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Chemotherapeutic agents used against Chagas disease and redox metabolism

The enzymatic deficiencies of T. cruzi against oxygen toxicity were correlated with their sensitivity to both intracellularly generated and phagocyte-derived by-products of O2 reduction.15,17 The chemotherapeutic potential of these enzyme deficiencies was first recognized during work on the mode of action of the trypanocidal o-naphthoquinone (3-lapachone and derivatives.15,17 These studies showed that the metabolism of these compounds by T. cruzi involved, at least in part, the generation of superoxide anion and hydrogen peroxide. H2O2 accumulated in the cells to cytotoxic levels and was also excreted.15,17

The chemotherapeutic implications of these deficiencies were also apparent in the case of nifurtimox (Fig. 17.1). One-electron reduction of nifurtimox to a nitro anion radical followed by autoxidation of this radical with generation of superoxide

anion and other oxygen reduction by-products, such as H2O2, and hydroxyl radical were implicated in the trypanocidal and mammalian toxic effects of this drug.17 More recent work in this area revealed the presence in T. cruzi of alternative activation mechanisms of nifurtimox by two enzymes that apparently catalyze its two- electron reduction. 0ne is the 0ld Yellow Enzyme, also known as prostaglandin F2a synthase170 and the other is a type I nitroreductase (TcNTR)171 that generates nitrile metabolites as final products. Interestingly, Kubata et al. could detect the one-electron reduction of naphthoquinones to semiquinones but not of nifurti- mox to nitro anion radicals using the recombinant 0ld Yellow Enzyme. These negative results, which were not shown, contradict the well-known ability of flavoproteins to catalyze reduction of naphthoquinones and nitrofurans equally well.173 Furthermore, no evidence was presented for the postulated two-electron reduction of nifurtimox.170 Detection of nitro anion radicals requires more strict anaerobic conditions than detection of semiquinones, which evidently were not obtained by the authors. However, these negative results were used by other authors172,174 to propose that two-electron reduction could be more relevant for T. cruzi toxicity, although not for mammalian toxicity, than the one-electron reduction catalyzed by type II nitroreductases, with generation of reactive oxygen species (ROS). It is important to also mention that these works172,174 were done with either the recombinant enzymes or with the epimastigote (culture) form of T. cruzi, while a nitro anion radical derivative of nifurtimox as well as redox cycling with generation of superoxide anion and H2O2 are easily detected using T. cruzi amastigote and trypomastigote homogenates.111 In addition, evidence of oxidative stress by nifurti- mox, as revealed by increased steady state concentration of H2O2 and lipid peroxidation in intact epimastigotes175 or depletion of low molecular weight thiols in different strains and life cycle stages of T. cruzi174,176 was presented by several authors. In the case of the studies with the recombinant TcNTR the authors172 found that 100 pM nifurtimox in the presence of NADH generated an increase in O2 consumption (compatible with redox cycling) although they did not test other concentrations, or the generation of ROS, or studied if it was possible to detect a nitro anion radical under anaerobic conditions.

In conclusion, there is evidence of one-electron reduction of nifurtimox with generation of ROS in live epimastigotes109,175 and by enzymes present in amasti- gotes and trypomastigotes.111 It is possible that two-electron reduction could also occur (in epimastigotes) but one-electron reduction of nifurtimox and subsequent redox cycling with generation of ROS in T. cruzi cannot be ruled out as a mechanism of toxicity against T. cruzi if nifurtimox accumulates to high levels in the parasite.

In contrast to nifurtimox, the direct involvement of oxygen reduction products in the trypanocidal action of benznidazole, which is a 2-nitroimidazole (Fig. 17.1), could be ruled out.17 As the rate of reduction of benznidazole is very low because of its lower reduction potential, redox cycling is considered a detoxification reaction that occurs by inhibition of the net reduction of the drug. The resultant low steady state concentration of superoxide anion might be easily detoxified by the superoxide dismutases present in T. cruzi.17,177 Reduction of benznidazole by recombinant TcNTR was reported to result in the generation of the cytotoxic metabolite glyoxal.178 However, recent studies

indicated that benznidazole treatment of epimastigotes resulted in significant decrease in redox active thiols, apparently by covalent adduct formation with reduced benznidazole, but no formation of glyoxal was detected.179

Reactive oxygen species are also involved in the photodynamic action of crystal violet that has been described in T. cruzi.180 Visible light causes photoreduction of crystal violet to a carbon-centered radical. Under aerobic conditions this free radical autooxidizes generating superoxide anion whose dismutation yields H2O2.180 Reducing agents known to enhance free radical formation from crystal violet in the presence of light enhance redox cycling of this dye.181 In contrast to other photosensitizers, irradiation of crystal violet with visible light does not generate detectable amount of singlet oxygen.181 The trypanocidal effect of crystal violet on T. cruzi epimastigotes and trypomastigotes is also enhanced by light.180 The chemo- prophylactic potential of the photodynamic action of crystal violet for the prevention of blood transmission of Chagas disease was also explored.182 It was demonstrated that photoreduction with visible light in the presence of ascorbate reduces the effective dose and time of contact of the dye with T. cruzi-infected blood.182 The scheme shown in Fig. 17.6 has been proposed to explain the enhancement of the cytotoxicity of crystal violet against T. cruzi by ascorbate.182 In reaction A, ascorbate anion reduces crystal violet under illumination. Under aerobic conditions, the crystal violet carbon-centered free radical then reduces O2 to superoxide anion (reaction B); dismu- tation of superoxide anion produces H2O2 (reaction C). Superoxide dismutase increases the rate of H2O2 formation by catalyzing reaction C. The oxidation of ascorbate by superoxide anion contributes to the formation of H2O2 and is responsible for the generation of the ascorbyl radical that is detected in incubations of T. cruzi- infected blood upon illumination.182 When catalase is present (as occurs in red and white blood cells but not in T. cruzi) H2O2 is detoxified. Formation of H2O2 may explain the photodynamic action of crystal violet/ascorbate on T. cruzi182 since the sensitivity of different T. cruzi stages to reagent H2O2, enzymatically generated H2O2, H2O2-generating drugs, and H2O2-generating phagocytic cells has been well documented.17

Mechanism of ascorbate enhancement of crystal violet toxicity

Figure 17.6 Mechanism of ascorbate enhancement of crystal violet toxicity. Light (hv) catalyzes the conversion of crystal violet into a carbon-centered radical (CV) in the presence of ascorbate (AH2), which generates ascorbyl radical (A2) (reaction A). Autoxidation of the carbon-centered radical generates superoxide anion (O22) (reaction B) that dismutates to H2O2 (reaction C) in the presence of SOD or reacts with ascorbate to generate more ascorbyl radical. H2O2 can be decomposed by catalase (CAT).

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