M n + O2 M n + H2O2 M n + 1 + OH- + OH. O2 + H2O2 O2 + OH- +u OH. A newly proposed mechanism for hydroxyl radical production is without transition metal ions and it involves the production of peroxynitrite (ONOO-) arising from the reaction of nitric oxide (NO.) with superoxide (O2-) as below: NO. + O2 ONOO ONOO- + H + ONOOH ONOOH OH. + NO2. The combination of NO. with superoxide competes with the dismutation of superoxide: 2H + + O2-+ O2- H2O2 + O2 Peroxynitrite (pKa of 7.5) is substantially protonated at physiological pH, to produce peroxynitrous acid (ONOOH). One route for the decomposition of this molecule is by homolytic cleavage to produce hydroxyl radical (OH.) and nitrogen dioxide (NO2.). All of these toxic oxygen species (hydroxyl, superoxide, peroxynitrate and peroxide) are potent oxidizers and attack many targets in the pathogen. Example of Nitric Oxide NO reacts with biomolecules (such as enzymes) containing iron-sulfur centers and inactivates them. For example, leishmania parasites utilize an iron-containing superoxide dismutase (Bukrinsky, M. I. et al, 1995), and inactivation of this enzyme could considerably weaken the ability of the parasites to withstand attack by reactive oxygen intermediates.

Effect of oxygen on some parasites Parasite Oxygen living condition Rumen ciliates and Termite Obligate anaerobes flagellates

Remarks/effects of oxygen Lack defenses against reactive oxygen species hence readily killed by high oxygen tensions Higher oxygen tensions decrease respiration rapidly leading to toxicity Trichomonas vaginalis microaerophilic is

Other rumen protozoa

Other protozoa Entamoeba spp. Trichomonas spp. Trypanosomatids Parasitic Helminths

Can respire under low oxygen tensions hence survive at low oxygen concentrations of rumen fluid e.g. Less sensitive to oxygen and damage. Can be cultivated indefinitely under anaerobic conditions Can survive anaerobically Facultatively aerobic

Cannot multiply in anaerobic conditions Show various degrees of tolerance to lack of oxygen

Fasciola hepatica and large Predominantly anaerobic Live in oxygen poor intestinal helminths (Ascris, environments Monieza e.t.c) REACTIVE OXYGEN SPECIES IN SPECIFIC PARAS REACTIVE OXYGEN SPECIES IN SPECIFIC PARASITESS Reactive oxygen species play important roles in many biochemical reactions that maintain normal cell functions. They are produced as normal intermediates in mitochondrial and microsomal electron-transport systems, and some enzymes that produce substantial amounts of O2 or H2O2 have been identified in several parasites (Docampo, R. et al, 1984). Tritrichomonas foetus aerobically excretes H2O2 (Ninomiya, M. and Ziro, S., 1952). In contrast, respiring Trypanosoma cruzi does not release either O2 or H2O2 to the suspending medium, although T. cruzi homogenates supplemented with reduced nicotinamide adenine dinucleotides are effective sources of O2 and H2O2. H2O2 formation by mitochondrial preparations of the protozoan C. fasciculata is qualitatively the same as that by mitochondria from vertebrate tissues. Mitochondrial succinate oxidase, in large helminths such as F. hepatica, Ascaris lumbricoides and Monieza expansa, forms H2O2 from oxygen instead of water and is insensitive to cyanide, azide and antimycin. These large helminths also contain oxidases: cytochrome o and a3. The utilization of NAD(P)H and succinate by Hymenolepis diminuta mitochondrial membranes also results in H2O2 formation.

ENZYMATIC DEFENSES OF PARASITES AGAINST REACTIVE OXYGEN SPECIES 1. Catalase Catalase plays two roles (1) decomposition of H2O2 to give H2O and O2 (catalatic activity) and (2) oxidation of electron donors, e.g., methanol, ethanol, formic acid, phenols, with the consumption of 1 mol of peroxide (peroxidatic activity): 2H2O2 2H2O + O2 (catalatic activity) ROOH + AH2 H2O + ROH + A (peroxidatic activity) Catalase is present in the cytoplasm of T. foetus (Beaulieu, Jr, B. B, et al, 1981) but absent from other Trichomonas species, Giardia lamblia and E. histolytica. Although catalase is absent in most pathogenic trypanosomatids such as T. brucei, T. cruzi, and most Leishmania spp., it is present in other mammalian trypanosomes as well as avian, amphibian and insect trypanosomes. The 3,3'-diamionobenzidine reaction is a cytochemical technique used to demonstrate peroxidase activity in microbodies. 3,3'-diaminobenzidine-positive microbodies are present in lower trypanosomatids. For example, Crithidia fasciculate possesses a high activity of catalase and their catalasecontaining microbodies occupy about 2.5% of the total cell volume. Toxoplasma gondii tachyzoites also have an abundant catalase activity. Catalase activity is low in trematodes, cestodes and nematodes, although it has been detected in significant amounts in the nematodes Ancylostoma ceylanicum and Nippostrongylus brasiliensis. It is undetectable in newborn larvae, adult worms, and muscle larvae of Trichinella spiralis as well as in F. hepatica and H. diminuta. In contrast Helioomosomoides polygyrus contains detectable levels of the enzyme. Surprisingly, a substantial catalase activity has been found in isolated A. Suum mitochondria. 2. Glutathione Peroxidase and Other Peroxidase Activities Glutathione peroxidase catalyzes the reaction of hydroperoxides (ROOH) with reduced glutathione (GSH) to form oxidized glutathione disulfide (GSSG) and the reduction product of the hydroperoxides (ROH): ROOH + 2GSH ROH + GSSG + H2O Since many of the radical or non-radical reactions in cells may lead to thiol oxidation to the disulfide, i.e., the oxidation of glutathione to form GSSG, the regenerative reaction of rereduction to GSH catalyzed by GSSG reductase can become pivotal in antioxidant defense: GSSG + NADPH + H + 2GSH + NADP + No glutathione peroxidase activity has been detected in E. histolytica and G. Lamblia. Therefore E. histolytica, G. duodenalis and T.foetus lack glutathione metabolism, and cysteine is the main thiol component present in thse parasites. T. vaginalis also lacks detectable GSSG reductase activity. Other thiol-dependent peroxidase and reductase activities are present in G. intestinalis but the nature of the thiol cofactors is not known.

Trypanosomatids do not have glutathione peroxidase. Some of them have a peroxidase activity dependent on a glutathione-spermidine cofactor termed trypanothione (T(SH)2). Oxidation of trypanothione leads to the formation of trypanothione disulfide (T(S)2). Trypanothione reductase has been found only in the cytosol of T. brucei and it catalyzes the NADPH-dependent reduction of trypanothione, but not glutathione. Ultimately, dihydrotrypanothione can undergo a rapid non-enzymatic disulfide exchange reaction with intracellular disulfides (RSSR), among them oxidized glutathione and cysteine (Frappier, F., Azoulay, M. and Leroy, J.-P., 1988). T(SH)2 + RSSR T(S)2 + 2RSH H2O2 decomposition in T. cruzi is due to non-enzymatic reactions of endogenous reduced thiols with peroxides, a true trypanothione peroxidase is absent in these cells. Other protozoa lack Trypanothione. A glutathione peroxidase activity is present in T. gondii tachyzoites (Yawetz, A. and Agosin, M.,1979). Glutathione peroxidase and glutathione reductase are present P. falci parum, P. vinckei and P. Berghei but this could have been derived from erythrocytes. F. hepatica lacks glutathione peroxidase. Glutathione peroxidase activity in Schistosoma mansoni increases significantly as worms mature in their host and is positively correlated to the resistance to antioxidants. Newborn larvae of T. Spiralis lack detectable glutathione peroxidase, whereas, adult worms and muscle larvae contain a high activity of this enzyme. Ancylostoma ceylanicum and Nippostrongylus brasiliensis contain significant glutathione peroxidase activities. Other peroxidases less studied are an ascorbate peroxidase present in T. cruzi, cytochrome c peroxidases in mitochondrial preparations of C. fasciculata and in A. lumbricoides and F. hepatica, as well as peroxidases found in F. hepatica, H. diminuta, M. expansa, the hemocele fluid of A. lumbricoides, and the miracidium of S. mansoni. Their role in H2O2 detoxification is unknown. 3. Superoxide Dismutase Superoxide dismutase (SOD) is a widely distributed enzyme that exists in a variety of forms. Amino acid sequence homologies indicate two families of superoxide dismutases. i.e. The copper-zinc enzyme (Cu, ZnSOD) ( primarily in cytosol of eukaryotic cells) and a family of manganese-containing enzyme (MnSOD)(mitochondrial) and ferrienzyme (FeSOD) in bacteria. These superoxide dismutases catalyze the same reaction (2H + + O2- + O2- H2O2 + O2) and with comparable efficiency. In general, protozoa, as eukaryotic algae, lack the Cu, Zn enzyme. E. dispar and E. histolytica lysates contain FeSODs. T. cruzi contains a cyanide-sensitive SOD activity. Cyanide insensitive SODs are also present in trypanosomatids, such as C. fasciculata and Trypanosoma brucei. The two SOD activities in T. brucei are insensitive to 1 mM cyanide. The SOD activities of T. brucei, L. tropica and T. cruzi are also cyanide-insensitive but peroxide- and azide-sensitive.

SOD of C. Fasciculate is located in the cytosol and exists in three forms. P. falciparum contain peroxide-sensitive, apparently manganese-containing SOD. Two Babesia species (B. hylomysci and B. divergens) also contain an endogenous superoxide dismutase that is cyanide-insensitive and inhibited by H2O2 indicating that iron is the cofactor metal. A similar situation has been found in T. gondii where the enzyme(s) are inhibited by azide and peroxide but not by cyanide. S. mansoni possess a Cu, ZnSOD. Extracts of F. hepatica show appreciable SOD activity. SOD activities are also present in H. diminuta extracts and in the A. lumbricoides but they have not been characterized. A cyanide-sensitive SOD has been purified from metacestodes of Taenia taeniformis. SOD activity is present in newborn larvae, adult worms, and muscle larvae of T. spiralis, the activity of newborn larvae being significantly lower than that of the other stages (Clark, A. G., 1989). A Cu, ZnSOD has been isolated and purified from muscle larvae of T. spiralis that is apparently secreted extracellularly. A from Onchocerca volvulus contains a Cu, ZnSOD. Other nematodes, such as Trichostrongylus vitrinus, Trichostrongylus colubriformis, Nematodiurus battus, Teladorsagia circumcincta, Nippostrongylus brasiliensis and Haemonchus contortus have higher superoxide dismutase activities in the third larval stages of each specie with marked interspecies variations in isoenzyme profiles. NON-ENZYMATIC DEFENSES OF PARASITES AGAINST REACTIVE OXYGEN SPECIES Detoxication of reactive oxygen species is also possible through the action of several lowmolecular-weight compounds. Some are water soluble, such as ascorbic acid, thiols, pyruvate and urate, while others are lipid soluble, such as vitamin E and fl-carotone. GSH is absent in some parasites such as E. histolytica , Tritrichomonasfoetus and Giardia duodenalis where cysteine is the main thiol present. However GSH is present in most parasites at varying concentrations e.g. it is low in trypanosomatids such as T. cruzi . In most trypanosomatids investigated thus far dihydrotrypanothione (T(SH2)) is present in concentrations of about 0.15 mM (Holy, J. M.,et al, 1989). T(SH2) is the principal intracellular thiol during exponential growth of C. fasciculata, whereas monoglutathionylspermidine is the major thiol in stationary phase. The net result of this biochemical reshuffle of metabolites can be described as follows: 2GSH-SPD T(SH2) + spermidine which results in no net change in free GSH, but in the release of unconjugated spermidine. Trypanosomatids, as well as G. lamblia and Trichomonas spp. lack xanthine oxidase and therefore uric acid, another free radical scavenger, is absent. In contrast, a xanthine oxidase activity has been reported in Ancylostoma ceylanicum and Nippostrongylus brasiliensis. There are no reports of the presence of vitamin E and fl-carotene in parasites.

Therapeutic applications Oxidants and Antioxidants Biological and Pharmacological consideration Since most biochemical processes are common to all organisms, the efficacy of an antiparasitic agent generally is a balance between its potency against the parasite's metabolism and its activity against the human host's metabolism. This latter is generally described as toxicity of the compound and the net effect of the two processes is termed the therapeutic index. In general, the more specific the compound for the metabolism of the parasite the greater the therapeutic index. 1. Trypanothione Trypanothione metabolism in the hemoflagellates may emerge as an important chemotherapeutic target. Trypanothione is essential for the survival of the parasite and is absent from its host (hosts have Glutathione). Glutathione is the major intracellular thiol in aerobic organisms where it functions to provide the cell with a reducing environment. In trypanosomatids, greater than 70% of the total intracellular glutathione is present as the spermidine conjugate trypanothione. The levels of this compound are maintained by the activity of trypanothione reductase. Inhibitors of this enzyme may serve as potent chemotherapeutic agents for the trypanosomatids. Biosynthesis of trypanothione is another potential target for chemotherapy since it utilizes enzymes which are not normally active in the mammalian cell. 2. Anaerobic energy metabolism Anaerobic parasites, such as T. vaginalis and E. histolytica, lack functional mitochondria. Pyruvate generated by the glycolytic pathway is further metabolized within the cytosol of Giardia and Entamoeba species and in the hydrogenosome of these parasites, a subcellular organelle unique to trichomonads. Under anaerobic conditions, pyruvate is converted into acetyl Co-A and CO2 by pyruvate:ferredoxin oxidoreductase with the concomitant reduction of ferredoxin. Metronidazole and other nitroimidazole derivatives are toxic to these anaerobes after reduction of the nitro moiety by ferredoxin. As these compounds are not reduced in mamalian cells, selectivity of their antiparasitic effects is assured. Carbohydrate metabolism in African trypanosomes is compartmentalized within the glycosome. Many structural and kinetic differences exist between the glycosomal and cytoplasmic glycolytic pathways. The high isoelectric point of the glycosomal enzymes has been postulated to be the mechanism by which suramin exerts its antitrypanosomal effect. Reducing equivalents produced by glycolysis withinthe glycosome are transferred to a mitochondrial glycerol-3phosphate oxidase complex. Inhibitors of this complex, such as salicylhydroxamic acid (SHAM) and esters of dihydroxybenzoate, in conjunction with glycerol, have exhibited good antitrypanosomal activity in vitro and in animal models but not in clinical trials.

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